Proteome analysis of functionally differentiated bovine (Bos indicus) mammary epithelial cells isolated from milk

Mammary gland is made up of a branching network of ducts that end in alveoli. Terminally differentiated mammary epithelial cells (MECs) constitute the innermost layer of aveoli. They are milk‐secreting cuboidal cells that secrete milk proteins during lactation. Little is known about the expression profile of proteins in the metabolically active MECs during lactation or their functional role in the lactation process. In the present investigation, we have reported the proteome map of MECs in lactating cows using 2DE MALDI‐TOF/TOF MS and 1D‐Gel‐LC‐MS/MS. MECs were isolated from milk using immunomagnetic beads and confirmed by RT‐PCR and Western blotting. The 1D‐Gel‐LC‐MS/MS and 2DE‐MS/MS based approaches led to identification of 431 and 134 proteins, respectively, with a total of 497 unique proteins. Proteins identified in this study were clustered into functional groups using bioinformatics tools. Pathway analysis of the identified proteins revealed 28 pathways (p < 0.05) providing evidence for involvement of various proteins in lactation function. This study further provides experimental evidence for the presence of many proteins that have been predicted in annotated bovine genome. The data generated further provide a set of bovine MEC‐specific proteins that will help the researchers to understand the molecular events taking place during lactation.     

Trypanosoma brucei mitochondrial respiratome: composition and organization in procyclic form

The mitochondrial respiratory chain is comprised of four different protein complexes (I–IV), which are responsible for electron transport and generation of proton gradient in the mitochondrial intermembrane space. This proton gradient is then used by F_oF₁-ATP synthase (complex V) to produce ATP by oxidative phosphorylation. In this study, the respiratory complexes I, II, and III were affinity purified from Trypanosoma brucei procyclic form cells and their composition was determined by mass spectrometry. The results along with those that we previously reported for complexes IV and V showed that the respiratome of Trypanosoma is divergent because many of its proteins are unique to this group of organisms. The studies also identified two mitochondrial subunit proteins of respiratory complex IV that are encoded by edited RNAs. Proteomics data from analyses of complexes purified using numerous tagged component proteins in each of the five complexes were used to generate the first predicted protein-protein interaction network of the Trypanosoma brucei respiratory chain. These results provide the first comprehensive insight into the unique composition of the respiratory complexes in Trypanosoma brucei, an early diverged eukaryotic pathogen.Mitochondria are dynamic organelles essential for cellular life, death, and differentiation of virtually every eukaryotic cell. They house systems for energy production through oxidative phosphorylation, synthesis of key metabolites, and iron-sulfur cluster assembly. The oxidative phoshorylation system of eukaryotic mitochondria comprises five major complexes located in the mitochondrial (mt)¹ inner membrane, and often abbreviated as mt complexes I–V. The redox energy of the substrates NADH and succinate is first converted into an electrochemical proton potential across the inner mt membrane by respiratory complexes I (NADH:ubiquinone reductase), II (SDH, succinate:ubiquinone reductase), III (bc₁, ubiquinone:cytochrome c reductase), and IV (cytochrome c oxidase). The electrochemical proton potential is then used by complex V (F_oF₁-ATP synthase) to synthesize ATP from ADP and inorganic phosphate, a mechanism that has essentially remained unchanged from bacteria to human (1). However, parasitic organisms have exploited unique energy metabolic pathways by adapting to their natural host habitats (2). Indeed, the respiratory systems of parasites typically show greater diversity in electron transfer pathways than those of their host, and Trypanosoma brucei is no exception to this rule (3).T. brucei, the causative agent of human African trypanosomiasis (HAT), or sleeping sickness, is a blood-borne pathogenic parasite transmitted by tsetse flies. It has a complex life cycle that alternates between the bloodstream forms (BF) in the mammalian host and several stages in the insect vector starting with the procyclic form (PF) in the midgut. During T. brucei differentiation between the distinct life-cycle stages, the mitochondrion undergoes morphological and functional changes, and the parasite switches its energy metabolism from amino acid to glucose oxidation (4). BF cells, which live in sugar-rich environment, use energy metabolism predominantly through the glycolytic pathway (5). They contain no cytochrome-mediated respiratory chain and they possess a unique electron transport chain in the mitochondria, the glycerol-3-phosphate dehydrogenase and the salicyl hydroxamic acid (SHAM)-sensitive alternative oxidase, which is known as the trypanosome alternative oxidase (TAO) (6). Despite the absence of complete cytochrome-containing complexes III and IV in BF trypanosomes, a mt membrane potential is maintained and involves the hydrolytic activity of the F_oF₁-ATP synthase complex (7). Conversely, PF cells are dependent on the cytochrome-containing respiratory chain and ATP generated by conventional function of the F_oF₁-ATP synthase complex for their energy production (8, 9). The branched electron-transport chain contains four complexes that donate electrons to the ubiquinone pool, two NADH:ubiquinone oxidoreductases (complex I and a rotenone-insensitive enzyme), complex II, and glycerol-3-phosphate dehydrogenase. Reduced ubiquinol can be reoxidized by the transfer of electron to either the TAO, which does not translocate protons, or to the cytochrome-containing complexes III and IV that produce a proton motive force by translocation of protons and thus create essential membrane potential (10).Although the T. brucei genome has been sequenced (11), little information is available on the subunit composition of mt complexes I–V based on similarity searches. However, some respiratory complexes have been partially characterized in other trypanosomatids such as Crithidia fasciculata, T. cruzi, and Leishmania tarentolae (12–15). In recent studies, we have determined the protein composition of complexes IV and V, and part of complex I purified from mitochondria of T. brucei PF cells (8, 16, 17, 25). These analyses revealed the uniqueness of respiratory complexes in trypanosomes, where large numbers of component proteins have no homologs outside of the Kinetoplastida.In this study, we focus on the comprehensive characterization of all respiratory complexes in T. brucei, collectively termed the respiratome. We report the composition of complexes II and III from PF cells, and extend the characterization of complex I by identifying additional protein constituents. This included the identification of two subunits of the respiratory complex IV, both encoded by mt edited RNAs. We also present a predicted protein-protein interaction network of the respiratome, which was generated using proteomics data collected from numerous tagged proteins in each of the complexes I–V. Our results provide a comprehensive insight into the unique composition of the respiratory complexes in one of the life-cycle stages of T. brucei.     

Bacterial and archaea community present in the Pine Barrens Forest of Long Island, NY: unusually high percentage of ammonia oxidizing bacteria

Of the few preserved areas in the northeast of United States, the soil in the Pine Barrens Forests presents a harsh environment for the microorganisms to grow and survive. In the current study we report the use of clustering methods to scientifically select the sampling locations that would represent the entire forest and also report the microbial diversity present in various horizons of the soil. Sixty six sampling locations were selected across the forest and soils were collected from three horizons (sampling depths). The three horizons were 0-10 cm (Horizon O); 11-25 cm (Horizon A) and 26-40 cm (Horizon B). Based on the total microbial substrate utilization pattern and K-means clustering analysis, the soil in the Pine Barrens Forest can be classified into four distinct clusters at each of the three horizons. One soil sample from each of the four clusters were selected and archaeal and bacterial populations within the soil studied using pyrosequencing method. The results show the microbial communities present in each of these clusters are different. Within the microbial communities present, microorganisms involved in nitrogen cycle occupy a major fraction of microbial community in the soil. High level of diversity was observed for nitrogen fixing bacteria. In contrast, Nitrosovibrio and Nitrosocaldus spp are the single bacterial and archaeal population respectively carrying out ammonia oxidation in the soil.     

Paromomycin affects translation and vesicle-mediated trafficking as revealed by proteomics of paromomycin -susceptible -resistant Leishmania donovani

Leishmania donovani is a protozoan parasite that causes visceral leishmaniasis (VL) and is responsible for significant mortality and morbidity. Increasing resistance towards antimonial drugs poses a great challenge in chemotherapy of VL. Paromomycin is an aminoglycosidic antibiotic and is one of the drugs currently being used in the chemotherapy of cutaneous and visceral leishmaniasis. To understand the mode of action of this antibiotic at the molecular level, we have investigated the global proteome differences between the wild type AG83 strain and a paromomycin resistant (PRr) strain of L. donovani. Stable isotope labeling of amino acids in cell culture (SILAC) followed by quantitative mass spectrometry of the wild type AG83 strain and the paromomycin resistant (PRr) strain identified a total of 226 proteins at ≥ 95% confidence. Data analysis revealed upregulation of 29 proteins and down-regulation of 21 proteins in the PRr strain. Comparative proteomic analysis of the wild type and the paromomycin resistant strains showed upregulation of the ribosomal proteins in the resistant strain indicating role in translation. Elevated levels of glycolytic enzymes and stress proteins were also observed in the PRr strain. Most importantly, we observed upregulation of proteins that may have a role in intracellular survival and vesicular trafficking in the PRr strain. Furthermore, ultra-structural analysis by electron microscopy demonstrated increased number of vesicular vacuoles in PRr strain when compared to the wild-type strain. Drug affinity pull-down assay followed by mass spectrometery identified proteins in L. donovani wild type strain that were specifically and covalently bound to paromomycin. These results provide the first comprehensive insight into the mode of action and underlying mechanism of resistance to paromomycin in Leishmania donovani.     

Biofouling and microbial corrosion problem in the thermo-fluid heat exchanger and cooling water system of a nuclear test reactor

This article discusses aspects of biofouling and corrosion in the thermo-fluid heat exchanger (TFHX) and in the cooling water system of a nuclear test reactor. During inspection, it was observed that >90% of the TFHX tube bundle was clogged with thick fouling deposits. Both X-ray diffraction and Mössbauer analyses of the fouling deposit demonstrated iron corrosion products. The exterior of the tubercle showed the presence of a calcium and magnesium carbonate mixture along with iron oxides. Raman spectroscopy analysis confirmed the presence of calcium carbonate scale in the calcite phase. The interior of the tubercle contained significant iron sulphide, magnetite and iron-oxy-hydroxide. A microbiological assay showed a considerable population of iron oxidizing bacteria and sulphate reducing bacteria (10⁵ to 10⁶ cfu g^?1 of deposit). As the temperature of the TFHX is in the range of 45–50°C, the microbiota isolated/assayed from the fouling deposit are designated as thermo-tolerant bacteria. The mean corrosion rate of the CS coupons exposed online was ～2.0 mpy and the microbial counts of various corrosion causing bacteria were in the range 10³ to 10⁵ cfu ml^?1 in the cooling water and 10⁶ to 10⁸ cfu ml^?1 in the biofilm.     

Response of antioxidant enzymes to high NaCl concentration in different salt-tolerant plants

The effects of NaCl on the H₂O₂ content and the activities of catalase (CAT) and superoxide dismutase (SOD) were studied in diverse group of plants, such as a unicellular alga, Chlorella sp., an aquatic macrophyte, Najas graminea, and a mangrove plant, Suaeda maritima, all showing high tolerance to NaCl. Significant accumulation of H₂O₂ was observed in all the tested plants upon their exposure to 255 mM NaCl. The activity of both CAT and SOD increased significantly in response to the NaCl treatment. Growing the plants in presence of 255 mM NaCl also resulted in the synthesis of new isoforms of both CAT and SOD.     

Regulatory mechanisms across networks of the circadian clock and senescence pathways

Journal of Plant Biochemistry and Biotechnology - The circadian clock serves the fitness of higher plants by controlling various aspects of plant growth and development ranging from photosynthesis...     

Cross-genera amplification of Cajanus spp. specific SSR markers in Clitoria ternatea (L.) and their application in genetic diversity studies

Clitoria ternatea (L.) is a medicinal leguminous plant and is cultivated to cater the need of herbal industries and asthetic purposes. The unavailability of steady molecular marker impedes the genetic improvement of C. ternatea. In the present study, transferability of 98 pairs of Cajanus spp. specific SSR primers were assessed among 14 genotypes of C. ternatea, varied for their flower color, floral architecture and bio-metabolite (taraxerol and delphinidin) content, and out of them 43 had successfully amplified the fragments. Among them, 36 pairs of primers showed 100% transferability, whereas rest seven varied from 42.86 to 92.85% transferability. The transferable 43 pairs of SSR primers generated 196 alleles across the 14 genotypes and the AMOVA analysis showed moderate genetic variation (55.1%) among the genotypes of C. ternatea, which was also reinforced by Nei’s genetic distance and gene identity estimates derived haplotype matrix. Similarly, both the principal coordinate analysis and dendrogram grouped these 14 genotypes of C. ternatea into two major clusters based on SSR allele distribution and frequency, and the clustering pattern is in accordance with petal color but in contrast to floral architecture. MCheza based outlier analysis revealed 16 alleles for balancing selection, which are putatively involved in the maintenance of genetic polymorphism in C. ternatea. Moreover, the estimates of molecular diversity and bio-metabolite content revealed the possible use of these genotypes in future breeding programme of this species.Electronic supplementary materialThe online version of this article (10.1007/s12298-020-00907-x) contains supplementary material, which is available to authorized users.     

Structure and energy of non-canonical basepairs: comparison of various computational chemistry methods with crystallographic ensembles

Different types of non-canonical basepairs, in addition to the Watson-Crick ones, are observed quite frequently in RNA. Their importance in the three dimensional structure is not fully understood, but their various roles have been proposed by different groups. We have analyzed the energetics and geometry of 32 most frequently observed basepairs in the functional RNA crystal structures using different popular empirical, semi-empirical and ab initio quantum chemical methods and compared their optimized geometry with the crystal data. These basepairs are classified into three categories: polar, non-polar and sugar-mediated, depending on the types of atoms involved in hydrogen bonding. In case of polar basepairs, most of the methods give rise to optimized structures close to their initial geometry. The interaction energies also follow similar trends, with the polar ones having more attractive interaction energies. Some of the C-H...O/N hydrogen bond mediated non-polar basepairs are also found to be significantly stable in terms of their interaction energy values. Few polar basepairs, having amino or carboxyl groups not hydrogen bonded to anything, such as G:G H:W C, show large flexibility. Most of the non-polar basepairs, except A:G s:s T and A:G w:s C, are found to be stable; indicating C-H...O/N interaction also plays a prominent role in stabilizing the basepairs. The sugar mediated basepairs show variability in their structures, due to the involvement of flexible ribose sugar. These presumably indicate that the most of the polar basepairs along with few non-polar ones act as seed for RNA folding while few may act as some conformational switch in the RNA.