Evaluation of semen DNA integrity and related parameters with COVID-19 infection: a prospective cohort study.

BACKGROUND: In the context of Corona Virus Disease 2019 (COVID-19) global pandemic, Its impact on male reproductive function should be concerned. METHODS: Our study is a prospective cohort study that recruited participants infected or uninfected with COVID-19 between December 2022 and March 2023. All laboratory tests and questionnaire data were completed at the First Affiliated Hospital of Nanchang University. A total of 132 participants were enrolled, with 78 COVID-19 positive patients as the positive group and 54 COVID-19 negative participants as the negative group. Semen quality was assessed by the fifth World Health Organization criteria. The general characteristics of semen samples were assessed using CASA (computer-assisted sperm analysis). DNA damage and the high density stainability was assessed by sperm chromatin structure analysis (SCSA) based on flowcytometry. RESULTS: The sperm concentration, progressive motility and motility in COVID-19 negative group were significantly higher than positive group. In the following DNA damage analysis, a remarkably lower sperm DNA fragmentation index (DFI) in the COVID-19 negative group. In the positive group, unhealthy lifestyles had no significant effect on semen parameters, DNA fragmentation and nuclear compaction. CONCLUSIONS: After excluding the interference of unhealthy lifestyle, the COVID-19 infection can have a significant impact on the quality of semen, especially the DFI,. Therefore, it shows that COVID-19 can adversely affects male fertility, and this result provides advisory guidance for clinicians.

Clinical and molecular features of two diabetes families carrying mitochondrial ND1 T3394C mutation.

BACKGROUND: Mutations in mitochondrial DNA (mtDNA) are found to be associated with type 2 diabetes mellitus (T2DM). However, the molecular pathogenesis of these mutations in T2DM is still poorly understood. METHODS: In this study, we report here the molecular features of two Han Chinese families with maternally transmitted T2DM. The matrilineal relatives are undergoing clinical, biochemical, genetic evaluations, and molecular analysis. Furthermore, the entire mitochondrial genomes of these matrilineal relatives are screened by PCR-Sanger sequencing. RESULTS: The age at onset of T2DM of these participants varies from 28 to 71 years, with an average of 43 years. Molecular analysis of mitochondrial genomes identifies the existence of ND1 T3394C mutation in both families, together with sets of variants belonging to mitochondrial haplogroup Y2 and M9a. The m.T3394C mutation is localized at very conserved tyrosine at position 30 of ND1, may result the failure in ND1 mRNA metabolism, and lead to mitochondrial dysfunction. Moreover, sequence analysis of matrilineal relatives in Family 1 identifies the m.A14693G mutation which occurs in the TΨC-loop of tRNAGlu (position 54), and is critical to the structural formation and stabilization of this tRNA. Thus, m.A14693G mutation may cause the impairment in tRNA metabolism, thereby worsens the mitochondrial dysfunction caused by ND1 T3394C mutation. However, no functional mtDNA variants are identified in Family 2 which suggest that mitochondrial haplogroup may not play an important role in diabetes expression. CONCLUSIONS: Our study indicates that mitochondrial ND1 T3394C mutation is involved in the pathogenesis of maternally inherited T2DM in these families.

Structure-Based Design and Preclinical Characterization of Selective and Orally Bioavailable Factor XIa Inhibitors: Demonstrating the Power of an Integrated S1 Protease Family Approach.

The serine protease factor XI (FXI) is a prominent drug target as it holds promise to deliver efficacious anticoagulation without an enhanced risk of major bleeds. Several efforts have been described targeting the active form of the enzyme, FXIa. Herein, we disclose our efforts to identify potent, selective, and orally bioavailable inhibitors of FXIa. Compound 1, identified from a diverse library of internal serine protease inhibitors, was originally designed as a complement factor D inhibitor and exhibited submicromolar FXIa activity and an encouraging absorption, distribution, metabolism, and excretion (ADME) profile while being devoid of a peptidomimetic architecture. Optimization of interactions in the S1, S1ß, and S1' pockets of FXIa through a combination of structure-based drug design and traditional medicinal chemistry led to the discovery of compound 23 with subnanomolar potency on FXIa, enhanced selectivity over other coagulation proteases, and a preclinical pharmacokinetics (PK) profile consistent with bid dosing in patients.

MAA868, a novel FXI antibody with a unique binding mode, shows durable effects on markers of anticoagulation in humans.

A large unmet medical need exists for safer antithrombotic drugs because all currently approved anticoagulant agents interfere with hemostasis, leading to an increased risk of bleeding. Genetic and pharmacologic evidence in humans and animals suggests that reducing factor XI (FXI) levels has the potential to effectively prevent and treat thrombosis with a minimal risk of bleeding. We generated a fully human antibody (MAA868) that binds the catalytic domain of both FXI (zymogen) and activated FXI. Our structural studies show that MAA868 traps FXI and activated FXI in an inactive, zymogen-like conformation, explaining its equally high binding affinity for both forms of the enzyme. This binding mode allows the enzyme to be neutralized before entering the coagulation process, revealing a particularly attractive anticoagulant profile of the antibody. MAA868 exhibited favorable anticoagulant activity in mice with a dose-dependent protection from carotid occlusion in a ferric chloride-induced thrombosis model. MAA868 also caused robust and sustained anticoagulant activity in cynomolgus monkeys as assessed by activated partial thromboplastin time without any evidence of bleeding. Based on these preclinical findings, we conducted a first-in-human study in healthy subjects and showed that single subcutaneous doses of MAA868 were safe and well tolerated. MAA868 resulted in dose- and time-dependent robust and sustained prolongation of activated partial thromboplastin time and FXI suppression for up to 4 weeks or longer, supporting further clinical investigation as a potential once-monthly subcutaneous anticoagulant therapy.

Effect of AXL on the epithelial-to-mesenchymal transition in non-small cell lung cancer.

Non-small-cell lung cancer (NSCLC) is the leading cause of cancer-associated mortality in the United States. AXL, which is a member of the receptor tyrosine kinases, has been established as a strong candidate for the targeted therapy of cancer. Therefore, the present study aimed to investigate the role of AXL in NSCLC; in particular the molecular mechanisms underlying the involvement of AXL in the epithelial-to-mesenchymal transition (EMT). Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis demonstrated that AXL, EMT-inducing Twist and the mesenchymal marker N-cadherin were upregulated, and the epithelial markers E-cadherin and ß-cadherin were downregulated, in the PC9 NSCLC cell line. Furthermore, downregulation of AXL expression by RNA interference was shown to inhibit cell growth by inducing the apoptosis of PC9 cells, as demonstrated by MTT and flow cytometry analyses. Notably, inhibition of AXL attenuated the regulation of EMT-associated genes, specifically downregulating Twist and N-cadherin, and upregulating E-cadherin and ß-cadherin. Conversely, downregulation of Twist did not affect the expression levels of AXL. These results suggested that AXL may inhibit the EMT by the regulation of EMT-associated genes in the PC9 cell line. The results of the present study indicated that AXL may have a role in the regulation of EMT and the cell cycle of the PC9 cells; thus suggesting that AXL may have clinical significance in the design of therapeutic strategies targeting NSCLC and EMT signaling pathways.

Association between polymorphisms in selected inflammatory response genes and the risk of prostate cancer.

Inflammation represents an important event which facilitates prostate carcinogenesis. Genetic variations in inflammatory response genes could affect the level and function of the protein products, resulting in the differential prostate cancer risk among carriers of different variants. This study attempted to investigate the association of IL-4 rs2243250, IL-6 rs10499563, IL-8 rs4073, as well as NFKBIA rs2233406 and rs3138053 polymorphisms with prostate cancer risk in the Chinese population. Genotyping of the polymorphisms was performed by using polymerase chain reaction-restriction fragment length polymorphism technique on 439 prostate cancer patients and 524 controls, and the association of each polymorphic genotype with prostate cancer risk was evaluated by using logistic regression analysis based on allele, heterozygous, and homozygous comparison models, with adjustment to age and smoking status. We showed that the C allele of IL-4 rs2243250 polymorphism could increase prostate cancer risk (heterozygous comparison model: odds ratio [OR] =1.434, 95% confidence interval [CI] =1.092-1.881, P=0.009; homozygous comparison model: OR =2.301, 95% CI =1.402-3.775, P=0.001; allele comparison model: OR =1.509, 95% CI =1.228-1.853, P<0.001). On the other hand, the C allele of rs10499563 polymorphism could decrease prostate cancer risk (heterozygous comparison model: OR =0.694, 95% CI =0.525-0.918, P=0.010; homozygous comparison model: OR =0.499, 95% CI =0.269-0.926, P=0.028; allele comparison model: OR =0.692, 95% CI =0.553-0.867, P=0.001). No association was observed for the other polymorphisms. In conclusion, IL-4 rs2243250 and IL-6 rs10499563 polymorphisms could serve as potential predictive biomarkers for prostate cancer risk in the Chinese population.

Pioglitazone protects against renal ischemia-reperfusion injury by enhancing antioxidant capacity.

BACKGROUND: In our previous study, we showed that pioglitazone exerts protective effects on renal ischemia-reperfusion injury (IRI) in mice by abrogating renal cell apoptosis. Oxidative stress due to excessive production of reactive oxygen species and subsequent lipid peroxidation plays a critical role in renal IRI. The purpose of the current study is to demonstrate the effect of pioglitazone on renal IRI by modulation of oxidative stress. MATERIALS AND METHODS: IRI was induced by bilateral renal ischemia for 45 min followed by reperfusion. Thirty healthy male Balb/c mice were randomly assigned to one of the following groups: phosphate buffer solution (PBS) + IRI, pioglitazone + IRI, PBS + sham IRI, pioglitazone + sham IRI. Kidney function tests and kidney antioxidant activities were determined 24 h after reperfusion. RESULTS: Pretreatment with pioglitazone produced reduction in serum levels of blood urea nitrogen and creatinine caused by IRI. Pretreatment with pioglitazone before IRI resulted in a higher level of kidney enzymatic activities of superoxide dismutase, glutathione, catalase, and total antioxidant capacity than in the PBS-pretreated IRI group. CONCLUSIONS: Our results indicate that pioglitazone can provide protection for kidneys against IRI by enhancing antioxidant capacity. Therefore, pioglitazone could be a potential therapeutic approach to prevent renal IRI relevant to various clinical conditions.

Protective effects of pioglitazone on renal ischemia-reperfusion injury in mice.

BACKGROUND: Renal ischemia-reperfusion injury (IRI) is a complex pathophysiologic process involving cell apoptosis and oxidant damages that leads to acute renal failure in both native kidneys and renal allografts. Pioglitazone is a novel class of oral antidiabetic agents currently used to treat type 2 diabetes mellitus. Pioglitazone exerts protective effects on acute myocardial ischemia and acute cerebral ischemia. The aim of this study was to investigate the possible beneficial effects of pioglitazone on renal IRI in mice. METHODS: IRI was induced by bilateral renal ischemia for 45 min followed by reperfusion. Fifty-five healthy male Balb/c mice were randomly assigned to one of the following groups: PBS + IRI, pioglitazone + IRI, PBS + sham IRI, pioglitazone + sham IRI. Kidney function tests, histopathologic examination, renal cell Bcl-2, and Bax expression were determined 24 h after reperfusion. Animals' survival was examined 7 days after operation. RESULTS: Animals pretreated with pioglitazone had lower plasma levels of blood urea nitrogen and creatinine caused by IRI, lower histopathologic scores, and improved survival rates following IRI. Renal cell apoptosis induced by IRI was abrogated in kidneys of mice pretreated by pioglitazone, with an increase in Bcl-2 expression and a decrease in Bax expression. Furthermore, pioglitazone pretreatment protected against lethal renal IRI. CONCLUSIONS: Peroxisome proliferator-activated receptor activation by pioglitazone exerts protective effects on renal IRI in mice by abrogating renal cell apoptosis. Thus, pioglitazone could be a novel therapeutic tool in renal IRI.

MAPK phosphatase-3 promotes hepatic gluconeogenesis through dephosphorylation of forkhead box O1 in mice.

Insulin resistance results in dysregulated hepatic gluconeogenesis that contributes to obesity-related hyperglycemia and progression of type 2 diabetes mellitus (T2DM). Recent studies show that MAPK phosphatase-3 (MKP-3) promotes gluconeogenic gene transcription in hepatoma cells, but little is known about the physiological role of MKP-3 in vivo. Here, we have shown that expression of MKP-3 is markedly increased in the liver of diet-induced obese mice. Consistent with this, adenovirus-mediated MKP-3 overexpression in lean mice promoted gluconeogenesis and increased fasting blood glucose levels. Conversely, shRNA knockdown of MKP-3 in both lean and obese mice resulted in decreased fasting blood glucose levels. In vitro experiments identified forkhead box O1 (FOXO1) as a substrate for MKP-3. MKP-3-mediated dephosphorylation of FOXO1 at Ser256 promoted its nuclear translocation and subsequent recruitment to the promoters of key gluconeogenic genes. In addition, we showed that PPARγ coactivator-1α (PGC-1α) acted downstream of FOXO1 to mediate MKP-3-induced gluconeogenesis. These data indicate that MKP-3 is an important regulator of hepatic gluconeogenesis in vivo and suggest that inhibition of MKP-3 activity may provide new therapies for T2DM.

Exposure to lanthanum compound diminishes LPS-induced inflammation-associated gene expression: involvements of PKC and NF-kappaB signaling pathways.

Lanthanum chloride, a rare earth compound, possesses antibacterial and cellular immunity regulating properties. However, the underlying molecular mechanisms remain largely unknown. In this study, we examined the effects of lanthanum chloride on the production of nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha), the expression of inducible NO synthase (iNOS) and TNF-alpha in RAW 264.7 cells, a mouse macrophage cell line. We found that the LPS-elicited excessive production of NO and TNF-alpha in RAW 264.7 cells was inhibited significantly in the presence of lanthanum chloride, and the attenuation of iNOS and TNF-alpha occurred at mRNA level. Furthermore, the possible signaling components affected by lanthanum chloride in the pathway that lead to LPS-induced iNOS and TNF-alpha expression were explored. The results indicated the involvements of PKC/Ca(2+) and NF-kappaB in the attenuation of NO and pro-inflammatory cytokine production by lanthanum chloride. Our observations suggest a possible therapeutic application of this agent for treating inflammatory diseases.

PPAR{delta} agonism activates fatty acid oxidation via PGC-1{alpha} but does not increase mitochondrial gene expression and function.

PPARdelta (peroxisome proliferator-activated receptor delta) is a regulator of lipid metabolism and has been shown to induce fatty acid oxidation (FAO). PPARdelta transgenic and knock-out mice indicate an involvement of PPARdelta in regulating mitochondrial biogenesis and oxidative capacity; however, the precise mechanisms by which PPARdelta regulates these pathways in skeletal muscle remain unclear. In this study, we determined the effect of selective PPARdelta agonism with the synthetic ligand, GW501516, on FAO and mitochondrial gene expression in vitro and in vivo. Our results show that activation of PPARdelta by GW501516 led to a robust increase in mRNA levels of key lipid metabolism genes. Mitochondrial gene expression and function were not induced under the same conditions. Additionally, the activation of Pdk4 transcription by PPARdelta was coactivated by PGC-1alpha. PGC-1alpha, but not PGC-1beta, was essential for full activation of Cpt-1b and Pdk4 gene expression via PPARdelta agonism. Furthermore, the induction of FAO by PPARdelta agonism was completely abolished in the absence of both PGC-1alpha and PGC-1beta. Conversely, PGC-1alpha-driven FAO was independent of PPARdelta. Neither GW501516 treatment nor knockdown of PPARdelta affects PGC-1alpha-induced mitochondrial gene expression in primary myotubes. These results demonstrate that pharmacological activation of PPARdelta induces FAO via PGC-1alpha. However, PPARdelta agonism does not induce mitochondrial gene expression and function. PGC-1alpha-induced FAO and mitochondrial biogenesis appear to be independent of PPARdelta.

Transducer of regulated CREB-binding proteins (TORCs) induce PGC-1alpha transcription and mitochondrial biogenesis in muscle cells.

PGC-1alpha (peroxisome proliferator-activated receptor gamma coactivator 1alpha) is a master regulator of mitochondrial biogenesis and plays an important role in several other aspects of energy metabolism. To identify upstream regulators of PGC-1alpha gene transcription, 10,000 human full-length cDNAs were screened for induction of the PGC-1alpha promoter. A number of activators of PGC-1alpha transcription were found; the most potent activator was the transducer of regulated CREB (cAMP response element-binding protein) binding protein (TORC) 1, a coactivator of CREB. The other two members of the TORC family, TORC2 and TORC3, also strongly activated PGC-1alpha transcription. TORCs dramatically induced PGC-1alpha gene transcription through CREB. Forced expression of TORCs in primary muscle cells induced the endogenous mRNA of PGC-1alpha and its downstream target genes in the mitochondrial respiratory chain and TCA cycle. Importantly, these changes in gene expression resulted in increased mitochondrial oxidative capacity measured by cellular respiration and fatty acid oxidation. Finally, we demonstrated that the action of TORCs in promoting mitochondrial gene expression and function requires PGC-1alpha. Previous studies had indicated that TORCs function as a calcium- and cAMP-sensitive coincidence detector and mediate individual and synergistic effects of these two pathways. Our results, together with previous findings, strongly suggest that TORCs play a key role in linking these external signals to the transcriptional program of adaptive mitochondrial biogenesis by activating PGC-1alpha gene transcription.

[Repair of skin damage with mesenchymal stem cells-poly (lactic-co-glycolic acid) scaffolds: experimental study with rabbits].

OBJECTIVE: To evaluate the feasibility and effect of mesenchymal stem cells (MSCs)-poly (lactic-co-glycolic acid) (PLGA) scaffold as transplant in repair of skin damage. METHODS: MSCs were isolated from the bone marrow of femur of a one-month-old New Zealand rabbit, cultured, and labeled with diamidino-phenyl-indole (DAPI). Porous foam scaffolds were made with PLGA. MSCs of 2 - 3 passages were seeded on the scaffolds. Fluorescence microscopy and scanning electron microscopy were used to observe the growth of the MSCs. Six pieces of skin 2 cm x 2 cm in size were cut from the backs of five 5-month-old new Zealand rabbits and then 4 pieces of MSCs- PLGA scaffolds and 2 pieces of porous foam PLGA scaffolds of the size similar to these of the cut skin were transplanted to the skin wounds. The wound healing was observed. Five days after the operation, samples of newly-grown skin were taken to undergo HE staining, VG staining, and microscopy. Immunofluorescence histochemistry was used to detect the cytokeratin AE1/AE3. RESULTS: Scanning electron microscopy showed that holes were distributed evenly on the surface of and inside the porous foam PLGA scaffolds Fluorescence microscopy and scanning electron microscopy showed that the MSCs grew well on the porous foam PLGA scaffolds and the number of MSCs increased gradually. Animal experiment showed that with the degradation of the polymer scaffolds the wounds were gradually covered by newly grown skin similar to the normal skin. Immunofluorescence histochemistry showed fluorescence positive cells in the stratum corneum and follicles. The wounds transplanted only with porous foam PLGA scaffolds formed new skin too, however, in the dermis of the new skin only thickened fibrous scars and a few follicles were seen. CONCLUSION: The compound of MSCs-PLGA polymer is effective in wound healing.

Mice deleted for fatty acid transport protein 5 have defective bile acid conjugation and are protected from obesity.

BACKGROUND & AIMS: Fatty Acid Transport Protein 5 (FATP5) is a liver-specific member of the FATP/Slc27 family, which has been shown to exhibit both fatty acid transport and bile acid-CoA ligase activity in vitro. Here, we investigate its role in bile acid metabolism and body weight homeostasis in vivo by using a novel FATP5 knockout mouse model. METHODS: Bile acid composition was analyzed by mass spectroscopy. Body weight, food intake, energy expenditure, and fat absorption were determined in animals fed either a low- or a high-fat diet. RESULTS: Although total bile acid concentrations were unchanged in bile, liver, urine, and feces of FATP5 knockout mice, the majority of gallbladder bile acids was unconjugated, and only a small percentage was conjugated. Primary, but not secondary, bile acids were detected among the remaining conjugated forms in FATP5 deletion mice, suggesting a specific requirement for FATP5 in reconjugation of bile acids during the enterohepatic recirculation. Fat absorption in FATP5 deletion mice was largely normal, and only a small increase in fecal fat was observed on a high-fat diet. Despite normal fat absorption, FATP5 deletion mice failed to gain weight on a high-fat diet because of both decreased food intake and increased energy expenditure. CONCLUSIONS: Our findings reveal an important role for FATP5 in bile acid conjugation in vivo and an unexpected function in body weight homeostasis, which will require further analysis. FATP5 deletion mice provide a new model to study the intersection of bile acid metabolism, lipid metabolism, and body weight regulation.

Dual specificity MAPK phosphatase 3 activates PEPCK gene transcription and increases gluconeogenesis in rat hepatoma cells.

Insulin is a key hormone that controls glucose homeostasis. In liver, insulin suppresses gluconeogenesis by inhibiting the transcriptions of phosphoenolpyruvate carboxylase (PEPCK) and glucose-6-phosphatase (G6Pase) genes. In insulin resistance and type II diabetes there is an elevation of hepatic gluconeogenesis, which contributes to hyperglycemia. To search for novel genes that negatively regulate insulin signaling in controlling metabolic pathways, we screened a cDNA library derived from the white adipose tissue of ob/ob mice using a reporter system comprised of the PEPCK promoter placed upstream of the alkaline phosphatase gene. The mitogen-activated dual specificity protein kinase phosphatase 3 (MKP-3) was identified as a candidate gene that antagonized insulin suppression on PEPCK gene transcription from this screen. In this study, we showed that MKP-3 was expressed in insulin-responsive tissues and that its expression was markedly elevated in the livers of insulin-resistant obese mice. In addition, MKP-3 can activate PEPCK promoter in synergy with dexamethasone in hepatoma cells. Furthermore, ectopic expression of MKP-3 in hepatoma cells by adenoviral infection increased the expression of PEPCK and G6Pase genes and led to elevated glucose production. Taken together, our data strongly suggests that MKP-3 plays a role in regulating gluconeogenic gene expression and hepatic gluconeogenesis. Therefore, dysregulation of MKP-3 expression and/or function in liver may contribute to the pathogenesis of insulin resistance and type II diabetes.