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Conventional type 1 dendritic cells (cDC1s) are critical for innate sensing of cancer, yet they are scarce in the tumor microenvironment (TME). Here, we present a protocol to identify and isolate cDC1 subsets from murine implantable tumors for subsequent transcriptomic profiling using a flow sorting-based strategy. We describe steps for cell culture of mouse tumors, tumoral growth, dissociation and isolation of tumoral cells, extracellular staining, and cell sorting. We then detail procedures for RNA isolation, mRNA library preparation, and sequencing. For complete details on the use and execution of this protocol, please refer to Papadas et al.1.

To determine the evolution of microbial community and microbial shift under anaerobic processes, this study investigates the use of denaturing gradient gel electrophoresis (DGGE). In the DGGE, short- and medium-sized DNA fragments are separated based on their melting characteristics, and this technique is used in this study to understand the dominant bacterial community in mesophilic and thermophilic anaerobic digestion processes. Dairy manure is known for emitting greenhouse gases (GHGs) such as methane, and GHG emissions from manure is a biological process that is largely dependent on the manure conditions, microbial community presence in manure, and their functions. Additional efforts are needed to understand the GHG emissions from manure and develop control strategies to minimize the biological GHG emissions from manure. To study the microbial shift during anaerobic processes responsible for GHG emission, we conducted a series of manure anaerobic digestion experiments, and these experiments were conducted in lab-scale reactors operated under various temperature conditions (28 °C, 36 °C, 44 °C, and 52 °C). We examined the third variable region (V3) of the 16S rRNA gene fingerprints of bacterial presence in anaerobic environment by PCR amplification and DGGE separation. Results showed that bacterial community was affected by the temperature conditions and anaerobic incubation time of manure. The microbial community structure of the original manure changed over time during anaerobic processes, and the community composition changed substantially with the temperature of the anaerobic process. At Day 0, the sequence similarity confirmed that most of the bacteria were similar (>95%) to Acinetobacter sp. (strain: ATCC 31012), a Gram-negative bacteria, regardless of temperature conditions. At day 7, the sequence similarity of DNA fragments of reactors (28 °C) was similar to Acinetobacter sp.; however, the DNA fragments of effluent of reactors at 44 °C and 52 °C were similar to Coprothermobacter proteolyticus (strain: DSM 5265) (similarity: 97%) and Tepidimicrobium ferriphilum (strain: DSM 16624) (similarity: 100%), respectively. At day 60, the analysis showed that DNA fragments of effluent of 28 °C reactor were similar to Galbibacter mesophilus (strain: NBRC 10162) (similarity: 87%), and DNA fragments of effluent of 36 °C reactors were similar to Syntrophomonas curvata (strain: GB8-1) (similarity: 91%). In reactors with a relatively higher temperature, the DNA fragments of effluent of 44 °C reactor were similar to Dielma fastidiosa (strain: JC13) (similarity: 86%), and the DNA fragments of effluent of 52 °C reactor were similar to Coprothermobacter proteolyticus (strain: DSM 5265) (similarity: 99%). To authors knowledge, this is one of the few studies where DGGE-based approach is utilized to study and compare microbial shifts under mesophilic and thermophilic anaerobic digestions of manure simultaneously. While there were challenges in identifying the bands during gradient gel electrophoresis, the joint use of DGGE and sequencing tool can be potentially useful for illustrating and comparing the change in microbial community structure under complex anaerobic processes and functionality of microbes for understanding the consequential GHG emissions from manure.

Inherited macular dystrophies (iMDs) are a group of genetic disorders, which affect the central region of the retina. To investigate the genetic basis of iMDs, we used single-molecule Molecular Inversion Probes to sequence 105 maculopathy-associated genes in 1352 patients diagnosed with iMDs. Within this cohort, 39.8% of patients were considered genetically explained by 460 different variants in 49 distinct genes of which 73 were novel variants, with some affecting splicing. The top five most frequent causative genes were ABCA4 (37.2%), PRPH2 (6.7%), CDHR1 (6.1%), PROM1 (4.3%) and RP1L1 (3.1%). Interestingly, variants with incomplete penetrance were revealed in almost one-third of patients considered solved (28.1%), and therefore, a proportion of patients may not be explained solely by the variants reported. This includes eight previously reported variants with incomplete penetrance in addition to CDHR1:c.783G>A and CNGB3:c.1208G>A. Notably, segregation analysis was not routinely performed for variant phasing-a limitation, which may also impact the overall diagnostic yield. The relatively high proportion of probands without any putative causal variant (60.2%) highlights the need to explore variants with incomplete penetrance, the potential modifiers of disease and the genetic overlap between iMDs and age-related macular degeneration. Our results provide valuable insights into the genetic landscape of iMDs and warrant future exploration to determine the involvement of other maculopathy genes.

Non-invasively evaluating gene expression products in human pre-implantation embryos remains a significant challenge. Here, we develop a non-invasive method for comprehensive characterization of the extracellular RNAs (exRNAs) in a single droplet of spent media that was used to culture human in vitro fertilization embryos. We generate the temporal extracellular transcriptome atlas (TETA) of human pre-implantation development. TETA consists of 245 exRNA sequencing datasets for five developmental stages. These data reveal approximately 4,000 exRNAs at each stage. The exRNAs of the developmentally arrested embryos are enriched with the genes involved in negative regulation of the cell cycle, revealing an exRNA signature of developmental arrest. Furthermore, a machine-learning model can approximate the morphology-based rating of embryo quality based on the exRNA levels. These data reveal the widespread presence of coding gene-derived exRNAs at every stage of human pre-implantation development, and these exRNAs provide rich information on the physiology of the embryo.

For over a century, early researchers sought to study biological organisms in a laboratory setting, leading to the generation of both in vitro and in vivo model systems. Patient-derived models of cancer (PDMCs) have more recently come to the forefront of preclinical cancer models and are even finding their way into clinical practice as part of functional precision medicine programs. The PDMC Consortium, supported by the Division of Cancer Biology in the National Cancer Institute of the National Institutes of Health, seeks to understand the biological principles that govern the various PDMC behaviors, particularly in response to perturbagens, such as cancer therapeutics. Based on collective experience from the consortium groups, we provide insight regarding PDMCs established both in vitro and in vivo, with a focus on practical matters related to developing and maintaining key cancer models through a series of vignettes. Although every model has the potential to offer valuable insights, the choice of the right model should be guided by the research question. However, recognizing the inherent constraints in each model is crucial. Our objective here is to delineate the strengths and limitations of each model as established by individual vignettes. Further advances in PDMCs and the development of novel model systems will enable us to better understand human biology and improve the study of human pathology in the lab.

Myelodysplastic syndromes (MDS) is a highly heterogeneous myeloid neoplastic disease, which needs personalized evaluation and therapy. To analyze the features and significance of gene mutations for MDS patients with normal karyotype (NK) at diagnosis, targeted sequencing was conducted on 616 MDS patients with NK, alongside 457 MDS cases with abnormal karyotype (AK). The results showed that the incidence of somatic mutation reached 70.3% and 83.8% in the NK and AK group, respectively. Initial mutation including ASXL1, DNMT3A and TET2 were common in NK group, which is the same as AK group. Some karyotype-associated gene mutations, such as TP53 and U2AF1, were relatively rare in NK group. Moreover, 34 out of 91 samples who progressed to acute myeloid leukemia (AML) underwent repeat sequencing during follow-up. 25 cases were checked out with newly emerged mutations. The AML-associated genetic alterations mainly involved with active signaling and transcription factors. In patients with NK, serial targeted sequencing was employed for minimal residual disease (MRD) monitoring, indicating the efficacy and relapse of the patients. In summary, MDS with NK showed distinct mutation features from those with AK. High-frequency gene mutations together with the mutational evolution suggested the diagnostic and monitoring significance of next generation sequencing for NK-MDS. Trial registration number and date: ChiCTR2300074952, August 21, 2023 (retrospectively registered). [ABSTRACT FROM AUTHOR]

The main aim of this study was to determine expanded sequence types (eSTs) of Ureaplasma species (U. spp.). DNA isolated from the amniotic fluid of pregnancies complicated by preterm prelabor rupture of membranes (PPROM) using an expanded multilocus sequence typing scheme. Additionally, the study sought to examine whether phylogenetic subgroups of U. spp. DNA differ with respect to maternal demographic and clinical parameters and selected aspects of short-term neonatal morbidity. This retrospective cohort study was focused on singleton pregnancies complicated by PPROM occurring between the gestational ages of 24+0 and 36+6 weeks, where amniocentesis was conducted to assess the intra-amniotic environment and the presence of U. spp. DNA in the amniotic fluid samples was confirmed. The stored aliquots of U. spp. DNA were used to assess differences in nucleotide sequences in six U. spp. genes (ftsH, rpL22, valS, thrS,ureG, and mba-np1) using the eMLST scheme. The expanded multilocus sequence typing scheme was performed in 73 samples of U. spp. DNA isolated from pregnancies complicated by PPROM. In total, 33 different U. spp. DNA eSTs were revealed, 21 (#20, 233–244, 248–251, 253, 255, 259, and 262) of which were novel. The most frequently identified eST was #41, identified in 18% (13/73) of the aliquots. Based on their genetic relationships, the U. spp. DNA was divided into two clusters and four subgroups [cluster I (U. parvum): A, 43% (n = 31); B, 15% (n = 11); and C, 26% (n = 19); cluster II (U. urealyticum): 1; 16% (n = 12)]. Cluster II had a higher rate of polymicrobial findings than cluster I (58% vs 16%; p = 0.005), while subgroup A had the highest rate of concomitant Mycoplasma hominis in the amniotic fluid samples (66%; p = 0.04). In conclusion, Ureaplasma spp. DNA obtained from PPROM consisted of 33 different eSTs of U. spp. DNA. No differences in maternal and neonatal characteristics were found among the phylogenetical subgroups of U. spp. DNA, except for a higher rate of polymicrobial amniotic fluid findings in those with U. urealyticumand the concomitant presence of M. hominis in the amniotic fluid in those with the presence of U. parvum. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) is a leading cause of morbidity and mortality worldwide. Detection before metastasis and efficient treatment of disease significantly improve patient survival and quality of life. However, limitations in diagnosis and postoperative surveillance are associated with low CRC detection and survival rates. Thus, this project aimed to evaluate the molecular profile of patients diagnosed with CRC, as molecular biomarkers constitute a new frontier for diagnosis, treatment and prognosis. Methods and Results: 42 patients were included in the study, predominantly male (59.5%), with a median age of 63 years (SD: 10.0; min: 41; max: 83). The majority of primary tumors were located in the rectum (38.1%), in the sigmoid (33.3%) and in the ascending (21.4%) colon. We evaluated the genes KRAS, NRAS, BRAF, EGFR and TP53 using Sanger sequencing. Somatic and germline mutations were found in the KRAS, EGFR and TP53 genes, with the most common somatic alteration being rs121913529 in KRAS. This variant was also strongly associated with alcoholism (p = 0.002). Furthermore, patients with somatic mutations in TP53 had significantly higher mortality compared to those with wild-type alleles (OR: 11.2; 95% CI 1.25–2.45). Conclusions: Our findings support a relationship between alcohol consumption and the rs121913529 mutation, which is classified as pathogenic for colorectal cancer. Thus, further studies investigating the link between alcohol consumption, colorectal carcinogenesis and tumor progression ought to be conducted. [ABSTRACT FROM AUTHOR]

Daqu, a crucial fermentation starter in the production of various Chinese fermented foods, plays a pivotal role in shaping complex enzyme profiles and diverse flavour precursors. This review aims to elucidate the microbial communities within Daqu, focusing on their functionalities in the context of flavour development. We delve into the detection methods of microorganisms and flavour substances in Daqu, employing advanced technologies including high-performance liquid chromatography, gas chromatography, pseudo-targeted metabolomics, and headspace solid-phase microextraction-gas chromatography-mass spectrometry. This review explores high throughput sequencing technologies, including pyrosequencing, clonal library sequencing, metaproteomic, and metagenomics, to gain a comprehensive understanding of the intricate microbial dynamics. Additionally, we discuss the metabolic pathways involved in flavour substance production within Daqu. By synthesizing information on Daqu types, microorganisms present, detection methodologies, and flavour substance metabolic pathways, this review contributes to a deeper comprehension of the intricate processes underpinning the Flavors of Chinese fermented foods. [Display omitted] • Crucial role of Daqu in enzyme profiles and flavor precursors in Chinese foods. • Microbial communities in Daqu and their role in flavor development. • High throughput sequencing: pyrosequencing, clonal library sequencing, metaproteomic, metagenomics. • Comprehensive synthesis of Daqu types, microorganisms, and metabolic pathways. [ABSTRACT FROM AUTHOR]

Field‐collected specimens were used to obtain nine high‐quality genome assemblies from a total of 10 insect species native to prairies and savannas of central Illinois (USA): Mellilla xanthometata (Lepidoptera: Geometridae), Stenolophus ochropezus (Coleoptera: Carabidae), Forcipata loca (Hemiptera: Cicadellidae), Coelinius sp. (Hymenoptera: Braconidae), Thaumatomyia glabra (Diptera: Chloropidae), Brachynemurus abdominalus (Neuroptera: Myrmeleontidae), Catonia carolina (Hemiptera: Achilidae), Oncometopia orbona (Hemiptera: Cicadellidae), Flexamia atlantica (Hemiptera: Cicadellidae) and Stictocephala bisonia (Hemiptera: Membracidae). Sequencing library preparation from single specimens was successful despite extremely small DNA yields (<0.1 μg) for some samples. Additional sequencing and assembly workflows were adapted to each sample depending on the initial DNA yield. PacBio circular consensus (CCS/HiFi) or continuous long reads (CLR) libraries were used to sequence DNA fragments up to 50 kb in length, with Illumina sequenced linked‐reads (TellSeq libraries) and Omni‐C libraries used for scaffolding and gap‐filling. Assembled genome sizes ranged from 135 MB to 3.2 GB. The number of assembled scaffolds ranged from 47 to >13,000, with the longest scaffold per assembly ranging from ~23 to 439 Mb. Genome completeness was high, with BUSCO scores ranging from 85.5% completeness for the largest genome (Stictocephala bisonia) to 98.8% completeness for the smallest genome (Coelinius sp.). The unique content was estimated using RepeatMasker and GenomeScope2, which ranged from 50.7% to 75.8% and roughly decreased with increasing genome size. Structural annotation predicted a range of 19,281–72,469 protein models for sequenced species. Sequencing costs per genome at the time ranged from US$3–5k, averaged ~1600 CPU‐hours on a high‐performance cluster and required approximately 14 h of bioinformatics analyses with samples using PacBio HiFi data. Most assemblies would benefit from further manual curation to correct possible scaffold misjoins and translocations suggested by off‐diagonal or depleted signals in Omni‐C contact maps. [ABSTRACT FROM AUTHOR]

Amplicon sequencing has long served as a robust method for characterising microbial communities, and despite inherent resolution limitations, it remains a preferred technique, offering cost‐ and time‐effective insights into bacterial compositions. Here, we introduce ONT‐AmpSeq, a user‐friendly pipeline designed for processing amplicon sequencing data generated from Oxford Nanopore Technology (ONT) devices. Our pipeline enables efficient creation of taxonomically annotated operational taxonomic unit (OTU) tables from ONT sequencing data, with the flexibility to multiplex amplicons on the same barcode. The pipeline encompasses six main steps—statistics, quality filtering, alignment, clustering, polishing, and taxonomic classification—integrating various state‐of‐the‐art software tools. We provide a detailed description of each step, along with performance tests and robustness evaluations using both test data and a ZymoBIOMICS® Microbial Community Standard mock community dataset. Our results demonstrate the ability of ONT‐AmpSeq to effectively process ONT amplicon data, offering valuable insights into microbial community composition. Additionally, we discuss the influence of polishing tools on taxonomic insight and the impact of taxonomic annotation methods on the derived microbial composition. Overall, ONT‐AmpSeq represents a comprehensive solution for analysing ONT amplicon sequencing data, facilitating streamlined and reliable microbial community analysis. The pipeline, along with test data, is freely available for public use. [ABSTRACT FROM AUTHOR]

Background: Autism spectrum disorder (ASD) is a neurodevelopmental disorder (NDD) characterized by significant impairments in social, communicative, and behavioral abilities. However, only a limited number of studies address the genetic basis of ASD in the African population. This study aims to document the genes associated with ASD in Africa and the techniques used to identify them. Additionally, genes identified elsewhere but not yet in Africa are also noted. Methods: Online databases such as Wiley Online Library, PubMed, and Africa Journal Online were used. The review was conducted using the keyword related to genetic and genomic ASD study in the African population. Result: In this scoping review, 40 genetic studies on ASD in Africa were reviewed. The Egyptian and South African populations were the most studied, with 25 and 5 studies, respectively. Countries with fewer studies included Tunisia (4), East African countries (3), Libya (1), Nigeria (1), and Morocco (1). Some 61 genes responsible for ASD were identified in the African population: 26 were identified using a polymerase chain reaction (PCR)-based method, 22 were identified using sequencing technologies, and 12 genes and one de novo chromosomal aberration were identified through other techniques. No African study identified any ASD gene with genome-wide association studies (GWAS). Notably, at least 20 ASD risk genes reported in non-African countries were yet to be confirmed in Africa's population. Conclusion: There are insufficient genetic studies on ASD in the African population, with sample size being a major limitation in most genetic association studies, leading to inconclusive results. Thus, there is a need to conduct more studies with large sample sizes to identify other genes associated with ASD in Africa's population using high-throughput sequencing technology. [ABSTRACT FROM AUTHOR]

Whole-genome sequencing (WGS) and whole-exome sequencing (WES) are crucial within the context of breast cancer (BC) research. They play a role in the detection of predisposed genes, risk stratification, and identification of rare single nucleotide polymorphisms (SNPs). These technologies aid in the discovery of associations between various syndromes and BC, understanding the tumour microenvironment (TME), and even identifying unknown mutations that could be useful in future for personalised treatments. Genetic analysis can find the associated risk of BC and can be used in early screening, diagnosis, specific treatment plans, and prevention in patients who are at high risk of tumour formation. This article focuses on the application of WES and WGS, and how uncovering novel candidate genes associated with BC can aid in treating and preventing BC. [ABSTRACT FROM AUTHOR]

Background: Malaria in pregnancy is a major public health issue, particularly among vulnerable populations in malaria-endemic sub-Saharan African countries. To mitigate its risks, WHO recommends sulphadoxinepyrimethamine (SP) for chemoprevention and artemisinin-based combination therapy (ACT) to treat uncomplicated Plasmodium falciparum malaria. These interventions have helped to alleviate the risk associated with malaria in pregnancy; however, in the context of the emergence of SP- and ACTresistant P. falciparum, maintained efficacy is under threat. Molecular surveillance is a reliable tool to monitor the emergence of resistance where molecular markers are known. Thus, the objective of the study was to use a multiplexed amplicon Oxford Nanopore sequencing approach to assess the molecular markers for antimalarial resistance among pregnant women in Nigeria. Methods: Dried blood spots (DBS) were collected from pregnant women who received IPTp-SP at the enrollment and follow-up visits. P. falciparum genomic DNA was extracted by the Chelex® method and Pf18S qPCR was used to detect parasite DNA in each sample. With nested PCR assays, fragments of Pfdhps, Pfdhfr, Pfmdr1, Pfcrt, Pfk13 and Pfama1 genes were amplified and multiplexed amplicon-based sequencing was conducted on the minION Oxford Nanopore Technology. Result: In total, 251 pregnant women were enrolled in the study and 457 DBS samples were collected. P. falciparum genomic DNA was detected in 12% (56/457) of the samples, 31 at baseline and the remaining during the follow-up visits. Pfama1, pfk13, Pfdhps, Pfdhfr, Pfmdr1 and Pfcrt were successfully sequenced in a single run. Notably, k13 artemisinin resistance mutations were absent, the frequencies of Pfdhfr and Pfdhps SP resistance haplotypes, IRN for pyrimethamine resistance and ISGKA/IAGKA associated with sulphadoxine resistance were 82% (36/44) and 64% (27/42), respectively, and the Pfcrt CVIET resistant haplotype was at approximately 22% (7/32). Conclusion and recommendations: Here a multiplexed amplicon-based ONT assay established that triple mutant Pfdfhr-IRN, double mutant Pfdhps-SG haplotypes and the chloroquine sensitive strain were prevalent among pregnant women in Nigeria. [ABSTRACT FROM AUTHOR]

DNA lesions are linked to cancer, aging, and various diseases. The recognition and sequencing of special DNA lesions are of great interest but highly challenging. In this paper, an unnatural‐base‐pair‐promoting method for sequencing highly mutagenic ethenodeoxycytidine (εC) DNA lesions that occurred frequently is developed. First, a promising unnatural base pair of dεC–dNaM to recognize εC lesions is identified, and then a conversion PCR is developed to site‐precise transfer dεC–dNaM to dTPT3–dNaM for convenient Sanger sequencing. The low sequence dependence of this method and its capacity for the enrichment of dεC in the abundance of as low as 1.6 × 10–6 nucleotides is also validated. Importantly, the current method can be smoothly applied for recognition, amplification, enrichment, and sequencing of the real biological samples in which εC lesions are generated in vitro or in vivo, thus offering the first sequencing methodology of εC lesions at single‐base resolution. Owing to its simple operations and no destruction of inherent structures of DNA, the unnatural‐base‐pair strategy may provide a new platform to produce general tools for the sequencing of DNA lesions that are hardly sequenced by traditional strategies. [ABSTRACT FROM AUTHOR]

BackgroundCase PresentationConclusionNeurofibromatosis type-2-related schwannomatosis (NF2-SWN, formerly neurofibromatosis type 2) is a rare genetic disorder marked by the development of multiple nervous system tumors.We report a 21-month-old female patient who presented for left eye deviation. Upon examination, intermittent exotropia and a fundus mass were detected. Wide field fundus examination revealed the presence of a combined hamartoma involving the optic nerve and retina. This finding was supported by MRI highlighting the lesion’s characteristics. The patient’s father and other relatives on the paternal side displayed symptoms of NF2-SWN, evident through the presence of acoustic neuroma, although they did not exhibit any ocular symptoms. DNA analysis revealed a novel loss-of-function mutation in exon 15 of the NF2 gene (NM_000268.3: c.1627_1628del, p.Lys543Aspfs *21) in both the patient and her father at a heterozygous state. By the age of three, her vision worsened, and optical coherence tomography showed vitreomacular traction and intraretinal fluid surrounding the lesion.This case underscores the need to consider NF2- SWN in peripapillary hamartoma diagnoses and highlights the importance of genetic testing for early detection and management. [ABSTRACT FROM AUTHOR]

The increasing reliance on fossil fuels and the growing accumulation of organic waste necessitates the exploration of sustainable energy alternatives. Anaerobic digestion (AD) presents one such solution by utilizing secondary biomass to produce biogas while reducing greenhouse gas emissions. Given the crucial role of microbial activity in anaerobic digestion, a deeper understanding of the microbial community is essential for optimizing biogas production. While metagenomics has emerged as a valuable tool for unravelling microbial composition and providing insights into the functional potential in biodigestion, it falls short of interpreting the functional and metabolic interactions, limiting a comprehensive understanding of individual roles in the community. This emphasizes the significance of expanding the scope of metagenomics through innovative tools that highlight the often-overlooked, yet crucial, role of microbiota in biomass digestion. These tools can more accurately elucidate microbial ecological fitness, shared metabolic pathways, and interspecies interactions. By addressing current limitations and integrating metagenomics with other omics approaches, more accurate predictive techniques can be developed, facilitating informed decision-making to optimize AD processes and enhance biogas yields, thereby contributing to a more sustainable future. [ABSTRACT FROM AUTHOR]

Background: Chikungunya cases was reported to be on the rise in Malaysia from 2019 to 2021. Although potential endemicity was described previously, genotype shift during 2008 outbreak originating from the 2004 Indian Ocean Islands outbreak presents the probability of current CHIKV spread from neighboring countries. This is due to the prevalence of the new IOL sub-lineage which consists of E1-226A wildtype or reverted strains that are circulating in the Indian subcontinent before spreading to neighboring Thailand during 2018–2019 outbreak. Method: In this study, samples received mostly from the Tangkak, Johor were analyzed. A total 56 CHIKV positive serum samples received in 2021 by Institute of Medical Research Malaysia (IMR), were collected based on sample selection criteria. Selected samples were subjected to total RNA extraction, whole-genome sequencing as well as bioinformatic analysis such as phylogenetic, variant and mutation analysis. Results: Based on the genomic and phylogenetic analysis, the CHIKV samples from 2021 outbreak were of ECSA-IOL genotype. Genome similarity analysis also revealed that these CHIKVs were highly similar to 2018–2019 outbreak strain from Thailand. In comparison to the 2008 outbreak CHIKV isolate, the current CHIKVs lacked the E1-A226V mutation and harbored the new E1-K211E/E2-V264A sub-linage mutation. Since the E1-K211E/E2-V264A mutation facilitates adaptation to Ae. aegypti as opposed to the E1-A226V mutation which improves adaptation to Ae. albopictus, the emergence 2021 CHIKV outbreak in Malaysia can be postulated due to vector shift. Interestingly, a novel nsP3-T441A/V mutation detected in this study, may also play a role in virus transmission, pathogenicity, fitness and vector adaptation. Conclusion: In summary, the current CHIKV outbreak are strains originated from the Indian subcontinent through Thailand which may have capitalized on vector shifting by adapting to Ae. aegypti. The presence of novel nsP3-T441A/V mutation may also contribute to the spread of this virus across peninsular Malaysia. [ABSTRACT FROM AUTHOR]

Background: Reticuloendotheliosis virus (REV) is an oncogenic immunosuppressive retrovirus that infects different kinds of avian species; posing significant economic losses to the poultry industry worldwide. Methods: In Egypt, there is an unidentified disease associated with the runting-stunting syndrome with neoplasia, suspected to be REV, that has been continuously monitored in several breeder flocks. To diagnose and analyze REV by cell cultures, enzyme-linked immunosorbent assay (ELISA), histopathological investigation, the polymerase chain reaction (PCR) test, and sequencing analysis, 200 blood samples, and 50 tissue specimens were collected. The current study targets the occurrence and genetic characteristics of a viral neoplastic disease, resembling REV infection, circulating in breeder flocks from 2022 to 2023 in the Ismailia, El-Sharqia, and El-Dakahliya governorates. Result: Here, REV was isolated on chicken embryo fibroblast cell culture; exhibiting cell aggregation, rounding, and cell detachments. Collectively, only 70 serum samples were positive for anti‐REV antibodies with seroprevalence rates of 35% based on the ELISA test. The histopathological observation demonstrated lymphoreticular tumors in the liver, spleen, and other examined organs. The immunohistochemical staining method confirmed the REV-positive signals in all examined organs (liver, kidney, spleen, bursa, ovaries) except for the heart. The PCR assay of the LTR gene assessed 370 base pairs with only 5 positive samples with a percentage of 16.6%. Three positive samples were further sequenced and submitted to the Genbank under accession numbers (PP763709, PP763710, PP763711). Phylogenetic analysis of the REV-LTR gene showed that our three isolates (Sharquia-1-REV, Ismilia-2-REV, Mansoura-3-REV) are REV subtype III which predominantly circulated in breeders in Egypt. These three isolates are highest similar to American, Chinese, and Taiwanese REV reference strains, and other Egyptian strains with nucleotide identity percentages of 100%, 99%, and 99%; respectively, and on the amino acid identity level were with (99–100%), (98%, 99%), (99%, 100%); respectively. Conclusions: This study established that REV infection was extensively distributed in the breeders and became one of the causes of the clinical outbreaks of tumors, raising awareness of REV as the causative agent of avian oncogenic disease in Egypt. [ABSTRACT FROM AUTHOR]

Genomic resources have yielded unprecedented insights into ecological and evolutionary processes, not to mention their importance in economic and conservation management of specific organisms. However, the field of macroalgal genomics is hampered by difficulties in the isolation of suitable DNA. Even when DNA that appears high quality by standard metrics has been isolated, such samples may not perform well during the sequencing process. We here have compared Oxford Nanopore long‐read sequencing results for three species of macroalgae to those of nonmacroalgal species and determined that when using macroalgal samples, sequencing activity declined rapidly, resulting in reduced sequencing yield. Chemical analysis of macroalgal DNA that would be considered suitable for sequencing revealed that DNA derived from dried macroalgae was enriched for polyphenol–DNA adducts (DNA with large polyphenols chemically attached to it), which may have led to sequencing inhibition. Of note, we observed the strongest evidence of sequencing inhibition and reduced sequence output when using samples dried using silica gel—suggesting that such storage approaches may not be appropriate for samples destined for Oxford Nanopore sequencing. Our findings have wide‐ranging implications for the generation of genomic resources from macroalgae and suggest a need to develop new storage methods that are more amenable to Oxford Nanopore sequencing or to use fresh flash‐frozen tissue wherever possible for genome sequencing. [ABSTRACT FROM AUTHOR]

Recently, several bispecific antibodies (BsAbs) have been approved for the treatment of relapsed multiple myeloma (MM) after early phase trials in heavily pre-treated patients demonstrated high response rates and impressive progression-free survival with monotherapy. These BsAbs provide crucial treatment options for relapsed patients and challenging decisions for clinicians. Evidence on the optimal patient population, treatment sequence, and duration of these therapeutics is unknown and subject to active investigation. While rates of cytokine release syndrome and neurotoxicity appear to be lower with BsAbs than with CAR T-cells, morbidity from infection is high and novel pathways of treatment resistance arise from the longitudinal selection pressure of chronic BsAb therapy. Lastly, a wealth of novel T-cell engagers with unique antibodystructures and antigenic targets are under active investigation with promising early outcome data. In this review, we examine the mechanism of action, therapeutic targets, combinational approaches, sequencing and mechanisms of disease relapse for BsAbs in MM. [ABSTRACT FROM AUTHOR]

Chiari malformation type 1 (CM1) is a structural defect that involves the herniation of the cerebellar tonsils through the foramen magnum, causing mild to severe neurological symptoms. Little is known about the molecular and developmental mechanisms leading to its pathogenesis, prompting current efforts to elucidate genetic drivers. Inherited genetic disorders are reported in 2–3% of CM1 patients; however, CM1, including familial forms, is predominantly non-syndromic. Recent work has focused on identifying CM1-asscoiated variants through the study of both familial cases and de novo mutations using exome sequencing. This article aims to review the current understanding of the genetics of CM1. We discuss three broad classes of CM1 based on anatomy and link them with genetic lesions, including posterior fossa-linked, macrocephaly-linked, and connective tissue disorder-linked CM1. Although the genetics of CM1 are only beginning to be understood, we anticipate that additional studies with diverse patient populations, tissue types, and profiling technologies will reveal new insights in the coming years. [ABSTRACT FROM AUTHOR]

Gene expression is a tightly regulated process that involves multiple layers of control, including transcriptional, post-transcriptional, and translational regulation. To gain a comprehensive understanding of gene expression dynamics and its functional implications, it is crucial to compare translatomic, transcriptomic, and proteomic data. The two most common analysis methods, Ribo-seq and RNC-Seq, were used to analyze the translatome of the same sample, whose datasets were downloaded from the TranslatomeDB database. The resulting translatome maps obtained for three cell lines (HBE, A549, and MCF-7) using these two methods were comparatively analyzed. The two methods of translatome analysis were shown to provide comparable results and can be used interchangeably. The obtained mRNA translation patterns were annotated in the transcriptome and proteome context for the same sample, which may become the basis for the reconstruction of the molecular mechanisms of pathological process development in the future. [ABSTRACT FROM AUTHOR]

A study published in Acta Neuropathologica explores the use of targeted whole-viral genome sequencing from formalin-fixed paraffin-embedded (FFPE) neuropathology specimens. The researchers conducted a proof-of-concept study using a viral nucleic acid enrichment protocol on 23 FFPE samples with confirmed viral infections. They found that the viral enrichment panel led to a significant enrichment of viral sequences, resulting in a high genome coverage and improved detection of viral integration sites. The study demonstrates that metagenomic sequencing of virus-enriched libraries from FFPE specimens is a valuable method for viral whole-genome sequencing, even in low-quality and low-biomass samples. [Extracted from the article]

Background: The myostatin gene has played an important role in the genetic improvement of the main species of economic importance; however, it has not yet been described for some Neotropical fish essential for aquaculture. This study aimed to characterize the myostatin gene of pacu, Piaractus mesopotamicus, and investigate the association of a microsatellite marker in this gene with the weight of fish. Methods and results: The myostatin gene sequence was obtained after following a RACE-PCR strategy based on a partial mRNA sequence available in the GenBank database and the alignment of myostatin sequences from other fish species. The obtained sequence for the P. mesopotamicus gene was analyzed for short tandem repeats, and one dinucleotide was observed at the 3´untranslated region. A short tandem repeat polymorphism was verified in a wild population. Subsequently, the STR was evaluated in a test population of 232 animals in two 220 m² concrete tanks at the Aquaculture Center of Unesp. Eight alleles and 22 genotype combinations were identified. A significant association was observed between microsatellite marker polymorphisms and the weight traits (WEIGHT1 and WEIGHT2). Alleles 210, 222, 226, and 230 were found to favor weight gain. Conclusions: In summary, this study contributes to the characterization of the myostatin gene in pacu fish and identifies an association between a STR and weight traits. Thus, this gene could be used as a target for genetic breeding using molecular strategies such as CRISPR and quantitative strategies such as marker-assisted selection, which would contribute to improving the production of the species. [ABSTRACT FROM AUTHOR]

Background: Diarrheal disease remains one of the leading causes of deaths in children below five years of age. The risk factors associated with diarrhea include poor hygiene practices such as open defecation and consumption of contaminated water and food. However, exposure of domestic animal is equally a potential risk factor for diarrhea disease in children. Methodology: We characterized animal-related exposures in a subset of households (n=73) by collecting faecal samples from 150 children with diarrhoea and 100 food animals (30 cattle, 30 chicken, 25 goats and 15 pigs). Escherichia coli was isolated from the faecal samples and biochemically confirmed using conventional microbiological techniques. The deoxyribonucleic acid (DNA) of each E. coli isolate was extracted and amplified by multiplex PCR to identify three diarrheagenic E. coli pathotypes. The amplified products were sequenced, and genetic relatedness of the isolates was determined through phylogenetic analysis. Results: We isolated and identified a total of 32 (12.8%) diarrheagenic E. coli (DEC) from the 250 faecal samples, 26 (17.3%) of which were from the 150 children with diarrhea while 6 (6.0%) were from the 100 food animals (OR=3.285, 95% CI=1.299-8.305, p=0.011). Three DEC pathotypes were confirmed by PCR in 16 DEC strains, with 9 enteroaggregative E. coli (EAEC), 2 enterotoxigenic E. coli (ETEC), 2 enteropathogenic E. coli (EPEC), 1 EAEC/ETEC, 1 EAEC/EPEC and 1 ETEC/EPEC mixed strains. The phylogenetic analysis showed that 6 DEC isolates had genetic similarity ranging between 31% to 90%. Isolates S04 originating from animal and S02 from a child with diarrhoea of the same household were closely related, with 55% similarity. Moreover, isolate S05 from animal origin and S06 of diarrheic child origin were closely related, with similarity degree as high as 82% even though they were not paired. Twenty four of the 26 (92.3%) DEC isolates from diarrhoeic children showed multidrug resistance (MDR) pattern to antibiotics but none of the 6 isolates from food animals was multi-drug resistant. Conclusion: The high degree of genetic relationship between DEC isolate S04 and S02 from animal and human origin indicated the high potency of zoonotic transmission. Further studies investigating animal husbandry practices and zoonotic transmission of DEC are needed. [ABSTRACT FROM AUTHOR]

American plum line pattern virus (APLPV) is the most infrequently reported Ilarvirus infecting stone fruit trees and is of sufficient severity to be classified as an EPPO quarantine A1 pathogen. In late spring, yellow line pattern symptoms were observed on leaves in a few flowering cherries (Prunus serrulata Lindl.) grown in a public garden in Northwest Italy. RNA extracts from twenty flowering cherries were submitted to Ilarvirus multiplex and APLPV-specific RT-PCR assays already reported or developed in this study. One flowering cherry (T22) with mixed prunus necrotic ringspot virus (PNRSV) and prune dwarf virus (PDV) infection also showed infection with APLPV. Blastn analysis of PCR products of the full coat protein (CP) and movement protein (MP) genes obtained from flowering cherry T22 showed 98.23% and 98.34% nucleotide identity with reference APLPV isolate NC_003453.1 from the USA. Then, a LAMP-specific assay was designed to facilitate the fast and low-cost identification of this virus either in the laboratory or directly in the field. The developed assay allowed not only the confirmation of APLPV (PSer22IT isolate) infection in the T22 flowering cherry but also the identification of APLPV in an asymptomatic flowering cherry tree (TL1). The LAMP assay successfully worked with crude flowering cherry extracts, obtained after manually shaking a single plant extract in the ELISA extraction buffer for 3–5 min. The developed rapid, specific and economic LAMP assay was able to detect APLPV using crude plant extracts rather that RNA preparation in less than 20 min, making it suitable for in-field detection. Moreover, the LAMP assay proved to be more sensitive in APLPV detection in flowering cherry compared to the specific one-step RT-PCR assay. The new LAMP assay will permit the estimation of APLPV geographic spread in the territory, paying particular attention to surrounding gardens and propagated flowering cherries in ornamental nurseries. [ABSTRACT FROM AUTHOR]

Within neurosurgery, it has always been important to individualize patient care. In recent years, however, technological advances have brought a new dimension to personalized care as developing methods, including next-generation sequencing, have enabled us to molecularly profile pathologies with increasing scale and resolution. In this review, the authors discuss the history and advances in precision medicine and neurosurgery, focusing both on neuro-oncology, as well as its extension to other neurosurgical subspecialties. They highlight the important roles of neurosurgeons in past work and future work, with the extension of tissue collection and precision medicine principles to additional sample types and disease indications. [ABSTRACT FROM AUTHOR]

Recent developments in technology have led to rapid advances in precision medicine, especially due to the rise of next-generation sequencing and molecular profiling. These technological advances have led to rapid advances in research, including increased tumor subtype resolution, new therapeutic agents, and mechanistic insights. Certain therapies have even been approved for molecular biomarkers across histopathological diagnoses; however, translation of research findings to the clinic still faces a number of challenges. In this review, the authors discuss several key challenges to the clinical integration of precision medicine, including the blood-brain barrier, both a lack and excess of molecular targets, and tumor heterogeneity/escape from therapy. They also highlight a few key efforts to address these challenges, including new frontiers in drug delivery, a rapidly expanding treatment repertoire, and improvements in active response monitoring. With continued improvements and developments, the authors anticipate that precision medicine will increasingly become the gold standard for clinical care. [ABSTRACT FROM AUTHOR]

Sequencing of phosphorodiamidate morpholino oligomers (PMOs) by hydrophilic interaction chromatography (HILIC) coupled to tandem mass spectrometry (MS/MS) is reported. The MS/MS analysis was performed using a quadrupole/time-of-flight (Q-ToF) mass analyzer and collision induced dissociation (CID) in negative ion mode. To improve MS sensitivity in negative ion mode, HILIC conditions, including the separation column, mobile phases, and MS parameters, were optimized. Using the developed HILIC–CID-MS/MS method, 100% sequence coverage was achieved for PMOs ranging from 18-mer to 25-mer. Additionally, the method was successfully applied to identifying positional isomers of n – 1 deletion impurities present in PMO drug substances. [ABSTRACT FROM AUTHOR]

Despite the numerous sequencing methods available, the diversity in RNA size and chemical modification makes it difficult to capture all RNAs in a cell. We developed a method that combines quasi-random priming with template switching to construct sequencing libraries from RNA molecules of any length and with any type of 3′ modifications, allowing for the sequencing of virtually all RNA species. Our ligation-independent detection of all types of RNA (LIDAR) is a simple, effective tool to identify and quantify all classes of coding and non-coding RNAs. With LIDAR, we comprehensively characterized the transcriptomes of mouse embryonic stem cells, neural progenitor cells, mouse tissues, and sperm. LIDAR detected a much larger variety of tRNA-derived RNAs (tDRs) compared with traditional ligation-dependent sequencing methods and uncovered tDRs with blocked 3′ ends that had previously escaped detection. Therefore, LIDAR can capture all RNAs in a sample and uncover RNA species with potential regulatory functions. [Display omitted] • LIDAR combines template switch and quasi-random hexamers to clone all types of RNA • LIDAR uncovered long 3′ tDRs missed by conventional cloning protocols • Long 3′ tDRs are found in multiple mouse tissues with specific expression patterns • Some long 3′ tDRs are internally modified and blocked at their 3′ termini Scacchetti et al. develop a ligation-independent cloning method (LIDAR) to sequence RNAs irrespective of their size or 3′ modifications. LIDAR reveals a class of long 3′ tRNA-derived fragments, present in cultured cells, tissues, and sperm and characterized by internal chemical modifications and blocked 3′ ends. [ABSTRACT FROM AUTHOR]

During the COVID-19 pandemic, wastewater surveillance was used to monitor community transmission of SARS-CoV-2. As new genetic variants emerged, the need for timely identification of these variants in wastewater became an important focus. In response to increased reports of Omicron transmission across the United States, the Oklahoma Wastewater Surveillance team utilized allele-specific RT-qPCR assays to detect and differentiate variants, such as Omicron, from other variants found in wastewater in Oklahoma. The PCR assays showed presence of the Omicron variant in Oklahoma on average two weeks before official reports, which was confirmed through genomic sequencing of selected wastewater samples. Through continued surveillance from November 2021 to January 2022, we also demonstrated the transition from prevalence of the Delta variant to prevalence of the Omicron variant in local communities. We further assessed how this transition correlated with certain demographic factors characterizing each community. Our results highlight RT-qPCR assays as a rapid, simple, and cost-effective method for monitoring the community spread of SARS-CoV-2 genetic variants in wastewater. Additionally, they demonstrate that specific demographic factors such as ethnic composition and household income can correlate with the timing of SARS-CoV-2 variant introduction and spread. [ABSTRACT FROM AUTHOR]

Objective: To develop a high‐throughput sequencing panel for the diagnosis of developmental and epileptic encephalopathy in Tunisia and to clarify the frequency of disease‐causing genes in this region. Methods: We developed a custom panel for next‐generation sequencing of the coding sequences of 116 genes in individuals with developmental and epileptic encephalopathy from the Tunisian population. Segregation analyses and in silico studies have been conducted to assess the identified variants' pathogenicity. Results: We report 12 pathogenic variants in SCN1A, CHD2, CDKL5, SZT2, KCNT1, GNAO1, PCDH19, MECP2, GRIN2A, and SYNGAP1 in patients with developmental and epileptic encephalopathy. Five of these variants are novel: "c.149delA, p.(Asn50MetfsTer26)" in CDKL5; "c.3616C > T, p.(Arg1206Ter)" in SZT2; "c.111_113del, p.(Leu39del)" in GNAO1; "c.1435G>C, p.(Asp479His)" in PCDH19; and "c.2143delC, p.(Arg716GlyfsTer10)" in SYNGAP1. Additionally, for four of our patients, the genetic result facilitated the choice of the appropriate treatment. Significance: This is the first report of a custom gene panel to identify genetic variants implicated in developmental and epileptic encephalopathy in the Tunisian population as well as the North African region (Tunisia, Egypt, Libya, Algeria, Morocco) with a diagnostic rate of 30%. This high‐throughput sequencing panel has considerably improved the rate of positive diagnosis of developmental and epileptic encephalopathy in the Tunisian population, which was less than 15% using Sanger sequencing. The benefit of genetic testing in these patients was approved by both physicians and parents. [ABSTRACT FROM AUTHOR]

The aim of the present study was to confirm the presence of the polled intersex syndrome (PIS) deletion in hermaphroditic individuals and polled bucks, transmitting at least one of its copies to PIS individuals. Two sex-reversed animals (Czech White Improved and Polish White Improved), two polled and two horned bucks were examined. Three primer pairs were designed for the identification of the PIS deletion in the sex-reversed individuals and polled bucks. Additional 24 primer pairs were used for the identification of the deletion region in sex-reversed individuals. Selected PCR products were sequenced. The identification of the PIS deletion in hermaphroditic individuals and polled bucks based on the primers designed on the reference template was impossible. Therefore, an attempt was made to determine the reasons for the failure of deletion identification in these individuals. For the polled bucks, the deletion region was amplified similarly to the horned ones, except for an initial sequence. In the two sex-reversed individuals, four inserts were identified in the deletion region. Despite the fact that PIS in goats has been known to breeders and scientists for several decades, many of its aspects, including the molecular reason, are still controversial and require further detailed research. [ABSTRACT FROM AUTHOR]

Background: Peste des Petits Ruminants (PPR) is an economically significant transboundary viral disease of sheep and goats caused by the PPRV virus, affecting annual losses of 1.45–2.10 billion US dollars globally. We designed the current study to evaluate the positive cases, molecular characterization, phylogenetic analysis, and risk factors correlated with the disease in various districts of Khyber Pakhtunkhwa, Pakistan, with the aim of contributing to these strategies. Methods and results: A total of 384 samples from three selected districts, i.e., Peshawar, Charsadda and Chitral (n = 128 each), were collected, and the virus was investigated by using the sandwich ELISA, while the N gene of the virus was used as a target for molecular detection via RT-PCR. The confirmed samples were then sequenced, and phylogenetic analysis was performed. According to our findings, the highest positive cases was found in district Peshawar (50.87%), followed by Charsadda and Chitral (24.56%), respectively, while risk factor analysis showed that certain categories, such as species, sex, and age less than two years, have higher risk (P < 0.05) in contrast to their respective categories. Furthermore, sequencing and phylogenetic analysis of representative samples showed that the PPRV strains in the current study clustered in lineage IV, which is circulating in the small ruminant population of Asia, the Middle East, and African countries. Comparative residue analysis highlighted the mutation by representing 242 variable sites out of 371 locations. Conclusions: PPRV has foremost importance in Pakistan because the virus was detected in a considerable number of samples, and most of which were sourced from subsidiary areas where veterinary services are not prioritized. [ABSTRACT FROM AUTHOR]

The purpose of the current study was to examine the occurrence and genetic characteristics of Bartonella henselae (B. henselae) and Bartonella clarridgeiae (B. clarridgeiae) in dogs from West Azerbaijan province, Iran. Blood samples were obtained from 400 dogs, and their gender, age, reproductive status, ownership status, and geographical origin were documented. Positive samples were identified using PCR and sequencing techniques, and the gene sequences of the ftsZ (for B.henselae) and gltA (for B.clarridgeiae) genes were examined using BioEdit software. The gene sequences acquired demonstrated a minimum similarity of 100.00% when compared to the reference sequences in the GenBank® database. Additionally, a phylogenetic tree was built using MEGA11. The findings of the study indicated that 8.5% (p<0.05; 95%, CI: 6.15%–11.64%) of the tested dogs were positive for B. henselae, and 3.25% (p<0.05; 95%, CI: 1.91%–5.48%) were positive for B. clarridgeiae. The results for both Bartonella species showed a significant difference (p=0.001) between neutered and nonneutered dogs, as well as a significant difference (p=0.001 and p=0.004) between stray and pet dogs. The study's findings highlight the significant role that dogs could potentially engage as the origins of Bartonella infection, as a zoonotic agent, in the region. [ABSTRACT FROM AUTHOR]

ABSTRACT Uveal melanoma (UM) and nonacral cutaneous melanoma (CM) are distinct entities with varied genetic landscapes despite both arising from melanocytes. There are, however, similarities in that they most frequently affect people of European ancestry, and high penetrance germline variants in BAP1, POT1 and CDKN2A have been shown to predispose to both UM and CM. This study aims to further explore germline variants in patients affected by both UM and CM, shedding light on the underlying genetic mechanism causing these diseases. Using exome sequencing we analysed germline DNA samples from a cohort of 83 Australian patients diagnosed with both UM and CM. Eight (10%) patients were identified that carried pathogenic mutations in known melanoma predisposition genes POT1, MITF, OCA2, SLC45A2 and TYR. Three (4%) patients carried pathogenic variants in genes previously linked with other cancer syndromes (ATR, BRIP1 and MSH6) and another three cases carried monoallelic pathogenic variants in recessive cancer genes (xeroderma pigmentosum and Fanconi anaemia), indicating that reduced penetrance of phenotype in these individuals may contribute to the development of both UM and CM. These findings highlight the need for further studies characterising the role of these genes in melanoma susceptibility. [ABSTRACT FROM AUTHOR]

Background: Comprehensive genomic profiling (CGP) has gained an important role in patients with advanced prostate cancer following the introduction of PARP inhibitors in daily clinical practice. Here, we report an overview of CGP results, specifically of BRCA1 and BRCA2 HRD-repair system genes, from patients with prostate cancer analyzed in our institution, and we compare our results with those available from more recent scientific literature. Methods: The study cohort consisted of 70 patients. Somatic DNA was extracted from Formalin-Fixed Paraffin-Embedded (FFPE) tissue using a MagCore Genomic DNA FFPE One-Step Kit for MagCore System. The DNA was quantified by EasyPGX® Real-Time qPCR and EasyPGX® Analysis Software (version 4.0.13). Tissue somatic DNA libraries were prepared with Myriapod® NGS BRCA1-2 panel-NG035 and sequenced in a Mi-Seq® System. The sequence alignment in hg19 and the variant calling were performed using Myriapod® NGS Data Analysis Software version 5.0.8 NG900-SW 5.0.8 with a software detection limit (LoD) of 95%. Variants with a coverage of 500 and VAF% ≥ 5 were evaluated. Results: Tumor tissue NGS was unsuccessful in 46/70 patients (66%). Mutations of the BRCA2 gene were detected in 4 of the samples: (1) BRCA2 ex10 c.1244A>G p.His415Arg VAF = 51.03%; (2) BRCA2 ex11 c.5946delT p.Ser1982fs VAF = 72.1%; (3) BRCA2 ex11 c.3302A>G p.His1101Arg VAF = 52.9%; and (4) BRCA2 ex11 c.3195_3198delTAAT p.Asn1066fs VAF = 51.1%. Conclusions: The results from our internal overview seem to support the data and to confirm the performance of the technical issues reported in the literature. Considering the advanced age of our patients, with 84% of men over the age of 65, the application of alternative and less invasive procedures such as liquid biopsy, could be a more suitable solution for some cases. [ABSTRACT FROM AUTHOR]

Progressive multifocal leukoencephalopathy (PML) is a rare neurological condition associated with reactivation of dormant JC polyomavirus (JCPyV). In this study, we characterized gene expression and JCPyV rearrangements in PML brain tissue. Infection of white matter astrocytes and oligodendrocytes as well as occasional brain cortex neurons was shown. PML brain harbored exclusively rearranged JCPyV variants. Viral transcripts covered the whole genome on both strands. Strong differential expression of human genes associated with neuroinflammation, blood-brain barrier permeability, and neurodegenerative diseases was shown. Pathway analysis revealed wide immune activation in PML brain. The study provides novel insights into the pathogenesis of PML. [ABSTRACT FROM AUTHOR]

Dielectric barrier discharge (DBD) plasma regulates the levels of reactive oxygen species (ROS), which are critical for sperm quality. MicroRNAs (miRNAs) are non-coding single-stranded RNA molecules encoded by endogenous genes, which regulate post-transcriptional gene expression in animals. At present, it is unknown whether DBD plasma can regulate sperm ROS levels through miRNAs. To further understand the regulatory mechanism of DBD plasma on sperm ROS levels, miRNAs in fresh boar spermatozoa were detected using Illumina deep sequencing technology. We found that 25 known miRNAs and 50 novel miRNAs were significantly upregulated, and 14 known miRNAs and 74 novel miRNAs were significantly downregulated in DBD plasma-treated spermatozoa. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that target genes of differentially expressed miRNAs were involved in many activities and pathways associated with antioxidants. We verified that DBD plasma significantly increased boar sperm quality and reduced ROS levels. These results suggest that DBD plasma can improve sperm quality by regulating ROS levels via miRNAs. Our findings provide a potential strategy to improve sperm quality through miRNA-targeted regulation of ROS, which helps to increase male reproduction and protect cryopreserved semen in clinical practice. • DBD plasma improves boar sperm quality by regulating ROS levels. • DBD plasma affects sperm ROS levels via differentially expressed miRNAs. • DBD plasma regulates sperm ROS levels via miRNA-targeted antioxidant enzyme genes. • DBD plasma regulates sperm ROS levels via miRNAs targeting the KEAP1/NRF2 pathway. [ABSTRACT FROM AUTHOR]

This paper asks whether, and if so how, it is possible to design a system characterised by coordination across sectors and levels of governance aimed at governing AMR. The ambition is, firstly, to analyse how coordination problems materialise in the governing of the AMR problem, and secondly, with an emphasis on the structure of decision-making and communication processes, to probe into how coordination of AMR governance is achieved. The paper’s focus is on Norway, which stands out as one of the better performing countries for AMR governance. Drawing on literature on coordination and governance, the paper argues that effective coordination of AMR governance is more likely to follow a ‘bottom-up’ sequencing pattern. It thus provides a study of the systems for governing AMR in a multi-level setting. Through public documents, literature and interviews with key officials involved in AMR management, the paper illustrates the importance of – and organisational barriers to – inter-sectoral cooperation and coordinated strategies and actions at different levels of governance. [ABSTRACT FROM AUTHOR]

Pancreatic ductal adenocarcinoma (PDAC) remains the gastrointestinal cancer with the worst prognosis. When metastases are present (mPDAC), stage the most common at diagnosis, the treatment lays on supportive cares to improve denutrition, pain and physical alteration associated with polychemotherapy regimens: FOLFIRINOX (FFX), gemcitabine plus NabPaclitaxel (GNP –Not reimbursed in France). Important gastrointestinal and hematological side effects associated with that regimens reserves them for patients with good performans status. Second line (L2) is generally in mirror with the first one (L1) with administration of 5-FU based regimen plus oxaliplatin or irinotecan (that can be the nanoliposmal form NALIRI – not reimbursed in France) for patient treated with GNP in L1 and gemcitabine +/-(Nab) paclitaxel for those treated with FFX in L1. Main innovations in mPDAC have occurred in specific subgroups identified using the tumors molecular portrait. PDAC with constitutional mutation of BRCA (4%-8%) is accessible to olaparib in maintenance after four months of platinum-based regimen and without progressive disease. Patients with MSI-H/dMMR (1%) or mutated for KRASG12C (< 1%) tumors can be treated by anti-PD1 or specific inhibitors in clinical trials. Huge effort of researchers and clinicians to identify PDAC biomarkers driven subgroups and to personalized treatment strategies for them will probably be the key to improve PDAC's prognosis. [ABSTRACT FROM AUTHOR]

Background: Glioblastoma is one of the most malignant brain tumors, which is also known as primary neuroepithelial tumors. Glioblastoma malignant tumors include 20-40% of brain tumors. The LMO1 gene is located at position 11P15.4 and is introduced as an oncogene in some cancers. This study was conducted for the first time in Iran to identify and investigate the relationship between LMO1 gene mutations and glioblastoma. Materials and Methods: In this research, the Touchdown PCR technique and DNA sequencing method were used in 35 blood samples of people with glioblastoma multiforme and 40 control samples. Bioinformatics analyses were also performed to investigate the pathogenic effect of nucleotide changes in this gene. Results: In this study, four point mutations were identified, of which two of the new mutations were misense and led to amino acid changes in one of the important domains of the protein (p.M135K and p.N148H), which indicates their potential pathogenicity. Our results of bioinformatics databases predicted that both of these mutations affect protein function, such that they can disrupt this domain and impair its function. Also, a nucleotide change was observed in the 3'UTR region of this gene (c.*74A>G), which is located at the binding site of two regulatory miRNAs and is expected to disrupt the binding of these miRNAs to the target sequence. Conclusion: These findings predict that any mutations in the LIM-sensitive domains in the LMO1 gene are significantly related to the pathogenesis of glioblastoma and most likely have a significant impact on the function of this transcriptional cofactor. [ABSTRACT FROM AUTHOR]

A quartz crystal microbalance (QCM) systematic evolution of ligands by the exponential enrichment (SELEX) technique was developed to screen out aptamers with high affinity for arsenic (III). Α random single strand DNA library was designed and fixed on the mercaptoethylamine-modified crystal plate with arsenic (III) as the target, and the free aptamer was captured in the solution, and the QCMSELEX screening method was constructed. After 6 rounds of screening, the secondary library was sequenced with high throughput method, and the 6S1 dissociation coefficient Kd value was 0.36 µmol/L based on QCM resonance frequency. Using 6S1 as a probe, the QCM biosensor was constructed for the detection of arsenic (III). The sensor has a good linear relationship in the range of 0.01 µmol/L~0.2 µmol/L, and the detection limit of arsenic (III) is 5.2 nmol/L(30), indicatinggood selectivity. [ABSTRACT FROM AUTHOR]

Simple Summary: Validation of plant pathogens' diagnostic assays requires positive and negative controls. High Throughput Sequencing (HTS) has shown capabilities for detecting multiple pathogens simultaneously. However, accurate pathogen identification from HTS data depends on the bioinformatic pipeline used. There is currently no consensus on the best pipeline, leading to inconsistent results. To address this, standardized artificial HTS datasets are proposed as benchmarks to evaluate the performance of bioinformatic pipelines used in detection scenarios using HTS. These datasets will aid in resolving challenges like unknown sensitivity and specificity of the bioinformatic tool, contributing to advancing plant pathogen detection using HTS. The validation of diagnostic assays in plant pathogen detection is a critical area of research. It requires the use of both negative and positive controls containing a known quantity of the target pathogen, which are crucial elements when calculating analytical sensitivity and specificity, among other diagnostic performance metrics. High Throughput Sequencing (HTS) is a method that allows the simultaneous detection of a theoretically unlimited number of plant pathogens. However, accurately identifying the pathogen from HTS data is directly related to the bioinformatic pipeline utilized and its effectiveness at correctly assigning reads to their associated taxa. To this day, there is no consensus about the pipeline that should be used to detect the pathogens in HTS data, and results often undergo review and scientific evaluation. It is, therefore, imperative to establish HTS resources tailored for evaluating the performance of bioinformatic pipelines utilized in plant pathogen detection. Standardized artificial HTS datasets can be used as a benchmark by allowing users to test their pipelines for various pathogen infection scenarios, some of the most prevalent being multiple infections, low titer pathogens, mutations, and new strains, among others. Having these artificial HTS datasets in the hands of HTS diagnostic assay validators can help resolve challenges encountered when implementing bioinformatics pipelines for routine pathogen detection. Offering these purely artificial HTS datasets as benchmarking tools will significantly advance research on plant pathogen detection using HTS and enable a more robust and standardized evaluation of the bioinformatic methods, thereby enhancing the field of plant pathogen detection. [ABSTRACT FROM AUTHOR]

This study focuses on the detection and differentiation of Canine Parvovirus 2 (CPV-2) strains in India using High Resolution Melt (HRM) curve analysis. CPV-2 is a highly contagious virus causing acute haemorrhagic enteritis and myocarditis in dogs, with a high mortality rate. A total of 45 faecal swab samples were collected from suspected CPV cases, out of which 20 tested samples were positive for CPV using PCR targeting the VP2 gene. These positive samples were further analyzed using HRM, which predicted them to belong to the CPV2a strain based on their melting temperature (71.4° or less). Two CPV2a positive samples were sequenced, they were found to belong to CPV2b strains, indicating a discrepancy between HRM prediction and sequencing results. Single nucleotide polymorphic variations were observed at positions 4062 and 4064, confirming their classification as CPV2b strains. Phylogenetic analysis of VP2 capsid gene showed that these field samples were closely related to CPV2c strains, suggesting potential genetic diversity among circulating CPV strains in India. The study highlights the importance of developing geographically specific CPV strain primers for accurate detection and differentiation using HRM. It raises concerns about potential co-infections or the emergence of new strains, indicated by varying melting temperatures in field samples. [ABSTRACT FROM AUTHOR]

The Fabaceae genus Trifolium comprises around 250 species widely distributed worldwide, with the temperate Northern Hemisphere exhibiting the highest variety. The plants in this genus are widely used as livestock fodder crops and are particularly significant economically. This study's objective included isolating bacteria from the root nodules of the Trifolium repens plant and diagnosing it at the molecular and microbiological levels. T. repens root nodules were used as the source of an endophytic bacteria isolated on Yeast Extract Mannitol (YEM) media that had solidified and diagnosed at the molecular level by DNA Sequencing technique for analysis of the sequence of the nitrogenous bases of 16S rRNA gene with the global database. The isolated bacteria were characteristic of greyish-white color after 48 hours of growth and appeared as a circular shape, slightly convex and gram-negative. The bacteria were resistant to the antibiotics 20µg/ml Aztreonam. The DNA sequencing technique for analysis of the sequence of the nitrogenous bases of 16S rRNA gene with the global database of the National Center for Biotechnology Information (NCBI) showed that the isolated bacteria was at least 96.22% similar to the species Acinetobacter baumannii As a result, it was recorded for the first time as Acinetobacter sp. AZS1 strain in NCBI. [ABSTRACT FROM AUTHOR]

Background: Building on our prior work that RNA alternative splicing modulates the druggability of kinase fusions, this study probes the clinical significance of sole reciprocal fusions. These rare genomic arrangements, despite lacking kinase domains at the DNA level, demonstrated potential RNA‐level druggability in sporadic cases from our prior research. Methods: Utilizing the large‐scale multicenter approach, we performed RNA sequencing and clinical follow‐up to evaluate a broad spectrum of kinase fusions, including ALK, ROS1, RET, BRAF, NTRK, MET, NRG1, and EGFR, in 1943 patients. Results: Our findings revealed 51 instances (2.57%) of sole reciprocal fusions, predominantly in lung (57%), colorectal (14%), and glioma (10%) cancers. Comparative analysis with an MSKCC cohort confirmed the prevalence in diverse cancer types and identified unique fusion partners and chromosomal locales. Cross‐validation through RNA‐NGS and FISH authenticated the existence of functional kinase domains in subsets including ALK, ROS1, RET, and BRAF, which correlated with positive clinical responses to targeted kinase inhibitors (KIs). Conversely, fusions involving EGFR, NRG1, and NTRK1/2/3 generated nonfunctional transcripts, suggesting the need for alternative therapeutic interventions. Conclusion: This inaugural multicenter study introduces a novel algorithm for detecting and treating sole reciprocal fusions in advanced cancers, expanding the patient population potentially amenable to KIs. [ABSTRACT FROM AUTHOR]

Genetic alterations are well known to be related to the pathogenesis and prognosis of papillary thyroid carcinoma (PTC). Some miRNA expression dysregulations have previously been described in the context of cancer development including thyroid carcinoma. In our study, we performed original molecular diagnostics on tissue samples related to our own patients. We aimed to identify all dysregulated miRNAs in potential association with PTC development via sequencing much higher numbers of control-matched PTC tissue samples and analyzing a wider variety of miRNA types than previous studies. We analyzed the expression levels of 2656 different human miRNAs in the context of 236 thyroid tissue samples (118 tumor and control pairs) related to anonymized PTC cases. Also, KEGG pathway enrichment analysis and GO framework analysis were used to establish the links between miRNA dysregulation and certain biological processes, pathways of signaling, molecular functions, and cellular components. A total of 30 significant differential miRNA expressions with at least ±1 log2 fold change were found related to PTC including, e.g., miR-551b, miR-146b, miR-221, miR-222, and miR-375, among others, being highly upregulated, as well as miR-873 and miR-204 being downregulated. In addition, we identified miRNA patterns in vast databases (KEGG and GO) closely similar to that of PTC including, e.g., miRNA patterns of prostate cancer, HTLV infection, HIF-1 signaling, cellular responses to growth factor stimulus and organic substance, and negative regulation of gene expression. We also found 352 potential associations between certain miRNA expressions and states of clinicopathological variables. Our findings—supported by the largest case number of original matched-control PTC–miRNA relation research—suggest a distinct miRNA expression profile in PTC that could contribute to a deeper understanding of the underlying molecular mechanisms promoting the pathogenesis of the disease. Moreover, significant miRNA expression deviations and their signaling pathways in PTC presented in our study may serve as potential biomarkers for PTC diagnosis and prognosis or even therapeutic targets in the future. [ABSTRACT FROM AUTHOR]

Leminorella grimontii strain LG-KP-E1-2-T0 was isolated from Zophobas morio larvae. It showed a susceptibility phenotype compatible with the expression of an inducible extended-spectrum β-lactamase. The presence of a chromosomal bla gene encoding for the class A GRI-1 β-lactamase was revealed by whole-genome sequencing. GRI-1 shared the highest amino acid identity with RIC-1 and OXY-type β-lactamases (76–80%). Analysis of six further publicly-available L. grimontii draft genomes deposited in NCBI revealed that blaGRI−1 was always present. Core-genome analysis indicated that LG-KP-E1-2-T0 was unique from other strains. We provided the first complete genome of L. grimontii and new insights on its chromosomal β-lactamases. [ABSTRACT FROM AUTHOR]