[Effects of high-fat and low-carbohydrate diet combined with radiotherapy on tumor microenvironment of Lewis lung cancer bearing mice].

Objective: To investigate the effect of high-fat and low-carbohydrate diet combined with radiotherapy on the tumor microenvironment of mice with lung xenografts. Methods: C57BL/6J mice were selected to establish the Lewis lung cancer model, and they were divided into the normal diet group, the high-fat and low-carbohydrate diet group, the normal diet + radiotherapy group, and the high-fat and low-carbohydrate diet + radiotherapy group, with 18 mice in each group. The mice in the normal diet group and the normal diet + radiotherapy group were fed with the normal diet with 12.11% fat for energy supply, and the mice in the high-fat and low-carbohydrate diet group and the high-fat and low-carbohydrate diet + radiotherapy group were fed with high-fat and low-carbohydratediet with 45.00% fat for energy. On the 12th to 14th days, the tumor sites of the mice in the normal diet + radiotherapy group and the high-fat and low-carbohydrate diet + radiotherapy group were treated with radiotherapy, and the irradiation dose was 24 Gy/3f. The body weight, tumor volume, blood glucose and blood ketone level, liver and kidney function, and survival status of the mice were observed and monitored. Immunohistochemical staining was used to detect the tumor-associated microangiogenesis molecule (CD34) and lymphatic endothelial hyaluronan receptor 1 (LYVE-1), Sirius staining was used to detect collagen fibers, and multiplex immunofluorescence was used to detect CD8 and programmed death-1 (PD-1). Expression of immune cell phenotypes (CD3, CD4, CD8, and Treg) was detected by flow cytometry. Results: On the 27th day after inoculation, the body weigh of the common diet group was(24.78±2.22)g, which was significantly higher than that of the common diet + radiotherapy group [(22.15±0.48)g, P=0.030] and high-fat low-carbohydrate diet + radiotherapy group [(22.02±0.77)g, P=0.031)]. On the 15th day after inoculation, the tumor volume of the high-fat and low-carbohydrate diet + radiotherapy group was (220.88±130.05) mm3, which was significantly smaller than that of the normal diet group [(504.37±328.48) mm3, P=0.042)] and the high-fat, low-carbohydrate diet group [(534.26±230.42) mm3, P=0.016], but there was no statistically significant difference compared with the normal diet + radiotherapy group [(274.64±160.97) mm3]. In the 4th week, the blood glucose values of the mice in the high-fat and low-carbohydrate diet group were lower than those in the normal diet group, with the value being (8.00±0.36) mmol/L and (9.57±0.40) mmol/L, respectively, and the difference was statistically significant (P＜0.05). The blood ketone values of the mice in the high-fat and low-carbohydrate diet group were higher than those in the normal diet group, with the value being (1.00±0.20) mmol/L and (0.63±0.06) mmol/L, respectively, in the second week. In the third week, the blood ketone values of the two groups of mice were (0.90±0.17) mmol/L and (0.70±0.10) mmol/L, respectively, and the difference was statistically significant (P＜0.05). On the 30th day after inoculation, there were no significant differences in aspartate aminotransferase, alanine aminotransferase, creatinine, and urea between the normal diet group and the high-fat, low-carbohydrate diet group (all P＞0.05). The hearts, livers, spleens, lungs, and kidneys of the mice in each group had no obvious toxic changes and tumor metastasis. In the high-fat and low-carbohydrate diet + radiotherapy group, the expression of CD8 was up-regulated in the tumor tissues of mice, and the expressions of PD-1, CD34, LYVE-1, and collagen fibers were down-regulated. The proportion of CD8+ T cells in the paratumoral lymph nodes of the high-fat and low-carbohydrate diet + radiotherapy group was (25.13±0.97)%, higher than that of the normal diet group [(20.60±2.23)%, P＜0.050] and the normal diet + radiotherapy group [(19.26±3.07)%, P＜0.05], but there was no statistically significant difference with the high-fat and low-carbohydrate diet group [(22.03±1.75)%, P＞0.05]. The proportion, of CD4+ T cells in the lymph nodes adjacent to the tumor in the normal diet + radiotherapy group (31.33±5.16)% and the high-fat and low-carbohydrate diet + radiotherapy group (30.63±1.70)% were higher than that in the normal diet group [(20.27±2.15)%, P＜0.05] and the high-fat and low-carbohydrate diet group (23.70±2.62, P＜0.05). Treg cells accounted for the highest (16.58±5.10)% of T cells in the para-tumor lymph nodes of the normal diet + radiotherapy group, but compared with the normal diet group, the high-fat and low-carbohydrate diet group, and the high-fat and low-carbohydrate diet + radiotherapy group, there was no statistically significant difference (all P＞0.05). Conclusion: High-fat and low-carbohydrate diet plus radiotherapy can enhance the recruitment and function of immune effector cells in the tumor microenvironment, inhibit tumor microangiogenesis, and thus inhibit tumor growth.

[Analysis of the therapeutic effect of aspiration thrombectomy for early carotid stent thrombosis].

Objective: To investigate the clinical characteristics and the efficacy of thrombus aspiration in patients with early intrastent thrombosis (EST) following carotid artery stenting (CAS). Methods: This study is a retrospective case series, collecting clinical data of five patients who developed EST after CAS in the Department of Neurosurgery, Beijing Chaoyang Hospital, Capital Medical University from January 2021 to September 2023.All patients were male, with an age of (64.0±11.9) years (range:48 to 77 years), accounting for 2.0% (5/244) of CAS procedures during the same period.Among them, three patients did not receive standard dual antiplatelet therapy before the procedure, and one had an inadequate ADP inhibition rate (45.6%).Four patients received XACT carotid stents, while one received a Wallstent carotid stent.All five patients showed significant residual stenosis ranging from 43% to 55% after CAS.Emergency thrombus aspiration was performed in all cases, and data regarding perioperative conditions, vascular patency, and clinical outcomes were collected. Results: The interval between CAS and the occurrence of EST ranged from 3 hours to 14 days.The main clinical symptoms included sudden onset of consciousness disorders and contralateral limb weakness.None of the patients received preoperative intravenous thrombolysis, and thrombus aspiration was performed during the procedure to restore vascular patency.Four cases underwent balloon angioplasty during the procedure, and two cases utilized overlapping stents.Two patients experienced intraoperative embolization of thrombus to the C2 segment.In one case, the embolized thrombus was retrieved using an intracranial thrombectomy stent, while in another case, it was aspirated using a guiding catheter.Postoperatively, all patients had a thrombolysis in cerebral infarction grade of 3, and symptoms improved in four cases.One patient showed no improvement in symptoms, and MRI revealed extensive new infarction in the right frontal and insular regions, adjacent to the right lateral ventricle.Regular follow-up examinations after discharge did not reveal restenosis or embolism within the stent.The follow-up period ranged from 7.6 to 21.2 months, with modified Rankin scale scores of 0 to 1 point in four cases and 2 points in one case, indicating good recovery in all patients. Conclusions: Acute intrastent thrombosis is a rare complication after carotid artery stenting.The combined use of percutaneous thrombus aspiration and endovascular techniques, such as balloon angioplasty and stent overlapping, can rapidly restore vessel patency with favorable outcomes.However, further large-scale clinical studies are needed to confirm the effectiveness of these treatments for acute intrastent thrombosis.

[Expression of lnc-MyD88 and its relationship with the prognosis of patients with epithelial ovarian cancer].

Objective: To explore the expression of long non-coding RNA-myeloid differentiation factor 88 (lnc-MyD88) and its relationship with the prognosis of patients with epithelial ovarian cancer (EOC). Methods: A total of 70 EOC patients who underwent initial cytoreductive surgery and platinum-based drugs combined with paclitaxel for 6 to 8 courses were selected at Sichuan Cancer Hospital from January 2016 to January 2019. The fresh cancer tissue specimens were collected. In addition, 28 fresh normal ovarian tissues from patients who underwent surgery for benign gynecological diseases during the same period were collected as control group. Reverse transcription (RT) and real-time quantitative polymerase chain reaction (qPCR) were used to detect the expression of lnc-MyD88 and myeloid differentiation factor 88 (MyD88) mRNA in EOC tissues and normal ovarian tissues. The correlation between the expression of lnc-MyD88 and MyD88 mRNA in EOC was analyzed by Pearson's correlation coefficient. The relationship between lnc-MyD88 expression and clinicopathological characteristics of patients with EOC was analyzed. Kaplan-Meier method was used to calculate the survival rate of patients. The log-rank test was used for univariate survival analysis, and Cox proportional hazard model was used for multivariate survival analysis. Results: (1) RT-qPCR showed that the relative expression level of lnc-MyD88 and MyD88 mRNA in EOC were 0.009 (0.000-0.049) and 0.001 (0.000-0.006), respectively, which were significantly higher than those of normal ovarian tissues (all P<0.01); Pearson's correlation coefficient showed that the expression of lnc-MyD88 and MyD88 mRNA in EOC was positively correlated (r2=0.610, P<0.01). (2) The high expression rate of lnc-MyD88 in EOC patients with lymph node metastasis, distant metastasis and chemotherapy resistance (71%, 64% and 70%, respectively) were significantly higher than the patients in control group (41%, 40% and 35%, respectively; all P<0.05). There were no statistically significant in the high expression rate of lnc-MyD88 in EOC patients with different ages, pathological types, pathological grades, surgical pathological stages, postoperative residual lesion size, and ascites cancer cells (all P>0.05). (3) Univariate analysis showed that surgical pathological staging, lymph node metastasis, distant metastasis, postoperative residual tumor size, and high expression of lnc-MyD88 and MyD88 mRNA significantly affected the progression-free survival (PFS) and overall survival (OS) of EOC patients (all P<0.05), ascites cancer cells were the risk factors that significantly affected PFS in EOC patients (P=0.040); multivariate analysis showed that surgical pathological staging and high expression of lnc-MyD88 and MyD88 mRNA were independent factors affecting PFS and OS in EOC patients (all P<0.05), the size of residual lesions after surgery was an independent factor affecting PFS in EOC patients (P=0.001). Conclusions: The level of lnc-MyD88 expression in ovarian cancer tissues was significantly increased. Lnc-MyD88, as a molecular marker for the poor prognosis of EOC, is related to the expression of MyD88 in EOC, and may be involved in its expression regulation, thereby affecting the survival and prognosis of EOC patients.

[Application of indocyanine green fluorescence visualization in surgical resection of abdominal wall endometriosis].

Objective: To investigate the feasibility, effectiveness and safety of indocyanine green (ICG) navigation in the surgical resection of abdominal wall endometriosis (AWE). Methods: Seven women undergoing surgery for AWE in First Affiliated Hospital of Sun Yat-sen University (from July 1, 2021 to October 1, 2021) were collected. After exposure of the focus, ICG were used intravenously (0.25 mg/kg) as fluorescent dye for the intraoperative evaluation of AWE vascularization. Resection of the AWE was guided by direct visualization of the focus under standard laparoscopy with a near-infrared (NIR) camera head. Surgical margin around the AWE (3, 6, 9 and 12 point) and the margin under the focus were obtained for postoperative pathological examination of endometriosis. Time from injection to fluorescence visualization, the proportion of fluorescence visualization, time of fully resection of AWE, side effects related to the use of ICG, perioperative complications as well as the pathological result of the surgical margins were recorded. Results: ICG fluorescence of the AWE were seen in 5 patients (5/7). The mean time from injection to fluorescence visualization was (46.7±9.8) s. The mean time of fully resection of AWE was (16.4±7.0) minutes. There were no side effects related to the use of ICG. The rate of class-A wound healing was 7/7. All of the surgical margins were confirmed endometriosis-negative by postoperative pathological examination. Conclusion: ICG fluorescence visualization could conduct accurate resection of AWE, which is clinically safe and effective.

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.

Identification of biomarkers for lymph node metastasis in early-stage cervical cancer by tissue-based proteomics.

BACKGROUND: Pelvic lymph node metastasis (PLNM) is the key to determining the treatment and prognosis of early-stage cervical cancer (CC, I-IIst). The aim of this study was to identify biomarkers for PLNM of CC, I-IIst. METHODS: Two-dimensional fluorescence difference gel electrophoresis and matrix-assisted laser desorption/ionisation-time-of-flight mass spectrometry (MALDI-TOF/TOF MS) were used to identify differentially expressed proteins in primary CC, I-IIst tissue with (n=8) and without (n=10) PLNM. The expression levels of three differential proteins (FABP5, HspB1, and MnSOD) were validated using western blotting and immunohistochemistry. An independent cohort of 105 CC, I-IIst patients was analysed to assess the correlation of FABP5, HspB1, and MnSOD with clinicopathologic factors and clinical outcomes. RESULTS: Forty-one differential proteins were identified. Upregulation of FABP5, HspB1, and MnSOD in CC, I-IIst with PLNM was confirmed and was significantly correlated with PLNM. FABP5, HspB1, and MnSOD were significant predictors of PLNM in univariate analysis. FABP5, HspB1, and lymphovascular space invasion (LVSI) were independent predictors of PLNM in multivariate analysis. Survival curves indicated that CC, I-IIst patients with FABP5, HspB1, and MnSOD upregulation had poor prognosis. CONCLUSIONS: FABP5, HspB1, and MnSOD may be potential biomarkers for PLNM of CC, I-IIst and may have important roles in the pathogenesis of PLNM.

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.

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.

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.

Arsenic trioxide induced apoptosis in retinoblastoma cells by abnormal expression of microRNA-376a.

Arsenic trioxide (ATO) has been demonstrated to induce apoptosis in retinoblastoma cells, however, mechanisms responsible for this phenomenon are not fully understood. In the present study, we determined whether ATO induced apoptosis by abnormal expression of microRNA. In an apoptosis model of retinoblastoma cells subjected to 4 µM ATO for 72 hours, we found 14 miRNAs changed more than 2-fold by using miRNA microarray analysis. Most of these aberrantly expressed miRNAs were confirmed by quantitative RT-PCR. MiR-376a, a significantly down-regulated miRNA, was selected for further study. The overexpression of miR-376a resulting from miR-376a mimic transfection significantly inhibited ATO-induced apoptosis. By contrast, miR-376a deficiency resulting from miR-376a inhibitor transfection aggravated ATO-induced apoptosis. Using bioinformatic algorithms, caspase-3, a key apoptosis executioner, was predicted as a putative target of miR-376a. The quantitative RT-PCR showed no effects of miR-376a mimic or inhibitor on caspase-3 mRNA level. However, the amount of caspase-3 proteins was reduced by miR-376a mimic, whereas increased by miR-376a inhibitor. Furthermore, the luciferase reporter assay confirmed caspase-3 to be a target of miR-376a, and the apoptosis caused by miR-376a inhibitor were abolished by a caspase-3 inhibitor. These results suggest that ATO -induced apoptosis in retinoblastoma cells is part mediated by decreasing expression of miR-376a, which subsequently increased caspase-3 expression.

Novel splice variants of the bovine PCK1 gene.

Phosphoenolpyruvate carboxykinase 1 (PCK1), also named PEPCK-C, is a multiple-function gene that is involved in gluconeogenesis, glyceroneogenesis, reproduction, female fertility, and development of obesity and diabetes. How its many functions are regulated was largely unknown. Therefore, we investigated mRNA expression and possible splice variants of PCK1 by screening cDNA in nine tissues from Holstein bulls and cows. PCK1 mRNA was highly expressed in the liver, kidney, ovary and testis; expression levels were low in the heart, spleen, and lung tissues. Expression of this gene was not detected in skeletal muscle. This led to the discovery of five novel bovine splice variants, named PCK1-AS1-PCK1-AS5. In PCK1-AS1, 51 nucleotides in the interior of exon 2 were spliced out. In PCK1-AS2, exons 2 and 3 were altered by the alternative 3' and 5' splice sites, respectively. PCK1-AS3 was truncated from the 3' end of exon 2 to the 5' end of exon 4. In PCK1-AS4, exon 5 was completely spliced out. In PCK1-AS5, exons 5 and 6 and the 5' end of exon 7 were spliced out. These splice variants (PCK1-AS1-PCK1-AS5) potentially encoded shorter proteins (605, 546, 373, 246 and 274 amino acids, respectively), when compared to the complete protein (622 amino acids). Considering the functional domains of the PCK1 protein, it is likely that these splice variants considerably affect the function of this protein; alternative splicing could be one of the mechanisms by which the diverse functions of PCK1 are regulated.

Meta-analysis demonstrates lack of a relationship between XRCC1-399 gene polymorphisms and susceptibility to hepatocellular carcinoma.

XRCC1-399 allele polymorphisms have been reported to be associated with susceptibility to hepatocellular carcinoma (HCC), but the conclusions of the various studies have been inconsistent. We conducted a meta-analysis of available studies to determine whether XRCC1-399 alleles influence susceptibility to hepatocellular carcinoma. We searched English-language databases, including PubMed, Medline and Embase, using terms such as "hepatocellular carcinoma" (or "HCC"), "X-ray repair cross-complementing group 1" (or "XRCC1") and "genetic polymorphism" (or "SNP"), among others; we also searched Chinese-language databases, including CNKI, VIP, Wanfang Data, and CBM, using terms such as "ganai", "ganxibaoai", "ganzhongliu", "duotaixing", and "X-xian xiufu jiaocha hubu jiyin 1". Eight independent studies, including 1604 HCC cases and 2185 controls, were included. The pooled odds ratio for XRCC1-399 was 0.99 (95% confidence interval = 0.75-1.31). We conclude that XRCC1- 399 gene polymorphisms are unrelated to risk for HCC.

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.

[Regulatory effects of the Nocardia rubra cell wall skeleton on the biological function of human neutrophils].

Objective: To investigate the regulatory effects and mechanism of Nocardia rubra cell wall skeleton (Nr-CWS) on the biological function of human neutrophils. Methods: The experimental research method was used. Fifteen healthy adult volunteers (7 males and 8 females, aged 24 to 45 years) were recruited from Suzhou Physical Examination Center for physical examination from May to October 2022, the peripheral venous blood was collected, and neutrophils were extracted by immunomagnetic bead sorting. The cells were divided into normal control group without any treatment, Nr-CWS alone group treated with Nr-CWS of final mass concentration 60 ng/mL alone, endotoxin/lipopolysaccharide (LPS) alone group stimulated with LPS of final mass concentration 1 µg/mL alone, and LPS+Nr-CWS group stimulated with LPS first and then treated with Nr-CWS as before. After 1 h of culture, the chemotaxis distance, chemotactic cell percentage, chemotactic index, maximum chemotactic speed, and chemotactic function score of neutrophils were detected using the modified agarose chemotactic model; the proportion and fluorescence intensity of phagocytosis cells, the level of reactive oxygen species (ROS), the protein expression levels of granular protein CD35, CD66b, and CD63, and the concentrations of inflammatory cytokines of interleukin 2 (IL-2), IL-4, IL-6, IL-10, IL-17A, tumor necrosis factor alpha (TNF-α), and interferon-γ in cell culture supernatant were detected by flow cytometry. The number of samples in each group in the above experiments was 15. Data were statistically analyzed with analysis of variance for factorial design and independent sample t test. Results: After 1 h of culture, the chemotactic function score of cells in normal control group, Nr-CWS alone group, LPS alone group, and LPS+Nr-CWS group were 15.0, 14.5±0.5, 1.5±0.5, 12.0±1.5, respectively. Compared with those in normal control group, the chemotaxis distance, chemotactic cell percentage, chemotactic index, maximum chemotactic speed, and chemotactic function score of cells were significantly decreased in LPS alone group and LPS+Nr-CWS group (with t values of 18.36, 18.88, 54.28, 18.36, 46.77, 10.58, 14.74, 6.84, 10.58, and 4.24, respectively, P<0.05); compared with those in LPS alone group, the five chemotactic function indexes as above in LPS+Nr-CWS group were significantly increased (with t values of 11.47, 14.65, 11.62, 11.47, and 13.75, respectively, P<0.05). After 1 h of culture, compared with those in normal control group, the proportion and fluorescence intensity of phagocytosis cells were significantly increased in Nr-CWS alone group (with t values of 6.86 and 6.73, respectively, P<0.05), and the above two indexes were significantly decreased in LPS alone group (with t values of 7.35 and 22.72, respectively, P<0.05) and LPS+Nr-CWS group (with t values of 21.37 and 13.10, respectively, P<0.05). After 1 h of culture, compared with that in normal control group, the level of ROS of cells in LPS alone group was significantly increased (t=6.64, P<0.05); compared with that in LPS alone group, the level of ROS of cells in LPS+Nr-CWS group was significantly decreased (t=5.46, P<0.05). After 1 h of culture, compared with those in normal control group, the protein expressions of CD35, CD66b, and CD63 of cells were significantly increased in LPS alone group and LPS+Nr-CWS group (with t values of 16.75, 17.45, 10.82, 5.70, 19.35, and 15.37, respectively, P<0.05); compared with those in LPS alone group, the protein expressions of CD35, CD66b, and CD63 of cells were significantly decreased in LPS+Nr-CWS group (with t values of 4.92, 5.72, and 3.18, respectively, P<0.05). After 1 h of culture, compared with those in normal control group, the concentrations of IL-2, IL-4, IL-6, IL-10, IL-17A, TNF-α, and interferon-γ in cell culture supernatant were significantly increased in LPS alone group (with t values of 22.10, 9.50, 7.21, 10.22, 24.88, 8.43, and 47.48, respectively, P<0.05), and the concentrations of IL-6, IL-10, IL-17A, TNF-α, and interferon-γ in cell culture supernatant were significantly increased in LPS+Nr-CWS group (with t values of 4.68, 5.12, 8.02, 5.58, and 7.13, respectively, P<0.05); compared with those in LPS alone group, the concentrations of IL-2, IL-4, IL-6, IL-10, IL-17A, TNF-α, and interferon-γ in cell culture supernatant were significantly decreased in LPS+Nr-CWS group (with t values of 5.39, 2.83, 5.79, 2.90, 5.87, 4.88, and 39.64, respectively, P<0.05). Conclusions: Nr-CWS can enhance the phagocytosis ability of neutrophils in normal condition and improve the chemotactic function, ROS level, degranulation protein level, and inflammatory factor level of human neutrophils in infectious condition. Nr-CWS can enhance the anti-infection ability of human neutrophils by regulating its biological behavior in innate immunity.

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.

[Re-understanding the physiological and pathophysiological roles of neutrophils].

Neutrophils have always been considered as a short-lived and homogeneous cell type in the innate immune system, which have limited pro-inflammatory or anti-inflammatory effects. However, in recent 10 years, the understanding of neutrophils has been undergoing some kind of revival as researches progressed. The researches on the heterogeneity of neutrophils and the mechanism of their interaction with other immune cells have promoted the researchers to re-understand the physiological and pathophysiological roles of neutrophils. In the following decades, with the development of single-cell sequencing technology, spatial transcriptome sequencing technology, and multi-omics combined sequencing technology, researchers will have a better understanding of the biological behaviors of neutrophils. This paper briefly reviews the biological behaviors of neutrophils and their roles in various diseases in recent years.