High‐throughput genotyping of mannose‐binding lectin variants using high‐resolution DNA‐melting analysis

High Resolution Melting Analysis (HRMA) is a rapid and sensitive method for single nucleotide polymorphism (SNP) analysis. In the present study we present a novel HRMA assay to detect three SNPs in close proximity of each other in the first exon of the gene encoding mannose‐binding lectin (MBL), a key molecule of innate immunity. These SNPs have been selected for their known biological and clinical relevance. The three SNPs in MBL2 were simultaneously determined in sixty‐nine human DNA samples using HRMA and a single non‐fluorescent melting probe, without any post‐PCR processing of samples. Combining analyses from amplicon melting and probe melting, we have been able to discriminate ten exon 1 MBL2 genotypes with HRMA, making it a suitable tool for MBL genotyping. A second HRMA assay is presented to detect a relevant polymorphism (Y/X SNP) in the MBL2 promoter region. In conclusion, HRMA is a closed tube assay that is easy to setup and lends itself perfectly for high throughput genotyping of MBL2 variants. The present study thereby facilitates further clinical studies into the role of MBL in inflammatory and infectious disease. © 2010 Wiley‐Liss, Inc.     

Online data on opening hours of general practices in England: a comparison with telephone survey data

Background

The NHS Choices website (www.nhs.uk) provides data on the opening hours of general practices in England. If the data are accurate, they could be used to examine the benefits of extended hours.

Aim

To determine whether online data on the opening times of general practices in England are accurate regarding the number of hours in which GPs provide face-to-face consultations.

Design and setting

Cross-sectional comparison of data from NHS Choices and telephone survey data reported by general practice staff, for a nationally representative sample of 320 general practices (December 2013 to September 2014).

Method

GP face-to-face consultation times were collected by telephone for each sampled practice for each day of the week. NHS Choices data on surgery times were available online. Analysis was based on differences in the number of surgery hours (accounting for breaks) and the times of the first and last consultations of the day only between the two data sources.

Results

The NHS Choices data recorded 8.8 more hours per week than the survey data on average (40.1 versus 31.2; 95% confidence interval [CI] = 7.4 to 10.3). This was largely accounted for by differences in the recording of breaks between sessions. The data were more similar when only the first and last consultation times were considered (mean difference = 1.6 hours; 95% CI = 0.9 to 2.3).

Conclusion

NHS Choices data do not accurately measure the number of hours in which GPs provide face-to-face consultations. They better record the hours between the first and last consultations of the day.     

The effect of lifestyle-based education to women and their husbands on the anxiety and depression during pregnancy: a randomized controlled trial

Objective: The aim of this study was to determine the effect of lifestyle-based education to women and their husbands on anxiety and depression during pregnancy.

Method: This controlled trial was conducted on 189 pregnant women. Participants were allocated into three groups through block randomization. In the first intervention group, both women and their husbands, and in the second one, only women received the lifestyle-based education. In the control group, women received only the routine care. Edinburgh depression and Spielberger anxiety questionnaires were completed at baseline and 8 weeks after it.

Results: Compared to the control group, significant reductions were observed in the scores of depression (adjusted difference: ?4.7; 95% CI: ?5.9 to ?3.4), state anxiety (?8.1; ?11.3 to ?4.9) and trait anxiety (?8.3; ?11.9 to ?5.0) in the first intervention group, and in the scores of depression (?2.9; ?4.1 to ?1.6), state anxiety (?4.3; ?7.5 to ?1.1) and trait anxiety (?5.5; 95% CI: ?8.7 to ?2.3) in the second intervention group. Comparing the two intervention groups, mean scores of depression and state anxiety were significantly lower in the first group.

Conclusion: Results suggested the positive effect of lifestyle education to women and their husbands on depression and anxiety during pregnancy.     

Omega-3 fatty acid supplementation affects pregnancy outcomes in gestational diabetes: a randomized,double-blind,placebo-controlled trial

Objective: This study was designed to assess the effects of omega-3 fatty acid supplementation on inflammatory factors, biomarkers of oxidative stress, and pregnancy outcomes among pregnant women with gestational diabetes (GDM).

Methods: This randomized, double-blind, placebo-controlled clinical trial was performed among 56 women with GDM. Subjects were randomly selected to receive either 1000?mg omega-3 fatty acid supplements (containing 180?mg eicosapentaenoic acid and 120?mg docosahexanoic acid) (n?=?27) or a placebo (n?=?27) for 6 weeks. Fasting blood samples were taken at study baseline and after 6 weeks of intervention to quantify biochemical variables. Newborn’s weight, height, head circumference, Apgar score, and hyperbilirubinemia were determined.

Results: At the end of the 6 weeks, taking omega-3 fatty acid significantly decreased serum high-sensitivity C-reactive protein (hs-CRP) (change from baseline: ?245.1?±?1570.5 versus?+?913.9?±?2329.4?ng/mL, p?=?0.03) and plasma malondialdehyde (MDA) concentrations (?0.4?±?1.3 versus?+?0.6±2.3, p?=?0.04) compared with the placebo. Supplementation with omega-3 had a low incidence of hyperbilirubinemiain newborns (7.7% versus 33.3%, p?=?0.02) and decreased newborns’ hospitalization rate (7.7% versus 33.3%, p?=?0.02).

Conclusions: Taken together, omega-3 fatty acid supplementation in GDM women had beneficial effects on maternal serum hs-CRP, plasma MDA levels, incidence of newborn’s hyperbilirubinemia, and hospitalization.     

Association between the DRD2 A1 allele and opium addiction in the Iranian population.

Dysfunction of the central dopaminergic neurotransmission has been suggested to play an important role in the etiology of certain neuropsychiatric disorders such as drug abuse. It has been shown that the dopamine D2 receptor (DRD2) gene dysfunction is associated with multi-drug addiction. Addiction to opium is the most common form of drug abuse in Iran. We studied the allelic association between DRD2 Taq I A polymorphism in 100 opium-dependent Iranian patients and 130 unrelated controls. A 310 bp (base pair) region surrounding Taq I site at the DRD2 locus was amplified by polymerase chain reaction (PCR) and the PCR product was incubated with Taq I restriction enzyme. The A1 allele remained intact while the A2 allele was cut. Significant association was observed between A1 allele and addiction in the patients group (P < 0.0001). Moreover, the frequency of A1A1 genotype was significantly higher in opium users than controls (P < 0.0001). Our result indicates that DRD2 might be involved in the pathophysiology of opium addiction.     

Pharmacologic bronchodilation response to salbutamol in COPD patients

Background: Airway hyperresponsiveness (AHR) is the most characteristic feature of asthma, which is reported in COPD patients and smokers. Increased airway responsiveness to ?-agonists is also demonstrated in asthmatics as well as smokers. However, there is no report regarding AHR to ?-agonist drugs in COPD patients. Therefore, in this study pharmacologic bronchodilation response to salbutamol in COPD patients was examined. Materials and Methods: The threshold concentrations of inhaled salbutamol required for a 20% change in forced expiratory flow in 1 sec (FEV 1 ) as PC 20 , or a 35% change in specific airway conductance (sGaw) as PC 35 was measured in 14 COPD patients and 14 normal subjects. Results: Airway responsiveness to salbutamol in COPD patients (PC 20 = 14.14 ± 1.62 and PC 35 = 9.70 ± 1.48 mg/l) was significantly lower than normal subjects (PC 20 = 224.57 ± 16.62 and PC 35 = 81.87 ± 8.16 mg/l, P < 0.001 for both cases). The values of FEV 1 and sGaw in COPD patients (56.43 ± 14.45 and 0.081 ± 0.120 respectively) were significantly lower than those of normal subjects (104.07 ± 5.72 and 0.194 ± 0.041 respectively), (P < 0.001 for FEV 1 and P < 0.005 for sGaw). There was a significant correlation between FEV 1 with PC 20 salbutamol (r = 0.862, P < 0.001). The correlations between PC 20 and PC 35 was also statistically significant (r = 0.862, P < 0.001). Conclusion: These results showed increased airway responsiveness of most COPD patients to salbutamol which was highly correlated to airway caliber.     

Myeloperoxidase is critically involved in the induction of organ damage after renal ischemia reperfusion

In this study the role of myeloperoxidase (MPO) in a murine (C57BL/6) model of ischemia and reperfusion (I/R)-induced renal failure was investigated. The renal function after I/R was analyzed in MPO-deficient (Mpo(-/-)) mice and compared with wild-type (WT) controls. A significant reduction in renal function loss (blood urea nitrogen) was observed after 24 hours of reperfusion of ischemically damaged kidneys in Mpo(-/-) mice compared with I/R WT controls (I/R Mpo(-/-) = 31.3 +/- 1.7 mmol/L versus I/R WT = 42.8 +/- 2.1 mmol/L, sham = 7.0 +/- 0.5 mmol/L; P = 0.003). The early reperfusion phase (2 hours of reperfusion) was characterized by a substantial increase in apoptosis and early complement activation, surprisingly similar in Mpo(-/-) and WT mice. Improved renal function in Mpo(-/-) mice after extended reperfusion was accompanied by a reduced neutrophil influx (P = 0.017) compared with WT controls. Activation and deposition of complement was not significantly reduced in Mpo(-/-) mice compared with WT controls after 24 hours of reperfusion, indicating no specific in vivo role for MPO in activating complement after renal I/R. Taken together, these results demonstrated an important contribution of MPO in the induction of organ damage after renal I/R by influencing critical factors such as neutrophil extravasation but not complement activation.     

Human neutrophil peptide-1 inhibits both the classical and the lectin pathway of complement activation

Human neutrophil peptide-1 (HNP-1) is a member of the alpha-defensin family. Defensins are cationic antimicrobial peptides, which play an important role in the antimicrobial response to microorganisms. In addition, recent studies have revealed the involvement of defensins in inflammation, immunity and wound repair. Defensins are present in the azurophilic granules of neutrophils and are released upon neutrophil stimulation. Previous studies showed that HNP-1 binds to C1q and inhibits the classical complement pathway. In view of the structural and functional similarity between C1q and MBL, we have now examined the interactions between HNP-1 and MBL. We observed a dose-dependent binding of HNP-1 to MBL in calcium-free buffer, indicating that HNP-1 binds to MBL most likely via the collagenous domains. To identify the binding sites in HNP-1 involved in the binding to C1q and MBL, we used a series of overlapping synthetic linear peptides that spanned the entire HNP-1 sequence. Both MBL and C1q showed a dose-dependent binding to the same set of peptides, suggesting a similar binding site in HNP-1 for both MBL and C1q. Strongest binding was observed to peptides containing the C- or N-terminal part of the HNP-1 molecule. Using an ELISA based system, we demonstrated that HNP-1 inhibits activation of both the classical pathway and lectin pathway of complement. Furthermore, we demonstrated that C1q and MBL can form complexes with HNP-1 in solution. Together, the data indicate that HNP-1 interacts with both C1q and MBL efficiently resulting in inhibition of both the classical and the lectin pathway of complement. We conclude that HNP-1 may play a role in protection against tissue injury during inflammatory conditions by inhibiting the early phase of complement activation.