Increased Agrobacterium-mediated transformation of Saccharomyces cerevisiae after deletion of the yeast ADA2 gene.

Agrobacterium tumefaciens is the causative agent of crown gall disease and is widely used as a vector to create transgenic plants. Under laboratory conditions, the yeast Saccharomyces cerevisiae and other yeasts and fungi can also be transformed, and Agrobacterium-mediated transformation (AMT) is now considered the method of choice for genetic transformation of many fungi. Unlike plants, in S. cerevisiae, T-DNA is integrated preferentially by homologous recombination and integration by non-homologous recombination is very inefficient. Here we report that upon deletion of ADA2, encoding a component of the ADA and SAGA transcriptional adaptor/histone acetyltransferase complexes, the efficiency of AMT significantly increased regardless of whether integration of T-DNA was mediated by homologous or non-homologous recombination. This correlates with an increase in double-strand DNA breaks, the putative entry sites for T-DNA, in the genome of the ada2Δ deletion mutant, as visualized by the number of Rad52-GFP foci. Our observations may be useful to enhance the transformation of species that are difficult to transform.

Time to seroconversion of HBsAg to anti-HBs in individuals who lost HBsAg during follow-up.

To determine the time to appearance of antibody against hepatitis B surface antigen (anti-HBs) after clearance of hepatitis B surface antigen (HBsAg) in chronically infected individuals, we followed up 3963 cases with positive antibody against hepatitis B e antigen (anti-HBe) from 1991 to 2014. Of these, 101 (67 males, 34 females) lost HBsAg. These serocleared cases were checked every 6-month interval regarding HBsAg, anti-HBs, liver function tests, and liver sonography. Hepatitis B virus DNA was assessed at the time of seroclearance or the appearance of anti-HBs. The mean age of these patients at entry to this study was 34·4 ± 13 years. The mean follow-up duration until seroclearance of HBsAg was 6·6 ± 4·3 years. After the mean follow-up of 43·7 ± 45 months, anti-HBs appeared in 64 (63·4%) cases. The cumulative probabilities of anti-HBs appearance for 2, 5 and 10 years were 24·3%, 58% and 78·2%, respectively. The appearance of anti-HBs was associated with age ⩾35 years and seroclearance of HBsAg (hazard ratio 1·96, 95% confidence interval 1·32-3·38, P = 0·016) but not with sex. The results show that anti-HBs may develop in 78·2% of cases within 10 years of HBsAg clearance. Age ⩾35 years at HBsAg loss was associated with earlier development of anti-HBs.

Evaluation of CD4+ CD25+ FoxP3+ regulatory T cells during treatment of patients with brucellosis.

Cell-mediated immunity (CMI) plays a critical role in the control of brucellosis. Regulatory T cells (Tregs) have a functional character in modulating the balance between host immune response and tolerance, which can eventually lead to chronic infection or relapse. The aim of this study was to assess the alteration of Tregs in cases of brucellosis before and after treatment. Thirty cases of acute brucellosis with the mean age of 41.03±15.15 years (case group) and 30 healthy persons with the mean age of 40.63±13.95 years (control group) were selected and assessed. Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of all individuals. We analyzed the alteration of Treg cell count using flow cytometry for CD4, CD25, and FoxP3 markers. The level of CD4+ CD25+ FoxP3+ Treg cells was increased in active patients compared with controls (2.5±0.99% vs 1.6±0.84%, p= 0.0004), but it had declined in the treated cases (1.83±0.73%, p=0.02). The level of Tregs was elevated in three relapsed cases. The frequency of Tregs and Treg/Teff (effector T cell) ratio was correlated with inverse serum agglutination test (SAT) and, 2-mercaptoethanol (2-ME) titers as markers of treatment in brucellosis. Based on our findings, we suggest that regulatory cells, such as CD4+ CD25+ FoxP3+ Treg cells, may contribute to the development of infection processes involving immune responses in brucellosis, and evaluation of regulatory T-cell levels may be a potential diagnostic strategy for the treatment outcome in chronic and relapsed cases of brucellosis.

Comparison of the efficacy of gentamicin for 5 days plus doxycycline for 8 weeks versus streptomycin for 2 weeks plus doxycycline for 45 days in the treatment of human brucellosis: a randomized clinical trial.

OBJECTIVES: To compare the efficacy of gentamicin for 5 days plus doxycycline for 8 weeks with streptomycin for 2 weeks plus doxycycline for 45 days in the treatment of human brucellosis. METHODS: In each arm of the study, 82 patients older than 10 years randomly received 5 mg/kg gentamicin once daily for 5 days plus 100 mg of doxycycline twice daily for 8 weeks or 1 g of streptomycin intramuscularly for 2 weeks plus the same dose of doxycycline for 45 days. Therapeutic failure and relapse in these two treatment groups were compared. This study was registered in the Iranian Registry of Clinical Trials (www.irct.ir) with registration number ID: IRCT138708191441N1. RESULTS: The clinical manifestations in these two groups were similar. Therapeutic failure was seen in two (2.4%) patients in the gentamicin/doxycycline group and in four (4.9%) patients in the streptomycin/doxycycline group [relative risk (RR) = 0.5, 95% confidence interval (CI) 0.09-2.66, P = 0.68]. Relapse was seen in two (2.4%) cases in the gentamicin/doxycycline group and in five (6.1%) cases in the streptomycin/doxycycline group (RR = 0.4, 95% CI 0.08-2, P = 0.44). The efficacy with the gentamicin/doxycycline regimen was 95.12% and that with the streptomycin/doxycycline regimen was 89% (RR = 1.07, 95% CI 0.98-1.17, P = 0.25). Cox regression analyses showed no differences among the two treatment groups for patients who had relapse or therapeutic failure and those who had not regarding baseline covariates such as sex, duration of disease before diagnosis, positive blood culture and focal disease. CONCLUSIONS: The results show that the efficacy of gentamicin for 5 days plus doxycycline for 8 weeks is not superior to that of streptomycin for 2 weeks plus doxycycline for 45 days.

Response to hepatitis B virus vaccination in haemodialysis patients with and without hepatitis C infection.

The aim of this study was to determine the efficacy of hepatitis B virus (HBV) vaccination and the response to vaccine in individuals on haemodialysis with and without HCV infection. From April 2000 to September 2003 all haemodialysis patients referred to the haemodialysis department in a Babol hospital received 4 microg vaccine intramuscularly at 0, 1, and 6 months. All were negative for HBV infection markers (HBcAb, HBsAg and HBsAb). Of 62 patients, 53 (85.5%) responded to vaccination and 26 (49.1%) were high responders. All individuals with HCV infection responded to vaccination. Duration of haemodialysis had no effect on response to vaccination.

Prevalence of hepatitis A IgG in individuals with chronic hepatitis B infection in Babol.

To determine the prevalence of previous hepatitis A virus (HAV) infection in people chronically infected with hepatitis B virus (HBV), we assessed the prevalence of anti-HAV IgG in 392 patients. The study was carried out in Babol, northern Islamic Republic of Iran from September 2004 to March 2005. Prevalence in those aged 10-19 years was 59.4% and was significantly lower than that in those aged 20-29 years (89.8%) and those over 29 years (97.5%). There was no significant difference in prevalence according to sex or place of residence. A significant proportion of Iranian adolescents and young adults with chronic HBV infection are at risk of contracting HAV infection.

Histopathological features of liver and its relation to serum transaminase levels in 91 cases of anti-HBe-positive chronic hepatitis B.

For assessing the histopathological features and its relation to alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in anti-HBe-positive chronic hepatitis B in Babol, north of Iran, liver biopsy samples of 91 anti-HBe-positive chronic hepatitis B virus (HBV) infection with raised ALT were scored according to histological hepatitis activity index (HAI) from March 2000 to July 2003. Seventy-seven males and 14 females with a mean age of 32.24 (+/-11.92) years were studied. Mean ALT and AST levels were 112.5 (+/-98.9) and 69.57 (+/-60.6) IU/l, respectively. Minimal, mild, moderate and severe chronic hepatitis were seen in 35 (38.5%), 49 (53.8%), seven (7.7%) and 0 cases, respectively. There was not any significant difference between HAI and ALT levels of < 60, between 60-79 and > or = 80 IU/l (p = 0.299), but with regard to AST levels, this difference was significant (p = 0.005). This study showed that more than 90% of our cases are in the early stages of the liver disease. Although both serum ALT and AST levels are associated with the activity of hepatitis in HBV carriers, the AST is a better laboratory screening test for finding the severity of liver injury than ALT.

Epidemiological features and clinical manifestations in 469 adult patients with brucellosis in Babol, Northern Iran.

The epidemiological features and clinical manifestations of adult cases of brucellosis admitted to the Department of Infectious Diseases, Babol Medical University, Iran from 1997 to 2002 were investigated. Of 469 cases, 267 (56.9%) were males. The mean age of cases was 36.9 +/- 15 years. Most (60.8%) were from rural areas. Two thirds of cases (306, 66.3%) presented during spring or summer. Fresh cheese (22.4%), animal husbandry (11.3%), laboratory worker (8.1%) and veterinary profession (1.5%) were the main risk factors. Forty-five families (9.6%) had two cases. Sweating, fever, and arthralgia were the most frequent clinical symptoms. Complications were documented in 105 males (39.5%) and 41 females (20.3%, P=0.0001). Peripheral arthritis was seen in 24 (9%) males and 19 (9.4%) females, with knees and hips being the most common sites of infection. Sacroiliitis and spondylitis were seen in 28 (6%) and 32 (6.8%) cases respectively with spondylitis more common in males (P=0.023). Epididymo-orchitis was seen in 29 (10.9%) males. There were three cases each of endocarditis (0.6%) and neurological complications (0.6%). Most patients with brucellosis did not have any of the known risk factors for brucellosis. Thus consumption of unsafe dairy products could be the main route of infection. The disease manifested with a diversity of clinical manifestations and complications. Complications were more frequent in males than females.