Epididymal epithelial degeneration and lipid metabolism impairment account for male infertility in occludin knockout mice.

Occludin (OCLN) is a tight junction protein and Ocln deletion mutation causes male infertility in mice. However, the role of OCLN in male reproductive system remains unknown. In this study, we used an interdisciplinary approach to elucidate the underlying mechanism of male infertility in related to OCLN function, including Ocln knockout mice as well as a combined omics analysis and immunofluorescent labelling. Our results showed that the epididymis of Ocln-null mice displayed a phenomenon resembling epididymal sperm granuloma, which occurred especially in the junctional region between caput and corpus epididymidis. Sperm motility and fertilisation capacity were also impaired in these Ocln-null mice, accompanied by enlarged tubules in the proximal regions and degeneration in the distal regions of epididymis. Cellular localization analysis showed that OCLN immunofluorescence was enriched only in the apical junction of epithelial principal cells in the proximal regions of epididymis. Integrative omics analysis revealed the downregulation of gene clusters enriched in acid secretion and fatty acid metabolism in the Ocln-null epididymis, especially the enzymes related to the unsaturated arachidonic acid pathway. The number of proton-pump V-ATPase-expression clear cells, a key player of luminal acidification in the epididymis, declined drastically from prepubertal age before sperm arrival but not in the early postnatal age. This was accompanied by programmed cell death of clear cells and increased pH in the epididymal fluid of OCLN-deficient mice. The lipidomics results showed significantly increased levels of specific DAGs conjugated to unsaturated fatty acids in the Ocln-mutant. Immunofluorescent labelling showed that the arachidonic acid converting enzyme PTGDS and phospholipase PLA2g12a were prominently altered in the principal cells and luminal contents of the Ocln-mutant epididymis. Whereas the carboxylate ester lipase CES1, originally enriched in the WT basal cells, was found upregulated in the Ocln-mutant principal cells. Overall, this study demonstrates that OCLN is essential for maintaining caput-to-corpus epithelial integrity, survival of acid-secreting clear cells, and unsaturated fatty acid catabolism in the mouse epididymis, thereby ensuring sperm maturation and male fertility.

Effect of silver diammine fluoride on micro-ecology of plaque from extensive caries of deciduous teeth - in vitro study.

BACKGROUND: The mechanism of action of silver diammine fluoride (SDF) on plaque micro-ecology is seldom studied. This study investigated micro-ecological changes in dental plaque on extensive caries of deciduous teeth after topical SDF treatment. METHODS: Deciduous teeth with extensive caries freshly removed from school children were collected in clinic. Unstimulated saliva collection and initial plaque sampling were done before tooth extraction, then each caries was topically treated with 38% SDF in vitro. After intervention, each tooth was stored respectively in artificial saliva at 37 °C. Repeated plaque collections were done at 24 h and 1 week post-intervention. Post-intervention micro-ecological changes including microbial diversity, microbial metabolism function as well as species correlations were analyzed and compared after pyrosequencing of the DNA from the plaque sample using Illumina MiSeq platform. RESULTS: After SDF application, microbial diversity decreased (P > 0.05), although not statistically significant. Microbial community composition post-intervention was noticeably different from that of supragingival and pre-intervention plaque as well as saliva. At 1 week post-intervention, the relative content of Pseudomonas, Fusobacterium and Pseudoramibacter were higher than before, while most of the other bacteria were reduced, although the changes were not statistically significant (P > 0.05). The inter-microbial associations became more complex, much more positive associations among survived bacteria were observed than negative ones. COG function classification diagram showed carbohydrate transportation and metabolic functions in the plaque were significantly reduced at 24 h and 1 week post-intervention. CONCLUSIONS: SDF has extensive antimicrobial effect on dental plaque, which may reduce carbohydrate metabolism in dental plaque and help promote new balance of the plaque flora.

Effect of Silver Fluoride in Preventing the Formation of Artificial Dentinal Caries Lesions in vitro.

OBJECTIVE: To investigate the effect of silver fluoride in preventing the development of artificial caries lesions in root dentine using an artificial mouth system (AMS). METHODS: A total of 34 extracted intact human premolars were embedded individually in blocks with one root dentine surface exposed. Among these, 32 were randomly divided into four groups (eight each) and the remaining two tooth blocks were used in the baseline evaluation of the cariogenic biofilm after bacterial inoculation. The interventions (topical application of 2.36 M solutions) were applied after inoculation as follows: group-1, silver fluoride (AgF); group-2, potassium fluoride (KF); group-3, silver nitrate (AgNO3); and group-4, deionised water (control). Subsequently, the tooth blocks were transferred into the AMS to start an artificial caries challenge (5% sucrose was delivered three times per day). After 10 days, the formed biofilm was assessed via colony forming unit (CFU) counts, confocal laser scanning microscopy, and scanning electron microscopy. The artificial dentinal caries lesion was evaluated using micro-computed tomography. RESULTS: After the interventions and the 10-day challenge in the AMS, the median CFUs (AgF < AgNO3 < KF = control, P < 0.001) and the median live-to-dead bacteria ratios (AgF < AgNO3 < KF = control, P < 0.005) of the biofilm differentiated significantly among the groups. Scattered bacterial cells were found in the tooth blocks of the AgF and AgNO3 groups, while a confluent biofilm layer was observed in the tooth blocks of the KF and control groups. The median lesion depth in the AgF group was significantly lower than in the AgNO3 (P = 0.016), KF (P = 0.016) and control (P = 0.009) groups. CONCLUSION: The combined use of silver and fluoride ions in the AgF solution significantly protected dentine against the development of caries lesions.

Vitamin K2-Dependent GGCX and MGP Are Required for Homeostatic Calcium Regulation of Sperm Maturation.

A low-calcium microenvironment is essential for spermatozoa to mature in the epididymis; however, it remains unclear how dysregulation of epididymal luminal calcium is associated with male infertility. Using a warfarin-induced vitamin K2 deficiency rat model, we found that vitamin-K-dependent γ-glutamyl carboxylase (GGCX) and matrix Gla protein (MGP) were essential in extracellular calcium signaling of the intercellular communication required for epididymal sperm maturation. We found that GGCX and MGP co-localized in the vesicular structures of epididymal cells and spermatozoa. Calcium-regulated MGP binds to proteins in a biphasic manner; sub-millimolar calcium enhances, whereas excessive calcium inhibits, the binding. Bioinformatic analysis of the calcium-dependent MGP-bound proteome revealed that vesicle-mediated transport and membrane trafficking underlie the intercellular communication networks. We also identified an SNP mutation, rs699664, in the GGCX gene of infertile men with asthenozoospermia. Overall, we revealed that the GGCX-MGP system is integrated with the intercellular calcium signaling to promote sperm maturation.

Glass ionomer ART sealant and fluoride-releasing resin sealant in fissure caries prevention--results from a randomized clinical trial.

BACKGROUND: The relative performance of ART sealant and fluoride-releasing resin sealant in preventing fissure caries in permanent molars was compared in a randomized clinical trial conducted in southern China (ClinicalTrials.gov NCT01829334). METHODS: After obtaining ethical approval, healthy schoolchildren who had permanent first molars with occlusal fissures which were sound but deep or presented with only incipient caries were recruited for the study. Included molars were randomly allocated into one of four parallel study groups in units of left/right teeth per mouth. Two of the four groups adopted the methods of ART or fluoride-releasing resin sealant placement while the other two groups adopted the topical fluoride application methods. Fissure status of the molars in each group was evaluated every 6 months. Development of dentine caries and sealant retention over 24 months in the molars in the two sealant-using groups was compared in this report. Outcome on cost-effectiveness of all four groups over 36 months will be reported elsewhere. RESULTS: At baseline, a total of 280 children (383 molars) with mean age 7.8 years were involved for the two sealant groups. After 24 months, 261 children (357 molars) were followed. Proportions of molars with dentine caries were 7.3% and 3.9% in the ART sealant and fluoride-releasing resin sealant groups, respectively (chi-square test, p = 0.171). Life-table survival analysis showed that sealant retention (full and partial) rate over 24 months for the resin sealant (73%) was significantly higher than that (50%) for the ART sealant (p < 0.001). Molar survival (no development of dentine caries) rates in the ART sealant (93%) and fluoride-releasing resin sealant (96%) groups were not significantly different (p = 0.169). Multilevel logistic regression (GEE modeling) accounting for the effects of data clustering and confounding factors confirmed this finding. CONCLUSIONS: Though the retention of fluoride-releasing resin sealant was better than that of the ART sealant, their effectiveness in preventing fissure caries in permanent molars did not differ significantly over 24 months. ART sealants could be a good alternative when and where resources for resin sealant placement are not readily available.

[Investigation on rat-shaped animals and insect vectors on their body surface at Ningde Harbor].

OBJECTIVE: To understand the composition, seasonal variation, as well as carrying insect vectors (including tick, flea, mite) of rat-shaped animals in Ningde Harbor area, so as to provide the evidence for prevention and control of rodent-borne diseases. METHODS: From July 2009 to June 2010, 2 dockyards were investigated according to the monitoring methods of The Industry Standard Inspection and Quarantine of China. RESULTS: A total of 171 rat-shaped animals (4 species, 2 genera, 2 families, and 2 orders) were captured, among which Rattus norvegicus was the dominant species. The overall rat density was 3.58%. The indexes of flea, mite and tick were 0.01, 0.33, and 0.05, respectively. CONCLUSIONS: The rat density at Ningde Harbor has not reached the national standard (< 1%), which suggests that the measures of rat monitoring at dockyards in Ningde Harbor should be enforced.

Down-regulation of zinc transporter 8 in the pancreas of db/db mice is rescued by Exendin-4 administration.

Recent human genetic studies have revealed that common variants of the zinc transporter 8 (ZnT-8) gene are strongly associated with type 2 diabetes mellitus (T2DM). ZnT-8 has been suggested as a potential candidate in the regulation of insulin secretion in pancreatic ß-cells. In this study, we aimed to explore the expression of ZnT-8 in the development of T2DM. The expression of ZnT-8 was investigated in the pancreas and adipose tissue of homozygous db/db mice compared to heterozygous sibling db/+ mice (n=6-8). Furthermore, the effect of Exendin-4 (an analogue of glucagon-like peptide-1) on ZnT-8 expression was examined in the db/db mice. Exendin-4 or vehicle (control) was administered (i.p., 1 nmol/kg) to diabetic 8-week-old db/db mice daily for 14 days (n=8). The results revealed that ZnT-8 protein levels in the pancreas of db/db mice were reduced, accompanied by a decrease in ZnT-8 mRNA. ZnT-8 mRNA and protein levels were also significantly reduced in the epididymal and visceral fat of the db/db mice. Treatment with Exendin-4 up-regulated ZnT-8 gene expression in the pancreas of the db/db mice, but did not affect its expression in adipose tissue. These findings suggest that ZnT-8 synthesis in the pancreas and adipose tissue is down-regulated in db/db mice. Reduced ZnT-8 production in the pancreas may advance defects in insulin secretion in diabetes. These may be reversed, at least partially, by the administration of Exendin-4.

[Early diagnosis of female genital tuberculosis by phage amplified biological assay].

OBJECTIVE: To establish an early diagnostic method for detecting female genital tuberculosis. METHODS: Eighty-six women with genital tuberculosis during January 2005-September 2007 were examined by phage amplified biological assay, and the results were compared with those from leucorrhea culture, smear and PCR. RESULTS: Forty-five patients were tuberculosis positive with 100% of specificity identified by phage amplified biological assay. Twenty patients were tuberculosis positive by PCR. Five patients were culture-positive tuberculosis and no case had smear-positive tuberculosis. CONCLUSION: Phage amplified biologically assay is sensitive and specific, which could be used for the early diagnosis of female genital tuberculosis.

[Value of coagulation function and fibrinolytic system assessment in patients with gestational diabetes mellitus].

OBJECTIVE: To study the clinical implications of changes of coagulation function and fibrinolytic system in patients with gestational diabetes mellitus (GDM). METHODS: Twenty non-pregnant women, 20 with normal pregnancy and 46 with GDM were enrolled in this study for examinations of platelet alpha-granule membrane protein (GMP-140), Von Willebrand factor (vWF), antithrombin III activity (AT-III), plasminogen activity (PLG), activity of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI). RESULTS: vWF, GMP-140, PLG, D-dimer, PAI were obviously elevated while t-PA was lower in GDM patients as compared with the measurement in non-pregnant women and women with normal pregnancy (P<0.01). AT-III and ProC measurement showed no significant differences between GDM patients and women of the other two groups. CONCLUSION: GDM patients may have elevated platelet activation and fibrinolyic activity as well as vascular endothelial injuries, and antenatal assessment of the coagulation function can be of value for prevention and treatment of GDM.

[Changes in serum endothelin and in pregnant rabbits with pulmonary embolism].

OBJECTIVE: To study change of serum endothelin (ET) and in serum endothelin and in pregnant rabbit with acute pulmonary embolism. METHODS: In 17 pregnant rabbits, autologous blood clots were administered into the right atrium via the femoral vein to establish the animal models of acute pulmonary embolism, and another 10 pregnant rabbits received injection with saline to serve as the control group. Serum levels of ET and CGRP were measured before, 0, 2, 4, 6 and 8 h after the injection. RESULTS: Acute pulmonary embolism was induced in all the rabbits receiving the injection of autologous blood clots with a success rate of 100%. In these rabbit models of pulmonary embolism, serum ET increased 0 h after the embolism and kept rising till 6 h, whereas CGRP level underwent immediate decrease after the embolism followed then by increase during the period from 4 to 8 h, showing significant difference from the levels of the control group. CONCLUSION: Significant difference in serum ET and CGRP levels occur in the event of acute pulmonary embolism in pregnant rabbits.

Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 transcriptional activator Rta is an oligomeric DNA-binding protein that interacts with tandem arrays of phased A/T-trinucleotide motifs.

Kaposi's sarcoma associated herpesvirus (KSHV)/human herpesvirus 8 (HHV-8) encodes an immediate early transcriptional activator, Rta, which mediates viral reactivation from latency and lytic viral replication. Here we report the purification and characterizations of HHV-8 Rta and its interaction with Rta-responsive DNA elements. The Rta response element (RtaRE) in the promoter of the KSHV/HHV-8 K8 open reading frame was mapped to a 47-bp sequence (RtaRE1) and a 60-bp sequence (RtaRE2) upstream of the TATA motif. A comparison of the K8 RtaREs with other viral RtaREs revealed a pattern of multiple A/T triplets spaced with a periodicity of 10 or 20 bp. Substitutions of the in-phase A/T trinucleotides of the RtaRE1 with G/C bases greatly diminished Rta responsiveness and Rta binding. By contrast, base substitutions in an out-of-phase A/T-trinucleotide sequence had no effect. Importantly, multimers of (A/T)(3)N(7) and N(5)(A/T)(5)N(6)(A/T)(4) motifs supported a strong Rta response in a copy number-dependent manner. No specific sequence motifs in the spacer regions could be discerned. Potent Rta response, however, was obtained with phased A/T trinucleotides with 7-bp spacers of arbitrary sequences with high G/C content. Lengthening of the phased A/T motifs or lowering of the G/C content of the spacers resulted in a reduction in Rta response. Finally, Escherichia coli-derived Rta is an oligomer of 440 kDa in molecular size and binds RtaRE as an oligomer. These results support a model of Rta transactivation wherein the subunits of the Rta oligomer make multiple contacts with a tandem array of phased A/T triplets in the configuration of (A/T)(3)(G/C)(7) repeats.

[Changes of relative variables about prethrombosis state and its clinical significance in patients with pregnancy induced hypertension].

OBJECTIVE: To explore the changes of the hemorrheology coagulation and fibrinolytic system in patients with pregnancy induced hypertension (PIH). METHODS: To examine the platelet alphagranule membrane protein (GMP-140), Von-Willebrand factor (vWF), antithrombin III activity (AT-III), plasminogen activity (PLG), the activity of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI) in 30 non-pregnancy women, 30 normal late pregnancy women and 50 pregnancy induced hypertension women. RESULTS: In PIH group vWF [(156 +/- 23)%], GMP-140 [(23.9 +/- 3.6) microg/L], Fbg [(15.5 +/- 0.8) g/L], PLG [(141 +/- 9)%], D-Dimer [(1.42 +/- 0.32) mg/L], PAI [(9.2 +/- 0.8) kU/L] were obviously higher than those of control group. AT-III, ProC in PIH group were lower than that of the control group (P < 0.01). t-PA has no significant differences (P > 0.05). CONCLUSIONS: Prethrombotic state was shown in normal late pregnancy women. Obvious prethrombotic state was shown in patients with pregnancy induced hypertension women.

[Assessment of variables relative to prethrombotic state after operation in patients with gynecological malignancies].

OBJECTIVE: To study the changes in the variables in patients with gynecological malignancies after operation. METHOD: Platelet alphagranule membrane protein (GMP-140), von Willebrand Factor (vWF), antithrombin III activity (AT-III), protein C-dependent partial thromboplastin activation time, plasminogen activity (PLG), the activity of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI), D-Dimer were examined in 20 normal non-pregnant women and 38 patients after operation with gynecological malignancies. RESULTS: vWF, GMP-140, PLG, D-Dimer, and PAI of patients with malignancies before operation were obviously higher than those of the healthy women (P<0.01). After operation the parameters were obviously elevated in the patients (P<0.01). AT-III and partial thromboplastin activation time were significantly reduced in comparison with the healthy subjects (P<0.01). t-PA showed no significant difference between the groups (P>0.05). CONCLUSIONS: Obvious prethrombotic state characterizes the patients with gynecological malignancies after operation.

Human T-lymphotropic virus type I tax activates I-kappa B kinase by inhibiting I-kappa B kinase-associated serine/threonine protein phosphatase 2A.

I-kappa B kinase (IKK) is a serine/threonine kinase that phosphorylates I-kappa B alpha and I-kappa B beta and targets them for polyubiquitination and proteasome-mediated degradation. IKK consists of two highly related catalytic subunits, alpha and beta, and a regulatory gamma subunit, which becomes activated after serine phosphorylation of the activation loops of the catalytic domains. The human T-lymphotropic retrovirus type-I trans-activator, Tax, has been shown to interact directly with IKK gamma and activates IKK via a mechanism not fully understood. Here we demonstrate that IKK binds serine/threonine protein phosphatase 2A (PP2A), and via a tripartite protein-protein interaction, Tax, IKK gamma, and PP2A form a stable ternary complex. In vitro, PP2A down-regulates active IKK prepared from Tax-producing MT4 cells. In the presence of Tax, however, the ability of PP2A to inactivate IKK is diminished. Despite their interaction with IKK gamma, PP2A-interaction-defective Tax mutants failed to activate NF-kappa B. Our data support the notion that IKK gamma-associated PP2A is responsible for the rapid deactivation of IKK, and inhibition of PP2A by Tax in the context of IKK x PP2A x Tax ternary complex leads to constitutive IKK and NF-kappa B activation.