Sequencing Methods Research Articles

Amplifiable protein identification via residue-resolved barcoding and composition code counting.

Ultrasensitive protein identification is of paramount importance in basic research and clinical diagnostics but remains extremely challenging. A key bottleneck in preventing single-molecule protein sequencing is that, unlike the revolutionary nucleic acid sequencing methods that rely on the polymerase chain reaction (PCR) to amplify DNA and RNA molecules, protein molecules cannot be directly amplified. Decoding the proteins via amplification of certain fingerprints rather than the intact protein sequence thus represents an appealing alternative choice to address this formidable challenge. Herein, we report a proof-of-concept method that relies on residue-resolved DNA barcoding and composition code counting for amplifiable protein fingerprinting (AmproCode). In AmproCode, selective types of residues on peptides or proteins are chemically labeled with a DNA barcode, which can be amplified and quantified via quantitative PCR. The operation generates a relative ratio as the residue-resolved 'composition code' for each target protein that can be utilized as the fingerprint to determine its identity from the proteome database. We developed a database searching algorithm and applied it to assess the coverage of the whole proteome and secretome via computational simulations, proving the theoretical feasibility of AmproCode. We then designed the residue-specific DNA barcoding and amplification workflow, and identified different synthetic model peptides found in the secretome at as low as the fmol/L level for demonstration. These results build the foundation for an unprecedented amplifiable protein fingerprinting method. We believe that, in the future, AmproCode could ultimately realize single-molecule amplifiable identification of trace complex samples without further purification, and it may open a new avenue in the development of next-generation protein sequencing techniques.

Depletion-assisted multiplexed cell-free RNA sequencing reveals distinct human and microbial signatures in plasma versus extracellular vesicles.

Cell-free long RNAs in human plasma and extracellular vesicles (EVs) have shown promise as biomarkers in liquid biopsy, despite their fragmented nature. To investigate these fragmented cell-free RNAs (cfRNAs), we developed a cost-effective cfRNA sequencing method called DETECTOR-seq (depletion-assisted multiplexed cell-free total RNA sequencing). DETECTOR-seq utilised a meticulously tailored set of customised guide RNAs to remove large amounts of unwanted RNAs (i.e., fragmented ribosomal and mitochondrial RNAs) in human plasma. Early barcoding strategy was implemented to reduce costs and minimise plasma requirements. Using DETECTOR-seq, we conducted a comprehensive analysis of cell-free transcriptomes in both whole human plasma and EVs. Our analysis revealed discernible distributions of RNA types in plasma and EVs. Plasma exhibited pronounced enrichment in structured circular RNAs, tRNAs, Y RNAs and viral RNAs, while EVs showed enrichment in messenger RNAs (mRNAs) and signal recognition particle RNAs (srpRNAs). Functional pathway analysis highlighted RNA splicing-related ribonucleoproteins (RNPs) and antimicrobial humoral response genes in plasma, while EVs demonstrated enrichment in transcriptional activity, cell migration and antigen receptor-mediated immune signals. Our study indicates the comparable potential of cfRNAs from whole plasma and EVs in distinguishing cancer patients (i.e., colorectal and lung cancer) from healthy donors. And microbial cfRNAs in plasma showed potential in classifying specific cancer types. Our comprehensive analysis of total and EV cfRNAs in paired plasma samples provides valuable insights for determining the need for EV purification in cfRNA-based studies. We envision the cost effectiveness and efficiency of DETECTOR-seq will empower transcriptome-wide investigations in the fields of cfRNAs and liquid biopsy. DETECTOR-seq (depletion-assisted multiplexed cell-free total RNA sequencing) enabled efficient and specific depletion of sequences derived from fragmented ribosomal and mitochondrial RNAs in plasma. Distinct human and microbial cell-free RNA (cfRNA) signatures in whole Plasma versus extracellular vesicles (EVs) were revealed. Both Plasma and EV cfRNAs were capable of distinguishing cancer patients from normal individuals, while microbial RNAs in Plasma cfRNAs enabled better classification of cancer types than EV cfRNAs.

Мутации в генах KRAS/NRAS и эффективность индукционной терапии по трехкомпонентным схемам на основе бортезомиба у пациентов с впервые диагностированной множественной миеломой

AIM. To identify the KRAS and NRAS gene mutations in patients with newly diagnosed multiple myeloma (ММ) and to classify them according to the depth of antitumor response to bortezomib-based triplet induction therapy. MATERIALS & METHODS. The trial enrolled 89 patients with newly diagnosed MM prior to chemotherapy. Among them, there were 45 women and 44 men aged 30–82 years (median 58.5 years). ММ was diagnosed according to IMWG criteria (2014). Bone marrow (BM) plasma cells were isolated from the aspirate using gradient method with subsequent immunomagnetic CD138 marker selection. The KRAS and NRAS gene mutations in BM CD138+ cells were identified with Sanger sequencing method. The proteomic programs MutationTaster, Polyphen2, and FATHMM-XF were used for mutation analysis in the KRAS and NRAS genes. All patients received bortezomib-based triplet chemotherapy as first-line treatment. The response depth was assessed after completing 6 cycles of PAD and VCD regimens. Antitumor response was evaluated according to IMWG (2016) criteria. RESULTS. The mutation rate in the gene family RAS was 42 % (37/89). The analysis focused on the data from 33 patients with mutations detected and response identified after 6 cycles of treatment. In 22 out of 33 patients, deep response was not achieved, whereas 11 patients showed complete remission (CR) + very good partial remission (VGPR). In the group of patients without mutations in the gene family RAS, the response to therapy meeting the CR + VGPR criteria was 64 % (27/42). The differences appeared to be significant (p = 0.008). The clinical data and the evaluation of primary treatment outcomes provided the basis for distinguishing a group of 9 prognostically unfavorable mutations: NRAS Gly13Asp, Gln61His; KRAS Gly12Ala, Gly12Asp, Gly12Val, Gly13Asp, Gln61Arg, Gln61His, and Ala146Val. CONCLUSION. The mutations in KRAS and NRAS belonging to the gene family RAS had a negative effect on the efficacy of the bortezomib-based triplet induction therapy. Mutation variants in the RAS family genes differed in prognostic significance. The analysis results helped to identify the mutation variants associated with the worse response to therapy: NRAS Gly13Asp, Gln61His; KRAS Gly12Ala, Gly12Asp, Gly12Val, Gly13Asp, Gln61Arg, Gln61His, and Ala146Val.

Combined Metagenomic Viral Detection and Donor-Derived Cell-Free DNA Quantification in Plasma From Kidney Transplant Recipients

BackgroundKidney transplant recipients require potent immunosuppression and are predisposed to opportunistic infections, many of which have a viral etiology. Currently, viral assays detect and quantify single pathogens using PCR or qPCR. An unbiased sequencing method with comparable accuracy would allow simultaneous monitoring of multiple viral pathogens and nonpathogenic Anelloviridae. The quantification of donor-derived cell-free DNA (dd-cfDNA) is an established method for the detection of allograft rejection, and a single workflow combining dd-cfDNA quantification and viral detection represents an opportunity to improve patient monitoring and management. MethodsWhole genome sequencing of cell-free DNA was performed using 1,980 plasma samples from 256 subjects enrolled in a multi-center study. Non-human sequences underwent reference-assisted assembly and taxonomic annotation of the viral DNA pathogens. ResultsOf the 1,980 samples tested, 1,453 (73.4%) had ≥1 viral detection(s), either a known viral pathogen or torque teno virus (TTV), with positivity rates generally declining 12–18 months post-transplant. Concordance of metagenomic NGS (mNGS) viral detection with qPCR detection was 97.7% (94.1% sensitivity, 98.2% specificity), and a linear relationship was demonstrated between mNGS viral quantitation and qPCR results. BK virus, cytomegalovirus, and Epstein-Barr virus were detected by sequencing up to 60 days prior to independently established clinical diagnoses. ConclusionsWhole-genome sequencing allows simultaneous quantification of dd-cfDNA as well as sensitive and early detection of viral infection through secondary analysis of the same sequencing results. In combination with dd-cfDNA, mNGS viral detection may provide additional pathogen surveillance results and serve as a useful biomarker for both over- and under-immunosuppression.

Molecular Identification of Mycobacterium leprae in the Leprosy Patients.

Several discoveries about leprosy indicate that Mycobacterium leprae transmission mainly occurs by inhalation, and the nose is a major port of entry and exit. Molecular probes have shown certain potential for the detection and identification of M. leprae in patients. The aim of this study was to identify M. leprae in nasal swab specimens using polymerase chain reaction (PCR)-based assays followed by gene sequencing methods. This observational study examines 64 anterior nasal swab samples taken from pretreatment leprosy patients, on-treatment and completed leprosy treatment in Bulukumba, South Sulawesi, Indonesia. samples were analyzed by molecular detection methods according to the standard methods at the Clinical Microbiology Laboratory of Hasanuddin University. Descriptive statistics were utilized to summarize patient demographics and outcomes. This study uses PCR to detect the M. leprae deoxyribonucleic acid (DNA) from nasal swab specimens. Data were collected from 64 patients with a percentage of male patients 51.54%. Based on the age category, the group 45-46 years was the most frequent (39.05%). PCR detection proline-rich antigen gene of a 531 bp DNA fragment from M. leprae, was positive in eight patients, and they were multibacillary. Furthermore, PCR was positive in 5 (31.25%) of 16 new leprosy patients, 2 (8.69%) of 23 on-treatment patients, and 1 (4%) of 25 treatment completed patients. Based on the results of the phylogenetic tree and analysis of 8 positive results detected by M. leprae from leprosy patients, almost all samples have a level of similarity, except for sample Ua7. M. leprae cannot grow in vitro, so molecular diagnostic tools were used to confirm the disease. This study predominantly of males with the age above 45 years of age being the most common. Eight M. leprae were positive from nasal swab leprosy patients. The sequencing findings provide insight into the genetic diversity of the genus M. leprae, so it is necessary to consider the detection of whole-genome sequence.

Behavior of Spoilage Bacterial Communities in Different Cuts of Enshi Black Pork under Refrigerated Storage (4 °C).

The Enshi black pig is a Chinese native breed known for its rich nutrition content and high quality, which has notable traction in the consumer market. In this study, the potential impact of the main commercial cuts from Enshi black pork carcasses (ham, loin, and belly) on the bacteria community of spoiled meat under refrigerated storage (4 °C) was assessed by using a high-throughput sequencing method. Moreover, the spoilage potential of isolated strains from spoiled pork was investigated. The results demonstrated significant differences (p < 0.05) in bacterial community diversity and composition among spoiled ham, loin, and belly samples. Linear discriminant analysis effect size (LEfSe) analysis revealed a total of 20 significantly different potential bacterial biomarkers, with the dominant genera of Pseudomonas, Psychrobacter, Shewanella and Carnobacterium. Additionally, C. divergens THT1-5, isolated from spoiled ham samples, displayed cold adaptation and higher spoilage potential in Enshi black pork. These findings are helpful for identifying key factors contributing to spoilage in Enshi black pork and developing strategies to inhibit bacterial growth during preservation.

Diversity and Isolation of Endophytic Fungi in Panax japonicus and Biotransformation Activity on Saponins.

This study reports the diversity and community structure differences of the endophytic fungi of Panax japonicus of different ages to obtain novel endophytic fungi with glycoside hydrolytic activity for rare saponins production. This study used the high-throughput sequencing method to analyze the diversity and community structure of endophytic fungi of P. japonicus. The endophytic fungi were processed by traditional isolation, culture, conservation, and ITS rDNA sequence analyses. Then the total saponins of P. japonicus were used as the substrate to evaluate the glycoside hydrolytic activity. The composition analysis of the community structure showed that the abundance, evenness, and diversity of endophytic fungi of nine-year-old P. japonicus were the best among all samples. A total of 210 endophytic fungi were isolated from P. japonicus samples and further annotated by sequencing the internal transcribed spacer. Then the biotransformation activity of obtained strains was further examined on total saponins of P. japonicus (TSPJ), with a strain identified as Fusarium equiseti (No.30) from 7-year-old P. japonicus showing significant glycoside hydrolytic activity on TSPJ, including ginsenoside Ro→zinglbroside R1, pseudoginsenoside RT1→pseudoginsenoside RP1, chikusetsusaponin IV→tarasaponin VI and chikusetsusaponin IVa →calenduloside E. These results reveal the diversity and community structure differences of the endophytic fungi of P. japonicus with different ages and establish a resource library of endophytic fungi of P. japonicus. More importantly, we identified a valuable endophytic fungus with glycoside hydrolytic activity and provided a promising convenient microbial transformation approach to produce minor deglycosylated ginsenosides.

Temporal effects of lascufloxacin on human gut and salivary microbiota: Analysis using next-generation sequencing method

IntroductionAntimicrobial treatment disrupts human microbiota.The effects of lascufloxacin (LSFX), a new fluoroquinolone, on human microbiota remains unknown. Therefore, in this study, we aimed to evaluate the effects of LSFX administration on the gut and salivary microbiota of healthy participants and those with pneumonia. MethodsLSFX (75 mg, once a day, orally) was administered to healthy adults (healthy group) and adult patients with pneumonia (pneumonia group), and fecal and saliva samples were collected at five time points (Days 0, 3, 7, 14, and 28). Using the collected samples, α- and β-diversity indices, as well as bacterial composition of the gut microbiota and salivary microbiota were analyzed using next-generation sequencing. ResultsIn the healthy group, α-diversity indices of the gut and salivary microbiota were reduced and the lowest values on Day 3. For the gut microbiota, the Chao1 index (richness) recovered on Day 28, whereas the Shannon index (evenness) did not. In the salivary microbiota, the Chao1 and Shannon indices did not recover within the 28 day period. The β-diversity indices changed after LSFX administration and subsequently recovered on Day 28. After LSFX administration, the abundance of the Lachnospiraceae family decreased in the gut microbiota, and the abundance of Granulicatella, Streptococcus, Prevotella, Absconditabacteriales(SR1), and Saccharimonadales decreased in the salivary microbiota. In the pneumonia group, the α-diversity indices were lowest on Day 14 after LSFX administration. ConclusionsWe elucidated that LSFX administration differentially affected the gut and salivary microbiota; however, the richness and beta diversity recovered within 28 days.

The establishment of a molecular diagnostic platform for mitochondrial diseases: from conventional to next-generation sequencing

BackgroundThe aim of this study was to create a molecular diagnostic platform and establish a diagnostic pipeline for patients highly suspected of mitochondrial disorders. The effectiveness of three methods, namely, traditional restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR), Sanger sequencing for hotspot detection and whole mitochondrial DNA (mtDNA), and third-generation (Nanopore) whole mtDNA sequencing, will be compared in diagnosing patients with suspected primary mitochondrial diseases (PMDs). The strengths and limitations of different methods are also discussed. Material and methodsA single-center prospective cohort study was conducted to validate the diagnostic pipeline for suspected mitochondrial diseases. In the first stage, a PCR-based method with five sets of primers was used to screen for eight hotspots (m.3243A>G, m.3460G>A, m.8344A>G, m.8993T>G, m.9185T>C, m.11778G>A, m.13513G>A, and m.4977deletion) using either RFLP or direct Sanger sequencing. Sanger sequencing was also used to confirm the RFLP-positive samples. In the second stage, for samples with negative screening results for the eight hotspots, mitochondrial whole-genome sequencing was performed using Sanger sequencing or third-generation nanopore sequencing. ResultsBetween June 2020 and May 2023, 30 patients from ages 0 to 63 with clinically suspected mitochondrial disease were enrolled. The positive yield for the diagnosis of PMDs was 8/30=26.7%, and the sensitivity of the heteroplasmy level for the RFLP-based method was approximately 5%. The remaining 22 patients who tested negative at the first stage were tested using Sanger sequencing or the third-generation sequencing Nanopore, and all tested negative for pathological mtDNA mutations. Compared to the Sanger sequencing method, the results of RFLP-PCR were compromised by the limitations of incomplete RFLP enzyme digestion. For whole-genome sequencing of mtDNA, Sanger sequencing, instead of nanopore sequencing, is preferred at our institution because of its cost-effectiveness. Conclusions: In our highly selective cohort, most tested positive in the first stage of the 8 hot spots screen. Sanger sequencing is a conventional and accurate method for mitochondrial disease screening, at least for the most common hot spots in the region. The results revealed that Sanger sequencing is an accurate method with the benefit of being more cost-effective. This integral platform of molecular diagnosis bears the advantages of being relatively low cost and having a shorter reporting time, facilitating crucial identification of patients with clinical evidence of such disorders. This diagnostic flowchart has also been translated into routine clinical use in the tertiary hospital.

The Role of Gut Microbiota in Inflammatory Bowel Disease

Background: The complicated result of immune response dysregulation, genetic predisposition, and external factors combines to cause a condition called inflammatory bowel disease (IBD). An increasing number of studies have lately connected the onset and progression of bowel inflammation to the gut flora.Aims and Objectives: This study aimed to look at gut function microbiota in IBD, elucidate its mechanisms, identify potential therapeutic targets, and explore future directions in this rapidly evolving field.Methodology: To examine the function of the gastrointestinal tract A cross-sectional study was carried out at the Pakistan Academy of Medicine and Dentistry (PIMS) in Islamabad between April 2023 and March 2024. We recruited a total of 72 participants who had received a diagnosis of inflammatory bowel illness. (IBD), and we used next-generation sequencing methods to collect stool samples for microbiota characterization. Demographic information, disease characteristics, and dietary habits were assessed through structured questionnaires.Results: Analysis of stool samples revealed alterations in gut microbiota composition in IBD patients compared to healthy controls, including a reduction in microbial diversity (Shannon Index: 2.8 ± 0.6 in IBD patients vs. 4.2 ± 0.8 in healthy controls) and shifts in the relative abundance of major phyla (Proteobacteria: 25.7% ± 5.2% in IBD patients vs. 12.1% ± 3.5% in healthy controls; Firmicutes: 41.3% ± 8.1% in IBD patients vs. 58.9% ± 7.3% in healthy controls). Dietary elements, illness severity, and gut microbiota composition were linked in IBD patients. Promising anti-inflammatory compounds and regulating the diversity of bacteria were two of the prospective goals for therapy found.Conclusion: Our research establishes a dysbiotic gut microbiota in IBD and shows how it relates to dietary variables and the severity of the illness. A potential treatment strategy for IBD is to target the gut microbiota, but more investigation is required to fully understand the intricate relationships that exist between the gut microbiota, host characteristics, and environmental effects.Keywords: Inflammatory Bowel Disease (IBD) refers to a group of chronic inflammatory conditions that affect the digestive tract. The gut microbiota, which refers to the community of microorganisms that reside in the gastrointestinal tract, plays a significant role in the development and progression of IBD, dysbiosis, microbial diversity, disease severity, therapeutic targets

Isolation, and Characterization of Petroleum Oil-Degrading Bacteria from Petroleum Contaminated Soil

Petroleum serves as a vital energy source, but extensive exploration can lead to frequent accidents, resulting in soil contamination from oil spills, which poses risks to both ecosystems and human health. Bioremediation emerges as a potent method for addressing polluted environments, involving the deliberate selection and application of microorganisms. In the present study, bacteria present in the petroleum oil contaminated soil were isolated by enrichment culture technique using Bushnall Hass (BS) media supplemented with petroleum oil as a sole carbon source. The isolated bacterial strains were characterized by morphological and biochemical tests and further characterized through molecular tools ie, sequencing method. Three bacterial isolates were morphologically characterized as gram-positive, spherical, cocci, and rod shaped further 16S rRNA sequence analysis revealed that the isolates are closely related to Staphylococcus hominis, Staphylococcus sp., and Bacillus pumilus, respectively. The optimum growth was at pH 7.0 and temperature 37 °C. All strains were susceptible to various antibiotics, and resistant to various heavy metals and have no antagonistic effect with oil degrading bacteria. All strains were grown in five different concentrations of petroleum oil (1%, 1.50%, 2%, 2.50%, and 3%), respectively. Study revealed that all the isolates showed their degradation efficacy in 3% v/v petroleum oil. So, this research clearly demonstrates that the isolated bacterial strains could be effectively used for bioremediation of petroleum-contaminated areas.

Single Nucleotide Polymorphism (SNP) Exon 4 Proclatine Gene on Local Duck

This study uses the sequencing method to determine the genetic diversity of the prolactin gene (PRL) in the exon four region in Bayang ducks. This study used 118 blood samples of Bayang ducks. Blood samples were isolated and then sequenced using a pair of primers L: 5'- GCA CAG TTG TTC TTA CTA GTT CG -3' and R: 5'- TCT GAG AAC TTT GCA GCT ATC T -3', which produced a 586 bp fragment in the Prolactin (PRL) gene exon 4. The amplification product was sequenced using First Base Singapore's services. The results showed that there were 16 polymorphisms in the exon four region and parts of introns 3 and 4 in the Bayang duck sample, namely two deletions at position -223C&gt;del and -218A&gt;del and 14 mutations at position -136G&gt;A, 157G&gt;A, +9T&gt;G, +11G&gt;A, +16T&gt;C, +20A&gt;C, +25T&gt;C, +29A&gt;C, +35T&gt;A, +38A&gt;G, +43C&gt;A, +71G&gt;A, +84G&gt;A, +91T&gt;A. Based on the study results, it can be concluded that the diversity of the prolactin gene (PRL) of Bayang ducks in the exon four region is polymorphic and can be used as a candidate for marker-assisted selection in Bayang ducks.

Neurodevelopmental Disorders in Children with Hereditary Diseases (Review of Literature, Clinical Case Report)

Introduction. The majority of studies on disturbed neurodevelopment in children focus on psychiatric or psychological-pedagogical issues, but the genetic component of pathology also occupies an important place, in which is important to conduct genetic investigation to verify hereditary pathology, and to identify target organs inherent in particular hereditary disease. The aim of the study. To conduct a review of current literature dedicated to the problem of impaired neurodevelopment in children with hereditary diseases, to describe a clinical case of A. Rett genetic syndrome, accompanied by impaired neurodevelopment in a child. Materials and methods. The method of systematic and comparative analysis, as well as the biblio-semantic method of studying modern views on the influence of hereditary diseases in the disruption of neurodevelopment in children were used. As much as 25 recent publications were analyzed. A clinical case of A. Rett syndrome in a child is described, where the analysis of clinical symptoms and laboratory-instrumental examinations were used, the main of which is the molecular genetic method of next generation sequencing (NGS). Results. Based on the literature analysis it was estimated that clinical cases of rare A. Rett syndrome in children occur with a frequency of 1:10.000-1:15.000. This syndrome is caused by a mutation in the MECP2 gene associated with X-linked A. Rett syndrome/atypical A. Rett syndrome (UID MedGen: 48441) or X-linked MECP2 duplication syndrome (MedGen: 337496). Conclusions. For a long time, the connection of impaired neurodevelopment with other clinical symptoms was not payed much attention, but the enhanced frequency of cases when a child with impaired neurodevelopment was diagnosed with other symptoms or a multisystem lesion stimulated research in this area. This is the story how the term ″syndromal autism″ was born. This term means developmental delay or autism spectrum disorder in children with symptoms of another hereditary disease.

Multivariate short-term wind speed prediction based on PSO-VMD-SE-ICEEMDAN two-stage decomposition and Att-S2S

To counter the challenges posed by the unpredictability of wind velocities on wind energy production, a wind speed prediction model combining chaotic mapping-based particle swarm optimization with variational modal decomposition (CPSO-VMD), sample entropy (SE), improved completely integrated empirical modal decomposition with adaptive noise (ICEEMDAN) two-layer decomposition, and attention mechanism-based sequence to sequence (Att-S2S) method is proposed. Firstly, the Lasso method is employed to filter the features that significantly contribute to the wind speed data, fully considering hidden relevant information and eliminating redundant data to improve the prediction accuracy; Secondly, CPSO optimized by introducing chaotic mapping determines the parameter pairs for VMD. This facilitates an adaptive VMD breakdown of the wind speed sequence, aiding in noise removal and selection of the intrinsic mode function (IMF) with the highest sample entropy for further decomposition using ICEEMDAN. In light of this, a sequence-to-sequence method based on the attention mechanism is suggested, which may highlight the influence features' effects on IMF; Finally, the proposed model is implemented at both the Paso Robles wind farm and the Oasis wind farm for practical assessment and benchmarked against other models. Overall, the proposed model outperforms the other models in these trials.

Single-cell RNA sequencing and multiple bioinformatics methods to identify the biomarkers of ischemic stroke to alzheimer’s disease

Ranked as the world’s second leading cause of mortality, ischemic stroke (IS) imposes a significant global burden, marked by its substantial incidence and economic impact across nations and regions, chiefly attributed to its elevated disability rates. Alzheimer’s disease (AD) is linked to a heightened susceptibility to IS. However, few studies have been reported to explain the potential correlation between IS and AD. We investigated the relationship and relevant mechanisms between IS and AD using single-cell RNA sequencing and multiple bioinformatics approaches. By intersecting differentially expressed genes and WGCNA critical module genes, we obtained 15 IS related AD genes. According to the KEGG pathway analysis, the IS related AD disease genes were significantly enriched in the parkinson disease pathway, oxidative phosphorylation pathway, thermogenesis pathway, alzheimer disease pathway and neurodegeneration pathway. Finally, five genes associated with immunity (TXN, HINT1, TOMM7, COX7C, RPL17) were identified in IS related AD. Significantly, it was found that all hub genes were mainly highly expressed in epithelial cells, endothelial cells, monocytes and oligodendrocytes by single cell RNA sequencing. We believe these hub genes may regulate the immune pathway which may contribute to the development of IS related AD. In addition, these genes may be potential targets for the treatment of IS related AD.

Optimizing single-cell RNA sequencing methods for human colon biopsies: droplet-based vs. picowell-based platforms.

Single-cell RNA sequencing (scRNA) has empowered many insights into gastrointestinal microenvironments. However, profiling human biopsies using droplet-based scRNA (D-scRNA) is challenging since it requires immediate processing to minimize epithelial cell damage. In contrast, picowell-based (P-scRNA) platforms permit short-term frozen storage before sequencing. We compared P- and D-scRNA platforms on cells derived from human colon biopsies. Endoscopic rectosigmoid mucosal biopsies were obtained from two adults and conducted D-scRNA (10X Chromium) and P-scRNA (Honeycomb HIVE) in parallel using an individual's pool of single cells (> 10,000 cells/participant). Three experiments were performed to evaluate 1) P-scRNA with cells under specific storage conditions (immediately processed [fresh], vs. frozen at -20C vs. -80C [2 weeks]); 2) fresh P-scRNA versus fresh D-scRNA; and 3) P-scRNA stored at -80C with fresh D-scRNA. Significant recovery of loaded cells was achieved for fresh (80.9%) and -80C (48.5%) P-scRNA and D-scRNA (76.6%), but not -20C P-scRNA (3.7%). However, D-scRNA captures more typeable cells among recovered cells (71.5% vs. 15.8% Fresh and 18.4% -80C P-scRNA), and these cells exhibit higher gene coverage at the expense of higher mitochondrial read fractions across most cell types. Cells profiled using D-scRNA demonstrated more consistent gene expression profiles among the same cell type than those profiled using P-scRNA. Significant intra-cell-type differences were observed in profiled gene classes across platforms. Our results highlight non-overlapping advantages of P-scRNA and D-scRNA and underscore the need for innovation to enable high-fidelity capture of colonic epithelial cells. The platform-specific variation highlights the challenges of maintaining rigor and reproducibility across studies that use different platforms.

Bacterial single-cell RNA sequencing captures biofilm transcriptional heterogeneity and differential responses to immune pressure.

Biofilm formation is an important mechanism of survival and persistence for many bacterial pathogens. These multicellular communities contain subpopulations of cells that display vast metabolic and transcriptional diversity along with high recalcitrance to antibiotics and host immune defenses. Investigating the complex heterogeneity within biofilm has been hindered by the lack of a sensitive and high-throughput method to assess stochastic transcriptional activity and regulation between bacterial subpopulations, which requires single-cell resolution. We have developed an optimized bacterial single-cell RNA sequencing method, BaSSSh-seq, to study Staphylococcus aureus diversity during biofilm growth and transcriptional adaptations following immune cell exposure. We validated the ability of BaSSSh-seq to capture extensive transcriptional heterogeneity during biofilm compared to planktonic growth. Application of new computational tools revealed transcriptional regulatory networks across the heterogeneous biofilm subpopulations and identification of gene sets that were associated with a trajectory from planktonic to biofilm growth. BaSSSh-seq also detected alterations in biofilm metabolism, stress response, and virulence that were tailored to distinct immune cell populations. This work provides an innovative platform to explore biofilm dynamics at single-cell resolution, unlocking the potential for identifying biofilm adaptations to environmental signals and immune pressure.

The Relevance of β-Thalassemia Heterozygosity in Pediatric Clinical Practice: Croatian Experience.

(1) Background: Thalassemia syndromes are common monogenic disorders that represent a significant global health issue. No systematic epidemiological or molecular investigations on thalassemias in the Croatian population have been reported to date. (2) Methods: This prospective study included 70 children with a presumptive diagnosis of thalassemia and their 42 first-degree relatives. Molecular characterization was performed using direct sequencing and gap-PCR methods. (3) Results: We identified 46 (30 children and 16 first-degree relatives) β-thalassemia heterozygous carriers from 24 unrelated families, carrying eight different mutations and one hemoglobin variant. Five variants account for approximately 85% of all affected β-globin alleles: Hb Lepore-Boston-Washington (32.6%), HBB:c.93-21G>A (19.6%), HBB:c.315+1G>A (13.1%), HBB:c.92+1G>A (10.9%), and HBB:c.92+6T>C (8.7%) variants. (4) Conclusions: β-thalassemia carriers need more detailed genetic profiling since genetic modifiers can significantly impact their phenotype. Our study provides important new insights into the relevance of β-thalassemia heterozygosity in pediatric clinical practice.

CapTrap-seq: a platform-agnostic and quantitative approach for high-fidelity full-length RNA sequencing

Long-read RNA sequencing is essential to produce accurate and exhaustive annotation of eukaryotic genomes. Despite advancements in throughput and accuracy, achieving reliable end-to-end identification of RNA transcripts remains a challenge for long-read sequencing methods. To address this limitation, we develop CapTrap-seq, a cDNA library preparation method, which combines the Cap-trapping strategy with oligo(dT) priming to detect 5’ capped, full-length transcripts. In our study, we evaluate the performance of CapTrap-seq alongside other widely used RNA-seq library preparation protocols in human and mouse tissues, employing both ONT and PacBio sequencing technologies. To explore the quantitative capabilities of CapTrap-seq and its accuracy in reconstructing full-length RNA molecules, we implement a capping strategy for synthetic RNA spike-in sequences that mimics the natural 5’cap formation. Our benchmarks, incorporating the Long-read RNA-seq Genome Annotation Assessment Project (LRGASP) data, demonstrate that CapTrap-seq is a competitive, platform-agnostic RNA library preparation method for generating full-length transcript sequences.

Huangqin Qingre Chubi Capsule inhibits rheumatoid arthritis by regulating intestinal flora and improving intestinal barrier.

Changes in intestinal flora and intestinal barrier in patients with preclinical and diagnosed rheumatoid arthritis (RA) suggest that intestinal flora and intestinal barrier play an important role in the induction and persistence of RA. Huangqin Qingre Chubi Capsule (HQC) is a clinically effective herbal formula for the treatment of RA, but its therapeutic mechanism has not been fully clarified. In this study, real-time qPCR (RT-qPCR), 16SrRNA sequencing, Western blot (WB), immunofluorescence and other methods were used to investigate whether HQC inhibited RA. Based on research in collages-induced arthritis (CIA) model in mice, human colon cancer cell line (Caco-2), and fibroblast-like synoviocytes (FLS) from RA patients, we found that intestinal flora was disturbed in CIA model group, intestinal barrier was damaged, and lipolyaccharide (LPS) level was increased, and HQC could regulate intestinal flora and intestinal barrier and reduce LPS translocation into blood. Antibiotic depletion weakened the anti-RA effect of HQC, and HQC fecal microbiota transplantation alleviated RA pathology. In addition, LPS increased the expression of RA pathologic factors MMP3, Fibronectin and inflammatory factors IL-6, TNF-α, IL-1β and IL-8, indicating that elevated peripheral blood level of LPS was related to RA pathology. The dysregulation of intestinal flora and the disruption of intestinal barrier are significant factors in the development of RA. HQC improves RA by regulating intestinal flora, intestinal barrier and inhibiting LPS translocation into blood. The study unveiles RA's new pathogenesis and laid a scientific groundwork for advancing HQC therapy for RA.