Three-dimensional hydrogel structures as optical sensor arrays, for the detection of specific DNA sequences

The fabrication and characterization of surface-attached PEG-diacrylate hydrogel structures and their application as sensing platforms for the detection of specific target sequences are reported. Hydrogel structures were formed by a photopolymerization process, using substrate-bound Eosin Y molecules for the production of free radicals. We have demonstrated that this fabrication process allows for control over hydrogel growth down to the micrometer scale. Confocal imaging revealed relatively large pore structures for 25% (v/v) PEG-diacrylate hydrogels, which appear to lie in tightly packed layers. Our data suggest that these pore structures decrease in size for hydrogels with increasing levels of PEG-diacrylate. Surface coverage values calculated for hydrogels immobilized with 21-mer DNA probe sequences were significantly higher compared to those previously reported for 2- and 3-dimensional sensing platforms, on the order of 10(16)molecules cm(-2). Used as sensing platforms in DNA hybridization assays, a detection limit of 3.9 nM was achieved for hybridization reactions between 21-mer probe and target sequences. The ability of these hydrogel sensing platforms to discriminate between wild-type and mutant allele sequences was also demonstrated, down to target concentrations of 1-2 nM. A reduction in the hybridization time down to a period of 15 min was also achieved, while still maintaining confident results, demonstrating the potential for future integration of these sensing platforms within Lab-on-Chip or diagnostic devices.     

Bioengineered Nisin A Derivatives with Enhanced Activity against Both Gram Positive and Gram Negative Pathogens

Nisin is a bacteriocin widely utilized in more than 50 countries as a safe and natural antibacterial food preservative. It is the most extensively studied bacteriocin, having undergone decades of bioengineering with a view to improving function and physicochemical properties. The discovery of novel nisin variants with enhanced activity against clinical and foodborne pathogens has recently been described. We screened a randomized bank of nisin A producers and identified a variant with a serine to glycine change at position 29 (S29G), with enhanced efficacy against S. aureus SA113. Using a site-saturation mutagenesis approach we generated three more derivatives (S29A, S29D and S29E) with enhanced activity against a range of Gram positive drug resistant clinical, veterinary and food pathogens. In addition, a number of the nisin S29 derivatives displayed superior antimicrobial activity to nisin A when assessed against a range of Gram negative food-associated pathogens, including E. coli, Salmonella enterica serovar Typhimurium and Cronobacter sakazakii. This is the first report of derivatives of nisin, or indeed any lantibiotic, with enhanced antimicrobial activity against both Gram positive and Gram negative bacteria.     

Ivabradine reduces heart rate while preserving metabolic fluxes and energy status of healthy normoxic working hearts

Heart rate reduction (HRR) is an important target in the management of patients with chronic stable angina. Most available drugs for HRR, such as β-blockers, have adverse effects, including on cardiac energy substrate metabolism, a well-recognized determinant of cardiac homeostasis. This study aimed at 1) testing whether HRR by ivabradine (IVA) alters substrate metabolism in the healthy normoxic working heart and 2) comparing the effect of IVA with that of the β-blocker metoprolol (METO). This was assessed using our well-established model of ex vivo mouse heart perfusion in the working mode, which enables concomitant evaluation of myocardial contractility and metabolic fluxes using (13)C-labeled substrates. Hearts were perfused in the absence (controls; n = 10) or presence of IVA (n = 10, 3 μM) with or without atrial pacing to abolish HRR in the IVA group. IVA significantly reduced HR (35 ± 5%) and increased stroke volume (39 ± 9%) while maintaining similar cardiac output, contractility, power, and efficiency. Effects of IVA on HR and stroke volume were reversed by atrial pacing. At the metabolic level, IVA did not impact on substrate selection to citrate formation, rates of glycolysis, or tissue levels of high-energy phosphates. In contrast, METO, at concentrations up to 40 μM, decreased markedly cardiac function (flow: 25 ± 6%; stroke volume: 30 ± 10%; contractility: 31 ± 9%) as well as glycolysis (2.9-fold) but marginally affected HR. Collectively, these results demonstrate that IVA selectively reduces HR while preserving energy substrate metabolism of normoxic healthy working mouse hearts perfused ex vivo, a model that mimics to some extent the denervated transplanted heart. Our results provide the impetus for testing selective HRR by IVA on cardiac substrate metabolism in pathological models.     

Saturation mutagenesis of selected residues of the α‐peptide of the lantibiotic lacticin 3147 yields a derivative with enhanced antimicrobial activity

The lantibiotic lacticin 3147 consists of two ribosomally synthesized and post‐translationally modified antimicrobial peptides, Ltnα and Ltnβ, which act synergistically against a wide range of Gram‐positive microorganisms. We performed saturation mutagenesis of specific residues of Ltnα to determine their functional importance. The results establish that Ltnα is more tolerant to change than previously suggested by alanine scanning mutagenesis. One substitution, LtnαH23S, was identified which improved the specific activity of lacticin 3147 against one pathogenic strain, Staphylococcus aureus NCDO1499. This represents the first occasion upon which the activity of a two peptide lantibiotic has been enhanced through bioengineering.     

HSV-2 Infection as a Cause of Female/Male and Racial/Ethnic Disparities in HIV Infection

Objectives

To examine the potential contribution of herpes simplex virus 2 (HSV-2) infection to female/male and racial/ethnic disparities in HIV among non-injecting heroin and cocaine drug users. HSV-2 infection increases susceptibility to HIV infection by a factor of two to three.

Methods

Subjects were recruited from entrants to the Beth Israel drug detoxification program in New York City 2005-11. All subjects reported current use of heroin and/or cocaine and no lifetime injection drug use. A structured questionnaire was administered and serum samples collected for HIV and HSV-2 testing. Population-attributable risk percentages (PAR%s) were calculated for associations between HSV-2 infection and increased susceptibility to HIV.

Results

1745 subjects were recruited from 2005-11. Overall HIV prevalence was 14%. Females had higher prevalence than males (22% vs. 12%) (p<0.001), African-Americans had the highest prevalence (15%), Hispanics an intermediate prevalence (12%), and Whites the lowest prevalence (3%) (p<.001). There were parallel variations in HSV-2 prevalence (females 86%, males 51%, African-Americans 66%, Hispanics 47%, Whites 36%), HSV-2 prevalence was strongly associated with HIV prevalence (OR  =  3.12 95% CI 2.24 to 4.32). PAR%s for HSV-2 as a cause of HIV ranged from 21% for Whites to 50% for females. Adjusting for the effect of increased susceptibility to HIV due to HSV-2 infection greatly reduced all disparities (adjusted prevalence  =  males 8%, females 11%; Whites 3%, African-Americans 10%, Hispanics 9%).

Conclusions

Female/male and racial/ethnic variations in HSV-2 infection provide a biological mechanism that may generate female/male and racial/ethnic disparities in HIV infection among non-injecting heroin and cocaine users in New York City. HSV-2 infection should be assessed as a potential contributing factor to disparities in sexually transmitted HIV throughout the US.     

Leucine-rich diet supplementation modulates foetal muscle protein metabolism impaired by Walker-256 tumour

Background

Cancer-cachexia induces a variety of metabolic disorders of protein turnover and is more pronounced when associated with pregnancy. Tumour-bearing pregnant rats have impaired protein balance, which decreases protein synthesis and increases muscle breakdown. Because branched-chain amino acids, especially leucine, stimulate protein synthesis, we investigated the effect of a leucine-rich diet on protein metabolism in the foetal gastrocnemius muscles of tumour-bearing pregnant rats.

Methods

Foetuses of pregnant rats with or without Walker 256 tumours were divided into six groups. During the 20 days of the experiment, the pregnant groups were fed with either a control diet (C, control rats; W, tumour-bearing rats; Cp, rats pair-fed the same normoprotein-diet as the W group) or with a leucine-rich diet (L, leucine rats; LW, leucine tumour-bearing rats; and Lp, rats pair-fed the same leucine-rich diet as the LW group). After the mothers were sacrificed, the foetal gastrocnemius muscle samples were resected, and the protein synthesis and degradation and tissue chymotrypsin-like, cathepsin and calpain enzyme activities were assayed. The muscle oxidative enzymes (catalase, glutathione-S-transferase and superoxide dismutase), alkaline phosphatase enzyme activities and lipid peroxidation (malondialdehyde) were also measured.

Results

Tumour growth led to a reduction in foetal weight associated with decreased serum protein, albumin and glucose levels and low haematocrit in the foetuses of the W group, whereas in the LW foetuses, these changes were less pronounced. Muscle protein synthesis (measured by L-[3H]-phenylalanine incorporation) was reduced in the W foetuses but was restored in the LW group. Protein breakdown (as assessed by tyrosine release) was enhanced in the L and W groups, but chymotrypsin-like activity increased only in group W and tended toward an increase in the LW foetuses. The activity of cathepsin H was significantly higher in the W group foetuses, but the proteolytic calcium-dependent pathway showed similar enzyme activity. In parallel, an intense oxidative stress process was observed only in the group W foetuses.

Conclusions

These data suggested that the proteasomal and lysosomal proteolytic pathways and oxidative stress are likely to participate in the process of foetal muscle catabolism of Walker’s tumour-bearing pregnant rats. The present work shows that foetal muscle can be protected by supplementation with a leucine-rich diet.