Polymerase Chain Reaction Sequencing Research Articles

Nkx1.2 deletion decreases fat production in zebrafish.

This study aimed to investigate the role of Nkx1-2, a transcription factor with the NK homeobox domain, in the regulation of fat production. Gene expression was analyzed using quantitative real-time polymerase chain reaction or transcriptome sequencing. CRISPR/Cas9 technology was employed to generate nkx1.2 knockout zebrafish and nkx1.2-deleted 3T3-L1 cells. Lipid droplet production in zebrafish larvae was visually quantified using Nile red staining, whereas lipid droplets in 3T3-L1 cells were stained with Oil red O. The binding of Nkx1-2 to the promoter was verified through an electrophoretic mobility shift assay experiment. Nkx1-2 plays crucial roles in the regulation of fat production in zebrafish. Knockout of nkx1.2 in zebrafish leads to weight loss, accompanied by significantly reduced lipid droplet production and decreased visceral and liver fat content. Furthermore, genes related to lipid biosynthesis are significantly downregulated. In 3T3-L1 preadipocytes, Nkx1-2 induces differentiation into mature adipocytes by binding to the cebpa promoter, thereby activating its transcription. Additionally, the expression of nkx1.2 is regulated by the p38 MAPK, JNK, or Smad2/3 signaling pathways in 3T3-L1 cells. Our findings suggest that Nkx1-2 functions as a positive regulator of fat production, playing a critical role in adipocyte differentiation and lipid biosynthesis.

Surface material analysis for human papillomavirus detection in nail Bowen's disease caused by HPV type 58.

Over the past few years, cases of human papillomavirus (HPV) infection in nail Bowen's disease have been reported. This disease presents diagnostic challenges due to its similarity to nail malignant melanoma, particularly with respect to the clinical manifestation of black nail streaks. While skin biopsy is usually employed for diagnosis, it is an invasive procedure. We report the case of a 52-year-old healthy Japanese male with a pigmented streak on the nail of the fourth finger of his right hand, which had extended from the central to the lateral nail fold within 4 months. Dermoscopic examination revealed a dark-brown pigmented band with splinter microhemorrhage. Clinically, nail Bowen's disease was suspected. The lesion was excised in strips under local anesthesia. Histopathological examination revealed hyperkeratosis, parakeratosis, papillomatosis, and dyskeratotic cells with atypical nuclei irregularly arranged. Immunohistochemistry using anti-HPV L1 antibody detected HPV-positive cells in the upper epidermis and stratum corneum of the nail matrix. Mucosal high-risk HPV type 58 DNA was detected from brush cytology of the keratotic surface prior to surgery, which was confirmed in formalin-fixed, paraffin-embedded excised samples using polymerase chain reaction (PCR) and subsequent direct DNA sequencing. Our case highlights HPV type 58 as a potential causative agent of nail Bowen's disease and shows that brush cytology of the surface material prior to excision may be a useful and less invasive way for mucosal high-risk HPV detection. PCR analysis of the nail surface could serve as a supplementary diagnostic tool for nail Bowen's disease.

Unraveling the Role of BRCA1 variants in Dysregulation of Transcriptional and Post-Transcriptional Mechanisms in Breast Cancer

Objective: To screen BRCA1 gene variants and predict potential role of the identified variants in breast cancer. Method: This case-control study included two hundred and fifty breast cancer patients and equal healthy individuals from the Federal Breast Cancer Screening Centre, Pakistan Institute of Medical Sciences, Islamabad from March 2021- January 2023. Demographic data was collected through questionnaires and clinical data was assessed using mammograms, ultrasound, histopathology and immunohistochemistry reports. Polymerase chain reaction and Sanger sequencing approach were used to detect variants in BRCA1 gene. In-silico analyses were carried out to predict mutation effect, miRNA binding site alterations and change in mRNA structure and stability. Results: Invasive ductal carcinoma was the most prevalent type of breast cancer. Old age [OR: 2.8149 (1.5995 to 4.9538) p value = 0.0003] and family history [OR: 4.3186 (1.7336 to 10.7581) p value = 0.001] were significant breast cancer risk. Six variants were identified. Two novel missense variants, Chr17:43082553A>T and Chr17:43093710A>T were predicted deleterious as these disrupted interaction with PALB2 and importin alpha’s NLS2 site, respectively. In silico analysis predicted the loss of hsa-miR-1179 binding site due to variant Chr17:43093220T>C. Moreover, four variants were predicted to affect the mRNA structure and stability. Conclusion: Two novel variants were predicted to be pathogenic. In-silico analysis predicted the loss of miRNA binding site along with change in mRNA secondary structure plus stability, possible mechanisms for oncogenesis. Further, expressional studies are required to confirm BRCA1 gene dysregulation in breast cancer due to these variants. doi: https://doi.org/10.12669/pjms.40.6.8761 How to cite this: Majeed AI, Zinan S, Faryal R. Unraveling the Role of BRCA1 variants in Dysregulation of Transcriptional and Post-Transcriptional Mechanisms in Breast Cancer. Pak J Med Sci. 2024;40(6):1093-1098. doi: https://doi.org/10.12669/pjms.40.6.8761 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Detection of fucosylated extracellular vesicles miR-4732-5p related to diagnosis of early lung adenocarcinoma by the electrochemical biosensor

Our preliminary investigation has identified the potential of serum fucosylated extracellular vesicles (EVs) miR-4732-5p in the early diagnosis of lung adenocarcinoma (LUAD) by a fucose-captured strategy utilizing lentil lectin (LCA)-magnetic beads and subsequent screening of high throughput sequencing and validation of real-time quantitative polymerase chain reaction (RT-qPCR). Considering the relatively complicated procedure, expensive equipment, and stringent laboratory condition, we have constructed an electrochemical biosensor assay for the detection of miR-4732-5p. miR-4732-5p is extremely low in serum, down to the fM level, so it needs to be detected by highly sensitive electrochemical methods based on the Mg2+-dependent DNAzyme splitting nucleic acid lock (NAL) cycle and hybridization chain reaction (HCR) signal amplification. In this study, signal amplification is achieved through the dual amplification reactions using NAL cycle in combination with HCR. In addition, hybridized DNA strands bind to a large number of methylene blue (MB) molecules to enhance signaling. Based on the above strategy, we further enhance our signal amplification strategies to improve detection sensitivity and accuracy. The implementation of this assay proceeded as follows: initially, miR-4732-5p was combined with NAL, and then Mg2+-dependent DNAzyme splitted NAL to release auxiliary DNA (S1) strands, which were subsequently captured by the immobilized capture probe DNA (C1) strands on the electrode surface. Following this, abundant quantities of DNA1 (H1) and DNA2 (H2) tandems were generated by HCR, and S1 strands then hybridized with the H1 and H2 tandems through base complementary pairing. Finally, MB was bonded to the H1 and H2 tandems through π–π stacking interaction, leading to the generation of a signal current upon the detection of a potential capable of inducing a redox change of MB by the electrode. Furthermore, we evaluated the performance of our developed electrochemical biosensor assay. The results demonstrated that our assay is a reliable approach, characterized by its high sensitivity (with a detection limit of 2.6 × 10−17 M), excellent specificity, good accuracy, reproducibility, and stability. Additionally, it is cost-effective, requires simple operation, and is portable, making it suitable for the detection of serum fucosylated extracellular vesicles miR-4732-5p. Ultimately, this development has the potential to enhance the diagnostic efficiency for patients with early-stage LUAD.

Subgingival microbiome in periodontal disease and comorbid pathology (meta-analysis)

The problem of chronic periodontitis (CP) is actively discussed due to the recognition of the fact that periodontal microbial damage is closely related to a number of systemic diseases and probably plays an important role in the occurrence of comorbid pathology. The aim of the meta-analysis was to characterize the composition of the subgingival microbiome and to determine the peculiarities of the formation of associations of the new periodontal pathogen Filifactor alocis with other I and II order periodontal pathogenic bacteria, as well as with the commensal bacteria colonizing this biotope. The study presents data of patient examination with obligatory use of polymerase chain reaction methods and sequencing of 16S rRNA genes in 1529 healthy individuals and 2394 patients with CP, 136 patients with CP and concomitant atherosclerosis, 258 patients with CP and concomitant type 2 diabetes mellitus. It was confirmed that the basis of the oral microbiome under normal circumstances is composed of representatives of microaerophilic streptococci, corynebacteria, lactobacilli, as well as representatives of the Veillonella and Sphingobacterium genera. 16S sequencing and bioinformatic analysis allowed us to specify the taxonomic place of the new pathogen F. alocis, as well as representatives of normal microbiota in CP and comorbid somatic pathology.

Influence of UGT2B7, UGT1A4 and ABCG2 Polymorphisms on the Pharmacokinetics and Therapeutic Efficacy of Lamotrigine in Patients with Epilepsy.

A substantial inter-individual variability has been observed in the pharmacokinetics of lamotrigine. The aim of the study was to investigate the impact of genetic polymorphism of the metabolizing enzymes (UGT2B7, UGT1A4) and transporter (ABCG2) on the pharmacokinetics and therapeutic efficacy of lamotrigine in patients with epilepsy. The genetic analysis of single-nucleotide polymorphisms was conducted using polymerase chain reaction sequence. High-performance liquid chromatography/tandem mass spectrometry was employed to measure the plasma concentrations of lamotrigine. The efficacy of lamotrigine was assessed by evaluating the reduction rate of epileptic seizure frequency. This study included a cohort of 331 patients who were treated with lamotrigine as monotherapy. A linear correlation was observed between the lamotrigine concentration and daily dose taken (r = 0.58, p < 2.2e-16). Statistically significant differences were found in both the median plasma concentration and dose-adjusted concentration (C/D ratio) when comparing the ineffective to the effective group (p < 0.05). Multivariate analysis showed that UGT1A4 rs2011425, ABCG2 rs2231142 polymorphisms and age had a significant relationship with the lamotrigine concentrations (p < 0.05). Age was a predictive factor for C/D ratio (p < 0.001). Lamotrigine concentration and weight were good predictive factors for effective seizure outcomes (odds ratio [OR] = 0.715, 95% CI 0.658-0.776, p < 0.001; OR = 0.926, 95% CI 0.901-0.951, p < 0.001, respectively). The cut-off values of lamotrigine trough concentrations for clinical outcomes in the age-related groups were determined as 2.49μg/ml (area under the receiver-operating characteristic curve [AUC]: 0.828, 95% CI 0.690-0.966), 2.70μg/ml (AUC: 0.805, 95% CI 0.745-0.866) and 3.25μg/ml (AUC: 0.807, 95% CI 0.686-0.928) for the adult group, adolescent group, and toddler and school-age group, respectively. UGT1A4 rs2011425 and ABCG2 rs2231142 were correlated with lamotrigine concentrations. Lower lamotrigine trough concentration was found in the ineffective group and the troughs were associated with seizure outcomes.

Insights into the abundance, expression and diversity of key denitrification genes in an ecologically managed greenhouse agricultural soil

Understanding the bacteria associated with nitrification and denitrification is crucial for comprehending the processes that lead to nitrous oxide emissions in agricultural greenhouse soils. Therefore, it is important to determine their abundance and expression to gain insight into these processes.The aim of this study was to explore the bacterial communities associated with denitrification in a greenhouse agricultural soil amended with crop residues and manure for six years. For this purpose, we proceeded to detect and quantify the genes nirK and nirS and the gene nosZ through clone library construction, sequencing, phylogenetic analysis, and quantitative polymerase chain reaction (qPCR). Sequence analysis based on the clone library revealed that many of the nirS or nirK genes detected were not closely related to known denitrifier bacteria, but some of the nosZ sequences were related to the genera such as Pseudomonas, Halomonas, and Marinobacter. Furthermore, the qPCR revealed a high abundance of DNA copies in nirK, 6.08 × 109 ± 1.16 × 109, while nirS and nosZ showed lower values, 9.05 × 106 ± 1.65 × 106 and 8.71 × 106 ± 1.44 × 106, respectively. However, the highest expression rate was observed for nirS (mRNA/DNA ratio = 3.10 × 10− 3), while nirK and nosZ showed 10-fold lower expression rates (4.4 × 10− 4 and 3.5 × 10− 4, respectively). The results of this work provide a preliminary overview of the diversity, abundance and expression of key genes associated with the denitrification process in this type of soil and are a starting point for further studies to understand how this type of soil management can influence the denitrification process.

Clonotypic VDJ rearrangements in Mixed Phenotype Acute Leukemia can be Successfully Utilized to Track Minimal Residual Disease (MRD).

Multiplex polymerase chain reaction (PCR) and next-generation sequencing (NGS) can both be used to identify a neoplastic clonotype by targeting CDR3 and assessing rearrangements in IgH, IgK, IgL, TCR-β, and TCR-gamma loci. The clonotypic sequence can be robustly used to track minimal residual disease (MRD). The ability to track MRD by NGS in mixed phenotype acute leukemia (MPAL) is unknown and warrants investigation. Institutional Review Board (IRB) approval was obtained. Central Moffitt Cancer Center (MCC) database was searched to locate any patients with MPAL from over 600,000 entries. Patient charts were manually curated to identify those with clonoSEQ data, and clinical data was procured from the electronic medical record (EMR). Twenty-nine patients with MPAL were identified. Only 2 patients with clonoSEQ testing were found. Both demonstrated a B/myeloid phenotype, and both were bilineal. NGS (clonoSEQ) identified 4 dominant (IGH) (patient A; 8/2019) and 2 dominant sequences (patient B; 10/2019), respectively. In both patients, clonoSEQ testing successfully tracked minimal residual disease and mirrored clinical disease burden. This report is the first to confirm the utility of NGS-based MRD tracking in patients with MPAL and shows increased sensitivity of NGS over MRD flow cytometry.

Mapping Transposon Insertion Sites by Inverse Polymerase Chain Reaction and Sanger Sequencing.

Inverse polymerase chain reaction (PCR) is a method designed to amplify a segment of DNA for which only a portion of the sequence is known. The method consists of circularizing the DNA fragment by self-ligation and performing PCR with primers annealing inside the known sequence but pointing away from each other (hence the technique is also called "inside-out PCR"). Here we describe how inverse PCR can be used to identify the site of transposon insertion in the bacterial chromosome. This protocol, implemented here with a class of transposons generating reporter gene fusions, involves (i) preparing genomic DNA from the strain harboring the unknown insertion, (ii) cleaving the genomic DNA with a restriction enzyme, (iii) performing a ligation reaction under conditions favoring circularization of the DNA fragments, and (iv) performing inverse PCRs with inside-out primers annealing near either or both termini of the transposon. This last step results in the amplification of the chromosomal sequences immediately adjacent to the transposon, which can then be identified by Sanger sequencing. The protocol can be performed in parallel on several strains providing an effective and economic way for rapidly identifying multiple transposon insertion sites.

Screening for antifolate and artemisinin resistance in Plasmodium falciparum dried-blood spots from three hospitals of Eritrea

Background Antimalarial drug resistance is a major challenge hampering malaria control and elimination. About three-quarters of Eritrea’s population resides in the malaria-endemic western lowlands of the country. Plasmodium falciparum, the leading causative parasite species, has developed resistance to basically all antimalarials. Continued surveillance of drug resistance using genetic markers provides important molecular data for treatment policies which complements clinical studies, and strengthens control efforts. This study sought to genotype point mutations associated with P. falciparum resistance to sulfadoxine-pyrimethamine and artemisinin, in dried-blood spots from three hospitals in the western lowlands of Eritrea. Methods Dried-blood spot samples were collected from patients visiting Adi Quala, Keren and Gash Barka Hospitals, between July and October, 2014. The patients were followed up after treatment with first line artesunate-amodiaquine, and dried-blood spots were collected on day three after treatment. Nested polymerase chain reaction and Sanger sequencing techniques were employed to genotype point mutations in the Pfdhfr (PF3D7_0417200), Pfdhps (PF3D7_0810800) and PfK13 (PF3D7_1343700) partial gene regions. Results Sequence data analyses of PCR-positive isolates found wild-type artemisinin haplotypes associated with resistance (Y493Y, R539R, I543I) in three isolates, whereas four mutant antifolate haplotypes associated with resistance were observed in six isolates. These included the triple-mutant Pfdhfr (S108N, C59R, N51I) haplotype, the double-mutant Pfdhfr (N51I, S108N) haplotype, the single-mutant Pfdhfr (K540E) haplotype, and the mixed-mutant Pfdhfr-Pfdhps (S108N, N51I + K540E) haplotype. Other findings observed were, a rare non-synonymous Pfdhfr V45A mutation in four isolates, and a synonymous Pfdhps R449R in one isolate. Conclusions The mutant antifolate haplotypes observed indicate a likely existence of full SP resistance. Further studies can be carried out to estimate the prevalence of SP resistance. The wild-type artemisinin haplotypes observed suggest artemisinin is still an effective treatment. Continuous monitoring of point mutations associated with delayed parasite clearance in ART clinical studies is recommended.

Abstract PO4-20-06: Impact of iatrogenic menopause on vaginal health and sexuality

Abstract Background Due to the lowering of circulating estrogens symptoms of genitourinary syndrome of menopause (GSM) may arise with premenopausal breast cancer patients who become menopausal due to breast cancer treatment. Clinical signs of GSM are i.a. vaginal dryness, dyspareunia, loss of libido, dysfunction of arousal and/or orgasm: this may negatively impact vaginal health, sexual activity and quality of life. The composition and function of the vaginal microbiome may also change due to changes of gonadal hormones during the menopausal transition. This change may contribute to vaginal discomfort and problems during sexual activity. With this prospective research we aim to uncover the relationship between vaginal health and sexuality, it gives an unique opportunity to investigate the vaginal changes and possible shift of vaginal microbiome when menopause occurs. This is a first in time study where longitudinal samples of the vaginal microbiome in one patient are investigated before and after menopause. The vaginal microbiome might be a future target to treat vaginal discomfort after treatment for breast cancer. Trial design This is a prospective minimal invasive study where participants are asked to undergo a baseline gynecologic examination, again 3 and 6 months after menopause. During this examination we measure vaginal pH, describe vaginal hydration and anatomy (using vaginal-, and vulvar assessment scale (VAS, VuAS)) and sample the vaginal microbiome (analyzed with 16S rRNA gene sequencing and quantitative real-time polymerase chain reaction (qPCR)). At the same time, a blood test is performed (Estradiol, FSH) and participants complete a survey (demographic information, medical history, current well-being using World Health Organisation- Five Well-Being Index (WHO-5), sexual functioning using the Female Sexual Functioning index (FSFI), Female Sexual Dysfunction Scale – revised (FSDS-R) and Short Sexual Functioning scale (SSFS)). Eligibility criteria - Patients (&amp;gt;18 years) with Stage I, IIA, IIB, IIIA, IIIB, IIIC breast cancer - Reproductive age or menopausal transition according to STRAW+10 criteria - Iatrogenic menopause caused by ovarian function suppression (OFS), bilateral salpingo-ovariectomy (BSO) or by starting chemotherapy - Any partner status - Latest cervical cytology is normal - No prior history of hysterectomy - Patients who take oral contraceptives or have a Levonorgestrel- containing IUD can participate. Oral contraceptives should be stopped or the Levonorgestrel-containing IUD should be removed before systemic breast cancer therapy Specific aims To accurately describe what vaginal changes occur during breast cancer treatment To evaluate if the onset of menopause is related to changes in vaginal microbiome To evaluate if there is a relation between the objective vaginal changes and the subjective complaints related to GSM To evaluate if there is a relation between the objective vaginal changes and a possible alteration in sexuality Statistical methods Using multiple comparison procedures in ANOVA (analysis of Variance) model, the clinical findings (VAS, VuAS, pH, composition of microbiome) and validated questionnaires (WHO-5, FSFI, FSDR-r, SSFS) are linked to analyze if vaginal changes are related to changes in sexual wellbeing. The interpretation of the results of this investigation should be interpreted with caution due to the explorative nature of the study. Present accrual/Target accrual 23/60 participants are included (JULY 2023) Citation Format: Nynke Willers, Sileny Han, Patrick Neven, Paul Enzlin, Sarah Lebeer, Sarah Ahannach. Impact of iatrogenic menopause on vaginal health and sexuality [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-20-06.

Conditional knockout mouse model reveals a critical role of peroxiredoxin 1 in oral leukoplakia carcinogenesis

Peroxiredoxin 1 (Prx1) is an antioxidant protein that may promote the carcinogenesis in oral leukoplakia (OLK). To investigate the effect of Prx1 on the oral mucosal epithelium of OLK, we generated a Prx1 conditional knockout (cKO) mouse model. The mRNA and gRNA were generated using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) technique. An infusion cloning method was used to construct a homologous recombination vector. To obtain the F0 generation mice, fertilized eggs of C57BL/6J mice were microinjected with Cas9 mRNA, gRNA, and a donor vector. Polymerase chain reaction (PCR) amplification and sequencing were used to identify F1 generation mice. Using the cyclization recombination-enzyme-locus of the X-overP1 (Cre-loxP) system, we created a Prx1 cKO mouse model, and the effectiveness of the knockout was confirmed through immunohistochemistry. We examined the influence of Prx1 knockout on the occurrence of OLK in mice by constructing a model of tongue mucosa carcinogenesis induced by 4-nitroquinoline-1-oxide (4NQO). Prx1 modification was present in the F1 generation, as evidenced by PCR amplification and sequencing. Prx1flox/flox: Cre + mice exhibited normal growth and fertility. Immunohistochemical analysis revealed that tongue epithelial cells in Prx1flox/flox: Cre + mice displayed a distinct deletion of Prx1. An examination of the heart, liver, spleen, lung, and kidney tissues revealed no visible histological changes. Histological analysis showed a reduction in the occurrence of the malignant transformation of OLK in the tongue tissues of Prx1flox/flox: Cre + mice. Ki67 immunostaining showed that Prx1 knockout significantly inhibited cell proliferation in the tongue epithelial. Our research developed a conditional knockout mouse model for Prx1. The obtained results provide insights into the function of Prx1 in the development of oral cancer and emphasize its potential as a therapeutic target for precancerous oral lesions.

Microscopic and molecular epidemiology of gastrointestinal nematodes in dairy and beef cattle in Pak Chong district, Nakhon Ratchasima province, Thailand

Background and Aim: Gastrointestinal (GI) nematode infection remains an important problem in livestock, particularly cattle. The infection may lead to serious health complications and affect animal products. The objective of this study was to investigate GI nematode infection and its associated risk factors in dairy and beef cattle farmed in Pak Chong District of Nakhon Ratchasima Province, northeast Thailand. Materials and Methods: Fecal specimens were collected from 101 dairy cattle and 100 beef cattle. Formalin-ethyl acetate concentration techniques were used to process the samples and the samples were observed under a light microscope. Samples were subjected to molecular identification of specific genera using conventional polymerase chain reaction and DNA sequencing. Results: The overall prevalence of GI nematode infection was 33.3%. The strongyle nematode was the most significant GI nematode in this area with a prevalence of 28.4%. The prevalence of strongyle nematodes was 58.0% in beef cattle and only 7.9% in dairy cattle. Trichuris spp. was another nematode found in both types of cattle with an overall prevalence of 5.0% with 9.0% in beef cattle and 1.0% in dairy cattle. The results of the epidemiological study indicate that the age of cattle, food, water sources, farming system, and housing floor are the most important risk factors. Among the strongyle nematodes, Ostertagia spp. was the most prevalent (82.0%), followed by Haemonchus spp. (62.3%) and Trichostrongylus spp. (8.2%), respectively. Conclusion: Infection with GI nematodes still exists in this area, particularly in beef cattle. Our reported data may benefit local parasitic control policies in the future. Keywords: beef cattle, dairy cattle, gastrointestinal nematode, strongyle nematodes.

Gastric adenocarcinoma predictive long intergenic noncoding RNA (GAPLINC) promotes oral cancer stemness by acting as a molecular sponge of miR331-3p

Background/purposeAccumulating evidence has suggested that treatment failure of cancer therapy can be attributed to cancer stem cells (CSCs). Among numerous regulators of cancer stemness, non-coding RNAs (ncRNAs) have gained significant attention recently. In this study, we examined the role of gastric adenocarcinoma predictive long intergenic noncoding RNA (GAPLINC) in oral CSCs (OCSCs). Materials and methodsRNA Sequencing and quantitative real-time polymerase chain reaction (qRT-PCR) were used to determine the expression of GAPLINC. Flow cytometry and sphere-forming assay were exploited to isolate OCSCs. Measurement of aldehyde dehydrogenase 1 (ALDH1) activity, CD44 expressing cells, and various phenotypic assays, such as self-renewal, migration, invasion, and colony-forming abilities, were conducted in CSCs of two types of oral cancer cells (SAS and GNM) following the knockdown of GAPLINC. A luciferase reporter was also carried out to validate the direct interaction between GAPLINC and microRNA (miR)-331-3p. ResultsOur results showed that GAPLINC was overexpressed in OCSCs from patient-derived and oral cancer cell lines. We demonstrated that silencing of GAPLINC in OCSCs downregulated various CSC hallmarks, such as ALDH1 activity, percentage of CD44-expressing cells, self-renewal capacity, and colony-forming ability. Moreover, our results revealed that the effect of GAPLINC on cancer stemness was mediated by direct repression of miR-331-3p. ConclusionThese data have potential clinical implications in that we unraveled the aberrant upregulation of GAPLINC and demonstrated that suppression of GAPLINC may reduce cancer stemness via sequestering miR-331-3p.

Da-Jian-Zhong decoction alleviates diarrhea-predominant irritable bowel syndrome via modulation of gut microbiota and Th17/Treg balance

Ethnopharmacological relevanceDa-Jian-Zhong decoction (DJZD) is a herbal formula clinically used for abdominal pain and diarrhea induced by spleen-Yang deficiency syndrome. Recently, treatment of diarrhea-predominant irritable bowel syndrome (IBS-D) with DJZD has received increasing attention, but the underlying mechanism of action remains elusive. Aim of the studyWe aimed to evaluate the therapeutic effect of DJZD on IBS-D rats and to elucidate the underlying mechanisms. Materials and methodsAn IBS-D rats model was constructed using a two-factor superposition method of neonatal maternal separation and Senna folium aqueous extract lavage. Moreover, the effect of DJZD was evaluated based on the body weight, rectal temperature, abdominal withdrawal reflex (AWR), and Bristol stool scale score (BSS). The factors that regulate the DJZD effects on IBS-D were estimated using whole microbial genome, transcriptome sequencing (RNA-Seq), flow cytometry, and quantitative reverse transcription polymerase chain reaction (RT-qPCR) analyses. ResultsWe found that DJZD alleviated the symptoms of IBS-D rats, with the low-dose (2.4 g/kg) as the better ones, as shown by the higher body mass and lower AWR score and BSS. At the phylum level, the relative abundance of Bacteroidetes was obviously increased, and at the genus level, Bacteroides was increased, while that of Firmicutes_bacterium_424 and Ruminococcus gnavus was decreased in DJZD group. The significantly enriched GO terms after treatment with DJZD mainly included the immune response, positive regulation of activated T cell proliferation, and positive regulation of interleukin-17 (IL-17) production. Importantly, flow cytometry analysis further revealed that the T helper cell type 17/regulatory T cell (Th17/Treg) balance contributed to the DJZD-induced alleviation of IBS-D symptoms, as DJZD downregulated Th17/Treg ratio and Th17 cell-related cytokines IL-17 and IL-6 levels in the colon. ConclusionsThese results demonstrate that DJZD has a good therapeutic effect on IBS-D rats, probably by maintaining the homeostasis of gut microbiota and regulating Th17/Treg balance and its related inflammatory factors.

Identification of Periostin Positive Cell Population during the Long-Term Culture of Mouse Tendon-Derived Cells in Late Passage.

Tendon-derived cells exhibit phenotypic changes and gradually lose their proliferative capacity during serial passages in vitro. This study aimed to characterize the changes in the growth and stem cell characteristics of tendon-derived cells over a long-term culture. Mouse flexor digitorum profundus tendon-derived cells were obtained by enzymatic digestion and seeded at an initial density of 5,000/cm2. Cells were characterized by morphology, growth, senescence staining, trilineage differentiation assays, real-time polymerase chain reaction, immunocytochemistry, flow cytometry, and RNA sequencing analysis. Tendon-derived cells underwent a proliferative stage in the first three passages, followed by a gradual senescence. However, a novel cell population expressing periostin (Postn+) emerged during the long-term culture from passages 5-8, which possessed a high rate of proliferation without significant senescence over successive passages. Compared to early passage cells, Postn+ cells exhibited enhanced osteogenic differentiation potential and attenuated chondrogenic differentiation potential, decreased expression of SSEA-1, Oct3/4, tenomodulin, scleraxis, CD90.2, CD73, CD105, Sca-1, and CD44, and increased expression of collagen III and CD34. RNA-sequencing analysis of Postn+ and early passage cells identified 908 differentially expressed genes, with extracellular matrix-receptor interaction and focal adhesion as the top pathways, and integrins as hub genes. This study highlights the dynamics of tendon-derived cells during serial passages. We identify a Postn+ cell population during long-term culture in late passages, with high proliferative ability and prominent osteogenic differentiation potential. Further investigations are needed to elucidate the origin and potential applications of Postn+ tendon-derived cells.

Differential expression and analysis of extrachromosomal circular DNAs as serum biomarkers in pulmonary arterial hypertension

BackgroundExtrachromosomal circular DNAs (eccDNAs) have been reported to play a key role in the occurrence and development of various diseases. However, the characterization and role of eccDNAs in pulmonary arterial hypertension (PAH) remain unclear.MethodsIn the discovery cohort, we first explored eccDNA expression profiles by Circle-sequencing analysis. The candidate eccDNAs were validated by routine polymerase chain reaction (PCR), TOPO-TA cloning and Sanger sequencing. In the validation cohort, 30 patients with PAH and 10 healthy controls were recruited for qPCR amplification to detect the candidate eccDNAs. Datas at the baseline were collected, including clinical background, biochemical variables, echocardiography and hemodynamic factors. Receiver operating characteristic curve was used to investigate the diagnostic effect of the eccDNA.ResultsWe identified a total of 21,741 eccDNAs in plasma samples of 3 IPAH patients and 3 individuals in good health, and the expression frequency, GC content, length distribution, and genome distribution of the eccDNAs were thoroughly characterized and analyzed. In the validation cohort, 687 eccDNAs were differentially expressed in patients with IPAH compared with healthy controls (screening threshold: |FC|≥2 and P < 0.05). Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the specific eccDNAs in IPAH were significantly enriched in calcium channel activity, the mitogen-activated protein kinase pathway, and the wnt signaling pathway. Verification queue found that the expression of eccDNA-chr2:131208878–131,424,362 in PAH was considerably higher than that in healthy controls and exhibited a high level of accuracy in predicting PAH with a sensitivity of 86.67% and a specificity of 90%. Furthermore, correlation analysis disclosed a significant association between serum eccDNA-chr2:131208878–131,424,362 and mean pulmonary artery pressure (mPAP) (r = 0.396, P = 0.03), 6 min walking distance (6MWD) (r = -0.399, P = 0.029), N-terminal pro-B-type natriuretic peptide (NT-proBNP) (r = 0.685, P < 0.001) and cardiac index (CI) (r = − 0.419, P = 0.021).ConclusionsThis is the first study to identify and characterize eccDNAs in patients with PAH. We revealed that serum eccDNA-chr2:131208878–131,424,362 is significantly overexpressed and can be used in the diagnosis of PAH, indicating its potential as a novel non-invasive biomarker.

The Influence of Tacrolimus on Cellular Morphology, Cellular Viability, Osteogenic Differentiation, and mRNA Expression within Stem Cell Spheroids.

Background and Objectives: Tacrolimus is a macrolide lactone compound derived from the bacterium Streptomyces tsukubensis, widely known as an immunosuppressant. In basic research, the effects of tacrolimus on osteogenic differentiation have been tested using mesenchymal stem cells. In this study, tacrolimus's effects on the cellular survival and osteogenic differentiation of stem cell spheroids were investigated. Materials and Methods: Concave microwells were used to form stem cell spheroids in the presence of tacrolimus at final concentrations of 0 μg/mL, 0.1 μg/mL, 1 μg/mL, 10 μg/mL, and 100 μg/mL. A microscope was used to test cellular vitality qualitatively, and an assay kit based on water-soluble tetrazolium salt was used to measure cellular viability quantitatively. Alkaline phosphatase activity and an anthraquinone dye test for measuring calcium deposits were used to assess osteogenic differentiation. To assess the expression of osteogenic differentiation, a quantitative polymerase chain reaction, Western blot, and RNA sequencing were performed. Results: Spheroids across all concentrations maintained a relatively uniform and spherical shape. Cell viability assay indicated that tacrolimus, up to a concentration of 100 μg/mL, did not significantly impair cell viability within spheroids cultured in osteogenic media. The increase in calcium deposition, particularly at lower concentrations of tacrolimus, points toward an enhancement in osteogenic differentiation. There was an increase in COL1A1 expression across all tacrolimus concentrations, as evidenced by the elevated mean and median values, which may indicate enhanced osteogenic activity. Conclusions: This study showed that tacrolimus does not significantly impact the viability of stem cell spheroids in osteogenic media, even at high concentrations. It also suggests that tacrolimus may enhance osteogenic differentiation, as indicated by increased calcium deposition and COL1A1 expression. These findings advance our understanding of tacrolimus's potential roles in tissue repair, regeneration, and stem cell-based therapeutic applications.

Prevalence and Molecular Analysis of Encephalomyocarditis Virus-2 in the Hazel Dormouse.

The hazel dormouse (Muscardinus avellanarius) population in the UK continues to decline due to habitat loss, despite reintroductions of captive-bred individuals being conducted nationally for over 30years. Disease surveillance of captive-bred and wild dormice is performed to identify novel and existing disease threats which could impact populations. In this study, we firstly investigated cause of death in seven hazel dormice found dead in England, through next-generation sequencing identifying a virus closely related to a wood mouse encephalomyocarditis virus-2 (EMCV-2). Subsequently, lung tissue samples from 35 out of 44 hazel dormice tested positive for EMCV-2 RNA using a reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and Sanger sequencing methods developed in this study. Formalin-fixed tissues available for nine hazel dormice which tested positive for EMCV-2 RNA were examined microscopically. Three cases showed moderate interstitial pneumonia with minimal to mild lymphoplasmacytic myocarditis, but no evidence of encephalitis. However, the presence of possible alternative causes of death in these cases means that the lesions cannot be definitively attributed to EMCV-2. Here, we report the first detection of EMCV-2 in hazel dormice and conclude that EMCV-2 is likely to be endemic in the hazel dormouse population in England and may be associated with clinical disease.

Assessing intracellular and extracellular distribution of antibiotic resistance genes in the commercial organic fertilizers

Compost-based organic fertilizers often contain high levels of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs). Previous studies focused on quantification of total ARGs and MGEs. For a more accurate risk assessment of the dissemination risk of antibiotic resistance, it is necessary to quantify the intracellular and extracellular distribution of ARGs and MGEs. In the present study, extracellular ARGs and MGEs (eARGs and eMGEs) and intracellular ARGs and MGEs (iARGs and iMGEs) were separately analyzed in 51 commercial composts derived from different raw materials by quantitative polymerase chain reaction (qPCR) and metagenomic sequencing. Results showed that eARGs and eMGEs accounted for 11–56% and 4–45% of the total absolute abundance of ARGs and MGEs, respectively. Comparable diversity, host composition and association with MGEs were observed between eARGs and iARGs. Contents of high-risk ARGs were similar between eARGs and iARGs, with high-risk ARGs in the two forms accounting for 6.7% and 8.2% of the total abundances, respectively. Twenty-four percent of the overall ARGs were present in plasmids, while 56.7% of potentially mobile ARGs were found to be associated with plasmids. Variation partitioning analysis, null model and neutral community model indicated that the compositions of both eARGs and iARGs were largely driven by deterministic mechanisms. These results provide important insights into the cellular distribution of ARGs in manure composts that should be paid with specific attention in risk assessment and management.