Molecular Biology Reports最新文献

Background: The role and relevance of macrophages both as causes and therapeutics of cellular senescence is rapidly emerging. However, current knowledge regarding the extent and depth of senescence in macrophages in vivo is limited and controversial. Further, acute models of stress-induced senescence in transformed/cancerous macrophage cell lines are being used although their efficacy and relevance are not characterized.

Methods and results: The present study sought to address these aspects by first comparing prevalent senescence in naturally aged murine peritoneal macrophages, and then assessing the effects of two different stressors (LPS and H₂O₂) in inducing premature senescence in young peritoneal macrophages. Next, RAW264.7 cell line was exposed to respective stressors and their efficiency in recapitulating the effects of natural senescence markers was characterized. We observed strong upregulation of primary markers of senescence such as SA-β-gal activity, p53, p21, p16^Inka4a, Rb, ATM, and Lamin B1in naturally aged mice along with increased SASP proteins (IL-6/TNF-α/MCP-1) and redox stress (ROS and NO). Aged macrophages also demonstrated severely reduced phagocytosis. Exposure to both LPS and H₂O₂ in young macrophages invoked the expression of all primary markers of senescence although SASP protein expression was exaggerated in LPS stimulation. Similarly, ROS and NO expression increased while phagocytosis decreased. Stimulation of RAW264.7 cells generally revealed a similar trend although the depth of all measured parameters was ostensibly stronger in young peritoneal macrophages. Among the two stressors, LPS stimulation appeared to be relatively more potent.

Conclusion: Overall, this study emphasizes that LPS exposure to young peritoneal macrophages more strongly recapitulates in vivo cellular senescence in macrophages.

Background: Sugarcane is cultivated globally and affected by more than 125 pathogens, which lead to various plant diseases. In recent years, high-throughput sequencing (HTS)-based genome analyses have been broadly adopted for the discovery of both characterized and un-characterized viruses from plant samples. In this study, the HTS data of sugarcane pooled sample retrieved from sequence read archive (SRA) were de novo re-assembled using CLC Genomic Workbench.

Methods and results: The genomic sequence of a novel dsRNA totivirus, 5,384 nucleotides (nt) long, excluding the 5' untranslated region (UTR) and 3' UTR, was discovered and named sugarcane totivirus 1 (STV 1). The genome contains two open reading frames (ORFs): a putative coat protein (CP) encoding 866 amino acids (aa) and RNA-dependent RNA polymerase (RdRp) encoding 824 aa. Phylogenetic studies based on the genomic sequences (nt), and the aa sequences of CP as well as RdRp regions revealed that STV 1 is closely related to other members of the genus Totivirus. Pairwise sequence identity of CP and RdRp aa sequences showed 30.0-51.5% and 26.3-47.2% similarity, respectively with other members of the family Totiviridae. The HTS results were further validated and confirmed through OneStep RT-PCR assay and Sanger sequencing.

Conclusion: A novel totivirus (STV 1) in the genus Totivirus, family Totiviridae has been identified. This is the first report of dsRNA totivirus STV 1 associated with sugarcane from India.

Background: Exosomes are extracellular vesicles released by cells that mediate intercellular communication and actively participate in cancer progression, metastasis, and regulation of immune response within the tumour microenvironment. Inhibiting exosome release from cancer cells could be employed as a therapeutic against cancer.

Methods and results: In the present study, we have studied the effects of Acorus calamus in inhibiting exosome secretion via targetting Rab27a and neutral sphingomyelinase 2 (nSMase2) in HER2-positive (MDA-MB-453), hormone receptor-positive (MCF-7) and triple-negative breast cancer (MDA-MB-231) cells. We observed that treatment with A. calamus significantly downregulated the expression of Rab27a and nSMase2 in all tested cells. NTA analysis showed that inhibition of Rab27a and nSMase2 reduced exosome secretion from breast cancer cells. We conducted metabolic profiling of A. calamus extract to reveal the phytochemicals present and docked them on Rab27a and nSMase2 to decipher the compounds responsible for protein inhibition. Molecular dynamic simulations were conducted on lead compounds, and we observed that calcitriol lactone showed the most stable binding interactions with nSMase2. Treatment of breast cancer cells with calcitriol lactone significantly downregulated nSMase2 expression.

Conclusions: Our study demonstrates that A. calamus significantly inhibits exosome secretion in HER2-positive, hormone receptor-positive, and triple-negative breast cancer cells by targeting key regulatory proteins Rab27a and neutral sphingomyelinase 2 (nSMase2). These findings suggest that A. calamus holds therapeutic potential in inhibiting exosome-mediated cancer progression by targeting the exosome secretion pathway. Further investigations are warranted to explore the clinical applications of these findings in breast cancer treatment.

Dengue virus (DENV) poses a considerable threat to public health on a global scale, since about two-thirds of the world's population is currently at risk of contracting this arbovirus. Being transmitted by mosquitoes, this virus is associated with a range of illnesses and a small percentage of infected individuals might suffer from severe vascular leakage. This leakage leads to hypovolemic shock syndrome, generally known as dengue shock syndrome, organ failure, and bleeding complications. The severe form of this disease is believed to be, at least partially, associated with inflammatory and thrombotic states. These issues are significantly affected by the activation of platelets and leukocytes, as well as their interactions, which may influence its prognosis. The platelets present in a thrombus are able to attract leukocytes to the site of injury. The intricate process leads to the significant accumulation, activation, and migration of leukocytes, thereby promoting thrombotic events and triggering inflammatory responses. The occurrence of these events, combined with the direct viral infection of endothelial cells, leads to vascular endothelialitis, the disruption of cellular membranes, and the subsequent release of DAMPs. As a result, considerable damage occurs in the endothelium, which activates neutrophils and platelets; thisleads to their interaction and initiates the process of Netosis. Collectively, these processes exacerbate inflammatory and thrombotic conditions. In this respect, current research has focused on understanding whether effective anti-inflammatory protocols can prevent thrombotic events or, conversely, if efficient anticoagulant regimens may lead to a reduction in cytokine storms and tissue damage. This review article aims to illuminate the platelet leukocyte crosstalk, detailing the mechanisms through which platelets may play a role in the pathogenesis of DENV. The research outputs are particularly important in severe cases, in which case their interactions with leukocytes can exacerbate both inflammation and thrombosis in a mutually reinforcing manner.

Background: The identification of helminth parasites in Schizothorax spp. from Kashmir, including Schyzocotyle acheilognathi, Pomphorhynchus kashmirensis, and Adenoscolex oreini, is hindered by morphological limitations and high intraspecific variation. While previous studies have relied on morphological diagnosis, a comprehensive molecular characterization is lacking. To address these limitations and achieve more accurate species identification, this study employs molecular techniques such as DNA sequencing and phylogenetic analysis.

Method: Genomic DNA was extracted using standardized protocols. Partial amplification of 18S, ITS, and 28S rRNA was carried out using specific primer sets, followed by Sanger sequencing. Genetic characteristics were analyzed using bioinformatic approaches, focusing on Nucleotide divergence and Phylogenetic relationships.

Results: Phylogenetic analysis confirmed the monophyly of S. acheilognathi, revealing close relationships with S. nayarensis. Interspecific variation ranged from 2.0 - 4.1% for 18S and 0.8 - 2.2% for 28S rDNA, while P. kashmirensis showed high genetic similarity to P. tereticollis and P. laevis with variations ranging from 0.1 - 1.5% for 18S and 2.3-6.0% for ITS rDNA. A. oreini forms a monophyletic lineage separate from Caryophyllaeides and Caryophyllaeus species.

Conclusion: Our study resolved persistent taxonomic ambiguities within the order Bothriocephalidea, resulting in the reassignment of Schyzocotyle acheilognathi (previously Bothriocephalus acheilognathi) to the reinstated genus Schyzocotyle. This study underscores the utility of ribosomal gene sequencing in characterizing fish parasites and highlights the need for taxonomic re-evaluation of A. oreini within the Capingentidae family.

Background: Modulation of protein synthesis according to the physiological cues is maintained through tight control of Eukaryotic Elongation Factor 2 (eEF2), whose unique translocase activity is essential for cell viability. Phosphorylation of eEF2 at its Thr56 residue inactivates this function in translation. In our previous study we reported a novel mode of post-translational modification that promotes higher efficiency in T56 phosphorylation. Cyclin A/CDK2-mediated phosphorylation of eEF2 at the S595 residue is required for more potent phosphorylation at the Thr56, suggesting CDK2 takes a role in robust suppression of protein synthesis.

Methods and results: In the current study, we analyzed the cell cycle, proliferation, cell death, migration, colony formation, autophagy, and response to Cisplatin properties of the point-mutant variant of HCT116 cells that express the CDK2 mutant (S595A-eEF2) of eEF2. The knocked in S595A mutation resulted in decreased levels of T56 phosphorylation of eEF2, which appears to have similar biological consequences to other experimental manipulations such as silencing the activity of the kinase for the Thr56 residue, eEF2 Kinase (eEF2K).

Conclusion: Our findings indicate that interfering with the inhibition of eEF2 results in elevated protein synthesis in HCT116 cells and is associated with the progression of malignancy in the colorectal cancer cell line, where eEF2K activity could provide a tumor suppressive role.

Background: LncRNA PCAT-1 is known to promote cancer proliferation, invasion, and metastasis. However, its significance in HNSCC is not fully understood. This research investigates how the PCAT-1 / miR-145-5p / FSCN-1 axis promote HNSCC.

Methods and results: We analyzed the gene expression patterns on 238 fresh-frozen samples, comparing tumors with their normal adjacent tissues (NATs). HNSCC samples showed higher PCAT-1 and FSCN-1 expression compared to NATs (p < 0.001 and p < 0.001, respectively). In contrast, miR-145-5p expression was markedly downregulated compared to NATs (p < 0.001). Notably, ROC curve analysis revealed exceptional diagnostic power, with an AUC of 0.83 for PCAT-1, 0.95 for miR-145-5p, and 0.91 for FSCN-1. Pearson correlation analysis unveiled a significant positive correlation between PCAT-1 and FSCN-1 expression levels (r = 0.084, p < 0.001) and negative correlations between FSCN-1 and miR-145-5p (r = -0.710, p < 0.001) as well as between PCAT-1 and miR-145-5p (r = -0.759, p < 0.001). Distinct molecular profiles were observed in the levels of PCAT-1, miR-145-5p, and FSCN-1 between HPV (-) and HPV ( +) 16 and 18 genotypes (p = 0.007, p = 0.027, and p = 0.002). MiR-145-5p expression showed significant differences between HPV (-) and HPV ( +) other genotypes (p = 0.035). FSCN-1 expression showed notable distinctions between HPV ( +) 18 & 16 and HPV ( +) other genotypes (p = 0.031).

Conclusions: Elevated levels of lncRNA PCAT-1 promote HNSCC through the miR-145-5p/FSCN-1 axis and are associated with poor prognosis and reduced immune cell infiltration levels.

Background: La-related protein 7 (LARP7) is a key regulator of RNA metabolism and is thought to play a role in various cellular processes. LARP7 gene autosomal recessive mutations are the cause of Alazami syndrome, which presents with skeletal abnormalities, intellectual disabilities, and facial dysmorphisms. This study aimed to determine the role of LARP7 in modulating gene expression dynamics during osteogenesis.

Methods and results: First, the temporal expression profile of the LARP7 gene during various stages of osteogenesis was examined. Then, RNA interference-mediated knockdown of LARP7 was implemented and high-throughput RNA-seq analysis was performed in order to identify global gene expression changes associated with knockdown of LARP7. The findings show there were significant alterations in the overall gene expression profile. The observed down-regulation in extracellular matrix (ECM) component genes suggests that it might lead to impairments in the structure and function of the bone matrix. Additionally, modulation of alternative splicing events were observed, especially in the RUNX2 and SPP1, indicating the potential contribution of LARP7 to the phenotypic features observed in Alazami syndrome.

Conclusion: Overall, the findings clarify the regulatory mechanisms of LARP7 in osteogenic differentiation and illuminate potential avenues for therapeutic interventions in patients with skeletal disorders.

MicroRNAs (miRNAs) and transfer RNA-derived stress-induced RNAs (tiRNAs) have emerged as crucial players in the post-transcriptional regulation of gene expression in various cellular processes, including immunity and host defense against infections. In recent years, increasing evidence has highlighted their complex role in influencing the host response during viral and bacterial infections. miRNAs have been shown to play multiple roles in host-pathogen interaction like TLR activation and altered disease virulence during bacterial infections. In the context of viral infections, miRNAs are involved in regulating viral replication, pathogenesis, and immune evasion. Similarly, tiRNAs have recently emerged as novel players in bacterial and viral infections such as modulating bacterial growth, adaptation to stress conditions, host antiviral responses, and impacting viral replication and pathogenesis. This review provides a comprehensive analysis of the potential of miRNA expression profiles as diagnostic biomarkers to differentiate between bacterial and viral infections. Further discusses the key pathways through which small RNAs regulate bacterial and viral infection-related diseases.

Background: Extracellular matrix (ECM) proteins play a crucial role in regulating the biological properties of adherent cells. For cryopreserved fibroblasts, a favourable ECM environment can help restore their natural morphology and function more rapidly, minimizing post-thaw stress responses.

Methods and results: This study explored the functional responses of cryopreserved enriched caprine adult dermal fibroblast (cadFibroblast) cells to structural [collagen-IV and rat tail collagen (RTC)] and adhesion ECM proteins (laminin, fibronectin, and vitronectin) under in vitro culture conditions. The cryopreserved cadFibroblasts were evaluated for cell morphology, confluence, viability, cell adhesion, colony-forming units, proliferation, population doubling time migration (scratch wound healing assay), and quantitative real-time PCR for gene expression analyses when cultivated on surfaces coated with ECM proteins. A significantly (p < 0.05) higher cellular responses regarding cell adhesion ability, proliferation, CFUs, migration rate and differential gene expression of cell adhesion (β integrin, β tubulin, and E-cadherin) associated marker genes were observed with adhesion ECM proteins (laminin, fibronectin, and vitronectin) than with structural ECM proteins (collagen-IV and RTC). RT-PCR analyses revealed ECM-dependent differential template expression in cultured post-thawed cadFibroblasts.

Conclusions: Overall, while comparing different types of ECM proteins, adhesion ECM proteins (laminin, fibronectin, and vitronectin) provide a relatively better niche that supports adhesion, growth, proliferation, and migration of post-thawed cadFibroblasts compared with structural ECM proteins (collagen-IV and RTC) and the culture without ECM. The outcomes of the present study are crucial for replicating physiological conditions in experimental models for future research involving cryopreserved dermal fibroblasts or any other cell type, which can lead to future developments in tissue engineering and regenerative medicine.