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This article discusses the use of CRISPRi technology, which stands for clustered regularly interspaced short palindromic repeats interference, in pathogenic streptococcal and enterococcal species. CRISPRi involves using a catalytically dead Cas effector (dCas) to bind to a gene and reduce its transcription, resulting in a knockdown of gene expression. The article provides examples of how CRISPRi has been applied in Enterococcus faecalis, Streptococcus pyogenes, Streptococcus agalactiae, and Streptococcus pneumoniae, and discusses future applications of CRISPRi technology, such as direct genome editing, gene silencing, and CRISPRseq. The article highlights the potential of CRISPRi technology in various bacterial species and suggests that it may have valuable applications in fields such as agriculture. [Extracted from the article]

Replication of the complex retrovirus mouse mammary tumor virus (MMTV) is antagonized by murine Apobec3 (mA3), a member of the Apobec family of cytidine deaminases. We have shown that MMTV-encoded Rem protein inhibits proviral mutagenesis by the Apobec enzyme, activation-induced cytidine deaminase (AID) during viral replication in BALB/c mice. To further study the role of Rem in vivo, we have infected C57BL/6 (B6) mice with a superantigen-independent lymphomagenic strain of MMTV (TBLV-WT) or a mutant strain that is defective in Rem and its cleavage product Rem-CT (TBLV-SD). Compared to BALB/c, B6 mice were more susceptible to TBLV infection and tumorigenesis. Furthermore, unlike MMTV, TBLV induced T-cell tumors in B6 μMT mice, which lack membrane-bound IgM and conventional B-2 cells. At limiting viral doses, loss of Rem expression in TBLV-SD-infected B6 mice accelerated tumorigenesis compared to TBLV-WT in either wild-type B6 or AID-knockout mice. Unlike BALB/c results, high-throughput sequencing indicated that proviral G-to-A or C-to-T mutations were unchanged regardless of Rem expression in B6 tumors. However, knockout of both AID and mA3 reduced G-to-A mutations. Ex vivo stimulation showed higher levels of mA3 relative to AID in B6 compared to BALB/c splenocytes, and effects of agonists differed in the two strains. RNA-Seq revealed increased transcripts related to growth factor and cytokine signaling in TBLV-SD-induced tumors relative to TBLV-WT-induced tumors, consistent with another Rem function. Thus, Rem-mediated effects on tumorigenesis in B6 mice are independent of Apobec-mediated proviral hypermutation. Author summary: Retroviruses cause lifelong infections resulting from their ability to thwart innate immunity. The Apobec family of cytidine deaminases are part of the innate immune response that recognizes and mutates foreign nucleic acids, including those from multiple viruses. Retroviral antagonists of Apobecs have been identified, including mouse mammary tumor virus (MMTV)-encoded Rem protein. Previous experiments have shown that Rem-null MMTV or closely related TBLV proviruses from BALB/c tumors accumulate G-to-A and C-to-T mutations typical of Apobecs compared to wild-type proviruses expressing Rem. The difference in mutations between Rem-expressing and non-expressing MMTV strains largely disappeared in mice lacking the Apobec family member, activation-induced cytidine deaminase (AID). These results suggested that Rem is an AID antagonist. In this study, we attempted to study AID-mediated mutations of TBLV proviruses lacking Rem expression obtained from tumors in C57BL/6 (B6) wild-type and AID-knockout backgrounds. Surprisingly, no differences in G-to-A mutations were observed in TBLV proviruses regardless of Rem expression, yet such mutations were significantly reduced in proviruses obtained from mA3/AID-double knockout mice relative to those from wild-type B6 or AID-knockout mice. Many cellular mRNAs involving the innate immune response, but not Apobecs, were elevated in the absence relative to the presence of Rem expression on the B6 AID-knockout background. These results revealed that Apobec-mediated mutagenesis is dependent on mouse strain and suggested a second means of Rem-dependent immune evasion. [ABSTRACT FROM AUTHOR]

R-loops are trimeric nucleic acid structures that form when an RNA molecule hybridizes with its complementary DNA strand, displacing the opposite strand. These structures regulate transcription as well as replication, but aberrant R-loops can form, leading to DNA breaks and genomic instability if unresolved. R-loop levels are elevated in many cancers as well as cells that maintain high-risk human papillomaviruses. We investigated how the distribution as well as function of R-loops changed between normal keratinocytes and HPV positive cells derived from a precancerous lesion of the cervix (CIN I). The levels of R-loops associated with cellular genes were found to be up to 10-fold higher in HPV positive cells than in normal keratinocytes while increases at ALU1 elements increased by up to 500-fold. The presence of enhanced R-loops resulted in altered levels of gene transcription, with equal numbers increased as decreased. While no uniform global effects on transcription due to the enhanced levels of R-loops were detected, genes in several pathways were coordinately increased or decreased in expression only in the HPV positive cells. This included the downregulation of genes in the innate immune pathway, such as DDX58, IL-6, STAT1, IFN-β, and NLRP3. All differentially expressed innate immune genes dependent on R-loops were also associated with H3K36me3 modified histones. Genes that were upregulated by the presence of R-loops in HPV positive cells included those in the DNA damage repair such as ATM, ATRX, and members of the Fanconi Anemia pathway. These genes exhibited a linkage between R-loops and H3K36me3 as well as γH2AX histone marks only in HPV positive cells. These studies identify a potential link in HPV positive cells between DNA damage repair as well as innate immune regulatory pathways with R-loops and γH2AX/H3K36me3 histone marks that may contribute to regulating important functions for HPV pathogenesis. Author summary: R-loops are trimeric RNA: DNA hybrids that regulate transcription but can also lead to DNA breaks. R-loop levels are elevated in many cancers as well in precancerous cells containing high-risk HPVs. How the distribution and function of R-loops changes due to the presence of viral genomes was examined by comparing cells derived from an HPV 31 positive precancer (CIN 612) to normal keratinocytes. Up to 10-fold higher R-loop levels were detected on cellular genes in CIN 612 cells, with over 500-fold increases at ALU elements. Analysis of the changes in R-loop landscape between normal and HPV positive cells identified a functional association between R-loop formation, gene expression, and chromatin states. R-loop formation in HPV postive cells was equally associated with negatively and positively regulated gene expression; however, coordinated expression of genes in specific pathways by R-loops was observed. This included repression of genes in the innate immune surveillance pathway, while those regulating DNA damage repair and metabolism were activated. [ABSTRACT FROM AUTHOR]

The ability to both sensitively and specifically assess the sequence composition of a nucleic acid strand is an ever-growing field. Designing a detection scheme that can perform this function when the sequence of the target being detected deviates significantly from the canonical sequence however is difficult in part because probe/primer design is based on established Watson-Crick base-pairing rules. We present here a robust and tunable toehold-based exchange probe that can detect a sequence with a variable number of SNPs of unknown identity by inserting a series of controlled, sequential mismatches into the protector seal of the toehold probe, in an effort to make the protector seal "sloppy". We show that the mismatch-tolerant system follows predicted behavior closely even with targets containing up to four mismatches that thermodynamically deviate from the canonical sequence by up to 15 kcal/mole. The system also performs faithfully regardless of the global mismatch position on either the protector seal or target. Lastly, we demonstrate the generalizability of the approach by testing the increasingly mismatch-tolerant protectors on HIV clinical samples to show that the system is capable of resolving multiple, iteratively mutated sequences derived from numerous HIV sub-populations with remarkable precision. [ABSTRACT FROM AUTHOR]

Tongue swabs hold promise as a non-invasive sample for diagnosing tuberculosis (TB). However, their utility as replacements for sputum has been limited by their varied diagnostic performance in PCR assays compared to sputum. The use of silica-based DNA extraction methods may limit sensitivity due to incomplete lysis of Mycobacterium tuberculosis (MTB) cells and co-extraction of non-target nucleic acid, which may inhibit PCR. Specificity may also be compromised because these methods are labor-intensive and prone to cross-contamination. To address these limitations, we developed a sample preparation method that combines sonication for MTB lysis and a sequence-specific MTB DNA capture method using hybridization probes immobilized on magnetic beads. In spiked tongue swabs, our hybridization capture method demonstrated a 100-fold increase in MTB DNA yield over silica-based Qiagen DNA extraction and ethanol precipitation. In a study conducted on clinical samples from South Africa, our protocol had 74% (70/94) sensitivity and 98% (41/42) specificity for detecting active pulmonary TB with sputum Xpert MTB/RIF Ultra as the reference standard. While hybridization capture did not show improved sensitivity over Qiagen DNA extraction and ethanol precipitation, it demonstrated better specificity than previously reported methods and was easier to perform. With integration into point-of-care platforms, these strategies have the potential to help enable rapid non-sputum-based TB diagnosis across key underserved patient populations. [ABSTRACT FROM AUTHOR]

Background: The SARS-CoV-2 Omicron variant, with the main subtypes BA.5.2 and BF.7 in China, led to off-target effects on the S and N genes from December 1, 2022, to January 31, 2023. The kits used for studying and developing these agents were not adequately and independently evaluated. It is important to verify the performance of commercial Real-Time quantitative PCR (RT-qPCR) tests. Objective: We conducted a clinical evaluation of two Real Time SARS-CoV-2 Omicron assays to verify their performance using various detection reagents and clinical specimens. Methods: We performed clinical evaluations of two existing Chinese SARS-CoV-2 Omicron RT-qPCR kits 2019-nCoV nucleic acid diagnostic kits (Fosun Biotechnology, National instrument registration 20203400299, Shanghai, China) and COVID-19 nucleic acid detection kits (eDiagnosis Biomedicine, National instrument registration 20203400212, Wuhan, China) and using BSD (Bondson) (Guangzhou Bondson Biotechnology Co. Ltd, batch number 2022101), quality controls provided by the inspection center and a large number of clinically confirmed specimens. Results: The concordance rates for the Fosun and eDiagnosis kits were 95% and 100%, respectively. The detection limit for the Fosun and eDiagnosis kits was verified to be 300 copies/mL and 500 copies/mL. The Fosun assay exhibited the largest coefficient of variation (CV) for ORF1ab and N gene at the detection limit concentration (4.80%, 3.49%), whereas eDiagnosis showed a smaller CV (0.93%, 1.10%). In the reference product from the Hangzhou Clinical Laboratory Center test, it was found that Fosun had the lowest sensitivity of 93.47% and a specificity of 100%, while eDiagnosis exhibited 100% for both sensitivity and specificity. The lowest single target gene detection rate of Fosun reagents was 68.7% for the ORF1ab gene and 87.5% for the N gene, while eDiagnosis detection rate was 100%. Among the clinical group S specimens, the missed detection rate of the Fosun reagent was 10.9%, which was higher than the 3.9% of eDiagnosis. However, there was no significant difference in the clinical diagnostic efficiency of the two reagents. Conclusions: The ORF1ab and N assays of SARS-CoV-2 Omicron on the eDiagnosis platform yielded higher values compared to those on the Fosun platform. Consequently, the eDiagnosis kit has also been used as standard detection reagents. Considering that the Fosun reagent has a relatively low detection limit and targets three single genes, it is more advantageous as a confirmatory reagent for the new museum. [ABSTRACT FROM AUTHOR]

Reactive carbonyl and oxygen species (RCS/ROS), often generated as metabolic byproducts, particularly under conditions of pathology, can cause direct damage to proteins, lipids, and nucleic acids. Glyoxal oxidases (Gloxs) oxidize aldehydes to carboxylic acids, generating hydrogen peroxide (H2O2). Although best characterized for their roles in lignin degradation, Glox in plant fungal pathogens are known to contribute to virulence, however, the mechanism underlying such effects are unclear. Here, we show that Glox in the insect pathogenic fungus, Metarhizium acridum, is highly expressed in mycelia and during formation of infection structures (appressoria), with the enzyme localizing to the cell membrane. MaGlox targeted gene disruption mutants showed RCS and ROS accumulation, resulting in cell toxicity, induction of apoptosis and increased autophagy, inhibiting normal fungal growth and development. The ability of the MaGlox mutant to scavenge RCS was significantly reduced, and the mutant exhibited increased susceptibility to aldehydes, oxidative and cell wall perturbing agents but not toward osmotic stress, with altered cell wall contents. The ΔMaGlox mutant was impaired in its ability to penetrate the host cuticle and evade host immune defense resulting in attenuated pathogenicity. Overexpression of MaGlox promoted fungal growth and conidial germination, increased tolerance to H2O2, but had little to other phenotypic effects. Transcriptomic analyses revealed downregulation of genes related to cell wall synthesis, conidiation, stress tolerance, and host cuticle penetration in the ΔMaGlox mutant. These findings demonstrate that MaGlox-mediated scavenging of RCS is required for virulence, and contributes to normal fungal growth and development, stress resistance. Author summary: The ability of organisms to detoxify reactive metabolic byproducts is crucial for normal development, stress resistance, and where applicable, pathogenesis. Here, using the insect pathogenic fungus, Metarhizium acridum, we show that a glyoxal oxidase acts to detoxify cellular reactive carbonyl species (RCS) as well as leading to decreased reactive oxygen species (ROS) levels by alleviating aldehydes accumulation which can trigger ROS production. Mutants deficient in MaGlox showed increased levels of RCS/ROS leading to increased autophagy and apoptosis, resulting in impaired fungal growth, development, and reduced virulence. These findings link Glox mediated RCS/ROS detoxification to cell degradation/turnover processes and cell death, impacting fungal pathogenicity and development. [ABSTRACT FROM AUTHOR]

RNA helicases are involved in the innate immune response against pathogens, including bacteria and viruses; however, their mechanism in the human airway epithelial cells is still not fully understood. Here, we demonstrated that DEAH (Asp-Glu-Ala-His) box polypeptide 35 (DHX35), a member of the DExD/H (Asp-Glu-x-Asp/His)-box helicase family, boosts antiviral innate immunity in human airway epithelial cells. DHX35 knockdown attenuated the production of interferon-β (IFN-β), IL6, and CXCL10, whereas DHX35 overexpression increased their production. Upon stimulation, DHX35 was constitutively expressed, but it translocated from the nucleus into the cytosol, where it recognized cytosolic poly(I:C) and poly(dA:dT) via its HELICc domain. Mitochondrial antiviral signaling protein (MAVS) acted as an adaptor for DHX35 and interacted with the HELICc domain of DHX35 using amino acids 360–510. Interestingly, DHX35 interacted with retinoic acid-inducible gene 1 (RIG-I), enhanced the binding affinity of RIG-I with poly(I:C) and poly(dA:dT), and formed a signalsome with MAVS to activate interferon regulatory factor 3 (IRF3), NF-κB-p65, and MAPK signaling pathways. These results indicate that DHX35 not only acted as a cytosolic nucleic acid sensor but also synergized with RIG-I to enhance antiviral immunity in human airway epithelial cells. Our results demonstrate a novel molecular mechanism for DHX35 in RIG-I-mediated innate immunity and provide a novel candidate for drug and vaccine design to control viral infections in the human airway. Author summary: RIG-I plays an important role in defending against RNA viral infections, such as SARS-CoV-2, influenza virus, and vesicular stomatitis virus (VSV). DHX35 is a member of the Asp-Glu-x-Asp/His (DExD/H)-box helicase family. This study revealed a novel function of DHX35 in regulating activation of the RIG-I signaling pathway. DHX35 translocates from the nucleus into cytosol where it recognizes cytosolic nucleic acids upon stimulation, and interacts with MAVS to activate downstream signaling pathways. More importantly, DHX35 interacts with RIG-I to enhance its binding affinity to nucleic acids. Taken together, our findings demonstrate a novel molecular mechanism of RIG-I-mediated innate immunity in human airway epithelial cells. [ABSTRACT FROM AUTHOR]

This study evaluated the use of endemic enteric coronaviruses polymerase chain reaction (PCR)-negative testing results as an alternative approach to detect the emergence of animal health threats with similar clinical diseases presentation. This retrospective study, conducted in the United States, used PCR-negative testing results from porcine samples tested at six veterinary diagnostic laboratories. As a proof of concept, the database was first searched for transmissible gastroenteritis virus (TGEV) negative submissions between January 1st, 2010, through April 29th, 2013, when the first porcine epidemic diarrhea virus (PEDV) case was diagnosed. Secondly, TGEV- and PEDV-negative submissions were used to detect the porcine delta coronavirus (PDCoV) emergence in 2014. Lastly, encountered best detection algorithms were implemented to prospectively monitor the 2023 enteric coronavirus-negative submissions. Time series (weekly TGEV-negative counts) and Seasonal Autoregressive-Integrated Moving-Average (SARIMA) were used to control for outliers, trends, and seasonality. The SARIMA's fitted and residuals were then subjected to anomaly detection algorithms (EARS, EWMA, CUSUM, Farrington) to identify alarms, defined as weeks of higher TGEV-negativity than what was predicted by models preceding the PEDV emergence. The best-performing detection algorithms had the lowest false alarms (number of alarms detected during the baseline) and highest time to detect (number of weeks between the first alarm and PEDV emergence). The best-performing detection algorithms were CUSUM, EWMA, and Farrington flexible using SARIMA fitted values, having a lower false alarm rate and identified alarms 4 to 17 weeks before PEDV and PDCoV emergences. No alarms were identified in the 2023 enteric negative testing results. The negative-based monitoring system functioned in the case of PEDV propagating epidemic and in the presence of a concurrent propagating epidemic with the PDCoV emergence. It demonstrated its applicability as an additional tool for diagnostic data monitoring of emergent pathogens having similar clinical disease as the monitored endemic pathogens. [ABSTRACT FROM AUTHOR]

The Burkholderia cepacia complex (Bcc) is the number one bacterial complex associated with contaminated Finished Pharmaceutical Products (FPPs). This has resulted in multiple healthcare related infection morbidity and mortality events in conjunction with significant FPP recalls globally. Current microbiological quality control of FPPs before release for distribution depends on lengthy, laborious, non-specific, traditional culture-dependent methods which lack sensitivity. Here, we present the development of a culture-independent Bcc Nucleic Acid Diagnostic (NAD) method for detecting Bcc contaminants associated with Over-The-Counter aqueous FPPs. The culture-independent Bcc NAD method was validated to be specific for detecting Bcc at different contamination levels from spiked aqueous FPPs. The accuracy in Bcc quantitative measurements was achieved by the high degree of Bcc recovery from aqueous FPPs. The low variation observed between several repeated Bcc quantitative measurements further demonstrated the precision of Bcc quantification in FPPs. The robustness of the culture-independent Bcc NAD method was determined when its accuracy and precision were not significantly affected during testing of numerous aqueous FPP types with different ingredient matrices, antimicrobial preservative components and routes of administration. The culture-independent Bcc NAD method showed an ability to detect Bcc in spiked aqueous FPPs at a concentration of 20 Bcc CFU/mL. The rapid (≤ 4 hours from sample in to result out), robust, culture-independent Bcc NAD method presented provides rigorous test specificity, accuracy, precision, and sensitivity. This method, validated with equivalence to ISO standard ISO/TS 12869:2019, can be a valuable diagnostic tool in supporting microbiological quality control procedures to aid the pharmaceutical industry in preventing Bcc contamination of aqueous FPPs for consumer safety. [ABSTRACT FROM AUTHOR]

Nucleic Acid Lateral Flow Assays (NALFAs) are a promising solution for the point-of-care detection of viruses like SARS-CoV-2. However, they show some drawbacks, such as the great dependency on the use of antibodies and the need for post-amplification protocols that enable the preparation of amplicons for effective readings, as well as low sensitivity. Here, we developed amplicons of a specific SARS-CoV-2 gene tailed with single-strand DNA (ssDNA) sequences to hybridize with DNA probes immobilized on the NALFA strips, thus overcoming the aforementioned problems. Results have shown that tailed primers have not compromised the amplification efficiency and allowed the correct detection of the amplicons in the lateral flow strip. This approach has presented a limit of detection (LOD) of 25 RNA copies /reaction mix (1 copy/μL) and the test of cross-reactivity with other related viruses has not shown any cross-reactivity. Twenty clinical samples were evaluated by NALFA and simultaneously compared with the gold standard RT-qPCR protocol, originating equal results. Although the number of clinical specimens tested being relatively small, this indicates a sensitivity and specificity both of 100%. In short, an alternative NALFA was successfully implemented, rendering an accurate route for SARS-CoV-2 diagnosis, compatible with low-resource settings. [ABSTRACT FROM AUTHOR]

Background: Diagnosis of visceral leishmaniasis (VL) in resource-limited endemic regions is currently based on serological testing with rK39 immunochromatographic tests (ICTs). However, rK39 ICT frequently has suboptimal diagnostic accuracy. Furthermore, treatment monitoring and detection of VL relapses is reliant on insensitive and highly invasive tissue aspirate microscopy. Miniature direct-on-blood PCR nucleic acid lateral flow immunoassay (mini-dbPCR-NALFIA) is an innovative and user-friendly molecular tool which does not require DNA extraction and uses a lateral flow strip for result read-out. This assay could be an interesting candidate for more reliable VL diagnosis and safer test of cure at the point of care. Methodology/Principle findings: The performance of mini-dbPCR-NALFIA for diagnosis of VL in blood was assessed in a laboratory evaluation and compared with the accuracy of rK39 ICTs Kalazar Detect in Spain and IT LEISH in East Africa. Limit of detection of mini-dbPCR-NALFIA was 650 and 500 parasites per mL of blood for Leishmania donovani and Leishmania infantum, respectively. In 146 blood samples from VL-suspected patients from Spain, mini-dbPCR-NALFIA had a sensitivity of 95.8% and specificity 97.2%, while Kalazar Detect had a sensitivity of 71.2% and specificity of 94.5%, compared to a nested PCR reference. For a sample set from 58 VL patients, 10 malaria patients and 68 healthy controls from Ethiopia and Kenya, mini-dbPCR-NALFIA had a pooled sensitivity of 87.9% and pooled specificity of 100% using quantitative PCR as reference standard. IT LEISH sensitivity and specificity in the East African samples were 87.9% and 97.4%, respectively. Conclusions/Significance: Mini-dbPCR-NALFIA is a promising tool for simplified molecular diagnosis of VL and follow-up of treated patients in blood samples. Future studies should evaluate its use in endemic, resource-limited settings, where mini-dbPCR-NALFIA may provide an accurate and versatile alternative to rK39 ICTs and aspirate microscopy. Author summary: Visceral leishmaniasis (VL) is a severe infectious disease caused by unicellular parasites of the Leishmania donovani complex. As VL is fatal when left untreated, early and confirmatory laboratory diagnosis is crucial for adequate patient management. However, currently available serological assays, such as the rK39 rapid diagnostic test, fall short in terms of sensitivity and cannot be used for detection of relapsing infections. Therefore, we evaluated an innovative and user-friendly diagnostic assay called mini-dbPCR-NALFIA, which is based on the direct detection of Leishmania kinetoplast DNA in human blood samples. We found that mini-dbPCR-NALFIA can detect down to 500 parasites per millilitre of blood, which is lower than the parasite loads generally seen in untreated VL patients. Furthermore, we assessed the accuracy of mini-dbPCR-NALFIA using a set of Spanish and East-African samples from VL patients and control groups. In the Spanish samples, sensitivity of mini-dbPCR-NALFIA was very high and somewhat better than in the East-African samples. The specificity of mini-dbPCR-NALFIA was excellent in both sample sets. In conclusion, considering its robust performance and simplicity, the mini-dbPCR-NALFIA is a promising diagnostic assay that has the potential to improve VL diagnosis and follow-up of treated patients, especially in resource-limited situations. [ABSTRACT FROM AUTHOR]

Background: Plague, caused by the bacterium Yersinia pestis, is a zoonotic disease that poses considerable threats to human health. Nucleic acid tests are crucial for plague surveillance and the rapid detection of Y. pestis. However, inhibitors in complex samples such as soil and animal tissues often hamper nucleic acid detection, leading to a reduced rate of identifying low concentrations of Y. pestis. To address this challenge, we developed a sensitive and specific droplet digital polymerase chain reaction (ddPCR) assay for detecting Y. pestis DNA from soil and animal tissue samples. Methods: Three genes (ypo2088, caf1, and pla) from Y. pestis were used to develop a multi-target ddPCR assay. The limits of detection (LoD), reproducibility, and specificity were assessed for bacterial genomic DNA samples. The ability of the assay to detect low concentrations of Y. pestis DNA from simulated soil and mouse liver tissue samples was respectively evaluated and compared with that of quantitative real-time PCR (qPCR). Results: The results showed that the ddPCR LoDs ranged from 6.2 to 15.4 copies/reaction for the target genes, with good reproducibility and high specificity for Y. pestis. By testing 130 soil and mouse liver tissue samples spiked with Y. pestis, the ddPCR assay exhibited a better sensitivity than that of the qPCR assay used in the study, with LoDs of 102 colony forming units (CFU)/100 mg soil and 103 CFU/20 mg liver. Moreover, the assay presented good quantitative linearity (R2 = 0.99) for Y. pestis at 103–106 CFU/sample for soil and liver samples. Conclusion: The ddPCR assay presented good performance for detecting Y. pestis DNA from soil and mouse tissue samples, showing great potential for improving the detection rate of low concentrations of Y. pestis in plague surveillance and facilitating the early diagnosis of plague cases. Author summary: Yersinia pestis is the causative agent of three historic plague pandemics. Presently, the plague remains endemic or sporadic in many countries. The wide distribution of natural plague foci in Asia, Africa, and the Americas reminds us of the threat of a plague outbreak. The timely detection and surveillance of Y. pestis prevalence in animals and the environment in natural epidemic foci is essential for preventing and controlling plague outbreaks. In this study, we developed a sensitive and specific ddPCR assay for detecting low concentrations of Y. pestis DNA from soil and mouse tissue samples. The ddPCR assay presented a considerable improvement in sensitivity compared to the qPCR assay used in the study, thus providing a promising tool for detecting Y. pestis DNA from complex samples and facilitating the accurate diagnosis of plague cases. [ABSTRACT FROM AUTHOR]

To control the SARS-CoV-2 pandemic, healthcare systems have focused on ramping up their capacity for epidemiological surveillance through viral whole genome sequencing. In this paper, we tested the performance of two protocols of SARS-CoV-2 nucleic acid enrichment, an amplicon enrichment using different versions of the ARTIC primer panel and a hybrid-capture method using KAPA RNA Hypercap. We focused on the challenge of the Omicron variant sequencing, the advantages of automated library preparation and the influence of the bioinformatic analysis in the final consensus sequence. All 94 samples were sequenced using Illumina iSeq 100 and analysed with two bioinformatic pipelines: a custom-made pipeline and an Illumina-owned pipeline. We were unsuccessful in sequencing six samples using the capture enrichment due to low reads. On the other hand, amplicon dropout and mispriming caused the loss of mutation G21987A and the erroneous addition of mutation T15521A respectively using amplicon enrichment. Overall, we found high sequence agreement regardless of method of enrichment, bioinformatic pipeline or the use of automation for library preparation in eight different SARS-CoV-2 variants. Automation and the use of a simple app for bioinformatic analysis can simplify the genotyping process, making it available for more diagnostic facilities and increasing global vigilance. [ABSTRACT FROM AUTHOR]

Background: CircTADA2A has been demonstrated to play critical roles in the occurrence and development of human cancer. However, the expression pattern and biological mechanisms of circTADA2A in melanoma remains largely unknown. Methods: CircTADA2A were detected by quantitative real-time RT-PCR (qRT-PCR) and validated by Sanger sequencing. Function of circTADA2A and its protein partner in melanoma cells was investigated using RNA interference and overexpression assays. Interaction of circTADA2A, CCHC-type zinc finger nucleic acid binding protein (CNBP) and solute carrier family 38 member 1 (SLC38A1) was confirmed by RNA immunoprecipitation, RNA pull-down, and dual-luciferase reporter assay. The expression of genes and proteins were detected by qRT-PCR and western blot assays. Results: Data from the investigation showed that a novel circRNA (circTADA2A, hsa_circ_0043278) was markedly downregulated in melanoma cells. Functionally, circTADA2A repressed cell proliferation, migration, invasion in melanoma cells. Mechanistically, circTADA2A interacted with CNBP, acting to suppress the binding of CNBP to the SLC38A1 promoter and subsequently restrained SLC38A1 transcription, which resulting in repression of melanoma progression. Conclusions: CircTADA2A suppresses melanoma progression by regulating CNBP/SLC38A1 axis, indicating a potential therapeutic target in melanoma. [ABSTRACT FROM AUTHOR]

Accurate multiple sequence alignment (MSA) is imperative for the comprehensive analysis of biological sequences. However, a notable challenge arises as no single MSA tool consistently outperforms its counterparts across diverse datasets. Users often have to try multiple MSA tools to achieve optimal alignment results, which can be time-consuming and memory-intensive. While the overall accuracy of certain MSA results may be lower, there could be local regions with the highest alignment scores, prompting researchers to seek a tool capable of merging these locally optimal results from multiple initial alignments into a globally optimal alignment. In this study, we introduce Two Pointers Meta-Alignment (TPMA), a novel tool designed for the integration of nucleic acid sequence alignments. TPMA employs two pointers to partition the initial alignments into blocks containing identical sequence fragments. It selects blocks with the high sum of pairs (SP) scores to concatenate them into an alignment with an overall SP score superior to that of the initial alignments. Through tests on simulated and real datasets, the experimental results consistently demonstrate that TPMA outperforms M-Coffee in terms of aSP, Q, and total column (TC) scores across most datasets. Even in cases where TPMA's scores are comparable to M-Coffee, TPMA exhibits significantly lower running time and memory consumption. Furthermore, we comprehensively assessed all the MSA tools used in the experiments, considering accuracy, time, and memory consumption. We propose accurate and fast combination strategies for small and large datasets, which streamline the user tool selection process and facilitate large-scale dataset integration. The dataset and source code of TPMA are available on GitHub (https://github.com/malabz/TPMA). Author summary: Accurate multiple sequence alignment (MSA) is vital for comprehensive biological sequence analysis. However, as no single MSA tool consistently outperforms others across diverse datasets, researchers must invest significant time exploring multiple tools to identify the most suitable one for their specific dataset. To address this, researchers seek tools that can merge locally optimal results from diverse initial alignments into a globally optimal alignment. Our novel approach, Two Pointers Meta-Alignment (TPMA), employs a two-pointer to partition initial alignments into blocks, selecting those with the higher sum of pairs (SP) scores for integration into a globally optimal alignment. TPMA consistently outperforms M-Coffee, demonstrating superior aSP, Q, and total column (TC) scores, coupled with faster running times and lower memory consumption. We present comprehensive assessments of various MSA tools, proposing efficient combination strategies for diverse datasets. Our tool, TPMA, and associated resources are publicly available on GitHub (https://github.com/malabz/TPMA), offering a valuable contribution to the field of evolutionary biology and streamlining the selection process for users dealing with large-scale datasets. [ABSTRACT FROM AUTHOR]

Deformed wing virus (DWV) was first detected in dead honey bees in 1982 but has been in honey bees for at least 300 years. Due to its high prevalence and virulence, they have been linked with the ongoing decline in honey bee populations worldwide. A rapid, simple, semi-automated, high-throughput, and cost-effective method of screening colonies for viruses would benefit bee research and the beekeeping industry. Here we describe a semi-automated approach that combines an RNA-grade liquid homogenizer followed by magnetic bead capture for total virus nucleic acid extraction. We compare it to the more commonly applied nucleic acid column-based purification method and use qPCR plus Oxford Nanopore Technologies sequencing to evaluate the accuracy of analytical results for both methods. Our results showed high reproducibility and accuracy for both approaches. The semi-automated method described here allows for faster screening of viral loads in units of 96 samples at a time. We developed this method to monitor viral loads in honey bee colonies, but it could be easily applied for any PCR or genomic-based screening assays. [ABSTRACT FROM AUTHOR]

This article discusses the challenges and opportunities in detecting vector-borne pathogens using nucleic acid-based assays and metagenomic sequencing. The authors highlight the difficulties in distinguishing pathogens from closely related endosymbionts and the need for regular updating of detection approaches. They also emphasize the importance of reviewing and improving primer and probe sequences, as well as considering the integration of endosymbiont sequences into host genomes. The article further discusses the challenges in detecting and identifying viruses, as well as the limitations of NGS technologies and computational analysis in microbial detection. The authors caution that the biological relevance of microbial detection should be carefully considered, especially in vector and environmental samples, and that additional evidence may be required to establish causative associations with disease. They conclude by emphasizing the need for careful planning and consideration of surveillance goals within the One Health concept. [Extracted from the article]

Retinoic acid inducible gene I (Rig-I) is a cytosolic pattern recognition receptor canonically described for its important role in sensing viral RNAs. Increasingly, bacterially-derived RNA from intracellular bacteria such as Mycobacterium tuberculosis, have been shown to activate the same host Rig-I/Mitochondrial antiviral sensing protein (MAVS) signaling pathway to drive a type-I interferon response that contributes to bacterial pathogenesis in vivo. In M. tuberculosis, this response is mediated by the protein secretion system SecA2, but little is known about whether this process is conserved in other pathogenic mycobacteria or the mechanism by which these nucleic acids gain access to the host cytoplasm. Because the M. tuberculosis and M. marinum SecA2 protein secretion systems share a high degree of genetic and functional conservation, we hypothesized that Rig-I/MAVS activation and subsequent induction of IFN-β secretion by host macrophages will also be conserved between these two mycobacterial species. To test this, we generated a ΔsecA2 M. marinum strain along with complementation strains expressing either the M. marinum or M. tuberculosis secA2 genes. Our results suggest that the ΔsecA2 strain has a growth defect in vitro but not in host macrophages. These intracellular growth curves also suggested that the calculation applied to estimate the number of bacteria added to macrophage monolayers in infection assays underestimates bacterial inputs for the ΔsecA2 strain. Therefore, to better examine secreted IFN-β levels when bacterial infection levels are equal across strains we plated bacterial CFUs at 2hpi alongside our ELISA based infections. This enabled us to normalize secreted levels of IFN-β to a standard number of bacteria. Applying this approach to both WT and MAVS-/- bone marrow derived macrophages we observed equal or higher levels of secreted IFN-β from macrophages infected with the ΔsecA2 M. marinum strain as compared to WT. Together our findings suggest that activation of host Rig-I/MAVS cytosolic sensors and subsequent induction of IFN-β response in a SecA2-dependent manner is not conserved in M. marinum under the conditions tested. [ABSTRACT FROM AUTHOR]

Proteins are biological units whose essence is defined by their functional relationships with other proteins or biomolecules such as RNA, DNA, lipids, or carbohydrates. These functions encompass enzymatic, structural, regulatory, or physical interaction roles. The STRING database (Nucleic Acids Research, 8 Jan 2021;49(D1): D605-12) provides an index that defines the functional interaction networks between proteins in model organisms. To facilitate the identification, visualization, and evaluation of potential functional networks across organisms from different phylogenetic lineages, we have developed PhyloString (https://biocomputo.ibt.unam.mx/phylostring/), a web server that utilizes the indices of the STRING database. PhyloString decomposes these functional networks into modules, representing cohesive units of proteins grouped based on their similarity of STRING values and the phylogenetic origins of their respective organisms. This study presents and thoroughly discusses examples of such functional networks and their modules identified using PhyloString. [ABSTRACT FROM AUTHOR]

Next-generation sequencing demands high-quality nucleic acid, yet isolating DNA and RNA is often challenging, particularly from plant tissues. Despite advances in developing various kits and reagents, these products are tailored to isolation of nucleic acid from model plant tissues. Here we introduce a universal lysis buffer to separate nucleic acid from various plant species, including recalcitrant plants, to facilitate molecular analyses, such as quantitative PCR (qPCR), transcriptomics, and whole-genome sequencing (WGS). The protocol is a modification of the original CTAB methods, which leads to nucleic acid isolation from many plant species, including monocots and eudicots. The lysis buffer consists of hexadecyltrimethylammonium bromide (CTAB), sodium chloride (NaCl), Tris base, ethylenediaminetetraacetic acid (EDTA) and β-mercaptoethanol (βME). The modified CTAB method enables the isolation of nucleic acid from small amounts of plant tissues (e.g., 15–100 mg) in a timely manner, which is well-suited for a large number of samples and also when adequate sample collection is a limiting factor. The protocol isolates not only DNA from various plant species but also RNA. This makes it highly effective for molecular analyses compared to previously described CTAB methods optimised for DNA isolation. The appropriate concentration of the components enables high-quality DNA and RNA isolation from plant tissues simultaneously. Additionally, this protocol is compatible with commercially available columns. For DNA and RNA to be qualified for next-generation sequencing platforms, the protocol is supplemented with columns to purify either DNA or RNA from the same tissue to meet high standards for sequencing analyses. This protocol provides an ideal approach to overcome potential obstacles in isolating high-quality DNA or RNA from a wide range of plant species for downstream molecular analysis. [ABSTRACT FROM AUTHOR]

The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has swept the world and poses a serious threat to human health. In the post-pandemic-era, we must remain vigilant against the co-infection of SARS-CoV-2 and other respiratory viruses. More accurate and convenient detection methods are required for the diagnosis of SARS-CoV-2 due to its prolonged existence. In this study, the application value of a novel lyophilized-pellet-based 2019-nCoV nucleic acid diagnostic kit (PCoV-Kit) was evaluated by comparing it with a conventional liquid diagnostic kit (LCoV-Kit). We assessed the sensitivity, precision, accuracy, specificity, and amplification efficiency of PCoV-Kit and LCoV-Kit using diluted SARS-CoV-2 RNA reference materials. The results showed that both kits had high sensitivity, precision, accuracy, and specificity. A total of 2,033 oropharyngeal swab specimens collected during mass screening in Fuzhou in December 2022 were applied for the consistency analysis of the two reagents. In the detection of clinical oropharyngeal swab specimens, although the positive rate of PCoV-Kit (19.28%) was slightly lower than that of LCoV-Kit (20.86%), statistical analysis demonstrated a high degree of consistency between the test results obtained using both kit (χ2 = 1.57, P>0.05; Kappa coefficient = 0.90, 95%CI: 0.88–0.93). In conclusion, the use of lyophilized PCoV-Kit provides a non-inferior assay for the diagnosis of COVID-19. [ABSTRACT FROM AUTHOR]

Ribosomes are information-processing macromolecular machines that integrate complex sequence patterns in messenger RNA (mRNA) transcripts to synthesize proteins. Studies of the sequence features that distinguish mRNAs from long noncoding RNAs (lncRNAs) may yield insight into the information that directs and regulates translation. Computational methods for calculating protein-coding potential are important for distinguishing mRNAs from lncRNAs during genome annotation, but most machine learning methods for this task rely on previously known rules to define features. Sequence-to-sequence (seq2seq) models, particularly ones using transformer networks, have proven capable of learning complex grammatical relationships between words to perform natural language translation. Seeking to leverage these advancements in the biological domain, we present a seq2seq formulation for predicting protein-coding potential with deep neural networks and demonstrate that simultaneously learning translation from RNA to protein improves classification performance relative to a classification-only training objective. Inspired by classical signal processing methods for gene discovery and Fourier-based image-processing neural networks, we introduce LocalFilterNet (LFNet). LFNet is a network architecture with an inductive bias for modeling the three-nucleotide periodicity apparent in coding sequences. We incorporate LFNet within an encoder-decoder framework to test whether the translation task improves the classification of transcripts and the interpretation of their sequence features. We use the resulting model to compute nucleotide-resolution importance scores, revealing sequence patterns that could assist the cellular machinery in distinguishing mRNAs and lncRNAs. Finally, we develop a novel approach for estimating mutation effects from Integrated Gradients, a backpropagation-based feature attribution, and characterize the difficulty of efficient approximations in this setting. Author summary: The ribosome is an information-processing cellular machine that synthesizes proteins from messenger RNA (mRNA) transcripts in a process called translation. Here, we model this information-processing behavior of the ribosome as an intelligent system capable of predicting which RNA transcripts undergo translation and which do not. Modern artificial intelligence techniques can produce powerful models of biological sequence properties but sometimes fail to reflect the underlying biology despite high prediction accuracy. We show that training such a model to predict translated protein sequences from mRNA sequences improves its ability to distinguish mRNAs from long noncoding RNAs. This ability to predict translated RNAs also allows us to search for potential micropeptides, which are small translation products outside of conventional coding regions. We also introduce a neural network architecture to capture the 3-nucleotide periodicity that is known to occur in coding nucleic acids. Crucially, this novel setup helps to instill complex and biologically meaningful logic into the model, which we probe for clues as to the patterns that govern translation in living organisms. [ABSTRACT FROM AUTHOR]

The R132H isocitrate dehydrogenase one (IDH1) mutation is a prognostic biomarker present in a subset of gliomas and is associated with heightened survival when paired with aggressive surgical resection. In this study, we establish proof-of-principle for rapid colorimetric detection of the IDH1-R132H mutation in tumor samples in under 1 hour without the need for a nucleic acid extraction. Colorimetric peptide nucleic acid loop-mediated isothermal amplification (CPNA-LAMP) utilizes 4 conventional LAMP primers, a blocking PNA probe complementary to the wild-type sequence, and a self-annealing loop primer complementary to the single nucleotide variant to only amplify the DNA sequence containing the mutation. This assay was evaluated using IDH1-WT or IDH1-R132H mutant synthetic DNA, wild-type or IDH1-R132H mutant U87MG cell lysates, and tumor lysates from archived patient samples in which the IDH1 status was previously determined using immunohistochemistry (IHC). Reactions were performed using a hot water bath and visually interpreted as positive by a pink-to-yellow color change. Results were subsequently verified using agarose gel electrophoresis. CPNA-LAMP successfully detected the R132H single nucleotide variant, and results from tumor lysates yielded 100% concordance with IHC results, including instances when the single nucleotide variant was limited to a portion of the tumor. Importantly, when testing the tumor lysates, there were no false positive or false negative results. [ABSTRACT FROM AUTHOR]

Protein regions consisting of arrays of tandem repeats are known to bind other molecular partners, including nucleic acid molecules. Although the interactions between repeat proteins and DNA are already widely explored, studies characterising tandem repeat RNA-binding proteins are lacking. We performed a large-scale analysis of human proteins devoted to expanding the knowledge about tandem repeat proteins experimentally reported as RNA-binding molecules. This work is timely because of the release of a full set of accurate structural models for the human proteome amenable to repeat detection using structural methods. The main goal of our analysis was to build a comprehensive set of human RNA-binding proteins that contain repeats at the sequence or structure level. Our results showed that the combination of sequence and structural methods finds significantly more tandem repeat proteins than either method alone. We identified 219 tandem repeat proteins that bind RNA molecules and characterised the overlap between repeat regions and RNA-binding regions as a first step towards assessing their functional relationship. We observed differences in the characteristics of repeat regions predicted by sequence-based or structure-based methods in terms of their sequence composition, their functions and their protein domains. [ABSTRACT FROM AUTHOR]

Tumor-derived extracellular vesicles (EVs) are reported to contain nucleic acids, including DNA. Several studies have highlighted the potential of EV-derived DNA (evDNA) as a circulating biomarker, even demonstrating that evDNA can outperform cell-free DNA (cfDNA) in terms of sensitivity. Here, we evaluated EVs as a potential source of tumor-derived DNA in patients with advanced pancreatic cancer. evDNA from both DNase-treated and untreated EV samples was analyzed to determine whether the DNA was primarily located internally or outside (surface-bound) the EVs. To assess whether methodology affected the results, we isolated EVs using four different methods for small EV isolation and differential centrifugation for isolating large EVs. Our results indicated that the DNA content of EVs was significantly less than the cfDNA content isolated from the same plasma volume (p < 0.001). Most of the detected evDNA was also located on the outside of the vesicles. Furthermore, the fraction of tumor-derived DNA in EVs was similar to that found in cfDNA. In conclusion, our results suggest that quantification of evDNA, as a source of tumor-derived DNA, does not add information to that obtained with cfDNA, at least not in patients with advanced pancreatic cancer. [ABSTRACT FROM AUTHOR]

Accurate prediction of nucleic binding residues is essential for the understanding of transcription and translation processes. Integration of feature- and template-based strategies could improve the prediction of these key residues in proteins. Nevertheless, traditional hybrid algorithms have been surpassed by recently developed deep learning-based methods, and the possibility of integrating deep learning- and template-based approaches to improve performance remains to be explored. To address these issues, we developed a novel structure-based integrative algorithm called NABind that can accurately predict DNA- and RNA-binding residues. A deep learning module was built based on the diversified sequence and structural descriptors and edge aggregated graph attention networks, while a template module was constructed by transforming the alignments between the query and its multiple templates into features for supervised learning. Furthermore, the stacking strategy was adopted to integrate the above two modules for improving prediction performance. Finally, a post-processing module dependent on the random walk algorithm was proposed to further correct the integrative predictions. Extensive evaluations indicated that our approach could not only achieve excellent performance on both native and predicted structures but also outperformed existing hybrid algorithms and recent deep learning methods. The NABind server is available at http://liulab.hzau.edu.cn/NABind/. Author summary: Ten years ago we developed two hybrid algorithms (DNABind and RBRDetector) to predict nucleic acid binding residues by combining machine learning- and template-based strategies. However, this kind of algorithms have been surpassed by recent deep learning methods. Moreover, the interplay between deep learning- and template-based approaches has yet to be explored. We thus designed a new generation hybrid algorithm termed NABind, in which a deep learning module was established by using diversified sequence and structural descriptors and edge-featured graph attention networks, while a template module was created by exploiting the relationship between the query protein and its multiple templates for supervised learning. Afterward, a merging module based on the stacking strategy was adopted to integrate the above two modules, and a post-processing module dependent on the random walk algorithm was utilized to correct the integrative predictions. The new algorithm outperformed traditional hybrid methods by a large margin and showed better results than purely deep learning-based methods. [ABSTRACT FROM AUTHOR]

Similar tests using macrophages lacking the well-characterized GXM receptors TLR2 and CD14 showed that both the percentage of GXM-containing macrophages and the indices of GXM-derived fluorescence intensity were similar [[10]], suggesting that the molecular mass of GXM does not influence its interaction with host receptors. Fluorescence-based assays using monoclonal antibodies to GXM revealed that polysaccharide fractions of variable masses (10 to 100; 100 to 300; >300 kDa, and all of them in combination) produced similar levels of association with macrophages [[10]]. GXM fractions ranging from 1 to 100 kDa tended to accumulate at the cell division area, while fractions of larger masses (100 to 300 and >300 kDa) produced a dotted, punctuate pattern of GXM incorporation into the entire cell surface. I gattii i complexes, as concluded by immunofluorescence using anti-GXM antibodies, analysis of capsule size, and determination of extracellular GXM [[8]]. [Extracted from the article]

Deubiquitinating enzymes (DUBs) regulate antiviral immune response through targeting DNA sensor signaling pathway members. As one of the DNA sensors, interferon (IFN)-γ inducible protein 16 (IFI16) play a major role in response to virus infections through activating the canonical STING/TBK-1/IRF3 signaling pathway. Only a few studies discuss the function of DUBs in IFI16-mediated antiviral response. Ubiquitin-specific protease 12 (USP12), which is one of the major members of the USP family, participates in various biological functions. However, whether USP12 regulates the nucleic acid sensor to modulate antiviral immune responses has not yet been elucidated. In this study, we found that knockout or knockdown of USP12 impaired the HSV-1-induced expressions of IFN-β, CCL-5, IL-6, and downstream interferon-stimulated genes (ISGs). Moreover, USP12 deficiency increased HSV-1 replication and host susceptibility to HSV-1 infection. Mechanistically, USP12 inhibited the proteasome-dependent degradation of IFI16 through its deubiquitinase activity, thereby maintaining IFI16 stability and promoting IFI16-STING-IRF3- and p65-mediated antiviral signaling. Overall, our findings demonstrate an essential role of USP12 in DNA-sensing signaling and contribute to the understanding of deubiquitination-mediated regulation of innate antiviral responses. Author summary: Ubiquitin-specific peptidase 12 (USP12), one of the major members of the deubiquitinating enzyme family, participates in various biological functions, such as cell proliferation and differentiation, apoptosis, neurodegeneration, tumor promoting, and antiviral immune responses. In this study, we reported that USP12 promoted IFI16-mediated innate antiviral signaling. Knockout or knockdown of USP12 impaired the DNA virus-triggered activation of the IFI16-STING-IRF3/NF-κB pathway and expression of downstream genes. USP12 deficiency increases HSV-1 replication and host susceptibility to HSV-1 infection. Mechanistically, USP12 inhibited the proteasome-dependent degradation of IFI16 through its deubiquitinase activity. Our results promote understanding of varied regulation of USP12 in antiviral signaling. [ABSTRACT FROM AUTHOR]

Background: Chronic obstructive pulmonary disease (COPD) is progressive and irreversible chronic lung inflammatory disease. Cigarette smoke, the main cause of COPD, is often associated with double-stranded DNA release which potentially activates DNA-sensing pathways, such as STING. This study, therefore, analyzed the role of STING pathway in inducing pulmonary inflammation, steroid resistance, and remodeling in COPD. Methods: Primary cultured lung fibroblasts were isolated from healthy non-smoker, healthy smoker, and smoker COPD individuals. The expression of STING pathway, remodeling, and steroid resistance signatures were investigated in these fibroblasts upon LPS stimulation and treatment with dexamethasone and/or STING inhibitor, at both mRNA and protein levels using qRT-PCR, western blot, and ELISA. Results: At baseline, STING was elevated in healthy smoker fibroblasts and to a higher extent in smoker COPD fibroblasts when compared to healthy non-smoker fibroblasts. Upon using dexamethasone as monotherapy, STING activity was significantly inhibited in healthy non-smoker fibroblasts but showed resistance in COPD fibroblasts. Treating both healthy and COPD fibroblasts with STING inhibitor in combination with dexamethasone additively inhibited STING pathway in both groups. Moreover, STING stimulation triggered a significant increase in remodeling markers and a reduction in HDAC2 expression. Interestingly, treating COPD fibroblasts with the combination of STING inhibitor and dexamethasone alleviated remodeling and reversed steroid hyporesponsiveness through an upregulation of HDAC2. Conclusion: These findings support that STING pathway plays an important role in COPD pathogenesis, via inducing pulmonary inflammation, steroid resistance, and remodeling. This raises the possibility of using STING inhibitor as a potential therapeutic adjuvant in combination with common steroid treatment. [ABSTRACT FROM AUTHOR]

Background: Optineurin (OPTN) is associated with several human diseases, including amyotrophic lateral sclerosis (ALS), and is involved in various cellular processes, including autophagy. Optineurin regulates the expression of interferon beta (IFNβ), which plays a central role in the innate immune response to viral infection. However, the role of optineurin in response to viral infection has not been fully clarified. It is known that optineurin-deficient cells produce more IFNβ than wild-type cells following viral infection. In this study, we investigate the reasons for, and effects of, IFNβ overproduction during optineurin deficiency both in vitro and in vivo. Methods: To investigate the mechanism of IFNβ overproduction, viral nucleic acids in infected cells were quantified by RT-qPCR and the autophagic activity of optineurin-deficient cells was determined to understand the basis for the intracellular accumulation of viral nucleic acids. Moreover, viral infection experiments using optineurin-disrupted (Optn-KO) animals were performed with several viruses. Results: IFNβ overproduction following viral infection was observed not only in several types of optineurin-deficient cell lines but also in Optn-KO mice and human ALS patient cells carrying mutations in OPTN. IFNβ overproduction in Optn-KO cells was revealed to be caused by excessive accumulation of viral nucleic acids, which was a consequence of reduced autophagic activity caused by the loss of optineurin. Additionally, IFNβ overproduction in Optn-KO mice suppressed viral proliferation, resulting in increased mouse survival following viral challenge. Conclusion: Our findings indicate that the combination of optineurin deficiency and viral infection leads to IFNβ overproduction in vitro and in vivo. The effects of optineurin deficiency are elicited by viral infection, therefore, viral infection may be implicated in the development of optineurin-related diseases. [ABSTRACT FROM AUTHOR]

Satellites associated with plant or animal viruses have been largely detected and characterized, while those from mycoviruses together with their roles remain far less determined. Three dsRNA segments (dsRNA 1 to 3 termed according to their decreasing sizes) were identified in a strain of phytopathogenic fungus Pestalotiopsis fici AH1-1 isolated from a tea leaf. The complete sequences of dsRNAs 1 to 3, with the sizes of 10316, 5511, and 631 bp, were determined by random cloning together with a RACE protocol. Sequence analyses support that dsRNA1 is a genome of a novel hypovirus belonging to genus Alphahypovirus of the family Hypoviridae, tentatively named Pestalotiopsis fici hypovirus 1 (PfHV1); dsRNA2 is a defective RNA (D-RNA) generating from dsRNA1 with septal deletions; and dsRNA3 is the satellite component of PfHV1 since it could be co-precipitated with other dsRNA components in the same sucrose fraction by ultra-centrifuge, suggesting that it is encapsulated together with PfHV1 genomic dsRNAs. Moreover, dsRNA3 shares an identical stretch (170 bp) with dsRNAs 1 and 2 at their 5′ termini and the remaining are heterogenous, which is distinct from a typical satellite that generally has very little or no sequence similarity with helper viruses. More importantly, dsRNA3 lacks a substantial open reading frame (ORF) and a poly (A) tail, which is unlike the known satellite RNAs of hypoviruses, as well as unlike those in association with Totiviridae and Partitiviridae since the latters are encapsidated in coat proteins. As up-regulated expression of RNA3, dsRNA1 was significantly down-regulated, suggesting that dsRNA3 negatively regulates the expression of dsRNA1, whereas dsRNAs 1 to 3 have no obvious impact on the biological traits of the host fungus including morphologies and virulence. This study indicates that PfHV1 dsRNA3 is a special type of satellite-like nucleic acid that has substantial sequence homology with the host viral genome without encapsidation in a coat protein, which broadens the definition of fungal satellite. Author summary: A novel hypovirus belonging to the genus Alphahypovirus of the family Hypoviridae was identified in a phytopathogenetic P. fici isolated from a tea leaf and tentatively named Pestalotiopsis fici hypovirus 1 (PfHV1), which represents the first hypovirus from Pestalotiopsis fungi. Three RNA components (RNA 1 to 3 termed according to their decreasing sizes, dsRNA 1 to 3 termed for their replicative form are corresponding with RNA 1 to 3) associated with PfHV1 including the genomic RNA, defective RNA and satellite-like RNA (SatL-RNA) were determined, and their molecular and biological traits were characterized. The SatL-RNA negatively regulates the accumulation of PfHV1 RNA1, and represents a novel class of satellite nucleic acids since it has substantial sequence homology with the host viral genome and is not encapsidated in the coat protein of its helper virus. This study contributes to our better understanding of fungal satellites, as well as the molecular and biological traits of hypoviruses. [ABSTRACT FROM AUTHOR]

The type VI secretion system (T6SS) is an antibacterial weapon that is used by numerous Gram-negative bacteria to gain competitive advantage by injecting toxins into adjacent prey cells. Predicting the outcome of a T6SS-dependent competition is not only reliant on presence-absence of the system but instead involves a multiplicity of factors. Pseudomonas aeruginosa possesses 3 distinct T6SSs and a set of more than 20 toxic effectors with diverse functions including disruption of cell wall integrity, degradation of nucleic acids or metabolic impairment. We generated a comprehensive collection of mutants with various degrees of T6SS activity and/or sensitivity to each individual T6SS toxin. By imaging whole mixed bacterial macrocolonies, we then investigated how these P. aeruginosa strains gain a competitive edge in multiple attacker/prey combinations. We observed that the potency of single T6SS toxin varies significantly from one another as measured by monitoring the community structure, with some toxins acting better in synergy or requiring a higher payload. Remarkably the degree of intermixing between preys and attackers is also key to the competition outcome and is driven by the frequency of contact as well as the ability of the prey to move away from the attacker using type IV pili-dependent twitching motility. Finally, we implemented a computational model to better understand how changes in T6SS firing behaviours or cell-cell contacts lead to population level competitive advantages, thus providing conceptual insight applicable to all types of contact-based competition. Author summary: The Type VI Secretion System (T6SS) was discovered in Gram-negative bacteria and is a contact-dependent molecular nanomachine which injects antimicrobial toxins into competitors. The T6SS activity provides a competitive edge for an organism to prevail in a given niche. The number and biochemical activity of toxins injected could vary from one bacterial species to the other. The opportunistic pathogen Pseudomonas aeruginosa is quite prolific with more than 20 characterized and distinct T6SS toxins. Here we experimentally addressed the importance in the degree of T6SS activity and made the demonstration that every single of the many P. aeruginosa T6SS toxin counts. There are no redundancies and instead on occasion synergies, which support the effectiveness of injecting a cocktail of toxins rather than a subset of specific ones. Since contact is a key factor for T6SS delivery, we further observed that preys able to use surface motility skills to run away from bacterial attackers have better chances to multiply and survive. Furthering experimental approaches, we used computational modelling to place these data in the context of a mixed P. aeruginosa population. This way we contribute understanding to how highly local contact-based interactions between individuals shape the structure of whole bacterial communities. [ABSTRACT FROM AUTHOR]

Twenty-four species of RNA viruses contain members infecting economically important crops that are classified within the genus Emaravirus, family Fimoviridae. There are at least two other non-classified species that may be added. Some of these viruses are spreading rapidly and cause economically important diseases on several crops, raising a need for a sensitive diagnostic technique for taxonomic and quarantine purposes. High-resolution melting (HRM) has shown to be reliable for the detection, discrimination, and diagnosis of several diseases of plants, animals, and humans. This research aimed to explore the ability to predict HRM outputs coupled to reverse transcription-quantitative polymerase chain reaction (RT-qPCR). To approach this goal a pair of degenerate genus-specific primers were designed for endpoint RT-PCR and RT-qPCR-HRM and the species in the genus Emaravirus were selected to framework the development of the assays. Both nucleic acid amplification methods were able to detect in-vitro several members of seven Emaravirus species with sensitivity up to one fg of cDNA. Specific parameters for in-silico prediction of the melting temperatures of each expected emaravirus amplicon are compared to the data obtained in-vitro. A very distinct isolate of the High Plains wheat mosaic virus was also detected. The high-resolution DNA melting curves of the RT-PCR products predicted in-silico using uMeltSM allowed saving time while designing and developing the RT-qPCR-HRM assay since the approach avoided extensive searching for optimal HRM assay regions and rounds of HRM tests in-vitro for optimization. The resultant assay provides sensitive detection and reliable diagnosis for potentially any emaravirus, including new species or strains. [ABSTRACT FROM AUTHOR]

Saliva is an emerging source of disease biomarkers, particularly for cancers of the head and neck. Although analysis of cell-free DNA (cfDNA) in saliva holds promise as a liquid biopsy for cancer detection, currently there are no standardized methodologies for the collection and isolation of saliva for the purposes of studying DNA. Here, we evaluated various saliva collection receptacles and DNA purification techniques, comparing DNA quantity, fragment size, source, and stability. Then, using our optimized techniques, we tested the ability to detect human papillomavirus (HPV) DNA– a bona fide cancer biomarker in a subset of head and neck cancers– from patient saliva samples. For saliva collection, we found that the Oragene OG-600 receptacle yielded the highest concentration of total salivary DNA as well as short fragments <300 bp corresponding to mononucleosomal cell-free DNA. Moreover, these short fragments were stabilized beyond 48 hours after collection in contrast to other saliva collection receptacles. For DNA purification from saliva, the QIAamp Circulating Nucleic Acid kit yielded the highest concentration of mononucleosome-sized DNA fragments. Freeze-thaw of saliva samples did not affect DNA yield or fragment size distribution. Salivary DNA isolated from the OG-600 receptacle was found to be composed of both single and double-stranded DNA, including mitochondrial and microbial sources. While levels of nuclear DNA were consistent over time, levels of mitochondrial and microbial DNA were more variable and increased 48 hours after collection. Finally, we found that HPV DNA was stable in OG-600 receptacles, was reliably detected within the saliva of patients with HPV-positive head and neck cancer, and was abundant among mononucleosome-sized cell-free DNA fragments. Our studies have defined optimal techniques for isolating DNA from saliva that will contribute to future applications in liquid biopsy-based cancer detection. [ABSTRACT FROM AUTHOR]

Modification of cellular and immunological events due to porcine reproductive and respiratory syndrome virus (PRRSV) infection is associated with pathogenesis in lungs. PRRSV also causes female reproductive dysfunction and persistent infection which can spread to fetus, stillbirth, and offspring. In this study, changes in cellular and innate immune responses to PRRSV type 1 or type 2 infection, including expression of PRRSV mediators, mRNA expression of Toll-like receptors (TLRs) and cytokine, and cytokine secretion, were examined in primary porcine glandular endometrial cells (PGE). Cell infectivity as observed by cytopathic effect (CPE), PRRSV nucleocapsid proteins, and viral nucleic acids was detected as early as two days post-infection (2 dpi) and persisted until 6 dpi. A higher percentage of CPE and PRRSV-positive cells were observed in type 2 infections. PRRSV mediator proteins, CD151, CD163, sialoadhesin (Sn), integrin and vimentin, were upregulated following type 1 and type 2 infection. CD151, CD163 and Sn were upregulated by type 2. In both PRRSV types, mRNA expression of TLR1 and TLR6 was upregulated. However, TLR3 was upregulated by type 1, but TLR4 and TLR8 mRNA and protein were downregulated by type 2 only. Interleukin (IL)-1β, IL-6 and tumor necrotic factor (TNF)-α were upregulated by type 2, but IL-8 was upregulated by type 1. Both PRRSV type 1 and 2 stimulated IL-6 but suppressed TNF-α secretion. In addition, IL-1β secretion was suppressed only by type 2. These findings reveal an important mechanism underlying the strategy of PRRSV infection in the endometrium and associated with the viral persistence. [ABSTRACT FROM AUTHOR]

Recent advances in electronics and microfluidics have enabled several research groups to develop fully integrated, sample-to-result isothermal nucleic acid amplification test (NAAT) platforms for the point of care. However, high component counts and costs have limited translation of these platforms beyond the clinic to low-resource settings—including homes. Many NAATs include complex, multi-component heater electronics based on flex circuits or multiple printed circuit boards (PCBs) to support essential NAAT steps such as lysis, sample deactivation, and nucleic acid amplification. In contrast, current commercial assays for home use, such as those for pregnancy or ovulation that include electronics, typically have just one onboard PCB. This work describes a generalizable strategy to integrate all heaters and the electronics needed to control them onto a single low-cost, USB-powered PCB. We built a multiplexable disposable NAAT ("MD NAAT") platform that applies these principles, integrating small-area heaters that heat small regions to near-boiling (for pathogen lysis and deactivation) and large-area heaters (for amplification) on the same PCB. We show that both classes of heaters have high intra-board and inter-device reproducibility despite only heating a NAAT cartridge from below. We validated the small-area heaters by lysing methicillin-resistant Staphylococcus aureus (MRSA) cells and the large-area heaters by performing two types of isothermal NAATs (isothermal strand displacement amplification (iSDA) and loop-mediated isothermal amplification (LAMP)). These results demonstrate the merit of integrating NAAT heaters and control electronics onto a single printed circuit board and are a step toward translating NAATs to the home. [ABSTRACT FROM AUTHOR]

The promise of therapeutic nucleic acids has long been tempered by difficulty in overcoming biological barriers to their delivery. The past two decades have seen the development of ionizable lipid nanoparticles as a vehicle for nucleic acid delivery and their translation to the clinic. Over the past 20 years, lipid-based nanoparticles have been engineered to overcome the biological barriers to nucleic acid delivery. This Perspective looks at their development for use in vaccines, immunotherapies, protein replacement therapies, and gene therapies. [ABSTRACT FROM AUTHOR]

CRISPR-mediated interference relies on complementarity between a guiding CRISPR RNA (crRNA) and target nucleic acids to provide defense against bacteriophage. Phages escape CRISPR-based immunity mainly through mutations in the protospacer adjacent motif (PAM) and seed regions. However, previous specificity studies of Cas effectors, including the class 2 endonuclease Cas12a, have revealed a high degree of tolerance of single mismatches. The effect of this mismatch tolerance has not been extensively studied in the context of phage defense. Here, we tested defense against lambda phage provided by Cas12a-crRNAs containing preexisting mismatches against the genomic targets in phage DNA. We find that most preexisting crRNA mismatches lead to phage escape, regardless of whether the mismatches ablate Cas12a cleavage in vitro. We used high-throughput sequencing to examine the target regions of phage genomes following CRISPR challenge. Mismatches at all locations in the target accelerated emergence of mutant phage, including mismatches that greatly slowed cleavage in vitro. Unexpectedly, our results reveal that a preexisting mismatch in the PAM-distal region results in selection of mutations in the PAM-distal region of the target. In vitro cleavage and phage competition assays show that dual PAM-distal mismatches are significantly more deleterious than combinations of seed and PAM-distal mismatches, resulting in this selection. However, similar experiments with Cas9 did not result in emergence of PAM-distal mismatches, suggesting that cut-site location and subsequent DNA repair may influence the location of escape mutations within target regions. Expression of multiple mismatched crRNAs prevented new mutations from arising in multiple targeted locations, allowing Cas12a mismatch tolerance to provide stronger and longer-term protection. These results demonstrate that Cas effector mismatch tolerance, existing target mismatches, and cleavage site strongly influence phage evolution. CRISPR-Cas immune systems provide immunity against viruses using RNA-guided endonucleases like Cas9 and Cas12a. Viruses can evade these Cas endonucleases through the evolution of mutants that block cleavage by creating mismatches between the guiding RNA and the viral DNA. We show that although Cas12a can tolerate some mismatches, the presence and locations of these mismatches strongly influences the outcomes of further viral evolution. [ABSTRACT FROM AUTHOR]

Early detection of Zika virus (ZIKV) transmission within geographic regions informs implementation of community mitigation measures such as vector reduction strategies, travel advisories, enhanced surveillance among pregnant women, and possible implementation of blood and organ donor screening or deferral. Standardized, comparative assessments of ZIKV assay and testing lab performance are important to develop optimal approaches to ZIKV diagnostic testing and surveillance. We conducted an expanded blinded panel study to characterize and compare the analytical performance of fifteen diagnostic and blood screening ZIKV NAT assays, including detection among single- and multiplex assays detecting ZIKV, dengue virus (DENV) and chikungunya virus (CHIKV). A 300 member blinded panel was constructed, consisting of 11 serial half-log dilutions ranging from ~104 to 10−1 genome equivalents/mL in 25 replicates each of the Tahitian Asian ZIKV isolate in ZIKV-negative human serum. Additionally, clinical samples from individuals with DENV-like syndrome or suspected ZIKV infection in Brazil were evaluated. The majority of assays demonstrated good specificity. Analytical sensitivities varied 1–2 logs, with a substantially higher limit of detection (LOD) in one outlier. Similar analytical sensitivity for ZIKV RNA detection in singleplex and multiplex assays of the Grifols and ThermoFisher tests were observed. Coefficient of Assay Efficiency (CE), calculated to characterize assays' RNA extraction and amplification efficiency, ranged from 0.13 for the Certest VIASURE multiplex and 0.75 for the Grifols multiplex assays. In general, assays using transcription mediated amplification (TMA) technology had greater CE compared to assays using conventional PCR technology. Donor screening NAT assays were significantly more sensitive than diagnostic RT-qPCR assays, primarily attributable to higher sample input volumes. However, ideal assays to maximize sensitivity and throughput may not be a viable option in all contexts, with other factors such as cost, instrumentation, and regulatory approval status influencing assay availability and selection, particularly in resource constrained settings. Author summary: Having standardized, comparative studies of Zika virus (ZIKV) tests are important for implementing optimal diagnostic testing and disease surveillance. Detection of ZIKV nucleic acid is ideal for confirming infection; however, knowing the sensitivity and specificity of a test is needed for selecting the right test in a given laboratory. We looked at the analytical performance of 15 diagnostic and blood screening ZIKV molecular tests, including tests that detect ZIKV only and tests that detect ZIKV plus other arboviruses. We found donor screening NAT assays were significantly more sensitive than diagnostic RT-qPCR assays, mostly due to the higher sample input volumes. We also found no difference in analytical sensitivity between tests that only detected ZIKV compared to tests that detected ZIKV and related viruses. The ideal molecular test will depend on the testing context, with other factors such as cost, instrumentation, and regulatory approval status influencing assay availability and selection. [ABSTRACT FROM AUTHOR]

Cervical carcinoma (CC) is the fourth most common malignancy among women. Screening with Papanicolau smear is linked to a reduction in CC incidence rates when screening programs have been developed. However, this technique has several limitations, including moderate sensitivity rates for detection of cervical preneoplastic HPV-related lesions. In this real-world study, we proposed to evaluate the sensitivity and specificity rates of cobas® test, which amplifies target DNA fragments by polymerase chain reaction and hybridization of nucleic acids for the detection of 14 HR-HPV types in a single analysis) used as primary screening test for CC and preneoplastic lesions in women aged 25–65 years in a large University Hospital in Buenos Aires. A total of 1044 patients were included in the sample (median age: 46 years); sensitivity and specificity rates for the HR-HPV test used as primary screening test were 98.66% (95% confidence interval [95CI]: 97.67–99.3%) and 87.15% (95CI: 84.93–89.15%), respectively. The positive predictive value was 88.47% (95CI: 86.54%-90.42%) and the negative predictive value was 98.48% (95CI: 97.75%-99.23%). The cobas® HR-HPV testing was highly sensitive and specific for the detection of CC and preneoplastic lesions in real practice. [ABSTRACT FROM AUTHOR]

Although the COVID-19 pandemic began over three years ago, the virus responsible for the disease, SARS-CoV-2, continues to infect people across the globe. As such, there remains a critical need for development of novel therapeutics against SARS-CoV-2. One technology that has remained relatively unexplored in COVID-19 is the use of antisense oligonucleotides (ASOs)—short single-stranded nucleic acids that bind to target RNA transcripts to modulate their expression. In this study, ASOs targeted against the SARS-CoV-2 genome and host entry factors, ACE2 and TMPRSS2, were designed and tested for their ability to inhibit cellular infection by SARS-CoV-2. Using our previously developed SARS-CoV-2 bioassay platform, we screened 180 total ASOs targeting various regions of the SARS-CoV-2 genome and validated several ASOs that potently blocked SARS-CoV-2 infection in vitro. Notably, select ASOs retained activity against both the WA1 and B.1.1.7 (commonly known as alpha) variants. Screening of ACE2 and TMPRSS2 ASOs showed that targeting of ACE2 also potently prevented infection by the WA1 and B.1.1.7 SARS-CoV-2 viruses in the tested cell lines. Combined with the demonstrated success of ASOs in other disease indications, these results support further research into the development of ASOs targeting SARS-CoV-2 and host entry factors as potential COVID-19 therapeutics. [ABSTRACT FROM AUTHOR]

Post-Transcriptional Gene Silencing (PTGS) is a defense mechanism that targets invading nucleic acids of endogenous (transposons) or exogenous (pathogens, transgenes) origins. During plant infection by viruses, virus-derived primary siRNAs target viral RNAs, resulting in both destruction of single-stranded viral RNAs (execution step) and production of secondary siRNAs (amplification step), which maximizes the plant defense. As a counter-defense, viruses express proteins referred to as Viral Suppressor of RNA silencing (VSR). Some viruses express VSRs that totally inhibit PTGS, whereas other viruses express VSRs that have limited effect. Here we show that infection with the Turnip yellow mosaic virus (TYMV) is enhanced in Arabidopsis ago1, ago2 and dcl4 mutants, which are impaired in the execution of PTGS, but not in dcl2, rdr1 and rdr6 mutants, which are impaired in the amplification of PTGS. Consistently, we show that the TYMV VSR P69 localizes in siRNA-bodies, which are the site of production of secondary siRNAs, and limits PTGS amplification. Moreover, TYMV induces the production of the host enzyme RNASE THREE-LIKE 1 (RTL1) to further reduce siRNA accumulation. Infection with the Tobacco rattle virus (TRV), which also encodes a VSR limiting PTGS amplification, induces RTL1 as well to reduce siRNA accumulation and promote infection. Together, these results suggest that RTL1 could be considered as a host susceptibility gene that is induced by viruses as a strategy to further limit the plant PTGS defense when VSRs are insufficient. Author summary: RNA silencing is a conserved defense mechanism directed against viruses in various eukaryotic kingdoms. As a counter-defense, viruses generally express proteins referred to as viral suppressor of RNA silencing (VSR), which promote infection by inhibiting one or the other component of the RNA silencing machinery. So far, most of the work on VSRs has concentrated on those that strongly inhibit RNA silencing, causing severe infections and plant death. However, situations where VSRs only partially inhibit RNA silencing could be considered as advantageous for both partners of the infection because infected plants survive, flower and produce seeds despite virus multiplication. In this study, we show that Turnip yellow mosaic virus (TYMV) encodes a weak VSR, P69, which partially inhibits the amplification but not the execution of RNA silencing. In addition, TYMV induces the expression of the endogenous enzyme RNASE THREE-LIKE 1 (RTL1) to further reduce siRNA accumulation. Similarly, Tobacco rattle virus (TRV), which also encodes a weak VSR, induces RTL1 to reduce siRNA accumulation and promote infection. We propose that the limited effect of some VSRs on RNA silencing together with the ability of the corresponding viruses to induce the host RTL1 results in a tight balance between virus propagation and plant development, allowing a virus to propagate without killing its host. In the light of these results, RTL1 could be considered as a susceptibility gene induced by viruses encoding weak VSRs. [ABSTRACT FROM AUTHOR]

Quantitative real-time PCR (qPCR) is a powerful method for measuring nucleic acid levels and quantifying mRNA levels, even in single cells. In the present study, we compared the results of single-cell qPCR obtained by different quantification methods (relative and absolute) and different reverse transcription methods. In the experiments, we focused on the cerebral giant cell (CGC), a key neuron required for the acquisition of conditioned taste aversion in the pond snail Lymnaea stagnalis, and examined changes in the mRNA levels of 3 memory-related genes, cAMP-response element binding proteins (LymCREB1 and LymCREB2) and CREB-binding protein (LymCBP), during memory formation. The results obtained by relative quantification showed similar patterns for the 3 genes. For absolute quantification, reverse transcription was performed using 2 different methods: a mixture of oligo d(T) primers and random primers (RT method 1); and gene-specific primers (RT method 2). These methods yielded different results and did not show consistent changes related to conditioning. The mRNA levels in the samples prepared by RT method 2 were up to 3.3 times higher than those in samples prepared by RT method 1. These results suggest that for qPCR of single neurons, the efficacy and validity do not differ between relative and absolute quantification methods, but the reverse transcription step critically influences the results of mRNA quantification. [ABSTRACT FROM AUTHOR]

Nucleic acid amplification is a sensitive and powerful tool for allergen detection. However, it is limited due to the relatively cumbersome methods required to extract nucleic acids from single plant seed allergen (e.g. peanut and soybean). In view of this, an approach of extracting nucleic acid with untreated glass-fiber paper (paper-based microfluidics) was applied for nucleic acid capture and purification from plant seed allergen and commercial products. After cut by hollow cylindrical cutter, a certain size the paper chip it used to absorb DNA. And this paper-based microfluidics with DNA was directly applied for amplification by loop-mediated isothermal amplification (LAMP). To evaluate the adsorption performance of paper chip to DNA, CTAB and SDS method were used as comparisons. From amplification results, the established technique has good specificity, high repeatability (C.V. values are 4.41% and 6.17% for peanut and soybean) and favorable sensitivity (7.39 ng/μL or peanut and 6.6 ng/μL for soybean), and successfully used for commercial products (2 kinds of candy and 2 kinds of cakes containing peanut, and 2 kinds of drinks, candy and 2 kinds of biscuits containing soybean). This speed and flexible detection method makes it suit for applications in point-of-care (POC) detection at different scenario, such as custom house and import port. [ABSTRACT FROM AUTHOR]

Prion strains in a given type of mammalian host are distinguished by differences in clinical presentation, neuropathological lesions, survival time, and characteristics of the infecting prion protein (PrP) assemblies. Near-atomic structures of prions from two host species with different PrP sequences have been determined but comparisons of distinct prion strains of the same amino acid sequence are needed to identify purely conformational determinants of prion strain characteristics. Here we report a 3.2 Å resolution cryogenic electron microscopy-based structure of the 22L prion strain purified from the brains of mice engineered to express only PrP lacking glycophosphatidylinositol anchors [anchorless (a) 22L]. Comparison of this near-atomic structure to our recently determined structure of the aRML strain propagated in the same inbred mouse reveals that these two mouse prion strains have distinct conformational templates for growth via incorporation of PrP molecules of the same sequence. Both a22L and aRML are assembled as stacks of PrP molecules forming parallel in-register intermolecular β-sheets and intervening loops, with single monomers spanning the ordered fibril core. Each monomer shares an N-terminal steric zipper, three major arches, and an overall V-shape, but the details of these and other conformational features differ markedly. Thus, variations in shared conformational motifs within a parallel in-register β-stack fibril architecture provide a structural basis for prion strain differentiation within a single host genotype. Author summary: Prions are protein-based pathogens that can spread within and between hosts without carrying a pathogen-specific nucleic acid genome. Given this protein-base propagation mechanism, a long-standing mystery in the prion disease field has been the molecular basis of distinct, faithfully propagating strains in a single type of mammalian host. Here we provide a direct, high-resolution cryo-EM-based comparison of the structures of two highly infectious prion strains isolated from the brains of mice of a single genotype. We show in detail how these two prion strains are protein filaments of mouse PrP molecules that display distinct conformational templates for growth on their tips. Our results identify purely conformational, rather than sequence-based, underpinnings of infectious and deadly prion strains. [ABSTRACT FROM AUTHOR]

Combining diagnostic specimens into pools has been considered as a strategy to augment throughput, decrease turnaround time, and leverage resources. This study utilized a multi-parametric approach to assess optimum pool size, impact of automation, and effect of nucleic acid amplification chemistries on the detection of SARS-CoV-2 RNA in pooled samples for surveillance testing on the Hologic Panther Fusion® System. Dorfman pooled testing was conducted with previously tested SARS-CoV-2 nasopharyngeal samples using Hologic's Aptima® and Panther Fusion® SARS-CoV-2 Emergency Use Authorization assays. A manual workflow was used to generate pool sizes of 5:1 (five samples: one positive, four negative) and 10:1. An automated workflow was used to generate pool sizes of 3:1, 4:1, 5:1, 8:1 and 10:1. The impact of pool size, pooling method, and assay chemistry on sensitivity, specificity, and lower limit of detection (LLOD) was evaluated. Both the Hologic Aptima® and Panther Fusion® SARS-CoV-2 assays demonstrated >85% positive percent agreement between neat testing and pool sizes ≤5:1, satisfying FDA recommendation. Discordant results between neat and pooled testing were more frequent for positive samples with CT>35. Fusion® CT (cycle threshold) values for pooled samples increased as expected for pool sizes of 5:1 (CT increase of 1.92–2.41) and 10:1 (CT increase of 3.03–3.29). The Fusion® assay demonstrated lower LLOD than the Aptima® assay for pooled testing (956 vs 1503 cp/mL, pool size of 5:1). Lowering the cut-off threshold of the Aptima® assay from 560 kRLU (manufacturer's setting) to 350 kRLU improved the assay sensitivity to that of the Fusion® assay for pooled testing. Both Hologic's SARS-CoV-2 assays met the FDA recommended guidelines for percent positive agreement (>85%) for pool sizes ≤5:1. Automated pooling increased test throughput and enabled automated sample tracking while requiring less labor. The Fusion® SARS-CoV-2 assay, which demonstrated a lower LLOD, may be more appropriate for surveillance testing. [ABSTRACT FROM AUTHOR]

Background: The COVID-19 pandemic has led nucleic acid collection and detection became a measure to ensure normal life in China. Considering the huge detection demand, it has emerged that robots replace manual sample collection. However, the cost-effectiveness of nucleic acid collection by robots instead of humans remain unknown. Methods: This study was approved by the Ethics Committee of the Shenzhen Luohu District People's Hospital, number 2021-LHQRMYY-KYLL-031a. All participants signed the written informed consent of this study. 273 volunteers were recruited on December 1st 2021 from Shenzhen and divided into six groups: one group to be sampled by robots and the others to be sampled manually with varying specifications for swab rotation and insertion time. Questionnaires were distributed to the robot group to ask them sampling feeling. The effectiveness and safety of sampling were evaluated through the sampling efficiency, adverse events and sampling feeling of different groups. The economics of the different methods were judged by comparing the sampling cost for each. Results: The sampling efficiency of the robot group was 96.9%, and there was no statistically significant difference between the other five manually sampled groups (p = 0.586). There were no serious adverse events in any of the six groups, but nasal soreness and tearing did occur in all group. Of the volunteers who underwent robotic sampling, 85.94% reported that the experience was either no different or more comfortable than the manual sampling. In economic terms, a single robot used to replace medical staff for sample collection becomes economically advantageous when the working time is ≥ 455 days. If multiple robots are used to replace twice the number of manual collections, it becomes more economical at 137 days and remains so as long as the robot is used. Conclusions: It appears safe and effective for robots to replace manual sampling method. Implementation of robotic sampling is economical and feasible, and can significantly save costs when working over a long term. [ABSTRACT FROM AUTHOR]

Background: Rapid diagnosis of influenza is critical in preventing the spread of infection and ensuring patients quickly receive antiviral medication to reduce the severity and duration of influenza symptoms, whilst controlling the spread of the causative virus. In Japan patients are often administered anti-influenza medication following a positive rapid antigen detection test (RADT) result. However, the sensitivity of RADTs can lead to false negative results. The cobas® Influenza A/B Nucleic acid test for use on the cobas Liat® System (Liat) is a molecular point-of-care method that can provide a more sensitive alternative to RADTs for rapid influenza diagnosis and treatment. Methods: In this prospective multicenter study, diagnostic performance of the Liat test was compared with RADTs in patients presenting with influenza-like-illness. Test performance was also assessed by time since symptom onset. Results: Of 419 patients enrolled, 413 were evaluable for all designated tests. Most patients had type-A infection, and only one patient had influenza type B. In 413 patients, the sensitivity and specificity (95% CI) of the Liat test were 99.5% (97.2–99.9%) and 99.5% (97.4–99.9%), respectively, and were 79.7% (73.5–84.7%) and 95.4% (91.7–97.5%) for RADTs. For patients tested <12 hours from symptom onset, the Liat test had significantly higher sensitivity than RADTs (p<0.0001). Conclusion: Overall, compared with standard of care RADTs, the Liat test was more sensitive and specific in children and adults, particularly in the early stages of infection. Greater sensitivity can enable earlier diagnosis and may better inform appropriate antiviral treatment decisions. [ABSTRACT FROM AUTHOR]

Endophytic fungi from the Chilli were used to help okra plants exposed to cadmium (Cd) or chromium (Cr) stress. Initially, the strain Ch06 produced higher amounts of indole acetic acid (IAA) (230.5 μg/mL), sugar (130.7 μg/mL), proteins (128.2 μg/mL), phenolics (525.6 μg/mL) and flavonoids (98.4 μg/mL) in Czapek broth supplemented with Cd or Cr. The production of IAA and other metabolites in such a higher concentration suggested that Ch06 might improve plant growth under heavy metal stress. For this reason, an experiment was designed, in which biomass of Ch06 (at 2g/100g of sand) were applied to the okra plants exposed to Cd or Cr stress (at 100 or 500 μg/g). The results exhibited that Ch06 improved the total chlorophyll (36.4±0.2 SPAD), shoot length (22.6±0.2 cm), root length (9.1±0.6 cm), fresh weight (5±0.6 g), dry weight (1.25±0.01 g), sugars (151.6 μg/g), proteins (114.8 μg/g), proline (6.7 μg/g), flavonoids (37.9 μg/g), phenolics (70.7 μg/g), IAA (106.7 μg/g), catalase (0.75 enzyme units/g tissue) and ascorbic acid oxidaze (2.2 enzyme units/g tissue) of the associated okra plants. Similar observations have been recorded in Ch06 associated okra plants under Cd and Cr stress. Also, Ch06 association reduced translocation of Cd (35% and 45%) and Cr (47% and 53%) to the upper parts of the okra plants and thus reduced their toxicity. The internal transcribed spacer (ITS) region amplification of 18S rDNA (ribosomal deoxyribo nucleic acid) exhibited that the potent strain Ch06 was Aspergillus violaceofuscus. The results implied that A. violaceofuscus has the ability to promote host species growth exposed to Cd and Cr. Moreover, it helped the host plants to recover in Cd and Cr polluted soils, hence can be used as biofertilizer. [ABSTRACT FROM AUTHOR]