Evaluation of canine adipose-derived mesenchymal stem cells for neurological functional recovery in a rat model of traumatic brain injury.

BACKGROUND: Traumatic brain injury (TBI) is a common condition in veterinary medicine that is difficult to manage.Veterinary regenerative therapy based on adipose mesenchymal stem cells seem to be an effective strategy for the treatment of traumatic brain injury. In this study, we evaluated therapeutic efficacy of canine Adipose-derived mesenchymal stem cells (AD-MSCs)in a rat TBI model, in terms of improved nerve function and anti-neuroinflammation. RESULTS: Canine AD-MSCs promoted neural functional recovery, reduced neuronal apoptosis, and inhibited the activation of microglia and astrocytes in TBI rats. According to the results in vivo, we further investigated the regulatory mechanism of AD-MSCs on activated microglia by co-culture in vitro. Finally, we found that canine AD-MSCs promoted their polarization to the M2 phenotype, and inhibited their polarization to the M1 phenotype. What's more, AD-MSCs could reduce the migration, proliferation and Inflammatory cytokines of activated microglia, which is able to inhibit inflammation in the central system. CONCLUSIONS: Collectively, the present study demonstrates that transplantation of canine AD-MSCs can promote functional recovery in TBI rats via inhibition of neuronal apoptosis, glial cell activation and central system inflammation, thus providing a theoretical basis for canine AD-MSCs therapy for TBI in veterinary clinic.

Organic Fluorophores with Large Stokes Shift for the Visualization of Rapid Protein and Nucleic Acid Assays.

Organic small-molecule fluorophores, characterized by flexible chemical structure and adjustable optical performance, have shown tremendous potential in biosensing. However, classical organic fluorophore motifs feature large overlap between excitation and emission spectra, leading to the requirement of advanced optical set up to filter desired signal, which limits their application in scenarios with simple settings. Here, a series of wavelength-tunable small-molecule fluorescent dyes (PTs) bearing simple organic moieties have been developed, which exhibit Stokes shift up to 262 nm, molar extinction coefficients ranged 30,000-100,000 M-1 cm-1, with quantum yields up to 54.8 %. Furthermore, these dyes were formulated into fluorescent nanoparticles (PT-NPs), and applied in lateral flow assay (LFA). Consequently, limit of detection for SARS-CoV-2 nucleocapsid protein reached 20 fM with naked eye, a 100-fold improvement in sensitivity compared to the pM detection level for colloidal gold-based LFA. Besides, combined with loop-mediated isothermal amplification (LAMP), the LFA system achieved the visualization of single copy level nucleic acid detection for monkeypox (Mpox).

Influence of air pollution on human comfort in five typical Chinese cities.

It is known that air pollution is harmful to creatures, though until now most of the human thermal comfort indices that existed were calculated only with meteorological conditions. Therefore, a new index - meteorology and environment comfort (MEC) - was given out in this paper that considers both meteorology and air pollution conditions and presents the comprehensive and synergistic effects of meteorological and air pollution. The meteorology and air pollution data were used to establish the influence function of the five air pollutants (PM2.5, PM10, O3, NO2, and SO2) according to Fechner's law; then, we calculated the somatosensory temperature (ST, a class of human thermal comfort indices) and MEC values of five typical cities (Beijing, Xining, Nanjing, Kunming, and Guangzhou). The results showed average improvements of five cities on MEC as a new comprehensive human comfort index to new ST. In spring, the MEC comfort proportion fell by 29.25%. Besides, the extreme heat discomfort ratio in Nanjing and Kunming has increased over 20%. In summer, the comfort proportion fell 12.54%; the extreme heat discomfort proportion of Beijing increased 37.86% and Kunming increased 24.09%. Air pollution significantly raised discomfort stress in Beijing. In fall, the comfort proportion fell by 20.87%; and the extreme heat discomfort of Nanjing increased 23.67% caused by poor air quality. About winter, the comfort ratio decreased 12.72%, and the cold discomfort proportion of Nanjing increased 30.30%, signifying awful air quality in winter. Air pollution levels significantly affect the comfort levels in all seasons, which is more evident with good weather patterns. MEC can offer early warnings of extreme weather events and provide a basis for the better prevention and control of air pollution to protect human health basing on the predictions of meteorological and environmental impact factors.

Manufacturing and banking canine adipose-derived mesenchymal stem cells for veterinary clinical application.

BACKGROUND: Mesenchymal stem cells (MSCs) have generated a great amount of interest in recent years as a novel therapeutic application for improving the quality of pet life and helping them free from painful conditions and diseases. It has now become critical to address the challenges related to the safety and efficacy of MSCs expanded in vitro. In this study, we establish a standardized process for manufacture of canine adipose-derived MSCs (AD-MSCs), including tissue sourcing, cell isolation and culture, cryopreservation, thawing and expansion, quality control and testing, and evaluate the safety and efficacy of those cells for clinical applications. RESULTS: After expansion, the viability of AD-MSCs manufactured under our standardized process was above 90 %. Expression of surface markers and differentiation potential was consistent with ISCT standards. Sterility, mycoplasma, and endotoxin tests were consistently negative. AD-MSCs presented normal karyotype, and did not form in vivo tumors. No adverse events were noted in the case treated with intravenously AD-MSCs. CONCLUSIONS: Herein we demonstrated the establishment of a feasible bioprocess for manufacturing and banking canine AD-MSCs for veterinary clinical use.

Isolation and identification of exosomes from feline plasma, urine and adipose-derived mesenchymal stem cells.

BACKGROUND: Exosomes, internal proteins, lipids, and nucleic acids coated by phospholipid bilayer membranes, are one type of small extracellular vesicles, which can mediate cell-cell communication. In recent years, exosomes have gained considerable scientific interest due to their widely applied prospect in the diagnosis and therapeutics of human and animal diseases. In this study, we describe for the first time a feasible method designed to isolate and characterize exosomes from feline plasma, urine and adipose-derived mesenchymal stem cells. RESULTS: Exosomes from feline plasma, urine and adipose-derived mesenchymal stem cells were successfully isolated by differential centrifugation. Quantification and sizing of exosomes were assessed by transmission electron microscopy, flow nano analysis and western blotting. Detected particles showed the normal size (30-100 nm) and morphology described for exosomes, as well as presence of the transmembrane protein (TSG101, CD9, CD63, and CD81) known as exosomal marker. CONCLUSIONS: The results suggest that differential centrifugation is a feasible method for isolation of exosomes from different types of feline samples. Moreover, these exosomes can be used to further diagnosis and therapeutics in veterinary pre-clinical and clinical studies.

Beta defensin 3 enhances ovarian granulosa cell proliferation and migration via ERK1/2 pathway in vitro†.

Antimicrobial peptides (AMPs) are regarded as host defense peptides that possess bactericidal activity as well as immunomodulatory function. However, the role of AMP in the mammalian ovary is unknown. In the present study, porcine granulosa cells were utilized in a cell model to study the role of porcine beta defensin 2 (pBD2; pDEFB4B) and 3 (pBD3; pDEFB103A) during ovarian follicular development. Granulosa cells were cultured in the absence and presence of 1, 10, and 50 µg/ml of pDEFB4B or pDEFB103A. After 24 h of treatment, pDEFB103A but not pDEFB4B stimulated granulosa cell proliferation in a concentration-dependent manner (P < 0.05). This effect was dependent on the stage of follicular development. In addition, transwell cell migration assay showed that in the presence of pDEFB103A (10 µg/ml), a 2.5-fold increase in cell migration was achieved. Furthermore, further study revealed that pDEFB103A increased the mRNA levels of cyclin D1 (CCND1) and proliferating cell nuclear antigen (PCNA), both associated with cell proliferation. To study the potential pathway involved in pDEFB103A-induced cell proliferation and migration, western blots were performed. It was found that pDEFB103A significantly increased the phosphorylated-ERK1/2 to nonphosphorylated ratio. Moreover, pretreatment with the U0126, a specific ERK1/2 phosphorylation inhibitor, suppressed PDEFB103A inducing GCs ERK1/2 phosphorylation, as well as proliferation and migration, suggesting that PDEFB103A may act via activating the ERK1/2 pathway. Furthermore, using a signal transduction pathway Elk-1 trans-reporting system, the activation of ERK1/2 pathway by PDEFB103A was further confirmed. Our data suggest that AMP may play a physiological role in the mammalian ovary.

A Comparative Study of Biological Characteristics and Transcriptome Profiles of Mesenchymal Stem Cells from Different Canine Tissues.

Mesenchymal stem cells (MSCs) are the most promising seed cells for cell therapy. Comparing the biological and transcriptome gene characteristics of MSCs from different sources provides an important basis for the screening of clinically used cells. The main purpose of this experiment was to establish methods for the isolation and culture of MSCs from five different canine sources, including adipose tissue, bone marrow, umbilical cord, amniotic membrane, and placenta, and compare biological and transcriptome characteristics of MSCs, in order to provide a basis for the clinical application of canine MSCs. MSCs were isolated from Chinese pastoral dogs, and the following experiments were performed: (1) the third, sixth, and ninth generations of cells were counted, respectively, and a growth curve was plotted to calculate the MSC population doubling time; (2) the expression of CD34 and CD44 surface markers was studied by immunofluorescence; (3) the third generation of cells were used for osteogenetic and adipogenic differentiation experiments; and (4) MSC transcriptome profiles were performed using RNA sequencing. All of the five types of MSCs showed fibroblast-like adherent growth. The cell surface expressed CD44 instead of CD34; the third-generation MSCs had the highest proliferative activity. The average population doubling time of adipose mesenchymal stem cells (AD-MSCs), placenta mesenchymal stem cells (P-MSCs), bone marrow mesenchymal stem cells (BM-MSCs), umbilical cord mesenchymal stem cells (UC-MSCs), and amniotic mesenchymal stem cells (AM-MSCs) were 15.8 h, 21.2 h, 26.2 h, 35 h, and 41.9 h, respectively. All five types of MSCs could be induced to differentiate into adipocytes and osteoblasts in vitro, with lipid droplets appearing after 8 days and bone formation occurring 5 days after AD-MSC induction. However, the multilineage differentiation for the remaining of MSCs was longer compared to that of the AD-MSCs. The MSC transcriptome profiles showed that AD-MSC and BM-MSCs had the highest homology, while P-MSCs were significantly different compared to the other four types of MSCs. All the isolated MSCs had the main biological characteristics of MSCs. AD-MSCs had the shortest time for proliferation, adipogenesis, and osteogenic differentiation.

Efficient and practical synthesis of Fondaparinux.

Combining advantageous sequences of Alchemia and Sanofi methods of synthesis of Fondaparinux, a more efficient and practical synthetic strategy for the synthesis of corresponding protected pentasaccharide was developed. The protected pentasaccharide was smoothly converted into Fondaparinux in overall high yield (1%).

MiR-29b inhibits COC expansion and oocyte in vitro maturation via induction of ROS by targeting CYCS.

Cumulus-oocyte complex (COC) expansion and oocyte maturation are crucial processes for embryo development and fertility across species. Although miR-29b has been detected in porcine ovarian granulosa cells, its specific role in regulating oocyte maturation remains largely unknown. In this study, using the pig as a model, we report that over-expression of miR-29b lead to a decrease of COC expansion area and inhibits oocyte maturation (P<0.05). This suppression correlated with a decrease expression of COC-expansion-associated genes, including SHAS2, ADAMTS1, ADAMTS2, ADAMTS17 and PTX 3 in both mural granulosa cells (mGCs) and cumulus granulosa cells (cGCs). Further investigation revealed that miR-29b over-expression induces reactive oxygen species (ROS) accumulation in both mGCs and cGCs, conversely, knock-down of miR-29b reverses all these effects. Treatment with the antioxidant ß-mercaptoethanol alleviates ROS accumulation, rescues COC expansion and restores oocyte polar body formation impaired by miR-29b mimics. Computational analysis predicted CYCS, the gene encoding cytochrome C, as a potential target of miR-29b. Subsequent examination demonstrated that miR-29b downregulates CYCS at both mRNA and protein levels. Dual-luciferase reporter assays further confirmed that miR-29b interacts with the 3'-untranslated region (3'UTR) of CYCS. Over-expression of CYCS decreases ROS accumulation and promotes COC expansion (P<0.05). These results indicate that miR-29b regulates COC expansion and oocyte maturation in vitro by inducing ROS, likely through targeting of CYCS. This study sheds light on the role of miR-29b in oocyte maturation and provides insight into the regulatory function of miRNAs in ovarian physiology.

Enhancement of the solubility and oral bioavailability of altrenogest through complexation with hydroxypropyl-ß-cyclodextrin.

Altrenogest (ALT), a synthetic progestogen, serves a critical role in estrus synchronization among animals like gilts and mares. However, its practical application in animal husbandry is hampered due to its poor solubility and limited oral bioavailability. To address this challenge, a solvent evaporation method was employed to create an inclusion complex of ALT with hydroxypropyl-ß-cyclodextrin (ALT/HP-ß-CD). The formation of this inclusion complex was confirmed by scanning electron microscopy, power X-ray diffraction, differential scanning calorimetry, Fourier transform infrared spectroscopy, nuclear magnetic resonance spectroscopy, and docking calculations. In addition, we further conducted pharmacokinetic investigation involving gilts, comparing ALT/HP-ß-CD inclusion complex to an ALT oral solution. The physicochemical characterization results unveiled a transformation of ALT's crystal morphology into an amorphous state, with ALT effectively entering the cavity of HP-ß-CD. Compared with ALT, the solubility of ALT/HP-ß-CD inclusion complex increased by 1026.51-fold, and its dissolution rate demonstrated significant improvement. Pharmacokinetic assessments further revealed that the oral bioavailability of ALT/HP-ß-CD inclusion complex surpassed that of the ALT oral solution, with a relative bioavailability of 114.08 %. In conclusion, complexation with HP-ß-CD represents a highly effective approach to improve both the solubility and oral bioavailability of ALT.

Ultrastructural characteristics and morphological relationships of cardiomyocytes and telocytes in the myocardium of the bullfrog (Rana catesbeiana).

Telocytes (TCs) are distinctive interstitial cells due to their characteristic structures and heterogeneity. They are suggested to participate in tissue repair/regeneration. TCs have been identified in many organs of various mammals. However, data on TCs in lower animals are still very limited. In this work, TCs were identified in the myocardium of the bullfrog (Rana catesbeiana) by light and transmission electron microscopy (TEM). The structural relationships between TCs and neighbouring cell types were measured using the ImageJ (FiJi) morphometric software. TCs with slender Tps (telepodes) were located around cardiomyocytes (CMC). TEM revealed TCs with long Tps in the stroma between CMC. The homocellular tight junctions were observed between the Tps. The Tps were also very close to the neighbouring CMC. The distance between Tps and CMC was 0.15 ± 0.08 µm. Notably, Tps were observed to adhere to the periphery of the satellite cells. The Tps and the satellite cells established heterocellular structural connections by tight junctions. Additionally, Tps were frequently observed in close proximity to mast cells (MCs). The distance between the Tps and the MCs was 0.19 ± 0.09 µm. These results confirmed that TCs are present in the myocardium of the bullfrog, and that TCs established structural relationships with neighbouring cell types, including satellite cells and MCs. These findings provide the anatomical evidence to support the note that TCs are involved in tissue regeneration.

Choline supplementation regulates gut microbiome diversity, gut epithelial activity, and the cytokine gene expression in gilts.

Choline is an essential nutrient that is necessary for both fetal development and maintenance of neural function, while its effect on female ovarian development is largely unexplored. Our previous study demonstrated that choline supplementation promotes ovarian follicular development and ovulation, although its underlying mechanism was unclear. To uncover the potential regulation pathway, eighteen female Yorkshire × Landrace gilts were fed with either standard commercial diet (Control group, n = 9) or choline supplemented diet (Choline group, additional 500 mg/kg of control diet, n = 9) from day 90 of age to day 186. At day 186, feces samples were analyzed for effects on the gut microbiome using 16S ribosomal RNA gene V3-V4 region sequencing with Illumina MiSeq, serum samples were analyzed for trimethylamine (TMA) and trimethylamine-N-oxide (TMAO) using HILIC method, and jejunum tissues were analyzed for immune related gene expression using qRT-PCR. Our results show that choline supplementation did not alter the circulating level of TMA and TMAO (P > 0.05), but rather increased gut microbiome alpha diversity (P < 0.05). Beta diversity analysis results showed that the choline diet mainly increased the abundance of Firmicutes, Proteobacteria, and Actinobacteria, but decreased the abundance of Bacteroidetes, Spirochaetes, and Euryarchaeota at the phyla level. Meta-genomic analysis revealed that choline supplementation activated pathways in the gut microbiota associated with steroid hormone biosynthesis and degradation of infertility-causing environmental pollutants (bisphenol, xylene, and dioxins). To further verify the effect of choline on intestinal activity, a porcine intestine cell line (IPEC-J2) was treated with serial concentrations of choline chloride in vitro. Our data demonstrated that choline promoted the proliferation of IPEC-J2 while inhibiting the apoptotic activity. qRT-PCR results showed that choline significantly increased the expression level of Bcl2 in both IPEC-J2 cells and jejunum tissues. The expression of IL-22, a cytokine that has been shown to impact ovarian function, was increased by choline treatment in vitro. Our findings reveal the beneficial effect of choline supplementation on enhancing the gut microbiome composition and intestinal epithelial activity, and offer insights into how these changes may have contributed to the ovarian development-promoting effect we reported in our previous study.

Novel Evidence Showing the Possible Effect of Environmental Variables on COVID-19 Spread.

Coronavirus disease (COVID-19) remains a serious issue, and the role played by meteorological indicators in the process of virus spread has been a topic of academic discussion. Previous studies reached different conclusions due to inconsistent methods, disparate meteorological indicators, and specific time periods or regions. This manuscript is based on seven daily meteorological indicators in the NCEP reanalysis data set and COVID-19 data repository of Johns Hopkins University from 22 January 2020 to 1 June 2021. Results showed that worldwide average temperature and precipitable water (PW) had the strongest correlation (ρ > 0.9, p < 0.001) with the confirmed COVID-19 cases per day from 22 January to 31 August 2020. From 22 January to 31 August 2020, positive correlations were observed between the temperature/PW and confirmed COVID-19 cases/deaths in the northern hemisphere, whereas negative correlations were recorded in the southern hemisphere. From 1 September to 31 December 2020, the opposite results were observed. Correlations were weak throughout the near full year, and weak negative correlations were detected worldwide (|ρ| < 0.4, p ≤ 0.05); the lag time had no obvious effect. As the latitude increased, the temperature and PW of the maximum confirmed COVID-19 cases/deaths per day generally showed a decreasing trend; the 2020-year fitting functions of the response latitude pattern were verified by the 2021 data. Meteorological indicators, although not a decisive factor, may influence the virus spread by affecting the virus survival rates and enthusiasm of human activities. The temperature or PW threshold suitable for the spread of COVID-19 may increase as the latitude decreases.

Human umbilical cord mesenchymal stem cell-derived exosomes promote neurological function recovery in rat after traumatic brain injury by inhibiting the activation of microglia and astrocyte.

Traumatic brain injury (TBI) is a serious neurological disorder with increasing worldwide incidence. Emerging evidence has shown a significant therapeutic role of mesenchymal stem cells (MSCs) derived exosomes on traumatic brain injury with broad application prospects as a cell-free therapy. However, a comprehensive understanding of its underlying mechanism remained elusive. In this study, umbilical cord mesenchymal stem cells (UCMSCs)-derived exosomes (UC-MSCs-Exo) were isolated by ultracentrifugation and injected intraventricularly in a rat model of TBI. Our results showed that UC-MSCs-Exo promoted functional recovery and reduced neuronal apoptosis in TBI rats. Moreover, UC-MSCs-Exo inhibited the activation of microglia and astrocytes during brain injury, thereby promoting functional recovery. However, the effect of UC-MSCs-Exo on the content of plasma inflammatory factors in rats was not significant. Collectively our study suggested that UC-MSCs-Exo promotes the recovery of neurological function in TBI rats by inhibiting the activation of microglia and astrocytes, providing a theoretical basis for new therapeutic strategies for central nervous system diseases.

Protegrin-1 inhibits porcine ovarian granulosa cell apoptosis from H2O2-induced oxidative stress via the PERK/eIF2α/CHOP signaling pathway in vitro.

In mammals, oxidative stress-induced apoptosis of granulosa cells is one of the major causes of follicular atresia, affecting ovarian physiological function. Protegrin-1 (PG-1) is an antimicrobial peptide with effective antimicrobial activity, immunomodulatory function, and porcine growth-promoting effects. PG-1 has been detected in porcine ovaries follicles. This study aimed to investigate the effect of PG-1 on oxidative stress-induced apoptosis of porcine ovarian granulosa cells and the underlying molecular mechanism. Granulosa cells were obtained from porcine follicles and treated with H2O2 to establish the oxidative stress model, and then treated with or without PG-1 (10 µg/mL). PG-1 significantly suppressed H2O2-induced apoptosis in granulosa cells after 24 h of treatment. Furthermore, these results revealed that PG-1 increased the mRNA and protein expression of anti-apoptotic B cell lymphoma/leukemia 2 (BCL2) and the BCL2/Bcl-2-associated X protein (BAX) ratio while decreasing the expression of pro-apoptotic BAX and active caspase-3. Using Western blot analysis, it was found that PG-1 decreased the phosphorylation of RNA-like endoplasmic reticulum kinase (PERK) and the α-subunit of eukaryotic initiation factor 2 (eIF2α) as well as the protein expression level of CCAAT enhancer-binding protein homologous protein (CHOP), all of which were increased by H2O2. Moreover, inhibitors against PERK and phospho-eIF2ɑ both suppressed the H2O2-induced granulosa cells apoptosis and enhanced the anti-apoptosis effect of PG-1. Taken together, our findings demonstrated that PG-1 inhibited porcine ovarian granulosa cell apoptosis from oxidative stress via the PERK/eIF2α/CHOP signaling pathway in vitro, which suggests the novel regulatory function of the antimicrobial peptide in the ovary.

Umbilical cord mesenchymal stem cells promote the repair of trochlear groove reconstruction in dogs.

Trochlear groove reconstruction (TGR) is a common treatment for patellar luxation (PL) in dogs. Nevertheless, the prognosis of TGR is poor due to the cartilage damage and secondary inflammation. To study the repair effect of canine umbilical cord mesenchymal stem cells (UC-MSCs) after TGR, 10 experimental dogs were given TGR surgery and then randomized into two groups: Treatment group (1 ml suspension allogeneic UC-MSCs (106 cells/kg) was injected into the cavum articulare on days 0, 7, and 14 after TGR); and the Model group (injected with 1 ml of physiological saline as negative control). The therapeutic effect of UC-MSCs was studied by blood routine examination, inflammatory factor index detection, double-blind knee score, histopathology, and computed tomography (CT) scans. The results showed that the total number of white blood cells and neutrophils in the model group were significantly higher than those in the treatment group on both 7 days and 21 days, postoperatively (P < 0.05); there were no significant changes in the levels of IL-6, MMP-13, and TGF-ß1 between the model group and the treatment group throughout the days of testing. The double-blind knee scores of the treatment group were significantly lower than the model group on 1st, 4th, and 5th days postoperatively (P < 0.05). The treatment group showed low-pain sensation, stable gait, and fast recovery of muscle strength in the knee score, and the wound healing of the treatment group returned to normal on the 5th day after surgery; CT scans and gross observation showed that the cartilage growth in the treatment group was faster than that in the model group. Histological observation of cases showed that fibro chondrocytes were predominantly found in the treatment group, and the distribution of chondrocytes was uneven, while the model group showed a large number of fibrous tissue hyperplasia, fissures, and unequal matrix staining. Intra-articular injection of UC-MSCs after TGR has the effect of relieving pain and promoting the repair of bone defects, making the operative limb recover function earlier, making up for the deficiency of TGR, and improving the effect of PL treatment. Future studies should furthermore explore the dose and frequency of therapy based on the multiple advantages of UC-MSCs and the mechanism of cartilage repair in dogs.

Choline supplementation influences ovarian follicular development.

BACKGROUND: Female infertility is a health issue for both humans and animals and despite developments in medical interventions, there are still some conditions that cannot be treated successfully. It is important to explore other potential therapies or remedies that could improve reproductive health. Choline is an over-the-counter supplement and essential nutrient that has many health benefits. It has been suggested to be beneficial in various aspects of fertility, including fetal development and endocrine disorders like polycystic ovarian syndrome (PCOS). However, choline's impact on ovarian function has not been explored. METHODS: To study the effects of choline on ovarian development, 36 female Yorkshire × Landrace pigs were fed the following four supplemented diets between 90 and 186 days of age: (1) Control (corn and soybean meal-based diet that met estimated nutrient requirements, n = 9); (2) Choline (additional 500 mg choline per 1 kg of control diet, n = 8); (3) Omega-3 (additional 5556 mg Omega-3 per 1 kg control diet by introducing fish oil); (4) Choline + Omega-3 (500 mg choline + 5556 mg Omega-3 per 1 kg control diet). Pigs fed the choline-supplemented diet were compared to the control group and those fed diets supplemented with Omega-3 as fertility-promoting agent. RESULTS: It was found that the number of corpus luteum per ovary in the Choline (16.25 ± 2.88), Omega-3 (10.78 ± 1.71) and Choline + Omega-3 (14.89 ± 2.97) groups were all higher in comparison to that of the control group (5.56 ± 1.72, p < 0.05). The percentage of antral follicles in the Choline + Omega-3 group were higher compared to the control group (p < 0.05). To elucidate the potential molecular mechanism of choline on these improved ovarian phenotypes, the expression of a group of genes that are involved in ovarian development, including cytochrome P450 family 11 subfamily A member 1 (CYP11A1), follicle stimulating hormone receptor (FHSR) and luteinizing hormone receptor (LHR), was analyzed using RT-qPCR. The expression of both LHR and CYP11A1 was significantly upregulated in the choline-supplemented group (p < 0.05), while there are no differences in FSHR expression among all the groups. Additionally, the expression of miR-21, -378, -574, previously found to be important in ovarian function, were examined. Our data showed that miR-574 was upregulated in the Choline group while miR-378 was upregulated in the Choline + Omega-3 group in comparison to the control group (p < 0.05). Further, serum metabolite analysis showed that 1-(5Z, 8Z, 11Z, 14Z, 17Z-eicosapentaenoyl)-sn-glycero-3-phosphocholine, a form of phosphatidylcholine metabolite, was significantly increased in all the treatment groups (p < 0.05), while testosterone was significantly increased in both Omega-3 and Choline + Omega-3 groups (p < 0.05) and tended to be reduced in the choline-supplemented group (p = 0.08) compared to the control group. CONCLUSIONS: Our study demonstrated choline's influence on ovarian function in vivo, and offered insights into the mechanisms behind its positive effect on ovarian development phenotype.

Design and Synthesis of Lactose, Galactose and Cholic Acid Related Dual Conjugated Chitosan Derivatives as Potential Anti Liver Cancer Drug Carriers.

Cholic acid and galactose or lactose dual conjugated chitosan derivatives were designed and synthesized as potential anti liver cancer drug carriers, their structures were characterized through proton NMR spectra, elemental analysis, size distribution, zeta potential, and scanning electron microscope image studies. The ability of the dual conjugates to enhance the aqueous solubility of the cancer drug sorafenib was evaluated. The entrapment efficiency (EE%) and drug content (DC%) of sorafenib in the inclusion complexes were measured. The chitosan dual conjugate with cholic acid and galactose was found to be best in enhancing the aqueous solubility of sorafenib. The solubility of sorafenib in water has increased from 1.7 µg/mL to 1900 µg/mL which is equal to 1117-fold increase in its solubility due to the inclusion complex with chitosan conjugate.

Formation of inclusion complex of enrofloxacin with 2-hydroxypropyl-ß-cyclodextrin.

Enrofloxacin, a third-generation fluoroquinolone, is a broad-spectrum antimicrobial drug against a lot of veterinary bacterial diseases. However, bactericidal activity of enrofloxacin is concentration-dependent and its poor aqueous solubility and bitter taste limit its development and application. Meanwhile, 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD), a widely used cyclodextrin analog, is a safe and an effective drug carrier. It forms inclusion complexes with its drug substrates and improves their physiochemical and pharmacokinetic properties. Enrofloxacin was also found to form a stable inclusion complex with HP-ß-CD and different research groups have shown improved solubility for enrofloxacin by 32.5%, 9.25 and 165-fold. Our own efforts in this direction resulted in manifold improvement (916-fold) in its solubility compared to the previous studies. It was further shown that pharmaceutical properties, absorption and bioavailability, of enrofloxacin have also been significantly improved by complexation with HP-ß-CD.

Establishment of A Reversibly Inducible Porcine Granulosa Cell Line.

Granulosa cells (GCs) are the key components of ovarian follicles for regulating oocyte maturation. Previous established GC lines have allowed prolonged proliferation, but lost some physiological features owing to long-term immortalization. This study was to establish an induced immortal porcine GC line with reversible proliferation status by the tetracycline inducible (Tet-on) 3G system. Our conditional immortal porcine GCs (CIPGCs) line steadily propagated for at least six months and displayed primary GC morphology when cultured in the presence of 50 ng/mL doxycycline [Dox (+)]. Upon Dox withdrawal [Dox (-)], Large T-antigen expression, reflected by mCherry fluorescence, gradually became undetectable within 48 h, accompanied by less proliferation and size increase. The levels of estradiol and progesterone, and the expression of genes associated with steroid production, such as CYP11A1 (cytochrome P450 family 11), 3ß-HSD (3ß-hydroxysteroid dehydrogenase), StAR (steroidogenic acute regulatory protein), and CYP19A1 (cytochrome P450 family 19 subfamily a member 1), were all significantly higher in the Dox (-) group than Dox (+) group. The CIPGCs could switch into a proliferative state upon Dox induction. Interestingly, the expression of StAR and CYP19A1 in the CIPGCs (-Dox) was significantly increased by adding porcine follicular fluid (PFF) to mimic an ovary follicle environment. Moreover, PFF priming the CIPGCs in Dox (-) group resulted in similar estradiol production as that of primary GC, and enabled this cell line to respond to gonadotrophins in estradiol production. Collectively, we have established an inducible immortal porcine GC line, which offers a unique and valuable model for future research on the regulation of ovarian functions.