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Microctis Folium is the dried leaves of a plant (Microcos paniculata L.) used to improve the digestive system, alleviate diarrhoea, and relieve fever, but information regarding its chemical composition has rarely been reported. The traditional research approach of determining chemical composition has included isolating, purifying, and identifying compounds with high-cost and time-consuming processes. In this study, molecular networking (MN) and fingerprint analysis were integrated as a comprehensive approach to study the chemical composition of Microctis Folium by an ultra fast liquid chromatography-photo diode array detector-triple-time of flight-tandem mass spectrometry (UFLC-DAD-Triple TOF-MS/MS). Large numbers of mass spectrometric data were processed to identify constituents, and the identified compounds and their unknown analogues were comprehensively depicted as visualized figures comprising distinct families by MN. A validated fingerprint methodology was established to quantitatively determine compounds in Microctis Folium. Ultimately, 165 constituents were identified in Microctis Folium for the first time and the identified compounds and approximately five hundred unknown analogues were applied to create visualized figures by MN, indicating compound groups and their chemical structure analogues in Microctis Folium. The validated fingerprint methodology was indicated to be specific, repeatable, precise, and stable and was used to determine 15 batches of samples during three seasons in three districts. Furthermore, seasonal or geographic environmental influences on the chemical profile were estimated by principal coordinate analysis. The results can be used to control the quality of Microctis Folium, observe seasonal or geographic environmental influences on the chemical profiles, and provide a reference for further exploitation of potential active unknown analogues in the future.

Silicosis is a systemic disease characterized by chronic persistent inflammation and incurable pulmonary fibrosis with the underlying molecular mechanisms to be fully elucidated. In this study, we employed tandem mass tag (TMT) based on quantitative proteomics technology to detect differentially expressed proteins (DEPs) in lung tissues of silica-exposed rats. A total of 285 DEPs (145 upregulated and 140 downregulated) were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to predict the biological pathway and functional classification of the proteins. Results showed that these DEPs were mainly enriched in the phagosome, lysosome function, complement and the coagulation cascade, glutathione metabolism, focal adhesion and ECM-receptor interactions. To validate the proteomics data, we selected and analyzed the expression trends of six proteins including CD14, PSAP, GM2A, COL1A1, ITGA8 and CLDN5 using parallel reaction monitoring (PRM). The consistent result between PRM and TMT indicated the reliability of our proteomic data. These findings will help to reveal the pathogenesis of silicosis and provide potential therapeutic targets. Data are available via ProteomeXchange with identifier PXD020625.

Explaining and predicting animal distributions is one of the fundamental objectives in ecology and conservation biology. Animal habitat selection can be regulated by top-down and bottom-up processes, and is mediated by species interactions. Species varying in body size respond differently to top-down and bottom-up determinants, and hence understanding these allometric responses to those determinants is important for conservation. In this study, using two differently sized goose species wintering in the Yangtze floodplain, we tested the predictions derived from three different hypotheses (individual-area relationship, food resource and disturbance hypothesis) to explain the spatial and temporal variation in densities of two goose species. Using Generalized Linear Mixed Models with a Markov Chain Monte Carlo technique, we demonstrated that goose density was positive correlated with patch area size, suggesting that the individual area-relationship best predicts differences in goose densities. Moreover, the other predictions, related to food availability and disturbance, were not significant. Buffalo grazing probably facilitated greater white-fronted geese, as the number of buffalos was positively correlated to the density of this species. We concluded that patch area size is the most important factor determining the density of goose species in our study area. Patch area size is directly determined by water levels in the Yangtze floodplain, and hence modifying the hydrological regimes can enlarge the capacity of these wetlands for migratory birds.

We aimed to measure prevalence of sleep disturbance in patients with differentiated thyroid cancer (DTC) by calculating Pittsburgh Sleep Quality Index (PSQI), and compare these data with patients with benign thyroid nodules or normal participants.Three groups of patients participated in this cross-sectional study. In the first group, 162 patients with DTC received total thyroidectomy, and then 131I therapy. The second group consisted of 84 patients with benign thyroid nodules, who received partial thyroidectomy. The third group was 78 normal healthy control cases. PSQI was used to assess the sleep quality. Inter-group differences were analyzed by Kruskal-Wallis test or independent samples T test. χ2 test was also used to check prevalence differences of poor sleep quality among the groups. Differences of PSQI score and poor sleep quality prevalence before and after 131I therapy in the same group of DTC participants were analyzed by paired T test and Mcnemar's test.Higher PSQI score (7.59 ± 4.21) and higher rate of poor sleep quality (54.32%) were shown in DTC patients than in any other group. After 131I therapy, PSQI score and prevalence of poor sleep quality in DTC patients increased significantly to 8.78 ± 4.72 and 70.99%. Then DTC patients were divided into two subgroups based on their metastatic status. DTC patients with metastasis (87/162 cases, 53.70%) had significantly higher PSQI score (10.87 ± 5.18) and higher prevalence of poor sleep quality (79.31%).DTC patients suffer from sleep disturbance, 131I therapy and awareness of metastatic status could worsen sleep problem. Psychological fear of cancer, nuclear medicine therapy and metastasis could be one major underlying reason. Longitude and interventional studies are necessary for further investigations.

PURPOSE: To assess scintigraphic pattern, clinical indication and relevance of dual ectopic thyroid tissue (ETT). Literature is reviewed for such cases. METHODS: In this 5-year retrospective study, we reviewed all thyroid scintigraphies in our data base. Patients diagnosed with suspected ETT were identified. Literature is reviewed. Statistics were done by one-way analysis of variance and least significant difference test. RESULTS: From 11905 thyroid scintigraphies during the 5-year period, we retrieved 121 patients eligible for analysis. The top two indications were assessing a palpable front neck mass to determine whether it was an ETT, and primary hypothyroidism. Patients were divided into 3 groups. Group 1 with single ETT (83 cases); group 2 with dual ETT (6 cases) and group 3 with athyroid (32 cases). Age and thyroid hormones were highest in group 2, and lowest in group 3. Thyrotropin was highest in group 3, and lowest in group 2. Thyroxine was given to hypothyroid patients, while no surgery was performed. There were 42 published cases with dual ETT, most of whom were under 30 years old. 38.10% of them were euthyroid, 33.33% hypothyroid, and 21.43% subclinical hypothyroid. Most frequent ectopic positions included lingual (33.73%), sublingual (27.71%) and subhyoid (22.89%). CONCLUSIONS: In our cohort, incidence of dual ETT was 0.05% if the denominator was total number of thyroid scintigraphies. The incidence was 4.96% if the denominator was the number of patients with suspected ETT. Important clinical indication is a front neck palpable mass suggestive of an ETT. Important clinical relevance of recognizing the dual ETT pattern is to avoid inappropriate surgery. After reviewing all published cases, we find dual ETT is often seen in young patients. Most of such patients are euthyroid or mildly hypothyroid. Thyroid ectopia often resides in lingual, sublingual and subhyoid areas.

Formaldehyde (FA) is a well-known irritant, and it is suggested to increase the risk of immune diseases and cancer. The present study aimed to evaluate the distribution of major lymphocyte subsets and cytokine expression profiles in the peripheral blood of FA-exposed workers. A total of 118 FA-exposed workers and 79 controls were enrolled in the study. High performance liquid chromatography, flow cytometry, and cytometric bead array were used to analyze FA in air sample and formic acid in urine, blood lymphocyte subpopulations, and serum cytokines, respectively. The FA-exposed workers were divided into low and high exposure groups according to their exposure levels. The results showed that both the low and high FA-exposed groups had a significant increase of formic acid in urine when compared to the controls. Both the low and high exposure groups had a significant increase in the percentage of B cells (CD19+) compared to the control group (p

Hand, foot and mouth disease caused by enterovirus 71(EV71) leads to the majority of neurological complications and death in young children. While putative inactivated vaccines are only now undergoing clinical trials, no specific treatment options exist yet. Ideally, EV71 specific intravenous immunoglobulins could be developed for targeted treatment of severe cases. To date, only a single universally neutralizing monoclonal antibody against a conserved linear epitope of VP1 has been identified. Other enteroviruses have been shown to possess major conformational neutralizing epitopes on both the VP2 and VP3 capsid proteins. Hence, we attempted to isolate such neutralizing antibodies against conformational epitopes for their potential in the treatment of infection as well as differential diagnosis and vaccine optimization. Here we describe a universal neutralizing monoclonal antibody that recognizes a conserved conformational epitope of EV71 which was mapped using escape mutants. Eight escape mutants from different subgenogroups (A, B2, B4, C2, C4) were rescued; they harbored three essential mutations either at amino acid positions 59, 62 or 67 of the VP3 protein which are all situated in the "knob" region. The escape mutant phenotype could be mimicked by incorporating these mutations into reverse genetically engineered viruses showing that P59L, A62D, A62P and E67D abolish both monoclonal antibody binding and neutralization activity. This is the first conformational neutralization epitope mapped on VP3 for EV71.

Wogonin is a plant monoflavonoid which has been reported to inhibit cell growth and/or induce apoptosis in various tumors. The present study examined the apoptosis-inducing activity and underlying mechanism of action of wogonin in A549 cells. The results showed that wogonin was a potent inhibitor of the viability of A549 cells. Apoptotic protein changes detected after exposure to wogonin included decreased XIAP and Mcl-1 expression, increased cleaved-PARP expression and increased release of AIF and cytochrome C. Western blot analysis showed that the activity of c-Myc/Skp2 and HDAC1/HDAC2 pathways, which play important roles in tumor progress, was decreased. Quantitative PCR identified increased levels of c-Myc mRNA and decreased levels of its protein. Protein levels of Fbw7α, GSK3β and Thr58-Myc, which are involved in c-Myc ubiquitin-dependent degradation, were also analyzed. After exposure to wogonin, Fbw7α and GSK3β expression decreased and Thr58-Myc expression increased. However, MG132 was unable to prevent c-Myc degradation. The present results suggest that wogonin has multiple anti-cancer effects associated with degradation of c-Myc, SKP2, HDAC1 and HDAC2. Its ability to induce apoptosis independently of Fbw7α suggests a possible use in drug-resistance cancer related to Fbw7 deficiency. Further studies are needed to determine which pathways are related to c-Myc and Fbw7α reversal and whether Thr58 phosphorylation of c-Myc is dependent on GSK3β.

Bufalin is the primary component of the traditional Chinese herb "Chan Su". Evidence suggests that this compound possesses potent anti-tumor activities, although the exact molecular mechanism(s) is unknown. Our previous study showed that bufalin inhibited growth of human osteosarcoma cell lines U2OS and U2OS/MTX300 in culture. Therefore, this study aims to further clarify the in vitro and in vivo anti-osteosarcoma effects of bufalin and its molecular mechanism of action. We found bufalin inhibited both methotrexate (MTX) sensitive and resistant human osteosarcoma cell growth and induced G2/M arrest and apoptosis. Using a comparative proteomics approach, 24 differentially expressed proteins following bufalin treatment were identified. In particular, the level of an anti-apoptotic protein, heat shock protein 27 (Hsp27), decreased remarkably. The down-regulation of Hsp27 and alterations of its partner signaling molecules (the decrease in p-Akt, nuclear NF-κB p65, and co-immunoprecipitated cytochrome c/Hsp27) were validated. Hsp27 over-expression protected against bufalin-induced apoptosis, reversed the dephosphorylation of Akt and preserved the level of nuclear NF-κB p65 and co-immunoprecipitated Hsp27/cytochrome c. Moreover, bufalin inhibited MTX-resistant osteosarcoma xenograft growth, and a down-regulation of Hsp27 in vivo was observed. Taken together, bufalin exerted potent anti-osteosarcoma effects in vitro and in vivo, even in MTX resistant osteosarcoma cells. The down-regulation of Hsp27 played a critical role in bufalin-induced apoptosis in osteosarcoma cells. Bufalin may have merit to be a potential chemotherapeutic agent for osteosarcoma, particularly in MTX-resistant groups.

OBJECTIVE: To evaluate changes of nuclear factor-kappa B (NF-κB) during radioiodine 131 ((131)I) therapy and whether NF-κB inhibition could enhance (131)I-induced apoptosis in differentiated thyroid cancer (DTC) cells in a synergistic manner. METHODS: Three human DTC cell lines were used. NF-κB inhibition was achieved by using a NF-κB inhibitor (Bay 11-7082) or by p65 siRNA transfection. Methyl-thiazolyl-tetrazolium assay was performed for cell viability assessment. DNA-binding assay, luciferase reporter assay, and Western blot were adopted to determine function and expression changes of NF-κB. Then NF-κB regulated anti-apoptotic factors XIAP, cIAP1, and Bcl-xL were measured. Apoptosis was analyzed by Western blot for caspase 3 and PARP, and by flow cytometry as well. An iodide uptake assay was performed to determine whether NF-κB inhibition could influence radioactive iodide uptake. RESULTS: The methyl-thiazolyl-tetrazolium assay showed significant decrease of viable cells by combination therapy than by mono-therapies. The DNA-binding assay and luciferase reporter assay showed enhanced NF-κB function and reporter gene activities due to (131)I, yet significant suppression was achieved by NF-κB inhibition. Western blot proved (131)I could increase nuclear NF-κB concentration, while NF-κB inhibition reduced NF-κB concentration. Western blot also demonstrated significant up-regulation of XIAP, cIAP1, and Bcl-xL after (131)I therapy. And inhibition of NF-κB could significantly down-regulate these factors. Finally, synergism induced by combined therapy was displayed by significant enhancements of cleaved caspase 3 and PARP from Western blot, and of Annexin V positively staining from flow cytometry. The iodine uptake assay did not show significant changes when NF-κB was inhibited. CONCLUSION: We demonstrated that (131)I could induce NF-κB activation, which would attenuate (131)I efficacy in DTC cells. NF-κB inhibition by Bay 11-7082 or by p65 siRNA transfection was effective in suppressing NF-κB regulated anti-apoptotic changes and in combined regimen apoptosis was achieved synergistically.

BACKGROUND: Enterovirus 71 (EV71) is the main causative agent of Hand, Foot and Mouth disease (HFMD) and is associated with severe neurologic complications and mortalities. At present, there is no vaccine or therapeutic available for treatment. METHODOLOGY/PRINCIPAL FINDING: In this study, we generated two mAbs, denoted as mAb 51 and 53, both targeting the same linear epitope on VP1 capsid protein, spanning amino acids 215-219. In comparison, mAb 51 belonging to isotype IgM possesses neutralizing activity in vitro, whereas, mAb 53 belonging to isotype IgG1 does not have any neutralizing ability, even towards its homologous strain. When mAb 51 at 10 µg/g of body weight was administered to the 2-week-old AG129 mice one day prior to lethal challenge, 100% in vivo passive protection was observed. In contrast, the isotype control group mice, injected with an irrelevant IgM antibody before the challenge, developed limb paralysis as early as day 6 post-infection. Histological examination demonstrated that mAb 51 was able to protect against pathologic changes such as neuropil vacuolation and neuronal loss in the spinal cord, which were typical in unprotected EV-71 infected mice. BLAST analyses of that epitope revealed that it was highly conserved among all EV71 strains, but not coxsachievirus 16 (CA16). CONCLUSION: We have defined a linear epitope within the VP1 protein and demonstrated its neutralizing ability to be isotype dependent. The neutralizing property and highly conserved sequence potentiated the application of mAb 51 and 53 for protection against EV71 infection and diagnosis respectively.

[This corrects the article DOI: 10.1371/journal.ppat.1007484.].

Abnormal metabolism and uncontrolled angiogenesis are two important characteristics of malignant tumors. The occurrence of both events involves many key molecular changes including miRNA. However, EBV encoded miRNAs are rarely mentioned as capable of regulating tumor metabolism and tumor angiogenesis. Here, we reported that one of the key miRNAs encoded by EBV, EBV-miR-Bart1-5P, can significantly promote nasopharyngeal carcinoma (NPC) cell glycolysis and induces angiogenesis in vitro and in vivo. Mechanistically, EBV-miR-Bart1-5P directly targets the α1 catalytic subunit of AMP-activated protein kinase (AMPKα1) and consequently regulates the AMPK/mTOR/HIF1 pathway which impelled NPC cell anomalous aerobic glycolysis and angiogenesis, ultimately leads to uncontrolled growth of NPC. Our findings provide new insights into metabolism and angiogenesis of NPC and new opportunities for the development of targeted NPC therapy in the future.

Alternative splicing (AS) contributes to the complexity of the mammalian proteome and plays an important role in diseases, including infectious diseases. The differential AS patterns of these transcript sequences between the healthy (HS3A) and mastitic (HS8A) cows naturally infected by Staphylococcus aureus were compared to understand the molecular mechanisms underlying mastitis resistance and susceptibility. In this study, using the Illumina paired-end RNA sequencing method, 1352 differentially expressed genes (DEGs) with higher than twofold changes were found in the HS3A and HS8A mammary gland tissues. Gene ontology and KEGG pathway analyses revealed that the cytokine-cytokine receptor interaction pathway is the most significantly enriched pathway. Approximately 16k annotated unigenes were respectively identified in two libraries, based on the bovine Bos taurus UMD3.1 sequence assembly and search. A total of 52.62% and 51.24% annotated unigenes were alternatively spliced in term of exon skipping, intron retention, alternative 5' splicing and alternative 3' splicing. Additionally, 1,317 AS unigenes were HS3A-specific, whereas 1,093 AS unigenes were HS8A-specific. Some immune-related genes, such as ITGB6, MYD88, ADA, ACKR1, and TNFRSF1B, and their potential relationships with mastitis were highlighted. From Chromosome 2, 4, 6, 7, 10, 13, 14, 17, and 20, 3.66% (HS3A) and 5.4% (HS8A) novel transcripts, which harbor known quantitative trait locus associated with clinical mastitis, were identified. Many DEGs in the healthy and mastitic mammary glands are involved in immune, defense, and inflammation responses. These DEGs, which exhibit diverse and specific splicing patterns and events, can endow dairy cattle with the potential complex genetic resistance against mastitis.

Inner centromere protein (INCENP) plays an important role in mitosis and meiosis as the main member of chromosomal passenger protein complex (CPC). To investigate the functional markers of the INCENP gene associated with semen quality, the single nucleotide polymorphisms (SNPs) g.19970 A>G and g.34078 T>G were identified and analyzed. The new splice variant INCENP-TV is characterized by the deletion of exon 12. The g.19970 A>G in the exonic splicing enhancer (ESE) motif region results in an aberrant splice variant by constructing two minigene expression vectors using the pSPL3 exon capturing vector and transfecting vectors into MLTC-1 cells. INCENP-TV was more highly expressed than INCENP-reference in adult bull testes. The g.34078 T>G located in the binding region of bta-miR-378 could affect the expression of INCENP, which was verified by luciferase assay. To analyze comprehensively the correlation of SNPs with sperm quality, haplotype combinations constructed by g.19970 A>G and g.34078 T>G, as well as g.-692 C>T and g.-556 G>T reported in our previous studies, were analyzed. The bulls with H1H12 and H2H2 exhibited a higher ejaculate volume than those with H2H10 and H9H12, respectively (P < 0.05). Bulls with H11H11 and H2H10 exhibited higher initial sperm motility than those with H2H2 (P < 0.05). The expression levels of INCENP in bulls with H1H12 and H11H11 were significantly higher than those in bulls with H9H12 (P < 0.05), as determined by qRT-PCR. Findings suggest that g.19970 A>G and g.34078 T>G in INCENP both of which appear to change the molecular and biological characteristics of the mRNA transcribed from the locus may serve as a biomarkers of male bovine fertility by affecting alternative splicing mode and binding affinity with the target bta-miR-378.

[This corrects the article DOI: 10.1371/journal.pone.0159719.].

The development of a rapid, high-throughput and cost-effective HIV-1 drug resistance (HIV-DR) testing system is a challenge for areas consisting different HIV-1 strains. In this study, we established a broadly reactive multiplex assay that could simultaneously detect major drug resistance mutations at 8 loci, which are associated with resistance to commonly used nucleoside reverse transcriptase inhibitors (NRTIs) and Non-nucleoside reverse transcriptase inhibitors (NNRTIs), in specimens of HIV-1 CRF01_AE, CRF07_BC and subtype B, the three major circulating strains in China, using the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) provided by Sequenom MassARRAY® system. To establish the assay, pol gene fragments were prepared from the plasma viral RNA of 159 patients by nested PCR and the presence of wild type and mutant alleles at the 8 loci were analyzed by MALDI-TOF MS. In terms of loci, the detection rate of the alleles was greater than 97% for M41L, K65R, M184V and G190A, 91.2% for K101E/Q/P, 91.2% for T215F/Y, 89.9% for K103N/S and 80.5% for L210W. In terms of individuals, 80% of the alleles were detected in 95.4% CRF01_AE patients, 100% CRF07_BC patients and 83.3% subtype B patients. Importantly, the MALDI-TOF MS results were concordant to the drug resistance profiles of patients obtained from conventional sequencing analysis after excluded the failed detections. Using plasmid templates, the assay was estimated to be sensitive to detect drug resistant variants at level about 20% of the circulating viral population. The capability of this assay to detect mixed viral populations was further verified by two different patient specimens. In conclusion, this study evaluated the use of Sequenom MassARRAY® system for high-throughput detection of HIV-DR mutations towards the commonly used reverse transcriptase inhibitors in China.

OBJECTIVES:This paper aims to investigate whether equibiaxial tensile strain alters chondrocyte gene expression via controlling subcellular localization of histone deacetylase 4 (HDAC4). MATERIALS AND METHODS:Murine chondrocytes transfected with GFP-HDAC4 were subjected to 3 h cyclic equibiaxial tensile strain (CTS, 6% strain at 0.25 Hz) by a Flexcell® FX-5000™ Tension System. Fluorescence microscope and western blot were used to observe subcellular location of HDAC4. The gene expression was analyzed by real-time RT-PCR. The concentration of Glycosaminoglycans in culture medium was quantified by bimethylmethylene blue dye; Collagen II protein was evaluated by western blot. Cells phenotype was identified by immunohistochemistry. Cell viability was evaluated by live-dead cell detect kit. Okadaic acid, an inhibitor of HDAC4 nuclear relocation, was used to further validate whether HDAC4 nuclear relocation plays a role in gene expression in response to tension stimulation. RESULTS:87.5% of HDAC4 was located in the cytoplasm in chondrocytes under no loading condition, but it was relocated to the nucleus after CTS. RT-PCR analysis showed that levels of mRNA for aggrecan, collagen II, LK1 and SOX9 were all increased in chondrocytes subjected to CTS as compared to no loading control chondrocytes; in contrast, the levels of type X collagen, MMP-13, IHH and Runx2 gene expression were decreased in the chondrocytes subjected to CTS as compared to control chondrocytes. Meanwhile, CTS contributed to elevation of glycosaminoglycans and collagen II protein, but did not change collagen I production. When Okadaic acid blocked HDAC4 relocation from the cytoplasm to nucleus, the changes of the chondrocytes induced by CTS were abrogated. There was no chondrocyte dead detected in this study in response to CTS. CONCLUSIONS:CTS is able to induce HDAC4 relocation from cytoplasm to nucleus. Thus, CTS alters chondrocytes gene expression in association with the relocation of HDAC4 induced by CTS.

The 3-hydroxyisobutyrate dehydrogenase (HIBADH) is regarded as a human sperm-motility marker. However, the molecular mechanisms involved in the regulation of expression of the HIBADH gene in bulls remain largely unknown. HIBADH was detected in the testis, epididymis, and sperm via reverse transcription polymerase chain reaction and Western blot analysis. It is also expressed in the seminiferous epithelium, spermatids, and the entire epididymis, as detected by immunohistochemistry. Furthermore, HIBADH was expressed in the neck-piece and mid-piece of bull spermatids, as shown in the immunofluorescence assay. Using serially truncated bovine HIBADH promoters and luciferase constructs, we discovered an 878 bp (-703 bp to +175 bp) fragment that constitutes the core promoter region. One SNP g.-165 T>C of HIBADH was identified and genotyped in 307 Chinese Holstein bulls. Correlation analysis revealed that bulls with the TT genotype had higher initial sperm motility than those with the CC genotype (P < 0.05). Furthermore, the T- or C-containing loci (designated as pGL3-T and pGL3-C) were transiently transfected into MLTC-1 to test the effect of SNP on HIBADH expression. The luciferase reporter assay showed that the pGL3-T genotype exhibited 58% higher transcriptional activity than the pGL3-C genotype (P < 0.05). The bisulfite sequencing analysis revealed that the methylation pattern of the core promoter presented hypomethylation in the ejaculated semen in high-motility and low-motility bulls. The results demonstrated for the first time that the g.-165 T>C rather than methylation in the 5'-flanking region could affect the bovine sperm motility through the regulation of HIBADH gene transcriptional activity.

Neutrophil cytosolic factor 4 (NCF4) is component of the nicotinamide dinucleotide phosphate oxidase complex, a key factor in biochemical pathways and innate immune responses. In this study, splice variants and functional single-nucleotide polymorphism (SNP) of NCF4 were identified to determine the variability and association of the gene with susceptibility to bovine mastitis characterized by inflammation. A novel splice variant, designated as NCF4-TV and characterized by the retention of a 48 bp sequence in intron 9, was detected in the mammary gland tissues of infected cows. The expression of the NCF4-reference main transcript in the mastitic mammary tissues was higher than that in normal tissues. A novel SNP, g.18174 A>G, was also found in the retained 48 bp region of intron 9. To determine whether NCF4-TV could be due to the g.18174 A>G mutation, we constructed two mini-gene expression vectors with the wild-type or mutant NCF4 g.18174 A>G fragment. The vectors were then transiently transfected into 293T cells, and alternative splicing of NCF4 was analyzed by reverse transcription-PCR and sequencing. Mini-gene splicing assay demonstrated that the aberrantly spliced NCF4-TV with 48 bp retained fragment in intron 9 could be due to g.18174 A>G, which was associated with milk somatic count score and increased risk of mastitis infection in cows. NCF4 expression was also regulated by alternative splicing. This study proposes that NCF4 splice variants generated by functional SNP are important risk factors for mastitis susceptibility in dairy cows.