Agroinfection and nucleotide sequence of cloned wheat dwarf virus DNA

Cloned DNA of the geminivirus wheat dwarf virus (WDV) was successfully used to infect seedling wheat plants. The clone was derived from circular double-stranded viral DNA isolated from naturally infected tissue. The initiation of infection was mediated by Agrobacterium tumefaciens using cloned dimeric WDV genomes in a binary Agrobacterium vector. The WDV DNA which comprised the infectious clone was sequenced and is compared with the published sequence of a Swedish isolate of the same virus. The results confirm that the single WDV genome component of 2.75 kb carries all the information necessary for production of viral symptoms, virus particles and viral double- and single-stranded DNA forms.     

Measuring Hordein (Gluten) in Beer – A Comparison of ELISA and Mass Spectrometry

Background

Subjects suffering from coeliac disease, gluten allergy/intolerance must adopt a lifelong avoidance of gluten. Beer contains trace levels of hordeins (gluten) which are too high to be safely consumed by most coeliacs. Accurate measurement of trace hordeins by ELISA is problematic.

Methods

We have compared hordein levels in sixty beers, by sandwich ELISA, with the level determined using multiple reaction monitoring mass spectrometry (MRM-MS).

Results

Hordein levels measured by ELISA varied by four orders of magnitude, from zero (for known gluten-free beers) to 47,000 µg/mL (ppm; for a wheat-based beer). Half the commercial gluten-free beers were free of hordein by MS and ELISA. Two gluten-free and two low-gluten beers had zero ELISA readings, but contained significant hordein levels (p<0.05), or near average (60–140%) hordein levels, by MS, respectively. Six beers gave false negatives, with zero ELISA readings but near average hordein content by MS. Approximately 20% of commercial beers had ELISA readings less than 1 ppm, but a near average hordein content by MS. Several barley beers also contained undeclared wheat proteins.

Conclusions

ELISA results did not correlate with the relative content of hordein peptides determined by MS, with all barley based beers containing hordein. We suggest that mass spectrometry is more reliable than ELISA, as ELISA enumerates only the concentration of particular amino-acid epitopes; this may vary between different hordeins and may not be related to the absolute hordein concentration. MS quantification is undertaken using peptides that are specific and unique, enabling the quantification of individual hordein isoforms. This outlines the problem of relying solely on ELISA determination of gluten in beverages such as beer and highlights the need for the development of new sensitive and selective quantitative assay such as MS.     

Engineering high α‐amylase levels in wheat grain lowers Falling Number but improves baking properties

Late maturity α‐amylase (LMA) and preharvest sprouting (PHS) are genetic defects in wheat. They are both characterized by the expression of specific isoforms of α‐amylase in particular genotypes in the grain prior to harvest. The enhanced expression of α‐amylase in both LMA and PHS results in a reduction in Falling Number (FN), a test of gel viscosity, and subsequent downgrading of the grain, along with a reduced price for growers. The FN test is unable to distinguish between LMA and PHS; thus, both defects are treated similarly when grain is traded. However, in PHS‐affected grains, proteases and other degradative process are activated, and this has been shown to have a negative impact on end product quality. No studies have been conducted to determine whether LMA is detrimental to end product quality. This work demonstrated that wheat in which an isoform α‐amylase (TaAmy3) was overexpressed in the endosperm of developing grain to levels of up to 100‐fold higher than the wild‐type resulted in low FN similar to those seen in LMA‐ or PHS‐affected grains. This increase had no detrimental effect on starch structure, flour composition and enhanced baking quality, in small‐scale 10‐g baking tests. In these small‐scale tests, overexpression of TaAmy3 led to increased loaf volume and Maillard‐related browning to levels higher than those in control flours when baking improver was added. These findings raise questions as to the validity of the assumption that (i) LMA is detrimental to end product quality and (ii) a low FN is always indicative of a reduction in quality. This work suggests the need for a better understanding of the impact of elevated expression of specific α‐amylase on end product quality.     

An Augmented SMS Intervention to Improve Access to Antenatal CD4 Testing and ART Initiation in HIV-Infected Pregnant Women: A Cluster Randomized Trial

BackgroundLess than one-third of HIV-infected pregnant women eligible for combination antiretroviral therapy (ART) globally initiate treatment prior to delivery, with lack of access to timely CD₄ results being a principal barrier. We evaluated the effectiveness of an SMS-based intervention to improve access to timely antenatal ART.MethodsWe conducted a stepped-wedge cluster randomized trial of a low-cost programmatic intervention in 20 antenatal clinics in Gaborone, Botswana. From July 2011-April 2012, 2 clinics were randomly selected every 4 weeks to receive an ongoing clinic-based educational intervention to improve CD₄ collection and to receive CD₄ results via an automated SMS platform with active patient tracing. CD₄ testing before 26 weeks gestation and ART initiation before 30 weeks gestation were assessed.ResultsThree-hundred-sixty-six ART-naïve women were included, 189 registering for antenatal care under Intervention and 177 under Usual Care periods. Of CD₄-eligible women, 100 (59.2%) women under Intervention and 79 (50.6%) women under Usual Care completed CD₄ phlebotomy before 26 weeks gestation, adjusted odds ratio (aOR, adjusted for time that a clinic initiated Intervention) 0.87 (95% confidence interval [CI]0.47–1.63, P = 0.67). The SMS-based platform reduced time to clinic receipt of CD₄ test result from median of 16 to 6 days (P<0.001), was appreciated by clinic staff, and was associated with reduced operational cost. However, rates of ART initiation remained low, with 56 (36.4%) women registering under Intervention versus 37 (24.2%) women under Usual Care initiating ART prior to 30 weeks gestation, aOR 1.06 (95%CI 0.53–2.13, P = 0.87).ConclusionsThe augmented SMS-based intervention delivered CD₄ results more rapidly and efficiently, and this type of SMS-based results delivery platform may be useful for a variety of tests and settings. However, the intervention did not appear to improve access to timely antenatal CD₄ testing or ART initiation, as obstacles other than CD₄ impeded ART initiation during pregnancy.     

Loss from Treatment for Drug Resistant Tuberculosis: Risk Factors and Patient Outcomes in a Community-Based Program in Khayelitsha,South Africa

Background

A community based drug resistant tuberculosis (DR-TB) program has been incrementally implemented in Khayelitsha, a high HIV and TB burden community in South Africa. We investigated loss from treatment (LFT), and post treatment outcomes of DR-TB patients in this setting.

Methodology

LFT, defined as interruption of treatment for ≥2 consecutive months was assessed among patients initiating DR-TB treatment for the first time between January 2009 and July 2011. Patients were traced through routine data sources to identify those who subsequently restarted treatment and those who died. Additional information on patient status and survival after LTF was obtained from community DR-TB counselors and from the national death registry. Post treatment outcomes were observed until July 2013.

Results

Among 452 patients initiating treatment for the first time within the given period, 30% (136) were LFT, with 67% retention at 18 months. Treatment was restarted in 27 (20%) patients, with additional resistance recorded in 2/25 (8%), excluding two with presumed DR-TB. Overall, 34 (25%) patients died, including 11 who restarted treatment. Males and those in the age category 15-25 years had a greater hazard of LFT; HR 1.93 (95% CI 1.35-2.75), and 2.43 (95% CI 1.52-3.88) respectively. Older age (>35 years) was associated with a greater hazard of death; HR 3.74 (1.13- 12.37) post treatment. Overall two-year survival was 62%. It was lower (45%) in older patients, and was 92% among those who received >12 months treatment.

Conclusion

LFT was high, occurred throughout the treatment period and was particularly high among males and those aged 15-25 years. Overall long term survival was poor. High rates of LFT should however not preclude scale up of community based care given its impact in increasing access to treatment. Further research is needed to support retention of DR-TB patients on treatment, even within community based treatment programs.     

The urokinase plasminogen activator receptor as a gene therapy target for cancer

Urokinase plasminogen activator (uPA) and/or its receptor (uPAR) are essential for metastasis, and overexpression of these molecules is strongly correlated with poor prognosis in a variety of malignant tumours. Impairment of uPA and/or uPAR function, or inhibition of the expression of these components, impedes the metastatic potential of many tumours. Several approaches have been employed to target uPAR with the aim of disrupting its ligand-independent action or interaction with uPA, including the more recent antigene technology. This review discusses the in vivo use of antigene approaches for downregulating uPAR as a potential therapy for cancer. Preclinical studies are advancing towards the translational phase, provided that established orthotopic tumours, which mimic clinical progression and presentation, are treated using clinically acceptable modes of nucleic acid delivery.     

Inhibition of mammalian glycan biosynthesis produces non-self antigens for a broadly neutralising, HIV-1 specific antibody

The HIV envelope has evolved a dense array of immunologically "self" carbohydrates that efficiently protect the virus from antibody recognition. Nonetheless, one broadly neutralising antibody, IgG1 2G12, has been shown to recognise a cluster of oligomannose glycans on the HIV-1 surface antigen gp120. Thus the self carbohydrates of HIV are now regarded as potential targets for viral neutralisation and vaccine design. Here, we show that chemical inhibition of mammalian glycoprotein synthesis, with the plant alkaloid kifunensine, creates multiple HIV (2G12) epitopes on the surface of previously non-antigenic self proteins and cells, including HIV gp120. This formally demonstrates the structural basis for self/non-self discrimination between viral and host glycans, by a neutralising antibody. Moreover, this study provides an alternative protein engineering approach to the design of a carbohydrate vaccine for HIV-1 by chemical synthesis.