Highly stable binding proteins derived from the hyperthermophilic Sso7d scaffold

We have shown that highly stable binding proteins for a wide spectrum of targets can be generated through mutagenesis of the Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus. Sso7d is a small (∼ 7 kDa, 63 amino acids) DNA-binding protein that lacks cysteine residues and has a melting temperature of nearly 100 °C. We generated a library of 10⁸ Sso7d mutants by randomizing 10 amino acid residues on the DNA-binding surface of Sso7d, using yeast surface display. Binding proteins for a diverse set of model targets could be isolated from this library; our chosen targets included a small organic molecule (fluorescein), a 12 amino acid peptide fragment from the C-terminus of β-catenin, the model proteins hen egg lysozyme and streptavidin, and immunoglobulins from chicken and mouse. Without the application of any affinity maturation strategy, the binding proteins isolated had equilibrium dissociation constants in the nanomolar to micromolar range. Further, Sso7d-derived binding proteins could discriminate between closely related immunoglobulins. Mutant proteins based on Sso7d were expressed at high yields in the Escherichia coli cytoplasm. Despite extensive mutagenesis, Sso7d mutants have high thermal stability; five of six mutants analyzed have melting temperatures > 89 °C. They are also resistant to chemical denaturation by guanidine hydrochloride and retain their secondary structure after extended incubation at extreme pH values. Because of their favorable properties, such as ease of recombinant expression, and high thermal, chemical and pH stability, Sso7d-derived binding proteins will have wide applicability in several areas of biotechnology and medicine.     

Effect of iron supplementation on incidence of infectious illness in children: systematic review

ObjectiveTo evaluate the effect of iron supplementation on the incidence of infections in children.DesignSystematic review of randomised controlled trials.InterventionsOral or parenteral iron supplementation or fortified formula milk or cereals.OutcomesIncidence of all recorded infectious illnesses, and individual illnesses, including respiratory tract infection, diarrhoea, malaria, other infections, and prevalence of positive smear results for malaria.ResultsThe pooled estimate (random effects model) of the incidence rate ratio (iron v placebo) was 1.02 (95% confidence interval 0.96 to 1.08, P=0.54; P<0.0001 for heterogeneity). The incidence rate difference (iron minus placebo) for all recorded illnesses was 0.06 episodes/child year (−0.06 to 0.18, P=0.34; P<0.0001 for heterogeneity). However, there was an increase in the risk of developing diarrhoea (incidence rate ratio 1.11, 1.01 to 1.23, P=0.04), but this would not have an overall important on public health (incidence rate difference 0.05 episodes/child year, –0.03 to 0.13; P=0.21). The occurrence of other illnesses and positive results on malaria smears (adjusted for positive smears at baseline) were not significantly affected by iron administration. On meta-regression, the statistical heterogeneity could not be explained by the variables studied.ConclusionIron supplementation has no apparent harmful effect on the overall incidence of infectious illnesses in children, though it slightly increases the risk of developing diarrhoea.

What is already known on this topic

Iron supplementation is recommended to prevent iron deficiency, which is a major health problem, especially in the developing countriesConflicting data exist regarding the possibility of an increase in the incidence of infections with iron supplementation, resulting in concern about the safety of this intervention

What this study adds

Iron supplementation has no apparent harmful effect on the overall incidence of infectious illnesses in childrenIron administration increases the risk of developing diarrhoeaFortification of foods may be the safest and most beneficial mode of supplementation in relation to infectious illnesses     

Human neutrophil peptide defensins induce single strand DNA breaks in target cells

To investigate whether target cell DNA injury participates in cytolysis by human neutrophil defensins (HNP), we analyzed HNP-treated cells for single strand breaks by the alkaline unwinding assay and the activation of ADPribose polymerase, a DNA repair enzyme. Strand breaks and ADP-ribosylation were first detected in K562 and Raji targets 6-8 hr after incubation with HNP and increased to maximal levels by 18 hr. DNA was not degraded into nucleosome-sized fragments. To assess the impact of DNA injury on cytolysis, we increased strand breakage by coincubating targets with HNP and two inhibitors of ADPribose polymerase, 3-aminobenzamide, or nicotinamide. Concurrently with inhibiting polymerase activity and increasing DNA injury, these agents significantly enhanced HNP-mediated cytolysis. Enhancement occurred only at time points (over 6 hr) and in targets (only nucleated targets) where HNP-induced DNA injury could be occurring. These data indicate that neutrophil defensins can induce DNA injury in targets and suggest such injury may be involved in target cell death.     

Isolation of autosomal mutations in Drosophila melanogaster without setting up lines

Summary Mutants of Drosophila melanogaster are being used increasingly for studying different biological mechanisms. However, most attempts to identify new mutations have been restricted to the X-chromosome. It has been very difficult to identify new loci on the autosomes, as recessive mutations have to be made homozygous by setting up independent cultures for each mutagenized chromosome. We introduce a mutagenesis scheme which does not require setting up independent cultures. It uses meiotic recombination in compound autosomes to make recessive mutations homozygous and allows the screening of tens of thousands of mutagenized chromosomes with relatively little effort. In a pilot experiment, we tested about 33,300 chromosomes for temperature-sensitive paralytic mutations. We obtained 62 independent paralytic mutations and a large number of other mutations. Eight out of 25 of the paralytic mutations are on the autosomes. This method makes autosomes, which constitute about 80% of the Drosophila genome, more accessible for mutational analysis of various biological mechanisms.     

Protective effect of glutathione and selenium against alloxan induced lipid peroxidation and loss of antioxidant enzymes in erythrocytes

Alloxan is a diabetogenic drug and is known to induce diabetes through generation of free radicals. The toxic oxygen species can be detoxified by antioxidant enzyme system and thus reduce the deleterious effect of lipid peroxidation. Erythrocytes exposed to alloxan induced lipid peroxidationin vivo as well asin vitro. Although alloxan treatment produced a deleterious effect on antioxidant enzymes, pretreatment with glutathione and selenium led to a recovery of the activities of superoxide dismutase and glutathione peroxidase. However, catalase activity increased on alloxan treatment. Alloxan reduced blood glucose level significantly within 60 min but thereafter a slow and steady rise was observed.     

Selection of a substratum for composing biofilm system of a textile-effluent decolourizing bacteria

Summary Some abundantly and cheaply available substrates were used to develop biofilms of textile-effluent decolourizing bacteria. These biofilm systems were compared for their decolourization efficiency in packed bed system. Biofilm systems using mineral material, sea-shells or nylon web as substratum decolourized effluent (100%) in 9 –12 h without major release of free cells into the medium.     

Characterisation of recombinant thermostable manganese-superoxide dismutase (NeMnSOD) from Nerium oleander

Superoxide dismutase is one of the key antioxidant enzymes accountable for the eradication of free radicals generated during various metabolic processes. This is first study reporting a thermostable MnSOD obtained from a xerophytic plant, Nerium oleander. The full-length gene identified using Rapid amplification of cDNA ends revealed an open reading frame of 699 bp flanked by 5′UTR and 3′UTR of 134 bp and 198 bp respectively. The corresponding NeMnSOD protein was cloned and expressed in Escherichia coli. The purified protein yields a band of 25.4 kDa, which established a specific activity of 2617 units mg^?1 of protein and under native condition yield bands of 52 kDa and 110 kDa, confirming the dimeric and tetrameric state of the protein. The Km and V_max of 0.078?±?0.008 mM and 1052.3?±?33.59 units mg^?1 of protein, respectively. The purified enzyme demonstrated thermostability by retaining more than 20% activity at a temperature 70 ℃. The enzyme functioned at pH range of 4–9.0 with maximum activity at pH 7.4. Sodium azide, effectively inhibited the activity of enzyme confirming it to be MnSOD. The enzyme activity was least affected on treatment with strong denaturants (Urea, guanidine HCl and SDS) and harsh chemicals (DTT, CHAPS and β-mercapto-ethanol) These experimental data validated with Insilco analysis revealed that NeMnSOD possessed thermo as well as kinetically stable moiety which can be further exploited with its applications in the field of pharmaceutical, food and cosmetic industry, which urge for such thermostable enzyme.

Graphic Abstract

     

Isolation and genetic analysis of ethanol-sensitive mutants of thermotolerant Kluyveromyces marxianus

Six monogenic mutants of K. marxianus, affected in their ability to tolerate ethanol, were assigned to 6 loci etr1 through etr6, probably the hot spots for ethyl methane sulfonate as these loci were found mutated in each of the non-monogenic mutants as well. Differential ethanol tolerance of allelic hexagenic mutants suggests that even more than 6 genes may be involved in controlling ethanol tolerance in K. marxianus.     

A genome-wide search for wild-species alleles that increase horticultural yield of processing tomatoes

To identify QTLs associated with horticultural yield it is necessary to conduct replicated plot trials of the tested genotypes. The first step in the utilization of an introgression-line (IL) population of Lycopersicon pennellii in a processing-tomato variety (M82) for mapping such QTLs was to screen 51 ILs in a non-replicated plot trial. The results of this survey were compared to those obtained in a replicated trial of the same genotypes grown as single plants at wide spacing. Fruit characteristics were similar between the two stands, but yield was generally different. Eight lines that outperformed the control in the plot survey were subjected to detailed analysis in the following year. The effects of these introgressions, measured on single plants, were reproducible relative to the previous year's results. In a replicated plot trial of these ILs and their hybrids involving two genetic backgrounds, the product of yield and total soluble solids (horticultural yield) in seven of the eight hybrids was 7–13% higher than that of their nearly isogenic controls. The results revealed a consistent trend in the interaction between introgression effects and genetic background. Combining the two introgressions with the largest contribution to horticultural yield in plots resulted in a 20% increase relative to the control in the third year. This research highlights the potential of wild germ plasm for yield improvement and the ability of nearly isogenic populations to achieve this goal.