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Background: Protein-coding genes have been considered the functional part of the genome, although they represent only 2% of the genome. In contrast, more than 90% of the genome produces non-coding RNA (ncRNA), including antisense (AS) genes, a type of long non-coding genes (encoding transcripts > 200 nucleotides) located on the opposite strand of coding genes. Therefore, antisense RNA (asRNA) can be complementary to the counterpart sense RNA, supporting a regulatory role with potential pathogenic consequences, as their deregulation has been associated with cardiovascular disease, cancer, and diabetes. Results: We performed an in-depth review of AS genes in Ensembl and evaluated the expression of AS genes in human liver by third-generation RNA sequencing methods. Currently, 1656 AS genes overlapping with 1556 sense genes have been identified in the human genome. Coding genes with antisense counterparts were significantly larger and had higher transcriptional activity than genes without antisense counterparts. RNA nanopore sequencing of 15 human livers identified 185 transcripts (55 novel) from 150 known AS genes, and 1316 transcripts from 807 sense genes, with 111 sense-antisense pairs. Sense transcripts showed higher expression than antisense transcripts. Remarkably, RNA nanopore sequencing of human livers identified 146 transcripts (67 novel) corresponding to 118 possible novel AS genes. Conclusion: This study reveals the landscape of AS genes in the human genome and demonstrates the power of long-read RNA sequencing to identify novel transcripts and even novel AS genes, exploring the relationship between sense and AS gene expression as well. [ABSTRACT FROM AUTHOR]

Genomic structural variation (SV) refers to differences in gene sequences between individuals on a genomic scale. It is widely distributed in the genome, primarily in the form of insertions, deletions, duplications, inversions, and translocations. Due to its characterization by long segments and large coverage, SVs significantly impact the genetic characteristics and production performance of livestock, playing a crucial role in studying breed diversity, biological evolution, and disease correlation. Research on SVs contributes to an enhanced understanding of chromosome function and genetic characteristics and is important for understanding hereditary diseases mechanisms. In this article, we review the concept, classification, main formation mechanisms, detection methods, and advancement of research on SVs in the genomes of cattle, buffalo, equine, sheep, and goats, aiming to reveal the genetic basis of differences in phenotypic traits and adaptive genetic mechanisms through genomic research, which will provide a theoretical basis for better understanding and utilizing the genetic resources of herbivorous livestock. [ABSTRACT FROM AUTHOR]

Objectives: Purple blotch, caused by the necrotrophic pathogen Alternaria porri, is one of the most economically significant diseases of onion and allied crops. While the virulent nature of many Alternaria spp. has been identified, the pathogenic repertoire of A. porri is still unknown. The objective of this work was to sequence the genome of A. porri using the PacBio SMRT sequencing strategy and analyse the repertoire of CAZymes, secondary metabolites, secretome and effectors in A. porri. Our research group is working to identify onion germplasm with purple blotch resistance and to understand the genetics of the pathogen. The reported de-novo assembly will contribute to the analysis of potential variants and the gene repertoire contributing to the virulence and pathogenicity of the purple blotch pathogen. Data description: Long-read sequencing on a PacBio Sequel II system resulted in a 32.98 Mb (20 contigs) assembly with an N50 of 2, 657, 264 bp, the longest contig length of 5.05 Mb, and a GC content of 51.06%. The Benchmarking Universal Single-Copy Orthologs (BUSCO) analysis resulted in 99.7% genome completeness at the Dothideomycetes lineage, representing a high-quality genome assembly. AUGUSTUS ab initio analysis resulted in 9875 protein-coding genes. Of the 6776 pathogenicity-related genes, 537 genes with effector functions were identified. Likewise, the glycoside hydrolases (434) were the most dominant group of the total 837 predicted CAZymes. The assembled genome of A. porri showed distinctive similarities to the genomes of A. alternata and A. brassicicola, the causal agents of leaf blight of onion and leaf spot of Brassica crops, respectively. [ABSTRACT FROM AUTHOR]

Bronchiectasis is marked by bronchial dilatation, recurrent infections and significant morbidity, underpinned by a complex interplay between microbial dysbiosis and immune dysregulation. The identification of distinct endophenotypes have refined our understanding of its pathogenesis, including its heterogeneous disease mechanisms that influence treatment and prognosis responses. Next-generation sequencing (NGS) has revolutionised the way we view airway microbiology, allowing insights into the “unculturable”. Understanding the bronchiectasis microbiome through targeted amplicon sequencing and/or shotgun metagenomics has provided key information on the interplay of the microbiome and host immunity, a central feature of disease progression. The rapid increase in translational and clinical studies in bronchiectasis now provides scope for the application of precision medicine and a better understanding of the efficacy of interventions aimed at restoring microbial balance and/or modulating immune responses. Holistic integration of these insights is driving an evolving paradigm shift in our understanding of bronchiectasis, which includes the critical role of the microbiome and its unique interplay with clinical, inflammatory, immunological and metabolic factors. Here, we review the current state of infection and the microbiome in bronchiectasis and provide views on the future directions in this field. [ABSTRACT FROM AUTHOR]

Brain tumors and genomics have a long-standing history given that glioblastoma was the first cancer studied by the cancer genome atlas. The numerous and continuous advances through the decades in sequencing technologies have aided in the advanced molecular characterization of brain tumors for diagnosis, prognosis, and treatment. Since the implementation of molecular biomarkers by the WHO CNS in 2016, the genomics of brain tumors has been integrated into diagnostic criteria. Long-read sequencing, also known as third generation sequencing, is an emerging technique that allows for the sequencing of longer DNA segments leading to improved detection of structural variants and epigenetics. These capabilities are opening a way for better characterization of brain tumors. Here, we present a comprehensive summary of the state of the art of third-generation sequencing in the application for brain tumor diagnosis, prognosis, and treatment. We discuss the advantages and potential new implementations of long-read sequencing into clinical paradigms for neurooncology patients. [ABSTRACT FROM AUTHOR]

Background: Molecular diagnostic technology is the foundation of precision medicine, which has the advantages of good specificity, high sensitivity, strong targeting, rapiddiagnosis, etc. It has a wide range of applications in the field of pediatrics. However, molecular diagnostic technology is characterized by complicated experimental operation, high difficulty in data analysis and interpretation, in consistent standards among laboratories, and difficult standardization of technical processes. Enhancing the application value of molecular diagnostic technology in pediatrics and promoting the high‐quality development of the discipline requires careful consideration by relevant management and professionals. Methods: This study firstly outlines the development history of molecular diagnostic technology. Then, it analyzes the application of molecular diagnostic technology in the field of pediatrics. Finally, it explores the countermeasures for the management of molecular diagnostic laboratories. Results: This study highlights the importance of molecular diagnostic technology in providing information and decision‐making basis for disease prevention, prediction,diagnosis, treatment and regression. It has a wide range of applications in the molecular diagnosis of pediatric hereditary diseases, malignant tumors and infectious diseases. In addition, the countermeasures for the management of molecular diagnostic laboratories are proposed from the five aspects of laboratory, personnel team construction, standardized management,multidisciplinary cross‐discipline, research and translation, safety managementand ethical supervision, and management upgrading and modernization. Conclusions: Molecular diagnostic technology, as the basis of precision medicine, has become one of the important frontier fields in the development of contemporary pediatric medicine. Enhanced laboratory capacity in molecular diagnostic techniques can improve outcomes in the prevention, prediction, diagnosis, treatment, prognosis and research of pediatricdiseases, and lay the groundwork for child healthcare. [ABSTRACT FROM AUTHOR]

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HLA typing serves as a standard practice in hematopoietic stem cell transplantation to ensure compatibility between donors and recipients, preventing the occurrence of allograft rejection and graft-versus-host disease. Conventional laboratory methods that have been widely employed in the past few years, including sequence-specific primer PCR and sequencing-based typing (SBT), currently face the risk of becoming obsolete. This risk stems not only from the extensive diversity within HLA genes but also from the rapid advancement of next-generation sequencing and third-generation sequencing technologies. Third-generation sequencing systems like single-molecule real-time (SMRT) sequencing and Oxford Nanopore (ONT) sequencing have the capability to analyze long-read sequences that span entire intronic-exonic regions of HLA genes, effectively addressing challenges related to HLA ambiguity and the phasing of multiple short-read fragments. The growing dominance of these advanced sequencers in HLA typing is expected to solidify further through ongoing refinements, cost reduction, and error rate minimization. This review focuses on hematopoietic stem cell transplantation (HSCT) and explores prospective advancements and application of HLA DNA typing techniques. It explores how the adoption of third-generation sequencing technologies can revolutionize the field by offering improved accuracy, reduced ambiguity, and enhanced assessment of compatibility in HSCT. Embracing these cutting-edge technologies is essential to advancing the success rates and outcomes of hematopoietic stem cell transplantation. This review underscores the importance of staying at the forefront of HLA typing techniques to ensure the best possible outcomes for patients undergoing HSCT. Highlights of the Study: This review delves into the progression of HLA typing techniques, from serology to high-resolution sequencing methods, and emphasizes the pivotal role of HLA compatibility in stem cell transplantation. We discuss resolution levels for precise HLA typing and highlight the transformative potential of PacBio and nanopore sequencing. Precise HLA typing revolutionizes transplantation, improving donor-recipient matching and reducing ambiguities. [ABSTRACT FROM AUTHOR]

Diabetes mellitus is a chronic disease affecting glucose metabolism. The pathophysiological reactions underpinning the disease can lead to the development of late diabetes complications. The gut microbiota plays important roles in weight regulation and the maintenance of a healthy digestive system. Obesity, diabetes mellitus, diabetic retinopathy, diabetic nephropathy and diabetic neuropathy are all associated with a microbial imbalance in the gut. Modern technical equipment and advanced diagnostic procedures, including xmolecular methods, are commonly used to detect both quantitative and qualitative changes in the gut microbiota. This review summarises collective knowledge on the role of the gut microbiota in both types of diabetes mellitus and their late complications, with a particular focus on diabetic foot syndrome. [ABSTRACT FROM AUTHOR]

Background: Spinal muscular atrophy (SMA) is a rare autosomal recessive hereditary neuromuscular disease caused by survival motor neuron 1 (SMN1) gene deletion or mutation. Homozygous deletions of exon 7 in SMN1 result in 95% of SMA cases, while the remaining 5% are caused by other pathogenic variants of SMN1. Methods: We analyzed two SMA-suspected cases that were collected, with no SMN1 gene deletion and point mutation in whole-exome sequencing. Exon 1 deletion of the SMN gene was detected using Multiplex ligation-dependent probe amplification (MLPA) P021. We used long-range polymerase chain reaction (PCR) to isolate the SMN1 template, optimized-MLPA P021 for copy number variation (CNV) analysis within SMN1 only, and validated the findings via third-generation sequencing. Results: Two unrelated families shared a genotype with one copy of exon 7 and a novel variant, g.70919941_70927324del, in isolated exon 1 of the SMN1 gene. Case F1-II.1 demonstrated no exon 1 but retained other exons, whereas F2-II.1 had an exon 1 deletion in a single SMN1 gene. The read coverage in the third-generation sequencing results of both F1-II.1 and F2-II.1 revealed a deletion of approximately 7.3 kb in the 5' region of SMN1. The first nucleotide in the sequence data aligned to the 7385 bp of NG_008691.1. Conclusion: Remarkably, two proband families demonstrated identical SMN1 exon 1 breakpoint sites, hinting at a potential novel mutation hotspot in Chinese SMA, expanding the variation spectrum of the SMN1 gene and corroborating the specificity of isolated exon 1 deletion in SMA pathogenesis. The optimized-MLPA P021 determined a novel variant (g.70919941_70927324del) in isolated exon 1 of the SMN1 gene based on long-range PCR, enabling efficient and affordable detection of SMN gene variations in patients with SMA, providing new insight into SMA diagnosis to SMN1 deficiency and an optimized workflow for single exon CNV testing of the SMN gene. [ABSTRACT FROM AUTHOR]

Background and Objectives: The presence of blood subtypes may lead to difficulties in blood group identification; however, third‐generation sequencing (TGS) can help in accurately identifying difficult blood groups, and study the serological characteristics and molecular mechanism of Ael subtypes. Materials and Methods: ABO blood group was identified by the standard serological technique, weak blood group antigen was identified by adsorption‐elution experiments, ABH substance in the saliva was determined and glycosyltransferase activity of A and B was detected. The ABO gene full‐length sequence and promoter region were amplified by specific primers using single‐molecule real‐time sequencing, with the amplified products being sequenced directly and analysed in real time. Results: The patient was serologically identified as Ael subtype, and TGS analysis revealed new intron mutations in Ael patients (c.467C>T; c.29‐10T>A). Conclusion: The discovery of the new allele and the identification of ABO subtypes can be combined with serological characterization and molecular biological methods. [ABSTRACT FROM AUTHOR]

Avena fatua L. (wild oat) is one of the most harmful gramineous weeds that can affect the yield and quality of infiltrating crops. Bacillus altitudinis D30202 exhibits an excellent biocontrol activity against wild oat. To elucidate the biocontrol mechanisms of B. altitudinis D30202, the genome structure of this strain was assessed via whole-genome sequencing analysis. We predicted and analyzed secondary metabolite synthesis gene clusters to elucidate the mechanisms underlying the biocontrol of weeds. The whole-genome sequencing data indicated that B. altitudinis D30202 had the genome size and GC content of 3,777,154 bp and 41.32%, respectively, and 3809 coding genes were identified. Moreover, this strain could generate several compounds with bioherbicidal activity, including 4-hydroxy-3-methoxycinnamic acid and two indole derivatives. Bioinformatics prediction and comparative genomic analysis revealed that the strain had 6 secondary metabolite gene clusters. Furthermore, the taxonomic position of B. altitudinis D30202 was assessed, confirming its uniqueness and novelty within the Bacillus genus. Comparative genomic analysis showed differences in gene distribution, suggesting potential adaptations to different environments. In conclusion, B. altitudinis D30202 possesses a genome with unique characteristics, encoding enzymes and pathways related to herbicidal potential and biocontrol. This study provides a reference basis for understanding the molecular mechanisms of weed inhibition. [ABSTRACT FROM AUTHOR]

Precision medicine based on the driver genes mutation status is the current systemic therapeutic paradigm in patients with lung cancer. Next-generation sequencing (NGS) has emerged as a powerful platform for molecular diagnosis by virtue of high-throughput and massively parallel sequencing. Liquid biopsy also enabled the dynamic monitoring and comprehensive profiling of lung cancer in a noninvasive manner. However, challenges remain in the field of technology and clinical applications, especially in the era of immunotherapy. Here, we update the role of NGS in the context of lung cancer screening, molecular diagnosis, predictive and prognostic biomarkers, and guiding personalized treatment. The NGS application for actable genomic alternation has greatly changed the therapeutic landscape in patients with lung cancer including perioperative setting and advanced stage. Meanwhile, emerging evidence has shown the potential of other applications such as early screening and detection, and MRD. However, challenges remain such as the lack of standardized protocols across different platforms and bioinformatics analysis pipelines, and the complexity of interpreting and leveraging numerous genomic mutation messages for therapy selection. Future research is needed to overcome these challenges and expand the applications of NGS to other aspects such as immunotherapy. [ABSTRACT FROM AUTHOR]

This article provides an overview of emerging trends in clinical cancer genomic research, with a specific focus on gastric cancer. It discusses four key trends in gastric cancer research, including the shift towards a reference pangenome-based methodology, cross-race studies, microenvironment-combined research, and the use of third-generation sequencing with long reads. The article also presents findings from studies on genetic variations in gastric cancer, the impact of patient ethnicity on gene mutations, and the role of the tumor microenvironment in peritoneal metastasis. These trends and findings offer valuable insights for genetic intervention treatment and clinical medication. The article also explores the use of omental neutrophils and peritoneal resident macrophages in promoting peritoneal metastasis and suggests blocking Tim-4 as a potential treatment strategy. It further discusses the use of The Cancer Genome Atlas (TCGA) database to identify mutated genes associated with vascular metastasis in gastric cancer, as well as the use of genomic sequencing techniques for disease diagnosis and the detection of structural variants. The article concludes by acknowledging the limitations and challenges of these technologies in clinical use. [Extracted from the article]

Background: Ulcerative colitis is one of the two main forms of inflammatory bowel disease. Cuproptosis is reported to be a novel mode of cell death. Methods: We examined clusters of cuproptosis related genes and immune cell infiltration molecules in 86 ulcerative colitis samples from the GSE179285 dataset. We identified the differentially expressed genes according to the clustering method, and the performance of the SVM model, the random forest model, the generalized linear model, and the limit gradient enhancement model were compared, and then the optimal machine model was selected. To assess the accuracy of the learning predictions, the nomogram and the calibration curve and decision curve analyses showed that the subtypes of ulcerative colitis have been accurately predicted. Results: Significant cuproptosis-related genes and immune response cells were detected between the ulcerative colitis and control groups. Two cuproptosis-associated molecular clusters were identified. Immune infiltration analysis indicated that different clusters exhibited significant heterogeneity. The immune scores for Cluster2 were elevated. Both the residual error and root mean square error of the random forest machine model had clinical significance. There was a clear correlation between the differentially expressed genes in cluster 2 and the response of immune cells. The nomogram and the calibration curve and decision curve analyses showed that the subtypes of ulcerative colitis had sufficient accuracy. Conclusion: We examined the complex relationship between cuproptosis and ulcerative colitis in a systematic manner. To estimate the likelihood that each subtype of cuproptosis will occur in ulcerative colitis patients and their disease outcome, we developed a promising prediction model. [ABSTRACT FROM AUTHOR]

Marine sediments are one of the largest habitats on Earth, and their unique ecology, such as high salinity, high pressure, and hypoxia, may activate certain silent genes in marine microbes, resulting in microbes, enzymes, active products, and specific metabolic pathways that can adapt to these specific ecological environments. Marine sediment-derived microorganisms and their bioactive metabolites are of great significance and have potential commercial development prospects for food, pharmaceutical, chemical industries, agriculture, environmental protection and human nutrition and health. In recent years, although there have been numerous scientific reports surrounding marine sediment-derived microorganisms and their bioactive metabolites, a comprehensive review of their research progress is lacking. This paper presents the development and renewal of traditional culture-dependent and omics analysis techniques and their application to the screening of marine sediment-derived microorganisms producing bioactive substances. It also highlights recent research advances in the last five years surrounding the types, functional properties and potential applications of bioactive metabolites produced by marine sediment-derived microorganisms. These bioactive metabolites mainly include antibiotics, enzymes, enzyme inhibitors, sugars, proteins, peptides, and some other small molecule metabolites. In addition, the review ends with concluding remarks on the challenges and future directions for marine sediment-derived microorganisms and their bioactive metabolites. The review report not only helps to deepen the understanding of marine sediment-derived microorganisms and their bioactive metabolites, but also provides some useful information for the exploitation and utilization of marine microbial resources and the mining of new compounds with potential functional properties. [ABSTRACT FROM AUTHOR]

Colon cancer is one of the leading causes of cancer in the United States. Colon cancer develops from the many gene mutations found in the genomes of colon cancer cells. Long non-coding RNAs (lncRNAs) can cause the development and progression of many cancers, including colon cancer. LncRNAs have been and could be corrected through the gene-editing technology of the clustered repeats of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease 9 (CRISPR/Cas9) system to reduce the proliferation of cancer cells in the colon. However, many current delivery systems for transporting CRISPR/Cas9-based therapeutics in vivo need more safety and efficiency. CRISPR/Cas9-based therapeutics require a safe and effective delivery system to more directly and specifically target cancer cells present in the colon. This review will present pertinent evidence for the increased efficiency and safety of using plant-derived exosome-like nanoparticles as nanocarriers for delivering CRISPR/Cas9-based therapeutics to target colon cancer cells directly. [ABSTRACT FROM AUTHOR]

Background: Ulcerative colitis is one of the two main forms of inflammatory bowel disease. Cuproptosis is reported to be a novel mode of cell death. Methods: We examined clusters of cuproptosis related genes and immune cell infiltration molecules in 86 ulcerative colitis samples from the GSE179285 dataset. We identified the differentially expressed genes according to the clustering method, and the performance of the SVM model, the random forest model, the generalized linear model, and the limit gradient enhancement model were compared, and then the optimal machine model was selected. To assess the accuracy of the learning predictions, the nomogram and the calibration curve and decision curve analyses showed that the subtypes of ulcerative colitis have been accurately predicted. Results: Significant cuproptosis-related genes and immune response cells were detected between the ulcerative colitis and control groups. Two cuproptosis-associated molecular clusters were identified. Immune infiltration analysis indicated that different clusters exhibited significant heterogeneity. The immune scores for Cluster2 were elevated. Both the residual error and root mean square error of the random forest machine model had clinical significance. There was a clear correlation between the differentially expressed genes in cluster 2 and the response of immune cells. The nomogram and the calibration curve and decision curve analyses showed that the subtypes of ulcerative colitis had sufficient accuracy. Conclusion: We examined the complex relationship between cuproptosis and ulcerative colitis in a systematic manner. To estimate the likelihood that each subtype of cuproptosis will occur in ulcerative colitis patients and their disease outcome, we developed a promising prediction model. [ABSTRACT FROM AUTHOR]

With the advent of rapid automated in silico identification of biosynthetic gene clusters (BGCs), genomics presents vast opportunities to accelerate natural product (NP) discovery. However, prolific NP producers, Streptomyces, are exceptionally GC-rich (>80%) and highly repetitive within BGCs. These pose challenges in sequencing and high-quality genome assembly which are currently circumvented via intensive sequencing. Here, we outline a more cost-effective workflow using multiplex Illumina and Oxford Nanopore sequencing with hybrid long-short read assembly algorithms to generate high quality genomes. Our protocol involves subjecting long read-derived assemblies to up to 4 rounds of polishing with short reads to yield accurate BGC predictions. We successfully sequenced and assembled 8 GC-rich Streptomyces genomes whose lengths range from 7.1 to 12.1 Mb with a median N50 of 8.2 Mb. Taxonomic analysis revealed previous misrepresentation among these strains and allowed us to propose a potentially new species, Streptomyces sydneybrenneri. Further comprehensive characterization of their biosynthetic, pan-genomic and antibiotic resistance features especially for molecules derived from type I polyketide synthase (PKS) BGCs reflected their potential as alternative NP hosts. Thus, the genome assemblies and insights presented here are envisioned to serve as gateway for the scientific community to expand their avenues in NP discovery. [ABSTRACT FROM AUTHOR]

The timing of ice ages over the past ∼2,600 thousand years (kyr) follows pacing by cyclical changes in three aspects of Earth's orbit that influence the solar energy received as a function of latitude and season. Explaining the large magnitude of the climate changes is challenging, particularly so across the period of time from ∼1,250 to 750 ka—the Mid-Pleistocene Transition or MPT. The average repeat time of ice age cycles changed from an earlier 41-kyr rhythm to longer and more intense glaciations at a spacing of about 100 kyr. Explaining this change is very difficult because there was no corresponding change in the orbital pacing that would trigger a change in timing. While the first generation of hypotheses looked largely to changes in the behavior of Northern Hemisphere ice sheets, more recent work integrates ice behavior with new data capturing the evolution of other important aspects of past climate. A full explanation is still lacking, but attention increasingly focuses on the ocean carbon cycle and atmospheric CO2 levels as the crucial agents involved in the MPT. The pattern of climate changes connected to the ice ages of the past few million years changed radically between about 1,250 and 750 thousand years ago, a time known as the Mid-Pleistocene Transition or MPT. While the glacial cycles were ultimately triggered by cyclical changes in Earth's orbit, the changes across the MPT came from changes in the Earth system itself, most likely in the form of a change in the carbon cycle. The dramatic change in many essential aspects of climate—ice volume, temperature, rainfall on land, and many others—in the absence of an external change suggests how important feedbacks are to the climate system. [ABSTRACT FROM AUTHOR]

The capture of metagenomic DNA in large clone libraries provides the opportunity to study microbial diversity that is inaccessible using culture-dependent methods. In this study, we harnessed nuclease-deficient Cas9 to establish a CRISPR counter-selection interruption circuit (CCIC) that can be used to retrieve target clones from complex libraries. Combining modern sequencing methods with CCIC cloning allows for rapid physical access to the genetic diversity present in natural ecosystems. A CRISPRi method retrieves sequences of interest from large metagenomic libraries. [ABSTRACT FROM AUTHOR]

Background: The filaggrin (FLG) protein, encoded by the FLG gene, is an intermediate filament-associated protein that plays a crucial role in the terminal stages of human epidermal differentiation. Loss-of-function mutations in the FLG exon 3 have been associated with skin diseases. The identification of causative mutations is challenging, due to the high sequence homology within its exon 3 (12,753 bp), which includes 10 to 12 filaggrin tandem repeats. With this study we aimed to obtain the whole FLG exon 3 sequence through PacBio technology, once 13-kb amplicons have been generated. Methods and results: For the preparation of SMRTbell libraries to be sequenced using PacBio technology, we focused on optimizing a 2-step long-range PCR protocol to generate 13-kb amplicons covering the whole FLG exon 3 sequence. The performance of three long-range DNA polymerases was assessed in an attempt to improve the PCR conditions required for the enzymes to function properly. We focused on optimization of the input template DNA concentration and thermocycling parameters to correctly amplify the entire FLG exon 3 sequence, minimizing non-specific amplification. Conclusions: Taken together, our findings suggested that the PrimeSTAR protocol is suitable for producing the amplicons of the 13-kb FLG whole exon 3 to prepare SMRTbell libraries. We suggest that sequencing the generated amplicons may be useful for identifying LoF variants that are causative of the patients' disorders. [ABSTRACT FROM AUTHOR]

ETV4, one of ETS proteins overexpressed in prostate cancer, promotes migration, invasion, and proliferation in prostate cells. This study identifies a series of previously unknown ETV4 alternatively spliced transcripts in human prostate cell lines. Their expression has been validated using several unbiased techniques, including Nanopore sequencing. Most of these transcripts originate from an in-frame exon skipping and, thus, are expected to be translated into ETV4 protein isoforms. Functional analysis of the most abundant among these isoforms shows that they still bear an activity, namely a reduced ability to promote proliferation and a residual ability to regulate the transcription of ETV4 target genes. Alternatively spliced genes are common in cancer cells: an analysis of the TCGA dataset confirms the abundance of these novel ETV4 transcripts in prostate tumors, in contrast to peritumoral tissues. Since none of their translated isoforms have acquired a higher oncogenic potential, such abundance is likely to reflect the tumor deranged splicing machinery. However, it is also possible that their interaction with the canonical variants may contribute to the biology and the clinics of prostate cancer. Further investigations are needed to elucidate the biological role of these ETV4 transcripts and of their putative isoforms. [ABSTRACT FROM AUTHOR]

Background: Apicomplexa consist of numerous pathogenic parasitic protistan genera that invade host cells and reside and replicate within the parasitophorous vacuole (PV). Through this interface, the parasite exchanges nutrients and affects transport and immune modulation. During the intracellular life-cycle, the specialized secretory organelles of the parasite secrete an array of proteins, among which dense granule proteins (GRAs) play a major role in the modification of the PV. Despite this important role of GRAs, a large number of potential GRAs remain unidentified in Apicomplexa. Methods: A multi-view attention graph convolutional network (MVA-GCN) prediction model with multiple features was constructed using a combination of machine learning and genomic datasets, and the prediction was performed on selected Neospora caninum protein data. The candidate GRAs were verified by a CRISPR/Cas9 gene editing system, and the complete NcGRA64(a,b) gene knockout strain was constructed and the phenotypes of the mutant were analyzed. Results: The MVA-GCN prediction model was used to screen N. caninum candidate GRAs, and two novel GRAs (NcGRA64a and NcGRA64b) were verified by gene endogenous tagging. Knockout of complete genes of NcGRA64(a,b) in N. caninum did not affect the parasite's growth and replication in vitro and virulence in vivo. Conclusions: Our study showcases the utility of the MVA-GCN deep learning model for mining Apicomplexa GRAs in genomic datasets, and the prediction model also has certain potential in mining other functional proteins of apicomplexan parasites. [ABSTRACT FROM AUTHOR]

The gene products of the HLA/MHC and KIR multigene families are important modulators of the immune system and are associated with health and disease. Characterization of the genes encoding these receptors has been integrated into different biomedical applications, including transplantation and reproduction biology, immune therapies and in fundamental research into disease susceptibility or resistance. Conventional short‐read sequencing strategies have shown their value in high throughput typing, but are insufficient to uncover the entire complexity of the highly polymorphic HLA/MHC and KIR gene systems. The implementation of single‐molecule and real‐time sequencing platforms, offered by Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT), revolutionized the fields of genomics and transcriptomics. Using fundamentally distinct principles, these platforms generate long‐read data that can unwire the plasticity of the HLA/MHC and KIR genes, including high‐resolution characterization of genes, alleles, phased haplotypes, transcription levels and epigenetics modification patterns. These insights might have profound clinical relevance, such as improved matching of donors and patients in clinical transplantation, but could also lift disease association studies to a higher level. Even more, a comprehensive characterization may refine animal models in preclinical studies. In this review, the different HLA/MHC and KIR characterization approaches using PacBio and ONT platforms are described and discussed. [ABSTRACT FROM AUTHOR]

Next-generation sequencing (NGS) is a highly effective genetic diagnostic test used in disease diagnosis. Although the Sanger method is used as the traditional method in genome studies, the use of NGS methods has been increasing with the development of technology. The foundation of next-generation sequencing was laid with the methods developed by Allan Maxam--Walter Gilbert and 2 Nobel laureates, Frederick Sanger. Initially, first-generation sequencing methods completed a certain part of the DNA with great efforts in a few days, while in today's technology, the entire DNA of even the most complex organisms is sequenced in 1 day. Second- and third-generation sequencing methods have been developed with improvements in cost, time, and accuracy of sequencing. The data obtained from these methods are interpreted with bioinformatics and contributed to the development of next-generation sequencing technology. These developments have increased the interest in studies on the relationship between next-generation sequencing and DNA or RNA depending on diseases. In this review, past and present methods of next-generation sequencing technologies are mentioned in detail and the difficulties and conveniences of these methods are reviewed. [ABSTRACT FROM AUTHOR]

Background: Structural variants (SVs) play important roles in adaptation evolution and species diversification. Especially, in plants, many phenotypes of response to the environment were found to be associated with SVs. Despite the prevalence and significance of SVs, long insertions remain poorly detected and studied in all but model species. Results: We used whole-genome resequencing of paired reads from 80 Asian butternuts to detect long insertions and further analyse their characteristics and potential functional effects. By combining of mapping-based and de novo assembly-based methods, we obtained a multiple related species pangenome representing higher taxonomic groups. We obtained 89,312 distinct contigs totaling 147,773,999 base pair (bp) of new sequences, of which 347 were putative long insertions placed in the reference genome. Most of the putative long insertions appeared in multiple species; in contrast, only 62 putative long insertions appeared in one species, which may be involved in the response to the environment. 65 putative long insertions fell into 61 distinct protein-coding genes involved in plant development, and 105 putative long insertions fell into upstream of 106 distinct protein-coding genes involved in cellular respiration. 3,367 genes were annotated in 2,606 contigs. We propose PLAINS (https://github.com/CMB-BNU/PLAINS.git), a streamlined, comprehensive pipeline for the prediction and analysis of long insertions using whole-genome resequencing. Conclusions: Our study lays down an important foundation for further whole-genome long insertion studies, allowing the investigation of their effects by experiments. [ABSTRACT FROM AUTHOR]

The role of the tropical Pacific Ocean and its linkages to the southern hemisphere during the last deglacial warming remain highly controversial. Here we explore the evolution of Pacific horizontal and vertical thermal gradients over the past 30 kyr by compiling 340 sea surface and 7 subsurface temperature records, as well as one new ocean heat content record. Our records reveal that La Niña-like conditions dominated during the deglaciation as a result of the more intense warming in the western Pacific warm pool. Both the subsurface temperature and ocean heat content in the warm pool rose earlier than the sea surface temperature, and in phase with South Pacific subsurface temperature and orbital precession, implying that heat exchange between the tropical upper water column and the extratropical Southern Ocean facilitated faster warming in the western Pacific. Our study underscores the key role of the thermal coupling between the warm pool and the Southern Ocean and its relevance for future global warming. The mechanism of the last deglacial global warming is key for future climate. Here, the authors shed light on the pivotal role of the thermal coupling between the western Pacific warm pool and the Southern Ocean. [ABSTRACT FROM AUTHOR]

The Subantarctic Southern Ocean has long been thought to be an important contributor to increases in atmospheric carbon dioxide partial pressure (pCO2) during glacial-interglacial transitions. Extensive studies suggest that a weakened biological pump, a process associated with nutrient utilization efficiency, drove up surface-water pCO2 in this region during deglaciations. By contrast, regional influences of the solubility pump, a process mainly linked to temperature variations, have been largely overlooked. Here, we evaluate relative roles of the biological and solubility pumps in determining surface-water pCO2 variabilities in the Subantarctic Southern Ocean during the last deglaciation, based on paired reconstructions of surface-water pCO2, temperature, and nutrient utilization efficiency. We show that compared to the biological pump, the solubility pump imposed a strong impact on deglacial Subantarctic surface-water pCO2 variabilities. Our findings therefore reveal a previously underappreciated role of the solubility pump in modulating deglacial Subantarctic CO2 release and possibly past atmospheric pCO2 fluctuations. Using paired reconstructions of seawater pCO2, temperature, and nutrient utilization, Dai et al. show underappreciated influences of the solubility pump on deglacial Subantarctic surface-water pCO2 variabilities compared to the biological pump. [ABSTRACT FROM AUTHOR]

Background: Recent genome-wide studies revealed the molecular subtypes and mutational landscape of bladder cancer, which is the 10th most common cancer causing many deaths. ELF3 is one of the frequently mutated genes in bladder cancer with 14% alteration rate. It mainly functions as an epithelial transcription factor and its proper function is critical for the urothelium development. However, the impact of ELF3 mutations in bladder cancer is currently unknown. Methods and results: In this study, we analysed the gene expression data available for primary bladder cancer and bladder cancer cell lines according to the mutation status of ELF3. Our results show that de-regulated genes common in cell lines and primary tissue are primarily involved in ameboidal type cell migration and cell–cell junction organization. Additionally, we identify that ELF3-mutant cases in primary samples significantly overexpress PIK3C2B and ELF3 and PIK3C2B and ELF3 are significantly co-mutated in many cancer types. Our integrative analysis with existing Hi-C data further revealed the genes proximally located to ELF3, including PIK3C2B to be upregulated in ELF3 mutant cases, potentially as a result of truncated ELF3 protein product and subsequent changes in regulatory interactions. Conclusions: Our results provide important insights about how ELF3 mutation contributes to bladder tumorigenesis and uncover previously unknown dependencies. [ABSTRACT FROM AUTHOR]

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Reconstruction of last millennia Sea Surface Temperature (SST) evolution is challenging due to the difficulty retrieving good resolution marine records and to the several uncertainties in the available proxy tools. In this regard, the Roman Period (1 CE to 500 CE) was particularly relevant in the socio-cultural development of the Mediterranean region while its climatic characteristics remain uncertain. Here we present a new SST reconstruction from the Sicily Channel based in Mg/Ca ratios measured on the planktonic foraminifer Globigerinoides ruber. This new record is framed in the context of other previously published Mediterranean SST records from the Alboran Sea, Minorca Basin and Aegean Sea and also compared to a north Hemisphere temperature reconstruction. The most solid image that emerges of this trans-Mediterranean comparison is the persistent regional occurrence of a distinct warm phase during the Roman Period. This record comparison consistently shows the Roman as the warmest period of the last 2 kyr, about 2 °C warmer than average values for the late centuries for the Sicily and Western Mediterranean regions. After the Roman Period a general cooling trend developed in the region with several minor oscillations. We hypothesis the potential link between this Roman Climatic Optimum and the expansion and subsequent decline of the Roman Empire. [ABSTRACT FROM AUTHOR]

Ventilation of carbon stored in the deep ocean is thought to play an important role in atmospheric CO2 increases associated with Pleistocene deglaciations. The presence of this respired carbon has been recorded by an array of paleoceanographic proxies from various locations across the global ocean. Here we present a new sediment core from the Eastern Equatorial Pacific (EEP) Ocean spanning the last 180,000 years and reconstruct high-resolution 230Th-derived fluxes of 232Th and excess barium, along with redox-sensitive uranium concentrations to examine past variations in dust delivery, export productivity, and bottom-water oxygenation, respectively. Our bottom-water oxygenation record is compared to other similar high-resolution records from across the Pacific and in the Southern Ocean. We suggest that the deep Pacific is a site of respired carbon storage associated with periods of decreased global atmospheric CO2 concentration during the LGM, confirming the conclusions from a wealth of previous studies. However, our study is the first to show a similar relationship beyond the last glacial, extending to at least 70,000 years. [ABSTRACT FROM AUTHOR]

This study evaluates paleoclimate sensitivity over the past 800,000 years from proxy-based reconstructions of changes in global temperature, ice sheets and sea level, vegetation, dust, and greenhouse gases. This analysis uses statistical methods that are not biased by the variable (heteroscedastic) uncertainty in the reconstructions, and applies a Monte Carlo-style probabilistic framework to quantify several sources of measurement and structural uncertainty. Not addressing the heteroscedastic uncertainty would result in regression results that underestimate paleoclimate sensitivity by over 30%, and not using a probabilistic framework could underestimate the credible interval by fivefold. A comparison of changes in global temperature (ΔT) and changes in radiative forcing from greenhouse gases, ice sheets, dust, and vegetation (ΔR[GHG,LI,AE,VG]) over the past 800 kyr finds that the two are closely coupled across glacial cycles with a correlation of 0.81 (0.6 to 0.9, 95% credible interval). The variation of ΔT with ΔR over the past 800 kyr is non-linear, with lower correlation and lower responsiveness at colder temperatures. The paleoclimate sensitivity parameter estimates (S[GHG,LI,AE,VG]) are 0.84 °C/W/m2 (0.20 to 1.9 °C/W/m2, 95% interval) for interglacial periods and intermediate glacial climates and 0.53 °C/W/m2 (0.08 to 1.5 °C/W/m2, 95% interval) for full glacial climates, 37% lower at the median. The estimates of S[GHG,LI,AE,VG] and the pattern of state dependence are similar across glacial cycles over the past 800 kyr. This analysis explicitly includes several sources of uncertainty and is still able to provide a strong upper bound for the paleoclimate sensitivity parameter for interglacial periods and intermediate glacial climates: over 1.5 °C/W/m2 is < 10% probability, 1.7 °C/W/m2 is < 5% probability, and over 1.9 °C/W/m2 is < 2.5% probability. [ABSTRACT FROM AUTHOR]

Combining instrumental period evidence regarding equilibrium climate sensitivity with largely independent paleoclimate proxy evidence should enable a more constrained sensitivity estimate to be obtained. Previous, subjective Bayesian approaches involved selection of a prior probability distribution reflecting the investigators’ beliefs about climate sensitivity. Here a recently developed approach employing two different statistical methods—objective Bayesian and frequentist likelihood-ratio—is used to combine instrumental period and paleoclimate evidence based on data presented and assessments made in the IPCC Fifth Assessment Report. Probabilistic estimates from each source of evidence are represented by posterior probability density functions (PDFs) of physically-appropriate form that can be uniquely factored into a likelihood function and a noninformative prior distribution. The three-parameter form is shown accurately to fit a wide range of estimated climate sensitivity PDFs. The likelihood functions relating to the probabilistic estimates from the two sources are multiplicatively combined and a prior is derived that is noninformative for inference from the combined evidence. A posterior PDF that incorporates the evidence from both sources is produced using a single-step approach, which avoids the order-dependency that would arise if Bayesian updating were used. Results are compared with an alternative approach using the frequentist signed root likelihood ratio method. Results from these two methods are effectively identical, and provide a 5-95% range for climate sensitivity of 1.1-4.05 K (median 1.87 K). [ABSTRACT FROM AUTHOR]

The present review discusses different mass spectrometric techniques-viz, thermal ionization mass spectrometry (TIMS), inductively coupled plasma mass spectrometry (ICPMS), and secondary ion mass spectrometry (SIMS)-used to determine 11B/10B isotope ratio, and concentration of boron required for various applications in earth sciences, marine geochemistry, nuclear technology, environmental, and agriculture sciences, etc. The details of the techniques-P-TIMS, which uses Cs2BO2+, N-TIMS, which uses BO2−, and MC-ICPMS, which uses B+ ions for bulk analysis or B− and B+ ions for in situ micro-analysis with SIMS-are highlighted. The capabilities, advantages, limitations, and problems in each mass spectrometric technique are summarized. The results of international interlaboratory comparison experiments conducted at different times are summarized. The certified isotopic reference materials available for boron are also listed. Recent developments in laser ablation (LA) ICPMS and QQQ-ICPMS for solids analysis and MS/MS analysis, respectively, are included. The different aspects of sample preparation and analytical chemistry of boron are summarized. Finally, the future requirements of boron isotope ratios for future applications are also given. Presently, MC-ICPMS provides the best precision and accuracy (0.2-0.4‰) on isotope ratio measurements, whereas N-TIMS holds the potential to analyze smallest amount of boron, but has the issue of bias (+2‰ to 4‰) which needs further investigations. © 2016 Wiley Periodicals, Inc. Mass Spec Rev 36:499-519, 2017 [ABSTRACT FROM AUTHOR]

Infectious diseases are a major global cause of morbidity and mortality, seriously affecting public health and socioeconomic stability. Since infectious diseases can be caused by a wide variety of pathogens with similar clinical manifestations and symptoms that are difficult to accurately distinguish, selecting the appropriate diagnostic techniques for the rapid identification of pathogens is crucial for clinical disease diagnosis and public health management. However, traditional diagnostic techniques have low detection rates, long detection times and limited automation, which means that they do not meet the requirements for rapid diagnosis. Recent years have seen continuous developments in molecular detection technology, which has a higher sensitivity and specificity, shorter detection time and increased automation, and performs an important role in the early and rapid detection of infectious disease pathogens. The present study summarizes recent progress in molecular diagnostic technologies such as PCR, isothermal amplification, gene chips and high-throughput sequencing for the detection of infectious disease pathogens, and compares the technical principles, advantages and disadvantages, applicability and costs of these diagnostic techniques. [ABSTRACT FROM AUTHOR]

We apply a novel method based upon 'before' and 'after' relationships to investigate and quantify interconnections between global temperature anomaly (GTA), as response variable, and greenhouse gases (CO) and total solar irradiance (TSI) as candidate causal variables for the period 1880 to 2010. The most likely interpretations of our results for the 6 to 8 years cyclic components of the variables are that during the period 1929 to 1936, CO significantly leads GTA. However, during the period 1960-2003, GTA apparently leads CO, that is, the peaks (and troughs) in GTA are in front of, and close to, the peaks (and troughs) in CO For time windows outside these periods, we did not find significant before or after-relations. An alternative interpretation is that there is a shift between short (≈1.5 year) and long (≈5 years) durations between cause and effect. Relationships between GTA and TSI suggest that 'inertia' of the global sea, land, and atmosphere system leads to delays longer than half their common cycle length of about 10 years. Based on the interaction patterns between the variables GTA, CO, and TSI, we suggest the possibility that a new regime for how the variables interact started around 1960. From trend forms, and not considering physical mechanisms, we found that the trend in CO contributes ≈ 90 %, and the trend in TSI ≈ 10 %, to the trend in GTA during the last 130 years. [ABSTRACT FROM AUTHOR]

This paper purports the temporal and spatial variations of total electron content (TEC), outgoing longwave radiation (OLR) and sea surface temperature during the greater earthquake regime. Nighttime TEC variations have been observed to be lower than those of the daytime. They were in the range of 10-30 TECU ( µ = 16 TECU) over nighttime, whereas 12-40 TECU ( µ = 26 TECU) during daytime. The coefficient of variation (CV) is of 32 % during the daytime and 51 % over the nighttime by using the IRI-2012 model (as per the boundary condition and grid-level output during event period). Latitude and longitudinal variations of TEC values integrated over the preparatory zone with in 5-h limits are in the range of 8-48 TECU ( µ = 30 TECU, CV = 45 %) and 14-48 TECU ( µ = 28 TECU, CV = 37 %). Mapping for the 2 days anomalies of OLR have good influence with the effect of greater earthquake; except the three events, it has increased from 20 to 100 watts/m. The detailed synthesis of this paper has showered light on the necessity of spatial variation of the TEC apart from temporal variations. [ABSTRACT FROM AUTHOR]

Evaluating the relationships between climate, the environment, and human traits is a key part of human origins research because changes in Earth's atmosphere, oceans, landscapes, and ecosystems over the past 10 Myr shaped the selection pressures experienced by early humans. In Africa, these relationships have been influenced by a combination of high-latitude ice distributions, sea surface temperatures, and low-latitude orbital forcing that resulted in large oscillations in vegetation and moisture availability that were modulated by local basin dynamics. The importance of both climate and tectonics in shaping African landscapes means that integrated views of the ecological, environmental, and tectonic histories of a region are necessary in order to understand the relationships between climate and human evolution. [ABSTRACT FROM AUTHOR]

Examines the seals of the last Singamangaraja, high priest of the Batak people in North Sumatra, Indonesia. Origin of the seals; Shows of the North Sumatra map; Role of Ompu Pulo Batu as the Singamangaraja in uniting Batak people.