Search Results (159)

Background and Objectives: Colorectal cancer (CRC) is one of the most frequently diagnosed malignancies worldwide. Although chemotherapy is an effective treatment for colorectal cancer (CRC), its effectiveness is frequently hindered by the emergence of resistant cancer cells. Studies have demonstrated a linkage between drug resistance and the pregnane X receptor (PXR), which influences the metabolism and the transport of chemotherapeutic agents. Likewise, autophagy is also a well-established mechanism that contributes to chemotherapy resistance, and it is closely tied to tumor progression. This pre-clinical study aims to investigate the role of mtKRAS-dependent autophagy with PXR expression after treatment with Irinotecan in colorectal cancer. Methods: CRC lines were treated with specific inhibitors, such as 3-methyladeninee, hydroxychloroquine PI-103, and irinotecan hydrochloride, and subjected to various assays, including MTT for cell viability, Western blot for protein expression, siRNA-mediated PXR knock-out, and confocal microscopy for autophagic vacuole visualization. Protein quantification, gene knockdown, and subcellular localization studies were performed under standardized conditions to investigate treatment effects on autophagy and apoptosis pathways. Conclusions: Our experiments showed that PXR knockdown does not alter autophagy levels following Irinotecan treatment, but it promotes apoptotic cell death despite elevated autophagy. Moreover, late-stage autophagy inhibition reduces PXR expression, whereas induction through PI3K/AKT/mTOR inhibition leads to increased expression of PXR. Our experiments uncover a mechanism by which autophagy facilitates the nuclear translocation of the PXR, thereby promoting resistance to Irinotecan across multiple cell lines. Full article

Secondary methicillin-resistant Staphylococcus aureus (MRSA) infection causes high mortality in patients with influenza A virus (IAV). Our previous study observed that the relative abundance of Lactobacillus murinus (L. murinus) was significantly reduced in both the respiratory tract and gut of IAV-infected mice and negatively correlated with the severity of IAV–MRSA coinfection pneumonia, but the role of L. murinus remains unclear. Here, we supplemented the respiratory tract and gut of IAV-infected mice with live L. murinus and performed a secondary MRSA infection challenge to investigate the effects and potential mechanisms further. Data showed that L. murinus supplementation significantly reduced mortality and pathogen loads in IAV–MRSA coinfected mice and upregulated the lung T cell-independent (TI) IgA response in IAV-infected mice. The 16S rRNA gene sequencing results showed that L. murinus supplementation ameliorated microbiota composition disorder and regulated metabolic dysfunction in the gut of IAV-infected mice. The correlation analysis and antibiotic cocktail treatment experiment showed that the TI IgA response in lungs is dependent on gut microbiota. These findings demonstrated that L. murinus supplementation reduces susceptibility to secondary MRSA infection in IAV-infected mice by promoting the TI IgA response, and provide a new perspective on the use of probiotics to prevent secondary bacterial infection following IAV infection. Full article

Translation initiation site (TIS) prediction in mRNA sequences constitutes an essential component of transcriptome annotation, playing a crucial role in deciphering gene expression and regulation mechanisms. Numerous computational methods have been proposed and achieved acceptable prediction accuracy. In our previous work, we developed NeuroTIS, a novel method for TIS prediction based on a hybrid dependency network combined with a deep learning framework that explicitly models label dependencies both within coding sequences (CDSs) and between CDSs and TISs. However, this method has limitations in fully exploiting the primary structural information within mRNA sequences. First, it only captures label dependency within three neighboring codon labels. Second, it neglects the heterogeneity of negative TISs originating from different reading frames, which exhibit distinct coding features in their vicinity. In this paper, under the framework of NeuroTIS, we propose its enhanced version, NeuroTIS+, which allows for more sophisticated codon label dependency modeling via temporal convolution and homogenous feature building through an adaptive grouping strategy. Tests on transcriptome-wide human and mouse datasets demonstrate that the proposed method yields excellent prediction performance, significantly surpassing the existing state-of-the-art methods. Full article

Nano-titanium dioxide (nano-TiO₂) can alleviate oxidative damage in plants subjected to abiotic stress, interfere with related gene expression, and change metabolite content. Polylactic acid (PLA) microplastics can inhibit plant growth, induce oxidative stress in plant cells, and alter the biophysical properties of rhizosphere soil. In this study, untargeted metabolomics (LC-MS) and RNA-seq sequencing were performed on radish root cells exposed to nano-TiO₂ and PLA. The results showed that nano-TiO₂ alleviated the growth inhibition of radish roots induced by PLA. Nano-TiO₂ alleviated PLA-induced oxidative stress, and the activities of SOD and POD were decreased by 28.6% and 36.0%, respectively. A total of 1673 differentially expressed genes (DEGs, 844 upregulated genes, and 829 downregulated genes) were detected by transcriptome analysis. Metabolomics analysis showed that 5041 differential metabolites were involved; they mainly include terpenoids, fatty acids, alkaloids, shikimic acid, and phenylpropionic acid. Among them, phenylpropanoid biosynthesis as well as flavone and flavonol biosynthesis were the key metabolic pathways. This study demonstrates that nano-TiO₂ mitigates PLA phytotoxicity in radish via transcriptional and metabolic reprogramming of phenylpropanoid biosynthesis. These findings provide important references for enhancing crop resilience against pollutants and underscore the need for ecological risk assessment of co-existing novel pollutants in agriculture. Full article

The Baculovirus Expression Vector System (BEVS) is an important protein and complex biologics production platform. The baculovirus GP64 protein is the major envelope glycoprotein that aids in virus entry and is required for cell-to-cell transmission in cell culture. Several studies have developed strategies around gp64 gene disruption in an attempt to minimize baculovirus co-production. Here, we investigate the result of transiently targeting the baculovirus gp64 gene with CRISPR-Cas9 during infection. Because not all genomes are effectively disrupted, we describe a variant calling methodology that allows the detection of the targeted mutations in gp64 even though these mutations are not the dominant sequences. Using a transfection-infection assay (T-I assay), the AcMNPV gp64 gene was targeted at six different locations to evaluate the effects of single and multiple targeting sites, and we demonstrated a reduction in the levels of baculovirus vectors while maintaining or enhancing foreign protein production when protein was driven by a p6.9 promoter. Viral genomes were subsequently isolated from the supernatant and cell pellet fractions, and our sequencing pipeline successfully detected indel mutations within gp64 for most of the single-guide RNA (sgRNA) targets. We also observed that 68.8% of variants found in the virus stock were conserved upon virus propagation in cell culture, thus indicating that they are not detrimental to viral fitness. This work provides a comprehensive assessment of CRISPR-Cas9 genome editing of baculovirus vectors, with potential applications in enhancing the efficiency of the BEVS. Full article

Utilizing high-throughput Illumina sequencing, we examined how small RNA (sRNA) profiles vary in Chinese white poplar (Populus tomentosa) across two pivotal infection stages by the rust fungus Melampsora larici-populina: the biotrophic growth phase (T02; 48 h post infection) and the urediniospore development and dispersal phase (T03; 168 h), both essential for plant colonization and prolonged biotrophic engagement. Far exceeding random expectations, siRNA clusters predominantly arose from transposon regions, with pseudogenes also contributing significantly, and infection-stage-specific variations were notably tied to these transposon-derived siRNAs. As the infection advanced, clusters of 24 nt siRNAs in transposon and intergenic regions exhibited pronounced abundance shifts. An analysis of targets indicated that Populus sRNAs potentially regulate 95% of Melampsora larici-populina genes, with pathogen effector genes showing heightened targeting by sRNAs during the biotrophic and urediniospore phases compared to controls, pointing to selective sRNA-target interactions. In contrast to conserved miRNAs across plant species, Populus-specific miRNAs displayed a markedly greater tendency to target NB-LRR genes. These observations collectively highlight the innovative roles of sRNAs in plant defense, their evolutionary roots, and their dynamic interplay with pathogen coevolution. Full article

As a tropomyosin-binding component, troponin T (TnT) is essential for the Ca²⁺ regulation of striated muscles’ contraction and locomotion activity, but its impacts on the growth and development of insects have rarely been reported. In this study, TnT was identified and functionally characterized in Tribolium castaneum by RNA interference (RNAi) and transcriptome analysis. The TnT of T. castaneum contained a 1152 bp open reading frame encoding 383 amino acids. It displayed the highest expression in late pupae and was highly expressed in the integument and CNS. Both the larval and early pupal injection of dsTnT led to 100% cumulative mortality before the pupal–adult transition. Late pupal RNAi caused 26.01 ± 4.29% pupal mortality; the survivors successfully became adults, but 49.71 ± 6.51% died in 10 days with a dried and shriveled abdomen, poorly developed reproductive system and no offspring. Additionally, RNA sequencing results indicated that key ecdysteroid and juvenile hormone biosynthesis genes (CYP314A1, aldehyde dehydrogenase family 3 member B1 and farnesol dehydrogenase) were affected, as well as several cuticle protein, nutrition metabolism and immune-related genes, suggesting that TnT may play prominent roles in development, metabolism and reproduction by affecting these pathways. This study could provide a brand-new target gene in the RNAi strategy for pest control. Full article

Orthobunyaviruses are a diverse group of segmented RNA viruses with significant but underexplored public and veterinary health implications. This study provides a genomic, phylogenetic, and ecological analysis of neglected Orthobunyaviruses using next-generation sequencing and computational predictions. We identified unique phylogenetic relationships, with Tanga virus forming a distinct lineage linked to zoonotic, human-associated, or non-vertebrate viruses across segments. GC content analysis revealed segment-specific patterns: higher GC content in the S segment suggests genomic stability and immune evasion, while lower GC content in the L segment reflects host-vector adaptation. Phylogenetic ties to well-characterized pathogenic viruses, such as Ilesha virus with Cache Valley virus and Bwamba virus with California encephalitis virus, indicate potential neurotropism. Ingwavuma virus clustered with Oropouche virus, suggesting risks of systemic febrile illnesses. Within the Simbu serogroup, Sango and Sabo viruses show teratogenic risks to livestock. Vector and host predictions implicate rodents, artiodactyls, and primates in Orthobunyavirus transmission, emphasizing complex ecological dynamics and zoonotic potential. These findings advance the understanding of Orthobunyavirus diversity, linking genomic features to pathogenicity and ecological adaptation, while providing a foundation for future surveillance and intervention strategies targeting these neglected viruses. Full article

Aging is associated with detrimental bone loss, often leading to fragility fractures, which may be driven by oxidative stress. In this study, the outcomes of comparing the differences among young, adult and aged C57BL/6J mice found that the trabecular bone volume was significantly lower in the aged mice compared to young mice, and the bone characteristics were significantly correlated with the oxidative status. To counteract the adverse effects of aging, methyl sulfonyl methane (MSM), a stable metabolite of dimethyl sulfoxide, was used to supplement the drinking water (400 mg/kg/day) of the aged mice (73 weeks old) for 8 weeks. The MSM supplementation improved the maximum load, bone microarchitecture, and mRNA levels of osteocyte-specific genes in the tibia. Furthermore, MSM reduced the serum level of the C-terminal telopeptide of type I collagen, a marker of bone resorption, and downregulated the mRNA levels of genes related to osteoclast proliferation and activity. MSM also decreased the levels of pro-inflammatory cytokines in both the serum and bone marrow. Importantly, the MSM-treated mice exhibited an enhanced antioxidant status, characterized by increased glutathione peroxidase (GPx) activity and glutathione concentration in plasma, erythrocytes and bone marrow. These improvements were linked to the activation of the nuclear factor E2 related factor 2 (Nrf2) pathway and its downstream antioxidant gene expression, including that of superoxide dismutase and GPx. These findings suggested that age-related bone loss is closely tied to oxidative stress, and MSM supplementation effectively reverses bone loss by mitigating oxidative stress-mediated bone resorption. Full article

Metal 3D printing is increasingly being used to manufacture titanium–aluminum–vanadium (Ti6Al4V) implants. In vitro studies using 2D substrates demonstrate that the osteoblastic differentiation of bone marrow stromal cells (MSCs) on Ti6Al4V surfaces, with a microscale/nanoscale surface topography that mimics an osteoclast resorption pit, involves non-canonical Wnt signaling; Wnt3a is downregulated and Wnt5a is upregulated, leading to the local production of BMP2 and semaphorin 3A (sema3A). In this study, it was examined whether the regulation of MSCs in a 3D environment occurs by a similar mechanism. Human MSCs from two different donors were cultured for 7, 14, or 21 days on porous (3D) or solid (2D) constructs fabricated by powder-bed laser fusion. mRNA and secretion of osteoblast markers, as well as factors that enhance peri-implant osteogenesis, were analyzed, with a primary focus on the Wnt family, sema3A, and microRNA-145 (miR-145) signaling pathways. MSCs exhibited greater production of osteocalcin, latent and active TGFβ1, sema3A, and Wnt16 on the 3D constructs compared to 2D, both of which had similar microscale/nanoscale surface modifications. Wnt3a was reduced on 2D constructs as a function of time; Wnt11 and Wnt5a remained elevated in the 3D and 2D cultures. To better understand the role of Wnt16, cultures were treated with rhWnt16; endogenous Wnt16 was blocked using an antibody. Wnt16 promoted proliferation and inhibited osteoblast differentiation, potentially by reducing production of BMP2 and BMP4. Wnt16 expression was reduced by exogenous Wnt16 in 3D cells. Addition of the anti-Wnt16 antibody to the cultures reversed the effects of exogenous Wnt16, indicating an autocrine mechanism. Wnt16 increased miR-145-5p, suggesting a potential feedback mechanism. The miR-145-5p mimic increased Wnt16 production and inhibited sema3A in a 3D porous substrate-specific manner. Wnt16 did not affect sema3A production, but it was reduced by miR-145-5p mimic on the 3D constructs and stimulated by miR-145-5p inhibitor. Media from 7-, 14-, and 21-day cultures of MSCs grown on 3D constructs inhibited osteoclast activity to a greater extent than media from the 2D cultures. The findings present a significant step towards understanding the complex molecular interplay that occurs in 3D Ti6Al4V constructs fabricated by additive manufacturing. In addition to enhancing osteogenesis, the 3D porous biomimetic structure inhibits osteoclast activities, indicating its role in modulating bone remodeling processes. Our data suggest that the pathway mediated by sema3A/Wnt16/miR145-5p was enhanced by the 3D surface and contributes to bone regeneration in the 3D implants. This comprehensive exploration contributes valuable insights to guide future strategies in implant design, customization, and ultimately aims at improving clinical outcomes and successful osseointegration. Full article

In previous studies, titanium peroxide nanoparticles (PAA-TiOx NPs) with surfaces functionalized using polyacrylic acid (PAA) and hydrogen peroxide (H₂O₂) demonstrated a synergistic effect when combined with X-ray irradiation. The combination generated H₂O₂ and reactive oxygen species (ROS) that enhanced the irradiation efficacy. In the present study, we examined the relationship between catalase and PAA-TiOx NPs sensitization to X-ray radiation because catalase is the primary antioxidant enzyme that converts H₂O₂ to water and oxygen. Catalase-knockout PANC-1 (dCAT) cells were generated using the CRISPR/Cas9 system, which was confirmed by the suppression of catalase expression in mRNA and protein levels that resulted in an 81.7% decrease in catalase activity compared with levels in wild-type cells. Catalase deficiency was found to increase the production of ROS, particularly in hypoxia. Also, the combination of PAA-TiOx NPs and X-ray 5 Gy resulted in a 7-fold decrease in the survival fraction (SF; p < 0.01) of dCAT cells compared with rates documented in wild-type cells. Interestingly, the combination treatment with X-ray 3 Gy in dCAT cells resulted in an SF similar to that observed in wild-type cells treated with the same combination but at a higher radiation dose (5 Gy). These results suggest that a strategy of catalase inhibition could be used to establish an advanced combination treatment of PAA-TiOx NPs and X-ray irradiation for pancreatic cancer cells. Full article

Background: Mesoporous titanium dioxide nanoparticles (mTiNPs) are known for their chemical stability, non-toxicity, antimicrobial and anticancer effects, as well as for their photocatalytic properties. When this material is subjected to UV radiation, its electronic structure shifts, and during that process, reactive oxygen species are generated, which in turn exert apoptotic events on the cancer cells. Objectives: We evaluated the cytotoxic effects of UV-irradiated mTiNPs on prostate cancer (PCa) cell line PC3 with the aim of demonstrating that the interaction between UV-light and mTiNPs positively impacts the nanomaterial’s cytotoxic efficiency. Moreover, we assessed the differential expression of key oncomiRs and tumor suppressor (TS) miRNAs, as well as their associated target genes, in cells undergoing this treatment. Methods: PBS-suspended mTiNPs exposed to 290 nm UV light were added at different concentrations to PC3 cells. Cell viability was determined after 24 h with a crystal violet assay. Then, the obtained IC₅₀ concentration of UV-nanomaterial was applied to a new PC3 cell culture, and the expression of a set of miRNAs and selected target genes was evaluated via qRT-PCR. Results: The cells exposed to photo-activated mTiNPs required 4.38 times less concentration of the nanomaterial than the group exposed to non-irradiated mTiNPs to achieve the half-maximal inhibition, demonstrating an improved cytotoxic performance of the UV-irradiated mTiNPs. Moreover, the expression of miR-18a-5p, miR-21-5p, and miR-221-5p was downregulated after the application of UV-mTiNPs, while TS miR-200a-5p and miR-200b-5p displayed an upregulated expression. Among the miRNA target genes, PTEN was found to be upregulated after the treatment, while BCL-2 and TP53 were underexpressed. Conclusions: Our cytotoxic outcomes coincided with previous reports performed in other cancer cell lines, strongly suggesting UV-irradiated mTiNPs as a promising nano-therapeutic approach against PCa. On the other hand, to the best of our knowledge, this is the first report exploring the impact of UV-irradiated mTiNPs on key onco- and TS microRNAs in PCa cells. Full article

The surface topography and chemistry of titanium–aluminum–vanadium (Ti6Al4V) implants play critical roles in the osteoblast differentiation of human bone marrow stromal cells (MSCs) and the creation of an osteogenic microenvironment. To assess the effects of a microscale/nanoscale (MN) topography, this study compared the effects of MN-modified, anodized, and smooth Ti6Al4V surfaces on MSC response, and for the first time, directly contrasted MN-induced osteoblast differentiation with culture on tissue culture polystyrene (TCPS) in osteogenic medium (OM). Surface characterization revealed distinct differences in microroughness, composition, and topography among the Ti6Al4V substrates. MSCs on MN surfaces exhibited enhanced osteoblastic differentiation, evidenced by increased expression of RUNX2, SP7, BGLAP, BMP2, and BMPR1A (fold increases: 3.2, 1.8, 1.4, 1.3, and 1.2). The MN surface also induced a pro-healing inflammasome with upregulation of anti-inflammatory mediators (170–200% increase) and downregulation of pro-inflammatory factors (40–82% reduction). Integrin expression shifted towards osteoblast-associated integrins on MN surfaces. RNA-seq analysis revealed distinct gene expression profiles between MSCs on MN surfaces and those in OM, with only 199 shared genes out of over 1000 differentially expressed genes. Pathway analysis showed that MN surfaces promoted bone formation, maturation, and remodeling through non-canonical Wnt signaling, while OM stimulated endochondral bone development and mineralization via canonical Wnt3a signaling. These findings highlight the importance of Ti6Al4V surface properties in directing MSC differentiation and indicate that MN-modified surfaces act via signaling pathways that differ from OM culture methods, more accurately mimicking peri-implant osteogenesis in vivo. Full article

Crown gall disease in plants is caused by “Agrobacteria”, bacteria belonging to the Rhizobiaceae family, which carry a tumor-inducing (Ti) plasmid. Unexpectedly, we found evidence that a natural isolate from a rose crown gall, called NBC51/LBA8980, was a bacterium that did not belong to the Rhizobiaceae family. Whole-genome sequencing revealed that this bacterium contained three large DNA circles with rRNA and tRNA genes, representing one chromosome and two chromids, respectively, and two megaplasmids, including a Ti plasmid. Average nucleotide identity (ANIb, ANIm) and genome-to-genome distance (GGDC) values above the thresholds of 96% and 70%, respectively, showed that NBC51/LBA8980 belonged to the species Brucella (Ochrobactrum) pseudogrignonense. Its Ti plasmid was almost identical to certain succinamopine Ti plasmids previously identified in Agrobacterium strains, suggesting that this Ti plasmid may have been recently acquired by NBC51/LBA8980 in the tumor environment. Full article

Background: Yellow fever (YF) is an acute hemorrhagic disease endemic to Africa and Latin America; however, no cases have been reported in Asian regions with high Aedes aegypti infestation. Factors such as environmental conditions and genetic variations in the yellow fever virus (YFV) strains and mosquito populations may explain this absence. Mosquito populations have undergone strong selective pressure owing to the excessive use of insecticides. This pressure has led to the spread of alterations, such as knockdown-resistant mutations (kdr), which, while conferring resistance to pyrethroids, also induce various physiological side effects in the insect. Therefore, it is important to investigate whether the presence of kdr mutations influences the infectivity of YFV mosquitoes. This study evaluated the susceptibility of Ae. aegypti from Pakistan with distinct kdr genotypes to different YFV strains under laboratory conditions. Methods: Ae. aegypti from a Pakistani colony were exposed to YFV strains (PR4408/2008 and ES504/2017) along with the Rockefeller strain. After 14 days, RNA and DNA were extracted for viral RNA detection (qPCR) and kdr genotyping (TaqMan qPCR and HRM for T1520I and F1534C SNPs). Results: Pakistani Ae. aegypti were orally susceptible to YFV, with infection rates of 83.7% (PR4408/2008) and 61.3% (ES504), respectively, similar to Rockefeller. Two kdr genotypes (II + CC and TI + FC) were identified, with no significant differences in viral infection or dissemination rates. Conclusions: The Ae. aegypti population from Asia is capable of YFV infection and dissemination, regardless of kdr genotype. Full article