RT-PCR Research Articles

Rhabdomyosarcoma Harboring NRAS or HRAS Mutation Arising in Giant Congenital Melanocytic Nevus: Report of 2 Cases.

Nonmelanoma malignancies associated with congenital melanocytic nevi (CMN) are extremely rare, with only 12 reported cases of rhabdomyosarcoma (RMS) to date. We present 2 additional cases of RMS arising in giant CMN, with immunohistochemical and molecular biologic investigations. The first case was a 32-year-old woman with a personal history of melanoma in giant CMN who, after successful treatment and long remission, presented with a new 1-cm nodule within the CMN. Microscopically, the atypical areas exhibited a round cell/alveolar morphology with immunoreactivity for desmin and myogenin, and lacked PAX3/7::FOXO1 fusions typical for alveolar RMS on a reverse transcription polymerase chain reaction analysis. An identical NRAS p.Q61R mutation with comparable variant allele frequency (32% and 44%) was identified in both the nevus and the RMS tissue by next-generation sequencing. The second patient was a 5-year-old girl with a rapidly growing, bleeding, ulcerated 3 × 4 cm interscapular mass within a giant CMN that histologically seemed as a proliferation of pleomorphic spindle, polygonal and epithelioid cells with marked pleomorphism immunoreactive for myogenin, muscle-specific actin, and smooth muscle actin. Next-generation sequencing yielded an HRAS p.Q61R mutation with limited variant allele frequency (7%) in the RMS component, while ATRX p.Q2193* variant was detected in the nevus. Our study is apparently the first report of NRAS and HRAS mutations in tumors with RMS phenotype arisen in CMN.

Neutralizing IL-15 Inhibits Tissue-Damaging Immune Response in Ex Vivo Cultured Untreated Celiac Intestinal Mucosa

In celiac disease (CeD), interleukin 15 (IL-15) affects the epithelial barrier by acting on intraepithelial lymphocytes, promoting interferon γ (IFN-γ) production and inducing strong cytotoxic activity as well as eliciting apoptotic death of enterocytes by the Fas/Fas ligand system. This study investigates the effects of a monoclonal antibody neutralizing the effects of IL-15 (aIL-15) on tissue-damaging immune response in untreated CeD patients by using an organ culture system. Jejunal biopsies from 10 untreated CeD patients were cultured ex vivo with or without aIL-15. Epithelial expressions of CD95/Fas, HLA-E and perforin were analyzed by immunohistochemistry. Apoptosis was detected in the epithelium by using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. Additionally, the surface epithelium compartment of ex vivo cultured biopsy samples was isolated by laser capture microdissection (LCM). RNA from each LCM sample was extracted and the relative expression of IFN-γ was evaluated by quantitative reverse transcriptase-PCR (qRT-PCR). Biopsies cultured with the aIL-15 antibody showed a reduction in Fas, HLA-E and perforin epithelial expression, as well as a decrease in epithelial TUNEL+ cells compared to biopsies cultured without the aIL-15 antibody. Moreover, downregulation of epithelial IFN-γ expression was recorded in biopsies incubated with aIL-15, compared to those cultured without aIL-15. Our findings suggest that neutralizing the effects of IL-15 in ex vivo cultured untreated CeD intestinal mucosa could block apoptosis by downregulating Fas and HLA-E expression and the release of cytotoxic proteins, such as perforin. Furthermore, it can dampen the hyperactive immune response by reducing IFN-γ expression. More generally, our study provides new evidence for the effects of anti-IL-15 neutralizing monoclonal antibodies in preventing or repairing epithelial damage and further supports the concept that IL-15 is a meaningful therapeutic target in CeD, or inflammatory diseases associated with the upregulation of IL-15.

Stachydrine targeting tumor-associated macrophages inhibit colorectal cancer liver metastasis by regulating the JAK2/STAT3 pathway

IntroductionColorectal cancer (CRC) represents the third most prevalent form of cancer worldwide, with liver metastasis representing a significant contributor to mortality. The interaction between tumor-associated macrophages (TAMs) and tumor cells plays a pivotal role in the development of colorectal cancer liver metastases (CRLM) and represents a promising avenue for therapeutic intervention. Stachydrine (STA), a compound derived from the Leonurus heterophyllus plant, has been shown to effectively inhibit tumor growth through a range of mechanisms.MethodsThe study employed imaging and histopathology to evaluate the efficacy of STA monotherapy in preventing CRLM. The inhibition of M2 macrophage polarization by STA was confirmed through the use of flow cytometry and immunofluorescence. Subsequently, a series of assays, including quantitative reverse transcription polymerase chain reaction (qRT-PCR), flow cytometry, scratch, invasion, and tube formation assays, were conducted to confirm STA’s capacity to impede tumor cell migration, invasion, and angiogenesis in vitro. Western blotting and flow cytometry were employed to elucidate the mechanisms through which STA exerts its effects on tumor metastasis.ResultsIn our research, STA has been shown to attenuate liver metastasis in CRC mouse models by inhibiting the polarization of macrophages to the M2 phenotype. This anti-metastatic effect is dependent on the presence of macrophages. In vitro, STA has been found to impede tumor cell migration, invasion, and angiogenesis by preventing TAMs from polarizing to the M2 phenotype via the JAK2/STAT3 signaling pathway. Moreover, the combination of STA with anti-PD-1 therapy has been observed to restore immune infiltration within the tumor microenvironment and inhibit tumor progression.ConclusionThe findings of this study demonstrate that STA exerts an inhibitory effect on colorectal cancer liver metastasis by targeting macrophages and impeding their M2 polarization via the JAK2/STAT3 pathway. Furthermore, the combination of STA with anti-PD-1 therapy has been observed to enhance the effectiveness of immune checkpoint blockade and reduce tumor spread, indicating the potential of STA to improve the efficacy of immunotherapy for liver metastases.

Identification of Lipophagy-Related Gene Signature for Diagnosis and Risk Prediction of Alzheimer’s Disease

Background: Recent research indicates that lipid metabolism and autophagy play crucial roles in the development of Alzheimer’s disease (AD). Investigating the relationship between AD diagnosis and gene expression related to lipid metabolism, autophagy, and lipophagy may improve early diagnosis and the identification of therapeutic targets. Methods: Transcription datasets from AD patients were obtained from the Gene Expression Omnibus (GEO). Genes associated with lipid metabolism, autophagy, and lipophagy were sourced from the Gene Set Enrichment Analysis (GSEA) database and the Human Autophagy Database (HADb). Lipophagy-related hub genes were identified using a combination of Limma analysis, weighted gene co-expression network analysis (WGCNA), and machine learning techniques. Based on these hub genes, we developed an AD risk prediction nomogram and validated its diagnostic accuracy using three external validation datasets. Additionally, the expression levels of the hub genes were assessed through quantitative reverse transcription polymerase chain reaction (qRT-PCR). Results: Our analysis identified three hub genes—ACBD5, GABARAPL1, and HSPA8—as being associated with AD progression. The nomogram constructed from these hub genes achieved an area under the curve (AUC) value of 0.894 for AD risk prediction, with all validation sets yielding AUC values greater than 0.8, indicating excellent diagnostic efficacy. qRT-PCR results further corroborated the associations between these hub genes and AD development. Conclusions: This study identified and validated three lipophagy-related hub genes and developed a reliable diagnostic model, offering insights into the pathology of AD and facilitating the diagnosis of AD patients.

FXR1 modulates the expression of oncogenes and tumor suppressor genes associated with poor cancer prognosis

Post-transcriptional regulation by RNA-binding proteins (RBPs) is critical for mRNA stability, localization, and translation, primarily through interaction with the 3’-untranslated region. Dysregulation of RBPs has been associated with various cancers, with fragile X-related protein 1 (FXR1) emerging as a critical RBP involved in tumorigenesis through its interactions with target mRNAs. Despite its significance, the specific role of FXRI in cancer progression remains underexplored. In this study, we investigated FXR1’s function using SH-SY5Y cells. RNA immunoprecipitation (RNA-IP) assay was employed to isolate RNA complexes associated with FXR1. We generated stable cell lines with either FXR1 overexpression or silencing to assess the impact of FXRI binding to mRNA complexes. Subsequent analyses, including quantitative reverse transcription polymerase chain reaction, correlation analysis, gene expression profiling, and survival analysis, were performed to validate the interactions of FXR1 with target mRNAs. Our RNA-IP analysis identified several mRNAs significantly enriched in FXR1-bound RNA complexes, including SHISAL1, SLC43A3, NBAT1, PDZK1IP1, ACKR3, KCNN3, NECAB2, ANO5, ATOH8, IGFBP7, LEMD1, GPR35, WNT7A, and F2RL3. Notably, we observed changes in the expression levels of these genes following FXR1 overexpression or depletion, indicating FXR1-mediated functional regulation. Co-expression analysis further supported FXR1’s association with these target mRNAs. These findings highlight the significant role of FXR1 in regulating the stability and expression of key mRNAs implicated in malignancies. The dysregulation of FXR1 and its interaction with these target transcripts suggest that FXR1 plays a critical role in tumor biology, potentially offering new avenues for therapeutic interventions. This study provides a deeper understanding of FXR1’s involvement in cancer and underscores the importance of RBPs in the post-transcriptional regulatory landscape of cancer progression.

First detection and molecular characterization of Jingmen tick virus with a high occurrence in Rhipicephalus (Boophilus) microplus collected from livestock in Cameroon (2024)

BackgroundJingmen tick virus (JMTV) is a novel tick-borne virus detected for the first time in Rhipicephalus (Boophilus) microplus in China. To date, there is no information regarding the circulation of JMTV in ticks collected from livestock in Cameroon. As part of the surveillance for arboviral circulation, this study aimed to assess the presence of JMTV in ticks collected from livestock (cattle and sheep) in an area of the Akonolinga health district, Center Region, Cameroon.MethodsA cross sectional study was carried out during the dry season between 5 and 14 March 2024. Ticks were collected from cattle and sheep in six sampling sites in an area approximately 30 km long and 18 km wide along the Nyong River, in central Cameroon. Ticks were identified morphologically and molecularly. Total RNA/DNA was extracted from tick pools and screened for JMTV RNA using a segment 2 RT-qPCR system. Positive JMTV pools were sequenced for partial JMTV-Segment 1 and full genome analyses.ResultsA total of 622 ticks, organized into 251 pools were collected from 155 cattle and nine sheep. They consisted of five species covering three genera: R. (B.) microplus (472; 75.9%), Amblyomma variegatum (118; 19.0%), Hyalomma truncatum (13; 2.1%), Hyalomma rufipes (2; 0.3%), and other Rhipicephalus spp. (17; 2.7%). The quantitative reverse transcription polymerase chain reaction (qRT-PCR) screening of 251 tick pools yielded 61 JMTV-positive pools, of which 58 corresponded to R. (B.) microplus. Multiple sequence analysis revealed that JMTV from the Akonolinga area shared > 95% identity with strains from Guinea, and that these strains clustered phylogenetically together.ConclusionsWe provide molecular evidence of the presence of JMTV in R. (B.) microplus and A. variegatum collected from cattle and sheep from an area not yet recognized as endemic for this virus, confirming its wide geographical distribution.Graphical

The Expression of Neuroendocrine Markers in a Small Subset of Ameloblastoma with Implications of Clusterin

Immunohistochemically, ameloblastomas often express CD56; however, novel neuroendocrine markers such as synaptophysin (SYP), insulinoma-associated protein 1 (INSM1), and chromogranin A (CgA) remain unexplored. We analyzed 36 ameloblastoma specimens for CD56, SYP, CgA, and clusterin (CLU) and examined limited samples for INSM1 expression by performing immunohistochemistry, transmission electron microscopy, and reverse transcriptase-polymerase chain reaction. Our findings indicate that the limited cells were positive for CD56, SYP, CgA, INSM1, and CLU expression in 72% (26/36), 14% (5/36), 0% (0/40), 80% (4/5), and 22% (8/36) of the cases, respectively. CD56 expression correlated with older age, but not with subtype, SYP, and CLU expression. However, SYP-positive cases were exclusively found in CD56- and CLU-positive cases, and SYP and CLU expression were significantly correlated. Selected cases had dense-core granules and NCAM1 and SYP mRNA expression. This study is the first to suggest neuroendocrine differentiation in ameloblastomas, as indicated by SYP and INSM1 immunoexpression and the presence of dense-core granules, which are consistent with the recent World Health Organization classification of Head and Neck Tumors guidelines. SYP-positive and CgA-negative phenotypes may characterize neuroendocrine differentiation in ameloblastoma. Although the underlying molecular mechanism remains unclear, CLU expression may be associated with neuroendocrine differentiation.

First report of Brugmansia latent virus on Angel’s Trumpet (Brugmansia sp.) in Florida, USA

Angel’s trumpet (Brugmansia sp.) is a perennial shrub that is widely propagated as an ornamental and landscape plant around the world. In November 2022, Brugmansia sp. leaves with severe mosaic symptoms were collected from a nursery in Duval County, Florida, USA and sent to the Florida Department of Agriculture and Consumer Services Division of Plant Industry for pathogen identification. Based on serology, reverse-transcription PCR, and sequencing, the virus was identified as Brugmansia latent virus (BrLV). To our knowledge, this is the first report of BrLV in Florida from any host.

Unraveling the association and regulatory role of miR-146b-5p in coronary artery disease

BackgroundCoronary artery disease (CAD), one of the most prevalent cardiovascular diseases, is a critical health issue that affects millions of individuals worldwide. It has been reported that miR-146b-5p exhibited a strong correlation with inflammatory responses and atherosclerosis. However, its association with the incidence and severity of CAD has not been substantiated in a large cohort. In the study, we focus on the expression of miR-146b-5p in peripheral blood mononuclear cells (PBMCs) of patients with CAD and preliminarily investigate its function and underlying mechanism.Methods and resultsThe study encompassed a total of 452 participants, consisting 295 patients with CAD and 157 individuals without CAD. Quantitative reverse transcription–polymerase chain reaction (qRT–PCR) was performed to assess miR-146b-5p expression in PBMCs. We found that miR-146b-5p was significantly increased in PBMCs of patients with CAD compared with the control group. Binary logistic regression revealed that miR-146b-5p was associated with CAD. Receiver Operation Characteristic (ROC) analysis showed that the sensitivity and specificity of miR-146b-5p in discriminating CAD patients from non-CAD patients were meaningful. Subsequent subgroup analysis showed that miR-146b-5p was related to the severity of CAD. Furthermore, gain- and loss-of-function experiments in THP-1 cells showed that miR-146b-5p inhibited inflammation, cell proliferation, and migration. Mechanically, miR-146b-5p was involved in the classical NF-κB inflammatory pathway by directly targeting IKKβ.ConclusionOur study revealed that miR-146b-5p was higher in the PBMCs of CAD patients than non-CAD individuals, and established a correlation between miR-146b-5p and occurrence and severity of CAD. In addition, the inflammatory role of miR-146b-5p is mediated by targeting IKKβ.

Unveiling miR-1468-5p: A New Prognostic Biomarker and Therapeutic Target in Lung Adenocarcinoma.

miR-1468-5p, a type of microRNA, is acknowledged for its crucial involvement in a variety of cancerous processes. Nonetheless, the specific impact of this microRNA on lung adenocarcinoma (LUAD) has not yet been clearly defined. Our aim was to investigate how miR-1468-5p influences LUAD. The Cancer Genome Atlas (TCGA) offered specimens for our research. Employing statistical techniques, we assessed the diagnostic and prognostic significance of miR-1468-5p, as well as its association with clinical characteristics. Our analysis delved into the target genes and the regulatory mechanisms influenced by miR-1468-5p. The expression levels of miR-1468-5p in LUAD cell lines were validated through quantitative reverse transcription polymerase chain reaction (qRT-PCR). The expression of miR-1468-5p varied significantly across different cancer types. The presence of reduced miR-1468-5p levels was correlated with a lower likelihood of overall survival in LUAD patients, with a statistically significant result (p = 0.005). miR-1468-5p demonstrated independent prognostic significance in LUAD and potentially contributes to disease progression via multiple pathways, including the HIF-1 signaling pathway and more. There was a significant reduction in miR-1468-5p expression in LUAD cell lines when compared to cells of the normal lung epithelium. miR-1468-5p may serve as a useful patent as a therapeutic intervention target and a prognostic indicator for LUAD patients.

Surfaces environmental monitoring of SARS-CoV-2: Loop mediated isothermal amplification (LAMP) and droplet digital PCR (ddPCR) in comparison with standard Reverse-Transcription quantitative polymerase chain reaction (RT-qPCR) techniques.

The persistence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) on substrates, and the impact of fomites on Coronavirus Disease 19 (COVID-19) transmission, is until now, widely discussed. Consequently, further investigations are required for a correct risk assessment in high-risk facilities such as hospitals, healthcare facilities (HCFs), and long-term care facilities (LTCFs). Therefore, appropriate surveillance and disinfection programs represent the best approach to guarantee the safety of these communities. This study proposes an environmental SARS-CoV-2 surfaces routine monitoring approach in HCF and communities' settings, to provide rapid and effective evaluation of surface hygienic conditions and the effectiveness of applied sanitization measures. Surfaces samples (n = 118) were collected using the SRK® kit (Copan Italia) from 2020 to 2023. Three molecular techniques were compared: Reverse Transcription Loop mediated isothermal AMPlification (RT-LAMP, Enbiotech), Reverse-Transcription quantitative polymerase chain reaction (RT-qPCR) (RT-qPCR, Seegene) and droplet digital PCR (ddPCR, Bio-Rad). For ddPCR, two RNA extraction methods were compared: TRIzol LS (Invitrogen) versus QIAmp Viral Mini kit (QIAGEN), showing how the latter is more suitable for surfaces. Regarding the quantitative ddPCR results, the ROC analysis allowed to reduce the manufacturer cut-off for droplets number (from 3 to 1) for the positive samples. Moreover, a new cut-off for the viral RNA copies' number/μL for each target (N1 and N2) on environmental monitoring was fixed at 2,82. The results obtained using the QIAmp kit, suggested that the N2 target is more stable in the environment and could be most suitable for the virus environmental detection. The percentage of positive samples was similar among the techniques (26% for RT-LAMP, 36% for ddPCR and 23% for RT-qPCR). Using RT-qPCR as reference method, a sensitivity (SE) of 30% for RT-LAMP and 41% for ddPCR was observed. By contrast, specificity (SP) was higher for RT-LAMP (75%) respect to ddPCR (66%). Comparing the faster RT-LAMP with the sensitive ddPCR the 26% and 74% of SE and SP for RT-LAMP, were reported. The low sensitivity for RT-LAMP and ddPCR could be explained with the use of clinical rather than environmental kits, other than the changing in the virus prevalence during the sampling campaign. Although the RT-LAMP requires improvements in term of SE and SP, this research presents an innovative environmental monitoring and prevention method for SARS-CoV-2, that could be extended to other pathogens that are under environmental surveillance.

Identification and functional analysis of a novel SMARCC2 splicing variant in a family with syndromic neurodevelopmental disorder

BackgroundTo determine the pathogenicity of a novel splicing variant in the SMARCC2 gene identified from a pair of adult male monozygotic twins with neurodevelopmental disorder, and to investigate the genotype-phenotype characteristics associated with SMARCC2 variants.MethodsWhole-exome sequencing (WES) was conducted on the proband, and candidate variants were validated using Sanger sequencing within the family. The effect of the identified splicing variant on SMARCC2 mRNA processing was analyzed using reverse transcription PCR (RT-PCR) and TA-clone sequencing using samples derived from the proband. The clinical features of the twins were collected and compared with the previously reported patients.ResultsThe twin adult males displayed comparable phenotypes, characterized by moderate developmental delay, intellectual and language delays, dense hair, craniofacial anomalies, scoliosis, cryptorchidism, hypotonia, behavioral abnormalities, allergic purpura and eczema, and drug allergies. WES unveiled a previously unreported heterozygous splice variant of the SMARCC2 gene (NM_003075.3: c.1496 + 1G > T). Sanger sequencing confirmed that the variant was de novo in both patients. TA-clone sequencing of the RT-PCR fragments showed that the canonical splicing variant resulted in two distinct aberrant splicing events in SMARCC2 mRNA. Specifically, approximately 80% of the mutant clones resulted from the in-frame insertion of 126 bases in intron 16, while the remaining 20% showed an in-frame deletion of exon 16 (c.1383_1496del). Crystal structure analysis showed that both in-frame alterations hindered the proper formation of the alpha helix structure within the SMARCC2 protein. An analysis of genotype-phenotype correlations indicated that our patients displayed neurological phenotypes of greater severity than those observed in patients with truncating variants, instead aligning more closely with the characteristics of the missense/in-frame variant group.ConclusionWe identified and reported a pair of twins suffering from syndromic neurodevelopmental disorders caused by a novel splicing variant of SMARCC2. Our findings further reinforce the notion that individuals harboring missense/in-frame variants in SMARCC2 are prone to experiencing more severe neurological phenotypes.

HDAC4 regulates apoptosis in Acan-CreERT2;HDAC4d /d mice with osteoarthritis by downregulating ATF4.

Several studies have previously shown that histone deacetylase 4 (HDAC4) can regulate endoplasmic reticulum stress-induced apoptosis through the activating transcription factor 4 (ATF4)/CAAT/enhancer binding protein homologous (CHOP) signaling pathway, thereby affecting the progression of osteoarthritis (OA). The present study investigated the regulatory mechanism of HDAC4 in chondrocyte apoptosis in OA using Acan-CreERT2;HDAC4fl/fl gene knockout mice. Forty mice were divided into four groups: TM-DMM group [tamoxifen (TM) injection at 2 months of age and destabilization of the medial meniscus (DMM) surgery at 3 months], TM-sham group (TM injection at 2 months of age and sham surgery at 3 months), no TM-DMM group (corn oil injection at 2 months of age and DMM surgery at 3 months) and no TM-sham group (corn oil injection at 2 months of age and sham surgery at 3 months). Apoptosis and cartilage damage were assessed through imaging, histological analysis, immunohistochemistry, reverse transcriptase-PCR and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining. HDAC4 knockdown resulted in increased osteophyte formation, significant narrowing of the joint space and increased articular cartilage damage. Furthermore, expression levels of key apoptosis-related markers, ATF4, CHOP, caspase-3 and caspase-9, were significantly higher in the TM groups than in their respective control groups. Taken together, our results suggest that HDAC4 deficiency leads to increased apoptosis induced by the ATF4/CHOP signaling pathway in the pathogenesis of OA. Therefore, upregulation of HDAC4 may represent a potential therapeutic strategy.

Identification of the BBX gene family in blueberry at different chromosome ploidy levels and fruit development and response under stress

BackgroundBlueberry (Vaccinium spp.) fruits are rich in flavonoids such as anthocyanins and have a high nutritional value. The zinc finger protein transcription factor B-box (BBX) plays important roles in plant growth and development, hormone response, abiotic stress, and anthocyanin accumulation. However, studies on the BBX family in blueberry are lacking.ResultsIn total, 83 VcBBX and 24 VdBBX genes were identified in tetraploid and diploid blueberry, respectively. A correlation was observed between the number of BBX genes in blueberry and chromosome ploidy. Gene loss and specific replication during blueberry evolution may lead to an imbalance of quantitative relationship between VcBBX and VdBBX genes. The analysis of transcriptome and quantitative reverse transcription–polymerase chain reaction data revealed that the expression pattern of BBX genes depended on the developmental stage of blueberry fruit. Gibberellin inhibited the expression of most VcBBX genes. Abscisic acid promoted the expression of some members of the BBX family. The expression levels of VcBBX15b4, VcBBX21a1, and VcBBX30a in blueberry leaves were significantly downregulated under blue light treatment, whereas that of VcBBX15c3 was significantly upregulated under red light treatment.ConclusionIn total, 83 VcBBX and 24 VdBBX genes were identified in 2 types of blueberries. Fruit development and transcription profiles under different stresses were analyzed. These findings will support further investigation of how BBX genes are involved in regulating hormone treatment and light stress during the growth and development of blueberry.

Electroacupuncture induces analgesia by regulating spinal synaptic plasticity via the AMPA/NMDA receptor in a model of cervical spondylotic radiculopathy: secondary analysis of an experimental study in rats.

Cervical spondylotic radiculopathy (CSR) is characterized by neuropathic pain (NP). Although the analgesic effect of electroacupuncture (EA) has been widely recognized in clinical practice, the mechanism of EA in the treatment of CSR remains unknown. We previously reported that 7 days of EA improved behavioral markers of NP, attenuated increases in α-synuclein, synapsin 1 and 2, postsynaptic density (PSD)-95 and growth-associated protein (GAP)-43, and improved ultrastructural changes within synapses in a rat model of CSR. Herein, we present supplemental data from the same cohort of animals examining the timing of behavioral improvement within the first week (through additional measurements at 3 and 5 days into the EA treatment) and new data on the effects of EA on α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) and N-methyl-d-aspartic acid receptor (NMDAR) levels. As previously reported, the rats were divided into four groups at random: normal, sham, CSR and CSR + EA. EA at bilateral LI4 and LR3 was administered once a day for 7 days (20 min each) in the CSR + EA group after the CSR model was established by inserting a fishing line under the laminae. Behavioral assessments were carried out prior to initiation of EA and at 3, 5 and 7 days into the 7-day treatment course. Concentrations ofγ-aminobutyric acid (GABA) and glutamate (Glu) were determined using enzyme-linked immunosorbent assay and ultraviolet colorimetry, respectively, and AMPAR (glutamate receptor (GluR)1 and GluR2 membrane protein) expression was determined using Western blotting. Immunohistochemistry was used to detect the protein expression and average optical density (AOD) of NMDAR1 (NR1), NMDAR2A (NR2A) and NMDAR2B (NR2B). Quantitative reverse transcription-polymerase chain reaction was used to detect the mRNA expression of NR1, NR2A and NR2B. Transmission electron microscopy was used to observe changes in synaptic ultrastructure. EA significantly improved the pressure pain threshold (PPT) and mechanical withdrawal threshold (MWT) 5 days into the intervention, although effects were less pronounced than at 7 days (at completion of treatment). However, significant effects on gait scores were not seen prior to 7 days. As previously reported, EA also improved markers of synaptic ultrastructure. In the spinal cord, GluR1 membrane protein expression was decreased, GluR2 membrane protein expression was increased, and the GluR1/GluR2 ratio was decreased. Protein and mRNA expression of NR1, NR2A and NR2B was significantly decreased. GABA concentration was markedly increased, while Glu concentration was markedly decreased. Evidence of EA analgesia (higher PPT and MWT scores) was seen after 5 days of EA, while positive effects on motor function required 7 days of treatment. The underlying mechanism may be related to inhibition of AMPAR and NMDAR expression, regulation of the concentration of related neurotransmitters and improvement of spinal cord synaptic plasticity. This study establishes a preliminary theoretical foundation for the use of EA in the clinical treatment of CSR.

Transcriptomics-Based Study of Immune Genes Associated with Subclinical Mastitis in Bactrian Camels

The significant increase in demand for camel milk has led to a rapid increase in the number of Bactrian camels. However, the widespread occurrence of mastitis significantly impacts the development of the Bactrian camel milk industry and poses a public health risk. Despite this, there is a lack of research on the transcriptional response, immune response pathways, and changes in core genes of Bactrian camels with subclinical mastitis. This study aimed to reveal the changes in immune-related response pathways and gene transcription levels in Bactrian camels with subclinical mastitis by analyzing the blood transcriptional response after the occurrence of subclinical mastitis in natural conditions. This study focused on 7-year-old Bactrian camels and collected 2 mL of blood from the camels that tested positive with a 4-peak California Mastitis Test (CMT) and those that tested negative with a 3-peak CMT. RNA sequencing (RNA-Seq) technology was used to analyze gene expression in the blood samples. Gene expression was verified using quantitative reverse transcription polymerase chain reaction (RT-qPCR). Overall, 1722 differentially expressed genes were sequenced in the blood samples of CMT-positive and CMT-negative Bactrian camels, including 1061 upregulated and 661 downregulated genes. After conducting gene ontology functional enrichment, 453 differentially expressed genes were identified. We also discovered pathways such as immune response, the G-protein-coupled receptor signaling pathway, and internal signal transmission. Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway enrichment detected 668 differentially expressed genes annotated onto 309 metabolic pathways, with significantly enriched immune pathways including cytokine–cytokine receptor interaction, complex and coalescence cascades, natural killer cell-mediated cytotoxicity, and T helper type 17 cell differentiation, among others. Through a STRING protein interaction database and cytoscape analysis, it was found that core differentially expressed genes related to immunity included IL10, CCL5, IL1B, OSM, TNFRSF1B, IL7, and CCR3, among others. The RT-qPCR results for six randomly selected core differentially expressed genes showed that the RT-qPCR expression pattern was consistent with the RNA Seq results. The immune-related genes in Bactrian camels affected by subclinical mastitis are primarily concentrated in the immune response and the cytokine–cytokine receptor interaction pathway. Given the importance of these pathways and the connections among related genes, the immune genes within these pathways may play a crucial role in the pathogenesis of subclinical mastitis in Bactrian camels. This study provides a valuable reference for investigating the immune regulatory mechanisms of subclinical mastitis in Bactrian camels.

Comparative study of nanohydroxyapatite-emdogain effects on apical papilla stem cell survival and differentiation.

The study was designed to explore the enhanced impact of nano-hydroxyapatite and emdogain on the survival and osteogenic/odontogenic differentiation of human stem cells isolated from the apical papilla (hSCAPs). In this in vitro trial, hSCAPS obtained from intact impacted immature third molars were confirmed to have characteristic cell surface markers, then exposed to nanohydroxyapatite, emdogain, and nanohydroxyapatite coated with emdogain for durations of 1-3days. The survival of apical papilla stem cells was measured using a methyl thiazolyl tetrazolium assay. The quantitative reverse transcription polymerase chain reaction, alkaline phosphatase activity (ALP) and Alizarin red staining were used to evaluate osteogenic-odontogenic differentiation. Analysis of the data was done using one-way ANOVA, t-test, and Mann-Whitney test (α = 0.05). At 1-3days, emdogain exhibited no significant impact on the survival of human stem cells from the apical papilla. In contrast, nanohydroxyapatite (α > 0.05) and nanohydroxyapatite coated with emdogain demonstrated a notable reduction in cell survival compared to the control group (α < 0.05). The expression of dentin sialophosphoprotein, dentin matrix protein 1, and bone sialoprotein genes demonstrated a notable increase in the group treated with nanohydroxyapatite coated with emdogain compared to the other groups (α < 0.05), and furthermore, this group exhibited more pronounced mineralized nodules than the other experimental groups. In contrast to nanohydroxyapatite, Emdogain did not demonstrate a detrimental effect on the survival of hSCAPs. Nanohydroxyapatite, emdogain, and nanohydroxyapatite coated with emdogain increased osteogenic/odontogenic differentiation of hSCAPs.

Rotavirus vaccine effectiveness and coverage among children younger than 5years old in Shanghai, China: A test-negative case control study.

The number of post-marketing studies assessing the vaccine effectiveness (VE) of the Lanzhou lamb rotavirus vaccine (LLR, licensed in 2000 exclusively in China) and the oral human attenuated pentavalent rotavirus vaccine (RotaTeq, licensed in China in 2018) in China is limited. A test-negative case-control study based on prospective surveillance was conducted among diarrhea patients aged 5years and younger at five hospitals in Shanghai, China. Cases and controls were defined based on the results of real-time fluorescent quantitative reverse transcription polymerase chain reaction (rRT-PCR) of fecal samples for rotavirus. Both matched and unmatched case-control study designs were employed using logistic regression models, with adjustments for age at onset age and the rotavirus epidemic season. In the LLR-specific analysis (247 cases, 2191 controls), the VE of partial LLR vaccination (2 doses) was 49.09% (95% CI: 1.69%∼73.64%) in multivariate analyses. In the RotaTeq-specific analysis (42 cases and 523 controls), the VE of complete RotaTeq vaccination was 87.13% (95% CI: 45.87%∼96.94%), 89.46% (95% CI: 55.03%∼97.53%), and 85.69% (95% CI: 33.43%∼96.93%) respectively in univariate, multivariate, and matched analyses, respectively. The vaccination coverage for any dose among 2893 patients with rotavirus-negative diarrhea born between 2011 and 2022 was 49.78%. Following the licensure of RotaTeq in 2018, this coverage increased from 45.02% to 61.77%. RotaTeq demonstrates a robust protective effectiveness, while LLR provides a certain level of protection against mild to moderate rotavirus diarrhea in children in Shanghai. For privately purchased (non-NIP) vaccines, we estimate that the coverage for rotavirus vaccines among children in Shanghai is high. Complete rotavirus vaccination is recommended for age-eligible children. Further post-marketing research on rotavirus vaccines is necessary to inform decision-making regarding the introduction of rotavirus vaccination in China.

A Novel Missense Variant in SORBS2 Is Causative With Familial Alzheimer's Disease.

Alzheimer's disease (AD) is a common neurodegenerative disorder with a substantial genetic component. Despite advances in elucidating the genetic underpinnings of AD, much of its heritability remains unexplained. Discovering novel genetic variants and understanding their pathogenic roles are crucial challenges in AD research. This study aimed to identify pathogenic genes and elucidate their role in familial early-onset AD (EOAD). Blood samples from an EOAD pedigree and Sorbin and SH3 Domain-Containing Protein 2 (SORBS2) T189M transgenic mice were analyzed. Cognitive function was assessed via the Morris water maze (MWM). Protein expression was evaluated by western blotting, while amyloid-β (Aβ) levels were quantified via immunohistochemistry and enzyme-linked immunosorbent assay. Inflammatory markers were measured using immunofluorescence and quantitative reverse transcription polymerase chain reaction (PCR). Neuronal morphology, including dendritic and spine alterations, was examined using Golgi staining. We identified a novel SORBS2 variant (c. 566C>T, p. T189M) in a Han Chinese family, segregating with AD in a Mendelian fashion. SORBS2 T189M transgenic mice exhibited cognitive deficits, cortical Aβ accumulation, and an increased Aβ42/Aβ40 ratio. Additionally, elevated levels of interleukin (IL)-1β, IL-6, tumor necrosis factor α (TNF-α), and ionized calcium-binding adaptor molecule 1 (Iba1)-positive microglia, along with neuronal loss, were observed in the brains of T189M mice. Our study suggest that the SORBS2 T189M variant is a novel candidate causal mutation associated with familial AD in a Chinese pedigree, contributing to AD pathogenesis by promoting neuroinflammation and neuronal injury. Notably, this study is the first to establish a link between SORBS2 mutations and AD.

Fluconazole induces cardiovascular toxicity in zebrafish by promoting oxidative stress, apoptosis, and disruption of key developmental genes.

This study systematically evaluated the toxic effects of fluconazole on the cardiovascular development of zebrafish. Zebrafish embryos were treated with different concentrations of fluconazole (200, 400, and 800μg/ml) to observe its impact on heart development, reactive oxygen species (ROS) generation, apoptosis, and hemoglobin production. The results showed that as the concentration of fluconazole increased, significant changes in zebrafish heart structure were observed, along with a notable reduction in heart rate. Pericardial edema and cardiac morphological abnormalities were particularly prominent in the high-dose group. In addition, fluconazole treatment significantly increased ROS levels and induced apoptosis in cardiac cells. Enzyme-linked immunosorbent assay (ELISA) results showed that fluconazole treatment significantly increased the malondialdehyde (MDA) content and reduced superoxide dismutase (SOD) and catalase (CAT) activity, suggesting that oxidative stress and cell death may play a key role in its cardiotoxicity. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis revealed that fluconazole treatment significantly affected the expression of several key genes related to heart development and function, particularly cardiac myosin light chain 2 (cmlc2), ventricular myosin heavy chain (vmhc), and myosin heavy chain 6 (myh6), whose expression changes were closely associated with alterations in heart morphology and function. Transcriptomic analysis showed that several signaling pathways related to cardiac development, apoptosis, and metabolism were affected. In summary, this study reveals the multifaceted cardiotoxic mechanisms of fluconazole in zebrafish and provides new insights into drug safety assessment.