Contiguous Sequence Research Articles

Complete genome of Xanthomonas citri pv. anacardii strain CCRMTAQ13 (causal agent of angular leaf spot in cashew) from Brazil using long-read Nanopore technology.

Here, we present a single contiguous genome sequence of Xanthomonas citri pv. anacardii (Xca; strain CCRMTAQ13), the causal agent of angular leaf spot in cashew, an important commodity crop in Brazil. Oxford Nanopore Sequencing Technology was used to assemble the genome of the Xca in a single contig of 5,086,757 bp with a 64.53% GC content. This genome sequence will provide a useful resource for studies on virulence mechanisms, including the identification of a single transcription activator-like effector gene of 3,359 bp.

Statistical design approach enables optimised mechanical lysis for enhanced long-read soil metagenomics

Metagenomic analysis has enabled insights into soil community structure and dynamics. Long-read sequencing for metagenomics can enhance microbial ecology by improving taxonomic classification, genome assembly, and functional annotation. However, protocols for purifying high-molecular weight DNA from soil are not yet optimised. We used a statistical design of experiments approach to enhance mechanical lysis of soil samples, increasing the length of purified DNA fragments. Low energy input into mechanical lysis improved DNA integrity, resulting in longer sequenced reads. Our optimized settings of 4 m s−1 for 10 s increased fragment length by 70% compared to the manufacturer’s recommendations. Longer reads from low intensity lysis produced longer contiguous sequences after assembly, potentially improving a range of down-stream analyses. Importantly, there was minimal bias exhibited in the microbial community composition due to lysis efficiency variations. We therefore propose a framework for improving the fragment lengths of DNA purified from diverse soil types, improving soil science research with long-read sequencing.

Removal of sequencing adapter contamination improves microbial genome databases

Advances in assembling microbial genomes have led to growth of reference genome databases, which have been transformative for applied and basic microbiome research. Here we show that published microbial genome databases from humans, mice, cows, pigs, fish, honeybees, and marine environments contain significant sequencing-adapter contamination that systematically reduces assembly accuracy and contiguousness. By removing the adapter-contaminated ends of contiguous sequences and reassembling MGnify reference genomes, we improve the quality of assemblies in these databases.

Where is the boundary of the human pseudoautosomal region?

A recent publication describing the assembly of the Y chromosomes of 43 males was remarkable not only for its ambitious technical scope but also for the startling suggestion that the boundary of the pseudoautosomal region 1 (PAR1), where the human X and Y chromosomes engage in crossing-over during male meiosis, lies 500 kb distal to its previously reported location. Where is the boundary of the human PAR1? We first review the evidence that mapped the PAR boundary, or PAB, before the human genome draft sequence was produced, then examine post-genomic datasets for evidence of crossing-over between the X and Y, and lastly re-examine contiguous sequence assemblies of the PAR-NPY boundary to see whether they support a more distal PAB. We find ample evidence of X-Y crossovers throughout the 500 kb in question, some as close as 246bp to the previously reported PAB. Our new analyses, combined with previous studies over the past 40 years, provide overwhelming evidence to support the original position and narrow the probable location of the PAB to a 201-bp window.

In vivo treatment with a non-aromatizable androgen rapidly alters the ovarian transcriptome of previtellogenic secondary growth coho salmon (Onchorhynchus kisutch).

Recent evidence suggests that androgens are a potent driver of growth during late the primary stage of ovarian follicle development in teleosts. We have previously shown that the non-aromatizable androgen, 11-ketotestosterone (11-KT), both advances ovarian follicle growth in vivo and dramatically alters the primary growth ovarian transcriptome in coho salmon. Many of the transcriptomic changes pointed towards 11-KT driving process associated with the transition to a secondary growth phenotype. In the current study, we implanted previtellogenic early secondary growth coho salmon with cholesterol pellets containing 11-KT and performed RNA-Seq on ovarian tissue after 3 days in order to identify alterations to the ovarian transcriptome in early secondary growth. We identified 8,707 contiguous sequences (contigs) that were differentially expressed (DE) between control and 11-KT implanted fish and were able to collapse those to 3,853 gene-level IDs, more than a 3-fold more DE contigs than at the primary growth stage we reported previously. These contigs included genes encoding proteins involved in steroidogenesis, vitellogenin and lipid uptake, follicle stimulating hormone signaling, growth factor signaling, and structural proteins, suggesting androgens continue to promote previtellogenic secondary growth.

Units containing telomeric repeats are prevalent in subtelomeric regions of a Mesorhabditis isolate collected from the Republic of Korea.

Mesorhabditis is known for its somatic genome being only a small portion of the germline genome due to programmed DNA elimination. This phenotype may be associated with the maintenance of telomeres at the ends of fragmented somatic chromosomes. To comprehensively investigate the telomeric regions of Mesorhabditis nematodes at the sequence level, we endeavored to collect a Mesorhabditis nematode in the Republic of Korea and acquire its highly contiguous genome sequences. We isolated a Mesorhabditis nematode and assembled its 108-Mb draft genome using both 6.3Gb (53 ×) of short-read and 3.0Gb (25 × , N50 = 5.7kb) of nanopore-based long-read sequencing data. Our genome assembly exhibits comparable quality to the public genome of Mesorhabditis belari in terms of contiguity and evolutionary conserved genes. Unexpectedly, our Mesorhabditis genome has many more interstitial telomeric sequences (ITSs), specifically subtelomeric ones, compared to the genomes of Caenorhabditis elegans and M. belari. Moreover, several subtelomeric sequences containing ITSs had 4-26 homologous sequences, implying they are highly repetitive. Based on this highly repetitive nature, we hypothesize that subtelomeric ITSs might have accumulated through the action of transposable elements containing ITSs. It still remains elusive whether these ITS-containing units are associated with programmed DNA elimination, but they may facilitate new telomere formation after DNA elimination. Our genomic resources for Mesorhabditis can aid in understanding how its distinct phenotypes have evolved.

An in silico design method of a peptide bioreceptor for cortisol using molecular modelling techniques

Cortisol is established as a reliable biomarker for stress prompting intensified research in developing wearable sensors to detect it via eccrine sweat. Since cortisol is present in sweat in trace quantities, typically 8–140 ng/mL, developing such biosensors necessitates the design of bioreceptors with appropriate sensitivity and selectivity. In this work, we present a systematic biomimetic methodology and a semi-automated high-throughput screening tool which enables rapid selection of bioreceptors as compared to ab initio design of peptides via computational peptidology. Candidate proteins from databases are selected via molecular docking and ranked according to their binding affinities by conducting automated AutoDock Vina scoring simulations. These candidate proteins are then validated via full atomistic steered molecular dynamics computations including umbrella sampling to estimate the potential of mean force using GROMACS version 2022.6. These explicit molecular dynamic calculations are carried out in an eccrine sweat environment taking into consideration the protein dynamics and solvent effects. Subsequently, we present a candidate baseline peptide bioreceptor selected as a contiguous sequence of amino acids from the selected protein binding pocket favourably interacting with the target ligand (i.e., cortisol) from the active binding site of the proteins and maintaining its tertiary structure. A unique cysteine residue introduced at the N-terminus allows orientation-specific surface immobilization of the peptide onto the gold electrodes and to ensure exposure of the binding site. Comparative binding affinity simulations of this peptide with the target ligand along with commonly interfering species e.g., progesterone, testosterone and glucose are also presented to demonstrate the validity of this proposed peptide as a candidate baseline bioreceptor for future cortisol biosensor development.

Identification of Sentence Stems Characteristic of Chinese Learner English Writing

Phraseological units in academic English texts have been a central focus in recent corpus linguistic research. This paper describes a special category of clause-level phraseological units, namely, Characteristic Sentence Stems (CSSs), with a view to describing their identifying criteria and their extraction method. CSSs are contiguous lexico-grammatical sequences which contain a subject-predicate structure and which are frame expressions characteristic of academic writing. The extraction method of a CSS consists of six steps: POS tagging, n-gram segmentation, structure identification, significance of occurrence calculation, text range calculation, and overlapping sequence reduction. The significance of occurrence calculation is the crux of this method. It includes the computing of both the internal association and the boundary independence of a CSS, and it tests the occurring significance of the CSS from both the inside and the outside perspectives. Our methods and results suggest that CSSs can be statistically defined and extracted from corpora and can employed in large-scale studies to more fully account for the phraseological features of non-native English academic writing.

Unlocking plant genetics with telomere-to-telomere genome assemblies.

Contiguous genome sequence assemblies will help us to realize the full potential of crop translational genomics. Recent advances in sequencing technologies, especially long-read sequencing strategies, have made it possible to construct gapless telomere-to-telomere (T2T) assemblies, thus offering novel insights into genome organization and function. Plant genomes pose unique challenges, such as a continuum of ancient to recent polyploidy and abundant highly similar and long repetitive elements. Owing to progress in sequencing approaches, for most crop plants, chromosome-scale reference genome assemblies are available, but T2T assembly construction remains challenging. Here we describe methods for haplotype-resolved, gapless T2T assembly construction in plants, including various crop species. We outline the impact of T2T assemblies in elucidating the roles of repetitive elements in gene regulation, as well as in pangenomics, functional genomics, genome-assisted breeding and targeted genome manipulation. In conjunction with sequence-enriched germplasm repositories, T2T assemblies thus hold great promise for basic and applied plant sciences.

Development of a Reference Transcriptome and Identification of Differentially Expressed Genes Linked to Salt Stress in Salt Marsh Grass (Sporobolus alterniflorus) along Delaware Coastal Regions.

Salt marsh grass (Sporobolus alterniflorus) plays a crucial role in Delaware coastal regions by serving as a physical barrier between land and water along the inland bays and beaches. This vegetation helps to stabilize the shoreline and prevent erosion, protecting the land from the powerful forces of the waves and tides. In addition to providing a physical barrier, salt marsh grass is responsible for filtering nutrients in the water, offering an environment for aquatic species and presenting a focal point of study for high salt tolerance in plants. As seawater concentrations vary along the Delaware coast from low to medium to high salinity, our study seeks to identify the impact of salt tolerance in marsh grass and to identify genes associated with salt tolerance levels. We developed more than 211,000 next-generation-sequencing (Illumina) transcriptomic reads to create a reference transcriptome from low-, medium-, and high-salinity marsh grass leaf samples collected from the Delaware coastline. Contiguous sequences were annotated based on a homology search using BLASTX against rice (Oryza sativa), foxtail millet (Setaria italica), and non-redundant species within the Viridiplantae database. Additionally, we identified differentially expressed genes related to salinity stress as candidates for salt stress qPCR analysis. The data generated from this study may help to elucidate the genetic signatures and physiological responses of plants to salinity stress, thereby offering valuable insight into the use of innovative approaches for gene expression studies in crops that are less salt tolerant.

P-564 Decoding ovarian response: the predictive power of FSH receptor gene expression in controlled ovarian stimulation

Abstract Study question Can profiling of variation in follicle-stimulating hormone receptor (FSHR) gene expression forecast response to Controlled Ovarian Stimulation (COS) in Assisted Reproductive Techniques? Summary answer Full-length FSHR transcript was the most common mRNA species. Nine distinct transcripts were characterised, but only one (deletion Exon 6) was associated with altered response. What is known already AMH levels and age are considered useful predictors of ovarian response to COS (high AMH and young age typically associated with high yields of oocytes; older ages and low AMH associated with fewer oocytes). However, it is not uncommon to see patients who do not fit this pattern, highlighting the limitations of these parameters in predicting response. Age-independent biomarkers capable of revealing patients at elevated risk of hyperstimulation, or those who might produce fewer than the optimal number of oocytes, are desirable. Alternative transcripts of the FSHR gene have been proposed as an explanation for variation in response to COS. Study design, size, duration FSHR splice variants in mRNA in granulosa cells from 126 patients were examined utilising long-read nanopore sequencing. This strategy permitted the sequencing of mRNA variants without fragmentation, allowing the detection of all splicing events in each mRNA strand. In addition to being categorised based on age and AMH levels, patients were also classified by the expression of FSHR transcript variants and their response to stimulation (high, low, or normal response). Participants/materials, setting, methods Patterns of FSHR mRNA splice variants were analysed in each of 126 patients. This involved extraction of RNA from the granulosa cells, followed by long-read nanopore sequencing, permitting analysis of whole transcripts in a single, contiguous sequence ‘read’. Patients were classified based upon their level of response to COS (low, normal, high), taking into account their age, AMH level and the number of oocytes retrieved. Patterns of FSHR transcript variation were compared to COS response. Main results and the role of chance Full-length FSHR transcript was detected in 97.6% of patients and skipping of Exon 6 was observed in 68%. Five novel transcripts were discovered, including partial insertion of ∼100bp segments of introns 1 or 2, seen in 6.55% of samples. In total, nine distinct FSHR mRNA variants were detected. As expected, increasing age was associated with higher FSH requirement per oocyte collected (p = 0.018), and AMH levels declined with age (2.8± 1.42 in women <38 versus 1.58 ±1.29 in women ≥38; p = 0.011). Consistent with previous analyses, AMH levels and amount of FSH per oocyte retrieved were negatively correlated (p < 0.001). One-third of the patients (33%) in this cohort displayed responses to COS that were not considered to be consistent with expectations based upon age and AMH levels. Expression of the novel FSHR transcript variants was not associated with FSH needed per oocyte retrieved (p = 0.78), nor the proportion of mature oocytes (p = 0.95).The only transcript potentially linked to an altered response to COS was the previously reported mRNA species, lacking exon 6, associated with significantly greater numbers of oocytes produced (p = 0.0087). Interestingly, women ≥38 expressed novel variants more frequently than their younger counterparts (p = 0.018), potentially indicative of an age-related decline in transcriptional fidelity. Limitations, reasons for caution It is possible that the use of controlled ovarian stimulation might alter the dynamics of transcription in cumulus cells. Therefore, it is possible that the spectrum of transcripts seen in this study might differ from those present in unstimulated cycles. Wider implications of the findings Expected associations between age, AMH levels and response to COS were observed. Skipping exon 6 may be associated with a superior response to COS, suggesting FSHR mRNA profiling might have a role in tailoring controlled ovarian stimulation protocols. Novel transcript variants were detected and seen most often in older women. Trial registration number not applicable

Fundamental Principles and Emerging Opportunities for Selectively‐Solvated Block Copolymer Networks in Nonpolar Media: A Perspective

AbstractBlock polymers remain an extensively studied class of macromolecules due to their ability to self‐organize spontaneously as a result of microphase separation into a variety of ordered nanostructures, depending on the number of contiguous sequences (“blocks”) present and their sequential arrangement. These polymers are classified as multifunctional since they exhibit two or more different property sets during application. In this work, the focus is on bicomponent block copolymers composed of soft and hard segments arranged as linear triblock or higher‐order multiblock copolymers and possessing the properties of a thermoplastic elastomer (TPE). Of particular interest are selectively‐solvated TPEs, designated as TPE gels (TPEGs), with precisely‐ and composition‐tunable properties. An important aspect of TPEs and their TPEG analogs is their elasticity, which reflects the ability of the soft block(s) to form a contiguous molecular network connected by dispersed microdomains composed of the hard block. Here, the origins of microphase separation and network formation in styrenic TPEs and TPEGs are explored, and experimental, theoretical, and simulation results are examined to elucidate chemistry‐structure‐property‐processing (CSPP) relationships in these self‐networking materials. Once such relationships are established, several unconventional technologies that can directly benefit from TPEGs, along with TPEGs fabricated from TPEs possessing different chemical moieties, are likewise considered.

A large fault partially reactivated during two contiguous seismic sequences in Central Italy: The role of geometrical and frictional heterogeneities

Earthquakes can rupture multiple fault segments as well as faults with complex geometry, or heterogeneous pre-stress and frictional properties. These observations have been documented mainly for moderate-to-large earthquakes by inverting geodetic and seismic data and by studying the influence of fault orientation and rheology within the regional stress field.In this work we have studied the Gorzano fault, GF, a large normal fault within the active fault system of Central Italy that during the last two largest Italian seismic sequences, L'Aquila (2009) and Amatrice-Visso-Norcia (2016–2017), was reactivated via a series of 5.0 < Mw < 6.0 events. We calculated moment tensor solutions for 134 M > 3 events and evaluated their normalized slip-tendency. Merging these results with high resolution earthquake catalogs, available M > 5 earthquake slip distributions, and frictional properties characterizing the activated fault, we develop a mechanical model and discuss potential earthquake rupture scenarios.The GF is an optimally oriented fault within the regional stress field and from, the reactivation via aftershock or mainshock slip of complementary fault portions from 2009 to 2017 indicates that the fault behaves as a single fault structure. The geometrical and mechanical heterogeneities suggest that the most likely slip behavior of GF is the reactivation of different fault portions with M > 5.0. However, due to favorable initial stress conditions, we suggest that a seismic rupture can produce the complete reactivation of the fault, resulting in a M 6.5–6.6 earthquake as documented in paleoseismological data.

Chromosome-Level Assemblies for the Pine Pitch Canker Pathogen Fusarium circinatum.

The pine pitch canker pathogen, Fusarium circinatum, is globally regarded as one of the most important threats to commercial pine-based forestry. Although genome sequences of this fungus are available, these remain highly fragmented or structurally ill-defined. Our overall goal was to provide high-quality assemblies for two notable strains of F. circinatum, and to characterize these in terms of coding content, repetitiveness and the position of telomeres and centromeres. For this purpose, we used Oxford Nanopore Technologies MinION long-read sequences, as well as Illumina short sequence reads. By leveraging the genomic synteny inherent to F. circinatum and its close relatives, these sequence reads were assembled to chromosome level, where contiguous sequences mostly spanned from telomere to telomere. Comparative analyses unveiled remarkable variability in the twelfth and smallest chromosome, which is known to be dispensable. It presented a striking length polymorphism, with one strain lacking substantial portions from the chromosome's distal and proximal regions. These regions, characterized by a lower gene density, G+C content and an increased prevalence of repetitive elements, contrast starkly with the syntenic segments of the chromosome, as well as with the core chromosomes. We propose that these unusual regions might have arisen or expanded due to the presence of transposable elements. A comparison of the overall chromosome structure revealed that centromeric elements often underpin intrachromosomal differences between F. circinatum strains, especially at chromosomal breakpoints. This suggests a potential role for centromeres in shaping the chromosomal architecture of F. circinatum and its relatives. The publicly available genome data generated here, together with the detailed metadata provided, represent essential resources for future studies of this important plant pathogen.

Identifying transgene insertions in Caenorhabditis elegans genomes with Oxford Nanopore sequencing.

Genetically modified organisms are commonly used in disease research and agriculture but the precise genomic alterations underlying transgenic mutations are often unknown. The position and characteristics of transgenes, including the number of independent insertions, influences the expression of both transgenic and wild-type sequences. We used long-read, Oxford Nanopore Technologies (ONT) to sequence and assemble two transgenic strains of Caenorhabditis elegans commonly used in the research of neurodegenerative diseases: BY250 (pPdat-1::GFP) and UA44 (GFP and human α-synuclein), a model for Parkinson's research. After scaffolding to the reference, the final assembled sequences were ∼102 Mb with N50s of 17.9 Mb and 18.0Mb, respectively, and L90s of six contiguous sequences, representing chromosome-level assemblies. Each of the assembled sequences contained more than 99.2% of the Nematoda BUSCO genes found in the C. elegans reference and 99.5% of the annotated C. elegans reference protein-coding genes. We identified the locations of the transgene insertions and confirmed that all transgene sequences were inserted in intergenic regions, leaving the organismal gene content intact. The transgenic C. elegans genomes presented here will be a valuable resource for Parkinson's research as well as other neurodegenerative diseases. Our work demonstrates that long-read sequencing is a fast, cost-effective way to assemble genome sequences and characterize mutant lines and strains.

Transcript Level and Sequence Matching Are Key Determinants of Off-Target Effects in RNAi.

Double-stranded RNA (dsRNA) pesticides, those based on RNA interference (RNAi) technology utilizing dsRNA, have shown potential for pest control. However, the off-target effects of dsRNA pose limitations to the widespread application of RNAi and raise concerns regarding potential side effects on other beneficial organisms. The precise impact and underlying factors of these off-target effects are still not well understood. Here, we found that the transcript level and sequence matching jointly regulate off-target effects of dsRNA. The much lower expressed target genes were knocked down to a lesser extent than genes with higher expression levels, and the critical sequence identity of off-target effects is approximately 80%. Moreover, off-target effects could be triggered by a contiguous matching sequence length exceeding 15 nt as well as nearly perfectly matching sequences with one or two base mismatches exceeding 19 nt. Increasing the dosage of dsRNA leads to more severe off-target effects. However, the length of mismatched dsRNA, the choice of different RNAi targets, and the location of target sites within the same gene do not affect the severity of off-target effects. These parameters can be used to guide the design of possibly selective sequences for RNAi, optimize the specificity and efficiency of dsRNA, and facilitate practical applications of RNAi for pest control.

Telomere-to-telomere and gap-free genome assembly of a susceptible grapevine species (Thompson Seedless) to facilitate grape functional genomics.

Grapes are globally recognized as economically significant fruit trees. Among grape varieties, Thompson Seedless holds paramount influence for fresh consumption and for extensive applications in winemaking, drying, and juicing. This variety is one of the most efficient genotypes for grape genetic modification. However, the lack of a high-quality genome has impeded effective breeding efforts. Here, we present the high-quality reference genome of Thompson Seedless with all 19 chromosomes represented as 19 contiguous sequences (N50 = 27.1Mb) with zero gaps and prediction of all telomeres and centromeres. Compared with the previous assembly (TSv1 version), the new assembly incorporates an additional 31.5Mb of high-quality sequenced data with annotation of a total of 30 397 protein-coding genes. We also performed a meticulous analysis to identify nucleotide-binding leucine-rich repeat genes (NLRs) in Thompson Seedless and two wild grape varieties renowned for their disease resistance. Our analysis revealed a significant reduction in the number of two types of NLRs, TIR-NB-LRR (TNL) and CC-NB-LRR (CNL), in Thompson Seedless, which may have led to its sensitivity to many fungal diseases, such as powdery mildew, and an increase in the number of a third type, RPW8 (resistance to powdery mildew 8)-NB-LRR (RNL). Subsequently, transcriptome analysis showed significant enrichment of NLRs during powdery mildew infection, emphasizing the pivotal role of these elements in grapevine's defense against powdery mildew. The successful assembly of a high-quality Thompson Seedless reference genome significantly contributes to grape genomics research, providing insight into the importance of seedlessness, disease resistance, and color traits, and these data can be used to facilitate grape molecular breeding efforts.

3D Carbonate Digital Rock Reconstruction by Self-Attention Network and GAN Structure

Amidst the rapid advancements in digital technology, the pursuit of simulating geologic and mineralogic samples in a digital domain has garnered considerable attention, becoming a linchpin in modern earth science and petrological research. This manuscript intricately explores the deployment of state-of-the-art generative models for the meticulous reconstruction of digital rock core samples. Central to this investigation was the innovative incorporation of the self-attention mechanism—a pioneering endeavor in the domain of digital rock core studies. By harnessing the prowess of this sophisticated model, we endeavored to produce samples that echo the nuanced geological and mineralogical attributes emblematic of authentic rock specimens. Distinguishing our approach, the generative architecture, bolstered by the self-attention mechanism, demonstrated unparalleled proficiency in replicating quintessential rock features, ranging from porosity and granular texture to contiguous core sequences. Additionally, the idiosyncrasies of carbonate rocks were meticulously captured, highlighting phenomena like dissolution. Empirical evaluations, rooted in stringent statistical analyses, attested to the model’s capability to generate outputs that resonate closely with genuine samples. This exploration not only amplifies the potential applications of our proposed model in geoscientific endeavors but also signals a transformative stride in digital rock physics, emphasizing the harmonious amalgamation of innovative computational models with profound geological insights.

Perceptions of Hospital Care for Persons With Dementia During the COVID-19 Pandemic: A Social Media Sentiment Analysis.

The coronavirus disease 2019 (COVID-19) pandemic led to many hospital service disruptions and strict visitor restrictions that affected care of older adult populations. This study investigates perceptions of hospital care for persons with dementia during the COVID-19 pandemic as shared on Reddit's social media platform. This study combined an Opinion Mining Framework with linguistic processing to conduct a sentiment analysis of word clusters and care-based content in a sample of 1,205 posts shared between February 2020 and March 2023 in Reddit's English-language corpus. Data were classified based on reoccurring contiguous sequences of 2 words from our text sample. Hospital dementia care discourse on Reddit advanced 4 negative sentiment themes: (1) fear of poor medication management, hydration, and hygiene, (2) loss of patient advocacy, (3) precipitation of advance directive discussions, and (4) delayed discharge and loss of nursing home bed. One positive sentiment theme also emerged: gratitude toward hospital staff. Negative sentiment Reddit posts constituted a larger share of the posts than positive posts regarding hospital care for persons with dementia. People who posted about their experiences shared their concerns about hospital care deficiencies and the importance of including informal caregivers in hospital settings, particularly in the context of a pandemic. Implications exist for dementia training, improved quality of care, advance care planning, and transitions in care policies.

Identification, Genome Sequencing, and Characterizations of Helicobacter pylori Sourced from Pakistan.

The stomach's colonization by Helicobacter pylori (H. pylori) results in gastritis, ulcers, and stomach cancer. Frequently, pain is treated with medication, but resistant H. pylori infections are not. Therefore, it is important to find pharmacological targets and improved treatments for resistant H. pylori strains. The aim of the current study was sampling, identification, drug susceptibility testing following genome sequencing and comparative genome-wide analysis of selected H. pylori strains from Pakistan with three representative strains for virulence and drug-resistant characteristics. Based on culture, biochemistry, and molecular biology, 84 strains of H. pylori were identified, which made up 47% of the enrolled cases. Among all H. pylori strains, the highest resistance was reported for metronidazole with 82 H. pylori strains (98%), followed by clarithromycin with 62 resistant strains (74%). Among metronidazole-resistant strains, 38 strains (46%) were also resistant to clarithromycin, contributing 61% of clarithromycin resistant cases. Two strains, HPA1 and HPA2, isolated from 'gastritis' and 'gastric ulcer' patients, respectively, were further processed for WGS. The draft genome sequences of H. pylori strains HPA1 and HPA2 encode 1.66 Mbp and 1.67 Mbp genome size, 24 and 4 contiguous DNA sequences, and 1650 and 1625 coding sequences, respectively. Both the genomes showed greater than 90% similarity with the reference strain H. pylori ATCC 43504/PMSS1. The antibiotic-resistant genes were identified among all the strains with overall similarity above 95%, with minor differences in the sequence similarity. Using the virulent gene data obtained from the Virulence Factor Database, 75 to 85 virulent genes were identified in the five genome assemblies with various key genes such as cytolethal distending toxin (cdt), type IV secretion system, cag PAI, plasticity region, cell-motility- and flagellar-associated genes, neutrophil-activating protein (HP-NAP), T4SS effector cytotoxin-associated gene A (cagA), and urease-associated genes ureA and ureB, etc. Sequence similarity between the virulence factors found in this study and reference genes was at least 90%. In summary, the results of our study showed the relationship between clinical results and specific H. pylori strains' (HPA1 and HPA2) genetics such as antibiotic resistance and specific virulence factors. These findings provide valued understanding of the epidemiology of H. pylori-associated diseases. Moreover, identification and genomics analysis have provided insights into the epidemiology, genetic diversity, pathogenicity, and potential drug resistance genes of H. pylori strains, offering a foundation for developing more targeted and effective medical interventions, including anti-virulent medications.