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Extrachromosomal DNA (ecDNA) is a central mechanism for focal oncogene amplification in cancer, occurring in ∼15% of early-stage cancers and ∼30% of late-stage cancers. ecDNAs drive tumor formation, evolution, and drug resistance by dynamically modulating oncogene copy number and rewiring gene-regulatory networks. Elucidating the genomic architecture of ecDNA amplifications is critical for understanding tumor pathology and developing more effective therapies. Paired-end short-read (Illumina) sequencing and mapping have been utilized to represent ecDNA amplifications using a breakpoint graph, in which the inferred architecture of ecDNA is encoded as a cycle in the graph. Traversals of breakpoint graphs have been used to successfully predict ecDNA presence in cancer samples. However, short-read technologies are intrinsically limited in the identification of breakpoints, phasing together complex rearrangements and internal duplications, and deconvolution of cell-to-cell heterogeneity of ecDNA structures. Long-read technologies, such as from Oxford Nanopore Technologies, have the potential to improve inference as the longer reads are better at mapping structural variants and are more likely to span rearranged or duplicated regions. Here, we propose Complete Reconstruction of Amplifications with Long reads (CoRAL) for reconstructing ecDNA architectures using long-read data. CoRAL reconstructs likely cyclic architectures using quadratic programming that simultaneously optimizes parsimony of reconstruction, explained copy number, and consistency of long-read mapping. CoRAL substantially improves reconstructions in extensive simulations and 10 data sets from previously characterized cell lines compared with previous short- and long-read-based tools. As long-read usage becomes widespread, we anticipate that CoRAL will be a valuable tool for profiling the landscape and evolution of focal amplifications in tumors.

HER2, encoded by the ERBB2 gene, is an important druggable driver of human cancer gaining increasing importance as a therapeutic target in urothelial carcinoma (UC). The genomic underpinnings of HER2 overexpression in ERBB2 nonamplified UC are poorly defined. To address this knowledge gap, we investigated 172 UC tumors from patients treated at the University of California San Francisco, using immunohistochemistry and next-generation sequencing. We found that GATA3 and PPARG copy number gains individually predicted HER2 protein expression independently of ERBB2 amplification. To validate these findings, we interrogated the Memorial Sloan Kettering/The Cancer Genome Atlas (MSK/TCGA) dataset and found that GATA3 and PPARG copy number gains individually predicted ERBB2 mRNA expression independently of ERBB2 amplification. Our findings reveal a potential link between the luminal marker HER2 and the key transcription factors GATA3 and PPARG in UC and highlight the utility of examining GATA3 and PPARG copy number states to identify UC tumors that overexpress HER2 in the absence of ERBB2 amplification. In summary, we found that an increase in copy number of GATA3 and PPARG was independently associated with higher ERBB2 expression in patient samples of UC. This finding provides a potential explanation for HER2 overexpression in UC tumors without ERBB2 amplification and a way to identify these tumors for HER2-targeted therapies.

The recently proposed concept of electric chiral magnonic resonator (ECMR) has been extended to include usage of spin–orbit torques (SOT). Unlike the original version of ECMR which was based on voltage controlled magnetic anisotropy (VCMA), the spin wave amplification power by this new version of ECMR (pumped by SOT) no longer depends on the phase of the incident wave, which is highly desirable from an application point of view. The performance of the SOT pumped ECMR has been compared with the case of amplification by applying SOT pumping directly to a waveguide (without any ECMR involved). It is argued that at the expense of narrowing the bandwidth (i.e., slower amplifier response), the advantage of the former configuration (amplification by a SOT pumped ECMR) over the latter (amplification by direct SOT pumping the waveguide) is to offer gain, while at the same time, maintaining system stability (avoidance of auto-oscillations). Non-linear behavior of the SOT pumped ECMR has been analyzed. It is demonstrated that by cascading a SOT ECMR operating in an off-resonance mode together with a VCMA biased passive ECMR, it is possible to produce a magnonic neuron with a transmitted signal magnitude larger than the input in the firing state. [ABSTRACT FROM AUTHOR]

UNLABELLED: 11q13 amplification is a frequent event in human cancer and in particular in squamous cell carcinomas (SCC). Despite almost invariably spanning 10 genes, it is unclear which genetic components of the amplicon are the key driver events in SCC. A combination of computational, in vitro, ex vivo, and in vivo models leveraging efficient primary human keratinocyte genome editing by Cas9-RNP electroporation, identified ORAOV1, CCND1, and MIR548K as the critical drivers of the amplicon in head and neck SCC. CCND1 amplification drives the cell cycle in a CDK4/6/RB1-independent fashion and may confer a novel dependency on RRM2. MIR548K contributes to epithelial-mesenchymal transition. Finally, we identify ORAOV1 as an oncogene that acts likely via its ability to modulate reactive oxygen species. Thus, the 11q13 amplicon drives SCC through at least three independent genetic elements and suggests therapeutic targets for this morbid and lethal disease. IMPLICATIONS: This work demonstrates novel mechanisms and ways to target these mechanisms underlying the most common amplification in squamous cell carcinoma, one of the most prevalent and deadly forms of human cancer.

Purpose: To determine the prevalent microbiological profile of biliary atresia (BA) patients at the time of its occurrence by studying their intestinal flora. Methods: A total of 118 gut microbiota samples from three groups of 43 BA patients, 33 disease controls (DC) with other cholestatic diseases and 42 healthy controls (HC), were analyzed by deep mining of public data. Subsequently, a total of 23 fecal samples from three groups of clinically collected patients (11 BA, 6 DC and 6 HC) were sequenced for 16S rRNA gene amplification and analyzed for serum butyrate (BU) level by liquid chromatography. Results: Taxonomic analysis revealed significant differences in the composition of the intestinal microbiota between BA patients and controls, with a reduction in diversity and a higher abundance of Proteobacteria, Streptococcus and Lactobacillus in the BA group. Database and clinical data analyses concluded that Streptococcus/Bacteroides (AUC = 0.9035, 95% CI 0.8347–0.9722, P < 0.0001) or Streptococcus/Eggerthella (AUC = 0.8333, 95% CI 0.6340–1.000, P = 0.027) was the best microbiota to differentiate between BA and DC. Serum butyrate levels were low in the BA and DC groups and differed from the HC group (P = 0.01, P = 0.04). Butyrate levels in BA were negatively correlated with jaundice clearance and cholangitis, but not statistically significant. Conclusions: Our study reveals changes in the composition of the gut microbiota in BA, especially the butyrate-producing microbiota, and suggests the potential for using gut microbiota as a noninvasive diagnostic benefit for BA. Low levels of serum butyrate in BA may indicate a poor prognosis. [ABSTRACT FROM AUTHOR]

Combined CDK4/6 inhibitor (CDK4/6i) and endocrine therapy significantly improves outcome for patients with estrogen receptor‐positive (ER+) metastatic breast cancer, but drug resistance and thus disease progression inevitably occur. Herein, we aimed to identify genomic alterations associated with combined CDK4/6i and endocrine therapy resistance, and follow the levels of specific mutations in longitudinal circulating tumor DNA (ctDNA) for early detection of progression. From a cohort of 86 patients with ER+ metastatic breast cancer we performed whole exome sequencing or targeted sequencing of paired tumor (N = 8) or blood samples (N = 5) obtained before initiation of combined CDK4/6i and endocrine therapy and at disease progression. Mutations in oncogenic genes at progression were rare, while amplifications of growth‐regulating genes were more frequent. The most frequently acquired alterations observed were PIK3CA and TP53 mutations and PDK1 amplification. Longitudinal ctDNA dynamics of mutant PIK3CA or private mutations revealed increased mutation levels at progression in 8 of 10 patients (80%). Impressively, rising levels of PIK3CA‐mutated ctDNA were detected 4–17 months before imaging. Our data add to the growing evidence supporting longitudinal ctDNA analysis for real‐time monitoring of CDK4/6i response and early detection of progression in advanced breast cancer. Further, our analysis suggests that amplification of growth‐related genes may contribute to combined CDK4/6i and endocrine therapy resistance. [ABSTRACT FROM AUTHOR]

This study aims to enhance the existing knowledge of the prevalence of genes responsible for beta-lactam resistance and aminoglycoside resistance in gram negative organisms by molecular detection of extended spectrum beta-lactamase and aminoglycoside modifying enzymes in multidrug-resistant gram-negative bacteria. Out of 864 gram-negative isolates, 710 were phenotypically identified as multidrug-resistant by antibiotic susceptibility testing. From the above isolates, 102 representative isolates as per sample size calculated were selected for further molecular studies. The presence of blaTEM, blaCTX-M blaSHV, and five AmpC genes was detected by real-time polymerase chain reaction (PCR). Conventional PCR was performed to detect seven aminoglycoside modifying enzyme genes namely aac(6')-Ib, aac(6')-Ic, aac(3)-Ia, aac(3)-Ib, aac(3)-IIa, ant(2'')-Ia, and ant(4'')-IIa. Most common multidrug-resistant isolate was Klebsiella pneumoniae (35%) followed by Escherichia coli (30%). Among the 102 selected isolates all harboured blaTEM gene, 71 (69.6%) harboured blaCTX-M gene and 48 (47%) blaSHV gene. Among the selected isolates 60% showed the presence of AmpC genes. Most common aminoglycosie modifying enzyme gene was AAC 6' Ib (51%) followed by ANT 2" Ia (36%). This study suggests a wider use of molecular methods using specific PCR amplification of resistance genes. It would be beneficial to perform the molecular identification of antimicrobial resistance genes to effectively monitor and manage antibiotic resistance, administer appropriate antimicrobial medication, practice antimicrobial stewardship and improve hospital infection control procedures. [ABSTRACT FROM AUTHOR]

The Almanagil province located in Gezira scheme, Gezira state, Sudan, represents a suitable environment for the breeding of malaria-carrying mosquitoes. An estimated 5.9% of Sudanese people suffer from malaria, with 87.6% of cases caused by Plasmodium falciparum and 12.4% by Plasmodium vivax. Clinical manifestation of malaria cases range from mild uncomplicated to severe and fatal complications and the genetic variants and multiplicity of falciparum infection can worsen the manifestations of malaria. The objective of this work is to determine the degree of genetic variation in P. falciparum infection in a high-transmission region of central Sudan by analyzing merozoite surface protein-1 (msp1) and merozoite surface protein-2 (msp2) variations. During the rainy season of 2022, Eighty-nine children with confirmed severe falciparum malaria whom admitted to Almanagil Pediatric Hospital were included in this study. Dry blood spots were used to extract the DNA and amplification of three msp1 and two of msp2 allelic subfamilies, namely K1, RO33 and MAD20 and FC27 and IC/3D7 respectively. The data was analyzed by using SPSS computer program (v 23.0). The three genetic subfamilies of msp1 (K1, RO33 and MAD20) and the two alleles of msp2 (FC27 and IC/3D7) were identified. Msp1 variants represent K1 (64/89, 71.9%), RO33 (56/89, 62.9%) and MAD20 (72/89, 80.9%), while msp2 diversity represents ICI/3D7 (52/89, 58.4%), FC27 (62/89, 69.6%) and ICI/3D7/FC27(33/89, 37.1%). The MAD20 and FC27 showed high genetic diversity among both genes, respectively. RO33 allele shows a strong association with severity of falciparum malaria (OR 2.572, P 0.045 ), while the K1 was the lowest risk factor for malaria severity. The allele subfamily K1 and MAD20 of msp1 were associated with hypoglycemia (OR 4.21 and 2.91), respectively. Our study revealed high genetic polymorphisms of msp1 and msp2. Among Central Sudanese children with high MOI of P. falciparum isolates, there was a significant frequency of msp1, a strong association between the K1 allele and hypoglycemia, and a substantial association between the RO33 and MAD20 alleles with the severity of the infection. These findings could help develop malaria control strategies. [ABSTRACT FROM AUTHOR]

Objective: Helicobacter pylori infection is acquired in childhood via the oral cavity, although its relationship with the characteristics of the oral microbiome has not been elucidated. In this study, we performed comprehensive analysis of the oral microbiome in children and adults with or without H. pylori in the oral cavity. Methods: Bacterial DNA was extracted from 41 adult and 21 child saliva specimens, and H. pylori was detected using PCR. 16S rRNA gene amplification was performed for next-generation sequencing. Bioinformatic analyses were conducted using Quantitative Insights into Microbial Ecology 2 (QIIME 2). Results: Faith's phylogenetic diversity analysis showed a significant difference between H. pylori-negative adult and child specimens in terms of α-diversity (p < 0.05), while no significant difference was observed between H. pylori-positive adult and child specimens. There was also a significant difference in β-diversity between H. pylori-positive and negative child specimens (p < 0.05). Taxonomic analysis at the genus level revealed that Porphyromonas was the only bacterium that was significantly more abundant in both H. pylori-positive adults and children than in corresponding negative specimens (p < 0.01 and p < 0.05, respectively). Conclusion: These results suggest unique oral microbiome characteristics in children with H. pylori infection in the oral cavity. [ABSTRACT FROM AUTHOR]

PURPOSE: The National Cancer Institute-Children's Oncology Group Pediatric Molecular Analysis for Therapy Choice trial assigned patients age 1-21 years with relapsed or refractory solid tumors, lymphomas, and histiocytic disorders to phase II treatment arms of molecularly targeted therapies on the basis of genetic alterations detected in their tumor. Patients with tumors that harbored prespecified genomic alterations in the cyclinD-CDK4/6-INK4a-Rb pathway with intact Rb expression were assigned and treated with the cdk4/6 inhibitor palbociclib. METHODS: Patients received palbociclib orally once daily for 21 days of 28-day cycles until disease progression, intolerable toxicity, or up to 2 years. The primary end point was objective response rate; secondary end points included safety/tolerability and progression-free survival. RESULTS: Twenty-three patients (median age, 15 years; range, 8-21) were enrolled; 20 received protocol therapy and were evaluable for toxicity and response. Of the evaluable patients, the most common diagnoses were osteosarcoma (n = 9) and rhabdomyosarcoma (n = 6). A single actionable gene amplification was found in 19 tumors (CDK4 , n = 11, CDK6 , n = 2, CCND3 , n = 6), with one tumor harboring two amplifications (CDK4 and CCND2). Hematologic toxicities were the most common treatment-related events. No objective responses were seen. Two patients with tumors harboring CDK4 amplifications (neuroblastoma and sarcoma) had best response of stable disease for six and three cycles. Six-month progression was 10% (95% CI, 1.7 to 27.2). CONCLUSION: The CDK4/6 inhibitor palbociclib at 75 mg/m2 orally daily was tolerable in this heavily pretreated cohort. No objective responses were observed in this histology-agnostic biomarker-selected population with treatment-refractory solid tumors, demonstrating that pathway alteration alone is insufficient in pediatric cancers to generate a response to palbociclib monotherapy. [ABSTRACT FROM AUTHOR]

PURPOSE: Panel-based comprehensive genomic profiling (CGP) is used in clinical practice worldwide; however, large real-world data (RWD) of patients with advanced small intestine cancer have not been characterized. We investigated differences in the prevalence of clinically relevant alterations across molecularly defined or age-stratified subgroups. PATIENTS AND METHODS: This was a collaborative biomarker study of RWD from CGP testing (Foundation Medicine, Inc). Hybrid capture was conducted on at least 324 cancer-related genes and select introns from up to 31 genes frequently rearranged in cancer. Overall, 1,364 patients with advanced small intestine cancer were available for analyses and were stratified by age (≥40 years/<40 years), microsatellite instability (MSI) status, tumor mutational burden (TMB) status (high ≥10/low <10 Muts/Mb), and select gene alterations. The frequency of alterations was analyzed using a chi-square test with Yate's correction. RESULTS: Genes with frequent alterations included TP53 (59.8%), KRAS (54.8%), APC (27.7%), and CDKN2A (22.4%). Frequent genes with amplifications were MYC (6.7%), MDM2 (5.9%), GATA6 (5.5%), and CCND1 (3.4%). Patients younger than 40 years had significantly lower frequency of APC mutations than those 40 years and older (10.4% v 28.7%; P =.0008). Druggable genomic alterations were detected in 22.3% of patients: BRAF V600E (1.2%), BRCA1 (1.8%), BRCA2 (3.2%), ERBB2 amplification (3.2%), KRAS G12C (3.3%), NTRK1/2/3 fusion (0.07%), MSI-high (7.0%), and TMB-high (12.2%), with no significant differences in the frequency according to age (<40 years v ≥40 years; 22.1% v 22.3%). TMB of 10-20 Mut/Mb was observed in 4.8% of patients, and TMB ≥20 Mut/Mb was seen in 7.3% of the cohort. CONCLUSION: RWD from clinical panel testing revealed the genomic landscape in small intestine cancer by subgroup. These findings provide insights for the future development of treatments in advanced small intestine cancer. [ABSTRACT FROM AUTHOR]

Trichotylenchus sp. is a migratory ectoparasitic nematode (PPN) that causes hypoplasia disease symptoms in economic plants, such as maize and banana. In this study, soil samples were collected from 6 locations in a tomato field in Khon Kaen Province, Thailand, for the purpose of nematode extraction using the Cobb's sieving and flotation-centrifugation techniques. Morphological and molecular characterization of the collected PPN identified it as Trichotylenchus sp., a PPN not previously found in Thai tomato fields. Morphologically, the nematode has a C-shaped body, a rounded lip region, robust stylet and dorsal gland orifice located 2.0 ± 0.5 µm from the base of basal knobs. The tail is conically shaped, and the cuticle exhibits annulations with 3 incisures in the lateral field. The morphological characteristics observed closely matched Trichotylenchus sp. To confirm the identity, diagnosis was done using Polymerase Chain Reaction (PCR) molecular technique. Nematode DNA amplification was conducted with different target genes using primer sets AB28/TW81 for ITS1-5.8S-ITS2 and D2A/D3B for D2-D3 region of the 28S rRNA. The PCR amplification showed DNA fragments of approximately 950 and 800 bp, respectively. The sequences obtained were compared with those in the NCBI GenBank, which showed a 98.0 - 99.4 % identity match with Trichotylenchus dispersus specimens previously documented in China. In addition, the phylogenetic trees reiterated a nematode grouping with T. dispersus, 100 % bootstrap value. To the best of our knowledge, this is the first report of the presence of T. dispersus in the soils of tomato field in Thailand. This nematode is likely to become a major factor limiting tomato production yields if not properly managed. [ABSTRACT FROM AUTHOR]

Research into various bacterial pathogens that can be transmitted between different animals and may have zoonotic potential has led to the discovery of different strains of Bartonella sp. in bats and their associated ectoparasites. Despite their enormous species diversity, only a few studies have focussed on the detection of bacterial pathogens in insectivorous bats of boreal forests and their associated Macronyssidae and Spinturnicidae mites. We collected and molecularly analysed mite samples from forest‐dwelling bat species distributed all along the boreal belt of the Palearctic, from Central Europe to Far East. Ectoparasitic mites were pooled for DNA extraction and DNA amplification polymerase chain reaction (PCRs) were conducted to detect the presence of various bacterial (Anaplasmataceae, Bartonella sp., Rickettsia sp., Mycoplasma sp.) and protozoal (Hepatozoon sp.) pathogens. Bartonella sp. DNA was detected in four different mite species (Macronyssidae: Steatonyssus periblepharus and Spinturnicidae: Spinturnix acuminata, Sp. myoti and Sp. mystacinus), with different prevalences of the targeted gene (gltA, 16‐23S ribosomal RNA intergenic spacer and ftsZ). Larger pools (>5 samples pooled) were more likely to harbour Bartonella sp. DNA, than smaller ones. In addition, cave‐dwelling bat hosts and host generalist mite species are more associated with Bartonella spp. presence. Spinturnicidae mites may transmit several distinct Bartonella strains, which cluster phylogenetically close to Bartonella species known to cause diseases in humans and livestock. Mites with ubiquitous presence may facilitate the long‐term maintenance (and even local recurrence) of Bartonella‐infestations inside local bat populations, thus acting as continuous reservoirs for Bartonella spp in bats. [ABSTRACT FROM AUTHOR]

Bacillus cereus is a common contaminating bacteria and conditioned pathogens in food. Immunological methods or molecular biology assays that are currently available cannot distinguish between live and dead bacterial cells, which may overestimate the number of bacteria and lead to false-positive results. To solve this problem, we introduced propidium monoazide (PMA) and calcein in this study and established a real-time visual loop-mediated isothermal amplification (LAMP) detection method to detect viable B. cereus. In addition, the results can be detected visually with calcein dye or quantitatively by monitoring the amplification curve of the fluorescence signal. In this study, the results showed that the visual PMA-LAMP method had high specificity, strong anti-interference ability, a sensitivity of 1.16 × 102 CFU/mL, and that the whole reaction could be completed within 80 min. This method not only has high sensitivity, but also has a shorter reaction time and is faster. In the detection of artificially contaminated food samples, the lowest detection limit reached 6 CFU/mL after 3 or 4 h of enrichment. Therefore, the visual PMA-LAMP technology can provide an efficient and rapid method for the detection of live B. cereus bacteria. [ABSTRACT FROM AUTHOR]

Fasciola hepatica is a parasitic liver trematode that causes fasciolosis in humans and dairy animals. Traditional detection of infection is based on a microscopic examination with low sensitivity. Therefore, this study was carried out to develop an accurate and rapid method to detect F. hepatica in Alborz province. In this cross-sectional study, 386 samples were collected from livers of cattle and sheep in three slaughterhouses of Alborz. DNA was extracted, and nested-PCR was carried out based on the F. hepatica ITS-2 sequence. DNA amplification showed first and second PCR products with expected size of 336 and 208 bp respectively, as unique for F. hepatica. The results showed no cross-reaction with the negative control. Thirty-five liver samples were positive for this parasitic infection. Totally, the prevalence of F. hepatica in the slaughterhouses of Alborz, Iran was 9.07%. This is the first study of the molecular detection of F. hepatica using nested-PCR in Alborz. It is a sensitive and rapid method that will help evaluate the prevalence rate of F. hepatica infection. [ABSTRACT FROM AUTHOR]

The function of spargel / dPGC-1 in Drosophila oogenesis has been unequivocally established. Here, we sought to assess whether Spargel protein or RNA is essential for developmentally competent eggs. The trans-heterozygotic combination of two spargel mutant alleles allowed us to decrease Spargel expression to very low levels. Using this model, we now demonstrated the requirement for Spargel in eggshell patterning and embryonic development, which led us to establish that spargel is a maternal effect gene. Further examination of Spargel's potential mechanism of action in eggshell biogenesis revealed that low levels of Spargel in the adult ovary cause diminished Cyclin E activity, resulting in reduced chorion gene amplification levels, leading to eggshell biogenesis defects. Thus, another novel role for spargel/dPGC-1 is exposed whereby, through Cyclin E activity, this conserved transcriptional coactivator regulates the chorion gene amplification process. [Display omitted] • Spargel, an ancestral PGC-1 ortholog, acts as a maternal effect gene. • Reduced Spargel leads to eggshell patterning and eggshell biogenesis defects. • Spargel deficiency impairs chorion synthesis associated with insufficient Cyclin E activity. [ABSTRACT FROM AUTHOR]

ABSTRACT Background Methods Results Conclusions Lip exposure to carcinogens lead to several disorders, such as actinic cheilitis (AC) and lower lip squamous cell carcinoma (LLSCC). Although several studies have described important pathways in lip carcinogenesis, the comprehension of association of target genes in this process and their association with tumor microenvironment still need to be better understood.Tissue samples of 30 AC and 17 LLSCC cases were included for histopathological analysis, immunohistochemical expression of CD4, CD8, and PD‐L1, and fluorescence in situ hybridization for AURKA, AURKB, TP53, PTEN, CCND1, and MYC. Non‐parametrical tests were done and p < 0.05 was considered significant.LLSCC patients presented higher amplifications of AURKA and AURKB, deletion of TP53, and PTEN and rearrangements of MYC than AC. AURKA, AURKB, TP53, PTEN, and CCND1 changes were correlated with PD‐L1 expression.AURKA and AURKB amplifications and other gene changes are pointed by their association of lip disorders. [ABSTRACT FROM AUTHOR]

Background: Accurate and comprehensive genomic annotation, including the full list of protein-coding genes, is vital for understanding the molecular mechanisms of human biology. We have previously shown that the genome contains a multitude of yet hidden functional exons and transcripts, some of which might represent novel mRNAs. These results resonate with those from other groups and strongly argue that two decades after the completion of the first draft of the human genome sequence, the current annotation of human genes and transcripts remains far from being complete. Results: Using a targeted RNA enrichment technique, we showed that one of the novel functional exons previously discovered by us and currently annotated as part of a long non-coding RNA, is actually a part of a novel protein-coding gene, InSETG-4, which encodes a novel human protein with no known homologs or motifs. We found that InSETG-4 is induced by various DNA-damaging agents across multiple cell types and therefore might represent a novel component of DNA damage response. Despite its low abundance in bulk cell populations, InSETG-4 exhibited expression restricted to a small fraction of cells, as demonstrated by the amplification-based single-molecule fluorescence in situ hybridization (asmFISH) analysis. Conclusions: This study argues that yet undiscovered human protein-coding genes exist and provides an example of how targeted RNA enrichment techniques can help to fill this major gap in our knowledge of the information encoded in the human genome. [ABSTRACT FROM AUTHOR]

Background: HER2-positivity is an essential marker for therapeutic decisions, while HER2 expression is heterogenous. In recent years, there has been increasing recognition of a subgroup of breast cancer patients who have low levels of HER2 expression, also known as HER2-low because trastuzumab deruxtecan offers clinical benefit for patients with HER2-low metastatic breast cancer. Despite the growing interest in HER2-low breast cancer, there is limited research on how multigene assays can help differentiate between HER2-low and HER2-negative breast cancer. Among HR + HER2- breast cancer, we compared genomic characteristics between HER2-low and HER2-zero using the 21-gene assay. Methods: A retrospective review of clinical records was performed in 2,295 patients who underwent Oncotype DX® test in two hospitals between 2013 and 2020. Patients were classified into two groups as the HER2-zero and HER2-low based on HER2 immunohistochemistry. In cases with HER2 2+, no amplification of HER2 gene was confirmed by silver in situ hybridization. High genomic risk was defined as cases with 21-gene recurrence score (RS) > 25. Multivariable binary logistic-regression analysis was performed. Results: Of these, 944 (41.1%) patients were assigned to the HER2-zero group, while 1351 (58.9%) patients were assigned to the HER2-low group. The average Recurrence Score (RS) was found to be 17.802 in the HER2-zero breast cancer group and 18.503 in the HER2-low group, respectively (p-value < 0.005). When comparing the proportion of high RS between the two groups, the HER2-zero group had a high RS rate of 12.4% (117 out of 944), while the HER2-low group had a high RS rate of 17.0% (230 out of 1351) (p = 0.002). The HER2 score identified by qRT-PCR was 8.912 in the HER2-zero group and 9.337 in the HER2-low group (p < 0.005). In multivariable analysis, HER2-low status was found to be an independent factor for high RS, with an odds ratio of 1.517 (1.172–1.964), independent of ER, PR, and Ki67. Within the subgroup of patients with invasive ductal carcinoma, the high RS rates were 19% in the HER2-low group and 14% in the HER2-zero group. However, when considering all patients, there were no significant differences observed in recurrence-free survival and overall survival between the HER2-low and HER2-zero groups. Conclusion: Within HR + HER2- breast cancer, HER2-low tumors are associated with high RS, especially for histologically invasive ductal carcinoma. A prognostic influence of HER2-low expression among HR + HER2- breast cancer remains as an area that requires further study. [ABSTRACT FROM AUTHOR]

ABSTRACT Fatty acid desaturase (FAD) is essential for plant growth and development and plant defence response. Although flax (Linum usitatissimum L.) is an important oil and fibre crop, but its FAD gene remains understudied. This study identified 43 LuFAD genes in the flax genome. The phylogenetic analysis divided the FAD genes into seven subfamilies. LuFAD is unevenly distributed on 15 chromosomes, and fragment duplication is the only driving force for the amplification of the LuFAD gene family. In the LuFAD gene promoter region, most elements respond to plant hormones (MeJA, ABA) and abiotic stresses (anaerobic and low temperature). The expression pattern analysis showed that the temporal and spatial expression patterns of all LuFAD genes in different tissues and the response patterns to abiotic stresses (heat and salt) were identified. Subcellular localisation showed that all LuFAD2‐GFP were expressed in the endoplasmic reticulum membrane. RT‐qPCR analysis revealed that LuFAD2 was significantly upregulated under cold, salt and drought stress, and its overexpression in Arabidopsis thaliana enhanced cold tolerance genes and reduced ROS accumulation. This study offers key insights into the FAD gene family's role in flax development and stress adaptation. [ABSTRACT FROM AUTHOR]

The clinical application of small molecule tyrosine kinase inhibitors (TKIs) has significantly improved the quality of life and prognosis of patients with non-small cell lung cancer (NSCLC) carrying driver genes. However, resistance to TKI treatment is inevitable. Bypass signal activation is one of the important reasons for TKI resistance. Although TKI drugs inhibit downstream signaling pathways of driver genes, key signaling pathways within tumor cells can still be persistently activated through bypass routes such as MET gene amplification, EGFR gene amplification, and AXL activation. This continuous activation maintains tumor cell growth and proliferation, leading to TKI resistance. The fundamental strategy to treat TKI resistance mediated by bypass activation involves simultaneously inhibiting the activated bypass signals and the original driver gene signaling pathways. Some clinical trials based on this combined treatment approach have yielded promising preliminary results, offering more treatment options for NSCLC patients with TKI resistance. Additionally, early identification of resistance mechanisms through liquid biopsy, personalized targeted therapy against these mechanisms, and preemptive targeting of drug-tolerant persistent cells may provide NSCLC patients with more sustained and effective treatment. [ABSTRACT FROM AUTHOR]

Background: Cystoisospora felis or Isospora felis is a ubiquitous apicomplexan protozoon parasite infecting domestic cats worldwide. Objectives of the study: this study aims to identify the causative agent of diarrhea in cats by determining several elevating stressors caused by these coccidian protozoans with molecular characterization. So, from January 2023 to April 2023, a total of 370 domestic cats were hospitalized at various clinics in the Cairo and Giza Governorates. Fecal samples were taken from these animals and examined by concentration floatation techniques using a saturated salt solution. The positive samples were sporulated to identify the collected oocyst. Venous blood was taken from the infected cats to evaluate the associated oxidative stress marker (lipid peroxidation products (MDA). Results: Out of 370 examined domestic cats, 27(7.29%) were positive for C. felis. The MDA levels increased with age, and females were higher than males. DNA was extracted from fecal samples for amplification of the ITS1 gene, followed by sequencing. The ITS1 gene was amplified and showed bands at 224 bp. The partial nucleotide sequence of the ITS1 gene was aligned with the reference sequences. In a conclusion: C. felis increases the free radicals, which in turn means the animals have stress and need a schedule to treat these animals with new, safe protocol drugs that give no resistance and are highly efficient. [ABSTRACT FROM AUTHOR]

Wild species of crops serve as a valuable germplasm resource for breeding of modern cultivars. Rice (Oryza sativa L.) is a vital global staple food. However, research on genome evolution and diversity of wild rice species remains limited. Here, we present nearly complete genomes of 13 representative wild rice species. By integrating with four previously published genomes for pangenome analysis, a total of 101,723 gene families are identified across the genus, including 9834 (9.67%) core gene families. Additionally, 63,881 gene families absent in cultivated rice species but present in wild rice species are discovered. Extensive structural rearrangements, sub-genomes exchanges, widespread allelic variations, and regulatory sequence variations are observed in wild rice species. Interestingly, expanded but less diverse disease resistance genes in the genomes of cultivated rice, likely due to the loss of some resistance genes and the fixing and amplification of genes encoding resistance genes to specific diseases during domestication and artificial selection. This study not only reveals natural variations valuable for gene-level studies and breeding selection but also enhances our understanding on rice evolution and domestication. The Oryza genus comprise two cultivated rice species and 20 extant wild species. Here the authors assemble genomes of 13 representative wild rice species, construct a super pangenome by integrating them with four previously reported genomes in the genus, and reveal the genome evolution and diversity within the genus. [ABSTRACT FROM AUTHOR]

Targeted treatment of advanced or metastatic urothelial carcinomas (UCs) requires the identification of druggable mutations. This study describes the development of a 3′ Rapid Amplification of cDNA Ends (3′ RACE)-based targeted RNA sequencing panel which accounts for the status of all genes relevant to UC treatment, namely, FGFR1-4, KRAS, NRAS, BRAF, ERBB2 (HER2), CD274 (PD-L1) and PIK3CA. FGFR2/3-activating point mutations or fusions were found in 54/233 (23.2%) tumors. FGFR3 rearrangements were identified in 11 patients, with eight of them being undetectable by commonly used PCR kits. In addition, one tumor contained a high-copy FGFR2 gene amplification accompanied by strong overexpression of the gene. Mutations in RAS/RAF genes were present in 30/233 (12.9%) UCs and were mutually exclusive with alterations affecting FGFR2/3 genes. On the contrary, activating events in the HER2 oncogene (point mutations and overexpression), as well as PIK3CA mutations, which were relatively common, occurred with similar frequencies in RAS/RAF- or FGFR2/3-positive vs. negative samples. High PD-L1 mRNA expression was associated with advanced disease stage and was not observed in tumors with increased HER2 mRNA expression or in UCs with evidence for FGFR2/3 activation. Three of the studied carcinomas had high-level microsatellite instability (MSI). Overall, more than half of the UCs had potentially druggable genetic alterations. The proposed NGS panel permits comprehensive and cost-efficient analysis of UC-specific molecular targets and may be considered in clinical routine. [ABSTRACT FROM AUTHOR]

SUMMARY As a result of extensive selection, two polyploid grass weeds, Hordeum glaucum (northern barley grass; 2n = 4x = 28) and Bromus diandrus (ripgut brome; 2n = 8x = 56), have evolved resistance to glyphosate, in Australia. Previous research suggested amplification of 5‐enolpyruvylshikimate‐3‐Phosphate synthase (EPSPS) gene confers resistance in these two weed species. The objective of this research was to investigate the genomic organization of the EPSPS gene in these two species through molecular cytogenetic analyses of fluorescence in situ hybridization (FISH) to understand possible mechanism of amplification of this gene. EPSPS copy number of H. glaucum and B. diandrus plants was estimated via quantitative polymerase chain reaction. The susceptible plants of both species had one copy of EPSPS, whereas the resistant plants of H. glaucum and B. diandrus had 14–17 and 16–32 copies, respectively. FISH analysis of glyphosate‐susceptible (Hg‐RWS) H. glaucum, revealed four faint signals of the EPSPS gene in two pairs of homologous chromosomes, at the telomeric region. The glyphosate‐resistant H. glaucum (Hg‐YP1) also showed amplification of EPSPS gene at telomeric regions in two pairs of homologous chromosomes, but the signals were brighter and appeared as cluster of EPSPS genes. Similarly, the glyphosate‐susceptible B. diandrus (Bd‐S) plants showed faint signals of EPSPS gene on two homologous chromosomes, at the telomeric position. However, samples of two glyphosate‐resistant, B. diandrus, Bd‐SA988 and Bd‐Vic showed much brighter hybridization signals of EPSPS gene, located at the telomere on two homologous chromosomes, suggesting an increase in EPSPS gene copies at this position. Overall, unequal crossover during meiosis may have triggered the initial EPSPS gene duplication sparking the evolution of glyphosate resistance. [ABSTRACT FROM AUTHOR]

AbstractOil spillage is an important environmental concern especially in areas near to the oil fields. Microorganisms present one of the most suitable and sustainable way of oil degradation and soil remediation for reuse. The present study was based on the identification of the oil degrading bacteria isolated from the oil contaminated soils located near the Shakar Dara oil fields of Kohat District, Pakistan. The rate of oil degradation by the selected bacterial isolates and the required optimum pH and temperature conditions were also assessed in the present study. Out of the total 15 bacterial isolates, 12 had noticeable oil degrading abilities. In the hydrocarbons sensitivity assays using 5% crude oil, 04 isolates denoted as M1, M2A, M3 and M7 displayed the maximum growth and were chosen for further optimization. The maximum growth was observed at pH 7 and at a mesophilic temperature range (37 °C–40 °C). The selected isolates M1, and M3 were identified as Stenotrophomonas, and Bacillus while, the isolates M2A and M7 were identified as Salmonella spp. respectively. The oil degrading efficiency of Stenotrophomonas sp. M1 was 43% followed by that of Bacillus sp. M3 as 34%. It was concluded that the selected indigenous bacterial strains specifically M1 and M3 aid in the removal of organic contaminants probably owing to some specific enzyme systems synthesized by bacteria. The novel strains being capable of degrading crude oils will help in understanding the activated enzyme machinery and further in the development of an indigenous technology for bioremediation of such contaminated areas. [ABSTRACT FROM AUTHOR]

Background: The increasing use of nanoparticles (NPs) necessitates investigation of their impact on wastewater treatment processes, particularly anammox, a critical biological nitrogen removal pathway. This study explored the effects of short-term exposure to TiO2, ZnO, and Ag-NPs on anammox activity in enriched cultures derived from River Nile sediments. Materials and methods: Anammox bacteria were identified and enriched, with activity confirmed through 16S rRNA and hydrazine oxidoreductase (hzo) gene amplification and sequencing. Activity assays demonstrated efficient ammonium removal by the enriched culture. Subsequently, the impact of different sized and concentrated NPs on anammox activity was assessed. Results: XRD analysis confirmed NP behavior within the microcosms: TiO2 transformed, ZnO partially dissolved, and Ag remained ionic. hzo gene expression served as a biomarker for anammox bacterial activity. Interestingly, 100 nm TiO2-NPs up-regulated hzo expression, potentially indicating a non-inhibitory transformed phase. Conversely, ZnO and Ag-NPs across all sizes and concentrations significantly down-regulated hzo expression, suggesting detrimental effects. Ag-NPs amended microcosms showed a significant reduction (79%) in hzo gene expression and a detrimental effect on bacterial populations. Overall, anammox activity mirrored hzo expression patterns, with TiO2 (21 and 25 nm, respectively) exhibiting the least inhibition, followed by ZnO and Ag-NPs. Conclusion: This study highlights the differential effects of NPs on anammox, with the order of impact being Ag > ZnO > TiO2. These findings provide valuable insights into the potential environmental risks of NPs on anammox-mediated nitrogen cycling in freshwater ecosystems. [ABSTRACT FROM AUTHOR]

Background and purpose: Infections caused by β-lactamase-producing strains of Staphylococcus aureus have become increasingly difficult to treat due to the expression of multiple virulence factors. This has heightened concerns about managing S. aureus-related infections. This study was conducted to characterize the blaZ gene and selected virulence determinants in β-lactam resistant S. aureus from human sources in three Nigerian tertiary hospitals. Materials and methods: Three hundred and sixty samples were collected for the study. S. aureus was isolated and characterized following standard microbiological protocols and nuc gene amplification. Antibiotic susceptibility and minimum inhibitory concentration tests were performed using the disk diffusion method and E-tests, respectively. Biofilm formation and β-lactamase production were assessed using Congo red agar and nitrocefin kits, while the blaZ gene was examined using conventional PCR. Capsular polysaccharide genotyping, accessory gene regulator (agr) detection, Panton-valentine leucocidin (PVL), and PVL proteins were performed using PCR and Western blotting. Results: S. aureus was recovered from 145 samples, 50 (34.5%) of these isolates exhibited multidrug resistance, with MICs ranging from 0.125 to 1.00 µg/mL, and showed significant resistance to aminoglycosides, fluoroquinolones, and β-lactams. Of these, 31 strains produced β-lactamases, 30 of which carried the blaZ gene in combination with cap8 (80%) or cap5 (20%). Biofilm formation and PVL gene were observed in 85% of the 20 randomly selected blaZ-positive multidrug-resistant (MDR) strains. The agr2 allele was predominant, found in 70% of the selected MDR strains. No significant difference in the occurrence of the blaZ gene was found among the three clinical sources (p ≤ α0.05). Conclusion: The co-occurrence of the blaZ gene with PVL, capsular polysaccharide genes, and agr alleles is associated with biofilm formation, indicating a high risk of β-lactam-resistant S. aureus infections. Our findings highlight the need for continuous molecular surveillance to enhance infection management, treatment options, and patient outcomes in the study locality. A limitation of this study is the random selection of MDR isolates, which may affect the comprehensiveness of the analyses. [ABSTRACT FROM AUTHOR]

The article discusses the discovery of an unidentified species of Hammerschmidtiella nematode isolated from the hindgut of Anthracophora rusticola beetles in Japan. Molecular profiling was conducted to determine its phylogenetic relationship, revealing its close relation to H. keeneyi found in the USA. The study suggests the existence of additional native Hammerschmidtiella species in Japan and explores the potential host switch or erratic parasitism of the nematode. Further research is recommended to deepen understanding of the nematode's host range and speciation. [Extracted from the article]

Objective: To investigate ear canal microflora's structure, composition and function in patients with chronic bacterial otitis externa. Methods: A case-control study design method was used to collect the ear canal secretions from 14 patients with chronic bacterial external otitis (CB group) and 14 healthy controls (H group) treated in the Department of Otolaryngology and Head and Neck Surgery in the First Affiliated Hospital of Hainan Medical University. 16S rRNA high-throughput sequencing technology was used to sequence the ear canal microflora's V3 ~ V4 region gene amplification products in the participating population. The α diversity of ear canal microflora in 2 groups was analyzed. Based on the weighted Unifrac distance, principal coordinate analysis was performed to compare the β diversity of ear tract microflora between the two groups. The differences in ear microflora at phylum and genus levels were analyzed. PICRUSt2 function prediction and BugBase phenotype prediction were also performed. Results: α diversity analysis showed that the diversity and richness of auricular microflora in the CB group were significantly lower than those in the H group. β diversity analysis showed that there were some differences between the two groups. At the phylum level, the relative abundance of Bdellovibrionota, Campylobacterota, and WPS-2 in the microbiota of patients in the CB group was significantly lower than that in the H group, and the differences were statistically significant. At the genus level, the relative abundance of Pseudomonas , Acinetobacter , Pelomonas , Sphingomonas , Bradyrhizobium , Brevundimonas , Enhydrobacter , Actinomyces , Paracoccus and Chryseobacterium in the ear canal of Group H is significantly higher than that of Group CB. Functional prediction of PICRUSt2 suggests that amino acid biosynthesis and bacterial microbiota may be related. In BugBase phenotypic prediction, the contribution of aerobic phenotype in group CB was significantly lower than that in group H. Conclusion: The diversity and abundance of the ear canal flora of patients with chronic bacterial otitis externa were significantly lower than those of the healthy population, and their bacterial colony structure was significantly altered. Dysbiosis of the ear canal flora may be an important cause of chronic bacterial otitis externa. [ABSTRACT FROM AUTHOR]

Background: Senecavirus A (SVA) is a newly pathogenic virus correlated with the acute death of piglets and vesicular lesions in pigs. The further prevalence of SVA will cause considerable economic damage to the global pig farming industry. Therefore, rapid and accurate diagnostic tools for SVA are crucial for preventing and controlling the disease. Methods: We designed multiple primer pairs targeting the most conserved region of the SVA 3D gene and selected those with the highest specificity. Nfo-probes were subsequently developed based on the highest specificity primer pairs. Subsequently, the recombinase-assisted amplification (RAA) reaction was completed, and the reaction temperature and duration were optimized. The RAA amplicons were detected using a lateral flow device (LFD). Finally, a rapid and intuitive RAA-LFD assay was established against SVA. Results: The SVA RAA-LFD assay can be performed under reaction conditions of 35°C within 17 minutes, with results observable to the naked eye. We then evaluated the performance of this method. It exhibited high specificity and no cross-reaction with the other common swine pathogens. The lowest detectable limits of this method for the plasmid of pMD18-SVA-3D, DNA amplification product, and viral were 3.86×101 copies/µL, 8.76×10-7 ng/µL, and 1×100.25 TCID50/mL, respectively. A total of 44 clinical samples were then tested using the RAA-LFD, PCR, and RT-qPCR methods. The results demonstrated a consistent detection rate between the RAA-LFD and RT-qPCR assays. Conclusion: The SVA RAA-LFD assay developed in our study exhibits excellent specificity, sensitivity, and time-saving attributes, making it ideally suited for utilization in lack-instrumented laboratory and field settings. [ABSTRACT FROM AUTHOR]

Lung cancer remains a significant health challenge, characterized by aberrant tissue growth within the pulmonary system. Early carcinogenic events often involve genomic instability and the emergence of a mutator phenotype. In this study, we aimed to explore the mutator phenotype in 89 patients diagnosed with non-small-cell lung cancer (NSCLC). RNA isolation from formalin-fixed paraffin-embedded (FFPE) tissue samples was performed using the Promega ReliaPrep RNA Miniprep System, facilitating gene amplification relevant to cancer through the Archer® FusionPlexComprehensiveThyroid and Lung (CTL) kit. Next-generation sequencing (NGS) on the Illumina NextSeq platform enabled comprehensive analysis of target areas. Utilizing Archer Analysis software, secondary analyses involving data cleansing, alignment, and variant/fusion identification were executed against the human reference genome hg19 (GRCh37). Expression patterns were visualized using HeatMap graphics. Our findings revealed a notable presence of KRAS gene mutations in approximately 20% of NSCLC patients. Among these mutations, the G12C variant was predominant at 50%, followed by G12V and G12D variants at 11.2% each. Notably, patients harboring the G12C variant responded favorably to sotorasib medication. These results underscore the importance of mutational profiling and targeted therapeutic approaches in managing NSCLC, particularly highlighting the promising efficacy of sotorasib in G12C-mutated cases. [ABSTRACT FROM AUTHOR]

Purposes: Osimertinib, one of the third-generation EGFR-tyrosine kinase inhibitors (TKIs) designed to target EGFR T790M mutation, significantly improves the prognosis of lung cancer. However, drug resistance still happens and MET amplification is responsible for one of the main causes. Fluorescence in situ hybridization (FISH) is the gold standard for MET amplification detection, but fundamentally limited by observer subjectivity. Herein, we assessed the value of next-generation sequencing (NGS) method in MET amplification detection in non-small cell lung cancer (NSCLC), as well as revealed the mutation profiling of NSCLC patients with osimertinib resistance to provide some valuable clues to the mechanisms of resistance. Methods: A total of 317 cancer tissue samples from 317 NSCLC patients at time of progression following osimertinib were submitted to NGS and only 96 tissues were tested by FISH simultaneously. With FISH results as gold standard, enumeration algorithm was applied to establish the optimal model for identifying MET amplification using gene copy number (GCN) data. Results: The optimal model for identifying MET amplification was constructed based on the GCN of MET, BRAF, CDK6 and CYP3A4, which achieved a 74.0% overall agreement with FISH and performed well in identifying MET amplification except polysomy with a sensitivity of 85.7% and a specificity of 93.9%. The inconsistency between NGS and FISH occurred mainly in polysomy subtype, while MET GCN ≥ 5 could be reliably recognized by NGS. Moreover, the most frequently mutated genes in NSCLC patients with osimertinib resistance were EGFR (59.94%), followed by TP53 (43.85%), NRG1 (9.46%), PIK3CA (6.31%), and ATM (5.36%). The known resistance mechanisms, including MET amplification, EGFR (C797S, L718Q/R), TP53, CDK4, CDK6, CDKN2A, BRAF, KRAS, NRAS and PIK3CA mutations were also disclosed in our cohort. Conclusions: NGS assay can achieve a high concordance with FISH in MET amplification detection and has advantages in portraying various genetic alterations, which is of worthy in clinical promotion. [ABSTRACT FROM AUTHOR]

The article published in the Annals of Internal Medicine explores the HIV reservoir landscape in breast milk from women living with HIV (WLWH) who have long-term viral suppression. The study found no intact or potentially replication-competent virus in breast milk samples from an exceptional elite controller (EEC) and a WLWH receiving antiretroviral treatment (ART). While the study had limitations in sample size and follow-up, it highlights the feasibility of evaluating the HIV landscape in breast milk and lays the groundwork for future research to inform guidelines on chestfeeding or breastfeeding for WLWH. This preliminary study is significant in addressing the evolving recommendations for WLWH who wish to chestfeed or breastfeed. [Extracted from the article]

Diagnosing the cause of death can be challenging, particularly for patients with no prior history of visits to the treating hospital. We encountered a case involving a 76‐year‐old male who was discovered in a state of cardiopulmonary arrest at his home and subsequently declared deceased in our hospital due to severe pneumonia. He had exhibited symptoms of fever over 37°C and severe coughing for several days. Despite consulting a primary care physician one day prior, his symptoms worsened. Autopsy findings revealed an increase in lung weight and diffuse changes in parenchyma. Histological analysis showed numerous inflammatory cells and exudate within the alveoli. Gram and Periodic acid‐Schiff staining were negative, but slight staining was observed in the cytoplasm of macrophages by Warthin‐starry and Gimenez stains. Tests using a pan bacterial/viral detection kit and qualitative polymerase chain reaction (PCR) for Legionella pneumophila were negative. However, using deoxyribonucleic acid extracted from formalin‐fixed paraffin‐embedded lung tissue, PCR amplification of the ssrA gene of congeneric Legionella species yielded positive results. The results suggest that the cause of death was likely due to bacterial pneumonia caused by Legionella species. [ABSTRACT FROM AUTHOR]

Loop-Mediated Isothermal Loop-Mediated Isothermal Amplification (LAMP) is a widely used technique for nucleic acid amplification due to its high specificity, sensitivity, and rapid results. Advances in microfluidic lab-on-chip (LOC) technology have enabled the integration of LAMP into miniaturized devices, known as μ -LAMP, which require precise thermal control for optimal DNA amplification. This paper introduces a novel thermal bed design using PCB copper traces and F R − 4 dielectric materials, providing a reliable, modular, and repairable heating platform. The system achieves accurate and stable temperature control, which is critical for μ -LAMP applications, with temperature deviations within ±1.0 °C. The thermal bed's performance is validated through finite element method (FEM) simulations, showing uniform temperature distribution and a rapid thermal response of 2.5 s to reach the target temperature. These results highlight the system's potential for applications such as disease diagnostics, biological safety, and forensic analysis, where precision and reliability are paramount. [ABSTRACT FROM AUTHOR]

Purpose: Loop-mediated isothermal amplification (LAMP) is a simple and rapid nucleic acid method for DNA amplification at a constant temperature. The "gold standard" culture method for yeast detection, has low sensitivity with severe consequences, increasing morbidity and mortality rates. Here, we report the development of a LAMP method for the specific detection of C. glabrata. Methodology: The specific LAMP primers for C. glabrata detection were designed and evaluated. Results: The LAMP assay accurately detected C. glabrata with no cross-reactivity with other Candida species. Conclusion: The developed molecular method would be a promising tool in the management of invasive candidiasis. [ABSTRACT FROM AUTHOR]

Background Ganaplacide, also known as KAF156, is among the new antimalarial drug candidates that have successfully reached Phase III clinical trials, and is proposed in combination with lumefantrine. This combination could replace the current front-line artemisinin-based combination therapies (ACTs) in case of Plasmodium falciparum resistance to both artemisinins and partner drugs. Indeed, the African continent, where the malaria burden is the highest, is currently experiencing worrying multiple emergences and spread of artemisinin resistance, which urges for the exploration of the antiparasitic properties of KAF156 in this context. Objectives and methods The objectives of this work were firstly to evaluate the risk of cross-resistance between artemisinins and KAF156 alone, and in combination with lumefantrine, using a panel of artemisinin-resistant strains carrying different pfk13 mutations and markers of other antiplasmodial drug resistances; secondly to explore in vitro the relevance of combining KAF156 and lumefantrine with artemisinins, based on the model of triple ACTs. Results Our results highlighted that KAF156 activity was not impaired by mutations in pfk13 , pfcrt , pfmdr1 , pfmdr2 , pfdhps and pfdhfr genes or by pfmdr1 amplification. Moreover, we demonstrated that KAF156 alone and in combination with lumefantrine was active against artemisinin-resistant parasites, including when they are quiescent. Conclusions All these in vitro results evidence that multi-drug resistant parasites currently in circulation in the field might not affect KAF156 efficacy, and are encouraging signs for KAF156 use in a triple ACT to preserve the use of artemisinins for as long as possible. [ABSTRACT FROM AUTHOR]

Background: Split hand/foot malformation (SHFM) is a congenital limb deficiency characterized by missing or shortened central digits. Several gene loci have been associated with SHFM. Identifying microduplications at the single-cell level is challenging in clinical practice, and traditional detection methods may lead to misdiagnoses in embryos and pregnant women. Results: In this research, we utilized a low cell count and whole-genome amplification products to employ single nucleotide polymorphism arrays, next-generation sequencing, and third-generation sequencing methods to detect copy number variants of microduplications in a SHFM3 case with limited DNA. Additionally, Karyomapping and combined linkage analysis were conducted to validate the results. Conclusions: This study establishes a new strategy for identifying microduplications or microdeletions at the single-cell level in clinical preimplantation genetic testing, enhancing the efficiency and accuracy of diagnosing microduplication or microdeletion diseases during IVF-PGT and prenatal diagnosis. [ABSTRACT FROM AUTHOR]

Context.--: Primary thoracic neoplasms are rare in children, whereas nonneoplastic mass lesions or cysts and metastases are more common, and there is a relative paucity of comprehensive histopathologic and molecular data. Objective.--: To define the clinicopathologic spectrum of neoplastic and nonneoplastic diseases observed in resected mass lesions in the chest of pediatric patients, and to identify somatic alterations observed in primary neoplasms. Design.--: Clinicopathologic features of thoracic mass lesions (n = 385) resected from 373 patients aged ≤21 years in a 25-year period (1993-2018) were included. Primary neoplasms having sufficient material were tested by a laboratory-developed comprehensive genomic profiling assay that assesses tumor mutational burden, microsatellite instability, somatic sequence variants, gene amplifications, fusions, and specific transcript variants. Results.--: The most commonly resected space-occupying lesions were nonneoplastic mass lesions and cysts or malformations, resected in 117 (31.4%) and 58 of 373 patients (15.5%) respectively. Metastatic neoplasms were observed in 169 of 373 patients (45.3%; mean age 14.4 years, range 1-21 years); the most common was osteosarcoma (68 of 169; 40.2% of metastases). Primary lung neoplasms occurred in 24 of 373 patients (6.4%; mean age 14.5 years, range 6 months-21 years), and 16 patients had primary extrapulmonary thoracic tumors. Carcinoid tumor was the most common primary lung neoplasm (7 typical, 3 atypical). Molecular testing showed a prevalence of somatic pathogenic or likely pathogenic mutations and copy-number alterations. No fusions or splice variants were identified. Tumors were microsatellite-stable with low tumor mutational burden. Conclusions.--: Resected pediatric thoracic mass lesions are more likely to be metastatic lesions, congenital cysts or malformations, or nonneoplastic lesions compared to primary thoracic neoplasms, which are encountered at a low frequency and tend to have relatively simple genetic profiles. [ABSTRACT FROM AUTHOR]

Polygalacturonases (PG) are the enzymes that cause depolymerization of pectin. In current study, bacterial strain was isolated from rotten sample and then purified. It was screened for endo-polygalacturonase activity using PSAM media. It had shown positive response for endo-PG activity. Bacterial DNA was isolated and 16 S rRNA gene amplification was done using universal primer pair of 8 F and 1492 R. Bacterial strain was also amplified by 16 S rRNA gene for sequencing. BLAST of gene sequence on NCBI database had shown that bacterial isolate was identified as Bacillus pumilus. Endo-polygalacturonase enzyme was produced by submerged fermentation to optimize culture conditions by RSM in JMP-12 software. The optimized parameters had shown maximum endo-polygalacturonase activity of 153.2 U/mL/min after using 3 g of orange peels as substrate, 3 mL of inoculum size, 6.5 pH buffer, 40°C temperature and 3 days of incubation. After optimizing fermentation conditions, endo-polygalacturonase was precipitated using 70 % concentration of ammonium sulfate. This partially precipitated sample was dialyzed with dialysis tube and used to find activity of endo-polygalacturonase (1620.6 U/mL/min). This sample was applied to gel filtration chromatography for further purification. Endo-polygalacturonase activity increased many times 2231.44 U/mL/min after gel filtration. The Molecular weight of endo-polygalacturonase was 46 kDa after SDS PAGE characterization. High Vmax value and alkaliphilic nature of endo-polygalacturonase will make it good candidate for food and feed industry near future. [Display omitted] • A bacterial strain was isolated from rotten sample. • Bacillus pumilus was identified through 16 S rRNA gene sequencing. • Maximum enzyme activity of 153.2 U/mL/min was achieved under optimized conditions. • The characterized endo-polygalacturonase has great potential in industrial applications. • Polygalacturonase had shown maximum activity after column chromatography. [ABSTRACT FROM AUTHOR]

This article reports on two cases of testicular sclerosing stromal tumors (SSTs) that show GLI1 gene alterations. SSTs are neoplasms that were first described in the ovary in 1973 and are characterized by lobulated microscopic appearance. The patients in these cases were aged 35 and 65 years and presented with testicular masses. The tumors demonstrated large areas of hypocellular myxoid/oedematous stroma with scattered hypercellular areas. Both tumors expressed nuclear GLI1. The article suggests that these findings support the existence of rare SSTs in the testis and their potential pathogenesis related to ovarian SSTs. [Extracted from the article]

Molecular features are incorporated into the integrated diagnostic system for adult diffuse gliomas. Of these, copy number variation (CNV) markers, including both arm‐level (1p/19q codeletion, +7/−10 signature) and gene‐level (EGFR gene amplification, CDKN2A/B homozygous deletion) changes, have revolutionized the diagnostic paradigm by updating the subtyping and grading schemes. Shallow whole genome sequencing (sWGS) has been widely used for CNV detection due to its cost‐effectiveness and versatility. However, the parallel detection of glioma‐associated CNV markers using sWGS has not been optimized in a clinical setting. Herein, we established a model‐based approach to classify the CNV status of glioma‐associated diagnostic markers with a single test. To enhance its clinical utility, we carried out hypothesis testing model‐based analysis through the estimation of copy ratio fluctuation level, which was implemented individually and independently and, thus, avoided the necessity for normal controls. Besides, the customization of required minimal tumor fraction (TF) was evaluated and recommended for each glioma‐associated marker to ensure robust classification. As a result, with 1× sequencing depth and 0.05 TF, arm‐level CNVs could be reliably detected with at least 99.5% sensitivity and specificity. For EGFR gene amplification and CDKN2A/B homozygous deletion, the corresponding TF limits were 0.15 and 0.45 to ensure the evaluation metrics were both higher than 97%. Furthermore, we applied the algorithm to an independent glioma cohort and observed the expected sample distribution and prognostic stratification patterns. In conclusion, we provide a clinically applicable algorithm to classify the CNV status of glioma‐associated markers in parallel. [ABSTRACT FROM AUTHOR]

MicroRNA (miR)-155-5p increases in innate and adaptive immune cells in response to IL-13 and is associated with the severity of asthma. However, little is known about its role in airway structural cells. Bronchial epithelial cells (BECs) isolated from healthy donors and patients with severe asthma were stimulated with IL-13. miR-155-5p expression and release were measured by real-time (RT)-PCR in BECs and in their derived exosomes. Modulation of miR-155-5p in BECs was performed using transfection of miR-155-5p inhibitor and mimic. IL-13 receptor α1 (IL-13Rα1), IL-13Rα2, MUC5AC, IL-8, and eotaxin-1 expression was measured by RT-PCR and Western blot analysis. The BEC repair process was assessed by a wound-healing assay. IL-13Rα1 and IL-13Rα2 expression and downstream pathways were evaluated by Western blot analysis. A dual luciferase assay was used to identify miR-155-5p target genes associated with IL-13R signaling. BECs from patients with severe asthma showed increased expression and exosomal release of miR-155-5p at baseline with amplification by IL-13 stimulation. BECs from patients with asthma expressed more IL-13Rα1 and less IL-13Rα2 than those from healthy donors, and IL-13Rα1 but not IL-13Rα2 induced miR-155-5p expression under IL-13 stimulation. miR-155-5p overexpression favored MUC5AC, IL-8, and Eotaxin-1 through the IL-13Rα1/SOCS1/STAT6 pathway while delaying the repair process by downregulating IL-13Rα2/MAPK14/c-Jun/c-fos signaling. The dual luciferase assay confirmed that miR-155-5p modulates both IL-13R pathways by directly targeting SOCS1, c-fos, and MAPK14. miR-155-5p is overexpressed in BECs from patients with severe asthma and regulates IL-13Rα1 and IL-13Rα2 expression and signaling, favoring expression of mucin- and eosinophil-related genes to the detriment of airway repair. These results show that miR-155-5p may contribute to airway epithelial cell dysfunction in patients with severe asthma. [ABSTRACT FROM AUTHOR]

Wolbachia are endosymbiotic bacteria that infect a wide range of arthropods and filarial nematodes, often manipulating host reproduction. The efficacy of Wolbachia-based interventions for dengue and chikungunya control has been validated through numerous field studies in recent years. This study aimed to investigate the diversity and prevalence of Wolbachia infections in sylvatic mosquitoes from two locations in Recife, Brazil. Multiple mosquito species were screened for Wolbachia using both target marker gene amplification coupled with Sanger sequencing and whole-genome sequencing (WGS) approaches. Phylogenetic analyses were conducted to classify Wolbachia strains into supergroups and assess their evolutionary relationships. Results revealed the presence of Wolbachia in eleven mosquito species examined, with different infection rates. Both supergroups A and B of Wolbachia strains were identified, with Aedes albopictus showing co-infection by both supergroups through the WGS approach. We also detected indirect evidence of Wolbachia horizontal transmission among mosquitoes and other distant host orders. This study provides valuable insights into the distribution and diversity of Wolbachia in sylvatic mosquitoes from Brazil and adds new important data about Wolbachia detection through target marker gene amplicon coupled with Sanger sequencing and WGS methods, highlighting its complementarity to ascertain the presence of Wolbachia in mosquito samples. [ABSTRACT FROM AUTHOR]

The rapid and accurate detection of SARS-CoV-2 in environmental settings is crucial for effective public health management during the COVID-19 pandemic. This study compares the performance of the Reverse Transcription quantitative polymerase chain reaction (RT-qPCR) and the Reverse Transcription loop-mediated isothermal amplification (RT-LAMP) for SARS-CoV-2 detection from 100 surface samples collected in healthcare environments. The reference method, RT-qPCR, identified a percentage of 25% of positive samples, while RT-LAMP detected a percentage of 27% of positive surfaces. Our findings reveal a sensitivity of 32% and specificity of 75% for RT-LAMP, with a positive predictive value of 30% and a negative predictive value of 77%. The overall accuracy and concordance with RT-qPCR was 64% for both methods. Despite its lower sensitivity compared to RT-qPCR, RT-LAMP had an advantage due to its rapid screening and environmental surveillance, which is particularly useful for confirming negative results. These results underscore the potential of RT-LAMP not only as a valuable method in the environmental monitoring of SARS-CoV-2 but also as a system to control the sanitation process in ordinary and emergency conditions, providing further optimization and validation for its reliability in routine surveillance and outbreak response efforts. [ABSTRACT FROM AUTHOR]

A portable multisample absorbance detection system for loop-mediated isothermal amplification using a positive temperature coefficient (PTC) heater was developed in this study. The system can simultaneously measure up to four samples and consists of four LEDs, four photodiodes, two PTC heaters, a sample holder, and a short-pass filter. The system successfully differentiated samples with and without human genomic DNA with photodiodes measuring absorbance in real time. We have also demonstrated that the quantitative analysis of human genomic DNA is possible at initial DNA concentrations ranging from 2.11 × 10² to 2.11 × 104 ng/µL. This device has potential use in on-site genetic testing owing to its compact size, portability, and capability to provide rapid results. [ABSTRACT FROM AUTHOR]

Cotton root rot caused by Macrophomina phaseolina pose a significant threat to cotton production, leading to substantial yield and quality losses. Early and accurate diagnosis of this pathogen in soil is crucial for effective disease management. This study presents a pioneering investigation into the utilization of the nit gene encoding nitrilase for the development of a molecular diagnostic assay aimed at the rapid detection of M. phaseolina in field soils. The methodology involved the design and validation of primers targeting the Nit gene sequence, followed by the optimization of PCR conditions for efficient amplification. Leveraging state‐of‐the‐art molecular techniques, the assay offers a novel protocol to accurately identify the presence of M. phaseolina in soil with high sensitivity and specificity. The specificity of the designed primers was confirmed through PCR amplification using DNA from M. phaseolina and other related fungi. Sensitivity tests demonstrated that the PCR assay reliably detected M. phaseolina DNA at concentrations as low as 1 ng. Furthermore, the performance of the diagnostic assay was rigorously evaluated using field soil samples with a known status of M. phaseolina infection, demonstrating its reliability and efficacy in real‐world scenarios. This study introduces a novel molecular marker for the detection of M. phaseolina and offers a rapid and efficient means for screening M. phaseolina in large soil samples with minimal time and manpower. [ABSTRACT FROM AUTHOR]

Enterovirus A71 (EVA71) is a leading causative agent of hand, foot, and mouth disease, posing a significant threat to the health of young children, particularly in the Asia‐Pacific region. Currently, there is no specific antiviral drug for EVA71 infection; therefore, early and rapid diagnosis is critical for disease prevention and control. Here, we report the development of a simple, rapid, and sensitive detection method for EVA71 infection using reverse transcription‐multiple cross displacement amplification (RT‐MCDA) combined with nanoparticle‐based lateral flow biosensors (LFB). In the RT‐MCDA system, a set of 10 primers was designed to target the highly conserved region of the VP1 gene of EVA71 and amplify the genes in an isothermal amplification device. The RT‐MCDA amplification reaction products could then be identified by visual detection reagent (VDR) and LFB without the need for specialized equipment. The results demonstrated that the optimal reaction condition for the EVA71‐RT‐MCDA assay was 65℃ for 40 min. The EVA71‐RT‐MCDA assay could detect as low as 40 copies of plasmid and 50 copies of pseudotyped virus in a reaction. No cross‐reaction was found between EVA71 strains and non‐EVA71 strains. For 125 clinical anal swab samples, with EVA71‐RT‐MCDA assay, 30 samples were positive, which was in consistent with the the conventional real‐time quantitative reverse transcription polymerase chain reaction assays. The entire procedure, including a 15‐min specimen processing step, a 40‐min MCDA reaction, and result reporting within 2 min, was completed in less than 60 min. In conclusion, the EVA71‐RT‐MCDA‐LFB assay targeting the VP1 gene is a rapid, highly sensitive, simple, and specific test that could be widely applied in point‐of‐care settings and basic medical facilities in rural areas. [ABSTRACT FROM AUTHOR]

BACKGROUND RESULTS CONCLUSION The identification of adulterated sweet potato vermicelli faces significant challenges, seriously hindering the development of the vermicelli industry. Herein, we investigate effects of energy‐gathered ultrasound (EGU) and energy‐divergent ultrasound (EDU) (30, 40 and 50 W  L−1) on structure, DNA extraction and adulterated quantification of sweet potato vermicelli and its starch, thereby exploring their potential in adulteration of sweet potato vermicelli.EGU‐assisted modified cetyltrimethylammonium bromide (CTAB) protocol with β‐mercaptoethanol significantly improved DNA extraction from sweet potato vermicelli (223.7–249.2 ng μL−1) and its starch (133.4–186.4 ng μL−1), followed by EDU‐assisted DNA extraction from sweet potato vermicelli (115.1–209.3 ng μL−1) and its starch (33.4–61.0 ng μL−1). Both EGU and EDU treatments resulted in the destruction of microstructure and crystalline structure, as well as changes in pasting and thermal properties of sweet potato vermicelli and its starch. Real‐time polymerase chain reaction (PCR) amplification results revealed that EGU and EDU enhanced the efficiency of DNA amplification, and EDU showed smaller cycle threshold (Ct) values than EGU. In addition, EDU‐assisted CTAB protocol combined with real‐time PCR could detect levels of less than 1% of cassava and maize starches in sweet potato vermicelli.EDU‐assisted CTAB protocol combined with real‐time PCR shows promise as a sensitive and reliable analytical tool for vermicelli adulteration. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]