The paramutagenic line niv-44 has a 5 kb insert, Tam 2, in the chalcone synthase gene of Antirrhinum majus

Summary Several alleles of the nivea locus of Antirrhinum majus, both stable and unstable, have been characterised genetically (Harrison and Carpenter 1973 a, b). In this work the niv-44 allele is characterised at the molecular level. It contains a 5kb insertion element, Tam 2, which has 14 base pair inverted repeats. There is a three base pair duplication at the target site, which is at the first intron-exon boundary of the chalcone synthase gene. Tam 2 homologous sequences are present in multiple copies in several A. majus lines, including niv-53, and most have at least a 2.9 kb sequence in common with the copy at the chalcone synthase gene. Possible reasons for the apparent stability of the niv-44 allele and molecular explanations for the role of this allele in paramutation in A. majus are discussed.Dedicated to Professor Georg Melchers to celebrate his 50-year association with the journal     

Microbiological Analyses of Traditional Alcoholic Beverage (Chhang) and its Starter (Balma) Prepared by Bhotiya Tribe of Uttarakhand,India

Present article depicts microbiology of starter (Balma) used in traditional solid-state fermentation of alcoholic beverage (Chhang) by Bhotiya tribe of Uttarakhand. It also highlights the importance of herbs in Balma preparation and kinetics of lactic acid and ethanol fermentation under Chhang preparation using Balma. Balma contains 214 × 10⁶ cfu/g yeasts, 2.54 × 10⁶ cfu/g lactic acid bacteria (LAB) and 1.4 × 10⁶ cfu/g other mesophilic bacteria. ITS sequence analysis revealed a rich diversity of yeast comprising of Saccharomyces cerevisiae, Saccharomycopsis fibuligera and Saccharomycopsis malanga in Balma. 16S rDNA sequence analysis revealed Lactobacillus pentosus and Pediococcus pentosaceus among LAB, while amylolytic Bacillus subtilis and B. aerophilus among other bacteria in Balma. Based on the results, it is speculated that herbs such as Inula cuspidata, Micromeria biflora, Origanum vulgare, Rubus sp. and Thymus linearis used earlier by Bhotiya in Balma preparation contribute as a source of yeasts, LAB and amylolytic bacilli. Study also demonstrates that Bhotiya tribe is rational in preparation of starter as they have circumvented the need of plants by using previous year Balma as inoculum and possibility of deficient quality of Balma due to weak colonization of phyllosphere and rhizosphere microbiota. Results suggest that simultaneous saccharification and lactic acid–ethanol fermentation take place in traditional cereal based Chhang fermentation system of Bhotiya.     

Histidine availability is decisive in ROS-mediated cytotoxicity of copper complexes of Aβ1–16 peptide

The binding of metal ions to Aβ peptide plays an important role in the etiology of AD. Copper coordinates chiefly to His residues and produces reactive oxygen species (ROS) upon redox cycling. ROS builds enormous burden on the normal functioning of neuronal cells and results into deleterious effects. Recently, two structurally distinct copper binding sites with contrasting redox properties were characterized. Here, we demonstrate for the first time the effect of binding of two equivalents of Cu²⁺ on redox properties and cytotoxicity of Aβ peptide. Our electrochemical data and ascorbate consumption assay suggest that in the presence of two equivalents of copper; Aβ peptide has higher propensity of H₂O₂ generation. The oxidation of Aβ1–16 peptide due to both gamma radiolysis and metal catalyzed oxidation in the presence of two equivalents of copper is inhibited confirming the binding of both equivalents of copper to peptide. The electrochemical and cytotoxicity study shows that negative shift in the reduction potential is reflected as slightly higher cytotoxicity in SH-SY5Y cell lines for Aβ1–16–Cu²⁺ (1:2) complex.     

Cloning and heterologous expression of ectoine biosynthesis genes from Bacillus halodurans in Escherichia coli

The genes involved in the biosynthetic pathway of ectoine (2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid) from Bacillus halodurans were cloned as an operon and expressed in E. coli. Analysis of the deduced ectoine biosynthesis cluster amino acid sequence revealed that the ectoine operon contain 2,389 bp, encoded by three genes; ectA, ectB and ectC that encode proteins of 189, 427 and 129 amino acids with deduced molecular masses of 21,048, 47,120 and 14,797 Da respectively. Extracts of induced cells showed two bands at 41 kDa and 17 kDa, possibly corresponding to the products of the later two genes. However the expression of ectA gene could not be ascertained by SDS-PAGE. The activity of the ectA protein was confirmed by an acylation assay. The transgenic E. coli accumulated upto 4.6 mg ectoine/l culture. This is the first report of an engineered E. coli strain carrying the ectoine genes of the alkaliphilic bacterium, B. halodurans.     

Living with a large predator: Assessing the root causes of Human–brown bear conflict and their spatial patterns in Lahaul valley,Himachal Pradesh

Brown bear‐mediated conflicts have caused immense economic loss to the local people living across the distribution range. In India, limited knowledge is available on the Himalayan brown bear (HBB), making human–brown bear conflict (HBC) mitigation more challenging. In this study, we studied HBC in the Lahaul valley using a semi‐structured questionnaire survey by interviewing 398 respondents from 37 villages. About 64.8% of respondents reported conflict in two major groups—crop damage (30.6%) and livestock depredations (6.2%), while 28% reported both. Conflict incidences were relatively high in summer and frequently occurred in areas closer to the forest (<500 m) and between the elevations range of 2700 m to 3000 m above sea level (asl). The dependency of locals on forest resources (70%) for their livelihood makes them vulnerable to HBC. The “upper lower” class respondents were most impacted among the various socioeconomic classes. Two of the four clusters were identified as HBC hot spots in Lahaul valley using SaTscan analysis. We also obtained high HBC in cluster II with a 14.35 km radius. We found that anthropogenic food provisioning for HBB, livestock grazing in bear habitats, and poor knowledge of animal behavior among the communities were the major causes of HBC. We suggest horticulture crop waste management, controlled and supervised grazing, ecotourism, the constitution of community watch groups, and others to mitigate HBC. We also recommend notifying a few HBB abundant sites in the valley as protected areas for the long‐term viability of the HBB in the landscape.     

Overexpression of Nmnat3 efficiently increases NAD and NGD levels and ameliorates age‐associated insulin resistance

Nicotinamide adenine dinucleotide (NAD) is an important cofactor that regulates various biological processes, including metabolism and gene expression. As a coenzyme, NAD controls mitochondrial respiration through enzymes of the tricarboxylic acid (TCA) cycle, β‐oxidation, and oxidative phosphorylation and also serves as a substrate for posttranslational protein modifications, such as deacetylation and ADP‐ribosylation by sirtuins and poly(ADP‐ribose) polymerase (PARP), respectively. Many studies have demonstrated that NAD levels decrease with aging and that these declines cause various aging‐associated diseases. In contrast, activation of NAD metabolism prevents declines in NAD levels during aging. In particular, dietary supplementation with NAD precursors has been associated with protection against age‐associated insulin resistance. However, it remains unclear which NAD synthesis pathway is important and/or efficient at increasing NAD levels in vivo. In this study, Nmnat3 overexpression in mice efficiently increased NAD levels in various tissues and prevented aging‐related declines in NAD levels. We also demonstrated that Nmnat3‐overexpressing (Nmnat3 Tg) mice were protected against diet‐induced and aging‐associated insulin resistance. Moreover, in skeletal muscles of Nmnat3 Tg mice, TCA cycle activity was significantly enhanced, and the energy source for oxidative phosphorylation was shifted toward fatty acid oxidation. Furthermore, reactive oxygen species (ROS) generation was significantly suppressed in aged Nmnat3 Tg mice. Interestingly, we also found that concentrations of the NAD analog nicotinamide guanine dinucleotide (NGD) were dramatically increased in Nmnat3 Tg mice. These results suggest that Nmnat3 overexpression improves metabolic health and that Nmnat3 is an attractive therapeutic target for metabolic disorders that are caused by aging.     

Blood-based lung cancer biomarkers identified through proteomic discovery in cancer tissues,cell lines and conditioned medium

Background

Support for early detection of lung cancer has emerged from the National Lung Screening Trial (NLST), in which low-dose computed tomography (LDCT) screening reduced lung cancer mortality by 20 % relative to chest x-ray. The US Preventive Services Task Force (USPSTF) recently recommended annual screening for the high-risk population, concluding that the benefits (life years gained) outweighed harms (false positive findings, abortive biopsy/surgery, radiation exposure). In making their recommendation, the USPSTF noted that the moderate net benefit of screening was dependent on the resolution of most false-positive results without invasive procedures. Circulating biomarkers may serve as a valuable adjunctive tool to imaging.

Results

We developed a broad-based proteomics discovery program, integrating liquid chromatography/mass spectrometry (LC/MS) analyses of freshly resected lung tumor specimens (n = 13), lung cancer cell lines (n = 17), and conditioned media collected from tumor cell lines (n = 7). To enrich for biomarkers likely to be found at elevated levels in the peripheral circulation of lung cancer patients, proteins were prioritized based on predicted subcellular localization (secreted, cell-membrane associated) and differential expression in disease samples. 179 candidate biomarkers were identified. Several markers selected for further validation showed elevated levels in serum collected from subjects with stage I NSCLC (n = 94), relative to healthy smoker controls (n = 189). An 8-marker model was developed (TFPI, MDK, OPN, MMP2, TIMP1, CEA, CYFRA 21–1, SCC) which accurately distinguished subjects with lung cancer (n = 50) from high risk smokers (n = 50) in an independent validation study (AUC = 0.775).

Conclusions

Integrating biomarker discovery from multiple sample types (fresh tissue, cell lines and conditioned medium) has resulted in a diverse repertoire of candidate biomarkers. This unique collection of biomarkers may have clinical utility in lung cancer detection and diagnoses.

Electronic supplementary material

The online version of this article (doi:10.1186/s12014-015-9090-9) contains supplementary material, which is available to authorized users.     

Regulatory Elements within the Prodomain of Falcipain-2, a Cysteine Protease of the Malaria Parasite Plasmodium falciparum

Falcipain-2, a papain family cysteine protease of the malaria parasite Plasmodium falciparum, plays a key role in parasite hydrolysis of hemoglobin and is a potential chemotherapeutic target. As with many proteases, falcipain-2 is synthesized as a zymogen, and the prodomain inhibits activity of the mature enzyme. To investigate the mechanism of regulation of falcipain-2 by its prodomain, we expressed constructs encoding different portions of the prodomain and tested their ability to inhibit recombinant mature falcipain-2. We identified a C-terminal segment (Leu¹⁵⁵–Asp²⁴³) of the prodomain, including two motifs (ERFNIN and GNFD) that are conserved in cathepsin L sub-family papain family proteases, as the mediator of prodomain inhibitory activity. Circular dichroism analysis showed that the prodomain including the C-terminal segment, but not constructs lacking this segment, was rich in secondary structure, suggesting that the segment plays a crucial role in protein folding. The falcipain-2 prodomain also efficiently inhibited other papain family proteases, including cathepsin K, cathepsin L, cathepsin B, and cruzain, but it did not inhibit cathepsin C or tested proteases of other classes. A structural model of pro-falcipain-2 was constructed by homology modeling based on crystallographic structures of mature falcipain-2, procathepsin K, procathepsin L, and procaricain, offering insights into the nature of the interaction between the prodomain and mature domain of falcipain-2 as well as into the broad specificity of inhibitory activity of the falcipain-2 prodomain.