Next-generation Sequencing Techniques Research Articles

A Preliminary Genomic Analysis of the Omicron Variants of SARS-CoV-2 in Central India During the third wave of the COVID-19 Pandemic

BackgroundOmicron was detected in South Africa for the first time at the month of November 2021, from then it expanded swiftly over the world, outcompeting other SARS-CoV-2 variants such as Delta. The toxicity, resistance to antiviral medicines, transmissibility, and vaccine-induced immunity of newly developed SARS-CoV-2 variants are major worldwide health concerns.Aim of studyThis study investigates the comprehensive explanation of all mutations and their evolutionary linkages between the Omicron variant and recently discovered SARS‐CoV‐2 variants.MethodOn Illumina MiniSeq Machine, 31 RNA isolates from clinical specimens were sequenced utilizing next-generation sequencing technique. Different bioinformatics tools have been used to analyze the mutations in omicron variant. A phylogenetic tree was constructed to determine Omicron's evolutionary relationships with other variants.ResultsIn our investigation, we discovered 79 distinct types of mutations in 31 fully vaccinated COVID-19 positive samples. Mostly mutations were found in non-spike region. According to the NJ approach of phylogenetic tree revels, the nearest variants were in the order listed, based on sequence identity: Omicron, Gamma, Alpha, Delta, Mu and Beta. On the other hand as per UPGMA approach, the Omicron variation creates a novel monophyletic clade that is distinct from previous SARS-CoV-2 variants.ConclusionDespite the fact that some of the mutations are prevalent in Omicron and other VOCs, there are several unique mutations that have been connected to the virus's transmissibility and immune evasion, indicating a substantial shift in SARS-CoV-2 evolution.

Critical care management of meningitis and encephalitis: an update.

Meningitis and encephalitis represent severe neurological syndromes associated with encephalopathy, seizures, focal deficits, and neurological sequelae in survivors. We update on the critical care management of adult patients with severe meningitis and encephalitis. Large multicenter studies conducted in the adult population improved current knowledge on the epidemiology and outcomes of patients with severe meningitis and encephalitis. An early multimodal diagnostic workup (including CSF analysis, brain MRI, EEG, and serum studies) is mandatory for diagnosis and prognostication in those patients.New diagnostic methods, including multiplex PCR and next-generation sequencing techniques, allow for a faster differential diagnosis of infectious causes that may require specific antimicrobial therapy. Autoimmune causes of encephalitis, which may require urgent immunotherapy, are also increasingly recognized in the ICU setting. Although observational data suggest that early combined immunotherapy is associated with better neurological outcomes in patients with autoimmune encephalitis, randomized clinical trials have yet to be performed. Our review highlights the importance of an early multimodal approach for diagnosing severe meningitis and encephalitis. Randomized clinical trials are needed to identify pharmacological interventions that may improve patients' outcomes.

Transcriptome Sequencing and Analysis of Male Fourfinger Threadfin, Eleutheronema tetradactylum (Shaw, 1804)

Fourfinger threadfin, Eleutheronema tetradactylum (Shaw, 1804), is one of the country’s most popular table fish species. This is a priority species for mariculture in India. Reproductive biology, life cycle, culture, and management are some budding research areas, while there is a lacuna in the species' genetic information. Using Illumina next-generation sequencing technique, 82,072 mRNA transcripts in the mature testis of fourfinger threadfin were obtained. The present study attempted to understand the expression of genes that facilitate sex determination, sex differentiation and gonadal maturation. From 82,072 transcripts, 50,943 were predicted by comparing them to proteins in UniProt using BLASTX. A total of 41 genes involved in sex determination and sex differentiation, spermatogenesis, steroid receptors in testis, steroidogenesis and gonadotropin-releasing hormone (GnRH) regulation are reported for the first time in E. tetradactylum. The entire raw sequencing reads of transcripts were made available in NCBI under BioProject PRJNA770837.

The bacteria inside human cancer cells: Mainly as cancer promoters.

The roles of the microbiome in human beings have become clearer with the development of next-generation sequencing techniques. Several pieces of evidence showed strong correlations between the microbiome and human health and disease, such as metabolic disorders, infectious diseases, digestive system diseases, and cancers. Among these diverse microbiomes, the role of bacteria in human cancers, especially in cancer cells, has received extensive attention. Latest studies found that bacteria widely existed in cancers, mainly in cancer cells and immune cells. In this review, we summarize the latest advances in understanding the role of bacteria in human cancer cells. We also discuss how bacteria are transported into cancer cells and their physiological significance in cancer progression. Finally, we present the prospect of bacterial therapy in cancer treatment.

Effect of sampling duration on culturable and viable bioaerosol determination when using Rutgers Electrostatic Passive Sampler (REPS)

Rutgers Electrostatic Passive Sampler (REPS) is a passive bioaerosol sampler composed of a permanently polarized polyvinylidene fluoride (PVDF) film wound into a 3D printed film holder. In order to investigate the effect of sampling duration on its performance, REPS was deployed in an indoor and outdoor location in Spring and Fall over continuing sampling periods ranging from 24 h to 21 days. Reference concentrations were determined by an active, culture-based sampler twice daily. REPS samples were analyzed for culturable bacteria and fungi as well as adenosine triphosphate (ATP) activity. The number of culture forming units (CFU) determined by REPS exhibited strong and positive correlations with sampling duration for culturable bacteria (Pearson r > 0.899, p < 0.05) and fungi (r > 0.902, p < 0.05) for both test locations. Similar results were observed for ATP activity in outdoor samples (r > 0.844, p < 0.05). REPS showed an equivalent flow rate (Qeq) as high as 4.86 ± 4.90 L/min when sampling bacteria outdoors. When sampling bacteria indoors, the Qeq was lower: 0.37 ± 0.78 L/min. When sampling fungi, the Qeq outdoors was also higher than that indoors. Overall, the study demonstrated that REPS is able to effectively capture airborne culturable bacteria and fungi by passive means of collection in indoor and outdoor environments while maintaining bioaerosol culturability for over 21 days of continuous sampling. Further studies will explore methods to improve the sample detection limit by using different sample concentration techniques as well as the sampler's integration with the next-generation sequencing techniques.

Inherited ichthyosis as a paradigm of rare skin disorders: Genomic medicine, pathogenesis, and management

Great advances have been made in the field of heritable skin disorders using next-generation sequencing (NGS) technologies (ie, whole-genome sequencing, whole-exome sequencing, whole-transcriptome sequencing, and disease-targeted multigene panels). When NGS first became available, the cost and lack of access to these technologies were limiting factors; however, with decreasing sequencing costs and the expanding knowledge base of genetic skin diseases, fundamental awareness of NGS has become prudent. The heritable ichthyoses comprise a genotypically and phenotypically heterogeneous group of monogenic keratinization disorders characterized by persistent scaling, with at least 55 distinct genes currently implicated in causing nonsyndromic and syndromic forms of the disease. By providing a simplified overview of available NGS techniques and applying them in the context of ichthyosis, one of the most common genodermatoses, we hope to encourage dermatologists to offer, when appropriate, genetic testing earlier in patients with unsolved presentations. With the aid of NGS, dermatologists can provide diagnostic certainty in cases of suspected genodermatoses and offer potentially life-changing genome-guided and targeted therapies as they become available.

Lung cancer coexisting with Papiliotrema flavescens infection diagnosed by next-generation sequencing: a case report

BackgroundPapiliotrema flavescens is a rare environmental yeast, which has been isolated from air, trees, kernels of wheat and corn, fermenting soya sauce, and cerebrospinal fluid of patient with AIDS. Additionally, it is also reported to cause subcutaneous infection in a dog. In this case, we describe primary lung adenocarcinoma coexisting with Papiliotrema flavescens infection in a female patient diagnosed by next-generation sequencing (NGS) technique, which is the first such reported case.Case presentationThe patient was a 52-year-old female with recurrent cough for 3 months. Chest CT examination revealed a ground glass nodule of 17 * 23 * 18 mm in the right upper lung, and 3 new pulmonary nodules appeared around it 2 months later. The patient underwent right upper lobe lobectomy and pathology confirmed that the primary 2-cm-lesion in the right upper lobe was invasive lung adenocarcinoma, and two of the three surrounding lung nodules were pathologically suggestive of pulmonary fungal infection (not known in specific fungal types). Hence, the patient received empirical anti-fungal treatments with fluconazole 400 mg/day for a week and follow-up CT scanning showed no tumor progression and no relapse of fungal infection. The specific pathogen was eventually identified as Papiliotrema flavescens by the next-generation sequencing of pathogen.Discussion and conclusionWe first reported that lung cancer coexisting with Papiliotrema flavescens infection in a female patient. The diagnosis of lung cancer with typical CT imaging features is relatively simple, while the diagnosis of lung cancer coexisting with rare fungal infection is challenging. NGS technique is an effective supplementary technique for clinical diagnosis of bacterial or fungal infectious diseases, enabling precise clinical decision-making and appropriate treatment. In this case, the lung cancer may result in a degree of immune suppression, at least locally, resulting in the formation of pulmonary fungal nodular lesions around the tumor.

Double heterozygous pathogenic variants prevalence in a cohort of patients with hereditary breast cancer.

Hereditary breast cancer (BC) corresponds to 5% of all BC and a larger parcel of early-onset disease. The incorporation of next-generation sequencing (NGS) techniques reduced the cost of molecular testing and allowed the inclusion of additional cancer predisposition genes in panels that are more comprehensive. This enabled the identification of germline pathogenic variants in carriers and the introduction of risk-reducing strategies. It also resulted in the identification of the co-occurrence of more than one germline pathogenic variant in BC genes in some families. This is a rare event, and there are few reports on its impact on cancer risk. We conducted a single-institution retrospective study in which 1,156 women with early onset BC and/or a family history of cancer were tested by a germline multi-gene hereditary cancer panel. Germline pathogenic variants in high- and/or moderate-penetrance BC genes were identified in 19.5% of the individuals (n = 226). The most frequent variants were found in TP53 (69 of 226; 55 of them represented by p.R337H), BRCA1 (47 of 226), and BRCA2 (41 of 226). Double heterozygous (DH) variants were detected in 14 cases, representing 1.2% of all individuals assessed. There were no significant differences in age of BC onset and risk for bilateral BC in DH carriers when compared with those with one germline variant.

Advances in Risk Stratification and Treatment of Polycythemia Vera and Essential Thrombocythemia.

Estimating and modifying thrombotic risk is currently the mainstay of care for patients with polycythemia vera (PV) and essential thrombocythemia (ET). In recent years, however, increased attention has shifted towards quality of life and disease modification. In this review, we discuss recent advances in risk stratification, present updated results for ruxolitinib and interferon randomized clinical trials, discuss new approaches in antiplatelet and anticoagulant treatment, and summarize early phase trials of novel agents and emerging therapeutic concepts for the treatment of PV and ET. International collaborations and novel technologies, i.e., next-generation sequencing and machine learning techniques, have demonstrated excellent abilities to improve thrombotic risk stratification in PV and ET. Updated results from ruxolitinib and interferon randomized clinical trials have confirmed excellent efficacy and safety of these agents, both as first- and second-line treatments. Early trials of novel agents (histone deacetylase inhibitors, telomerase inhibitors, lysine-specific demethylase-1 inhibitors, human double-minute 2 inhibitors, and hepcidin mimetics) have shown encouraging efficacy and safety in blood count control, reduction of splenomegaly, and alleviation of disease-related symptoms. Finally, accumulating evidence suggested that direct oral anticoagulants may be a valid therapeutic alternative to warfarin for prolonged thromboprophylaxis. International collaborations ("big data") with the help of new technologies represent an exciting new approach to analyze rare outcomes in rare diseases, especially for identifying novel prognostic biomarkers in PV and ET. Randomized clinical trials are also needed to fully elucidate whether novel agents may establish new standards of care.

Recent Advances in Sugarcane Genomics, Physiology, and Phenomics for Superior Agronomic Traits.

Advances in sugarcane breeding have contributed significantly to improvements in agronomic traits and crop yield. However, the growing global demand for sugar and biofuel in the context of climate change requires further improvements in cane and sugar yields. Attempts to achieve the desired rates of genetic gain in sugarcane by conventional breeding means are difficult as many agronomic traits are genetically complex and polygenic, with each gene exerting small effects. Unlike those of many other crops, the sugarcane genome is highly heterozygous due to its autopolyploid nature, which further hinders the development of a comprehensive genetic map. Despite these limitations, many superior agronomic traits/genes for higher cane yield, sugar production, and disease/pest resistance have been identified through the mapping of quantitative trait loci, genome-wide association studies, and transcriptome approaches. Improvements in traits controlled by one or two loci are relatively easy to achieve; however, this is not the case for traits governed by many genes. Many desirable phenotypic traits are controlled by quantitative trait nucleotides (QTNs) with small and variable effects. Assembling these desired QTNs by conventional breeding methods is time consuming and inefficient due to genetic drift. However, recent developments in genomics selection (GS) have allowed sugarcane researchers to select and accumulate desirable alleles imparting superior traits as GS is based on genomic estimated breeding values, which substantially increases the selection efficiency and genetic gain in sugarcane breeding programs. Next-generation sequencing techniques coupled with genome-editing technologies have provided new vistas in harnessing the sugarcane genome to look for desirable agronomic traits such as erect canopy, leaf angle, prolonged greening, high biomass, deep root system, and the non-flowering nature of the crop. Many desirable cane-yielding traits, such as single cane weight, numbers of tillers, numbers of millable canes, as well as cane quality traits, such as sucrose and sugar yield, have been explored using these recent biotechnological tools. This review will focus on the recent advances in sugarcane genomics related to genetic gain and the identification of favorable alleles for superior agronomic traits for further utilization in sugarcane breeding programs.

PGT for structural chromosomal rearrangements in 300 couples reveals specific risk factors but an interchromosomal effect is unlikely

Research questionWhat factors affect the proportion of chromosomally balanced embryos in structural rearrangement carriers? Is there any evidence for an interchromosomal effect (ICE)? DesignPreimplantation genetic testing outcomes of 300 couples (198 reciprocal, 60 Robertsonian, 31 inversion and 11 complex structural rearrangement carriers) were assessed retrospectively. Blastocysts were analysed either by array-comparative genomic hybridization or next-generation sequencing techniques. ICE was investigated using a matched control group and sophisticated statistical measurement of effect size (φ). Results300 couples underwent 443 cycles; 1835 embryos were analysed and 23.8% were diagnosed as both normal/balanced and euploid. The overall cumulative clinical pregnancy and live birth rates were 69.5% and 55.8%, respectively. Complex translocations and female age (≥35) were found to be risk factors associated with lower chance of having a transferable embryo (P < 0.001). Based on analysis of 5237 embryos, the cumulative de-novo aneuploidy rate was lower in carriers compared to controls (45.6% versus 53.4%, P < 0.001) but this was a ‘negligible’ association (φ < 0.1). A further assessment of 117,033 chromosomal pairs revealed a higher individual chromosome error rate in embryos of carriers compared to controls (5.3% versus 4.9%), which was also a ‘negligible’ association (φ < 0.1), despite a P-value of 0.007. ConclusionsThese findings suggest that rearrangement type, female age and sex of the carrier have significant impacts on the proportion of transferable embryos. Careful examination of structural rearrangement carriers and controls indicated little or no evidence for an ICE. This study helps to provide a statistical model for investigating ICE and an improved personalized reproductive genetics assessment for structural rearrangement carriers.

New Genetic Technologies in Diagnosis and Treatment of Cancer of Unknown Primary.

Cancer of unknown primary (CUP) represents a rare oncological and heterogeneous disease in which one or more metastases are present, but the location of the primary site is unknown. Pathological diagnosis, using immunohistochemistry, of such metastatic materials is challenging and frequently does not allow for determining the tissue of origin (ToO). The selection of systemic therapy in patients with CUP is usually based on empiric grounds, and the prognosis is generally unfavourable. New molecular techniques could identify the tissue of origin and be used to select systemic agnostic therapies in various malignancies with specific molecular abnormalities. Targetable driver mutations or gene rearrangements in cancer cells may be identified using various molecular assays, of which particularly valuable are next-generation sequencing techniques. These assays may identify tumour sources and allow personalized treatments. However, current guidelines for CUP management do not recommend routine testing of gene expression and epigenetic factors. This is mainly due to the insufficient evidence supporting the improvement of CUP's prognosis by virtue of this approach. This review summarizes the advantages and disadvantages of new genetic techniques in CUP diagnostics and proposes updating the recommendations for CUP management.

Spontaneous Remission of Acute Myeloid Leukemia: A Case Report

Spontaneous remissions (SRs) in acute myeloid leukemia (AML) are infrequent, poorly documented and transient. Similarly, morphological and cytogenetic complete remissions (CR) under azacitidine treatment are scarce. We report a 71-year-old man with a secondary AML arising from essential thrombocythemia (ET), who developed an SR after discontinuation of azacitidine following a respiratory infection (four courses were administered). The distinctive feature of our case is the depth of the achieved CR, documented by next-generation sequencing (NGS) techniques. We also detected persistence of molecular lesions that might already have been present in the previous ET clone. Our patient relapsed 5 months after achieving CR. We conclude that our patient showed a spontaneous remission of his AML rather than an exquisite response to azacitidine. We hypothesize that the concurrent respiratory infection, or any other unknown trigger, might have activated his immune system forcing the leukemic stem cell to enter a quiescent state through a yet unexplained mechanism.

Comparison Between Full-Length 16S rRNA Metabarcoding and Whole Metagenome Sequencing Suggests the Use of Either Is Suitable for Large-Scale Microbiome Studies.

Since the number of studies of the microbial communities related to food and food-associated matrices almost completely reliant on next-generation sequencing techniques is rising, evaluations of these high-throughput methods are critical. Currently, the two most used sequencing methods to profile the microbiota of complex samples, including food and food-related matrices, are the 16S ribosomal RNA (rRNA) metabarcoding and the whole metagenome sequencing (WMS), both of which are powerful tools for the monitoring of foodborne pathogens and the investigation of the microbiome. Herein, the microbial profiles of 20 bulk tank milk filters from different dairy farms were investigated using both the full-length 16S (FL-16S) rRNA metabarcoding, a third-generation sequencing method whose application in food and food-related matrices is yet in its infancy, and the WMS, to evaluate the correlation and the reliability of these two methods to explore the microbiome of food-related matrices. Metabarcoding and metagenomic data were generated on a MinION platform (Oxford Nanopore Technologies) and on a Illumina NovaSeq 6000 platform, respectively. Our findings support the greater resolution of WMS in terms of both increased detection of bacterial taxa and enhanced detection of diversity; in contrast, FL-16S rRNA metabarcoding has proven to be a promising, less expensive, and more practical tool to profile most abundant taxa. The significant correlation of the two technologies both in terms of taxa diversity and richness, together with the similar profiles defined for both highly abundant taxa and core microbiomes, including Acinetobacter, Bacillus, and Escherichia genera, highlights the possible application of both methods for different purposes. This study allowed the first comparison of FL-16S rRNA sequencing and WMS to investigate the microbial composition of a food-related matrix, pointing out the advantageous use of FL-16S rRNA to identify dominant microorganisms and the superior power of WMS for the taxonomic detection of low abundant microorganisms and to perform functional analysis of the microbial communities.

Batch effect detection and correction in RNA-seq data using machine-learning-based automated assessment of quality

BackgroundThe constant evolving and development of next-generation sequencing techniques lead to high throughput data composed of datasets that include a large number of biological samples. Although a large number of samples are usually experimentally processed by batches, scientific publications are often elusive about this information, which can greatly impact the quality of the samples and confound further statistical analyzes. Because dedicated bioinformatics methods developed to detect unwanted sources of variance in the data can wrongly detect real biological signals, such methods could benefit from using a quality-aware approach.ResultsWe recently developed statistical guidelines and a machine learning tool to automatically evaluate the quality of a next-generation-sequencing sample. We leveraged this quality assessment to detect and correct batch effects in 12 publicly available RNA-seq datasets with available batch information. We were able to distinguish batches by our quality score and used it to correct for some batch effects in sample clustering. Overall, the correction was evaluated as comparable to or better than the reference method that uses a priori knowledge of the batches (in 10 and 1 datasets of 12, respectively; total = 92%). When coupled to outlier removal, the correction was more often evaluated as better than the reference (comparable or better in 5 and 6 datasets of 12, respectively; total = 92%).ConclusionsIn this work, we show the capabilities of our software to detect batches in public RNA-seq datasets from differences in the predicted quality of their samples. We also use these insights to correct the batch effect and observe the relation of sample quality and batch effect. These observations reinforce our expectation that while batch effects do correlate with differences in quality, batch effects also arise from other artifacts and are more suitably corrected statistically in well-designed experiments.

Determination of Nucleotide Sequences within Promoter Regions Affecting Promoter Compatibility between Zymomonas mobilis and Escherichia coli.

A promoter plays a crucial role in controlling the expression of the target gene in cells, thus being one of the key biological parts for synthetic biology practices. Although significant efforts have been made to identify and characterize promoters with different strengths in various microorganisms, the compatibility of promoters within different hosts still lacks investigation. In this study, we chose the native Pgap promoter of Zymomonas mobilis to investigate nucleotide sequences within promoter regions affecting promoter compatibility between Escherichia coli and Z. mobilis. Pgap is one of the strongest promotors in Z. mobilis that has many excellent characteristics to be developed as microbial cell factories. Using EGFP as a reporter, a Z. mobilis-derived Pgap mutant library was constructed and sorted in E. coli, with candidate promoters exhibiting high fluorescence intensity collected. A total of 53 variants were finally selected and sequenced by Sanger sequencing. The sequencing results grouped these variants into 12 different Pgap variant types, among which seven types presented higher promoter strength than native Pgap in E. coli. The next-generation sequencing technique was then employed to identify key mutations within the Pgap promoter region that affect the promoter compatibility. Finally, six important sites were identified and confirmed to help increase Pgap strength in E. coli while keeping similar strength of native Pgap in Z. mobilis. Compared to native Pgap, synthetic promoters combining these sites had enhanced strength; especially, Pgap-6M combining all six sites exhibited 20-fold greater strength than native Pgap in E. coli. This study thus not only determined six important sites affecting promoter compatibility but also confirmed a series of Pgap promoter variants with strong promoter activity in both E. coli and Z. mobilis. In addition, a strategy was established in this study to investigate and determine nucleotide sequences in promoter regions affecting promoter compatibility, which can be applied in other microorganisms to help reveal universal factors affecting promoter compatibility and design promoters with desired strengths among different microbial cell factories.

Genetic Signatures of Chromosome 20 Mosaicism in Recurrent Pregnancy Loss

Recurrent pregnancy loss (RPL) causes an immeasurable physical, emotional, and economical impact on the couple. RPL is a complicated challenging scenario in front of gynaecologists working in the area of reprogenomics. Chromosomal aneuploidy and mosaicism are common causes of sporadic early pregnancy loss and play an important proportion in RPL in patients undergoing in vitro fertilization (IVF). In a single-center case series, we report five cases of RPL after implantation of embryos with mosaicism of chromosome 20. The female partners were of advanced maternal age and had a history of previous miscarriages. The couple’s blood karyotype reports were normal and there was no family history of any inherited disease. The chromosomal aneuploidies were screened in day 5 embryos with good morphological grades. The day five trophectoderm biopsies were subjected to Preimplantation genetic screening (PGS) using the next-generation sequencing (NGS) technique. The couples were genetically counseled and written informed consents were taken before implantation explaining all the associated risks. All the clinical parameters were normal and the patients were in close observation with regular follow-up throughout the treatment. These cases had chromosome 20 mosaic embryo transfer resulting in fetal loss in first trimester of pregnancy. Our findings strongly suggest that chromosomal mosaicism in embryos is one of the leading causes of RPL in females with previous miscarriages and advanced maternal age

P-520 Factors associated with embryonic mosaicism: a systematic review and meta-analysis

Abstract Study question What factors are involved in chromosomal mosaicism in human blastocysts? Summary answer Progenitors’ age and embryo biopsy day are associated with mosaicism rate. What is known already Chromosomal abnormalities are common in embryos analyzed in preimplantation genetic testing for aneuploidy (PGT-A) cycles. Mosaicism is a usual event in embryos derived from IVF cycles. With the development of Next-Generation Sequencing (NGS) techniques, the ability to detect mosaicism in embryos has been improved. Several studies show that mosaic embryos have reduced potential to reach term compared to euploid. Furthermore, the existing scientific evidence regarding the cause of embryonic mosaicism is scarce with conclusions generating considerable controversy. Study design, size, duration A systematic review and meta-analysis were conducted following the PRISMA guidelines on Medline and Google Scholar (January 2016 to December 2021). Studies addressing factors associated with embryo mosaicism were comprehensively analyzed. As inclusion criteria: mosaicism should have been detected from a blastocyst trophectoderm biopsy on day 5,6 o 7 and analyzed by NGS. Embryo quality, maternal and paternal age, day of biopsy and seminal quality were the outcomes variables included for analysis. Participants/materials, setting, methods A comprehensive database search resulted in 195 articles, 18 of which were included for abstract reading. Search, screen, and data extraction were performed by two reviewers independently based on inclusion criteria. The study by meta-analysis of the effect of the different factors associated with embryonic mosaicism was carried out using the inverse variance method. Depending on the existence of heterogeneity between studies, the fixed effects method or the random effects method were used. Main results and the role of chance After critically and thoroughly reading the selected papers, 10 of them were included in the meta-analysis. Data from our reproductive clinic were also enclosed in the analysis (2513 cycles, 7242 embryos). The results of the meta-analysis show that neither embryo quality nor seminal quality (male factor) were related to mosaic embryo rate (OR: 1.15; 95%CI: 0.98-1.35 and OR: 1.01; 95%CI: 0.80-1.27, respectively). In contrast, a positive association with embryo mosaicism was observed for the variable “biopsy day” (OR: 1.06; 95% CI: 1.01-1.11): in embryos biopsied on day 6 or 7 of embryonic development, a small increase in the mosaicism rate was observed, in comparison with day 5. A significative positive association was also observed when we studied “paternal age” factor (OR: 1.13; 95% CI: 1.02-1.26): embryo mosaicism increases with paternal age. On the other hand, maternal age showed a negative association with mosaicism (OR: 0.84; 95% CI: 0.74-0.95). Interestingly, in opposite to what happens with the aneuploidy rate, embryo mosaicism is higher in young women. Limitations, reasons for caution The main limitation is the low number of papers analyzed in the meta-analysis. Limitations also include the retrospective design and heterogeneity of studies, limiting comparison and pooling of data. Nonetheless, our conclusions were based on studies with low risk of bias. Wider implications of the findings Our result, combined with the body of medical evidence available suggest that embryo mosaicism rate is influenced by trophectoderm biopsy day and maternal and paternal age. This information will add in the knowledge for elucidating the uncertainties surrounding the factors by which mosaicism is generated in embryos. Trial registration number Not applicable

Molecular Genetic Analysis of Patients with Duchenne/Becker Muscular Dystrophy by Multiplex Ligation-Dependent Probe Amplification and Next-Generation Sequencing Techniques

Background: Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are neuromuscular genetic disorders, which are characterized by mutations in the dystrophin gene. Large deletions or duplications in the dystrophin gene are found in approximately 60 to 70% of cases, and the remaining have point mutations, small deletions, or insertions. Objectives: The aim of this study was to assess mutations in the dystrophin gene in clinically suspicious subjects of DMD/ BMD by multiplex ligation-dependent probe amplification (MLPA) and next-generation sequencing (NGS) techniques. Methods: This study consisted of 13 clinically suspected DMD/BMD patients. MLPA, NGS, and Sanger sequencing were used to determine mutations in the dystrophin gene. In order to minimize the time and cost, MLPA (SALSA P034/P035 DMD test kit) was performed as the first step to detect large deletions or duplications, followed by NGS and Sanger sequencing for MLPA-negative cases. Results: This study included nine males and four females with clinical suspicion of DMD or BMD. The MLPA was performed for all cases, among which ten cases were definitely diagnosed with DMD/BMD. NGS and Sanger sequencing were performed for the three remaining subjects to find out possible point mutations and small deletions, which could not be detected by MLPA. Nine intragenic deletions were identified, mostly in exons 46 to 47 and 4 to 7. Furthermore, an intragenic triplication was detected in exon 71 by MLPA. Conclusions: In agreement with other reports, our findings indicated that exonic deletions of the dystrophin gene are more frequent than duplications and point mutations in Duchenne/ Becker muscular dystrophy. Most deletions were demonstrated to occur in exons 46 to 47 and 4 to 7.

Gut microbiome and neurocritically ill patients

Since the times of Rokitansky and Cushing, we have been fascinated by the connections between the gut and the brain. Recent advances in next-generation sequencing techniques have shown that this relationship is even more complex and integral to our sense of self than previously imagined. As these techniques refine our understanding of the abundance and diversity of the gut bacterial microbiome, the relationship between the gut and the brain has been redefined. Now, this is understood as a complex symbiotic network with bidirectional communication, the gut-brain axis. The implication of this communication involves an intense focus of research on a variety of chronic psychiatric, neurological, neurodegenerative, and neuro-oncological diseases. Recently, the gut-brain axis has been studied in neurologically ill patients requiring intensive care. Preliminary studies have shown that acute brain injury changes the bacterial phenotype from one that is symbiotic with the host human to one that is pathologic, termed the “pathobiome.” This can contribute to nosocomial pneumonia and sepsis. The first studies in neurologically ill patients in the neurointensive care unit (NeuroICU) demonstrated changes in the gut microbiome between neuroICU patients and healthy matched subjects. Specifically, a decrease in short-chain fatty acid-producing bacteria and increase in harmful gut microbes have been associated with mortality and decreased function at discharge. Although these preliminary findings are exciting and have opened a new field of research in the complex NeuroICU population, there are several limitations and challenges. Further investigation is needed to confirm these correlations and understand their implications on patients in a complex intensive care environment.