Can SpRY recognize any PAM in human cells? / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

The application of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) can be limited due to a lack of compatible protospacer adjacent motif (PAM) sequences in the DNA regions of interest. Recently, SpRY, a variant of Streptococcus pyogenes Cas9 (SpCas9), was reported, which nearly completely fulfils the PAM requirement. Meanwhile, PAMs for SpRY have not been well addressed. In our previous study, we developed the PAM Definition by Observable Sequence Excision (PAM-DOSE) and green fluorescent protein (GFP)‍-reporter systems to study PAMs in human cells. Herein, we endeavored to identify the PAMs of SpRY with these two methods. The results indicated that 5'-NRN-3', 5'-NTA-3', and 5'-NCK-3' could be considered as canonical PAMs. 5'-NCA-3' and 5'-NTK-3' may serve as non-priority PAMs. At the same time, PAM of 5'-NYC-3' is not recommended for human cells. These findings provide further insights into the application of SpRY for human genome editing.

Cancer is associated with the severity and mortality of patients with COVID-19: a systematic review and meta-analysis

BackgroundCancer patients are considered a highly vulnerable population in the COVID-19 epidemic, but the relationship between cancer and the severity and mortality of patients with COVID-19 remains unclear. This study aimed to explore the prevalence of cancer in patients with COVID-19 and to examine whether cancer patients with COVID-19 may be at an increased risk of severe illness and mortality. MethodsA comprehensive electronic search in seven databases was performed, to identified studies reporting the prevalence of cancer in COVID-19 patients, or providing data of cancer between patients with severe or non-severe illness or between non-survivors and survivors. Meta-analyses were performed to estimate the pooled prevalence and odds risk (OR) using the inverse variance method with the random-effects model. ResultsThirty-four studies with 8080 patients were included. The pooled prevalence of cancer in patients with COVID-19 was 2.0% (95% CI: 2.0% to 3.0%). The prevalence in Italy (5.0%), France (6.0%), and Korea (4.0%) were higher than that in China (2.0%). Cancer was associated with a 2.84-fold significantly increased risk of severe illness (OR = 2.84, 95%CI: 1.75 to 4.62, P < 0.001) and a 2.60-fold increased risk of death (OR = 2.60, 95%CI: 1.28 to 5.26, P = 0.008) in patients with COVID-19. Sensitivity analyses showed that the results were stable after excluding studies with a sample size of less than 100. ConclusionsCancer patients have an increased risk of COVID-19 and cancer was associated with a significantly increased risk of severity and mortality of patients with COVID-19.

Construction and screening effective sequence of shRNA targeting human trefoil factor 3 / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#Trefoil factor 3 plays a pivot role in oncogenic transformation, growth and metastatic extension of solid tumours besides mucosal protection. We screened the best siRNA sequence targeting human TFF3 by the transient-transfection of the lentiviral mediated shRNA into thyroid carcinoma K1 cells which secrete TFF3 themselves.@*METHOD@#Four siRNA transcription template hairpin structure target potential sites in human TFF3 mRNA sequence(132,170,258 and 537 bp,seperately) were selected and synthesized,as well as one negative shRNA(shRNAC). After annealing in vitro, insert pLVX-shRNA-puro construct recombinant plasmid, then enzyme digestion and sequencing analysis. The lentiviral-shRNAs were transient-transfected into K1 cells. TFF3 mRNA and protein levels were test by real-time PCR and western blot respectively in K1 cells at 48h post transient-transfected.@*RESULT@#Genetic mutations in two sequences of shRNA1~2, so the follow-up study terminated. The TFF3 expression were obviously inhibited in K1 cells at 48 hours post transient-transfected of shRNA3 and shRNA4. TFF3 (258-276) showed the highest silencing efficiency (TFF3 mRNA reduced 60.67% and TFF3 protein reduced 56.44%, P < 0.01) when the transfection efficiency was 76.83%.@*CONCLUSION@#pLVX-shRNA-puro-TFF3 expression plamid were successfully constructed and the highest efficiency sequences were screened. All these laid a foundation for further study about the function of TFF3 gene.

Rhabdomyolysis After Out-of-Water Exercise in an Elite Adolescent Water Polo Player Carrying the IL-6 174C Allele Single-Nucleotide Polymorphism.

We present an adolescent elite water polo player who despite a genetic predisposition to develop exercise-induced severe muscle damage due to carrying the IL-6 174C allele single-nucleotide polymorphism, developed acute rhabdomyolysis only after a vigorous out-of-water training, suggesting that water polo training may be more suitable for genetically predisposed athletes.

Resting measures and physiological responses to exercise for the determination of prognosis in patients with chronic heart failure: useful tools for clinical decision-making.

Despite recent advances in the management of chronic heart failure (CHF), the prognosis of many of these patients remains dire. The need for an accurate prognosis in these patients has led to the identification of several indicators purported to represent the impact of the disease. Such indicators often are obtained at rest and are not always accurate at determining the clinical status of CHF patients. As a result, the relationship between prognostic indicators and clinical outcomes is frequently weak. On the other hand, physiological responses to acute exercise may unmask patients with the worst clinical status and identify those at increased risk of poor outcomes. Therefore, the present review appraises the value of several prognostic indicators for patients with CHF collected at rest and in response to exercise. In particular, it contrasts the value and accuracy of predictors of mortality widely used in clinical settings, such as oxygen uptake, ventilatory efficiency, and left ventricular ejection fraction, with new and more direct indicators of ventricular systolic and diastolic function.

The response of circulating brain natriuretic peptide to academic stress in college students.

Brain natriuretic peptide (BNP), a cardiac peptide, has been implicated in the regulation of hypothalamic-pituitary-adrenocortical (HPA) responses to psychological stressors. The influence of academic stress on circulating concentration of the N-terminal fragment of BNP precursor (NT-proBNP), and in relation to the stress hormone (cortisol) response was studied in 170 college students undergoing major examinations. Just prior to the examination, we measured self-estimated stress level, systolic, and diastolic blood pressure (SBP, DBP), heart rate (HR), plasma levels of cortisol, and NT-proBNP. These parameters were compared to the participants' baseline measurements, taken at the same hour of a different 'control day', without a major examination to induce stress. Hemodynamic variables (SBP, DBP, and HR) increased on the examination day compared with baseline values ( p < 0.001). Circulating cortisol concentration increased before examinations (+42%, p < 0.001). The response to stress was marked by a significant decrease in plasma NT-proBNP concentration (-40%, p < 0.001). We found in males a significant interaction between the cortisol elevation with examination stress and the NT-proBNP reduction ( p = 0.02). In response to academic stress, the plasma cortisol elevation was accompanied by a marked reduction in plasma NT-proBNP level. These data may indicate that mental stress entails an interface between the HPA axis and the peripheral natriuretic peptide system, leading to reciprocating changes in circulating levels of the corresponding hormones.

Is the interaction between HIF1A P582S and ACTN3 R577X determinant for power/sprint performance?

Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that regulates gene expression in response to hypoxia and has been associated with athletic performance. The aims of this study were (1) to determine the frequency distribution of HIF1A Pro582Ser (rs11549465) polymorphism among 155 Israeli athletes (sprinters and endurance athletes) and 240 healthy controls and (2) to analyze the influence of the interaction between HIF1A Pro582Ser and ACTN3 R577X (rs1815739) genotypes on sprint performance. There were no differences across the HIF1A genotype and allele frequencies among endurance athletes, sprinters, and controls. Similarly, no differences were found between the subgroups of top-level and national-level endurance athletes, or between top-level and national-level sprinters. Conversely, interaction effects were found between HIF1A Pro582Ser and ACTN3 R577X polymorphisms and sprinters. The proportion of HIF1A Pro/Pro + ACTN3 R/R genotypes was significantly higher in sprinters than in endurance athletes and healthy controls (P = .002). In addition, the odds ratio for HIF1A Pro/Pro + ACTN3 R/R genotype carriers being a sprinter was 2.25 (95% confidence interval, 1.24-4.1); and that for HIF1A Pro/Pro + ACTN3 R/R genotype carriers being an endurance athlete was 0.5 (95% confidence interval, 0.2-1.24). We conclude that HIF1A Pro582Ser polymorphism by itself is not critical in determining sprint performance. However, sprinter performance is determined by the interaction between the wild-type HIF1A Pro/Pro genotype and ACTN3 RR genotype.

Interaction between SNPs in the NRF2 gene and elite endurance performance.

Nuclear respiratory factor 2 (NRF2), a member of the Cap-N-Collar family of transcription factors, plays an important role in the mitochondrial biogenesis, and variants of NRF2 gene have been associated with endurance performance. The aims of the present study were 1) to compare NRF2 A/C (rs12594956) and NRF2 C/T (rs8031031) genotype and allele frequencies between athletes of sports with different demands (endurance vs. sprinters) as well as between competitive levels (elite level vs. national level) and 2) to analyze the interaction of these two polymorphisms and its influence on the level of endurance performance. One hundred and fifty-five track and field athletes (74 endurance athletes and 81 sprinters) and 240 nonathletic healthy individuals participated in this study. Endurance athletes presented a higher frequency of the AA (rs12594956) and CT (rs8031031) genotypes than sprinters and the control group, as well as higher A and T alleles, respectively. These differences did not appear between the sprinters and control subjects. The odds ratio for harboring the "optimal genotype" (NRF2 AA+ NRF2 CT) was 4.53 (95% confidence interval 1.23-16.6) in the whole cohort of endurance athletes and 6.55 (95% confidence interval 1.12-38.25) in elite-level endurance athletes, compared with control subjects and both levels of sprinters. In conclusion, our data indicate that the NRF2 A/C and NRF2 C/T single nucleotide polymorphisms (SNPs) are associated, separately and in combination, with elite endurance athletes, which supports the notion that these specific gene variants might belong to a growing group of SNPs that are associated with endurance performance.

Is there an interaction between PPARD T294C and PPARGC1A Gly482Ser polymorphisms and human endurance performance?

Functional Gly482Ser (rs8192678) and T294C (rs2016520) polymorphisms in the peroxisome proliferator-activated receptor gamma coactivator-1 (PPARGC1A) and peroxisome proliferator-activated receptor delta (PPARD) genes, respectively, have been associated with mRNA and/or protein activity. The aim of this study was to determine their frequency distribution among 155 Israeli athletes (endurance athletes and sprinters) and 240 healthy control subjects. There were no differences between the endurance athletes, the sprinters and the control group across the PPARD T294C genotypes (P = 0.62). Similarly, no statistical differences were found between the subgroups of elite-level endurance athletes (those who had represented Israel in a world track and field championship or in the Olympic Games) and national-level endurance athletes (P = 0.3), or between elite-level and national-level sprinters (P = 0.9). However, a combined influence of these two polymorphisms on endurance performance was found. The PPARD CC + PPARGC1A Gly/Gly genotypes were more frequently found in the elite endurance athletes than in national-level endurance athletes (P < 0.000). In the cohort of endurance athletes, the odds ratio of the 'optimal genotype' for endurance athletes (PPARD CC + PPARGC1A Gly/Gly + PPARGC1A Gly/Ser) being an elite-level athlete was 8.32 (95% confidence interval 2.2-31.4). In conclusion, the present study suggests that PPARD T294C is not associated with endurance performance. However, a higher frequency of the PPARGC1A Gly/Gly + PPARD CC genotype is associated with elite-level endurance athletes.

NRF2 intron 3 A/G polymorphism is associated with endurance athletes' status.

The aim of this study was to determine the frequency distribution of nuclear respiratory factor 2 (NRF2) intron 3 A/G polymorphism (rs7181866) among 155 Israeli athletes (endurance athletes and sprinters) and 240 healthy controls. Results showed that there was a significantly higher proportion of the AG genotype, rather than the AA genotype, in the group of endurance athletes compared with the sprinters (P = 0.014) and controls (P = 0.0008). However, the sprinters' genotype and allele frequencies were similar to those of the control group (P = 0.62 for genotype distribution percentage). These results were even more pronounced when we compared between the subgroups of 20 elite endurance athletes (those who had represented Israel in a world track-and-field championship or in the Olympic Games) and 54 national-level endurance athletes. In the group of elite endurance athletes the G allele was more frequent than in the national-level endurance athletes (P = 0.047). We conclude that 1) in Israeli athletes the NRF2 AG genotype is more frequent in elite endurance athletes than in sprinters, and 2) within the endurance group the NRF2 AG genotype and the G allele are more frequent in elite athletes, suggesting a positive association between the AG genotype, and possibly the G allele, and the likelihood of being an elite endurance athlete.

The guanine nucleotide binding protein beta polypeptide 3 gene C825T polymorphism is associated with elite endurance athletes.

A functional C825T polymorphism in the human guanine nucleotide binding protein beta polypeptide 3 (GNB3) gene has been associated with enhanced G protein activation. Since reports regarding the interaction between physical activity and the GNB3 C825T polymorphism are limited and inconsistent, the aim of this study was to determine the frequency of C825T alleles among 155 elite Israeli athletes (endurance athletes and sprinters) and 234 healthy control subjects. Genotyping for GNB3 C825T was performed using polymerase chain reaction on DNA from leucocytes. Results showed that there was a significant difference in GNB3 C825T polymorphism genotype frequencies between endurance athletes and sprinters (P = 0.045) as well as between endurance athletes and control subjects (P = 0.046). We also observed a significantly higher proportion of the GNB3 TT genotype in the group of endurance athletes (19%) compared with the sprinters (5%, P = 0.014) and the control subjects (8.5%, P = 0.026). In the group of athletes, the odds ratio of GNB3 TT genotype being an endurance athlete was 4.49 (95% confidence interval 1.4-14.3) and of GNB3 CC genotype was 0.91 (95% confidence interval 0.47-1.77). These results were even more pronounced when we compared between the subgroups of 20 top-level endurance athletes and 24 top-level sprinters. We conclude that in Israeli athletes the GNB3 TT genotype is higher in elite endurance athletes than it is in sprinters, and within the endurance group it is higher in top-level athletes, suggesting a positive association between the TT genotype and the likelihood of being an elite endurance athlete.

IL6 (-174) and TNFA (-308) promoter polymorphisms are associated with systemic creatine kinase response to eccentric exercise.

Exertional rhabdomyolysis is a complex and poorly understood entity. The inflammatory system has an important role in muscle injury and repair. Serum creatine kinase (CK) is often used as systemic biomarker representing muscle damage. Considerable variation exists in CK response between different subjects. Genetic elements may act as predisposition factors for exertional rhabdomyolysis. Based on their biological activity, we hypothesized that in healthy subjects IL6 G-174C and TNFA G-308A promoter polymorphisms would be associated with CK response to exercise. We determined serum CK activity pre- and post-maximal eccentric contractions of the elbow flexor muscles. IL6 G-174C and TNFA G-308A genotypes were analyzed for possible relationship with changes in serum CK activity. IL6 G-174C genotype was associated with CK activity in a dose-dependent fashion. Subjects with one or more of the -174C allele had a greater increase and higher peak CK values than subjects homozygous for the G allele (mean +/- SE U/L: GG, 2,604 +/- 821; GC, 7,592 +/- 1,111; CC, 8,403 +/- 3,849, ANOVA P = 0.0003 for GG + GC genotypes versus CC genotype, P = 0.0005 for linear trend). IL6-174CC genotype was associated with a greater than threefold increased risk of massive CK response (adjusted odds ratio 3.29, 95% confidence interval 1.27-7.85, P = 0.009). A milder association (P = 0.06) was noted between TNFA G-308A genotype and CK activity. In conclusion, we found a strong association of the IL6 G-174C genotype with systemic CK response to strenuous exercise. Data suggest that homozygosity for the IL6-174C allele is a clinically important risk factor for exercise-induced muscle injury, further supporting the central role of cytokines in the reactive inflammatory process of muscle damage and repair.

Clinical analysis of nosocomial infection in patients with non-Hodgkin lymphoma / 中华临床感染病杂志

Objective To identify risk factors and preventive measures of nosoconial infections in patients with non-Hodgkin lymphoma (NHL). Methods Clinical data of 65 NHL patients admitted from January to December 2007 were retrospectively analyzed. Results According to WHO classification (2001), 58 patients were with B-cell lymphoma, 7 were with T-cell lymphorna. All patients received CHOP regimen as initial chemotherapy and 23 of them were with nosecomial infections. Logistic regression analysis demonstrated that age, length of stay, pathological type, bone marrow involvement, levels of serum lactate dehydrogenase (LDH), beta2-microglobulin and invasive treatment were identified as risk factors of nosocomial infections. Respiratory tract infections and infections with gram-negative microorganisms were the most popular. Conclusion High nosocomial infection rate is found in NHL patients, and control of risk factors may effectively prevent nosocomial infections in NHL patients.

ACE ID genotype affects blood creatine kinase response to eccentric exercise.

Unaccustomed exercise may cause muscle breakdown with marked increase in serum creatine kinase (CK) activity. The skeletal muscle renin-angiotensin system (RAS) plays an important role in exercise metabolism and tissue injury. A functional insertion (I)/deletion (D) polymorphism in the angiotensin I-converting enzyme (ACE) gene (rs4646994) has been associated with ACE activity. We hypothesized that ACE ID genotype may contribute to the wide variability in individuals' CK response to a given exercise. Young individuals performed maximal eccentric contractions of the elbow flexor muscles. Pre- and postexercise CK activity was determined. ACE genotype was significantly associated with postexercise CK increase and peak CK activity. Individuals harboring one or more of the I allele had a greater increase and higher peak CK values than individuals with the DD genotype. This response was dose-dependent (mean +/- SE U/L: II, 8,882 +/- 2,362; ID, 4,454 +/- 1,105; DD, 2,937 +/- 753, ANOVA, P = 0.02; P = 0.009 for linear trend). Multivariate stepwise regression analysis, which included age, sex, body mass index, and genotype subtypes, revealed that ACE genotype was the most powerful independent determinant of peak CK activity (adjusted odds ratio 1.3, 95% confidence interval 1.03-1.64, P = 0.02). In conclusion, we indicate a positive association of the ACE ID genotype with CK response to strenuous exercise. We suggest that the II genotype imposes increased risk for developing muscle damage, whereas the DD genotype may have protective effects. These findings support the role of local RAS in the regulation of exertional muscle injury.

The ACE deletion allele is associated with Israeli elite endurance athletes.

An Alu insertion (I)/deletion (D) polymorphism in the angiotensin I converting enzyme (ACE) gene has been associated with ACE activity. Opposing effects on elite athletic performance have been proposed for the I and D alleles; while the D allele favours improved endurance ability, the I allele promotes more power-orientated events. We tested this hypothesis by determining the frequency of ACE ID alleles amongst 121 Israeli top-level athletes classified by their sporting discipline (marathon runners or sprinters). Genotyping for ACE ID was performed using polymerase chain reaction on DNA from leucocytes. The ACE genotype and allele frequencies were compared with those of 247 healthy individuals. Allele and genotype frequencies differed significantly between the groups. The frequency of the D allele was 0.77 in the marathon runners, 0.66 in the control subjects (P = 0.01) and 0.57 in the sprinters (P = 0.002). The ACE DD genotype was more prevalent among the endurance athletes (0.62) than among the control subjects (0.43, P = 0.004) and the power athletes (0.34, P = 0.004). In the group of elite athletes, the odds ratio of ACE DD genotype being an endurance athlete was 3.26 (95% confidence interval 1.49-7.11), and of ACE II genotype was 0.41 (95% confidence interval 0.14-1.19). We conclude that in Israeli elite marathon runners the frequency of the ACE D allele and ACE DD genotype seems to be higher than in sprinters, suggesting a positive association between the D allele and the likelihood of being an elite endurance athlete in some ethnic groups.

Effects of B7-H1 molecule blockade on tumor-infiltrating dendritic cell-mediated T-cell function / 中国肿瘤生物治疗杂志

Objective:To explore the expression of B7-1,B7-2 and B7-H1 on tumor-infiltrating dendritic cells(TIDC) and on splenic dendritic cells(SDC),and to investigate TIDC-mediated and SDC-mediated T-cell function after blocking B7-H1 expression in these dendritic cells.Methods: The TIDCs and SDCs were isolated from tumor-bearing mice using anti-mouse CD11c magnetic beads.The expression of B7-1,B7-2 and B7-H1 on TIDC and SDC was analyzed using flow cytometer.T cells were co-cultured with TIDCs or SDCs for the mixed lymphocyte reaction(MLR),and monoclonal antibodies to B7-H1 or the isotype control antibodies were added to the MLR cultures.T-cell proliferation was assessed using XTT method and the secretion of IL-10 was detected using ELISA.Results: B7-1 and B7-2 positive TIDCs were significantly less than SDCs(P0.05).T-cell proliferation stimulated by TIDCs was weaker than that stimulated by SDCs;T cells produced more IL-10 after TIDCs stimulation than after SDCs stimulation(P