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Simple Summary: Colorectal cancer (CRC) is the second leading cause of cancer deaths worldwide. Genetic alterations promote cancer development and its spread to distant sites. Medications that counteract the effects of these alterations/mutations, called targeted therapies, are used to treat CRC. Immunotherapy, another class of medications that promotes immune system mediated recognition and destruction of cancer cells, is used for treatment of a small subset of CRC patients. Here we review the current state of knowledge and ongoing research into biomarkers, targeted therapy, and immunotherapy for CRC treatment. Colorectal cancer (CRC) is the second leading cause of cancer deaths worldwide. Although the overall incidence of CRC is decreasing, the incidence of young-onset CRC, characterized by a diagnosis of CRC before age 50, is increasing. Outcomes for CRC patients are improving, partly due to comprehensive molecular characterization of tumors and novel therapeutic strategies. Advances in genomic and transcriptomic analyses using blood- and tumor-tissue-based sequencing have facilitated identification of distinct tumor subtypes harboring unique biological characteristics and therapeutic vulnerabilities. These insights have led to the development and incorporation of targeted therapies and immunotherapy in CRC treatment. In this review, we discuss the molecular landscape and key oncogenes/tumor suppressors contributing to CRC tumorigenesis, metastasis, and therapeutic resistance. We also discuss personalized therapeutic strategies for subsets of CRC patients and provide an overview of evolving novel treatments being evaluated in clinical trials. [ABSTRACT FROM AUTHOR]

Variants in KCNT1 are associated with a wide spectrum of epileptic phenotypes, including epilepsy of infancy with migrating focal seizures (EIMFS), non-EIMFS developmental and epileptic encephalopathies, autosomal dominant or sporadic sleep-related hypermotor epilepsy, and focal epilepsy. Here, we describe a girl affected by drug-resistant focal seizures, developmental delay and behavior disorders, caused by a novel, de novo heterozygous missense KCNT1 variant (c.2809A > G, p.S937G). Functional characterization in transiently transfected Chinese Hamster Ovary (CHO) cells revealed a strong gain-of-function effect determined by the KCNT1 p.S937G variant compared to wild-type, consisting in an increased maximal current density and a hyperpolarizing shift in current activation threshold. Exposure to the antidepressant drug fluoxetine inhibited currents expressed by both wild-type and mutant KCNT1 channels. Treatment of the proband with fluoxetine led to a prolonged electroclinical amelioration, with disappearance of seizures and better EEG background organization, together with an improvement in behavior and mood. Altogether, these results suggest that, based on the proband's genetic and functional characteristics, the antidepressant drug fluoxetine may be repurposed for the treatment of focal epilepsy caused by gain-of-function variants in KCNT1. Further studies are needed to verify whether this approach could be also applied to other phenotypes of the KCNT1-related epilepsies spectrum. [ABSTRACT FROM AUTHOR]

Background: Carnitine palmitoyltransferase II (CPT II) deficiency is a rare inborn error of mitochondrial fatty acid metabolism with autosomal recessive pattern of inheritance. Its phenotype is highly variable (neonatal, infantile, and adult onset) on the base of mutations of the CPT II gene. In affected subjects, long-chain acylcarnitines cannot be subdivided into carnitine and acyl-CoA, leading to their toxic accumulation in different organs. Neonatal form is the most severe, and all the reported patients died within a few days to 6 months after birth. Hereby, we report on a male late-preterm newborn who presented refractory cardiac arrhythmias and acute multiorgan (hepatic, renal, muscular) injury, leading to cerebral hemorrhage, hydrocephalus, cardiovascular failure and early (day 5 of life) to death. Subsequently, extended metabolic screening and target next generation sequencing (NGS) analysis allowed the CPT II deficiency diagnosis. Case presentation: The male proband was born at 36+ 4 weeks of gestation by spontaneous vaginal delivery. Parents were healthy and nonconsanguineous, although both coming from Nigeria. Family history was unremarkable. Apgar score was 9/9. At birth, anthropometric measures were as follows: weight 2850 g (47th centile, -0.07 standard deviations, SD), length 50 cm (81st centile, + 0.89 SD) and occipitofrontal circumference (OFC) 35 cm (87th centile, + 1.14 SD). On day 2 of life our newborn showed bradycardia (heart rate around 80 bpm) and hypotonia, and was then transferred to the Neonatal Intensive Care Unit (NICU). There, he subsequently manifested many episodes of ventricular tachycardia, which were treated with pharmacological (magnesium sulfate) and electrical cardioversion. Due to the critical conditions of the baby (hepatic, renal and cardiac dysfunctions) and to guarantee optimal management of the arrythmias, he was transferred to the Pediatric Cardiology Reference Center of our region (Sicily, Italy), where he died 2 days later. Thereafter, the carnitines profile evidenced by the extended metabolic screening resulted compatible with a fatty acid oxidation defect (increased levels of acylcarnitines C16 and C18, and low of C2); afterwards, the targeted next generation sequencing (NGS) analysis revealed the known c.680 C > T p. (Pro227Leu) homozygous missense mutation of the CPTII gene, for diagnosis of CPT II deficiency. Genetic investigations have been, then, extended to the baby's parents, who were identified as heterozygous carriers of the same variant. When we meet again the parents for genetic counseling, the mother was within the first trimester of her second pregnancy. Therefore, we offered to the couple and performed the prenatal target NGS analysis on chorionic villi sample, which did not detect any alterations, excluding thus the CPT II deficiency in their second child. Conclusions: CPTII deficiency may be suspected in newborns showing cardiac arrhythmias, associated or not with hypertrophic cardiomyopathy, polycystic kidneys, brain malformations, hepatomegaly. Its diagnosis should be even more suspected and investigated in cases of increased plasmatic levels of creatine phosphokinase and acylcarnitines in addition to kidney, heart and liver dysfunctions, as occurred in the present patient. Accurate family history, extended metabolic screening, and multidisciplinary approach are necessary for diagnosis and adequate management of affected subjects. Next generation sequencing (NGS) techniques allow the identification of the CPTII gene mutation, essential to confirm the diagnosis before or after birth, as well as to calculate the recurrence risk for family members. Our report broads the knowledge of the genetic and molecular bases of such rare disease, improving its clinical characterization, and provides useful indications for the treatment of patients. [ABSTRACT FROM AUTHOR]

Introduction: The Red Sea is a narrow rift basin characterized by latitudinal environmental gradients which shape the diversity and distribution of reefdwelling organisms. Studies on Symbiodiniaceae associated with select hard coral taxa present species- specific assemblages and concordant variation patterns from the North to southeast Red Sea coast at depths shallower than 30 m. At mesophotic depths, however, algal diversity studies are rare. Here, we characterize for the firsttime host-associated algal communities of a mesophotic specialist coral species, Leptoseris cf. striatus, along the Saudi Arabian Red Sea coast. Methods: We sampled 56 coral colonies spanning the eastern Red Sea coastline from the Northern Red Sea to the Farasan Banks in the South, and across two sampling periods, Fall 2020 and Spring 2022. We used Next Generation Sequencing of the ITS2 marker region in conjunction with SymPortal to denote algal assemblages. Results and discussion: Our results show a relatively stable coral species-specific interaction with algae from the genus Cladocopium along the examined latitudinal gradient, with the appearance, in a smaller proportion, of presumed thermally tolerant algal taxa in the genera Symbiodinium and Durusdinium during the warmer season (Fall 2020). Contrary to shallow water corals, our results do not show a change in Symbiodiniaceae community composition from North to South in this mesophotic specialist species. However, our study highlights for the first time that symbiont communities are subject to change over time at mesophotic depth, which could represent an important phenomenon to address in future studies. [ABSTRACT FROM AUTHOR]

The rapid identification of lactic acid bacteria, which are essential microorganisms in the food industry, is of great significance for industrial applications. The identification of lactic acid bacteria traditionally relies on the isolation and identification of pure colonies. While this method is well-established and widely used, it is not without limitations. The subjective judgment inherent in the isolation and purification process introduces potential for error, and the incomplete nature of the isolation process can result in the loss of valuable information. The advent of next generation sequencing has provided a novel approach to the rapid identification of lactic acid bacteria. This technology offers several advantages, including rapidity, accuracy, high throughput and low cost. Next generation sequencing represents a significant advancement in the field of DNA sequencing. Its ability to rapidly and accurately identify lactic acid bacteria strains in samples with insufficient information or in the presence of multiple lactic acid bacteria sets it apart as a valuable tool. The application of this technology not only circumvents the potential errors inherent in the traditional method but also provides a robust foundation for the expeditious identification of lactic acid bacteria strains and the authentication of bacterial powder in industrial applications. This paper commences with an overview of traditional and molecular biology methods for the identification of lactic acid bacteria. While each method has its own advantages as they are not without limitations in practical application. Subsequently, the paper provides an introduction of the principle, process, advantages, and disadvantages of next generation sequencing and also details its application in strain identification and rapid identification of lactic acid bacteria. The objective of this study is to provide a comprehensive and reliable basis for the rapid identification of industrial lactic acid bacteria strains and the authenticity identification of bacterial powder. [ABSTRACT FROM AUTHOR]

Purpose: To identify germline mutations related to azoospermia etiology and reproductive potential of surgically retrieved spermatozoa, and to investigate the feasibility of predicting seminiferous tubule function of nonobstructive azoospermic men by transcriptomic profiling of ejaculates. Materials and methods: Sperm specimens were obtained from 30 men (38.4 ± 6 years) undergoing epididymal sperm aspiration for obstructive azoospermia (OA, n = 19) acquired by vasectomy, or testicular biopsy for nonobstructive azoospermia (NOA, n = 11). To evaluate for a correlation with azoospermia etiology, DNAseq was performed on surgically retrieved spermatozoa, and cell-free RNAseq on seminal fluid (n = 23) was performed to predict spermatogenesis in the seminiferous tubule. Results: Overall, surgically retrieved sperm aneuploidy rates were 1.7% and 1.8% among OA and NOA cohorts, respectively. OA men carried housekeeping-related gene mutations, while NOA men displayed mutations on genes involved in crucial spermiogenic functions (AP1S2, AP1G2, APOE). We categorized couples within each cohort according to ICSI clinical outcomes to investigate genetic causes that may affect reproductive potential. All OA-fertile men (n = 9) carried mutations in ZNF749 (sperm production), whereas OA-infertile men (n = 10) harbored mutations in PRB1, which is essential for DNA replication. NOA-fertile men (n = 8) carried mutations in MPIG6B (stem cell lineage differentiation), whereas NOA-infertile individuals (n = 3) harbored mutations in genes involved in spermato/spermio-genesis (ADAM29, SPATA31E1, MAK, POLG, IFT43, ATG9B) and early embryonic development (MBD5, CCAR1, PMEPA1, POLK, REC8, REPIN1, MAPRE3, ARL4C). Transcriptomic assessment of cell-free RNAs in seminal fluid from NOA men allowed the prediction of residual spermatogenic foci. Conclusions: Sperm genome profiling provides invaluable information on azoospermia etiology and identifies gene-related mechanistic links to reproductive performance. Moreover, RNAseq assessment of seminal fluid from NOA men can help predict sperm retrieval during testicular biopsies. [ABSTRACT FROM AUTHOR]

The presence of a microbiome in the urinary system has been established through recent advancements in technology and investigation of microbial communities in the human body. The study of the taxonomic and genomic ecology of microbial communities has been greatly improved by the use of metagenomics. The research in this area has expanded our understanding of microbial ecosystems and shows that the urinary tract contains over 100 species from over 50 genera, with Lactobacillus, Gardnerella, and Streptococcus being the most common. Previous studies have suggested that the microbiota in the urinary tract may play a role in carcinogenesis by causing chronic inflammation and genotoxicity, but more research is needed to reach a definite conclusion. This is a narrative review. We conducted a search for relevant publications by using the databases Medline/PubMed and Google Scholar. The search was based on keywords such as "urinary microbiome," "bladder cancer," "carcinogenesis," "urothelial carcinoma," and "next-generation sequencing." The retrieved publications were then reviewed to study the contribution of the urinary microbiome in the development of bladder cancer. The results have been categorized into four sections to enhance understanding of the urinary microbiome and to highlight its role in the emergence of bladder cancer through alterations in the immune response that involve T-cells and antibodies. The immune system and microbiome play crucial roles in maintaining health and preventing disease. Manipulating the immune system is a key aspect of various cancer treatments, and certain gut bacteria have been linked to positive responses to immunotherapies. However, the impact of these treatments on the urinary microbiome, and how diet and lifestyle affect it, are not well understood. Research in this area could have significant implications for improving bladder cancer treatment and patient outcomes. [ABSTRACT FROM AUTHOR]

Kernels of cotton provide lint and linter for textiles, oil and protein for food and feed. Cotton seed is formed following fertilization between an ovule and a pollen grain. The seed coat is maternal in origin, whereas the embryo and attached cotyledonary leaves are hybrids of parental lines. The extraction of genomic DNA from an ungerminated whole, a portion or mixed seeds are prerequisite in genetic and genomic studies of cotton. As far as our knowledge, there is only one method of nondescriptive DNA extraction from ungerminated cotton seeds without affecting the seed germination capability, but it has technical difficulties and requires special equipment. Furthermore, the amount of DNA extracted using the published method is low and, therefore, it is only suitable for routine marker assisted selection studies. In this study, a DNA extraction protocol referred to as the CTAB-LiCl was developed for single whole cotton seed, a portion of cotton seed and bulked cotton seeds. This protocol uses a combination of CTAB and LiCl to lyse cells and deplete RNAs simultaneously. The CTAB-LiCl DNA extraction method was evaluated in ninety-six individuals of six different cotton cultivars along with two genetic standards of cotton, TM-1 (G. hirsutum L.), Pima 3–79 (G. barbadense L.), and several other plant species of different plant genera. Results revealed that this method produced high quality and amounts of DNA as confirmed by spectrophotometry, agarose gel, restriction enzyme digestion, polymerase chain reaction, and library production for next generation sequencing studies of whole genome bisulfite sequencing. It does not require the use of liquid nitrogen, RNase, proteinase K, or beta-mercaptoethanol and can be completed in approximately 2 h. Small tissues of the chalaza ends of ungerminated cotton seeds could be used to obtain high quality and quantity of DNA ranging from 14 to 28 µg without affecting the seeds' germination ability, allowing marker-assisted selection before planting and flowering. [ABSTRACT FROM AUTHOR]

In advanced pancreatic neuroendocrine neoplasms (PanNEN), there are little data detailing the frequency of genetic alterations identified in cell free DNA (cfDNA), plasma-tissue concordance of detected alterations, and clinical utility of cfDNA. Patients with metastatic PanNENs underwent cfDNA collection in routine practice. Next-generation sequencing (NGS) of cfDNA and matched tissue when available was performed. Clinical actionability of variants was annotated by OncoKB. Thirty-two cfDNA samples were analyzed from 25 patients, the majority who had well-differentiated intermediate grade disease (13/25; 52%). Genomic alterations were detected in 68% of patients and in 66% of all cfDNA samples. The most frequently altered genes were DAXX(28%), TSC2 (24%), MEN1 (24%), ARID1B (20%), ARID1A (12%), and ATRX(12%). Twenty-three out of 25 (92%) patients underwent tumor tissue NGS. Tissue-plasma concordance for select genes was as follows: DAXX(95.7%), ARID1A (91.1%), ATRX(87%), TSC2 (82.6%), MEN1 (69.6%). Potentially actionable alterations were identified in cfDNA of 8 patients, including TSC2 (4; level 3b), ATM (1; level 3b), ARID1A (2; level 4), and KRAS (1; level 4). An ETV6:NTRKfusion detected in tumor tissue was treated with larotrectinib; at progression, sequencing of cfDNA identified an NTRK3 G623R alteration as the acquired mechanism of resistance; the patient enrolled in a clinical trial of a second-generation TRK inhibitor with clinical benefit. In metastatic PanNENs, cfDNA-based NGS identified tumor-associated mutations in 66% of plasma samples with a high level of plasma-tissue agreement in PanNEN-associated genes. Clonal evolution, actionable alterations, and resistance mechanisms were detected through circulating cfDNA genotyping. [ABSTRACT FROM AUTHOR]

Lung diseases, including lung cancer, are rising causes of global mortality. Despite novel imaging technologies and the development of biomarker assays, the detection of lung cancer remains a significant challenge. However, the lung communicates directly with the external environment and releases aerosolized droplets during normal tidal respiration, which can be collected, stored and analzsed as exhaled breath condensate (EBC). A few studies have suggested that EBC contains extracellular vesicles (EVs) whose microRNA (miRNA) cargos may be useful for evaluating different lung conditions, but the cellular origin of these EVs remains unknown. In this study, we used nanoparticle tracking, transmission electron microscopy, Western blot analyses and super resolution nanoimaging (ONi) to detect and validate the identity of exhaled EVs (exh‐EVs). Using our customizable antibody‐purification assay, EV‐CATCHER, we initially determined that exh‐EVs can be selectively enriched from EBC using antibodies against three tetraspanins (CD9, CD63 and CD81). Using ONi we also revealed that some exh‐EVs harbour lung‐specific proteins expressed in bronchiolar Clara cells (Clara Cell Secretory Protein [CCSP]) and Alveolar Type II cells (Surfactant protein C [SFTPC]). When conducting miRNA next generation sequencing (NGS) of airway samples collected at five different anatomic levels (i.e., mouth rinse, mouth wash, bronchial brush, bronchoalveolar lavage [BAL] and EBC) from 18 subjects, we determined that miRNA profiles of exh‐EVs clustered closely to those of BAL EVs but not to those of other airway samples. When comparing the miRNA profiles of EVs purified from matched BAL and EBC samples with our three tetraspanins EV‐CATCHER assay, we captured significant miRNA expression differences associated with smoking, asthma and lung tumor status of our subjects, which were also reproducibly detected in EVs selectively purified with our anti‐CCSP/SFTPC EV‐CATCHER assay from the same samples, but that confirmed their lung tissue origin. Our findings underscore that enriching exh‐EV subpopulations from EBC allows non‐invasive sampling of EVs produced by lung tissues. [ABSTRACT FROM AUTHOR]

Background: Leptospirosis is a water-related zoonotic disease. The disease is primarily transmitted from animals to humans through pathogenic Leptospira bacteria in contaminated water and soil. Rivers have a critical role in Leptospira transmissions, while co-infection potentials with other waterborne bacteria might increase the severity and death risk of the disease. Methods: The water samples evaluated in this study were collected from four recreational forest rivers, Sungai Congkak, Sungai Lopo, Hulu Perdik, and Gunung Nuang. The samples were subjected to next-generation sequencing (NGS) for the 16S rRNA and in-depth metagenomic analysis of the bacterial communities. Results: The water samples recorded various bacterial diversity. The samples from the Hulu Perdik and Sungai Lopo downstream sampling sites had a more significant diversity, followed by Sungai Congkak. Conversely, the upstream samples from Gunung Nuang exhibited the lowest bacterial diversity. Proteobacteria, Firmicutes, and Acidobacteria were the dominant phyla detected in downstream areas. Potential pathogenic bacteria belonging to the genera Burkholderiales and Serratia were also identified, raising concerns about co-infection possibilities. Nevertheless, Leptospira pathogenic bacteria were absent from all sites, which is attributable to its limited persistence. The bacteria might also be washed to other locations, contributing to the reduced environmental bacterial load. Conclusion: The present study established the presence of pathogenic bacteria in the river ecosystems assessed. The findings offer valuable insights for designing strategies for preventing pathogenic bacteria environmental contamination and managing leptospirosis co-infections with other human diseases. Furthermore, closely monitoring water sample compositions with diverse approaches, including sentinel programs, wastewater-based epidemiology, and clinical surveillance, enables disease transmission and outbreak early detections. The data also provides valuable information for suitable treatments and long-term strategies for combating infectious diseases. [ABSTRACT FROM AUTHOR]

The 21st century, thanks to the development of molecular methods, including DNA barcoding, using Sanger sequencing, and DNA metabarcoding, based on next-generation sequencing (NGS), is characterized by flourishing research on the human microbiome. Microbial dysbiosis is perceived as a new pathogenetic factor for neonatal diseases. Fungi are crucial, but neglected, components of the neonatal microbiome, which, despite their low abundance, significantly impact morbidity and mortality rates of premature infants hospitalized in Neonatal Intensive Care Units (NICUs). The neonatal mycobiome's composition and effect on health remain poorly studied research areas. Our knowledge about neonatal mycobiome, composed of limited genera, is mainly based on research on the bacterial microbiome. We presume it is influenced by clinical factors, including prematurity, antibiotic therapy, and type of delivery. Understanding these risk factors may be useful in prevention strategies against dysbiosis and invasive fungal infections. Despite the methodological challenges resulting from the biology of the fungal cell, this topic is an attractive area of research that may contribute to more effective treatment, especially of newborns from risk groups. In this mini review, we discuss the current state of knowledge, research gaps, study difficulties, and future research directions on the neonatal mycobiome, concerning potential future clinical applications. [ABSTRACT FROM AUTHOR]

Arbuscular mycorrhizal fungi (AMF) live simultaneously inside and outside of host plant roots for a functional mycorrhizal symbiosis. Still, the year-round dynamics and relationships between soil properties and AMF communities of trees in forest ecosystems remain unclear. We collected paired root and soil samples of the same Cryptomeria japonica trees at two forest sites (five trees at each site) every 2 months over a year. Total DNA was extracted from roots and soil separately and soil physicochemical properties were measured. With Illumina's next-generation amplicon sequencing targeting the small subunit of fungal ribosomal DNA, we clarified seasonal dynamics of soil properties and AMF communities. Soil pH and total phosphorus showed significant seasonality while total carbon, nitrogen, and C/N did not. Only pH was a good predictor of the composition and dynamics of the AMF community. The total AMF community (roots + soil) showed significant seasonality because of variation from May to September. Root and soil AMF communities were steady year-round, however, with similar species richness but contained significantly different AMF assemblages in any sampling month. Despite the weak seasonality in the communities, the top two dominant OTUs showed significant but different shifts between roots and soils across seasons with strong antagonistic relationships. In conclusion, few dominant AMF taxa are dynamically shifting between the roots and soils of C. japonica to respond to seasonal and phenological variations in their microhabitats. AMF inhabiting forest ecosystems may have high environmental plasticity to sustain a functional symbiosis regardless of seasonal variations that occur in the soil. [ABSTRACT FROM AUTHOR]

In this study, Little Tern (Sternula albifrons) populations in Taiwan are examined based on two different types of data: mitochondrial control region DNA sequences and double digest restriction-site associated DNA (ddRAD) sequencing data. Feather samples were collected from 59 chicks across four known breeding colonies located on the eastern (Yilan and Hualien) and western (Penghu and Changhua) coasts of Taiwan. The results obtained are consistent in analyses and do not cluster into two geographical groups with respect to the eastern and western Taiwan. Furthermore, AMOVA analyses and pairwise ΦST/FST estimations based on both types of data reveal little to no differentiation among populations and between groups. The findings of this study suggest high population connectivity among Taiwan's breeding colonies. Additionally, control region sequences of Taiwan's Little Terns are compiled with those from Japan deposited in GenBank to compare genetic diversity and examine for phylogeographic breaks that could shape the diversity pattern of the species in eastern Asia. The resulting haplotype network does not clearly separate Taiwanese and Japanese populations, but the three most common haplotypes are prevalent for mainland Japan, Okinawa, and Taiwan. Little Tern populations may be frequently connected, but with some restrictions on gene flow causing moderate to great differentiation among the three. This is further supported by AMOVA analyses, pairwise ΦST estimations, and pattern of positive yet significant isolation by distance. [ABSTRACT FROM AUTHOR]

Purpose We examined the concordance of genetic mutations between pretreatment tumor tissue and posttreatment circulating tumor DNA (ctDNA) in patients with metastatic squamous cell carcinoma of the anal canal (SCCA) and assessed the impact of therapy on this concordance. Methods We analyzed next-generation sequencing reports from pretreatment tumor tissue and posttreatment ctDNA in 11 patients with metastatic SCCA treated at Vanderbilt University Medical Center between 2017 and 2021. Results Among the mutations identified in posttreatment ctDNA, 34.5% were also found in pretreatment tumor tissue, while 47.6% of pretreatment tumor tissue mutations were found in posttreatment ctDNA. Four patients had preservation of potentially actionable mutations in both pretreatment tissue and posttreatment ctDNA, while 7 patients had newly identified mutations in posttreatment ctDNA that were not present in pretreatment tumor tissue. Conclusion Patients with SCCA demonstrate a high degree of temporal mutational heterogeneity. This supports the hypothesis that ctDNA can serve as a real-time tracking mechanism for solid tumors' molecular evolution in response to therapy. Our findings highlight the potential of ctDNA in identifying emerging actionable mutations, supplementing information from tissue-based genomic assessments. Further research, ideally with larger and multi-institutional cohorts, is needed to validate our findings in this relatively rare tumor type. [ABSTRACT FROM AUTHOR]

Copyright of Advances in Biochemistry / Postepy Biochemii is the property of Polish Biochemical Society / Acta Biochimica Polonica and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Advances in Biochemistry / Postepy Biochemii is the property of Polish Biochemical Society / Acta Biochimica Polonica and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Anaesthesiologie & Intensivmedizin is the property of DGAI e.V. - Deutsche Gesellschaft fur Anasthesiologie und Intensivmedizin e.V. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

We evaluated the changes in the quality and microflora of yellowtail flesh cold-stored until spoilage. Based on the sensory evaluation, odor palatability was deemed unacceptable for dark muscle (DM) and the dorsal part of the ordinary muscle (OD) after >10 days and 14 of storage, respectively. Log 7 CFU/g in DM as well as OD was obtained on days 10 (Aeromonas spp.) and 14 (Enterobacteriaceae and Pseudomonas spp.) of storage, whereas log 5 (Brocothrix thermosphacta) and 6 (H2S-producing bacteria) CFU/g in them were obtained on day 14 of storage. In these bacteria, the viable bacterial counts of Pseudomonas spp. and Aeromonas spp. in DM were significantly higher than those in OD only at some storage times. Amplicon sequencing revealed that in both muscles, Pseudomonas became predominant after storage, with greater than 90% recorded after more than 10 days of storage. The relative abundances of Acinetobacter, Unclassified Gammaproteobacter, and Shewanella were relatively high in both muscles after more than 10 days of storage; however, these values were less than 5%. Ethyl butyrate in the OD and DM and 2,3-butanedione in the OD were first detected on days 14 and 10 of storage, respectively. Acetoin in the OD increased by 81-fold after 14 days of storage and was significantly increased in the DM after more than 10 days compared with the amount detected pre-storage. Volatiles, such as (E)-2-pentenal in the OD and 1-pentanol in the DM, decreased and increased linearly, respectively, throughout the 14-day storage period. Altogether, these volatile components may cause quality deterioration due to spoilage and/or lipid oxidation during cold storage of the OD and DM. [ABSTRACT FROM AUTHOR]

Hemoglobinopathies are the most common monogenic disorders in the world. Traditional diagnostic algorithms generated by conventional methods for thalassemia can be labor-intensive and time-consuming due to the complexities of the genes involved and the variability in disease-causing mutations. With the advantages of next-generation sequencing (NGS) technology, molecular analysis of highly complex diseases such as hemoglobinopathies has become easier. Next-generation sequencing is a highly sensitive and effective method due to its capacity to sequence many gene regions simultaneously while allowing good read depths. In this study, single nucleotide changes, small deletions and copy number variations in HBA1, HBA2 and HBB in 914 patients with suspected hemoglobinopathy were analysed with NGS. At least one HBA1, HBA2, HBB or HBD variant was detected in 483 (52.8%) patients. Ten novel variants were detected in HBA1 and HBA2, three in HBB, and one in HBD. From these variants, c.*76T > A, c.301–24 G > A, c.301-24G > C c.-41C > G, c.-37-40C > G, c.-9G > C, c. 95 + 9C > T, c.95 + 26C > A, c.95 + 38C > T and c.*18C > G variants were located in α-globin genes, c.-25T > C, c.*103T > C and c92 + 39A > G variants were located in β-globin genes, and c.-43C > A was located in HBD. This is the first comprehensive study using NGS for the molecular diagnosis of hemoglobinopathies in Turkey. Accurate molecular diagnosis is of critical importance in hemoglobinopathies which are a public health problem due to their increased prevalence, high burden to society, and lack of curative treatment. Currently, NGS appears to be an advanced option over conventional methods to detect all variants occurring by molecular mechanisms and simultaneously analyse many genomic sequences. [ABSTRACT FROM AUTHOR]

Background: Global developmental delay or intellectual disability usually accompanies various genetic disorders as a part of the syndrome, which may include seizures, autism spectrum disorder and multiple congenital abnormalities. Next-generation sequencing (NGS) techniques have improved the identification of pathogenic variants and genes related to developmental delay. This study aimed to evaluate the yield of whole exome sequencing (WES) and neurodevelopmental disorder gene panel sequencing in a pediatric cohort from Ukraine. Additionally, the study computationally predicted the effect of variants of uncertain significance (VUS) based on recently published genetic data from the country's healthy population. Methods: The study retrospectively analyzed WES or gene panel sequencing findings of 417 children with global developmental delay, intellectual disability, and/or other symptoms. Variants of uncertain significance were annotated using CADD-Phred and SIFT prediction scores, and their frequency in the healthy population of Ukraine was estimated. Results: A definitive molecular diagnosis was established in 66 (15.8%) of the individuals. WES diagnosed 22 out of 37 cases (59.4%), while the neurodevelopmental gene panel identified 44 definitive diagnoses among the 380 tested patients (12.1%). Non-diagnostic findings (VUS and carrier) were reported in 350 (83.2%) individuals. The most frequently diagnosed conditions were developmental and epileptic encephalopathies associated with severe epilepsy and GDD/ID (associated genes ARX, CDKL5, STXBP1, KCNQ2, SCN2A, KCNT1, KCNA2). Additionally, we annotated 221 VUS classified as potentially damaging, AD or X-linked, potentially increasing the diagnostic yield by 30%, but 18 of these variants were present in the healthy population of Ukraine. Conclusions: This is the first comprehensive study on genetic causes of GDD/ID conducted in Ukraine. This study provides the first comprehensive investigation of the genetic causes of GDD/ID in Ukraine. It presents a substantial dataset of diagnosed genetic conditions associated with GDD/ID. The results support the utilization of NGS gene panels and WES as first-line diagnostic tools for GDD/ID cases, particularly in resource-limited settings. A comprehensive approach to resolving VUS, including computational effect prediction, population frequency analysis, and phenotype assessment, can aid in further reclassification of deleterious VUS and guide further testing in families. [ABSTRACT FROM AUTHOR]

Objective: To improve the accuracy of preimplantation genetic testing (PGT) in deletional α-thalassemia patients. Design: Article. Patient(s): fifty-two deletional α-thalassemia couples. Intervention(s): Whole genome amplification (WGA), Next-generation sequencing (NGS) and PCR mutation loci detection. Main outcome measures: WGA, Single nucleotide polymorphism (SNP) and PCR mutation loci detection results; Analysis of embryo chromosome copy number variation (CNV). Results: Multiple Displacement Amplification (MDA) and Multiple Annealing and Looping-Based Amplification Cycles (MALBAC) methods for PGT for deletional α-thalassemia. Blastocyst biopsy samples (n = 253) were obtained from 52 deletional α-thalassemia couples. The results of the comparison of experimental data between groups MALBAC and MDA are as follows: (i) The average allele drop-out (ADO) rate, MALBAC vs. MDA = 2.27% ± 3.57% vs. 0.97% ± 1.4%, P=0.451); (ii) WGA success rate, MALBAC vs. MDA = 98.61% vs. 98.89%, P=0.851; (iii) SNP haplotype success rate, MALBAC vs. MDA = 94.44% vs. 96.68%, P=0.409; (iv) The result of SNP haplotype analysis is consistent with that of Gap-PCR/Sanger sequencing results, MALBAC vs. MDA = 36(36/72, 50%) vs. 151(151/181, 83.43%), P=0; (v) Valid SNP loci, MALBAC vs. MDA = 30 ± 9 vs. 34 ± 10, P=0.02; (vi) The mean CV values, MALBAC vs. MDA = 0.12 ± 0.263 vs. 0.09 ± 0.40, P=0.916; (vii) The average number of raw reads, MALBAC vs. MDA =3244259 ± 999124 vs. 3713146 ± 1028721, P=0; (viii) The coverage of genome (%), MALBAC vs. MDA = 5.02 ± 1.09 vs. 5.55 ± 1.49, P=0.008. Conclusions: Our findings indicate that MDA is superior to MALBAC for PGT of deletional α-thalassemia. Furthermore, SNP haplotype analysis combined with PCR loci detection can improve the accuracy and detection rate of deletional α-thalassemia. [ABSTRACT FROM AUTHOR]

Metagenomic sequencing has emerged as a transformative tool in infectious disease diagnosis, offering a comprehensive and unbiased approach to pathogen detection. Leveraging international standards and guidelines is essential for ensuring the quality and reliability of metagenomic sequencing in clinical practice. This review explores the implications of international standards and guidelines for the application of metagenomic sequencing in infectious disease diagnosis. By adhering to established standards, such as those outlined by regulatory bodies and expert consensus, healthcare providers can enhance the accuracy and clinical utility of metagenomic sequencing. The integration of international standards and guidelines into metagenomic sequencing workflows can streamline diagnostic processes, improve pathogen identification, and optimize patient care. Strategies in implementing these standards for infectious disease diagnosis using metagenomic sequencing are discussed, highlighting the importance of standardized approaches in advancing precision infectious disease diagnosis initiatives. [ABSTRACT FROM AUTHOR]

Introduction: Malignant ectomesenchymoma (MEM) is a soft tissue tumour, consisting of both malignant neuroectodermal elements and one or more mesenchymal elements. Case presentation and review of the literature: Here we describe the case of a 6-months-old male, previously treated in another hospital for abdominal rhabdomyosarcoma (RMS). Histological re-examination demonstrated that the tumour had mesenchymal and neuroectodermal elements components, with a new diagnosis of abdominal-pelvic MEM. A Next-Generation Sequencing (NGS) analysis was performed on a surgical tumour specimen and revealed the presence of a somatic mutation, already reported in MEM cases. We carried out a review of the literature and we found 33 new cases of MEM since the last review. We reported the clinic-pathologic features of new cases of MEM, highlighting the role of molecular studies in supporting the diagnosis of this ambiguous tumours. Conclusion: We promote the importance of a diagnosis based on an integrative morpho-molecular approach, that routinely include molecular analysis and the use of bioinformatic mutation detection tools, to support diagnostic and therapeutical queries and to highlight tumour biology and behaviour. [ABSTRACT FROM AUTHOR]

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for the COVID-19 pandemic, has been evolving rapidly causing emergence of new variants and health uncertainties. Monitoring the evolution of the virus was of the utmost importance for public health interventions and the development of national and global mitigation strategies. Here, we report national data on the emergence of new variants, their distribution, and dynamics in a 3-year study conducted from March 2020 to the end of January 2023 in the Republic of Serbia. Nasopharyngeal and oropharyngeal swabs from 2,398 COVID-19- positive patients were collected and sequenced using three different next generation technologies: Oxford Nanopore, Ion Torrent, and DNBSeq. In the subset of 2,107 SARS-CoV-2 sequences which met the quality requirements, detection of mutations, assignment to SARS-CoV-2 lineages, and phylogenetic analysis were performed. During the 3-year period, we detected three variants of concern, namely, Alpha (5.6%), Delta (7.4%), and Omicron (70.3%) and one variant of interest—Omicron recombinant “Kraken” (XBB1.5) (<1%), whereas 16.8% of the samples belonged to other SARS-CoV-2 (sub)lineages. The detected SARS-CoV-2 (sub)lineages resulted in eight COVID-19 pandemic waves in Serbia, which correspond to the pandemic waves reported in Europe and the United States. Wave dynamics in Serbia showed the most resemblance with the profile of pandemic waves in southern Europe, consistent with the southeastern European location of Serbia. The samples were assigned to sixteen SARS-CoV-2 Nextstrain clades: 20A, 20B, 20C, 20D, 20E, 20G, 20I, 21J, 21K, 21L, 22A, 22B, 22C, 22D, 22E, and 22F and six different Omicron recombinants (XZ, XAZ, XAS, XBB, XBF, and XBK). The 10 most common mutations detected in the coding and untranslated regions of the SARS-CoV-2 genomes included four mutations affecting the spike protein (S:D614G, S:T478K, S:P681H, and S:S477N) and one mutation at each of the following positions: 5′-untranslated region (5’UTR:241); N protein (N:RG203KR); NSP3 protein (NSP3:F106F); NSP4 protein (NSP4:T492I); NSP6 protein (NSP6: S106/G107/F108 - triple deletion), and NSP12b protein (NSP12b:P314L). This national-level study is the most comprehensive in terms of sequencing and genomic surveillance of SARS-CoV-2 during the pandemic in Serbia, highlighting the importance of establishing and maintaining good national practice for monitoring SARS-CoV-2 and other viruses circulating worldwide. [ABSTRACT FROM AUTHOR]

Simple Summary: In everchanging landscapes with intense land use for agriculture, forestry and settlements, songbirds need to adapt their feeding and breeding behaviour according to changing habitats and food supplies. Blue and Great Tits occur in various habitats from forest to park-like sites and strongly rely on caterpillars to feed their offspring. Using genetic analyses of Blue and Great Tit droppings, we compared the diet composition of nestlings among forest, urban parks, and meadows with scattered fruit trees (traditional orchards). Additionally, we compared the food availability in the trees of each habitat to the respective nestling diets. Both bird species showed a diverse diet dominated by butterflies in all habitats. Moth components in the diet were most similar between forest and orchard sites, while components of other insect-like prey were most similar between orchard and urban sites. Both bird species especially selected three major moth families at all sites and alongside two families of sawflies in the orchard and forest sites. Our results on nestling diet and prey preferences provide further insights in the feeding behaviours of Blue and Great Tits among changing anthropogenic landscapes. Moreover, our genetic approach using bird droppings provides a baseline to support insect monitoring. Increasing landscape transformations and urbanisation affect insectivorous bird populations in various ways such as food availability, breeding phenology, or reproductive success. Especially during the breeding season, many passerine birds rely on the availability of caterpillars as the main prey for their nestlings. Previous studies suggested that similar diet preferences of sympatric species may result in interspecific competition, as demonstrated for Blue and Great Tits in forest habitats. However, nestling diet and prey preferences in other habitats are not fully understood. Prey availability, especially caterpillars, is lower in cities than in forests, thus influencing prey choice and interspecific competition. Here we used faecal DNA metabarcoding to investigate if nestling diet composition of the two sympatric species Blue Cyanistes caeruleus and Great Tits Parus major varied among species and different habitats (forest, traditional orchards, and urban parks). Furthermore, we examined food availability by DNA barcoding of the arboreal arthropod communities among habitats and compared them to the nestling diet to infer parental prey selectivity. The study was carried out in central Germany from 2018 to 2019. Blue and Great Tits showed a diverse diet which was dominated by Lepidoptera in all habitats. Lepidopteran diet components were most similar between forest and orchard sites, as were the components with other arthropods between orchard and urban sites. Both tit species showed selectivity for the lepidopteran families Geometridae and Tortricidae in all habitats, and for Noctuidae (Lepidoptera), Tenthredinidae and Braconidae (Hymenoptera) in forest and orchard sites. As the tits showed preferences for mainly families of Lepidoptera, and Hymenoptera, our approach provides a baseline to support monitoring of these groups. [ABSTRACT FROM AUTHOR]

Shotgun sequencing is a high-throughput method used to detect copy number variants (CNVs). Although there are numerous CNV detection tools based on shotgun sequencing, their quality varies significantly, leading to performance discrepancies. Therefore, we conducted a comprehensive analysis of next-generation sequencing-based CNV detection tools over the past decade. Our findings revealed that the majority of mainstream tools employ similar detection rationale: calculates the so-called read depth signal from aligned sequencing reads and then segments the signal by utilizing either circular binary segmentation (CBS) or hidden Markov model (HMM). Hence, we compared the performance of those two core segmentation algorithms in CNV detection, considering varying sequencing depths, segment lengths and complex types of CNVs. To ensure a fair comparison, we designed a parametrical model using mainstream statistical distributions, which allows for pre-excluding bias correction such as guanine-cytosine (GC) content during the preprocessing step. The results indicate the following key points: (1) Under ideal conditions, CBS demonstrates high precision, while HMM exhibits a high recall rate. (2) For practical conditions, HMM is advantageous at lower sequencing depths, while CBS is more competitive in detecting small variant segments compared to HMM. (3) In case involving complex CNVs resembling real sequencing, HMM demonstrates more robustness compared with CBS. (4) When facing large-scale sequencing data, HMM costs less time compared with the CBS, while their memory usage is approximately equal. This can provide an important guidance and reference for researchers to develop new tools for CNV detection. [ABSTRACT FROM AUTHOR]

Structural variants drive tumorigenesis by disrupting normal gene function through insertions, inversions, translocations, and copy number changes, including deletions and duplications. Detecting structural variants is crucial for revealing their roles in tumor development, clinical outcomes, and personalized therapy. Presently, most studies rely on short-read data from next-generation sequencing that aligns back to a reference genome to determine if and, if so, where a structural variant occurs. However, structural variant discovery by short-read sequencing is challenging, primarily because of the difficulty in mapping regions of repetitive sequences. Optical genome mapping (OGM) is a recent technology used for imaging and assembling long DNA strands to detect structural variations. To capture the structural variant landscape more thoroughly in the human genome, we developed an integrated pipeline that combines Bionano OGM and Illumina whole-genome sequencing and applied it to samples from 29 pediatric B-ALL patients. The addition of OGM allowed us to identify 511 deletions, 506 insertions, 93 duplications/gains, and 145 translocations that were otherwise missed in the short-read data. Moreover, we identified several novel gene fusions, the expression of which was confirmed by RNA sequencing. Our results highlight the benefit of integrating OGM and short-read detection methods to obtain a comprehensive analysis of genetic variation that can aid in clinical diagnosis, provide new therapeutic targets, and improve personalized medicine in cancers driven by structural variation. [ABSTRACT FROM AUTHOR]

Genomic medicine is a powerful tool to improve diagnosis and outcomes for cancer patients by facilitating the delivery of the right drug at the right dose at the right time for the right patient. In 2023, a Canadian conference brought together leaders with expertise in different tumor types. The objective was to identify challenges and opportunities for change in terms of equitable and timely access to biomarker testing and reporting at the education, delivery, laboratory, patient, and health-system levels in Canada. Challenges identified included: limited patient and clinician awareness of genomic medicine options with need for formal education strategies; failure by clinicians to discuss genomic medicine with patients; delays in or no access to hereditary testing; lack of timely reporting of results; intra- and inter-provincial disparities in access; lack of funding for patients to access testing and for laboratories to provide testing; lack of standardized testing; and impact of social determinants of health. Canada must standardize its approach to biomarker testing across the country, with a view to addressing current inequities, and prioritize access to advanced molecular testing to ensure systems are in place to quickly bring innovation and evidence-based treatments to Canadian cancer patients, regardless of their place of residence or socioeconomic status. [ABSTRACT FROM AUTHOR]

MicroRNA is attracting attention as a therapeutic target for osteoarthritis. We focused on joint capsules and synovium in lumbar facet joint osteoarthritis. The purpose of this study was to identify microRNAs that are upregulated in lumbar facet joint capsules and synovium with osteoarthritis. We included patients who underwent spinal fusion for degenerative lumbar spine diseases. We selected patients who had both early-stage and late-stage facet joint osteoarthritis in a single individual. We extracted joint capsule and synovium samples from these patients and isolated microRNAs. During the screening phase, we compared early-stage and late-stage osteoarthritis samples from the same individual. We identified microRNAs with >2-fold change in expression in 75% or more of patients with late-stage osteoarthritis using next generation sequencing. During the technical validation phase, the same samples were used for real-time polymerase chain reaction. We identified microRNAs with >2-fold change in expression in 62.5% or more of patients with late-stage osteoarthritis. Of 40 patients who underwent spinal fusion, we selected eight patients with both early-stage and late-stage facet joint osteoarthritis. During the screening phase, we identified eight upregulated microRNAs out of 2274 microRNAs in late-stage OA. In late-stage OA, two microRNAs (miR-133a-5p and miR-144-3p) were upregulated in seven patients and six microRNAs (miR-133a-3p, miR-133b, miR-206, miR-20a-5p, miR-301a-3p, and miR-32-5p) were upregulated in six patients. During the technical validation phase, we found significant upregulation of miR-144-3p expression in late-stage osteoarthritis compared with early-stage osteoarthritis. Expression of the other microRNAs was not significantly different according to the paired-t test. However, miR-133a-3p, miR-133b, and miR-206 were upregulated >2-fold in 62.5% or more of patients with late-stage osteoarthritis. Some of the microRNAs identified in this study might be involved in joint capsule degeneration or synovitis. [ABSTRACT FROM AUTHOR]

Seminal stains acquired from fabric surfaces stand as pivotal biological evidence of utmost significance for elucidating sexual assault cases. The ability to determine the temporal aspect of a forensic incident via the analysis of a biological specimen found at the crime scene is crucial in resolving most cases. This study aimed to investigate the time-dependent change in the microbiota structure of human seminal stains exposed to indoor environmental conditions. Stains on polyester fabric generated using semen samples from five male volunteers were kept indoors for varying durations of up to 20 days, followed by sequencing of the V1–V9 regions of the 16S rRNA gene of the microbial DNA extracted from the stains. The acquired data provided the taxonomic composition, and microbial alterations across different days were examined. The most abundantly detected phyla in all samples were Firmicutes, Proteobacteria, and Bacteroidetes, and the relative abundances of bacteria were observed to change over time. Statistically significant changes at the species level were found for Treponema medium, Corynebacterium tuberculostearicum, Faecalibacterium prausnitzii, and Anaerostipes hadrus. Alterations observed in the samples between the analyzed time periods were investigated. The changes during the specified time periods were examined, identifying rare bacterial species that were initially present on certain days but later ceased to exist in the environment. Conversely, bacterial species that were absent before exposure but emerged at a later stage were also identified. The findings of this study demonstrate that species-level evaluations, in particular, can provide crucial insights into semen stain age. [ABSTRACT FROM AUTHOR]

B-cell neoplasms possess clonal B-cell receptor rearrangements (BCR clonotype lineages) that can be identified by sequencing the B-cell repertoire for use in diagnostics, risk stratification, and high-sensitivity monitoring. BCR somatic hypermutation (SHM) can result in clonality detection failure from point mutations in PCR primer binding regions, often necessitating splitting samples into multiple reactions which increases test costs, turnaround times, and sample requirements. We evaluated the Oncomine BCR Pan-Clonality Assay, a novel single-tube PCR reaction that simultaneously amplifies all BCR loci for next-generation DNA sequencing, using neoplastic B-cell lines and clinical research samples from multiple myeloma (MM) patients, a plasma cell neoplasm associated with high SHM levels. The assay showed a linear detection range down to 1 ng of clonal DNA input, sensitivity to 10−6 in a polyclonal background, and high reproducibility. Clonotype lineages were identified in 42/45 (93%) MM samples. Ion Reporter software packaged with the assay permitted straightforward identification of MM subgroups. As expected, SHM was identified in 94% of MM cases, but several unexpected subgroups were identified including biased IGHV3-11 or IGHV4-34 usage in 20% of MM samples, and two cases with very low levels of SHM. Evidence of intraclonal diversity/ongoing SHM was identified in 18% of samples, suggesting a possible germinal center origin for some MM cases. The single-tube Oncomine BCR Pan-Clonality assay efficiently detects BCR clonotype lineages at rates comparable to existing multiple reaction assays and permits their characterization for cell of origin studies and lymphoma classification. [ABSTRACT FROM AUTHOR]

Background: Hybridization capture-based targeted next generation sequencing (NGS) is gaining importance in routine cancer clinical practice. DNA library preparation is a fundamental step to produce high-quality sequencing data. Numerous unexpected, low variant allele frequency calls were observed in libraries using sonication fragmentation and enzymatic fragmentation. In this study, we investigated the characteristics of the artifact reads induced by sonication and enzymatic fragmentation. We also developed a bioinformatic algorithm to filter these sequencing errors. Results: We used pairwise comparisons of somatic single nucleotide variants (SNVs) and insertions and deletions (indels) of the same tumor DNA samples prepared using both ultrasonic and enzymatic fragmentation protocols. Our analysis revealed that the number of artifact variants was significantly greater in the samples generated using enzymatic fragmentation than using sonication. Most of the artifacts derived from the sonication-treated libraries were chimeric artifact reads containing both cis- and trans-inverted repeat sequences of the genomic DNA. In contrast, chimeric artifact reads of endonuclease-treated libraries contained palindromic sequences with mismatched bases. Based on these distinctive features, we proposed a mechanistic hypothesis model, PDSM (pairing of partial single strands derived from a similar molecule), by which these sequencing errors derive from ultrasonication and enzymatic fragmentation library preparation. We developed a bioinformatic algorithm to generate a custom mutation "blacklist" in the BED region to reduce errors in downstream analyses. Conclusions: We first proposed a mechanistic hypothesis model (PDSM) of sequencing errors caused by specific structures of inverted repeat sequences and palindromic sequences in the natural genome. This new hypothesis predicts the existence of chimeric reads that could not be explained by previous models, and provides a new direction for further improving NGS analysis accuracy. A bioinformatic algorithm, ArtifactsFinder, was developed and used to reduce the sequencing errors in libraries produced using sonication and enzymatic fragmentation. [ABSTRACT FROM AUTHOR]

Human beings possess trillions of microbial cells in a symbiotic relationship. This relationship benefits both partners for a long time. The gut microbiota helps in many bodily functions from harvesting energy from digested food to strengthening biochemical barriers of the gut and intestine. But the changes in microbiota composition and bacteria that can enter the gastrointestinal tract can cause infection. Several approaches like culture‐independent techniques such as high‐throughput and meta‐omics projects targeting 16S ribosomal RNA (rRNA) sequencing are popular methods to investigate the composition of the human gastrointestinal tract microbiota and taxonomically characterizing microbial communities. The microbiota conformation and diversity should be provided by whole‐genome shotgun metagenomic sequencing of site‐specific community DNA associating genome mapping, gene inventory, and metabolic remodelling and reformation, to ease the functional study of human microbiota. Preliminary examination of the therapeutic potency for dysbiosis‐associated diseases permits investigation of pharmacokinetic‐pharmacodynamic changes in microbial communities for escalation of treatment and dosage plan. Gut microbiome study is an integration of metagenomics which has influenced the field in the last two decades. And the incorporation of artificial intelligence and deep learning through "omics‐based" methods and microfluidic evaluation enhanced the capability of identification of thousands of microbes. [ABSTRACT FROM AUTHOR]

Classic Hodgkin lymphoma (cHL) consists of a heterogeneous group of haematological disorders that covers undifferentiated B cell neoplasms originating from germinal centre B cells. The HL molecular characterization still represents an ongoing challenge due to the low fraction of tumour Hodgkin and Reed–Sternberg cells mixed with a plethora of non‐tumour haematological cells. In this scenario, next generation sequencing of liquid biopsy samples is emerging as a useful tool in HL patients' management. In this review, we aimed to overview the clinical and methodological topics regarding the implementation of molecular analysis in cHL, focusing on the role of liquid biopsy in diagnosis, follow‐up, and response prediction. [ABSTRACT FROM AUTHOR]

Objective： To investigate the clinical characteristics of patients with chronic lymphocytic leukemia (CLL)carrying exportin 1 (XPO1)mutations, providing clues for clinical diagnosis and treatment. Methods： The clinical data of CLL patients with XPO1 mutations detected in the Department of Hematology of the First Affiliated Hospital of Nanjing Medical University from November 2006 and March 2022 were retrospectively analyzed. The clinical data, treatment responses, and survival outcomes of the treatment native (TN)and relapsed/refractory (R/R)patients with XPO1 mutation were compared. Results： Among 543 CLL patients, 15 patients (2.8%)tested positive with XPO1 mutations. The mutation rates in the TN group (368 cases)and R/R group (175 cases) were 2.4% (9 cases)and 3.4% (6 cases), respectively, with a hotspot mutation (E571K)identified. Most of the patients were in Rai Ⅲ/ Ⅳ stage and Binet B/C group, and had no mutations in the immunoglobulin heavy⁃chain variable region (IGHV). XPO1 gene mutation co⁃occurred with NOTCH1, SF3B1, KMT2D, TP53, and other gene mutations, with TP53 and XPO1 mutations more common in the R/ R group (TN：11.1%; R/R：50%). The median time to first treatment (TTFT)for patients with XPO1 mutations was 1.8 months, the median progression⁃free survival (PFS)was 19.8 months, and the median overall survival (OS)was 40.0 months. In the XPO1 non ⁃ mutated patients, TTFT was 8.1 months, PFS was 32.5 months, and OS was 49.8 months. Conclusion：XPO1 mutations in CLL are low⁃frequency mutations often occurring simultaneously with other gene mutations. R/R patients are more likely to carry XPO1 mutations than TN patients, with a higher tumor burden. XPO1 mutated patients tend to have shorter TTFT and PFS, compared with those without XPO1 mutations. [ABSTRACT FROM AUTHOR]

Background: While many molecular assays can detect mutations at low tumor purity and variant allele frequencies, complex biomarkers such as tumor mutational burden (TMB), microsatellite instability (MSI), and genomic loss of heterozygosity (gLOH) require higher tumor purity for accurate measurement. Scalable, quality-controlled, tissue-conserving methods to increase tumor nuclei percentage (TN%) from tumor specimens are needed for complex biomarkers and hence necessary to maximize patient matching to approved therapies or clinical trial enrollment. We evaluated the clinical utility and performance of precision needle-punch enrichment (NPE) compared with traditional razor blade macroenrichment of tumor specimens on molecular testing success. Methods: Pathologist-directed NPE was performed manually on formalin-fixed, paraffin embedded (FFPE) blocks. Quality control of target capture region and quantity of residual tumor in each tissue block was determined via a postenrichment histologic slide recut. Resultant tumor purity and biomarker status were determined by the computational analysis pipeline component of the FDAapproved next-generation sequencing (NGS) assay, FoundationOne®CDx. Following NPE implementation for real-world clinical samples, assay performance and biomarker (MSI, TMB, gLOH) detection were analyzed. Results: In real-world clinical samples, enrichment rate via NPE was increased to ~50% over a 2.5-year period, exceeding the prior use of razor blade macroenrichment (<30% of cases) prior to NPE implementation due to proven efficacy in generating high quality molecular results from marginal samples and the ease of use for both pathologist and histotechnologists. NPE was associated with lower test failures, higher computational tumor purity, and higher rates of successful TMB, MSI and gLOH determination when stratified by pre-enriched (incipient) tumor nuclei percentage. In addition, challenging cases in which tumor content was initially insufficient for testing were salvaged for analysis of biomarker status, gene amplification/deletion, and confident mutant or wild-type gene status determination. Conclusions: Pathologist-directed precision enrichment from tissue blocks (aka NPE) increases tumor purity, and consequently, yields a greater number of successful tests and complex biomarker determinations. Moreover, this process is rapid, safe, inexpensive, scalable, and conserves patient surgical pathology material. NPE may constitute best practice with respect to enriching tumor cells from low-purity specimens for biomarker detection in molecular laboratories. [ABSTRACT FROM AUTHOR]

Background: Intra-abdominal desmoplastic small round cell tumor (IDSRCT) is a rare entity (0.2-0.74 cases per million people per year), which predominantly occurs in young men. It may present as an abdominal mass with pain, distention, and constipation. IDSRCT has a very poor prognosis, with 5-year overall survival estimated at 15%-30%. Diagnosis is made with tissue biopsy. Case description: We present a case of a 28-year-old man with a history of schizophrenia and depression who presented to an emergency room (ER) in November 2022 with constipation and pelvic pain. The patient was sent home with a bowel regimen after radiography showed no obstruction. He re-presented for evaluation due to persistent pain. A computerized tomography scan of the abdomen and pelvis (CT A/P) revealed numerous pelvic masses with severe colitis, bilateral moderate hydronephrosis, and metastatic disease in the liver. A colonoscopy showed a mass extending 3 cm from the anus to 10 cm causing a partial obstruction. Biopsy was read as squamous cell carcinoma (SCC). The patient was subsequently admitted to our institution with pelvic pain, nausea, and vomiting. Colorectal surgery performed a colectomy with end-ileostomy due to colonic obstruction. He was evaluated by a medical oncologist, with previous slides requested for review. Initial review was concerning metastatic basaloid SCC with neuroendocrine features and a Ki67 of 70%. Given his recent abdominal surgeries, chemotherapy was delayed until February 2023 when he was started on reduced dose carboplatin and paclitaxel. Tumor specimen was sent for next generation sequencing (NGS) and programmed death-1 ligand 1 (PD-L1) testing. NGS results returned after the first dose of chemotherapy was given and showed a t(11;22) EWSR-WT1 translocation characteristic of desmoplastic small round cell tumor. The patient was supported in the hospital and discharged with oncology follow-up. Discussion: As seen in this case, pathology review is essential to ensuring correct diagnosis and appropriate treatment plan. This is especially true when the clinical scenario does not match the listed pathology. Additional diagnostics such as NGS are invaluable in establishing correct diagnosis. [ABSTRACT FROM AUTHOR]

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Circular economy recycling-derived fertilizers (RDF) have the potential to replace linear economy fertilizers such as unsustainable superphosphates. Here, effects of ash RDF treatments in Irish grassland cultivation were investigated in a simulated second growing season. Soil fertilized in a preceding pot trial with superphosphate (SP), poultry-litter ash (PLA) and sewage-sludge ash (SSA) at P concentration of 60 kg P ha−1 and a P-free control (SP0) was reused in a microcosm trial. Lolium perenne was cultivated for 54 days in six replicates with a full complement of micro- and macro-nutrients other than P. PLA treatments provided higher dry weight shoot yields than SP0, while SSA and SP overlapped with SP0 and PLA. Most probable number (MPN) analysis showed that phosphonate- and phytate-utilizing bacterial abundance was significantly increased in PLA. Alkaline (phoD) phosphomonoesterase gene fragments were significantly more abundant (qPCR) in the ashes than the superphosphate or P-free control. Bacterial communities were significantly affected by the P application. Similarly, a significant separation of treatments was confirmed in a canonical correspondence analysis of the phoD-harboring community. The genera Streptomyces and Xanthomonas were significantly higher in abundance in the ash RDFs. These results demonstrated the potential benefits of ash RDF treatments as an alternative P source. [ABSTRACT FROM AUTHOR]

Human astroviruses (HAstVs) are a significant etiological agent of acute gastroenteritis in children. In order to investigate the circulation of HAstVs during the COVID-19 pandemic, a 2-year environmental surveillance was conducted in Jinan between 2020 and 2021. A total of 24 sewage samples were collected and concentrated. Real-time PCR indicated a positive rate of 83.3%, 79.2% (19/24), and 62.5% for classic, MLB, and VA types of HAstV in sewage samples, respectively, with genomic copies ranging from 6.4 × 103 to 3.7 × 107, 3.2 × 104 to 2.2 × 106, and 1.2 × 104 to 1.6 × 107 l−1. Next-generation sequencing (NGS) analysis on complete ORF2 amplicons from each sewage concentrate revealed the presence of 11 HAstV types, including HAstV-1, -2, -4, -5, MLB1, and VA1 to VA6, as well as non-human animal astroviruses. The most abundant HAstV types were HAstV-1, -4, and -5, which accounted for 70.3%, 12.6%, and 9.1% of total HAstV reads, respectively. Phylogenetic analysis revealed that the sequences obtained in this study were segregated into multiple transmission lineages, yet exhibited less genetic divergence among themselves than with foreign strains. These findings provide insight into the genotype diversity and genetic characterization of HAstVs during the COVID-19 pandemic, and highlight the effectiveness of utilizing NGS approaches to investigate sewage HAstVs. [ABSTRACT FROM AUTHOR]

Objectives: To test whether Kangaroo mother care (KMC) aids in transfer of favourable skin microbiome from mother to infant by comparing the microbiome composition before and after KMC. Methods: A prospective cohort pilot study was conducted in a Level III neonatal intensive care unit (NICU) in South India, recruiting 30 preterm infants with gestation <32 wk from October 2020 through December 2020. Neonatal skin involving the area in contact with the mother during KMC i.e., axilla, chest and abdomen was swabbed at the end of first week of life, prior to initiation of KMC. The 2nd swab involving the same areas was taken following KMC for 7 d for at least 6 h a day. The swabs were analysed using Next Generation Sequencing (NGS) - 16sRNA and abundance of organisms isolated were mapped. Statistical analyses using t-test and PERMANOVA were performed to compare phyla and genera of bacterial abundance pre-KMC and post-KMC. Results: KMC at phyla level increased the relative abundance of Firmicutes (p=0.52) and significantly decreased Proteobacteria (p=0.02). At species level, KMC decreased pathogenic bacterial count of Escherichia (p=0.05), while counts of S. hemolyticus (p=0.01) and S. hominis (p=.002) significantly increased post KMC. Conclusions: KMC has a potential role in altering the neonatal skin microbiota towards a more favourable microenvironment. The clinical significance of these novel findings needs to be validated with larger studies. [ABSTRACT FROM AUTHOR]

In this study, total RNA high-throughput sequencing (HTS) of a single symptomatic Phytolacca americana plant enabled the obtention of a nearly complete genome of two new isolates of turnip yellows virus (TuYV), named TuYV-ITA1 and TuYV-ITA2, and revealed a mixed infection with a new variant of citrus exocortis viroid (CEVd), named CEVd-ITA1. The TuYV-ITA2 isolate diverged from the known virus isolates of TuYV and showed variability in the P0 and P5 readthrough domain. Recombination analysis revealed its recombinant nature between TuYV and an unidentified polerovirus. The putative recombination event was identified in the P5 readthrough domain of the TuYMV-ITA2 isolate. Our results thus represent the first report of TuYV in Italy and some molecular evidence for the possible natural co-existence of TuYV and CEVd in a new natural host for both infectious entities. This study is adding further knowledge about the role of weed plants as virus reservoirs, and thus additional biological and impact studies would be desirable to determine in particular the role of P. americana in the spread of TuYV and if this virus should be considered a new threat for the susceptible Italian crops.

IntroductionType 1 diabetes is an autoimmune disease with an significant genetic component, played mainly by the HLA class II genes. Although evidence on the role of HLA class I genes in developing type 1 diabetes and its onset have emerged, current HLA screening is limited to determining DR3 and DR4 haplotypes. This study aimed to investigate the role of HLA genes on type 1 diabetes risk and age of onset by extensive typing.MethodsThis study included 115 children and young adults with type 1 diabetes for whom typing of HLA-A, -B, -C, -DRB1, -DRB3/4/5, -DQA1, -DQB1, -DPA1 and -DPB1 genes was conducted using Next Generation Sequencing.ResultsWe observed that 13% of type 1 diabetes subjects had non-classical HLA haplotypes that predispose to diabetes. We also found that compared to type 1 diabetes subjects with classical HLA haplotypes, non-classical HLA subjects had a significantly higher frequency of HLA-B*39:06:02 (p-value=0.01) and HLA-C*07:02:01 (p-value=0.03) alleles, known to be involved in activating the immune response. Non-classical HLA subjects also presented peculiar clinical features compared to classical HLA subjects, such as multiple diabetic antibodies and the absence of other autoimmune diseases (i.e., coeliac disease and thyroiditis). We also observed that subjects with early onset had a higher frequency of DQ2/DQ8 genotype than late-onset individuals. Moreover, subjects with late-onset had a higher frequency of alleles HLA-B*27 (p-value=0.003), HLA-C*01:02:01 (p-value=0.027) and C*02:02:02 (p-value=0.01), known to be associated with increased protection against viral infections.DiscussionThis study reveals a broader involvement of the HLA locus in the development and onset of type 1 diabetes, providing insights into new possible disease prevention and management strategies.

Lawsonella clevelandensis is rare to associated with human infection, which may cause abscesses in abdominal cavity, liver, breast, and spine. Lawsonella clevelandensis is very difficult to be cultivated in regular manner; detection of 16S rRNA sequence is the main evidence for L. clevelandensis infection. The clinical manifestations of L. clevelandensis infection resemble other agents of Nocardia, Tuberculosis and non-tuberculous Mycobacterium (NTM) due to their morphologic similarities. Hidradenitis suppurativa (HS) is a chronic inflammatory disorder, which affects the intertriginous skin and is associated with numerous systemic comorbidities. HS eventually leads to severe pain, multiple abscesses, pus discharge, and irreversible tissue destruction. Lawsonella clevelandensis has not been reported to cause HS and systemic comorbidities. We presented the case of a 33-year-old male with widespread soft tissue infection and multiple abscesses secondary to HS (Hurley stage III) inducing septic shock caused by L. clevelandensis in China. He was diagnosed as HS and treated with intravenous antibiotic empirically. He developed multiple abscesses including lung and scrota. Bacterial and fungal cultures on blood and secretions from multiple skin lesions were all negative. Due to the misdiagnosis and progression of disease, the patient was transferred to intensive care unit, and he underwent drainage of the chest and right hemothorax removal under thoracoscopic. During the hospitalization, the patient developed septic shock and received mechanical ventilation. Computerized tomography (CT) scans revealed mediastinal emphysema, multiple subcutaneous emphysema, and severe pneumonia. Gene analysis of samples of incision and drainage of pus at the skin showed the rare infection of L. clevelandensis. Finally, the patients with recurrent soft tissue infections and multiple abscesses with negative microbiological culture results recovered after effective abscess drainage and antibiotic therapy. We suggested that NGS is a crucial supplementary diagnostic tool in individuals with recurrent skin infections and multiple abscesses, especially when conventional diagnostic methods are inconclusive.

R-loop, a chromatin structure containing one RNA:DNA hybrid and one unpaired single-stranded DNA, plays multiple biological roles. However, due to technical limitations, the landscapes and potential functions of R-loops during embryogenesis remain elusive. Here, we developed a quantitative and high-resolution ultra-low input R-loop profiling method, named ULI-ssDRIP-seq, which can map global R-loops with as few as 1000 cells. By using ULI-ssDRIP-seq, we reveal the R-loop dynamics in the zebrafish from gametes to early embryos. In oocytes, the R-loop level is relatively low in most regions of the nuclear genome, except maternal-inherited rDNA and mitochondrial genome. The correlation between R-loop and CG methylation dynamics during early development is relatively weak. Furthermore, either up- or down-regulation of global R-loops by knockdown or overexpression of RNase H1 causes a delay of embryonic development with dramatic expression changes in zygotic and maternal genes. This study provides comprehensive R-loop landscapes during early vertebrate embryogenesis and demonstrates the implication of R-loops in embryonic development.

BackgroundA novel approach for molecular residual disease (MRD) detection and treatment monitoring is needed in diffuse large B-cell lymphoma (DLBCL) to identify patients with a poor prognosis. We performed a retrospective evaluation of commercial ctDNA testing in patients with stage I-IV DLBCL to evaluate the prognostic and predictive role of tumor-informed ctDNA assessment.MethodsA personalized and tumor-informed multiplex PCR assay (Signatera™ bespoke mPCR NGS assay) was used for ctDNA detection and quantification.ResultsIn total, 50 patients (median age: 59 years; median follow-up: 12.68 months) were analyzed, of which 41 had pretreatment time points with ctDNA detected in 95% (39/41). Baseline ctDNA levels correlated with R-IPI scores and stage. ctDNA clearance during first-line therapy was predictive of improved therapy responses and outcomes (EFS, HR: 6.5, 95% CI: 1.9-22, p=0.003 and OS, HR: 22, 95% CI: 2.5-191, p=0.005). Furthermore, 48% (13/27) of patients cleared their ctDNA following the first cycle of treatment. Patients who cleared their ctDNA, irrespective of their R-IPI score, had superior outcomes compared to ctDNA-positive patients. ctDNA clearance outperformed other factors associated with EFS in multivariate analysis (HR: 49.76, 95% CI:1.1-2225.6, p=0.044). Finally, ctDNA clearance predicted complete response (CR)/no evidence of disease (NED) on average 97 days (range: 0-14.7 months) ahead of imaging/biopsy.ConclusionctDNA testing in patients with DLBCL is predictive of patient outcomes and may enable personalized surveillance, intervention, and/or trial options.

Abstract There are limited studies on mutation profiling for Peripheral T‐cell lymphomas (PTCL) in the Chinese population. We retrospectively analyzed the clinical and genetic landscape of 66 newly diagnosed Chinese patients. Targeted next‐generation sequencing (NGS) was performed for tissues from these patients. At least one mutation was detected in 60 (90.9%) patients, with a median number of 3 (0–7) mutations, and 32 (48.5%) cases detected with more than 4 mutations. The genes with higher mutation frequencies were TET2, RHOA, DNMT3A, IDH2, TP53, STAT3, and KMT2D respectively. When mutant genes are classified by functional group, the most prevalent mutations are related to epigenetics and signal transduction. IPI ≥2, PIT ≥2, and failure to achieve partial remission (PR) were factors for inferior progression‐free survival (PFS) and overall survival (OS). Multivariate analysis showed TP53 was an adverse factor for PFS (HR, 3.523; 95% CI, 1.262–9.835; p = 0.016), and KMT2D was an adverse factor for OS (HR, 10.097; 95% CI, 1.000–101.953; p = 0.048). Mutation profiling could help differentiate distinct types of PTCL and serve as a useful tool for determining treatment options and prognoses.