Ordering three microsatellites on porcine chromosome 12 by single sperm typing

Three microsatellite loci on porcine chromosome 12 were ordered by single sperm typing to expand the limited genetic map of this region. Individual sperm cells from a Chinese indigenous Qingping boar triply heterozygous at SW874, SW1350 and SW1553 were amplified using PEP and heminesting primer design at each locus. Analysis of the sperm typing data by the SPERM.FOR program showed that the most likely order was SW1553-SW1350-SW874.     

Artificial insemination in cattle with DNA‐treated sperm

Abstract

We have investigated the use of sperm cells as vectors for transferring exogenous DNA into the genome of cattle by artificial insemination with DNA‐treated sperm. First we demonstrated the DNA‐binding ability of cattle sperm with radioactively labeled DNA. For artificial insemination ejaculated semen was washed and incubated with 1 μg DNA/10⁶ sperm for one hour at 37°C. Three hundred synchronized heifers were inseminated once with a dose of 40×10⁶ sperm. Forty‐five calves and 41 fetuses were obtained. Southern analysis revealed in one calf a signal after probing with the 1 kb Pst I fragment of pSV2‐cat.     

The binding of exogenous DNA fragments to bovine spermatozoa

Abstract

The ability of mature, freeze‐thawed bovine sperm to bind exogenous end‐labelled or oligo‐labelled λ Hind III DNA restriction fragments was examined. Following 30 min. incubation of bovine sperm with P³² end‐labelled λ Hind III DNA and five washes with medium, approximately 5.8 ng DNA were bound to 10⁷ sperm. Agarose gel autoradiography revealed that all of the λ Hind III DNA bands were present following sperm washes except for the smaller 0.5 Kb and 0.125 Kb bands. Incubation of sperm with ³H oligo‐labelled λ Hind III DNA gave a much higher level of binding (138 ng/10⁷ sperm) than that found with end‐labelled DNA. This binding was entirely eliminated by DNase I. The separation of live and dead sperm fractions on Percoll gradients revealed that more oligo‐labelled λ Hind III DNA was found to be associated with the dead sperm fraction (31.2 ng/10⁷ sperm) rather than the live sperm fraction (2.7 ng/10⁷ sperm). Analysis of supravital stained, light microscopic autoradiographs confirmed that oligo‐labelled λ Hind III DNA bound to dead sperm in the post‐acrosomal region of the sperm head although other minor distribution patterns were observed.     

Short communication: Extraction of sp18 family membrane proteins on posterior head of bull sperm and the effect of anti‐sp18 IgG on sperm motility and murine in vitro fertilization

Abstract

Bovine sperm heads were separated via ultrasonic treatment and centrifugation. Anti‐bull sperm IgG was produced by immunizing rabbits with acrosome‐reacted bull sperm heads. SDS PAGE patterns revealed that the main membrane proteins on acrosome‐reacted bull sperm head were sp18 family, including 18, 16, and 14 kD, which represented about 64% of the total membrane proteins in bull sperm. Indirect immunofluorescence shown sp18 antigens primarily distributed in postacrosomal and proximal tail regions. Western blot analysis revealed that the anti‐bull sperm IgG reacted with sp18 antigens in acrosome‐reacted bull sperm head and bull seminal plasma. Anti‐bull sperm IgG also reacted with 14, 16, 18, 42, 57 and 60 kD proteins in fresh bull, mouse and rabbit sperm. Anti‐sp18 IgG caused agglutination of bull and rabbit sperm, but had no effect on murine sperm. In murine in vitro fertilization trials, preincubating capacitated sperm with 0.364 mg/ml of anti‐sp18 IgG resulted in a decrease in the fertilization rate from 75.6% in the controls to 50.8% in the experimental groups (p<0.001).     

The effect of tumor necrosis factor alpha (-308G/a) and interferon gamma (+874T/a) polymorphisms on susceptibility to coronary heart disease

Abstract

Background: Coronary heart disease (CHD) is a chronic inflammatory disease, which is still regarded as a major cause of morbidity and mortality worldwide. Several studies have suggested that polymorphisms in cytokine genes are associated with the pathogenesis of CHD. The genotype distribution of Tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) genes polymorphisms have been shown to be different in various ethnic populations. This study was aimed to investigate the association of TNF-α-308?G/A and IFN-γ?+?874T/A polymorphisms with risk of CHD in an Iranian population.

Methods: A total of 187 unrelated subjects comprised 96 CHD patients and 91 healthy controls were enrolled in this cross-sectional study. The TNF-α-308?G/A and IFN-γ?+?874T/A polymorphisms were genotyped using amplification refractory mutation system-PCR (ARMS-PCR). The chi-square and logistic regression tests were used to calculate the odds ratios (ORs) as a measure of differences in genotype frequencies.

Results: A significant differences in the allelic and genotypic distribution of TNF-α-308?G/A and IFN-γ?+?874T/A polymorphisms was found between CHD patients and healthy controls (P?=?0.017, P?=?0.011, P?=?0.006 and P?=?0.002, respectively). Logistic regression analyses were also revealed statistically significant risk for CHD with respect to TNF-α-308?A and IFN-γ?+?874?T carriers either in crude or after adjustment for potential confounders (P?=?0.003 and P?=?0.006, respectively).

Conclusion: This study provides strong evidence supporting the association of TNF-α-308G/A and IFN-γ?+?874T/A polymorphisms with the increased risk of CHD. Therefore, these two cytokine polymorphisms may play a role in predisposition to coronary heart disease.     

Internal Desynchronization of Circadian Rhythms and Tolerance to Shift Work

Intolerance to shift work may result from individual susceptibility to an internal desynchronization. Some shift workers (SW) who show desynchronization of their circadian rhythms (e.g., sleep‐wake, body temperature, and grip strength of both hands) exhibit symptoms of SW intolerance, such as sleep alteration, persistent fatigue, sleep medication dependence, and mood disturbances, including depression. Existing time series data previously collected from 48 male Caucasian French SW were reanalyzed specifically to test the hypothesis that internal synchronization of circadian rhythms is associated with SW intolerance and symptoms. The entry of the subjects into the study was randomized. Three groups were formed thereafter: SW with good tolerance (n=14); SW with poor tolerance, as evident by medical complaints for at least one year (n=19); and former SW (n=15) with very poor tolerance and who had been discharged from night work for at 1.5 yr span but who were symptom‐free at the time of the study. Individual and longitudinal time series of selected variables (self‐recorded sleep‐wake data using a sleep log, self‐measured grip strength of both hands using a Colin Gentile dynamometer, and oral temperature using a clinical thermometer) were gathered for at least 15 days, including during one or two night shifts. Measurements were performed 4–5 times/24 h. Power spectra that quantify the prominent period (τ) and t‐test, chi square, and correlation coefficient were used as statistical tools. The mean (±SEM) age of SW with good tolerance was greater than that of SW with poor tolerance (44.9±2.1 yrs vs. 40.1±2.6 yrs, p<.001) and of former SW discharged from night work (very poor tolerance; 33.4±1.7, p<.001). The shift-work duration (yrs) was longer in SW with good than poor tolerance (19.9±2.2 yrs vs. 15.7±2.2; p<0.002) and former SW (10.7±1.2; p<.0001). The correlation between subject age and shift-work duration was stronger in tolerant SW (r=0.97, p<.0001) than in non‐tolerant SW (r=0.80, p<0.001) and greater than that of former SW (r=0.72, p<.01). The mean sleep‐wake rhythm τ was 24 h for all 48 subjects. The number of desynchronized circadian rhythms (τ differing from 24 h) was greater in non‐tolerant than in tolerant SW (chi square=38.9, p<.0001). In Former SW (i.e., 15 individuals assessed in follow‐up studies done 1.5 to 20 yrs after return to day work), both symptoms of intolerance and internal desynchronization were reduced or absent. The results suggest that non‐tolerant SW are particularly sensitive to the internal desynchronization of their circadian time organization.     

Population paleoecology and comparative taphonomy of two edrioasteroid (echinodermata) pavements: Upper ordovician of kentucky and Ohio

Two strophomenid brachiopod shell pavements bearing abundant edrioasteroid epizoans occur stratigraphically within the top 4.5 m of the Corryville Formation (Upper Ordovician, Maysvillian) in Boone County, Kentucky, and Hamilton County, Ohio. Both assemblages are dominated by Isorophus cincinnatiensis and contain Carneyella pilea and Streptaster vorticellatus as lesser constituents. Pedicle exterior valves of Rafinesquina “alternata”; are the preferred substratum for all species at both sites, but the Kentucky population occurs mainly on unabraded, articulated shells and the Ohio population occupies abraded, disarticulated shells. The Kentucky population includes a greater size range of Isorophus (at least three cohorts) than the Ohio population (at least two cohorts), suggesting a greater time span of colonization for the Kentucky site. Host shells from Kentucky have anterior‐posterior axes strongly aligned NE‐SW whereas the Ohio shells are unoriented. Isorophus in Kentucky show a preference for the anterior margin of the Rafinesquina pedicle exterior valve, whereas Isorophus in Ohio show no preferred location on host shells. The Kentucky edrioasteroids are inferred to have attached to mainly live Rafinesquina in brachial‐valve‐up position. Host shells were inverted and oriented by a NE‐SW storm surge, then buried rapidly. Ohio edrioasteroids occupied mainly dead, already inverted host shells not oriented by storm surge possibly because of their more downslope (distal) location. Comparative taphonomic relationships between the two coeval populations are consistent with a model of proximal to distal distribution along a gentle shallow subtidal N‐S paleoslope subject to episodic storm activity.     

利用CRISPR/Cas9技术靶向KRAS或TP53突变抑制SW620细胞的增殖

摘要 目的：运用CRISPR/Cas9基因编辑工具研究靶向SW620细胞系中KRAS或TP53突变对细胞增殖活性的影响。方法：针对SW620细胞中KRAS和TP53的突变位点设计sgRNA,并利用TIDE法检测sgRNA的切割效率。通过细胞增殖实验检测靶向KRAS或TP53突变后SW620细胞增殖活性的改变,并应用Annexin V-FITC/PI双染法检测细胞凋亡水平的变化。结果：分别构建了靶向SW620细胞系中KRAS和TP53突变的sgRNA质粒,并通过TIDE分析验证了sgRNA的内源切割效率;细胞增殖实验及细胞凋亡检测显示,靶向突变的KRAS或TP53基因后,SW620细胞增殖活性明显减弱,凋亡水平明显升高（P<0.05）。结论：本研究基于CRISPR/Cas9技术实现了对SW620细胞系中突变的KRAS和TP53的基因编辑,发现靶向KRAS或TP53突变能够明显抑制SW620细胞的增殖活性并促进细胞凋亡,为结直肠癌相关靶点治疗提供了体外实验依据。     

Lactate Formation in Primary and Metastatic Colon Cancer Cells at Hypoxia and Normoxia

High glucose consumption and lactate synthesis in aerobic glycolysis are a hallmark of cancer cells. They can form lactate also in glutaminolysis, but it is not clear how oxygen availability affects this process. We studied lactate synthesis at various oxygen levels in human primary (SW480) and metastatic (SW620) colon cancer cells cultured with L‐Ser and/or L‐Asp. Glucose and lactate levels were determined colorimetrically, amino acids by HPLC, expression of AST1‐mRNA and AST2‐mRNA by RT‐PCR. In both lines glucose consumption and lactate synthesis were higher at 10% than at 1% oxygen, and lactate/glucose ratio was increased above 2.0 by L‐Asp. AST1‐mRNA expression was independent on oxygen and cell line, but AST2‐mRNA was lower at hypoxia in SW480. We conclude that, in both cell lines at 1% hypoxia, lactate is formed mainly from glucose but at 10% normoxia also from L‐Asp. At 10% normoxia, lactate synthesis is more pronounced in primary than metastatic colon cancer cells.     

稳定表达Cas9蛋白的SW620细胞株构建

摘要 目的：建立稳定表达Cas9蛋白的SW620人结肠癌细胞系的单克隆细胞株,提高基因编辑效率,为利用基于CRISPR/Cas9技术的高通量筛选结肠癌相关致病基因提供细胞工具。方法：用Cas9慢病毒侵染SW620细胞系,用致死剂量的puro筛选5-7天,通过有限稀释法获得单克隆细胞株。提取单克隆细胞基因组进行Sanger测序,筛选出含有Cas9基因序列的单克隆细胞株。利用基于SSA修复荧光素酶的报告系统检测单克隆细胞株中Cas9的编辑活性,并通过细胞增殖实验检测Cas9蛋白的表达是否影响细胞增殖。结果：获得了两个表达Cas9蛋白的SW620单克隆细胞株,并通过Sanger测序验证了Cas9序列;荧光素酶报告系统检测显示单克隆细胞株的Cas9蛋白有较高的编辑活性;细胞增殖实验显示Cas9蛋白的表达对SW620增殖活性影响不大。结论：本研究利用慢病毒感染的方式,构建了稳定表达Cas9蛋白的SW620单克隆细胞株,为后续大规模筛选与人结肠癌相关的基因突变提供了细胞工具。     

基于基因组序列的脑膜炎奈瑟菌分型方法

[目的]建立并评估1种适宜的脑膜炎奈瑟菌(Neisseria meningitidis,Nm)基因组分子分型方法.[方法]本研究以125株代表性Nm菌株的基因组序列为对象,建立了基于核心基因SNP的基因组分型方法,并与pubMLST网站公布的MLST和cgMLST分型方法进行比较.[结果]基于核心基因SNP的基因组分型...     

Mapping of an immotile short tail sperm defect in the Finnish Yorkshire on porcine Chromosome 16

An immotile short tail sperm defect has recently been identified as a hereditary disorder present within the Finnish Yorkshire pig population. The syndrome is inherited as an autosomal recessive disease exclusively expressed in male individuals as shorter sperm tail length and immotile spermatozoa. Based on the assumption of a recent common origin of the disease-causing mutation, a genome-wide search was performed with 228 evenly spaced microsatellites by homozygosity mapping of affected and unaffected DNA pools. One locus, SW2411 on Chr 16, demonstrated a significantly skewed allele distribution between the two pools. Linkage analysis of five markers in this region mapped the disease-causing gene within a 6-cM confidence interval region with a highest LOD score of 7.7 at marker SW419. It appears that three-marker haplotypes can be used for marker-assisted selection within analyzed pedigrees. Furthermore, future fine mapping may reveal a more precise population-wide associated haplotype and facilitate identification of a new gene affecting sperm tail development. Received: 5 July 2001 / Accepted: 13 September 2001     

Relationship between γ‐interferon gene polymorphisms and susceptibility to brucellosis infection

Interferon‐gamma (IFN‐γ) is a pro‐inflammatory cytokine that plays a pivotal role in the defense mechanism against Brucella infection. It was hypothesized that the IFN‐γ in (+874 A/T in intron 1) TT and +5644 T/A, TT genotypes, which are reportedly associated with high IFN production, are associated with susceptibility to brucellosis in Iranian subjects. Genotyping of these IFN‐γ variants by an allele‐specific polymerase chain reaction method was performed in 281 subjects, comprising 153 patients with active brucellosis and 128 healthy controls. It was found that the +874 minor allele (A) and homozygote genotype (AA) were significantly more frequently present in brucellosis patients than in controls (OR = 2.588; 95% CI, 1.313–5.104; P = 0.006 for the AA genotype; OR = 1.575; 95% CI, 1.124–2.216; P = 0.010 for the A allele). However, the allelic and genotypic distribution of the IFN‐γ polymorphism at position UTR5644 A>T did not differ significantly between patients and controls (P > 0.05). The distribution of haplotypes in this study suggests that the T/A haplotype (+874/UTR5644), which was present more frequently in controls than in patients, may protect subjects against Brucella infection. It is suggested that IFN‐γ +874 AA genotype and A allele are risk factors for developing brucellosis infection in Iranian subjects.     

Sperm cell mediated transgenesis: A review

Abstract

Transgenesis would be greatly streamlined if sperm cells could be used as transgene vectors. Attempts to ‘force’ sperm cells to take up transgenes may be more worthy of further study than ‘autouptake’ attempts. However, thorough proof of its effectiveness will be required if sperm cell‐mediated gene transfer is to be accepted, given the evolutionary implications associated with the possibility of sperm cells being able to transfer exogenous DNA.     

Order of bovine DRB3, DYA,and PRL determined by sperm typing

The order and recombination fractions () between the bovine major histocompatibility complex DRB3, DYA, and prolactin (PRL) genes were determined by typing of 254 sperm from a triply heterozygous bull. A recently developed method, primer extension preamplification (PEP), was used to amplify the bovine sperm genome prior to amplification of specific loci by the polymerase chain reaction (PCR). At least 28 copies of the DRB3, PRL, or DYA gene were obtained from 50 cycles of PEP. For sperm typing, alleles of each locus were discriminated by restriction endonuclease cleavage of PCR products and polyacrylamide gel electrophoresis of the restriction fragments. The most likely gene order is PRL-DRB3-DYA, with =0.025 (±0.012) and =0.150 (±0.024), respectively. The odds are 128:1 in favor of this order in comparison with the second most likely order DRB3-PRL-DYA. Our results demonstrate the power of sperm typing in concert with PEP for multilocus gene mapping.     

Fatty‐Acid Profile of Total and Polar Lipids in Cultured Rainbow Trout (Oncorhynchus mykiss) Raised in Freshwater and Seawater (Croatia) Determined by Transmethylation Method

Fatty acids from total lipids and polar lipids in cultured rainbow trout (Oncorhynchus mykiss) raised in seawater (SW) and freshwater (FW) were identified and quantified from the muscle samples in January, April, and July. The highest total lipid and polar lipid amounts were found in April. July contents of total lipids were low, but percent of the polyunsaturated fatty acids (PUFAs) was high in SW and FW environment (particularly n‐3 PUFAs). Variety of 17 fatty acids was identified by GC‐FID after transmethylation. The predominant fatty acids in rainbow trout from SW and FW were: docosahexaenoic acid among n‐3 PUFAs, palmitic acid among saturated fatty acids (SFAs), and oleic acid among monounsaturated fatty acids (MUFAs). Appreciably higher n‐3/n‐6 ratio was found in total lipids in April (6.40, FW fish) and in polar lipids in July (18.76; SW fish). High n‐3/n‐6 ratio in total lipids and polar lipids of rainbow trout from SW and FW, besides beneficial n‐3/n‐6 ratio in the commercial fish food, could be characteristic for the local environmental conditions (Croatia).     

基于逆转录病毒载体构建稳定表达人CD19和荧光蛋白的SW620细胞株

摘要 目的：构建表达人CD19和增强型绿色荧光蛋白（EGFP）和荧光素酶（LUC）的SW620细胞株。方法：构建MFG-CD19质粒,进行逆转录病毒载体包装,与MFG-EGFP-P2A-LUC逆转录病毒载体共同转导SW620细胞,筛选出稳定且强表达的单克隆细胞株SW620-CD19-EGFP-LUC并扩大培养。通过流式细胞术与qPCR技术检测细胞CD19的表达、通过荧光素酶法对细胞系表面荧光素酶的表达和细胞系功能进行鉴定。结果：流式检测构建完成的细胞株中CD19阳性的细胞占比为99.9%,EGFP阳性的细胞占比为99.2%;荧光显微镜下能够明显观察到细胞的绿色荧光;qPCR检测结果表明细胞株中CD19的表达水平相比原始细胞株明显上调,并且能够激活CD19 CAR-T细胞对其进行杀伤。结论：成功构建了能够稳定表达人CD19和荧光蛋白的SW620细胞株。     

Liposome‐mediated DNA transfer to chicken sperm cells

Abstract

The efficacy of using liposomes to transfer DNA to chicken sperm cells was investigated. Liposomes were prepared from dilauroyl (12:0) phosphatidylcholine (DLPC), dimyristoyl (14:0) phosphatidyl choline (DMPC), dipalmitoyl (16:0) phosphatidylcholine (DPPC), egg yolk phosphatidylcholine (EYPC) or lipids extracted from sperm cell membranes. The efficiency of trapping of DNA into the liposomes, transfer of the DNA from the liposomes to the sperm cells and the effect of the liposomes on the fertilizing ability of the sperm cells were determined. Increasing the concentration of lipid in the liposome preparations increased the trapping efficiency of DNA into liposomes but lowered the transfer of DNA to sperm. Including stearylamine (SA) in the liposomes increased the incorporation of DNA into the liposomes and the DNA transfer to sperm cells, while including lauroyllysophosphatidylcholine (LPC) along with SA resulted in the highest transfer efficiency from liposomes to sperm. The transfer of DNA from liposomes to sperm cells was lowered by increasing the number of sperm cells, while decreasing the number of sperm cells lowered the fertility. The sperm cells remained fertile after exposure to low levels of DPPC or lipofectin reagent or to high levels of SA and LPC. The best conditions for liposome‐mediated gene transfer to chicken sperm cells are thus using either lipofectin reagent at .006 to .06 μmol/ml and 5 × 10⁷ sperm or with DPPC liposomes comprised of 10 μmol/ml total lipid including 5 mol% SA and 20 mol% LPC with 2.5 × 10⁸ sperm cells. The use of liposomes to enhance the transfer of DNA to sperm cells may make the use of sperm cells as gene transfer vectors possible.     

Wide step width reduces knee abduction moment of obese adults during stair negotiation

PurposeAn increased likelihood of developing obesity-related knee osteoarthritis may be associated with increased peak internal knee abduction moments (KAbM). Increases in step width (SW) may act to reduce this moment. The purpose of this study was to determine the effects of increased SW on knee biomechanics during stair negotiation of healthy-weight and obese participants.MethodsParticipants (24: 10 obese and 14 healthy-weight) used stairs and walked over level ground while walking at their preferred speed in two different SW conditions – preferred and wide (200% preferred). A 2 × 2 (group × condition) mixed model analysis of variance was performed to analyze differences between groups and conditions (p < 0.05).ResultsIncreased SW increased the loading-response peak knee extension moment during descent and level gait, decreased loading-response KAbMs, knee extension and abduction range of motion (ROM) during ascent, and knee adduction ROM during descent. Increased SW increased loading-response peak mediolateral ground reaction force (GRF), increased peak knee abduction angle during ascent, and decreased peak knee adduction angle during descent and level gait. Obese participants experienced disproportionate changes in loading-response mediolateral GRF, KAbM and peak adduction angle during level walking, and peak knee abduction angle and ROM during ascent.ConclusionIncreased SW successfully decreased loading-response peak KAbM. Implications of this finding are that increased SW may decrease medial compartment knee joint loading, decreasing pain and reducing joint deterioration. Increased SW influenced obese and healthy-weight participants differently and should be investigated further.