Molecular Biology Reports最新文献

Background: Human sex development is a highly regulated process guiding undifferentiated gonads toward a testicular or ovarian fate. Disruptions in this pathway result in disorders of sexual development (DSD), characterized by atypical chromosomal, gonadal, or anatomical sex. These conditions usually appear as ambiguous genitalia at birth or as atypical pubertal development during adolescence. Different etiologic, phenotypic, and genotypic factors can cause DSD. Advances in next-generation sequencing (NGS) have significantly accelerated the identification of genetic variants through targeted panels, including both known genes involved in sex determination and differentiation, as well as newly discovered genes linked to DSD.

Methods and results: In this study, whole exome sequencing (WES) was performed on a Moroccan patient, born to non-consanguineous parents, who presented with severe hypospadias, micropenis, and cryptorchidism, and exhibited overlapping phenotypic features consistent with congenital disorder of glycosylation (CDG) and primary ciliary dyskinesia (PCD). After variant annotation and prioritization, two heterozygous variants in the MPI (c.305 C > T; p. Ser102Leu) and RSPH1 (c.471 C > G; p. His157Gln) genes were identified and confirmed by Sanger sequencing in family members. Their pathogenic effects on protein structures and functions were subsequently anticipated using bioinformatic tools and molecular dynamics (MD) simulations.

Conclusions: To our knowledge, this is the first report of these specific variants in the context of DSD, shedding light on a unique genotype-phenotype profile associated with the patient's complex clinical presentation. The high genetic variability underlying these disorders has made molecular diagnosis challenging. Yet, genomic approaches could expand our understanding of DSD landscape and improve diagnosis, personalized interventions, and patient management.

Purpose: Inflammatory bowel disease (IBD) is a chronic inflammatory disease characterized by damage to the epithelial barrier and intestinal inflammation; there is currently no standard treatment. Recent evidence suggests that worms and their by-products may have potential to treat IBD, owing to their immunomodulatory effects in humans. We investigated the effects of the recombinant Echinococcus granulosus antigen EgM123 on host immunity and inflammation.

Methods: We produced recombinant EgM123 to test its effects in the dextran sodium sulfate (DSS)-induced colitis mouse model. Mice were immunized with EgM123 three times via intraperitoneal injection and then challenged with 4% DSS in the drinking water for 1 week; they were observed daily for weight, mobility, and stool. After necropsy, fixed tissues/organs were sectioned and stained with hematoxylin and eosin to assess inflammation. Cytokine levels were detected by ELISA and reverse transcription polymerase chain reaction. Colitis severity was assessed by colon length, weight loss, and a semi-quantitative score of morphological changes.

Results: EgM123 had protective effects against colitis in mice. Histopathological analysis of the colon revealed a significant reduction in intestinal inflammation caused by DSS, which was associated with downregulation of the Th1/Th17 response in the colon. Administration of EgM123 before colitis induction limited the severity of intestinal inflammation and the disease index score, inhibited TNF-α secretion, and induced IL-10 expression.

Conclusions: EgM123 may alleviate colitis by inhibiting the Th1 response and inducing Th2 immunity. Immunotherapy with EgM123 may represent a strategy to modulate the inflammatory processes involved in IBD.

Background: Eurasian red squirrels (Sciurus vulgaris) are common in Europe and Asia, but are declining in many regions across their range. As continental European populations are now facing current and future threats from invasive species in addition to existing anthropogenic pressures it will be important to carefully monitor these populations. Non-invasive genetic sampling methods are a useful tool in conservation assessments, but often require techniques such as microsatellite markers that can be used with lower quality DNA. It remains helpful to increase the resolution of these assessments by identifying additional genetic markers. We describe new microsatellite markers developed from European red squirrels from France and use them to assess genetic diversity in populations in southern France.

Methods and results: We used Illumina sequencing to characterize microsatellites from tissue samples of S. vulgaris. Using 7 tissue samples we assessed amplification and polymorphism in 48 microsatellite inserts and further evaluated 16 of these microsatellite loci in hair samples from 120 individuals from four populations. In the 104 samples for which those loci amplified, there was an average of 6.1 alleles amplified per locus, with mean observed and expected heterozygosities of 0.44 and 0.59, respectively. Only one locus showed significant deviation from HWE across all populations. The same locus exhibited a likely presence of null alleles.

Conclusions: We describe 16 new microsatellite loci, with caution required for one locus in analyses sensitive to null alleles. These new loci can help provide increased resolution in population genetic assessments of red squirrels in continental Europe.

Diabetes-associated cognitive dysfunction (DACD), a serious central nervous system complication of diabetes mellitus, poses a heavy global burden due to its intricate pathogenesis and lack of effective therapies. Mounting evidence identifies programmed cell death (PCD) as a pivotal driver of DACD progression. This review systematically elaborates on the molecular mechanisms of various PCD modalities (including apoptosis, autophagy, pyroptosis, and ferroptosis) in DACD, with a particular focus on the role of ferroptosis as a core pathogenic mechanism. Hyperglycemia disrupts cerebral iron homeostasis, induces mitochondrial dysfunction and oxidative stress, leading to inhibition of glutathione peroxidase 4 (GPX4) activity and accumulation of lipid peroxides. This cascade ultimately triggers ferroptosis in neurons, glial cells, and the blood-brain barrier, resulting in impaired synaptic plasticity and cognitive decline. Furthermore, the review delineates the complex interactive regulatory network between ferroptosis and other PCD forms (e.g., autophagy, pyroptosis), which can converge into the coordinated cell death program of PANoptosis. Intervention strategies targeting ferroptosis, such as iron chelators (deferoxamine), antioxidants (N-acetylcysteine), GPX4 activators, natural products (resveratrol, curcumin), repurposed traditional medicines (liraglutide, metformin), and non-pharmaceutical interventions (exercise, electroacupuncture), have demonstrated significant potential in improving cognitive function in preclinical models. This review aims to provide novel insights into the pathophysiology of DACD and establish a theoretical foundation for developing precise therapeutic strategies centered on targeting ferroptosis.

Background: A crucial step in molecular oncology diagnostics is the acquisition of high-quality genomic DNA. However, DNA extracted from melanocytes frequently contains melanin a potent PCR inhibitor that co-purifies with nucleic acids. This contamination can significantly impair both research applications and molecular diagnostic assays essential for targeted therapies. To address the inhibitory impact of melanin on PCR amplification, particularly in DNA isolated from pigmented tissues such as melanoma lesions, we propose a rapid, efficient, and straightforward protocol for eliminating melanin contamination.

Methods and results: PCR and Sanger sequencing were performed on 15 pigmented tissue specimens for which prior molecular testing and genotyping had yielded inconclusive results. Pre-treatment of sample lysates with phenol prior to DNA extraction significantly enhanced the removal of PCR inhibitors, improved DNA purity, and resulted in higher PCR amplification and genotyping success rates.

Conclusions: This protocol enables simultaneous DNA extraction and purification, thereby enhancing the accuracy of genetic analyses, improving consistency in molecular biology and diagnostic research, and supporting more informed therapeutic strategies for patients with melanoma.

Background: The present study evaluated the diagnostic performance of conventional microscopy and polymerase chain reaction (PCR) based assays for the detection of Babesia infections in dogs, including semi-nested PCR (SN-PCR) targeting the 18 S rRNA gene and single-round PCR (SR-PCR) assays targeting the mitochondrial cytochrome b (cytb) and cytochrome c oxidase subunit 1 (cox1) genes for B. gibsoni and B. vogeli, respectively. Mitochondrial sequence variation was further assessed by integrating newly generated sequences from Jabalpur, Madhya Pradesh (central India), with global reference datasets.

Methods and results: A total of 100 blood samples from dogs suspected of having haemoprotozoan infections were analysed between June 2022 and May 2023. Microscopic examination of Giemsa-stained smears detected Babesia parasites in 13% of the samples, whereas the 18 S rRNA SN-PCR assay identified infections in 29%, comprising B. gibsoni (25%) and B. vogeli (4%). Representative sequences showed 98-99% identity with corresponding GenBank reference sequences. Representative sequences showed 98-99% identity with corresponding GenBank reference sequences. Compared with SN-PCR, microscopy demonstrated moderate sensitivity but perfect specificity, resulting in an overall diagnostic accuracy of 84.0% (p < 0.01). Mitochondrial SR-PCR assays detected B. gibsoni and B. vogeli in 5% and 4% of the samples, respectively. The cytb-based assay showed higher sensitivity and a significant diagnostic association (p < 0.01) than the cox1 assay, whereas the cox1 assay demonstrated lower sensitivity with a non-significant association (p > 0.05). All PCR assays showed 100% specificity and positive predictive value. Bayesian phylogenetic and haplotype analyses indicated that B. gibsoni cytb sequences formed a monophyletic lineage with limited regional structuring, with Indian isolates clustering within a distinct sub-lineage. In contrast, B. vogeli cox1 sequences exhibited low global diversity with a dominant shared haplotype across geographic regions.

Conclusions: The 18S rRNA SN-PCR assay showed the highest sensitive method for detecting Babesia infections in dogs. Mitochondrial markers (cytb and cox1) supported species confirmation and comparative phylogenetic assessment, highlighting the complementary value of nuclear and mitochondrial gene targets for molecular surveillance and control of canine babesiosis in India.