RES-Xre toxin-antitoxin locus knaAT maintains the stability of the virulence plasmid in Klebsiella pneumoniae.

Hypervirulent Klebsiella pneumoniae isolates have been increasingly reported worldwide, especially hypervirulent drug-resistant variants owing to the acquisition of a mobilizable virulence plasmid by a carbapenem-resistant strain. This pLVPK-like mobilizable plasmid encodes various virulence factors; however, information about its genetic stability is lacking. This study aimed to investigate the type II toxin-antitoxin (TA) modules that facilitate the virulence plasmid to remain stable in K. pneumoniae. More than 3,000 TA loci in 2,000 K. pneumoniae plasmids were examined for their relationship with plasmid cargo genes. TA loci from the RES-Xre family were highly correlated with virulence plasmids of hypervirulent K. pneumoniae. Overexpression of the RES toxin KnaT, encoded by the virulence plasmid-carrying RES-Xre locus knaAT, halts the cell growth of K. pneumoniae and E. coli, whereas co-expression of the cognate Xre antitoxin KnaA neutralizes the toxicity of KnaT. knaA and knaT were co-transcribed, representing the characteristics of a type II TA module. The knaAT deletion mutation gradually lost its virulence plasmid in K. pneumoniae, whereas the stability of the plasmid in E. coli was enhanced by adding knaAT, which revealed that the knaAT operon maintained the genetic stability of the large virulence plasmid in K. pneumoniae. String tests and mouse lethality assays subsequently confirmed that a loss of the virulence plasmid resulted in reduced pathogenicity of K. pneumoniae. These findings provide important insights into the role of the RES-Xre TA pair in stabilizing virulence plasmids and disseminating virulence genes in K. pneumoniae.

Antibiotic-induced degradation of antitoxin enhances the transcription of acetyltransferase-type toxin-antitoxin operon.

BACKGROUND: Bacterial toxin-antitoxin (TA) modules respond to various stressful conditions. The Gcn5-related N-acetyltransferase-type toxin (GNAT) protein encoded by the GNAT-RHH TA locus is involved in the antibiotic tolerance of Klebsiella pneumoniae. OBJECTIVES: To investigate the transcriptional mechanism of the GNAT-RHH operon kacAT under antibiotic stress. METHODS: The transcriptional level of the kacAT operon of K. pneumoniae was measured by quantitative real-time (qRT) PCR assay. The degradation of antitoxin KacA was examined by western blot and fluorescent protein. The ratio of [KacA]:[KacT] was calculated by the fluorescence intensity of KacA-eGFP and mCherry-KacT. Mathematical modelling predicted protein and transcript synthesis dynamics. RESULTS: A meropenem-induced increase in transcript levels of kacA and kacT resulted from the relief from transcriptional autoregulation of the kacAT operon. Meropenem induces the degradation of KacA through Lon protease, resulting in a reduction in the ratio of [KacA]:[KacT]. The decreased ratio causes the dissociation of the KacAT complex from its promoter region, which eliminates the repression of kacAT transcription. In addition, our dynamic model of kacAT expression regulation quantitatively reproduced the experimentally observed reduction of the [KacA]:[KacT] ratio and a large increase in kacAT transcript levels under the condition of strong promoter autorepression by the KacAT complex. CONCLUSIONS: Meropenem promotes the degradation of antitoxin by enhancing the expression of Lon protease. Degradation of antitoxin reduces the ratio of intracellular [antitoxin]:[toxin], leading to detachment of the TA complex from its promoter, and releasing repression of TA operon transcription. These results may provide an important insight into the transcriptional mechanism of GNAT-RHH TA modules under antibiotic stress.

The role of circular RNA hsa_circ_0001789 as a diagnostic biomarker in gastric carcinoma.

BACKGROUND: Circular RNA (circRNA) is a kind of endogenous non-coding RNAs and has shown diagnostic values in various cancers. This study aimed to explore whether hsa_circ_0001789 could be a novel biomarker for gastric cancer (GC). METHODS: Quantitative reverse transcriptase PCR was used to detect the expression of hsa_circ_0001789 in 108 paired GC and paracancerous tissues as well as in 24 paired plasma specimens. Possible associations between hsa_circ_0001789 expression and clinicopathologic factors of GC patients were examined using one-way ANOVA. A receiver operating characteristic (ROC) curve was established to investigate the diagnostic value of hsa_circ_0001789 in GC. RESULTS: GC tissues and plasma samples showed down-regulated hsa_circ_0001789 levels than their counterparts, which were closely correlated with the malignant characteristics of GC. The area under the ROC curve (AUC) of hsa_circ_0001789 in GC tissues was 0.82, while the cut-off value was 9.5, indicating a favorable diagnostic value. Compared with the traditional tumor biomarkers, hsa_circ_0001789 had preferred AUCs that reached 0.786 for predicting the stage of invasion, 0.603 for predicting the stage of lymphatic metastasis, 0.722 for predicting the stage of distant metastasis, and 0.786 for predicting TNM stage. CONCLUSIONS: Hsa_circ_0001789 may be a novel biomarker for the diagnosis of gastric carcinoma.

Comparative Analysis of Diverse Acetyltransferase-Type Toxin-Antitoxin Loci in Klebsiella pneumoniae.

Toxin-antitoxin (TA) modules containing a Gcn5-related N-acetyltransferase (GNAT) toxin domain regulate bacterial physiology under adverse environmental stresses. Multiple GNAT-ribbon-helix-helix domain (RHH) TA loci have been identified in single bacterial genomes. However, their diversity and interactions are still obscure. Our previous analysis showed that the GNAT toxin of Klebsiella pneumoniae, KacT, introduces antibiotic tolerance and the toxicity of GNAT is neutralized by KacA, an RHH antitoxin. We here present a phylogenetic analysis of GNAT toxins of more than 1,000 GNAT-RHH pairs detected in completely sequenced K. pneumoniae genomes, revealing that the GNAT toxins are diverse and grouped into four distinct clades. Overexpression of GNAT toxins representative of each of the four clades halts the cell growth of K. pneumoniae, while the coexpression of the cognate RHH antitoxin neutralizes GNAT toxicity. We also identify point mutations that inactivate the GNAT toxins. Moreover, we observe a cross-interaction between GNAT-RHH pairs encoded by different replicons, where a chromosomal toxin (KacT2) can be neutralized by its cognate RHH antitoxin (KacA2 on a chromosome) and another antitoxin (KacA3 on a plasmid). Finally, statistical analysis of the distribution of GNAT-RHH loci in K. pneumoniae strains shows pronounced deviation from random distribution within the same clades. Moreover, we also obtain statistically significant correlations between different clades, which we discuss in terms of the experimental results. IMPORTANCE Elucidating the roles of multifaceted GNAT-RHH TA loci is essential for understanding how these TAs interact among themselves. Recently, the reaction mechanisms and structures of several GNAT-RHH pairs have been reported. While bacterial strains can carry multiple GNAT-RHH loci with diverse origins, studies on the possible cross-interactions of these TA pairs are still limited. Here, we find that 1,000 predicted GNAT toxins of K. pneumoniae can be grouped into four distinct clades. The distributions of TA loci within these clades in K. pneumoniae strains are highly nonrandom, with the presence of a single locus of each clade per strain being highly overrepresented. Moreover, the toxicity of a GNAT toxin encoded by a chromosome was alleviated by a noncognate RHH antitoxin on a plasmid. These results might yield a profound understanding of the widespread GNAT-RHH TA pairs and the cross-interactions between noncognate TA pairs located on different replicons.

miR-654-5p promotes gastric cancer progression via the GPRIN1/NF-κB pathway.

BACKGROUND: Gastric carcinoma (GC) ranks the fifth most common cancer worldwide, with high incidence and mortality rates. Numerous microRNAs (miRNAs), including miR-654-5p, have been implicated in the pathophysiological processes of tumorigenesis. Nevertheless, the mechanism of miR-654-5p in GC is unclear. OBJECTIVES: Our study is devoted to exploring the function and molecular mechanism of miR-654-5p on the malignant cell behaviors of GC. METHODS: The gene expression was detected by reverse transcription quantitative polymerase chain reaction. GC cell proliferation and motion were assessed by colony formation assay and transwell assay. The binding capacity between miR-654-5p and G protein-regulated inducer of neurite outgrowth 1 (GPRIN1) was explored by luciferase reporter and RNA pulldown assays. The protein levels were detected by Western blotting. RESULTS: miR-654-5p expression was higher in GC cells and tissues than control cells and tissues. miR-654-5p promoted GC cell growth and motion. Moreover, our findings showed that miR-654-5p was bound with GPRIN1. Importantly, downregulation of GPRIN1 rescued the inhibitory influence of miR-654-5p knockdown on GC cell malignant behaviors. Additionally, miR-654-5p activated the nuclear factor kappa-B (NF-κB) pathway by regulation of GPRIN1. CONCLUSIONS: miR-654-5p facilitated cell proliferation, migration, and invasion in GC via targeting the GPRIN1 to activate the NF-κB pathway.

Hsa_circ_0001696 modulates cell proliferation and migration in colorectal cancer.

Circular RNAs (circRNAs) are a novel class of endogenous non-coding RNA molecules that are extensively expressed in a variety of species. Recently, increasing evidence suggests that circRNAs have vital functions indifferent types of human cancer, such as gastric cancer, papillary thyroid cancer and lung cancer. However, the roles of circRNAs in the development of colorectal cancer (CRC) remain unclear. The present study aimed to determine the molecular mechanism underlying hsa_circ_0001696 on the proliferation and migration of CRC cells. Reverse transcription-quantitative PCR analysis was performed to detect hsa_circ_0001696 expression in 18 paired CRC tissues and matched adjacent normal tissues. RNA interference was also performed to decrease hsa_circ_0001696 expression, and its biological effects were further assessed via flow cytometry, wound healing, colony formation and western blot assays. The results demonstrated that hsa_circ_0001696 expression was significantly lower in CRC tissues compared with adjacent normal tissues. Furthermore, hsa_circ_0001696 knockdown promoted cell proliferation and migration, and the number of cell colonies significantly increased. In addition, western blot analysis demonstrated that the protein expression levels of cyclin-dependent kinase 4 (CDK4), cyclin D, cyclin E and matrix metalloproteinase 9 (MMP9) increased. Taken together, the results of the present study demonstrated that hsa_circ_0001696 expression was downregulated in CRC tissues, and inhibition of hsa_circ_0001696 promoted cell proliferation and migration by regulating the levels of CDK4, cyclin D, cyclin E and MMP9.

miR-6838-5p Affects Cell Growth, Migration, and Invasion by Targeting GPRIN3 via the Wnt/ß-Catenin Signaling Pathway in Gastric Cancer.

Gastric cancer (GC) is a highly prevalent digestive malignant tumor, ranking second in the tumor-related mortality globally. The microRNAs have been confirmed to be connected with GC progression. Accumulative evidence has suggested that miR-6838-5p exerts a suppressive effect on human cancers. Nonetheless, whether miR-6838-5p is involved in the regulation of GC remains to be investigated. During our research, miR-6838-5p was downregulated in GC cells. Upregulated miR-6838-5p repressed GC cell cycle progression, proliferation, migration, and invasion. Furthermore, miR-6838-5p overexpression repressed the nuclear import of ß-catenin, thus inactivating Wnt/ß-catenin pathway. Moreover, we observed that GPRIN3 was targeted by miR-6838-5p in GC with luciferase reporter and RIP assays. GPRIN3 upregulation reversed the suppression of miR-6838-5p in GC cellular processes. These findings suggest miR-6838-5p restrains the malignant behaviors of GC cells via targeting GPRIN3 to repress Wnt/ß-catenin signaling pathway, which may provide novel targets for GC treatment.

Genomic Sequencing Reveals the Diversity of Seminal Bacteria and Relationships to Reproductive Potential in Boar Sperm.

A number of emerging studies suggest that pathogenic microorganisms in semen may cause a decline in the reproductive potential of spermatozoa, and the bacterial diversity and profile of ejaculated boar semen in different seasons are currently unknown. To explore the bacterial composition and changes in ejaculated boar semen from winter and summer, and the underlying mechanism of decline in sperm quality and fertility capacity in summer, 120 ejaculated semen samples were examined for bacterial communities using genomic sequencing technology, and the associations between microbial composition and sperm reproductive potential were investigated. The results showed that Proteobacteria (57.53%), Firmicutes (31.17%), Bacteroidetes (4.24%), and Actinobacteria (3.41%) are the dominant phyla in the ejaculated semen, and the dominant genera were Pseudomonas (34.41%) and Lactobacillus (19.93%), which belong to the phyla of Proteobacteria and Firmicutes, respectively. Interestingly, the higher diversity of bacteria in ejaculated semen of winter differs from that of summer semen, potentially due to seasonal changes related to changes in semen quality and sperm fertilizing capacity. Furthermore, the highly abundant Lactobacillus in winter samples were positively associated with sperm quality and reproductive performance obtained from sows inseminated with such semen samples, while in contrast, the highly abundant Pseudomonas in summer samples was negatively associated with sperm quality and reproductive potential. Additionally, our results strongly indicated that Lactobacillus is not only a potential probiotic for semen quality and fertility potential but also beneficial for restraining the negative influence of Pseudomonas. Overall, our findings significantly contribute to the current understanding of the phenotypes and etiology of male "summer infertility," and may represent a frontier in male reproductive disorders and possible early prevention against pathogenic bacteria.

Ca2+ ionophore A23187 inhibits ATP generation reducing mouse sperm motility and PKA-dependent phosphorylation.

Male infertility is a global problem in modern society of which capacitating defects are a major cause. Previous studies have demonstrated that Ca2+ ionophore A23187 can make mouse sperm capable of fertilizing in vitro, which may aid in clinical treatment of capacitating defects. However, the detailed role and mechanism of Ca2+ in the capacitating process are still unclear especially how A23187 quickly renders sperm immotile and inhibits cAMP/PKA-mediated phosphorylation. We report that A23187 induces a Ca2+ flux in the mitochondria enriched sperm tail and excess Ca2+ inhibits key metabolic enzymes involved in acetyl-CoA biosynthesis, TCA cycle and electron transport chain pathways resulting in reduced ATP and overall energy production, however this flux does not destroy the structure of the sperm tail. Due to the decrease in ATP production, which is the main phosphate group donator and the power of sperm, the sperm is rendered immobile and PKA-mediated phosphorylation is inhibited. Our study proposed a possible mechanism through which A23187 reduces sperm motility and PKA-mediated phosphorylation from ATP generation, thus providing basic data for exploring the functional roles of Ca2+ in sperm in the future.

Carbonic anhydrase IV inhibits cell proliferation in gastric cancer by regulating the cell cycle.

Carbonic anhydrase IV (CA4) is silenced in colorectal cancer. However, the effect of CA4 on the development of gastric cancer (GC) is poorly understood. The present study aimed to determine the role of CA4 in GC tumorigenesis and its underlying molecular mechanism. The levels of CA4 in GC cells and tissues were evaluated by reverse transcription-quantitative PCR and immunohistochemistry. CA4 expression was suppressed in GC cells and tissues compared with adjacent healthy tissues and normal human gastric epithelial cells, respectively. This reduced expression was significantly associated with tumor size, invasion and differentiation. Analyses with a real-time cell analyzer and clonogenic assays were conducted to validate the impact of CA4 on GC cell lines (AGS and HGC-27) and normal human gastric epithelial cell line (GES-1) proliferation. The effects of CA4 on the cell cycle in GC cells were determined by flow cytometry. The levels of CA4 and cell cycle-associated proteins were confirmed by western blotting. CA4 overexpression inhibited GC cell proliferation and reduced colony-forming ability, arrested the cell cycle in the G2/M phase, inhibited cyclin B1 and cyclin-dependent kinase 2 expression and induced p21 expression. These results indicate that CA4 may serve an important role in GC tumorigenesis by inhibiting cellular proliferation via regulating the expression of cell cycle-associated proteins. CA4 may serve as a diagnostic biomarker and a potential therapeutic target in GC.

Toxin-antitoxin operon kacAT of Klebsiella pneumoniae is regulated by conditional cooperativity via a W-shaped KacA-KacT complex.

Bacterial toxin-antitoxin pairs play important roles in bacterial multidrug tolerance. Gcn5-related N-acetyltransferase (GNAT) toxins inhibit translation by acetylation of aminoacyl-tRNAs and are counteracted by direct contacts with cognate ribbon-helix-helix (RHH) antitoxins. Our previous analysis showed that the GNAT toxin KacT and RHH antitoxin KacA of Klebsiella pneumoniae form a heterohexamer in solution and that the complex interacts with the cognate promoter DNA, resulting in negative autoregulation of kacAT transcription. Here, we present the crystal structure of DNA-bound KacAT complex at 2.2 Å resolution. The crystal structure revealed the formation of a unique heterohexamer, KacT-KacA2-KacA2-KacT. The direct interaction of KacA and KacT involves a unique W-shaped structure with the two KacT molecules at opposite ends. Inhibition of KacT is achieved by the binding of four KacA proteins that preclude the formation of an active KacT dimer. The kacAT operon is auto-regulated and we present an experimentally supported molecular model proposing that the KacT:KacA ratio controls kacAT transcription by conditional cooperativity. These results yield a profound understanding of how transcription GNAT-RHH pairs are regulated.

Nonalcoholic fatty pancreas disease is related independently to the severity of acute pancreatitis.

BACKGROUND: This study aimed to investigate the association between nonalcoholic fatty pancreas disease and the severity of acute pancreatitis (AP). PATIENTS AND METHODS: Among the 1662 AP patients admitted between August 2010 and August 2017, 82 eligible patients with moderately severe acute pancreatitis (SAP) and SAP were selected. Meanwhile, 164 mild AP patients were age-matched, sex-matched, and BMI-matched at a ratio of 1 : 2. Nonalcoholic fatty pancreas disease was estimated by mean pancreas attenuation by unenhanced computed tomography. Finally, 1662 patients were screened and 246 patients were analyzed. RESULTS: For the 246 patients, the mean pancreatic attenuation and pancreas-to-spleen attenuation ratio (P/S ratio) were significantly lower in the moderately SAP and SAP groups compared with those in the mild AP group (both, P<0.001). Pancreatic attenuation decreased with an increase in the rate of ICU transfer, AP severity, systemic complications, and prognostic factors of AP (Acute Physiology and Chronic Health Evaluation II score≥8; P<0.001). A decreased P/S ratio was correlated positively with the increased mortality of patients with AP (hazard ratio: 0.000; 95% confidence interval: 0.000-0.012; P<0.001), as determined by Cox proportional regression analysis adjusted for creatinine, calcium, and albumin levels. CONCLUSION: The pancreatic attenuation level and P/S ratio are correlated independently to severity, mortality, and systemic complications in patients with AP.

Impact of visceral adiposity on severity of acute pancreatitis: a propensity score-matched analysis.

BACKGROUND: The relationship between visceral adiposity and acute pancreatitis (AP) has not been completely elucidated. This study evaluated the significance of visceral adipose tissue (VAT) and the ratio of VAT to skeletal muscle tissue (VAT/SMT) in the prognosis of AP patients. METHODS: Based on a 1:2 propensity score matching, 306 hospitalized patients were enrolled in the study analysis from 2010 to 2017. VAT, subcutaneous adipose tissue (SAT), and SMT were measured using unenhanced computed tomography (CT). Cox proportional hazards models were applied for the analysis. RESULTS: VAT and the VAT/SMT ratio were significantly higher in the severe AP (SAP) and moderately severe AP (MSAP) groups compared to the mild AP (MAP) group (both p < 0.001). Intensive care transfer, AP severity, systemic complications, and prognostic scores (Acute Physiology and Chronic Health Evaluation II [APACHE-II] score ≥ 8, Ranson's score ≥ 3, Bedside Index of Severity in Acute Pancreatitis [BISAP] score ≥ 3, and the systemic inflammatory response syndrome [SIRS] score ≥ 2) significantly correlated with VAT and the VAT/SMT ratio in AP patients. The multivariate adjusted hazard ratios (HRs) for VAT and the VAT/SMT ratio in the relationship of body parameters and AP mortality were 1.042 (95% confidence interval (CI), 1.019-1.066) and 7.820 (95% CI, 1.978-30.917), respectively. Compared with other prognostic scores, VAT had the highest area under the curve of receiver operating characteristics (ROC) (0.943, 95% CI, 0.909-0.976). CONCLUSION: High VAT and VAT/SMT ratio are independent negative prognostic indicators of AP. TRIAL REGISTRATION: Clinical study registration number: NCT03482921 . Date of registration: 03/23/2018.

CircScd1 Promotes Fatty Liver Disease via the Janus Kinase 2/Signal Transducer and Activator of Transcription 5 Pathway.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is one of the most common liver diseases in affluent countries. Recent studies have reported that circular RNAs (circRNAs) are important regulators of hepatic steatosis. However, the role and mechanism of circRNA in NAFLD are poorly understood. AIMS: This study is to reveal the role and mechanism of circRNA in NAFLD. METHODS: Through NAFLD-related circRNA microarrays, we used real-time quantitative reverse transcription-polymerase chain reaction to screen circScd1 levels in control and test groups of mice fed a high-fat diet. RNA interference and over-expression plasmid vectors were used to manipulate the expression of circScd1, and the biological effects were evaluated by oil red staining, triglyceride detection, and western blot analysis. RESULTS: CircScd1 expression was significantly lower in NAFLD tissues than in control tissues. Moreover, over-expression of circScd1 significantly inhibited the formation of lipid droplets. Western blot analyses showed that the protein levels of Janus kinase 2 (JAK2) and signal transducer and activator of transcription 5 (STAT5) were significantly increased. However, knockdown of circScd1 significantly promoted the degree of hepatocellular lipidosis and reduced the expression levels of JAK2 and STAT5. CONCLUSIONS: Aberrant expression of circScd1 affects the extent of hepatocellular lipidosis in NAFLD and promotes fatty liver disease via the JAK2/STAT5 pathway.

Nonalcoholic Fatty Liver Disease Is Associated with Increased Atrial Fibrillation Risk in an Elderly Chinese Population: A Cross-Sectional Study.

BACKGROUND: Atrial fibrillation and nonalcoholic fatty liver disease are two pathological conditions that are highly prevalent worldwide and share multiple CVD risk factors. There are rare researches performed among elderly adults. AIMS: We conducted this cross-sectional analysis of elderly adults (≥65 years) to investigate the association between atrial fibrillation and nonalcoholic fatty liver disease. METHODS: We analyzed clinical data of the elderly adults (≥ 65 years) who had health examination in Zhenhai Lianhua Hospital, Ningbo, China, in 2014. RESULTS: 522 of the 1688 participants were diagnosed with nonalcoholic fatty liver disease, and 39 participants were diagnosed as having atrial fibrillation. Nonalcoholic fatty liver disease was associated with risk factors for AF in the elderly Chinese population (OR 1.95, 95% CI 1.03-3.69). Adjustments for age, gender, systolic blood pressure, fasting plasma glucose, γ-glutamyl transpeptidase, high-density lipoprotein, triglycerides, total cholesterol and albumin, nonalcoholic fatty liver disease, and prevalent atrial fibrillation remained statistically significant (OR 2.76, 95% CI 1.32-5.77). CONCLUSIONS: Our results show that nonalcoholic fatty liver disease is associated with an increased risk of atrial fibrillation in an elderly Chinese population.

Candidates for reproductive biomarkers: Protein phosphorylation and acetylation positively related to selected parameters of boar spermatozoa quality.

Protein post-translational modifications (PTMs) have been reported to be involved in various functions of sperm, yet the exact correlation between PTMs and sperm motility remains unclear. With the goal of contributing to this subject, motility variables were measured by computer-assisted sperm analysis system (CASA), and the amount of PTMs were evaluated using Western blot and immunofluorescence in fresh sperm and liquid stored sperm. Results of the present study indicate that the amount of the phosphorylated substrates of PKA (P-PKAs), protein tyrosine phosphorylation (PTP), global protein acetylation (Pan-Kac) and α-tubulin acetylation (Tub-Kac) was greater in sperm of fresh semen samples with relatively greater motility than in sperm of fresh semen samples with relatively lesser motility. Similarly, the amounts of phosphorylation and acetylation gradually decreased with the reduction in the motility of sperm in liquid stored semen samples. Interestingly, the P-PKAs (r = 0. 634, P < 0. 01) and Pan-Kac (r = 0. 380, P < 0. 05) were positively correlated with sperm motility in fresh semen, whereas only P-PKAs (r = 0.607, P < 0. 01) were positively correlated with sperm motility during liquid storage. Furthermore, it is noteworthy that the amounts of phosphorylation and acetylation were positively correlated with the acrosome integrity and mitochondrial membrane potential of fresh sperm and liquid stored sperm. This study is the first to explore the correlation between PTMs and sperm motility, and it may provide a new reproductive biomarker for evaluating semen quality and predicting sperm capacity for enhancing reproductive performance, which is meaningful for the pig breeding industry.

Quantitative proteomic profiling indicates the difference in reproductive efficiency between Meishan and Duroc boar spermatozoa.

The reproductive efficiency of Meishan pigs is higher than that of Duroc pigs, but the underlying molecular mechanism for this disparity remains unclear. No systematic quantitative proteomics studies, comparing global proteins in Meishan and Duroc boar spermatozoa have been reported. Therefore, we applied iTRAQ labeling coupled with mass spectrometry, and analyzed the differences in proteins between Meishan and Duroc sperm. In the present study, a total of 1597 proteins were quantified. Of these proteins, 190 showed statistically significant fold changes between Meishan and Duroc spermatozoa. Bioinformatics analysis revealed that these differentially abundant proteins were primarily involved in energy metabolism, sperm motility, capacitation and sperm-oocyte binding. Remarkably, SPAG6, ACR, LDHC, CALM, ACE and ENO1 which are positively related to high litter size, were more abundant in Meishan spermatozoa than in Duroc spermatozoa. Moreover, APOA1, NDUFS2 and RAB2A which are negatively related to farrowing rates, were less abundant in Meishan spermatozoa than in Duroc spermatozoa. Interestingly, essential enzymes in Glycolysis/Gluconeogenesis, such as HK1, ALDH2, LDHA and LDHC, were markedly up-regulated in Meishan spermatozoa compared to Duroc spermatozoa. In addition, we first demonstrated that the levels of protein phosphorylation in Meishan spermatozoa were higher than those in Duroc. Taken together, the physiologically and functionally differential proteins may be one main reason for explaining the high reproductive efficiency of Meishan boar.

Cadmium inhibits lysine acetylation and succinylation inducing testicular injury of mouse during development.

The toxic effects of cadmium (Cd) in the reproductive system have been confirmed, and lysine acetylation and succinylation play important roles in spermatogenesis. However, little attention determined whether Cd could affect lysine acylation and how it might have an impact on the reproductive system. Therefore, with the goal of contributing to this subject, we have examined the effects of Cd on lysine acetylation and succinylation of proteins in the germ cells of male mice testes during different developmental stages. We adopted intraperitoneal injection of cadmium chloride (1.2 mg/kg body weight) in mice once every 5 days from postnatal day 5-60. The results showed that Cd could restrict GAPDH activity, ATP and cAMP levels of germ cells to inhibit lysine acetylation and succinylation in the testes, inducing reproductive injuries. Cd also restricts acetylation of histone H4K5 and H4K12, which could result in failure of spermiogenesis. Remarkably, polarized acetylation occurs in meiosis, and high-level acetylation occurs earlier than high-level succinylation during spermatogenesis. Moreover, Cd has a limited effect on body weight but reduces the weight of the testis and litter size. Our research may provide a new way to reveal the mechanisms of Cd reproductive toxicity related to lysine acetylation and succinylation.

Vitamin C exerts novel protective effects against cadmium toxicity in mouse spermatozoa by inducing the dephosphorylation of dihydrolipoamide dehydrogenase.

Cadmium (Cd) has been reported to inhibit mouse sperm motility by inducing the tyrosine phosphorylation of dihydrolipoamide dehydrogenase (DLD). This study aimed to assess the potential effects of vitamin C (Vc) on ameliorating Cd-induced tyrosine phosphorylation of DLD and the specific underlying mechanism. Vc induced the dephosphorylation of DLD or inhibited the tyrosine phosphorylation of DLD. Accordingly, DLD activity, nicotinamide adenine dinucleotide hydrogen (NADH) levels, ATP levels and motility parameters were all restored to normal levels by Vc. Moreover, the effects of Vc on ameliorating these indicators had striking similarities to the effects of ethylenediaminetetraacetic acid (EDTA). In addition, neither the antioxidant melatonin nor the universal oxidant H2O2 influenced the tyrosine phosphorylation of DLD. Hence, the protective effects of Vc on the tyrosine phosphorylation of DLD might be attributed to its binding to Cd ions outside or inside sperm, and were not due to its antioxidant properties.