Polymerase Chain Reaction Sequencing Research Articles

Innovations in cancer diagnosis and treatment: prospects and challenges

The research relevance of cancer diagnostics and treatment is determined by its widespread occurrence and the lack of adequate modern diagnostic methods. The study aims to characterise new diagnostic methods, namely screenings in detecting cancer at early stages of development. To achieve this goal, the bibliosemantic and bibliographic methods were used. Main results. Cancer is the leading cause of morbidity and mortality after cardiovascular diseases and injuries in many countries around the world. Various diagnostic and treatment methods are used to combat this problem, including computed tomography, magnetic resonance imaging and positron emission tomography. In addition, modern diagnostic methods such as polymerase chain reaction, mass spectrometry and genomic sequencing play an important role in determining the types of cancer cells and their sensitivity to treatment. These advanced methods can be used to diagnose cancer more accurately and efficiently and choose the most appropriate treatment strategies for each patient. The practical significance of this topic is to ensure appropriate care for patients with cancer: providing high-quality, efficient, fast and minimally invasive diagnostics using the latest methods, as well as implementing a screening system.

The first report of prion protein gene sequences in Dybowski’s frog and the American bullfrog: high amyloid propensity of the frog prion protein

Prion diseases are fatal infectious neurodegenerative diseases caused by the proteinase K-sensitive form of prion protein (PrPSc). The exact origin of prion seeding and the transition factor of PrPSc has not been elucidated. The main hosts of prion diseases are herbivores, so the feces and corpses of Amphibians can seed PrPSc through ecosystems. The frog is an excellent candidate for transmission studies for this reason, but genetic analyses of the prion protein gene (PRNP) in the context of prion-related characteristics of frog species are lacking. We amplified frog PRNP gene sequences in Dybowski’s frog and the American bullfrog by polymerase chain reaction (PCR) and amplicon sequencing. In addition, we carried out multiple sequencing alignments and annotated major PrP components including signal peptide, tandem repeat domain, and PrPC-PrPSc interaction region of frog PrPs by bioinformatics tools. We predicted secondary and tertiary structures and amyloid propensities of frog PrPs using AlphaFold2 and AMYCO, respectively. We obtained DNA sequences of the PRNP gene in Dybowski’s frog and the American bullfrog, as well as a partially conserved palindromic sequence (PrPC-PrPSc interaction region) and absence of tandem repeat region of PrP in seven frog species. We analyzed protein structure of among these frog species and found that the high Himalaya frog has high aggregation propensity and the western clawed frog does not have the N-terminal signal peptide. To the best of our knowledge, this was the first comparative genetic study regarding prion-related features of frog species.

Use of FISH-FLOW as a Method for the Identification and Quantification of Bacterial Populations.

The gastrointestinal tract (GIT) harbors the largest group of microbiotas among the microbial communities of the human host. The resident organisms typical of a healthy gut are well adapted to the gastrointestinal environment while alteration of these populations can trigger disorders that may affect the health and well-being of the host. Various investigations have applied different tools to study bacterial communities in the gut and their correlation with gastrointestinal disorders such as inflammatory bowel disease (IBD), obesity, and diabetes. This study proposes fluorescent in situ hybridization, combined with flow cytometry (FISH-FLOW), as an alternative approach for phylum level identification of Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria and quantification of target bacteria from the GIT based on analysis of fecal samples, where results are validated by quantitative polymerase chain reaction (qPCR) and 16S ribosomal ribonucleic acid (16s rRNA) sequencing. The results obtained via FISH-FLOW experimental approach show high specificity for the developed probes for hybridization with the target bacteria. The study, therefore, suggests the FISH-FLOW as a reliable method for studying bacterial communities in the gut with results correlating well with those of metagenomic investigations of the same fecal samples.

Identification of the genetic background of laboratory rats through amplicon-based next-generation sequencing for single-nucleotide polymorphism genotyping

BackgroundLaboratory rats, as model animals, have been extensively used in the fields of life science and medicine. It is crucial to routinely monitor the genetic background of laboratory rats. The conventional approach relies on gel electrophoresis and capillary electrophoresis (CE) technologies. However, the experimental and data analysis procedures for both of these methods are time consuming and costly.ResultsWe established a single-nucleotide polymorphism (SNP) typing scheme using multiplex polymerase chain reaction (PCR) and next-generation sequencing (NGS) to address the genetic background ambiguity in laboratory rats. This methodology involved three rounds of PCR and two rounds of magnetic bead selection to improve the quality of the sequencing data. We simultaneously analysed 100 laboratory rats (including rats of 5 inbred strains and 2 in-house closed colonies), and the sequencing depth varied from an average of 108.25 to 5189.89, with sample uniformity ranging from 82.5 to 97.5%. A total of 98.9% of the amplicons were successfully genotyped (≥ 30 reads). Genetic background analysis revealed that all 38 experimental rats from the 5 inbred strains were successfully identified (without a heterozygous allele). For the 2 in-house closed colonies, the average heterozygosity (0.162 and 0.169) deviated from the typical range of 0.5–0.7, indicating a departure from the ideal heterozygosity level. Additionally, we employed multiplex PCR-CE to validate the NGS-based method, which yielded consistent results for all the rat strains. These results demonstrated that this approach significantly improves efficiency, saves time, reduces costs and ensures accuracy.ConclusionBy utilizing NGS technology, our developed method leverages SNP genotyping for genetic background identification in laboratory rats, demonstrating advantages in terms of labour efficiency and cost-effectiveness, thereby rendering it well suited for projects involving extensive sample cohorts.

HGGL-01 GLIOBLASTOMA MULTIFORM INVOLVING MULTIPLE BRAIN REGIONS ASSOCIATED WITH MUTATIONS IN EXON 4 OF ISOCITRATE DEHYDROGENASE 1 COMPLICATED BY ACUTE KIDNEY FAILURE: A CASE REPORT

Abstract Glioblastoma multiform (GBM), WHO grade 4, is the most aggressive primary brain tumor in adults associated with poor prognosis. We present a case of a 43-year-old female, Muyira, diagnosed with GBM involving multiple brain regions and harboring mutations in exon 4 of isocitrate dehydrogenase 1, who subsequently developed acute kidney failure. The patient initially presented with progressive neurological symptoms, including headache and cognitive decline. Multiplanar multiecho MRI of the brain was performed with IV contrast in orthogonal plane and revealed a moderate sized multilobulated heterogeneously and predominantly peripherally enhancing mass lesion with irregular margins and central non enhancing areas of necrosis arising from the right corona radiata, insular cortex, external capsule, lentiform nucleus, temporal, and frontal white matter with blood products. Perilesional edema was seen. Effacement of sulcal spaces. Right sylvian fissure, compression over right ventricle and midline shift of 8mm toward left side. There was dilatation of the left lateral ventricle with periventricular cerebrospinal fluid ooze. Uncal herniation was right side. On MR spectroscopy large choline, creatinine levels were seen in the lesion with reduced N-acetyl aspartate levels as compared to the normal parenchyma. Findings were suggestive of neoplastic lesion mostly high-grade glioma. Polymerase chain reaction sequencing confirmed the presence of missense mutation R132H in exon 4 of isocitrate dehydrogenase 1 gene. Despite maximal safe surgical resection, the patient developed acute kidney failure secondary to the nephrotoxic effects of gadolinium. Management of both the primary brain tumor and renal complication necessitated a multidisciplinary approach involving neurosurgery, oncology, nephrology, and supportive care. Despite aggressive interventions, the patient’s condition deteriorated, highlighting the challenges in managing concurrent life-threatening conditions in patients with advanced GBM.

Autism spectrum disorder: difficulties in diagnosis and microRNA biomarkers.

We performed a PubMed search for microRNAs in autism spectrum disorder that could serve as diagnostic biomarkers in patients and selected 17 articles published from January 2008 to December 2023, of which 4 studies were performed with whole blood, 4 with blood plasma, 5 with blood serum, 1 with serum neural cell adhesion molecule L1-captured extracellular vesicles, 1 with blood cells, and 2 with peripheral blood mononuclear cells. Most of the studies involved children and the study cohorts were largely males. Many of the studies had performed microRNA sequencing or quantitative polymerase chain reaction assays to measure microRNA expression. Only five studies had used real-time polymerase chain reaction assay to validate microRNA expression in autism spectrum disorder subjects compared to controls. The microRNAs that were validated in these studies may be considered as potential candidate biomarkers for autism spectrum disorder and include miR-500a-5p, -197-5p, -424-5p, -664a-3p, -365a-3p, -619-5p, -664a- 3p, -3135a, -328-3p, and -500a-5p in blood plasma and miR-151a-3p, -181b-5p, -320a, -328, -433, -489, -572, -663a, -101-3p, -106b-5p, -19b-3p, -195-5p, and -130a-3p in blood serum of children, and miR-15b-5p and -6126 in whole blood of adults. Several important limitations were identified in the studies reviewed, and need to be taken into account in future studies. Further studies are warranted with children and adults having different levels of autism spectrum disorder severity and consideration should be given to using animal models of autism spectrum disorder to investigate the effects of suppressing or overexpressing specific microRNAs as a novel therapy.

Waveguide-Enhanced Nanoplasmonic Biosensor for Ultrasensitive and Rapid DNA Detection.

DNA is fundamental for storing and transmitting genetic information. Analyzing DNA or RNA base sequences enables the identification of genetic disorders, monitoring gene expression, and detecting pathogens. Traditional detection techniques like polymerase chain reaction (PCR) and next-generation sequencing (NGS) have limitations, including complexity, high cost, and the need for advanced computational skills. Therefore, there is a significant demand for enzyme-free and amplification-free strategies for rapid, low-cost, and sensitive DNA detection. DNA biosensors, especially those utilizing plasmonic nanomaterials, offer a promising solution. This study introduces a novel DNA-functionalized waveguide-enhanced nanoplasmonic optofluidic biosensor using a nanogold-linked sorbent assay for enzyme-free and amplification-free DNA detection. Integrating plasmonic gold nanoparticles (AuNPs) with a glass planar waveguide (WG) and a microfluidic channel, fabricated through cost-effective, vacuum-free methods, the biosensor achieves specific detection of complementary target DNA sequences. Utilizing a sandwich architecture, AuNPs labeled with detection DNA probes enhance sensitivity by altering evanescent wave distribution and inducing plasmon resonance modes. The biosensor demonstrated exceptional performance in DNA detection, achieving a limit of detection (LOD) of 33.1 fg/mL (4.36 fM) with a rapid response time of approximately 8 min. This ultrasensitive, rapid, and cost-effective biosensor exhibits minimal background nonspecific adsorption, making it highly suitable for clinical applications and early disease diagnosis. The innovative design and fabrication processes offer significant advantages for mass production, presenting a viable tool for precise disease diagnostics and improved clinical outcomes.

Occurrence rate and species and subtypes of Cryptosporidium spp. in pet dogs in Yunnan Province, China

BackgroundCryptosporidium spp. is a ubiquitous, globally distributed intestinal protozoan infecting humans and at least 260 animal hosts. Due to close human contact with pet dogs and identification of zoonotic Cryptosporidium species and subtypes in these animals, dog health is not only a veterinarian issue but also a public health issue. This study aimed to understand occurrence and genetic characterization at both genotype and subtype levels in pet dogs in Yunnan Province, China.ResultsA total of 589 fresh fecal specimens were collected from adult pet dogs in the rural areas of eight cities/autonomous prefectures of Yunnan Province, China. 16 fecal specimens were positive for Cryptosporidium spp. by polymerase chain reaction (PCR) amplification and sequence analysis of the small subunit ribosomal RNA (SSU rRNA) gene, with an average occurrence rate of 2.7% (16/589) being observed. Three zoonotic Cryptosporidium species were identified: C. parvum (n = 7), C. suis (n = 5) and C. canis (n = 4). At the 60-kDa glycoprotein (gp60) locus, only three C. parvum and two C. canis specimens were successfully amplified and sequenced, with subtype IIaA17G2R1 (n = 3) and subtypes XXa4 (n = 1) and XXa5 (n = 1) being identified, respectively.ConclusionsThe present finding of three zoonotic Cryptosporidium species in dogs implied that dogs infected with Cryptosporidium spp. may pose a threat to human health. C. suis was identified in dogs in this study for the first time, expanding the host range of this species. Identification of C. parvum subtype IIaA17G2R1 and C. canis subtypes XXa4 and XXa5 will be helpful to explore the source attribution of infection/contamination and assess the transmission dynamics of C. parvum and C. canis in the investigated areas in the future.

Diversity, vertical stratification and detection of Leishmania DNA in sand flies (Diptera: Psychodidae) collected in the Mapinguari National Park, Brazilian Amazon

This study aimed to assess the diversity patterns of sand fly fauna across different strata and detect Leishmania DNA in these insects in the Mapinguari National Park in Rondônia and Amazonas states, Northern Brazil. Sand flies were collected with “HP” light traps in the canopy (15 m) and at ground level (1 m) on two trails, during August and November 2021 and March and August 2022. Polymerase chain reaction and DNA sequencing were used to identify Leishmania species. A total of 8,040 individuals (2,303♂ – 28.64 %, 5,737♀ – 71.36 %) were collected and 53 species and 13 genera were identified. The most abundant species were Psychodopygus chagasi (21.00 %), Trichophoromyia ubiquitalis (13.61 %) Psychodopygus davisi (11.69 %), Nyssomyia fraihai (10.58 %), Nyssomyia antunesi (6.68 %) and Nyssomyia richardwardi (5.55 %). Species diversity was greater in the canopy (Shannon index H' = 10.8 common species) when compared to ground level (H' = 10.5 common species). We observed a minimum infection rate of 0.45 % (22/4,868 females), in which Leishmania braziliensis DNA was found in Ps. chagasi and Le. lainsoni DNA in Ps. chagasi, Ny. richardwardi, Ps. ayrozai and Th. ubiquitalis. The minimum infection rate of Leishmania in the canopy was 0.47 % (19/4,031) and in the ground was 0.52 % (3/567). In the present study, we observed Le. lainsoni DNA in females of Ny. richardwardi for the first time. The data presented in this study contribute to understanding sand fly diversity and its distribution between the states of Rondônia and Amazonas. They may be useful for implementing targeted control measures to reduce the spread of leishmaniasis and implement entomological surveillance strategies.

Genome‑wide analysis of cotton SCAMP genes and functional characterization of GhSCAMP2 and GhSCAMP4 in salt tolerance

BackgroundSecretory carrier membrane proteins (SCAMPs) form a family of integral membrane proteins and play a crucial role in mediating exocytosis in both animals and plants. While SCAMP genes have been studied in several plant species, their functions in cotton, particularly in response to abiotic stress, have not yet been reported.ResultsIn this study, a total of 53 SCAMP genes were identified in G. arboreum, G. raimondii, G. hirsutum, and G. barbadense. These genes were classified into five groups based on a phylogenetic analysis with SCAMPs from Arabidopsis thaliana. The main factor driving the expansion of the SCAMP gene family in G. hirsutum is tandem and segmental duplication events. Using MEME, in addition to the conserved SCAMP domain, we identified 3–13 other domains in each GhSCAMP. The cis-element analysis suggested that GhSCAMPs were widely involved in cotton growth and development, and responses to abiotic stresses. RNA sequencing (RNA-Seq) and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) results showed that most GhSCAMPs were expressed highly in many tissues and had differential expression responses to drought, cold, and heat stresses. Knock-down of GhSCAMP2 and GhSCAMP4 by virus-induced gene silencing (VIGS) lead to a salt-sensitive phenotype and had a lower content of CAT, POD, and SOD.ConclusionsThis study identified SCAMP genes in four cotton species, enhancing our understanding of the potential biological functions of SCAMPs. Additionally, we demonstrated that GhSCAMP2 and GhSCAMP4 positively regulate cotton tolerance to salt stress.

Methylation alterations of imprinted genes in different placental diseases

BackgroundImprinted genes play important functions in placentation and pregnancy; however, research on their roles in different placental diseases is limited. It is believed that epigenetic alterations, such as DNA methylation, of placental imprinting genes may contribute to the different pathological features of severe placental diseases, such as pre-eclampsia (PE) and placenta accreta spectrum disorders (PAS).ResultsIn this study, we conducted a comparative analysis of the methylation and expression of placental imprinted genes between PE and PAS using bisulfite sequencing polymerase chain reaction (PCR) and quantitative PCR, respectively. Additionally, we assessed oxidative damage of placental DNA by determining 8-hydroxy-2′-deoxyguanosine levels and fetal growth by determining insulin-like growth factor 2 (IGF2) and cortisol levels in the umbilical cord blood using enzyme-linked immunosorbent assay. Our results indicated that methylation and expression of potassium voltage-gated channel subfamily Q member 1, GNAS complex locus, mesoderm specific transcript, and IGF2 were significantly altered in both PE and PAS placentas. Additionally, our results revealed that the maternal imprinted genes were significantly over-expressed in PE and significantly under-expressed in PAS compared with a normal pregnancy. Moreover, DNA oxidative damage was elevated and positively correlated with IGF2 DNA methylation in both PE and PAS placentas, and cortisol and IGF2 levels were significantly decreased in PE and PAS.ConclusionsThis study suggested that DNA methylation and expression of imprinted genes are aberrant in both PE and PAS placentas and that PE and PAS have different methylation profiles, which may be linked to their unique pathogenesis.

Molecular Testing for EGFR Mutation in Moroccan NSCLC Patients: CHU Hassan II-Fez Experience.

Epidermal growth factor receptor (EGFR) mutation screening in non-small cell lung cancer (NSCLC) is now used to guide treatment decisions to identify patients with EGFR positive mutations that predict response to EGFR tyrosine kinase inhibitors. This study aimed to explore with a prospective study the current testing practices and the predictive value of EGFR mutations in a series of 261 patients with NSCLC. EGFR mutation testing was conducted using 2 different assays: bidirectional Sanger sequencing of polymerase chain reaction (PCR) and real-time PCR on the Rotor-Gene Q instrument. Epidermal growth factor receptor mutation testing was performed for 261 patients with lung cancer. Exons 18 to 21 were successfully analyzed in 113 tumors by Direct sequencing and in 148 tumors by real-time PCR. The prevalence of positive EGFR-mutations in each method was 22.1% (N = 25) and 24.3% (N = 36), respectively (P = .3). In total, EGFR mutations were detected in 59 patients among 261 patients with NSCLC. A statistically significant association between female sex, nonsmoking history, nonsolid major pattern, and a higher EGFR mutation frequency. In this study, we investigated clinicopathological differences between tumors harboring exon 19del and those harboring L858R. We did not find any significant differences between the 2 mutations and gender or smoking features, interestingly, the prevalence of patients aged >60 years was significantly higher in the L858R group than in the exon 19del group (81.8% vs 55.8%, P = .05). A significant association was observed between exon 19 deletions and the papillary major pattern, but no correlation was detected between exon 21 mutation and any histological pattern. This prospective study documented the real-world clinical testing of EGFR mutation in Moroccan NSCLC patients. Our experience confirms the need to develop standards-based guidelines for the routine performance and evaluation of EGFR testing to improve clinical care for this subset of lung cancer. On the other hand, our study demonstrated that tumors with exon 19 deletions and L858R harbor specific clinicopathological features in NSCLC.

Characterization of Hemoglobin Malay Phenotypes in Tertiary Hospitals

Hemoglobin (Hb) Malay is a common β hemoglobinopathy in Malaysia caused by A > G mutation in codon 19 leading to β+-thalassemia phenotype. However, screening for Hb Malay is challenging as it is undetectable by routine capillary electrophoresis (CE) or high-performance liquid chromatograpy (HPLC) methods. This study aimed to determine the Hb Malay phenotypes. The study was done on 521 cases with presumed β thalassemia from UKMMC and Hospital Selayang as well as confirmed Hb Malay cases from Hospital Sultanah Bahiyah, Kedah in over a 5-year period. Hb analysis using CE or HPLC followed by multiplex amplification refractory mutation system polymerase chain reaction and DNA sequencing were performed. Significant differences in mean values of haematological parameters among Hb Malay carriers against β thalassemia carriers were determined using one-way ANOVA and ROC analysis. A total of 482/521 cases of β globin mutations were identified. Among these, 54 Hb Malay cases were identified whereby 21 Hb Malay cases were from UKMMC and Hospital Selayang whilst 33 Hb Malay cases were from Hospital Sultanah Bahiyah, Kedah. Fifty-two were Hb Malay carriers whereas two cases were compound heterozygotes. The mean hemoglobin, mean cell volume, mean cell hemoglobin, and HbA of Hb Malay carriers were significantly higher than β° thalassemia carriers. The HbA2 range of Hb Malay carriers was wider (3.5–5.5%) with median value of 3.9%. A new HbA2 cutoff value ≤4.6% (AUC 0.717, p < 0.001) was proposed. Compound heterozygous Hb Malay/IVS1-5(G > C) showed transfusion-dependent thalassemia phenotype. Hb Malay carriers have different red cell and electrophoretic parameters than classical β° thalassemia carriers with wider HbA2 range. HbA2 of ≤4.6% should prompt a molecular confirmation for Hb Malay carrier status.

Molecular genetic monitoring of HIV-1 variants circulating in St. Petersburg

The aim of the study: to assess the genetic diversity of HIV-1 variants circulating in St. Petersburg.Materials and methods. The study included 289 patients with virological ART failure in 2022 in the St. Petersburg AIDS Center. Fragments of the pol gene encoding integrase, reverse transcriptase and protease were analyzed by polymerase chain reaction and Sanger sequencing. Phylogenetic tree created by the Neighbor-joining method with 1000 repeats of nucleotide sequences, bootstrap values &gt;70. To assess the circulation of HIV genovariants in dynamics in St. Petersburg, sequences and clinical and laboratory parameters obtained from 544 patients since 2018. The total sample included 833 samples (289 were collected in 2022), compared with the results of a study from 1104 HIV-infected patients in 2006–2011.Results and discussion. Monovariants of HIV dominated in the examined patients (95.1%, 275 people), subtype A6 — 88.2% (255 people); subtype B — 5.9% (17 people); C — 0.3% (1 person); G — 0.7% (2 people), the proportion of recombinant forms — 4.9% (14 people). In the sample collection, the proportion of new HIV cases of non-A subtype was 13.3% in 2006–2011, and 11.1% in 2018–2022. A significant increase in the incidence of recombinant forms of HIV-1 was revealed from 1.6% to 3.5% (c2=6.111; p=0.014). In the group (2018–2022), the recombinant form of CRF63_02A6 was more common (15/29 people).Conclusion. Phylogenetic analyses makes it possible to determine HIV subtypes, but also to establish the potential geographical origin of the virus, to identify transmission clusters taking into account the socio-demographic indicators of HIV-infected patients. Molecular epidemiological monitoring can be used to develop and implement programs to counter the spread of HIV among the population. The dominant genetic variant of HIV circulating in St. Petersburg is sub-subtype A6, as it was 10 years ago. The proportion of new cases of infection with non-A subtype of HIV remains stable, with a downward trend. The increase in the frequency of detection of recombinant forms of HIV-1 is probably related to the migration processes of the population.

An L-type calcium channel blocker nimodipine exerts anti-fibrotic effects by attenuating TGF-β1 induced calcium response in an in vitro model of thyroid eye disease

BackgroundThyroid eye disease (TED) is a vision-threatening autoimmune disorder. Orbital tissue fibrosis leading to intractable complications remains a troublesome issue in TED management. Exploration of novel therapeutic targets and agents to ameliorate tissue fibrosis is crucial for TED. Recent work suggests that Ca2+ signaling participates in tissue fibrosis. However, whether an alteration of Ca2+ signaling has a role in fibrogenesis during TED remains unclear. In this study, we aimed to investigate the role of Ca2+ signaling in the fibrogenesis process during TED and the potential therapeutic effects of a highly selective inhibitor of the L-type calcium channel (LTCC), nimodipine, through a TGF-β1 induced in vitro TED model.MethodsPrimary culture of orbital fibroblasts (OFs) were established from orbital adipose connective tissues of patients with TED and healthy control donors. Real-time quantitative polymerase chain reaction (RT-qPCR) and RNA sequencing were used to assess the genes expression associated with LTCC in OFs. Flow cytometry, RT-qPCR, 5-ethynyl-2′-deoxyuridine (EdU) proliferation assay, wound healing assay and Western blot (WB) were used to assess the intracellular Ca2+ response on TGF-β1 stimulation, and to evaluate the potential therapeutic effects of nimodipine in the TGF-β1 induced in vitro TED model. The roles of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and signal transducer and activator of transcription 1 (STAT1) in fibrogenesis during TED were determined by immunohistochemistry, WB, flow cytometry and co-immunoprecipitation assay. Selective inhibitors were used to explore the downstream signaling pathways.ResultsLTCC inhibitor nimodipine blocked the TGF-β1 induced intracellular Ca2+ response and further reduced the expression of alpha-smooth muscle actin (α-SMA), collagen type I alpha 1 (Col1A1) and collagen type I alpha 2 (Col1A2) in OFs. Besides, nimodipine inhibited cell proliferation and migration of OFs. Moreover, our results provided evidence that activation of the CaMKII/STAT1 signaling pathway was involved in fibrogenesis during TED, and nimodipine inhibited the pro-fibrotic functions of OFs by down-regulating the CaMKII/STAT1 signaling pathway.ConclusionsTGF-β1 induces an LTCC-mediated Ca2+ response, followed by activation of CaMKII/STAT1 signaling pathway, which promotes the pro-fibrotic functions of OFs and participates in fibrogenesis during TED. Nimodipine exerts potent anti-fibrotic benefits in vitro by suppressing the CaMKII/STAT1 signaling pathway. Our work deepens our understanding of the fibrogenesis process during TED and provides potential therapeutic targets and alternative candidate for TED.

High serological and molecular prevalence of Ehrlichia canis and other vector-borne pathogens in dogs from Boa Vista Island, Cape Verde

Despite the high global impacts of canine vector-borne diseases (CVBD) due to their wide distribution and zoonotic potential, the current epidemiological situation of CVBD in many tropical and subtropical regions remains unknown. This study examines the seroprevalence and molecular prevalence of Ehrlichia canis and other pathogens causing CVBDs (Leishmania infantum, Dirofilaria immitis, Babesia spp., Anaplasma spp. and Hepatozoon canis) in dogs living on the island of Boa Vista (Cape Verde Republic). Blood samples and infesting ticks were taken from 150 dogs across the island (stray, shelter, and pet dogs). Serum samples were tested using a rapid immunochromatographic test (Uranotest® Quattro) that detects antibodies against E. canis, L. infantum, Anaplasma spp. and D. immitis antigen. Levels of serum antibodies against E. canis were measured using the immunofluorescence antibody test (IFAT). In addition, tick-borne pathogens in blood samples (Anaplasma spp., Babesia spp., Hepatozoon spp., and Ehrlichia canis) were detected by microscopy observation and/or PCR plus sequencing. The seroprevalence of E. canis was extremely high at 82% (123/150), as revealed by both immunochromatography and IFAT. Most dogs returning a seropositive test result (82.92%; 102/123) had antibody titres > 1:1280 but showed no clinical signs or notable laboratory abnormalities. Of the 123 animals testing seropositive for E. canis, 67 (54.47%) also presented antibodies against Anaplasma spp., and 13 (10.56%) showed the presence of Hepatozoon spp. gamonts in the blood smear. Ehrlichia canis infection was detected in 17.1% (25/146) of dogs tested by direct sequencing of polymerase chain reaction (PCR) products. Co-infections were detected in seven of these dogs: four dogs tested PCR-positive for both E. canis and A. platys, two dogs tested positive for E. canis and Hepatozoon spp., and one dog tested positive for E. canis, A. platys and Hepatozoon spp. Rhipicephalus sanguineus sensu lato was the only tick species found infesting the canine study population. The high prevalence of tick-borne pathogens detected in dogs from Boa Vista Island highlights a need for improved control measures designed to prevent the transmission of these pathogens.Graphical

Histopathologic and Clinical Characterization of Brentuximab Vedotin-associated Rash.

Rash is one of the commonly observed adverse events with brentuximab vedotin (BV), a CD30-targeted antibody-drug conjugate used to treat cutaneous T-cell lymphoma (CTCL). However, clinical and histopathologic characterization of BV-associated rash (BVAR) is limited. Distinguishing BVAR from a patient's underlying CTCL can be challenging and can lead to treatment interruptions or even premature drug discontinuation. We performed a thorough clinical and histopathologic retrospective characterization of BVAR from a single institution. Utilizing polymerase chain reaction (PCR) and T-cell receptor high-throughput sequencing (TCR-HTS), we were able to isolate skin biopsy specimens from rash clinically suggestive of BVAR that also lacked a dominant TCR clone. A retrospective evaluation was performed of 26 biopsy specimens from 14 patients. Clinical features of BVAR included predominantly morbilliform or maculopapular morphology, delayed onset, and the trend toward moderate to severe classification, often requiring oral steroids. Most histopathologic specimens (25/26) showed spongiotic dermatitis as the primary reaction pattern. Many cases showed subtle findings to support a background interface or lichenoid eruption. Langerhans cell microabscesses were seen in one-fourth of specimens, and eosinophils were present in over one-half of the specimens. There were focal features mimicking CTCL, but these were not prominent. In 17 specimens with immunohistochemistry, the CD4:CD8 ratio in intraepidermal lymphocytes was relatively normal (1-6:1) in 65% (11/17) and 1:1 in 35% (6/17), demonstrating a trend toward increased CD8-positive cells compared with baseline CTCL. We have identified features that can help distinguish BVAR from a patient's CTCL, which can, in turn, help guide appropriate clinical management.

Linkage of alternative exon assembly in Drosophila TrpA1 transcripts

Drosophila TrpA1 (transient receptor potential ankyrin 1) transcripts are alternatively spliced at two distinct sites each with a choice of mutually exclusive exons. The first site determines exon1 encoding the amino terminus to produce either nucleophile-, electrophile- and noxious temperature-gated TRPA1(A) or electrophile- and innocuous warmth-gated TRPA1(B). The second site selects for exon10, resulting in TrpA1 variants with either exon10a or exon10b encoding a domain between the N-terminal ankyrin repeats and the transmembrane segments. Although unbiased assembly would generate TRPA1 with four different domain combinations, the functional impact of these alternative domains remains to be thoroughly examined. Here, we find that there is a relatively strong linkage in mRNA splicing between the two sites in case of TrpA1(B), but not TrpA1(A), transcripts. Our semi-quantitative assay, consisting of reverse transcription polymerase chain reaction (RT-PCR) and Sanger sequencing, revealed that exon10b is little coupled with TrpA1(B) transcripts, suggesting that only three isoforms, TRPA1(A)-exon10a [denoted as TRPA1(A)], TRPA1(A)-exon10b [TRPA1(A)10b] and TRPA1(B)-exon10a [TRPA1(B)], are present at detectable levels using our method. Interestingly, heterologously expressed TRPA1(A)10b showed elevated sensitivity to low concentrations of N-methyl maleimide (NMM), a cysteine-modifying electrophile, compared with other isoforms. Equivalent isoforms in malaria-transmitting Anopheles gambiae displayed a similar pattern of isoform-dependent NMM dose dependences, suggesting that the chemosensory regulation by selective domain assembly is conserved in insect TRPA1s. Thus, alternative RNA splicing of exon10 is coordinated in conjunction with the first exons, regulating chemical sensitivity of insect TRPA1s.

DETECTION OF BABESIA CF. ODOCOILEI, BABESIA CAPREOLI, AND ANAPLASMA PHAGOCYTOPHILUM IN CERVIDS OF THE SCOTTISH HIGHLANDS, UNITED KINGDOM.

Outbreaks of suspected tick-borne disease (redwater fever) have been reported in captive deer of the Scottish Highlands. In this pilot study, polymerase chain reaction and amplicon sequencing were used to detect tick-borne pathogens in opportunistically collected blood and spleen samples from 63 (healthy, n = 44; diseased, n = 19) cervids, and 45 questing and feeding ticks (Ixodes ricinus) from the outbreak sites in 2021-2022. Potentially pathogenic Babesia species were detected in deer but not identified in ticks, Anaplasma phagocytophilum was detected in both deer and ticks, and Borrelia afzelii was detected in ticks but not in deer. Sequencing confirmed Babesia capreoli and Babesia cf. odocoilei parasitemia in clinically healthy red deer (Cervus elaphus), B. capreoli parasitemia in clinically healthy domestic reindeer (Rangifer tarandus tarandus), and two cases of B. cf. odocoilei-associated hemolytic anemia in white-lipped deer (Cervus albirostris), of which one was fatal despite imidocarb treatment. White-lipped deer appear to be highly susceptible to babesiosis caused by B. cf. odocoilei. This investigation highlights the importance of disease surveillance, including molecular diagnostics, for the detection of emerging tick-borne pathogens in managed populations of cervids.

Molecular genotyping and subgenotyping of duck circovirus at duck farms in Thailand

Background and Aim: Ducks worldwide are infected with duck circovirus (DuCV), which causes feather abnormality, emaciation, and poor growth performance. DuCV is similar to other circoviruses that induce immunosuppression due to the occurrence of the bursae of Fabricius (BF) and spleen atrophies. In Thailand, retarded ducks with feather losses were submitted for disease investigation. The ducks presented low body weight gain, had small BF and spleens, and were consistent with duck-infected DuCV. Our study investigated the possibility of DuCV infection in duck flocks in Thailand. We also analyzed the genetic characteristics of the virus. Materials and Methods: BF and spleen samples were collected from affected meat and layer ducks from six farms thought to have been infected with DuCV. These tissues were then subjected to histopathological examination and molecular identification using conventional polymerase chain reaction and nucleotide sequencing. To identify DuCV, phylogenetic trees were generated using MEGA version X software. Samples of tissues or swabs were collected to determine whether coinfections with bacteria and viruses existed. Results: Phylogenetic analysis using the entire genome (1995–1996 bp) and cap gene (762 bp) revealed that the DuCV isolates circulating in Thailand belonged to DuCV genotype I, which was further subdivided into two sub-genotypes: sub-genotype I b and an unclassified sub-genotype based on reference sub-genotypes. Thai isolates have variations in 10 amino acid residues in the capsid protein. Ducks infected with Thai DuCV were also coinfected with Riemerella anatipestifer, Escherichia coli, Pasteurella multocida, duck viral enteritis, and duck Tembusu virus, which is consistent with previous DuCV infection studies. Conclusion: Six DuCVs from ducks who were previously found to have feather loss, were underweight, had growth retardation, and had poor body condition were identified in this study as belonging to genotype Ⅰ and constituting at least two sub-genotypes. Due to the immunosuppressive effects of DuCV, coinfection of bacterial and viral pathogens was typically observed in Thai DuCV-infected ducks. Keywords: duck, duck circovirus, genetic characterization, immunosuppression, phylogenetic tree.