Background: Significant alterations in feeding, housing, and physiology are observed in dairy cows during the transition period (3 weeks pre- and post-calving), in addition to changes in the composition and abundance of the endometrial microbiota. Thus, this study aimed to evaluate any changes in the composition and predicted metabolic pathways in the cow uterine microbiome during this transition period. Methods: Scrapings were sampled from the endometrial surface of clinically healthy cows (n = 3) in dynamics as follows: in the 10 Days period before, and on Days 3, 5, and 20 after calving. Total DNA was isolated from the samples, and the composition of the microbial community was assessed using targeted next-generation sequencing (NGS) technology. Based on the subsequent NGS data, the dynamics of the predicted metabolic pathways of the microbiota were evaluated. Results: Seven superphyla and phyla of microorganisms were found in the endometrial microbiota of cows during the transition period. Among these, the phylum Firmicutes (with a dominant class of Clostridia) and the superphylum Fusobacteriota (represented by a single class of Fusobacteriia) can be considered the dominant bacteria in the endometrium, with representation noted from 25.2 to 68.2% and from 12.3 to 51.1%, respectively. The microbiome composition underwent significant changes (p < 0.05) during the transition period. In particular, the high abundance of the Fusobacteriaceae family (up to 68.2%) in the uterus of clinically healthy cows was unexpected, given the potential association of Fusobacteriaceae with the occurrence of metritis in cows. The numbers of microorganisms in two dominant classes, Fusobacteriia and Clostridia, showed generally opposite changes in their relative abundance during the transition period. The predicted functional potential level for 32 pathways in the endometrium changed (p < 0.05) in cows during the transition period. Indeed, the activity of the predicted pathways, such as pyridoxal 5′-phosphate biosynthesis I and teichoic acid (poly-glycerol) biosynthesis, was lowered on day 3 postpartum (p < 0.05). Conclusions: Microbiota composition and the activity of the predicted metabolic pathways in the cow endometrium underwent significant changes at different critical stages in the transition period. Moreover, even clinically healthy cows exhibited signs of dysbiotic disorders.
Background: Transient transformation is a convenient and less time-consuming method for investigating gene functions compared with production of stable transformants. Most systems for Agrobacterium-mediated plant transformation, regardless of transient or stable transformation, utilize a combination of Agrobacterium strains carrying a helper Ti-plasmid and a binary vector. However, the helper Ti-plasmids which are mega-plasmids with sizes of 200–250 kbp and are difficult to manipulate directly with conventional molecular cloning techniques due to their large sizes. Methods: A small helper Ti-plasmid, pCU307D with a size of 46 kbp was constructed from pTiEHA101 which is commonly used for plant genetic engineering. They consist of the virulence (vir) region, the mutated replication origin from pTiEHA101, the gentamicin resistance marker and the replication origin for E. coli cells. The abilities of T-DNA transfer were examined by transient expression in Arabidopsis cultured cells. Results: Efficiencies to transfer T-DNA to plant cells by EHA101 and C58C1 carrying pCU307D were similar, although pCU307D had 2.8-fold higher copy numbers than EHA101. The construction processes of pCU307D eliminated the A281virF gene encoding F-box-like protein which was located outside of the vir region. The A281virF gene was cloned into a binary vector and it was introduced into C58C1 cells with pCU307D which was named as CU307DF. T-DNA transfer efficiencies by EHA101 or CU307DF were examined by GUS activities from transiently transformed T-DNA with the CaMV35S::NLS-GUS cassette in Arabidopsis cells. Co-culture with CU307DF carrying the GUS gene conferred 3.2-fold higher GUS activities, compared to that with its parental strain EHA101. Conclusion: A new Agrobacterium strain CU307DF has higher capacity for T-DNA transfer, compared with its parental strain EHA101. pCU307D is as small as 46 kbp and can propagate in E. coli, and that may enable to add further modification to it for further improvement of T-DNA transfer by CU307DF.
Background: Adeno-associated viruses (AAVs) are established vectors for efficient gene delivery to the central nervous system (CNS). Increasingly, strategies aim to restrict transduction to specific neuronal subtypes defined by the associated functional properties, thereby enhancing precision and therapeutic potential. Methods: Recombinant AAV9 vectors carrying fluorescent reporters under the control of cytomegalovirus (CMV), human synapsin (hSyn), or homeobox 9 (Hb9) promoters were delivered intrathecally in Wistar rats. Transgene expression was evaluated 7 days post-injection by confocal microscopy. Neurons in laminae VII–X were quantified across cervical, thoracic, lumbar, and sacral spinal cord levels. Statistical analysis was performed using the Kruskal–Wallis test followed by Mann–Whitney U tests with Bonferroni correction. Results: In lamina VII, consistent neuronal expression was mediated by hSyn across all spinal levels, with significantly higher transduction at cervical compared to thoracic and lumbar regions (p < 0.01). CMV and Hb9 showed no detectable tropism for this lamina. In lamina VIII, CMV drove markedly higher expression than hSyn and Hb9, with a 2.8-fold difference at the lumbar level (p < 0.001). In lamina X, CMV expression exceeded hSyn at the lumbar and sacral levels (p < 0.05), while Hb9 showed no activity. In lamina IX, all promoters mediated motoneuron transduction, but only Hb9 restricted expression specifically to motoneuron somata. Notably, CMV induced off-target expression in glial cells. Conclusions: AAV9-mediated expression patterns in the spinal cord are strongly shaped by promoter choice and segmental level. Hb9 provides high motoneuron specificity, hSyn supports broad neuronal activation across laminae VII–X, whereas CMV drives robust but non-specific expression with significant off-target activity. These findings highlight the importance of rational promoter selection for spinal cord gene therapy and strategies aimed at functional recovery in motor system disorders.
Excessive fructose consumption has emerged as a critical driver of obesity and metabolic dysfunction, with far-reaching implications for multiple organ systems. This review synthesizes current evidence on the biochemical and molecular pathways underlying fructose induced disease mechanisms, discussing how fructose metabolism activates the “survival switch”, promotes fat storage, and generates uric acid, mitochondrial dysfunction, and oxidative stress, thereby disrupting energy homeostasis. Key organ-specific consequences are explored, including hepatic steatosis and progression to non-alcoholic fatty liver disease, pancreatic β-cell dysfunction, renal fibrosis, intestinal barrier disruption with microbial dysbiosis, cardiometabolic impairment, pulmonary inflammation, and neurocognitive decline with relevance to Alzheimer’s disease. Moreover, mechanistic insights highlight the role of fructokinase C activation, adenosine triphosphate (ATP) depletion, leptin resistance, pro-inflammatory signaling (mechanistic target of rapamycin complex-1 (mTORC1), renin angiotensin system (RAS), Toll-like receptor 4 (TLR4)), and cross-talk between fructose metabolism and organ-specific pathophysiology. Animal and human studies consistently reinforce the central role of fructose overload in driving obesity and associated complications. Meanwhile, this review frames fructose not merely as a caloric contributor but as a metabolic disruptor, thereby underscoring the urgent need for public health interventions, dietary regulation, and mechanistic research to mitigate fructose-driven metabolic disease.
Background: Nitrosylcobalamin (NO-Cbl) is a vitamin B12 analog designed to exploit the “Trojan horse” vulnerability created by the heightened need of cancer cells for cobalamin and one-carbon metabolism. Building on our recent biophysical studies confirming the affinity of NO-Cbl for intrinsic factor, this work aimed to investigate the mechanistic basis for the selective anticancer activity of NO-Cbl through the cobalamin transport axis and lysosomal processing. Materials and Methods: Human cancer cell lines (NIH-OVCAR-3, MCF-7, WM9, and DU145) were cultured and transfected to overexpress transcobalamin II (TCII). Cell proliferation and cytotoxicity were measured using the sulforhodamine B (SRB) assay. TCII-R (CD320) expression was quantified by flow cytometry. The impact of anti-CD320 antiserum and lysosomal alkalization (chloroquine) on NO-Cbl activity was assessed. Results: Antiserum inhibition of the TCII receptor resulted in dose-dependent inhibition of NIH-OVCAR-3 and MCF-7 cell proliferation. Lysosomal alkalinization by chloroquine pretreatment abrogated NO-Cbl-induced cytotoxicity in OVCAR-3 cells. Flow cytometric analysis demonstrated an inverse correlation between TCII-R (CD320) expression (MFI ratio) and NO-Cbl ID50. TCII overexpression significantly reduced NO-Cbl ID50 in NIH-OVCAR-3 cells. Conclusion: NO-Cbl utilizes tumor cell cobalamin transport and processing pathways to deliver nitric oxide selectively to cancer cells. These results, integrated with recent binding studies, validate NO-Cbl as a cobalamin-based targeted anticancer agent with efficacy in tumors expressing high levels of TCII and CD320.
