Acetate promotes lipogenesis in adipocytes but not in hepatocytes of chickens.

1.The role of acetate in lipogenesis of chickens remains largely unknown. This trial investigated the effect of sodium acetate (SA) on chicken fat metabolism via in vivo and in vitro experiments.2.The results indicated that supplementation of SA (1.0 g/kg feed) showed marginal to moderate stimulation on the area of the abdominal fat cells and triglyceride (TG) content in liver and adipose tissues. It increased the transcription of some genes involved in fat synthesis and deposition, but did not affect free fatty acid receptor 2 (FFAR2) expression in either liver or abdominal fat.3. In cultured hepatocytes treated with 0.01 mM to 5 mM SA, although mRNA levels of ACC1, PPAR, SREBP-1 c, and FFAR2 were upregulated with SA at certain concentrations, TG content and protein expression of lipogenic genes and FFAR2 were not altered at any dosages. In adipogenic differentiation of preadipocytes, high concentrations of SA (5 mM) exhibited significant increments in TG content and accumulated fat droplets, associated with stimulated transcription of FAS, LPL, AD, FABP4, and FFAR2, as well as elevated protein expression of FABP4 and FFAR2.4. The results showed that adipocytes were more sensitive to acetate than hepatocytes in chickens. While acetate played a minor role in hepatic fat metabolism, it promoted lipogenesis in adipocytes via FFAR2 with the involvement of FAS, LPL, and FABP4.

Association of serum galectin-3 with risks of death and vascular events in acute ischaemic stroke patients: the role of hyperglycemia.

BACKGROUND AND PURPOSE: Whether the association between galectin-3 and stroke outcome is modified by fasting plasma glucose (FPG) is unknown. The aim was to evaluate the prognostic effect of galectin-3 amongst ischaemic stroke patients stratified by FPG. METHODS: In all, 3082 ischaemic stroke patients were included in this study and serum galectin-3 was tested at baseline. The primary outcome was a composite outcome of death and vascular events, and secondary outcomes were death, stroke recurrence and vascular events within 1 year after stroke. RESULTS: Increased galectin-3 was significantly associated with the primary outcome, stroke recurrence and vascular events in the patients with hyperglycemia but not in those with normoglycemia (P for interaction < 0.05 for all). The multivariate-adjusted hazard ratios (95% confidence intervals) were 1.72 (1.05-2.84), 2.64 (1.14-6.12) and 2.68 (1.33-5.38) for the primary outcome, stroke recurrence and vascular events, respectively. A linear association between galectin-3 and the primary outcome was observed in hyperglycemic patients (P for linearity = 0.007). CONCLUSION: Increased galectin-3 was associated with the primary outcome, stroke recurrence and vascular events within 1 year after stroke in the patients with hyperglycemia, suggesting that galectin-3 may be an important prognostic factor for ischaemic stroke patients with hyperglycemia.

[Clinical observation of different reperfusion methods in patients with acute ischemic stroke with atrial fibrillation within 4.5 hours from onset].

Objective: To detect the ideal treatment for acute ischemic stroke (AIS) patients with atrial fibrillation (AF) within 4.5 hours from onset. Methods: A total of 95 AIS patients with AF was retrospectively analyzed from April 2014 to January 2019. Thirty patients (group A) were treated with endovascular treatment directly, 35 (group B) patients were treated with intravenous recombinant tissue plasminogen activator (rtPA) followed by endovascular treatment, and 30 (group C) patients were treated with intravenous rtPA only. There were no significant differences among the groups in baseline data as age, gender, underlying diseases, medication, National Institutes of Health Stroke Scale (NIHSS) score, time from onset to treatment. Modified thrombolysis in cerebral infarction (mTICI), Symptomatic hemorrhagic transformation (SICH), 90 d prognosis of modified Rankin Scale (mRS) and death were compared. Results: Recanalization (mTICI≥2b) was similar in group A and B (70.0% vs. 68.6%, P>0.05). SICH of group A (6.7%) was significantly lower than that of group B (31.4%, P<0.05), but similar with that of group C (13.7%, P>0.05). Prognosis (mRS≤2) was significantly better in group A (70.0%) than that in group B (37.1%) and group C (30.0%), both P<0.01. The mortality rate in group A (6.7%) was lower than that in group B (14.3%) and group C (20.0%) without statistically significant, both P>0.05. Conclusion: AIS patients with AF within 4.5 hours from onset should receive endovascular treatment directly.

[Effects of nasogastric and percutaneous endoscopic gastrostomy tube feeding on the susceptibility of pulmonary infection in long-term coma patients with stroke or traumatic brain injury].

Objective: To investigate the effects of nasogastric tube (NGT) and percutaneous endoscopic gastrostomy (PEG) tube feeding on the susceptibility of pulmonary infection in long-term coma patients with stroke or traumatic brain injury. Methods: A total of 295 candidates who were in long-term coma after stroke or traumatic brain injury but without pulmonary infection and eligible for PEG catheterization were screened prospectively between January 2014 and February 2018. The patients were divided into PEG group (86 patients) and NGT group (209 patients) according to the choice of next-of-kin. Data related to the susceptibility of pulmonary infection were collected and analyzed in the two groups one month after the catheterization. Results: After follow-up for one month, compared with NGT group, patients in PEG group had a lower incidence of pulmonary infection (23.3% vs 37.8%, P=0.023), a later occurrence of pulmonary infection (average time: 21 days vs 13 days, P=0.034), and a less severe pulmonary infection (7.0% vs 12.9%, P=0.029). The following characteristics made patients more susceptible to pulmonary infection: age ≥ 70 years (HR=1.619, 95% CI 1.054-2.172), Charlson comorbidity index ≥ 2 points (HR=1.647, 95% CI 1.043-2.485), using of proton pump inhibitor ≥ 7 days (HR=1.725, 95% CI 1.214-2.738), and number of pressure ulcers ≥ 3 (HR=2.109, 95% CI 1.128-3.844). However, serum albumin concentration ≥35 g/L (HR=0.670, 95% CI 0.375-0.963) was a protective factor for pulmonary infections. The number of consistent pathogens cultivated from saliva, gastric juice and sputum simultaneously in NGT and PEG group was 35 strains (27.8%) and 8 strains (13.3%), respectively (P=0.029). The mortality of pulmonary infection was similar in the two groups (3.5% vs 4.3%, P=1.000), but the death due to pulmonary infection in the PEG group occur later (median time: 20 days vs 11 days, P=0.012). Conclusions: PEG feeding was a preferred nutrition way which could reduce the risk of pulmonary infection more effectively than NGT feeding which might favored a retrograde gastro-pulmonary route by which pathogens colonized in stomach migrated to respiratory tract. Patients with characteristics mentioned above had the susceptibility of pulmonary infection, thus risk assessment of pulmonary infection should be conducted before selecting the catheterization method.

[Significance of monitoring the gradients between transcutaneous PCO(2) and end-tidal PCO(2) in patients with septic shock].

Objective: To investigate the significance of monitoring the gradients between transcutaneous PCO(2) and end-tidal PCO(2) [P(c-et)CO(2)] in patients with septic shock. Method: Thirty-five mechanically ventilated patients with early septic shock were enrolled as the study group and 18 non-septic shock patients with stable hemodynamics as the control group between May 2014 and October 2016. The patients with septic shock were treated by early goal-directed therapy (EGDT) within 6 hours since hospitalization. The differences of baseline level of P(c-et)CO(2) and arterial lactate concentration (LAC) between the two groups and the variations of these indexes after EGDT in the study group were compared respectively. Results: The baseline levels of P(c-et)CO(2) and LAC in patients with septic shock were significantly higher than those of the control group [(26.0±16.2) mmHg vs (11.0±5.6) mmHg (1 mmHg=0.133 kPa) and (4.0±1.7) mmol/L vs (1.6±0.6)mmol/L, all P=0.000]. The area under receiver operator characteristic (ROC) curve (AUC) for baseline P(c-et)CO(2) and LAC was 0.924 (95%CI: 0.851-0.996) and 0.931 (95%CI: 0.872-1.000), respectively. P(c-et)CO(2) >12.6 mmHg and LAC >2.5 mmol/L could discriminate septic shock patients from those without shock with the same sensibility of 97% and the specificity of 83% and 78% respectively. With regard to the prognosis (Day 28) of the patients with septic shock, AUC for baseline P(c-et)CO(2) and LAC was 0.709 (95%CI: 0.533-0.886) and 0.714 (95%CI: 0.545-0.883), respectively. P(c-et)CO(2) >20.0 mmHg and LAC>3.6 mmol/L could discriminate survivors from non-survivors with the same sensibility of 92% and the same specificity of 76%. All the patients in the study group completed EGDT within 6 hours after admission, 20 (57.1%) passed EGDT and 17 (85.0%) survived, 15 (42.9%) failed EGDT and 4 (26.7%) survived, and the survival rates were significantly different (F=9.844, P=0.001). After EGDT, P(c-et)CO(2) (21.0±9.5 mmHg) and LAC(3.3±2.5 mmol/L)reduced significantly compared with the baselines (P=0.008 and P=0.046), and the associated AUC was 0.905(95%CI: 0.792-1.000) and 0.747 (95%CI: 0.576-0.917)respectively. P(c-et)CO(2) > 16.5 mmHg and LAC > 3.1 mmol/L could discriminate survivors from non-survivors with the sensibility of 97% and 91%, and the specificity of 78% and 69%, respectively. Conclusions: P(c-et)CO(2) >12.6 mmHg could play the same role as LAC in recognizing early septic shock. EGDT was an effective therapy for the septic shock and P(c-et)CO(2) reflected efficacy. P(c-et)CO(2)>20 mmHg before EGDT and >16.5 mmHg after EGDT both could predict the 28 d prognosis of patients with septic shock, and the effect of the former was equal to that of LAC, but the latter was better than LAC.

Serum 25-hydroxyvitamin D deficiency predicts poor outcome amongst acute ischaemic stroke patients with low high density lipoprotein cholesterol.

BACKGROUND AND PURPOSE: Current observational studies indicate that a lower vitamin D level is associated with a higher risk of poor ischaemic stroke prognosis. Whether this association is affected by lipid levels is unclear. Our aim was to examine the effect of serum vitamin D especially its deficiency on the global outcome of ischaemic stroke stratified by individual lipid component level. METHODS: A total of 3181 ischaemic patients from China Antihypertensive Trial in Acute Ischaemic Stroke were included in this study and their baseline serum 25-hydroxyvitamin D levels were tested. They were prospectively followed up for death, major disability and vascular events for 3 months after acute ischaemic stroke. A multivariable logistic model was used to evaluate the association between serum 25-hydroxyvitamin D levels and clinical outcomes of ischaemic stroke in the 3-month period of follow-up in all patients and in different lipid-level subgroups. RESULTS: Vitamin D deficiency was associated with poor clinical outcomes only in ischaemic stroke patients with high density lipoprotein cholesterol (HDLC) <1.04 mmol/l rather than all patients. The multivariable adjusted odds ratios (95% confidence intervals) of major disability and composite adverse events were 1.98 (1.08-3.63) and 2.24 (1.22-4.12), respectively. There was a significant interaction effect between vitamin D and HDLC levels on major disability and the composite outcome (P for interaction < 0.05 for both). A significant linear trend existed between 25-hydroxyvitamin D and risk of poor prognosis (P = 0.03). CONCLUSIONS: Vitamin D deficiency may be merely an independent risk factor of poor prognosis in ischaemic stroke patients with low HDLC level.

Radiotherapy effects on brain/bone metastatic adenocarcinoma lung cancer and the importance of EGFR mutation test.

This study proposed to retrospectively analyze the efficacy of radiotherapy on brain/bone metastases in patients with stage IV lung adenocarcinoma and to evaluate the correlation between overall survival after radiotherapy and other factors including metastatic sites and EGFR mutation status. 115 patients with Stage IV lung adenocarcinoma admitted to our center from March, 2011 to December, 2013 were enrolled. They presented with metastases to no other solid organs except the bone or brain and had received no prior treatment. 50 patients received EGFR mutation test with 32 detected as EGFR mutant and 18 wild-type. Patients with brain metastases were treated with 40 Gy whole brain irradiation (WBI) in 2 Gy fractions; patients with bone metastases were treated with 30 Gy local irradiation in 3 Gy fractions or 40 Gy in 2 Gy fractions. All the patients received systemic therapy during or after radiotherapy and 68 received targeted therapy.The median overall survival of patients with solitary brain metastases, solitary bone metastases or combined brain and bone metastases were 8.50 months, 8.50 months and 9.50 months respectively, revealing no significant difference (p=0.57). The median overall survival of patients with EGFR mutations was 10.25 months, longer than the 8.75 months of patients without EGFR mutations, revealing no significant difference (p=0.57). The median overall survival of EGFR mutant patients with solitary bone metastases, solitary brain metastases or combined brain and bone metastases were 7.50 months, 10.50 months and 11.50 months respectively, revealing no significant difference (p=0.91). 36 patients with untested EGFR mutation status received EGFR-TKI. Among EGFR mutant patients, 10 didn't receive targeted therapy; 8 were administered Erlotinib and 14 Gefitinib with median overall survival of 10.25 months and 14.5 months, showing no significant difference (p=0.11) between the two drugs. When patients with stage IV lung adenocarcinoma have been treated by early radiotherapy, the overall survival doesn't correlate with metastatic sites. Radiotherapy could extend survival for EGFR mutant patients with stage IV lung adenocarcinoma. EGFR mutation test should be performed before treatment of the disease.

A novel splice variant of the bovine GALNTL5 gene identified in Chinese Holstein bull testis tissue and its mRNA expression.

The polypeptide N-acetylgalactosaminyltransferase-like protein 5 (GALNTL5) is a newly identified protein that is specifically expressed in testis tissue and participates in spermatogenesis. In this study, we characterized a novel bovine GALNTL5 splice variant, designated as GALNTL5-AS, by using real-time polymerase chain reaction (RT-PCR) and clone sequencing methods. The novel GALNTL5 isoform was derived from the complete transcript, GALNTL5-complete, via alternative splicing (AS). The pattern of the splice variant was exon skipping. Bovine GALNTL5 transcripts were expressed in the testis, as demonstrated by RT-PCR. The expression levels of both transcripts were higher in adult testes than in calf testes (P < 0.05). In addition, prediction analysis showed that the GALNTL5-AS transcript only encoded 122 amino acids and lost its glycosyltransferase 1 and Gal/GalNAc-T motifs, which may result in a dysfunctional protein compared with the predominant transcript GALNTL5-complete. This study improves our understanding of the bovine GALNTL5 gene function during bull sperm formation.

Identification of CD14 transcript in blood polymorphonuclear neutrophil leukocytes and functional variation in Holsteins.

Polymorphonuclear neutrophil (PMN) leukocytes are primary phagocytic cells of the bovine mammary gland and a first line of defense against invading pathogens during bovine mastitis infection. Cluster of differentiation 14 (CD14) is mainly expressed in macrophages and neutrophils and acts as a co-receptor that binds bacterial lipopolysaccharide (LPS) and recruits PMNs to CD14-LPS complexes in mammary epithelial cells. In this study, we identified a novel splice variant in PMNs, named CD14-SV, characterized by a deleted region from c.143-579 nt compared to the CD14 reference mRNA sequence. Moreover, a single nucleotide polymorphism (c.523 A>G) in exon 2 of CD14 was identified and found to modify the secondary structure and hydrophilicity of the CD14 protein. Association analysis also showed that the milk somatic cell score, an indicator of mastitis, of cows with the GG genotype was lower than that of cows with the AA and AG genotypes. Our findings suggest that the expression of CD14 in bovine blood PMNs is regulated by alternative splicing, and that CD14-SV is a candidate functional marker that may influence mastitis-resistance in dairy cows.

Alternative splicing, promoter methylation, and functional SNPs of sperm flagella 2 gene in testis and mature spermatozoa of Holstein bulls.

The sperm flagella 2 (SPEF2) gene is essential for development of normal sperm tail and male fertility. In this study, we characterized first the splice variants, promoter and its methylation, and functional single-nucleotide polymorphisms (SNPs) of the SPEF2 gene in newborn and adult Holstein bulls. Four splice variants were identified in the testes, epididymis, sperm, heart, spleen, lungs, kidneys, and liver tissues through RT-PCR, clone sequencing, and western blot analysis. Immunohistochemistry revealed that the SPEF2 was specifically expressed in the primary spermatocytes, elongated spermatids, and round spermatids in the testes and epididymis. SPEF2-SV1 was differentially expressed in the sperms of high-performance and low-performance adult bulls; SPEF2-SV2 presents the highest expression in testis and epididymis; SPEF2-SV3 was only detected in testis and epididymis. An SNP (c.2851G>T) in exon 20 of SPEF2, located within a putative exonic splice enhancer, potentially produced SPEF2-SV3 and was involved in semen deformity rate and post-thaw cryopreserved sperm motility. The luciferase reporter and bisulfite sequencing analysis suggested that the methylation pattern of the core promoter did not significantly differ between the full-sib bulls that presented hypomethylation in the ejaculated semen and testis. This finding indicates that sperm quality is unrelated to SPEF2 methylation pattern. Our data suggest that alternative splicing, rather than methylation, is involved in the regulation of SPEF2 expression in the testes and sperm and is one of the determinants of sperm motility during bull spermatogenesis. The exonic SNP (c.2851G>T) produces aberrant splice variants, which can be used as a candidate marker for semen traits selection breeding of Holstein bulls.

Common characteristics shared by different differential phase contrast imaging methods.

There are many variations of differential phase contrast imaging methods. Although these imaging methods are different in configuration, they are alike in imaging by extracting differential phase information through the evaluation of the refraction angles. In this paper, we investigate common characteristics shared by various different differential phase contrast imaging methods.

Regulatory mutations in the A2M gene are involved in the mastitis susceptibility in dairy cows.

Mutations, such as single nucleotide polymorphisms (SNPs), in the 5'-flanking and microRNA (miRNA) regulatory regions may result in altered gene expression levels and cause diseases. Alpha-2-macroglobulin (A2M) has the function of binding host or foreign peptides and particles, and thereby serves as a defense barrier against pathogens in the plasma and tissues of animals. To investigate the functional markers of the A2M gene associated with mastitis, the promoter was characterized and SNPs that affect promoter activity or binding affinity with the target miRNA were identified using the luciferase reporter assay and real-time quantitative PCR method. Results showed that the core promoter of A2M was found between the bases g.-2641 and g.-2479. Four novel SNPs (g.-724A>G, g.-665G>A, g.-535C>G and g.-520_-519insA) in the promoter region were completely linked. The activity of the mutant haplotype (GAGA) increased by 177% compared with that of the wild haplotype (AGC-). Bta-miR-2898 was upregulated by 6.25-fold in the mammary gland tissues of mastitis-infected cows compared with that of the healthy cows. One SNP (c.4659_4661delC) located in the 3'-untranslated region of the A2M gene may affect the binding affinity with the target bta-miR-2898. Five SNPs exhibited tight linkage. Association analysis showed that the milk somatic cell score for cows with the mutant haplotype (GAGA-) was lower than that for cows with the wild haplotype. Thus, the mutant type can be used as a potential functional marker for a mastitis resistance breeding program in dairy cows. Our findings provided the molecular basis for A2M transcriptional and post-transcriptional regulations. A close relationship between regulatory mutations and mastitis susceptibility of cows also was established.

Association between an alternative promoter polymorphism and sperm deformity rate is due to modulation of the expression of KATNAL1 transcripts in Chinese Holstein bulls.

Katanin p60 subunit A-like 1 (KATNAL1) is an ATPase that regulates Sertoli cell microtubule dynamics and sperm retention. We evaluated one novel splice variant and characterized the promoter and a functional single nucleotide polymorphism (SNP) of the bovine KATNAL1 gene to explore its expression pattern, possible regulatory mechanism and relationship with semen traits in Chinese Holstein bulls. A novel splice variant, KATNAL1 transcript variant 2 (KATNAL1-TV2) of the retained 68 bp in intron 2, was identified by RT-PCR and compared with KATNAL1 transcript variant 1 (KATNAL1-TV1, NM 001192918.1) in various tissues. Bioinformatics analyses predicted that KATNAL1 transcription was regulated by two promoters: P1 in KATNAL1-TV1 and P2 in KATNAL1-TV2. Results of qRT-PCR revealed that KATNAL1-TV1 had higher expression than did KATNAL1-TV2 in testes of adult bulls (P < 0.05). Promoter luciferase activity analysis suggested that the core sequences of P1 and P2 were mapped to the region of c.-575˜c.-180 and c.163-40˜c.333+59 respectively. One novel SNP (c.163-210T>C, ss836312085) located in intron 1 was found using sequence alignment. The SNP in P2 resulted in the presence of the DeltaE binding site, improving its basal promoter activity (P < 0.05); and we observed a greater sperm deformity rate in bulls with the genotype CC than in those with the genotype TT (P < 0.05), which indicated that different genotypes were associated with the bovine semen traits. Bioinformatics analysis of the KATNAL1 protein sequence predicted that the loss of the MIT domain in the KATNAL1-TV2 transcript resulted in protein dysfunction. These findings help us to understand that a functional SNP in P2 and subsequent triggering of expression diversity of KATNAL1 transcripts are likely to play an important role with regard to semen traits in bull breeding programs.

Identification and characterization of a novel splice variant of the PLCζ1 gene in bull testis tissues.

Phospholipase C zeta 1 (PLCζ1), which transcribes a key protein, has an important function in oocyte activation and embryo development because PLCζ1 can trigger a series of intracellular Ca2+ oscillations in mammals. In this study, a novel splice variant in the testis tissues of adult and fetal Chinese Holstein bulls was characterized by reverse transcription-polymerase chain reaction (RT-PCR) and sequencing analysis. The novel splice variant PLCζ1-sv1 was derived from the PLCζ1 complete transcript (PLCζ1-complete) by alternative splicing; the alternative splicing pattern exhibited alternative 5'-splice sites. The full-length transcript, PLCζ1-complete, is the main transcript found in fetal and adult cow testis tissue. Quantitative real-time PCR (qPCR) analysis demonstrated that the expression levels of the PLCζ1-complete transcript were significantly higher than those of the PLCζ1-sv1 splice variant in bovine testis tissues. PLCζ1 protein sequencing analysis showed that the amino acids at positions 453 to 457 were deleted in PLCζ1-sv1, thereby terminating transcription prematurely. In summary, this study provided information to elucidate the structure and function of the bovine PLCζ1 gene.

Association of novel single nucleotide polymorphisms of the CXCR1 gene with the milk performance traits of Chinese native cattle.

Mastitis is an economically devastating disease affecting the dairy industry. Dairy cows with mastitis give reduced milk yield and produce milk that is unfit for consumption. The chemokine receptor CXCR1 is an excellent prospective genetic marker for mastitis resistance in cattle because it regulates neutrophil migration, killing, and survival during infection. We detected 4 single nucleotide polymorphisms (SNPs) of the CXCR1 gene in Chinese native cattle and analyzed their associations with milk traits. Screening for genetic variations in CXCR1 among 648 Chinese Holstein, Luxi Yellow, and Bohai Black cattle by created restriction site polymerase chain reaction (PCR), nested PCR, and DNA sequencing revealed 4 new SNPs with allelic frequencies ranging from 0.676 to 0.821, 0.706 to 0.803, 0.647 to 0.824, and 0.558 to 0.581. All four CXCR1 gene SNPs were located in exon II. Two SNPs, c.337A>G and c.365C>T, were nonsynonymous mutations [ATC (Ile) > GTC (Val) and GCC (Ala) > GTC (Val)], whereas two, c.291C>T and c.333C>T, were synonymous mutations [TTC (Gly) > TTT (Gly) and GGC (Phe) > GGT (Phe)]. Statistical analyses revealed the significant association of c.337A>G and c.365C>T with the somatic cell score, which suggests the possible role of these SNPs in the host response against mastitis. Our data suggest that combined genotypes CCAC/CCGC, CCAC/CTAT, and CCAT/CTAT (lowest somatic cell scores); CTAC/CTAT (highest protein rate); CCAC/CTGC (highest fat rate); and CCAT/CTAT (highest 305-day milk yield) can be used as possible candidates for marker-assisted selection in dairy cattle breeding programs.

Novel SNPs of the mannan-binding lectin 2 gene and their association with production traits in Chinese Holsteins.

The mannan-binding lectin gene (MBL) participates as an opsonin in the innate immune system of mammals, and single nucleotide polymorphisms (SNPs) in MBL cause various immune dysfunctions. In this study, we detected SNPs in MBL2 at exon 1 using polymerase chain reaction single-strand conformation polymorphism analysis and DNA sequencing techniques in 825 Chinese Holstein cows. Four new SNPs with various allele frequencies were also found. The g.1164 G>A SNP was predicted to substitute arginine with glutamine at the N-terminus of the cysteine-rich domain. In the collagen-like domain, SNPs g.1197 C>A and g.1198 G>A changed proline to glutamine, whereas SNP g.1207 T>C was identified as a synonymous mutation. Correlation analysis showed that the g.1197 C>A marker was significantly correlated to somatic cell score (SCS), and the g.1164 G>A locus had significant effects on SCS, fat content, and protein content (P < 0.05), suggesting possible roles of these SNPs in the host response against mastitis. Nine haplotypes and nine haplotype pairs corresponding to the loci of the 4 novel SNPs were found in Chinese Holsteins. Haplotype pairs MM, MN, and BQ were correlated with the lowest SCS; MN with the highest protein yield; MM with the highest protein rate, and MN with the highest 305- day milk yield. Thus, MM, MN, and BQ are possible candidates for marker-assisted selection in dairy cattle breeding programs.

Increased expression of a novel splice variant of the complement component 4 (C4A) gene in mastitis-infected dairy cattle.

The complement system helps in the direct lysis of invading pathogens and modulates phagocytic, humoral and cellular immune responses. Complement 4 is a critical component in complement activity and protection against many bacterial pathogens because it is essential to classical and lectin activation pathways. We used reverse transcription and PCR to investigate alternative splicing and expression of the complement component 4 (C4A) gene in Chinese Holstein cattle. The PCR products were cloned and sequenced. A novel splice variant involving intron 10 was identified, which we named C4A-AS. To examine how C4A gene activity is affected by bovine mastitis, six Chinese Holstein cattle were divided into healthy (non-mastitic) and Staphylococcus aureus-induced mastitic groups. Real-time quantitative PCR (qRT-PCR) revealed that the C4A-complete and C4A-AS transcripts are expressed at significantly different levels in healthy cows, while there were no significant differences in the mastitic group (P = 0.257). Expression of C4A-AS increased significantly when mastitis developed. We also examined the expression of C4A-complete and C4A-AS in several tissues (liver, heart, spleen, lung, kidney, tongue, and muscle). The two transcripts were expressed in all of these tissues but there were no significant differences in expression between healthy and mastitic cows. We therefore conclude that the C4A-complete transcript is the main transcript under normal physiological conditions, while C4A-AS is augmented when mastitis develops.

Two splice variants of the bovine lactoferrin gene identified in Staphylococcus aureus isolated from mastitis in dairy cattle.

Bovine lactoferrin (bLF) is a member of the transferrin family; it plays an important role in the innate immune response. We identified novel splice variants of the bLF gene in mastitis-infected and healthy cows. Reverse transcription-polymerase chain reaction (RT-PCR) and clone sequencing analysis were used to screen the splice variants of the bLF gene in the mammary gland, spleen and liver tissues. One main transcript corresponding to the bLF reference sequence was found in three tissues in both healthy and mastitis-infected cows. Quantitative real-time PCR analysis showed that the expression levels of the LF gene's main transcript were not significantly different in tissues from healthy versus mastitis-infected cows. However, the new splice variant, LF-AS2, which has the exon-skipping alternative splicing pattern, was only identified in mammary glands infected with Staphylococcus aureus. Sequencing analysis showed that the new splice variant was 251 bp in length, including exon 1, part of exon 2, part of exon 16, and exon 17. We conclude that bLF may play a role in resistance to mastitis through alternative splicing mechanisms.

Three novel SNPs of the bovine Tf gene in Chinese native cattle and their associations with milk production traits.

Transferrin (Tf) is a ß-globulin protein that transports iron ions in mammalian cells. It contributes to innate immunity to microbial pathogens, primarily by limiting microbial access to iron. Thus, polymorphisms present in bovine Tf could potentially underlie inherited differences in mastitis resistance and milk production traits. We detected three novel single-nucleotide polymorphisms of the Tf gene in Chinese native cattle by screening for genetic variation of Tf in 751 individuals of three Chinese cattle breeds, namely China Holstein, Luxi Yellow and Bohai Black, using PCR-RFLP and DNA sequencing techniques. The three new SNPs, g.-1748G>A ss250608649, g.13942T>C ss250608650, and g.14037A>G ss250608651, had allele frequencies of 85.9, 86.3 and 92.5%, 64.5, 73.3 and 65.0%, and 67.6, 73.7 and 60.0%, respectively. SNP g.-1748G>A was located in the 5' flanking region of Tf. SNP g.14037A>G was located in intron 8 of Tf. SNP g.13942T>C, located in exon 8 of Tf, was a synonymous mutation (TTA > CTA), encoding a leucine (326 aa) in the Tf protein. Associations of the Tf SNPs with milk traits were also analyzed. Significant (P < 0.05) relationships among the Tf polymorphisms, somatic cell scores (SCS), and milk productive traits were observed. Cows with genotypes TT (g.13942T>C), GG (g.-1748G>A) and AG (g.14037A>G) had a lower SCS and higher protein levels and 305-day milk yield. Nineteen combinations of different haplotypes from the three SNPs were identified in Chinese Holstein cattle. The haplotype combination ATA/GCA, GCA/GCA and GCG/ GTA was dominant in cows with a lower SCS, a higher protein level and a higher 305-day milk yield, respectively. Moreover, the gene expression level of Tf was higher in mastitis-affected mammary tissues than in normal mammary tissues. These results suggest that the Tf gene affects milk production, as well as mastitis-resistance traits, in Chinese Holsteins.

Effect of lncRNA-BLACAT1 on drug resistance of non-small cell lung cancer cells in DDP chemotherapy by regulating cyclin D1 expression.

OBJECTIVE: The aim of this study was to investigate the effect of long non-coding ribonucleic acid (lncRNA)-bladder cancer associated transcript 1 (BLACAT1) on the drug resistance of non-small cell lung cancer (NSCLC) cells in cisplatin (DDP) chemotherapy by regulating the expression of Cyclin D1. MATERIALS AND METHODS: The analysis of the lncRNA expression profiles in 483 cases of NSCLC tissues and 347 cases of cancer-adjacent tissues in Gene Expression Omnibus (GEO) database revealed that lncRNA-BLACAT1 was differentially expressed in NSCLC and related to prognosis. In order to further study its mechanism of action on DDP-resistant cells, the expression level of lncRNA-BLACAT1 in normal human lung bronchial epithelial cell line BEAS-2B, NSCLC cell line A549, and DDP-resistant cell line A549 (A549/DDP) was detected by quantitative Polymerase Chain Reaction (qPCR). LncRNA-BLACAT1 small interfering RNA (siRNA) (si-BLACAT1) and lncRNA-BLACAT1 negative control (si-NC) were transfected into A549/DPP cells. Then, qPCR was carried out to detect the changes in the expression of lncRNA-BLACAT1 before and after transfection. Thereafter, cell cycle and cell growth rate were detected by flow cytometry and the cell growth curve. Besides, the changes in cell migration, cell apoptosis, and Cyclin D1 were detected via wound healing assay, flow cytometry, and Western blotting (WB). RESULTS: In GEO database, lncRNA-BLACAT1 was significantly overexpressed in NSCLC (p<0.05), and the prognosis of NSCLC in BLACAT1 low-expression group was better than that in the BLACAT1 high-expression group (p<0.0001). Compared with that in BEAS-2B cells, BLACAT messenger RNA (mRNA) was notably highly expressed in A549 cells (p<0.05), and compared with that in A549 cells, BLACAT1 mRNA in A549/DPP was significantly highly expressed in A549/DDP cells (p<0.05). Additionally, in comparison with that in the si-NC group, the content of lncRNA-BLACAT1 in si-BLACAT1 group was remarkably decreased (p<0.01). Moreover, flow cytometry detection of cell cycle revealed that compared with those in si-NC group, G0/G1 phase was markedly prolonged and S phase was shortened in si-BLACAT1 group. MTS assay manifested that the absorbance at 450 nm in si-BLACAT1 group was evidently decreased on the 3rd day compared with that in the si-NC group (p<0.05), and the difference between the two groups was the most significant on the 5th day (p<0.001). According to wound healing assay, compared with those in si-NC group, the distance between cells became larger, the cell migration ability was remarkably weakened (p<0.05), and cell apoptosis was prominently reduced in si-BLACAT1 group (p<0.05). WB results showed that compared with si-NC group, si-BLACAT1 group had significantly reduced Cyclin D1 (p<0.05) CONCLUSIONS: LncRNA-BLACAT1 regulates the expression of Cyclin D1, reduces the malignant phenotype of drug-resistant cells, and increases the sensitivity of lung cancer cells to DDP.