High-throughput identification of regulatory elements and functional assays to uncover susceptibility genes for nasopharyngeal carcinoma.

Large-scale genetic association studies have identified multiple susceptibility loci for nasopharyngeal carcinoma (NPC), but the underlying biological mechanisms remain to be explored. To gain insights into the genetic etiology of NPC, we conducted a follow-up study encompassing 6,907 cases and 10,472 controls and identified two additional NPC susceptibility loci, 9q22.33 (rs1867277; OR = 0.74, 95% CI = 0.68-0.81, p = 3.08 × 10-11) and 17q12 (rs226241; OR = 1.42, 95% CI = 1.26-1.60, p = 1.62 × 10-8). The two additional loci, together with two previously reported genome-wide significant loci, 5p15.33 and 9p21.3, were investigated by high-throughput sequencing for chromatin accessibility, histone modification, and promoter capture Hi-C (PCHi-C) profiling. Using luciferase reporter assays and CRISPR interference (CRISPRi) to validate the functional profiling, we identified PHF2 at locus 9q22.33 as a susceptibility gene. PHF2 encodes a histone demethylase and acts as a tumor suppressor. The risk alleles of the functional SNPs reduced the expression of the target gene PHF2 by inhibiting the enhancer activity of its long-range (4.3 Mb) cis-regulatory element, which promoted proliferation of NPC cells. In addition, we identified CDKN2B-AS1 as a susceptibility gene at locus 9p21.3, and the NPC risk allele of the functional SNP rs2069418 promoted the expression of CDKN2B-AS1 by increasing its enhancer activity. The overexpression of CDKN2B-AS1 facilitated proliferation of NPC cells. In summary, we identified functional SNPs and NPC susceptibility genes, which provides additional explanations for the genetic association signals and helps to uncover the underlying genetic etiology of NPC development.

Clinical and genome-wide association analysis of chemoradiation-induced hearing loss in nasopharyngeal carcinoma.

Chemoradiation-induced hearing loss (CRIHL) is one of the most devasting side effects for nasopharyngeal carcinoma (NPC) patients, which seriously affects survivors' long-term quality of life. However, few studies have comprehensively characterized the risk factors for CRIHL. In this study, we found that age at diagnosis, tumor stage, and concurrent cisplatin dose were positively associated with chemoradiation-induced hearing loss. We performed a genome-wide association study (GWAS) in 777 NPC patients and identified rs1050851 (within the exon 2 of NFKBIA), a variant with a high deleteriousness score, to be significantly associated with hearing loss risk (HR = 5.46, 95% CI 2.93-10.18, P = 9.51 × 10-08). The risk genotype of rs1050851 was associated with higher NFKBIA expression, which was correlated with lower cellular tolerance to cisplatin. According to permutation-based enrichment analysis, the variants mapping to 149 hereditary deafness genes were significantly enriched among GWAS top signals, which indicated the genetic similarity between hereditary deafness and CRIHL. Pathway analysis suggested that synaptic signaling was involved in the development of CRIHL. Additionally, the risk score integrating genetic and clinical factors can predict the risk of hearing loss with a relatively good performance in the test set. Collectively, this study shed new light on the etiology of chemoradiation-induced hearing loss, which facilitates high-risk individuals' identification for personalized prevention and treatment.

The interaction and mediation effects between the host genetic factors and Epstein-Barr virus VCA-IgA in the risk of nasopharyngeal carcinoma.

Previous studies have demonstrated strong associations between host genetic factors and Epstein-Barr virus (EBV) VCA-IgA with the risk of nasopharyngeal carcinoma (NPC). However, the specific interplay between host genetics and EBV VCA-IgA on NPC risk is not well understood. In this two-stage case-control study (N = 4804), we utilized interaction and mediation analysis to investigate the interplay between host genetics (genome-wide association study-derived polygenic risk score [PRS]) and EBV VCA-IgA antibody level in the NPC risk. We employed a four-way decomposition analysis to assess the extent to which the genetic effect on NPC risk is mediated by or interacts with EBV VCA-IgA. We consistently found a significant interaction between the PRS and EBV VCA-IgA on NPC risk (discovery population: synergy index [SI] = 2.39, 95% confidence interval [CI] = 1.85-3.10; replication population: SI = 3.10, 95% CI = 2.17-4.44; all pinteraction < 0.001). Moreover, the genetic variants included in the PRS demonstrated similar interactions with EBV VCA-IgA antibody. We also observed an obvious dose-response relationship between the PRS and EBV VCA-IgA antibody on NPC risk (all ptrend < 0.001). Furthermore, our decomposition analysis revealed that a substantial proportion (approximately 90%) of the genetic effects on NPC risk could be attributed to host genetic-EBV interaction, while the risk effects mediated by EBV VCA-IgA antibody were weak and statistically insignificant. Our study provides compelling evidence for an interaction between host genetics and EBV VCA-IgA antibody in the development of NPC. These findings emphasize the importance of implementing measures to control EBV infection as a crucial strategy for effectively preventing NPC, particularly in individuals at high genetic risk.

Peptidome-wide association analysis of Epstein-Barr virus identifies epitope repertoires associated with nasopharyngeal carcinoma.

Human leukocyte antigen (HLA) molecules are essential for presenting Epstein-Barr virus (EBV) antigens and are closely related to nasopharyngeal carcinoma (NPC). This study aims to systematically investigate the association between HLA-bound EBV peptides and NPC risk through in silico HLA-peptide binding prediction. A total of 455 NPC patients and 463 healthy individuals in NPC endemic areas were recruited, and HLA-target sequencing was performed. HLA-peptide binding prediction for EBV, followed by peptidome-wide logistic regression and motif analysis, was applied. Binding affinity changes for EBV peptides carrying high-risk mutations were analyzed. We found that NPC-associated EBV peptides were significantly enriched in immunogenic proteins and core linkage disequilibrium (LD) proteins related to evolution, especially those binding HLA-A alleles (p = 3.10 × 10-4 for immunogenic proteins and p = 8.10 × 10-5 for core LD proteins related to evolution). These peptides were clustered and showed binding motifs of HLA supertypes, among which supertype A02 presented an NPC-risk effect (padj = 3.77 × 10-4 ) and supertype A03 presented an NPC-protective effect (padj = 4.89 × 10-4 ). Moreover, a decreased binding affinity toward risk HLA supertype A02 was observed for the peptide carrying the NPC-risk mutation BNRF1 V1222I (p = 0.0078), and an increased binding affinity toward protective HLA supertype A03 was observed for the peptide carrying the NPC-risk mutation BALF2 I613V (p = 0.022). This study revealed the distinct preference of EBV peptides for binding HLA supertypes, which may contribute to shaping EBV population structure and be involved in NPC development.

Membrane-Bound EMC10 Is Required for Sperm Motility via Maintaining the Homeostasis of Cytoplasm Sodium in Sperm.

Endoplasmic reticulum membrane protein complex subunit 10 (EMC10) is an evolutionarily conserved and multifunctional factor across species. We previously reported that Emc10 knockout (KO) leads to mouse male infertility. Emc10-null spermatozoa exhibit multiple aspects of dysfunction, including reduced sperm motility. Two subunits of a Na/K-ATPase, ATP1A4 and ATP1B3, are nearly absent in Emc10 KO spermatozoa. Here, two isoforms of EMC10 were characterized in the mouse testis and epididymis: the membrane-bound (mEMC10) and secreted (scEMC10) isoforms. We present evidence that mEMC10, rather than scEMC10, is required for cytoplasm sodium homeostasis by positively regulating ATP1B3 expression in germ cells. Intra-testis mEMC10 overexpression rescued the sperm motility defect caused by Emc10 KO, while exogenous recombinant scEMC10 protein could not improve the motility of spermatozoa from either Emc10 KO mouse or asthenospermic subjects. Clinically, there is a positive association between ATP1B3 and EMC10 protein levels in human spermatozoa, whereas no correlation was proven between seminal plasma scEMC10 levels and sperm motility. These results highlight the important role of the membrane-bound EMC10 isoform in maintaining cytoplasm sodium homeostasis and sperm motility. Based on the present results, the mEMC10-Na, K/ATPase α4ß3 axis is proposed as a novel mechanism underlying the regulation of cytoplasmic sodium and sperm motility, and its components seem to have therapeutic potential for asthenospermia.

Soluble expression and purification of human ß-defensin DEFB136 in Escherichia coli and identification of its bioactivity.

Human ß-defensins are an important family of innate host defense peptides with pleiotropic activities. Human ß-defensin 36 (DEFB136) is a novel member of the ß-defensin family which have not been characterized so far. In the present research, the DEFB136 peptide was expressed successfully and purified using the IMPACT-TWIN 1 expression system. The purified DEFB136 peptide was identified by MALDI-TOF mass spectrometry and circular dichroism spectroscopy. While the recombinant DEFB136 peptide exhibited a broad spectrum of antimicrobial activity against E. coli, Staphylococcus aureus and Candida albicans strains, but had low cytotoxicity to human erythrocytes. In addition, the result of the octet assay showed that the DEFB136 had a high lipopolysaccharide (LPS)-binding affinity, suggesting the DEFB136 may be involved in immunoregulation through its LPS neutralization. These results may help lay the groundwork to understand better the complex interaction between innate host defense and the diversity of the defensin family.

A novel posttranslational modification of histone, H3 S-sulfhydration, is down-regulated in asthenozoospermic sperm.

Oxidative stress is one of the major causes leading to male infertility including asthenozoospermia. Hydrogen sulfide (H2S) has been widely recognized to be a potent antioxidant whose role is partially implemented by protein S-sulfhydration. However, protein S-sulfhydration has not been reported in germ cells. Therefore, we investigated whether asthenozoospermia could be associated with sperm protein S-sulfhydration. S-sulfhydrated proteins in human sperm were enriched via biotin-switch assay and analyzed using LC-MS/MS spectrometry. Two hundred forty-four S-sulfhydrated proteins were identified. Importantly, we validated that sperm histones H3.1 and H3.3 were the S-sulfhydrated proteins. Their S-sulfhydrated amino acid residue was Cysteine111. Abundances of S-sulfhydrated H3 (sH3) and S-sulfhydrated H3.3 (sH3.3) were significantly down-regulated in asthenozoospermic sperm, compared with the fertile controls, and were significantly correlated with progressive motility. Retinoic acid (RA) up-regulated level of sH3.3 in primary round spermatids and the C18-4 cells (a mouse spermatogonial stem cell line). Overexpression of the mutant H3.3 (Cysteine111 was replaced with serine) affected expression of 759 genes and raised growth rate of C18-4 cells. For the first time, S-sulfhydration H3 and H3.3 were demonstrated in the present study. Our results highlight that aberrant S-sulfhydration of H3 is a new pathophysiological basis in male infertility.

The impacts of multiple obesity-related interventions on quality of life in children and adolescents: a randomized controlled trial.

BACKGROUND AND PURPOSE: Obesity has become a serious public health problem and family- and school-based interventions including physical exercise and diet control have been widely applied to attempt to combat this issue. The purpose of our study was to verify the effectiveness of an obesity-related comprehensive intervention model aimed at improving quality of life (QoL) among adolescents. METHODS: A cluster randomized controlled trial (RCT) was conducted involving 948 subjects who were divided into an intervention group (n = 518) and a control group (n = 430). The intervention group received 1 year of obesity-related health education, physical exercise, and diet control. Their baseline body mass index (BMI) was calculated, and their QoL and basic information were assessed both before and after the intervention period using a self-designed Adolescent Quality of Life Scale and a basic information questionnaire. RESULTS: After the intervention, significant differences in the psychological, social, and pubertal dimensions, and in total QoL (P < 0.05) were observed in the intervention group relative to the control group. Improved psychological QoL in the intervention group was our most robust study finding, with increases in psychological (B = 1.883, SE = 0.646, P = 0.004), pubertal (B = 0.853, SE = 0.296, P = 0.004) and total (B = 3.024, SE = 1.214, P = 0.013) QoL all being higher in this group. This intervention effect was found to be more substantial in boys than in girls. CONCLUSIONS: Family-individual-school-based interventions combining obesity-related health education, physical exercise, and diet control can improve psychological, pubertal, and total QoL in children, with these effects being most pronounced in boys. TRIAL REGISTRATION: retrospectively registered NCT02343588 .

The impacts of peer education based on adolescent health education on the quality of life in adolescents: a randomized controlled trial.

PURPOSE: During adolescence, adolescents are more susceptible to internalizing and externalizing problems influencing quality of life (QoL). The purpose of the study is to verify the effectiveness of a peer education on improving QoL of adolescents. METHODS: A cluster randomized controlled trial (RCT) was conducted involving 1564 subjects who were divided into an intervention group (n = 714) and a control group (n = 850). The intervention group received 1-year peer education. Their QoL and basic information were assessed using a Adolescent Quality of Life Scale and a self-designed basic situation questionnaire. RESULTS: After the intervention, significant increases were found in the psychological, and social, pubertal dimensions, and in total QoL (P < 0.05) in the intervention group relative to the control group. Significant decrease was found in physical dimension (P < 0.05), but the change in the intervention group (0.74 decrease) was much less than that in the control group (1.94 decrease). The improvements of physical (B = 1.215, SE = 0.305, P < 0.001), psychological (B = 1.496, SE = 0.598, P = 0.013), pubertal (B = 0.828, SE = 0.244, P = 0.001), and total (B = 3.455, SE = 1.429, P = 0.016) QoL in the intervention group were higher than in the control group in mixed model. CONCLUSIONS: The peer education based on adolescent health education is effective in improving the physical, psychological, pubertal, and total QoL of adolescents, but no social QoL.

The association between sleep duration, bedtimes, and early pubertal timing among Chinese adolescents: a cross-sectional study.

BACKGROUND AND OBJECTIVE: Early pubertal timing is associated with sleep among Western adolescents, but little is known about this association in Chinese adolescents, especially with regard to the association between bedtimes and early pubertal timing. This paper aimed to identify the association between sleep duration, bedtimes, and early pubertal timing in Chinese adolescents. METHODS: An anonymous cross-sectional survey was conducted among primary and junior middle students (grades 3 to 9) from QiJiang District, ChongQing, China. Participants were recruited by applying stratified cluster sampling. Pubertal timing, sleep duration, and bedtimes were assessed using the Pubertal Development Scale and a self-designed sleep questionnaire. We utilized multivariable logistic linear regression (MLLR) to test the association between sleep duration, bedtimes, and pubertal timing. RESULTS: A total of 5461 adolescents were evaluated, with mean age and BMI values of 11.41 ± 2.05 and 18.03 ± 3.03, respectively, of whom 1257 (23.02%) were in early pubertal timing. In MLLR controlling for age, BMI, family economic status, and other covariates, sufficient sleep (b = - 0.214, P = 0.032, OR = 0.808, 95% CI 0.664-0.982) was negatively related to early pubertal timing, and later bedtime (b = 0.195, P < 0.001, OR = 1.215, 95% CI 1.104-1.338) was positively associated with early pubertal timing. CONCLUSION: Students with early pubertal timing had less sleep duration and later bedtimes, which may be the result of increased stress caused by physical and psychological changes. Therefore, more attention should be paid to pubertal health education for adolescents during puberty. Further longitudinal studies are needed to confirm the causality between sleep and early pubertal timing in Chinese adolescents.

Comparative analysis of human sperm glycocalyx from different freezability ejaculates by lectin microarray and identification of ABA as sperm freezability biomarker.

BACKGROUND: Semen cryopreservation has been widely applied in assisted reproductive technologies and sperm bank, but it causes considerable impairments on sperm quality. It is necessary to find an evaluation indicator for determining the sperm-freezing tolerance. METHODS: The glycocalyx of good freezability ejaculates was compared with poor freezability ejaculates by lectin microarray. The significant different lectins were validated by flow cytometry (FACS). To analyze the relationship between the potential biomarker and the tolerance of sperm to cryopreservation, 60 samples with different recovery rates were collected and detected the lectin-binding intensity by FACS. The receiver operating characteristic (ROC) curve was analyzed to test the capability of the lectin as a potential biomarker for detecting the sperm freezablility. RESULTS: ABA and DSL were found to develop significant differences between them. Further validation showed that ABA was significantly negative correlated with the sperm recovery rates (r = - 0.618, P < 0.000) and could be a potential biomarker for predicting sperm freezability (AUC = 0.733 ± 0.067, 95% CI 0.601 - 0.865, P < 0.01). CONCLUSION: ABA could be a potential biomarker for predicting sperm freezability. It will help to reduce sperm-freezing recovery tests and improve the efficiency of cryopreservation in human sperm bank.

A Human Lectin Microarray for Sperm Surface Glycosylation Analysis.

Glycosylation is one of the most abundant and functionally important protein post-translational modifications. As such, technology for efficient glycosylation analysis is in high demand. Lectin microarrays are a powerful tool for such investigations and have been successfully applied for a variety of glycobiological studies. However, most of the current lectin microarrays are primarily constructed from plant lectins, which are not well suited for studies of human glycosylation because of the extreme complexity of human glycans. Herein, we constructed a human lectin microarray with 60 human lectin and lectin-like proteins. All of the lectins and lectin-like proteins were purified from yeast, and most showed binding to human glycans. To demonstrate the applicability of the human lectin microarray, human sperm were probed on the microarray and strong bindings were observed for several lectins, including galectin-1, 7, 8, GalNAc-T6, and ERGIC-53 (LMAN1). These bindings were validated by flow cytometry and fluorescence immunostaining. Further, mass spectrometry analysis showed that galectin-1 binds several membrane-associated proteins including heat shock protein 90. Finally, functional assays showed that binding of galectin-8 could significantly enhance the acrosome reaction within human sperms. To our knowledge, this is the first construction of a human lectin microarray, and we anticipate it will find wide use for a range of human or mammalian studies, alone or in combination with plant lectin microarrays.

Dynamic cyclic compression modulates the chondrogenic phenotype in human chondrocytes from late stage osteoarthritis.

Human osteoarthritic chondrocytes (hOACs) are characterized by their "dedifferentiated" and catabolic phenotype and lack the ability for restoring their inherent functions by themselves. Here we investigated whether extrinsically supplemented mechanical signal via compression loading would affect the phenotype of hOACs. Specifically, we applied cyclic compression loading on cultured hOACs-collagen constructs and measured the expression of the major chondrogenic factors, cell-matrix interaction molecules and matrix degradation enzymes. Dynamic compression loading stimulates the expression and nuclear localization of sox9 in hOACs and reduces the catabolic events via downregulated expression of collagenases. These results contribute to better understanding towards mechanoregulation of hOACs.

Acetylproteomic analysis reveals functional implications of lysine acetylation in human spermatozoa (sperm).

Male infertility is a medical condition that has been on the rise globally. Lysine acetylation of human sperm, an essential posttranslational modification involved in the etiology of sperm abnormality, is not fully understood. Therefore, we first generated a qualified pan-anti-acetyllysine monoclonal antibody to characterize the global lysine acetylation of uncapacitated normal human sperm with a proteomics approach. With high enrichment ratios that were up to 31%, 973 lysine-acetylated sites that matched to 456 human sperm proteins, including 671 novel lysine acetylation sites and 205 novel lysine-acetylated proteins, were identified. These proteins exhibited conserved motifs XXXKYXXX, XXXKFXXX, and XXXKHXXX, were annotated to function in multiple metabolic processes, and were localized predominantly in the mitochondrion and cytoplasmic fractions. Between the uncapacitated and capacitated sperm, different acetylation profiles in regard to functional proteins involved in sperm capacitation, sperm-egg recognition, sperm-egg plasma fusion, and fertilization were observed, indicating that acetylation of functional proteins may be required during sperm capacitation. Bioinformatics analysis revealed association of acetylated proteins with diseases and drugs. Novel acetylation of voltage-dependent anion channel proteins was also found. With clinical sperm samples, we observed differed lysine acetyltransferases and lysine deacetylases expression between normal sperm and abnormal sperm of asthenospermia or necrospermia. Furthermore, with sperm samples impaired by epigallocatechin gallate to mimic asthenospermia, we observed that inhibition of sperm motility was partly through the blockade of voltage-dependent anion channel 2 Lys-74 acetylation combined with reduced ATP levels and mitochondrial membrane potential. Taken together, we obtained a qualified pan-anti-acetyllysine monoclonal antibody, analyzed the acetylproteome of uncapacitated human sperm, and revealed associations between functional protein acetylation and sperm functions.

Histone deacetylase inhibitor induces cell apoptosis and cycle arrest in lung cancer cells via mitochondrial injury and p53 up-acetylation.

The reversibility of non-genotoxic phenotypic changes has been explored in order to develop novel preventive and therapeutic approaches for cancer. Quisinostat (JNJ-26481585), a novel second-generation histone deacetylase inhibitor (HDACi), has efficient therapeutic actions on non-small cell lung cancer (NSCLC) cell. The present study aims at investigating underlying molecular mechanisms involved in the therapeutic activity of quisinostat on NSCLC cells. We found that quisinostat significantly inhibited A549 cell proliferation in dose- and time-dependent manners. Up-acetylation of histones H3 and H4 and non-histone protein α-tubulin was induced by quisinostat treatment in a nanomolar concentration. We also demonstrated that quisinostat increased reactive oxygen species (ROS) production and destroyed mitochondrial membrane potential (ΔΨm), inducing mitochondria-mediated cell apoptosis. Furthermore, exposure of A549 cells to quisinostat significantly suppressed cell migration by inhibiting epithelial-mesenchymal transition (EMT) process. Bioinformatics analysis indicated that effects of quisinostat on NSCLC cells were associated with activated p53 signaling pathway. We found that quisinostat increased p53 acetylation at K382/K373 sites, upregulated the expression of p21(Waf1/Cip1), and resulted in G1 phase arrest. Thus, our results suggest that the histone deacetylase can be a therapeutic target of NSCLC to discover and develop a new category of therapy for lung cancer.

Another functional frame-shift polymorphism of DEFB126 (rs11467497) associated with male infertility.

DEFB126 rs140685149 mutation was shown to cause sperm dysfunction and subfertility. Indel rs11467497 is another 4-nucleotide frame-shift mutation (151bp upstream of rs140685149) that leads to the premature termination of translation and the expression of peptide truncated at the carboxyl terminus. In the present study, we performed a comprehensive association study to check the contribution of rs140685149 and rs11467497 to male infertility. Our results confirmed the previous findings that there was no association between rs140685149 and sperm motility. In contrast, we found a significant association of another indel rs11467497 with male infertility. Moreover, rs11467497 was shown to be associated with higher number of round cells in the infertile males with low sperm motility. Surprisingly, the two mutations commonly existed in the sperm donors (n = 672), suggesting a potential application of the two indels in the screening for eligible sperm donors. Western blotting assays showed the sperms with rs140685149 2-nt deletion tended to have unstable DEFB126 protein in contrast of no DEFB126 protein expressed in the sperms with rs11467497 4-nt deletion, suggesting a more severe consequence caused by rs11467497 mutation. In conclusion, our study presented a significant contribution of another functional frame-shift polymorphism of DEFB126 (rs11467497) to male infertility.

Characterization of ß-defensin 42 expressed in principal cells at the initial segment of the rat epididymis.

ß-defensins, preferentially expressed in male reproductive tracts, particularly in the testes and epididymis with region-specific patterns, play an important role in both innate immunity and sperm fertility. Expressed in the caput region of epididymis, ß-defensins have been known to contribute to innate immunity, sperm motility initiation, and maintenance. However, ß-defensins of the initial region remain to be uncharacterized. In this study, rat ß-defensin 42 (Defb42) was revealed to be exclusively located in the principal cells at the initial segment of the rat epididymis and its sperm's acrosome. Furthermore, the expression of Defb42 was dependent on luminal testicular factors and developmental phases. The recombinant Defb42 was predominantly antimicrobial not against Candida albicans, but against Escherichia coli and Staphylococcus aureus. Based on these findings, Defb42 was suggested to play a dual role in sperm fertility and host defense in rat epididymis.

The novel human ß-defensin 114 regulates lipopolysaccharide (LPS)-mediated inflammation and protects sperm from motility loss.

Lipopolysaccharide (LPS) is an important pathological factor involved in serious inflammatory diseases and male reproductive impairments. Emerging evidence demonstrates that antimicrobial peptides possess protective activity in response to LPS-induced inflammation. However, the LPS-binding and/or immunosuppressive activity of ß-defensins (DEFBs) has been underestimated. In the present work, we characterized a novel human defensin, DEFB114, which was expressed predominantly in the epididymis and gingival cells at the RNA level. Homogenous recombinant DEFB114 peptides were prepared and characterized using mass spectrometry. DEFB114 protein exhibited a broad spectrum of antimicrobial activity with salt sensitivity against typical pathogenic microbes (i.e. Escherichia coli, Staphylococcus aureus, and Candida albicans). Interestingly, DEFB114 demonstrated novel LPS-binding activity in vitro and inhibited TNF-α release in RAW264.7 cultures through the inhibition of MAPK p42/44 when challenged with LPS. Moreover, DEFB114 could also rescue the LPS-induced reduction of human sperm motility in vitro and protect d-galactosamine-sensitized C57BL/6 mice from LPS-induced lethality in vivo. The protective activity of DEFB114 on RAW264.7, human sperm, and the d-galactosamine-sensitized mice was disulfide bond-dependent because alkylated DEFB114 lost its activity. The low cytotoxicity of the DEFB114 peptide toward human erythrocytes is indicative of its potential therapeutic use in the treatment of LPS-induced inflammation, LPS contamination, and potentially septic shock.

Comprehensive profiling of accessible surface glycans of mammalian sperm using a lectin microarray.

It is well known that cell surface glycans or glycocalyx play important roles in sperm motility, maturation and fertilization. A comprehensive profile of the sperm surface glycans will greatly facilitate both basic research (sperm glycobiology) and clinical studies, such as diagnostics of infertility. As a group of natural glycan binders, lectin is an ideal tool for cell surface glycan profiling. However, because of the lack of effective technology, only a few lectins have been tested for lectin-sperm binding profiles. To address this challenge, we have developed a procedure for high-throughput probing of mammalian sperm with 91 lectins on lectin microarrays. Normal sperm from human, boar, bull, goat and rabbit were collected and analyzed on the lectin microarrays. Positive bindings of a set of ~50 lectins were observed for all the sperm of 5 species, which indicated a wide range of glycans are on the surface of mammalian sperm. Species specific lectin bindings were also observed. Clustering analysis revealed that the distances of the five species according to the lectin binding profiles are consistent with that of the genome sequence based phylogenetic tree except for rabbit. The procedure that we established in this study could be generally applicable for sperm from other species or defect sperm from the same species. We believe the lectin binding profiles of the mammalian sperm that we established in this study are valuable for both basic research and clinical studies.

Production and characterization of recombinant human beta-defensin DEFB120.

Public health of human beings is threatened by superbugs. Novel human beta-defensins, which contribute to host defense against pathogen invasion and innate immune protection, might be a potent natural candidate pool for new antibiotic lead screening. In the present work, we successfully expressed and purified a novel human beta-defensin, DEFB120, using the IMPACT-TWIN system in Escherichia coli and identified the purified homogeneous proteins using MALDI-TOF mass spectrometry. Then, we performed the fundamental studies on the structure and biological functions for the DEFB120 peptide. The recombinant DEFB120 peptide showed wide antimicrobial effects against E. coli, Staphylococcus aureus and Candida albicans strains without significant hemolytic activity. Furthermore, the high lipopolysaccharide (LPS)-binding affinity in vitro indicated that DEFB120 might be associated with the inhibition of LPS-induced inflammatory response. These results may pave a way for exploiting the essential physiological functions of DEFB120 and also for the development of natural antibiotic pools.