PCR Sequencing Research Articles

Eight Years of Norovirus Surveillance in Urban Wastewater: Insights from Next-Generation

Human noroviruses (HNoVs) are a leading cause of acute gastroenteritis worldwide, with significant public health implications. In this study, wastewater-based epidemiology (WBE) was used to monitor the circulation and genetic diversity of HNoVs in Rome over an eight-year period (2017–2024). A total of 337 wastewater samples were analyzed using RT-nested PCR and next-generation sequencing (NGS) to identify genogroups GI and GII and their respective genotypes. The results showed that GII had higher detection rates (66.5%) compared to GI (50.7%), with significant variation between years. Detection rates peaked in 2019 before declining sharply in 2020, coinciding with the COVID-19 pandemic and rebounding after the pandemic in 2023. A total of 24 genotypes were identified (8 GI and 17 GII), including persistent variants GII.2, GII.3 and GII.4 and emerging genotypes such as GII.8, GII.10 and GII.14. Only two GII.4 variants, Sydney_2016 and Sydney_2012, were detected in the study. These results demonstrate the utility of WBE in tracking HNoVs circulation, identifying genotype diversity and capturing shifts in transmission dynamics. WBE provides a cost-effective and comprehensive tool for public health surveillance, particularly in regions with limited clinical surveillance. Sustained investment in WBE is crucial for advancing our understanding of HNoVs epidemiology and its long-term trends.

Histone Methyltransferase G9a in Primary Sensory Neurons Promotes Inflammatory Pain and Transcription of Trpa1 and Trpv1 via Bivalent Histone Modifications.

Transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1) channels are crucial for detecting and transmitting nociceptive stimuli. Inflammatory pain is associated with sustained increases in TRPA1 and TRPV1 expression in primary sensory neurons. However, the epigenetic mechanisms driving this upregulation remain unknown. G9a (encoded by Ehmt2) catalyzes H3K9me2 and generally represses gene transcription. In this study, we found that intrathecal administration of UNC0638, a specific G9a inhibitor, or G9a-specific siRNA, substantially reduced complete Freund's adjuvant (CFA)-induced pain hypersensitivity. Remarkably, CFA treatment did not induce persistent pain hypersensitivity in male and female mice with conditional Ehmt2 knockout in dorsal root ganglion (DRG) neurons. RNA sequencing and quantitative PCR analyses showed that CFA treatment caused a sustained increase in mRNA levels of Trpa1 and Trpv1 in the DRG. Ehmt2 knockout in DRG neurons elevated baseline Trpa1 and Trpv1 mRNA levels but notably reversed CFA-induced increases in their expression. Chromatin immunoprecipitation revealed that CFA treatment reduced G9a and H3K9me2 levels while increasing H3K9ac and H3K4me3-activating histone marks-at Trpa1 and Trpv1 promoters in the DRG. Strikingly, conditional Ehmt2 knockout in DRG neurons not only diminished H3K9me2 but also reversed CFA-induced increases in H3K9ac and H3K4me3 at Trpa1 and Trpv1 promoters. Our findings suggest that G9a in primary sensory neurons constitutively represses Trpa1 and Trpv1 transcription under normal conditions but paradoxically enhances their transcription during tissue inflammation. This latter action accounts for inflammation-induced TRPA1 and TRPV1 upregulation in the DRG. Thus, G9a could be targeted for alleviating persistent inflammatory pain.Significance statement This study demonstrates for the first time that G9a, a histone methyltransferase, in sensory neurons is crucial for the persistent pain development following inflammation. Inhibiting or knockdown of G9a at the spinal cord level reduced inflammation-induced pain hypersensitivity. Remarkably, mice lacking G9a in sensory neurons failed to develop persistent pain hypersensitivity after inflammation. Ablating G9a in sensory neurons increased baseline expression of the TRPA1 and TRPV1 ion channels but reversed their upregulation induced by inflammation. Additionally, G9a deletion blocked the inflammation-driven enrichment of activating histone marks at Trpa1 and Trpv1 promoters. These findings highlight a dual role of G9a in sensory neurons: suppressing Trpa1 and Trpv1 transcription under normal conditions while promoting their transcription during inflammation through bivalent histone modifications.

Linking LRP12 CGG repeat expansion to inherited peripheral neuropathy

BackgroundThe causative genes for over 60% of inherited peripheral neuropathy (IPN) remain unidentified. This study endeavours to enhance the genetic diagnostic rate in IPN cases by conducting screenings focused on...

Characterization of Streptococcus pyogenes Strains from Tonsillopharyngitis and Scarlet Fever Resurgence, 2023—FIRST Detection of M1UK in Bulgaria

Recently a resurgence of Streptococcus pyogenes infections has arisen, with concerns around the highly virulent M1UK lineage. Our aim was to characterize S. pyogenes, the immune responses it causes, and to determine the presence of the M1UK lineage in Sofia, Bulgaria. In our study, the infections were confirmed by culture testing or rapid antigen test. Identification was performed by MALDI-TOF and was followed up by antibiotic susceptibility testing (EUCAST). Virulence factors were identified using multiplex PCR and whole genome sequencing (WGS). Immune responses were measured through detection of serum complement levels, lymphocyte subsets, and cytokine profiling. Out of 82 children, 38 had scarlet fever and the rest had streptococcal pharyngitis. Strains were susceptible to penicillin (β-lactams), macrolides, clindamycin, tetracyclines, co-trimoxazole, fluoroquinolones, and linezolid. Superantigen profiles were identified: SpeA + SpeJ (45%), SpeC, and SpeI + SpeH (27.5% each). A novel Multilocus sequence typing (MLST) haplotype in the mutS gene (d90b) was found in four strains. The M1UK lineage was detected for the first time in Bulgaria. We observed an increase in complement fractions C3 and C4 and a decrease in T lymphocytes. A significant increase in the levels of IFN-γ, IL-6, and IL-10 with corresponding reduction in IL-17A were revealed. In conclusion, the studied S. pyogenes strains were characterized by their susceptibility to antibiotics and the predominance of SpeA superantigen; for the first time in Bulgaria the presence of M1UK and a novel SNP variation in the mutS gene (d90b) were found. A mixed pattern of pro- and anti-inflammatory immune responses in patients was observed.

Analysis of HIV transmission characteristics and intervention effects in Guangxi based on molecular networks.

This study evaluates changes in HIV transmission and the effectiveness of interventions after two rounds of the Guangxi AIDS Conquering Project (GACP) in Guangxi, China. Samples and epidemiological data from newly diagnosed people living with HIV (PLWH) between 2014-2020 were analyzed. Molecular networks were constructed using nested PCR amplification and sequencing of the pol region, and multivariable logistic regression identified factors associated with clustering and high-degree nodes. A total of 4,227 valid sequences (73.12% inclusion rate) were analyzed. Demographic changes included an increasing proportion of individuals aged ≥50 years (49.66%), with lower education (50.51%), peasants (76.82%), and heterosexual transmission (90.29%). The overall clustering rate was 86.89%, with higher clustering among individuals aged ≥50 (92.57%), those with primary school or below (89.09%), peasants (88.11%), and CRF08_BC infections (91.48%). Annual declines in cluster growth rate and clustering rates were observed, particularly among individuals aged <30, college graduates, men who have sex with men (MSM), and people who inject drugs (PWID). Key transmission hotspots were identified in Lingshan, particularly among older, less-educated individuals, and peasants. Factors associated with clustering included being male (aOR: 1.27), aged ≥50 (aOR: 3.84), and infected with CRF08_BC (aOR: 2.12). From 2017 to 2020, the risk of clustering and high-degree nodes was lower compared to 2014-2016, suggesting the effectiveness of interventions. Interventions in Guangxi effectively reduced HIV transmission among younger, high-degree populations. However, older, less-educated individuals remain at high risk, necessitating targeted strategies to address their specific needs and achieve better HIV control.

A Review of the Molecular Understanding of the Mpox Virus (MPXV): Genomics, Immune Evasion, and Therapeutic Targets

The Mpox virus (MPXV), a zoonotic pathogen from the Orthopoxvirus genus, has emerged as a significant global public health concern, especially after the unprecedented outbreak in 2022. This review synthesizes the MPXV’s molecular features, focusing on its genomic structure, replication mechanisms, immune evasion strategies, and implications for diagnostics and therapeutics. The study examines the virus’s genomic organization utilizing recent peer-reviewed literature, highlighting essential genes like OPG027 and D1L, which contribute to host adaptation, increased transmissibility, and immune evasion. Advances in molecular diagnostics, including real-time PCR and genome sequencing, are reviewed, emphasizing their critical role in outbreak monitoring and control. However, challenges persist, such as diagnostic limitations in resource-constrained settings and the lack of targeted vaccines and antivirals. This review discusses new antiviral candidates, confirmed through computational and in vitro techniques, identifying thymidine kinase and VP39 as key therapeutic targets. Emphasizing the need for genomic surveillance to track adaptive evolution, results show that particular mutations, such as in the OPG027 and D1L genes, increase the transmissibility and immune evasion of the MPXV. These molecular revelations highlight the urgent necessity for better diagnostics catered towards addressing present constraints and developing focused treatments that reduce the effect of the virus. This study emphasizes how these results underscore the need for combined public health plans to handle the changing MPXV epidemiology properly.

Biofilm Formation, Modulation, and Transcriptomic Regulation Under Stress Conditions in Halomicronema sp.

In nature, bacteria often form heterogeneous communities enclosed in a complex matrix known as biofilms. This extracellular matrix, produced by the microorganisms themselves, serves as the first barrier between the cells and the environment. It is composed mainly of water, extracellular polymeric substances (EPS), lipids, proteins, and DNA. Cyanobacteria form biofilms and have unique characteristics such as oxygenic photosynthesis, nitrogen fixation, excellent adaptability to various abiotic stress conditions, and the ability to secrete a variety of metabolites and hormones. This work focused on the characterization of the cyanobacterium Halomicronema sp. strain isolated from a brackish environment. This study included microscopic imaging, determination of phenolic content and antioxidant capacity, identification of chemicals interfering with biofilm formation, and transcriptomic analysis by RNA sequencing and real-time PCR. Gene expression analysis was centered on genes related to the production of EPS and biofilm-related transcription factors. This study led to the identification of wza1 and wzt as EPS biomarkers and luxR-05665, along with genes belonging to the TetR/AcrR and LysR families, as potential biomarkers useful for studying and monitoring biofilm formation under different environmental conditions. Moreover, this work revealed that Halomicronema sp. can grow even in the presence of strong abiotic stresses, such as high salt, and has good antioxidant properties.

Accelerating the diagnosis of Chinese cblC type MMA patients by multiplex PCR sequencing method.

CblC type methylmalonic aciduria (cblC disease) is the most common inborn error of vitamin B12 metabolism and due to mutations in the MMACHC gene. The earlier the diagnosis, the better the prognosis. Therefore, convenient and inexpensive detection method is needed. This study selected mutational hot-spot regions in the MMACHC gene which harbors more than 90% of mutant alleles responsible for cblC disease in China. Subsequently, a hot-spot regions multi-PCR Sanger sequencing method (HsRMSS) was designed. The accuracy and efficiency of HsRMSS was validated using samples from 20 cblC families with known MMACHC gene mutations and 50 healthy volunteers. In addition, patients' clinical phenotypes and molecular genetic features were analyzed. A total of 16 different mutations were identified in 20 cblC families. Among them, the most common mutations were c.609 G>A (26/80, 32.5%), c.567dupT (10/80, 12.5%), c.80A>G (8/80, 10.0%), c.658_660delAAG (8/80, 10.0%) and c.394C>T (6/80, 7.5%), which accounted for over 70% of disease alleles. The HsRMSS results were the same as the results using the whole exon sequencing, with a coincidence rate of 100%. The HsRMSS targeting the mutational hot-spots of MMACHC gene could be a promising tool to accurately and rapidly diagnose cblC disease in China. This study reported the development and validation of a hot-spot regions multi-PCR Sanger sequencing method for targeting hotspots which harbor most of the common MMACHC gene mutations reported in Chinese patients with cblC disease. The approach could have a potential clinical application as a rapid diagnosis and screening tool for suspected children with cblC type MMA and population carrier, owing to its high throughput, low cost, and high sensitivity and specificity.

Presence of the Anopheles culicifacies complex species A in southeast Iran

BackgroundThe Anopheles culicifacies complex is one of the most important malaria vectors in Southeast Asia and Southeastern Iran. Although the sibling species within this complex are morphologically indistinguishable, they differ significantly in their disease transmission potential, blood-feeding behaviour, and other biological traits. Cytogenetic and chromosomal studies have identified five sibling species within this complex: A, B, C, D, and E. Understanding the species composition and distribution of this complex is crucial for malaria control strategies.ObjectivesThis study aimed to identify the sibling species of the An. culicifacies complex in Qaleh Ganj County, Kerman Province, Southeastern Iran. Specifically, the study sought to determine the presence of species A, which is known to be a primary vector of malaria in the region.MethodsWe employed allele-specific PCR and sequencing PCR techniques to identify the sibling species. DNA was extracted from mosquito specimens, and the D3 region of the 28S rDNA gene and a segment of the COII gene from the mitochondrial genome (mtDNA) were targeted for amplification and sequencing.ResultsData analysis revealed a positive correlation between An. culicifacies s.l. specimens and altitude, with most specimens collected from mountainous areas. Both allele-specific PCR and sequencing PCR confirmed the presence of species A in the study areas of Kerman Province.ConclusionsGiven that species A is a primary malaria vector, the findings of this study provide valuable insights for guiding malaria control strategies in Southeastern Iran. Further studies are recommended to assess the vector competence and ecological dynamics of other species within the An. culicifacies complex in the region.

Study of the Prevalence of Human Papillomavirus Genotypes in Jeddah, Saudi Arabia

Human papillomavirus (HPV), a common sexually transmitted infection, includes over 200 types, some linked to genital warts and various cancers, including cervical, anal, penile, and oropharyngeal cancers. In Saudi Arabia, an estimated 10.7 million women aged 15 years and older are at risk of HPV-related cervical cancer. This study assessed HPV prevalence, genotype distribution, awareness, and health behaviors among females in Jeddah, Saudi Arabia. Pap smear samples were collected from 106 women attending gynecology clinics at King Abdulaziz University Hospital and were analyzed using nested PCR and Sanger sequencing. Results showed a 15.1% HPV positivity rate, with genotypes of HPV 16, 6, and 58 identified. HPV 16 was the most prevalent (43.75%), particularly among women aged 35–44. In addition, over 60% of the participants were unaware of the link between HPV and cervical cancer, or the availability of vaccines, indicating significant knowledge gaps in the population. These findings underscore the urgent need for comprehensive HPV surveillance and targeted public health interventions in Saudi Arabia. This study emphasizes the importance of HPV vaccination programs, increased screening, and educational campaigns to address the high prevalence of high-risk genotypes, particularly HPV 16. Tailored strategies are crucial to reducing HPV-related health risks, especially for high-risk age groups and under-informed populations. The data provide a foundation for improving HPV prevention and reducing the burden of related diseases in the Saudi population.

Case Report: Prenatal diagnosis of novel compound heterozygous variants in WDR35 gene causing short-rib thoracic dysplasia 7 with or without polydactyly

BackgroundWhole exome sequencing (WES) technology has been increasingly used for the etiological diagnosis of fetuses with ultrasound anomalies. In this article, we report a novel deletion compound combined with a causative variant in WDR35 gene leading to short-rib thoracic dysplasia 7 (SRTD7) with or without polydactyly using WES.MethodsThis study involved a Chinese fetus with clinical features of skeletal dysplasia on ultrasound imaging, in whom chromosome abnormalities and copy number variants (CNVs) were detected by chromosomal microarray analysis (CMA), and sequence variants were detected by WES. The obtained results were further verified by Sanger sequencing or real time quantitative PCR (qPCR).ResultsNo chromosomal abnormality or CNVs were identified in the fetus by CMA. However, WES result revealed a 14.38-kb large novel deletion compound covering exon 7 to exon 12 combined with a missense variant NM_001006657.2:c.932G&amp;gt;T(p.W311l) in WDR35. Both variants were thought of as pathogenic, which was further confirmed by Sanger sequencing and qPCR. In addition, two compound heterozygous variants NM_015102.5:c.[1196A&amp;gt;G(p.E399G)];[1972C&amp;gt;T(p.R658*)] in NPHP4 gene were also identified in the fetus, which may be partially responsible for fetal kidney hyperechogenicity and oligohydramnios.ConclusionThis is the first study reporting a novel deletion compound combined with a causative missense variant in WDR35 leading to SRTD7. This finding may broaden the spectrum of variants of WDR35 gene and provide a valuable reference for clinical counseling of related abnormalities in pregnancies.

A comparative template-switching cDNA approach for HTS-based multiplex detection of three viruses and one viroid commonly found in apple trees

Exclusion is a keystone of integrated pest management to prevent the introduction of pathogens. U.S. plant quarantine programs employ PCR and high-throughput sequencing (HTS) to test imported plants for viruses and viroids of concern. Achieving a low limit of detection in any HTS protocol could be challenging. Following a template-switching cDNA amplification protocol, seven cDNA synthesis treatments were used to test simultaneously the relative abundance and coverage of the three most commonly latent RNA viruses found in apples: apple chlorotic leaf spot virus, apple stem grooving virus, and apple stem pitting virus, as well as the viroid apple hammerhead viroid. Amplified double-stranded cDNAs were subjected to library preparation using Nanopore SQK-DCS109 and Illumina Nextera XT, and sequenced with MinION and NextSeq2000, respectively. Treatments with oligo d(T)23-VN or its combination with random hexamers yielded the highest relative reads for viruses, while treatments containing the reverse primer pool produced more relative reads for AHVd. These treatments and random hexamers also generated the highest genome coverages, which were typically similar in both HTS workflows. However, relative abundances of viruses determined with SQK-DCS109 were up to 2.22-fold higher compared to Nextera XT. In contrast, Nextera XT yielded viroid reads 3.30-fold higher than SQK-DCS109. A framework of considerations for expanding this sensitive approach to other targets and crops is discussed.

The Effect of Single Nucleotide Polymorphisms on Clinical Phenotypes of Sabahan Transfusion-Dependent β-Thalassemia Patients with Homozygous Filipino β0-Deletion

Sabah has the highest prevalence of β-thalassemia in Malaysia, with the Filipino β0-deletion as the predominant mutation. Patients with the homozygous Filipino β0-deletion exhibit phenotypic heterogeneity due to various genetic modifiers, yet the effects of these modifiers on the clinical phenotype remain poorly understood. This study investigated the effects of the coinheritance of α-thalassemia, XmnI-Gγ rs7482144, BCL11A rs766432, and 5’HS4 rs16912979 polymorphisms on the clinical phenotype of homozygous Filipino β0-deletion patients in Sabah. Molecular analyses were performed on 124 homozygous Filipino β0-deletion patients using gap-PCR, PCR-RFLP, multiplex PCR, ARMS-PCR, gel electrophoresis, and DNA sequencing. Data showed that the coinheritance of the –α3.7 deletion significantly affected the clinical phenotypes of homozygous Filipino β0-deletion patients (p < 0.05). Patients with the –α3.7/–α3.7 genotype (5.6%) had a less severe clinical phenotype compared to those with the αα/αα (71.8%) and –α3.7/αα (22.6%) genotypes. Our data further revealed that the MAFs of the XmnI-Gγ rs7482144 and BCL11A rs766432 polymorphisms in these patients were 0.032 and 0.194, respectively. Interestingly, none of these single nucleotide polymorphisms significantly influenced the clinical phenotype of the patients. The effect of the 5’HS4 rs16912979 polymorphism on the clinical phenotype could not be assessed due to its rarity (1.6%). However, a novel 5’HS4 c.733+G mutation was identified, warranting further investigation of its potential impact on β-thalassemia pathogenesis. Our findings indicate that the clinical phenotype of patients with the homozygous Filipino β0-deletion is strongly influenced by the coinheritance of the –α3.7 deletion, but not by the XmnI-Gγ rs7482144 and BCL11A rs766432 polymorphisms.

The distribution of pigeon adenoviruses in Northern Chinese pigeon and turtledove flocks provides further evidence of viral crosstransmission.

To understand the prevalence, genetic diversity, and potential pathogenicity of adenoviruses present in pigeon and turtledove populations. Nested PCR and Sanger sequencing methods were used to identify the genotype and percentage of various adenoviruses in the feces of pigeon (Columba) and turtledove (Streptopelia) populations. In Beijing, China, a total of 194 fresh feces samples from meat-use pigeons (C livia domestica), homing pigeons (C livia domestica), wild pigeons (C livia domestica), and turtledoves (S decaocto and S chinensis) were collected using noninvasive sampling collection techniques. Their partial DNA-dependent DNA polymerase gene sequences were obtained using nested PCR and double-ended Sanger sequencing, and their genotypes were then ascertained based on sequence alignment. A total of 6 genotypes of adenovirus were detected in pigeon and turtledove flocks, including pigeon adenovirus (PiAdV)-1, PiAdV-2A, PiAdV-3, PiAdV-4, PiAdV-5, and a novel adenovirus genotype (PiAdV-6). Among them, PiAdV-1 was found widespread in flocks of pigeons exhibiting extensive presentations of hepatic necrosis. Highly conserved PiAdV-4 and PiAdV-5 were found to be nonpathogenic and extensively distributed in all pigeon and turtledove groups. These findings imply the presence of diverse PiAdVs in pigeon and turtledove flocks, and the wild pigeons and wild turtledove birds are potentially serving as natural sources of these viruses. This study provides supportive evidence of the pathogenicity of different genotypes of adenovirus in pigeon flocks and also implies that stopping the transmission of the virus brought by wild pigeons and turtledoves may be important for the prevention of diseases associated with PiAdVs.

Chromatin Immunoprecipitation for Standard, Rare, or Weakly Binding Proteins.

Various proteins interact with specific genome regions, playing crucial roles in gene regulation. Chromatin Immunoprecipitation (ChIP) is the most commonly used method to study protein-DNA interactions in vivo. By combining ChIP with high-throughput sequencing, ChIP-seq allows for studying the genome-wide localization of proteins. Although several ChIP protocols are available for plant tissues, they are primarily designed for histone modifications and abundant proteins with high DNA-binding affinity, which are considered as the "standard targets." Here we describe a ChIP protocol for plant tissues not only optimized for the standard targets but also adapted for proteins with low abundances or weak DNA-binding ability. Successful execution of the protocol enables reliable generation of DNA templates for quantitative PCR or libraries for next-generation sequencing, which makes it an effective tool for analyzing genomic interactions of a wide range of proteins.

Assessing the prevalence of human enteric viruses in hospital wastewater to evaluate the effectiveness of wastewater treatment systems.

In this experiment, we employed Real-time PCR(RT-PCR) and metagenomic Next-Generation Sequencing (mNGS) techniques to detect the presence of Norovirus, Rotavirus Group A, Adenovirus Group F, and Astrovirus in untreated sewage from three major hospitals. A comparison with clinical lab test outcomes revealed Norovirus as having the highest infection rate, followed by Adenovirus Group F and Rotavirus Group A. Despite not testing for Astrovirus in clinical labs, its sewage detection rate was surpassed only by Norovirus, suggesting a potentially high clinical infection rate. Further analysis of these viruses in treated sewage revealed that chlorination failed to eliminate the virus, maintaining viral concentrations in the treated sewage between 10^2 and 10^3 copies/ml. Even though nucleic acid testing methods fail to detect viral actions, the possible danger they present to public safety should not be ignored. During this experiment, viral nucleic acid was extracted directly from the samples without prior concentration. This method, unlike conventional virus detection post-concentration, bypasses concerns such as recovery efficiency, offering a clearer representation of virus concentrations in water samples and facilitating easier operation.

MiR-143-3p/TET1 Axis Regulates GPC1 Through DNA Methylation and Impairs the Malignant Biological Behaviour of HCC via the Hippo Signalling Pathway.

Hepatocellular carcinoma (HCC) is a malignant tumour that poses a serious threat to human health and places a heavy burden on individuals and society. However, the role of GPC1 in the malignant progression of HCC is unknown. In this study, we analysed the expression of GPC1 in HCC, and its association with poor patient prognosis. The effects of GPC1 on the proliferation, invasion and migration of HCC were analysed through cellular functional experiments invitro and invivo. Mechanistically, DNA methylation of GPC1 was analysed by DNA extraction, methylation-specific PCR and bisulfite Sanger sequencing (BSP), and the target genes TET1 and miRNA regulating DNA methylation of GPC1 were found through the bioinformatics database. The results revealed that GPC1 was highly expressed in HCC, and its high expression was significantly associated with poor prognosis of HCC patients. Inhibiting the expression of GPC1 can inhibit the proliferation, invasion and migration of HCC cells. GPC1 was hypomethylated in HCC, and its methylation level was regulated by TET1. miR-143-3p can significantly regulated the expression of TET1 and affect the methylation level and protein expression of GPC1. Furthermore, GPC1 also affects the malignant biological behaviour of HCC by regulating the expression of Hippo signalling pathway. In summary, miR-143-3p regulates the expression of TET1, affects the expression of GPC1 through DNA methylation and regulates the malignant progression of HCC via Hippo signalling pathway.

Spotted fever group rickettsiae in black rats, pets, and humans in Zungarococha community, A rural area in the surroundings of Iquitos, Peru.

Rickettsiae are a family of ectoparasite-borne bacteria that can produce high morbidity and mortality among humans. There are scarce data on rickettsial ecology in rural areas of the Peruvian Amazon basin, where seroprevalence has not been determined, and the identities of animals acting as reservoirs of these bacteria are not known. We conducted a cross-sectional study in Zungarococha (between 2019 and 2021), a rural community located approximately 20 km away from Iquitos city. Blood samples were collected from humans (175), dogs (123), and cats (12). Blood samples and tissues were collected from black rats (84). Finally, we collected fleas from dogs and cats (222), ticks from dogs (91), and mites from black rats (32). Blood samples from humans, dogs, cats, and black rats were analyzed by indirect immunofluorescence assays (IFA) to detect IgG antibodies against rickettsias. We screened ectoparasites and black rat tissues by real-time-PCR (qPCR). Positive ectoparasites were further assessed by PCR and DNA amplicon sequencing. Non-parametric tests were used to evaluate factors associated with being seropositive among human adults. IgG seroprevalences were 38.3 %, 58.5 %, 16.7 % and 48.1 % among humans, dogs, cats, and rats, respectively. Among humans, only male gender was statistically associated with having IgG antibodies against Rickettsia spp. (p-value=0.049, chi-square test). Different ectoparasites were identified, including Ctenocephalides felis from cats and dogs, Rhipicephalus sanguineus s.l. from dogs, and Laelaps nuttalli from black rats. Rhipicephalus sanguineus s.l. (2/91 ticks) and Ct. felis (53/56 fleas and 55/55 flea pools) were qPCR-positive for Rickettsia spp. Recovered genetic material from 53 Ct. felis was sequenced and all were identified as Rickettsia asembonensis. All tissue samples from black rats were negative by qPCR. Humans, dogs, cats, and black rats are exposed to spotted fever group rickettsiae in rural areas surrounding Iquitos. As reported in urban areas, R. asembonensis is the main Rickettsia species circulating in rural areas surrounding Iquitos and Ct. felis appears to be the main vector.

The Role of HCN4 Variants in Human Systolic Heart Failure and Protein Interaction Network

Introduction: An elevated heart rate (HR) results in adverse outcomes in human systolic heart failure with a sinus rhythm. Genetic variants may alter HCN4 interactions with regulatory proteins and increase HR. This study aimed to generate a protein interaction network (PIN) associated with elevated HR and to determine HCN4 gene variants (718G&gt;A, 1571G&gt;A, and 2648C&gt;G) in patients with systolic heart failure, sinus rhythm, and elevated heart. Methods: STRING protein database was used to generate a PIN. Laboratory exploration was performed to identify HCN4 gene variants in patients with systolic heart failure using PCR and DNA sequencing. Results: PIN revealed eight nodes and 13 edges. STRING functional enrichment showed the essential proteins (ADRB1 and HCN channels) involved in HR regulation. GNAS and ADCY1 contributed to the regulation process. HCN4 gene variants (718G&gt;A, 1571G&gt;A, and 2648C&gt;G) alter the properties of HCN4 channel. STRING scores of protein-protein interactions that involved HCN4, ADRB1, GNAS, and ADCY1 were high (in the range of 0.879-0.979). The three gene variants were evaluated in 49 study participants with HR≥70 bpm after 10 mg bisoprolol therapy. However, only 718G&gt;A was identified in three of 49 patients. Conclusion: PIN revealed that the three essential proteins associated with HCN4 channels in elevating HR were ADRB1, GNAS, and ADCY1. Only HCN4 718G&gt;A was found in three out of 49 patients with systolic heart failure, sinus rhythm, and increased HR, according to a laboratory investigation on HCN4 gene variants

ALOX15-Driven Ferroptosis: The key target in Dihydrotanshinone I's epigenetic Battle in hepatic stellate cells against liver fibrosis.

It is known that ferroptosis promotes hepatic stellate cells (HSCs) inactivation. Arachidonate 15-Lipoxygenase (ALOX15), a ferroptosis driver gene, participates in disease progression. Dihydrotanshinone I (DHI), an active compound from Salvia miltiorrhiza, effectively regulates HSC inactivation. Nonetheless, there still needs to be clear understanding of how DHI affects HSC ferroptosis. This study primarily investigates DHI's protective effects on liver fibrosis in vivo and in vitro. Additionally, we explored the molecular mechanisms by which DHI promotes ferroptosis in HSCs. The relationship between ALOX15 level and methylation was examined. Molecular docking was performed to confirm the targeting between early growth response protein 1 (EGR1) and DHI. DHI exhibited a mitigating effect on liver fibrosis in vivo. DHI-induced inactivation of HSC by promoting ferroptosis, accompanied by an elevation in intracellular iron and reactive oxygen species (ROS) levels. Results of transcriptome sequencing and quantitative real-time PCR (qRT-PCR) confirmed the elevation of ALOX15 (a ferroptosis driver gene) in HSCs with DHI. Loss of ALOX15 inhibited DHI-induced ferroptosis. Interestingly, DNA methyltransferase 1 (DNMT1), an essential DNA methyltransferase, was downregulated by DHI. Overexpression of DNMT1 resulted in decreased ALOX15 expression in cells with DHI. Notably, transcription factor EGR1 was demonstrated to regulate DNMT1 expression. EGR1 deficiency led to an increase in DNMT1 expression, which inhibited DHI-induced ferroptosis. Molecular docking confirmed that EGR1 could serve as a direct pharmacological target of DHI. DHI upregulates EGR1 level, leading to decreased DNMT1 expression and increased ALOX15 demethylation, thereby promoting HSC ferroptosis and inactivation.