Partial ablation of adult Drosophila insulin-producing neurons modulates glucose homeostasis and extends life span without insulin resistance

In Drosophila melanogaster (D. melanogaster), neurosecretory insulin-like peptide-producing cells (IPCs), analogous to mammalian pancreatic β cells are involved in glucose homeostasis. Extending those findings, we have developed in the adult fly an oral glucose tolerance test and demonstrated that IPCs indeed are responsible for executing an acute glucose clearance response. To further develop D. melanogaster as a relevant system for studying age-associated metabolic disorders, we set out to determine the impact of adult-specific partial ablation of IPCs (IPC knockdown) on insulin-like peptide (ILP) action, metabolic outcomes and longevity. Interestingly, while IPC knockdown flies are hyperglycemic and glucose intolerant, these flies remain insulin sensitive as measured by peripheral glucose disposal upon insulin injection and serine phosphorylation of a key insulin-signaling molecule, Akt. Significant increases in stored glycogen and triglyceride levels as well as an elevated level of circulating lipid measured in adult IPC knockdown flies suggest profound modulation in energy metabolism. Additional physiological outcomes measured in those flies include increased resistance to starvation and impaired female fecundity. Finally, increased life span and decreased mortality rates measured in IPC knockdown flies demonstrate that it is possible to modulate ILP action in adult flies to achieve life span extension without insulin resistance. Taken together, we have established and validated an invertebrate genetic system to further investigate insulin action, metabolic homeostasis and regulation of aging regulated by adult IPCs.Key words: Drosophila melanogaster, insulin-producing cells (IPCs), drosophila insulin-like peptides (DILPs), type 2 diabetes, oral glucose tolerance test (OGTT), insulin sensitivity, energy metabolism, life span     

Consequences of daily administered parathyroid hormone on myeloma growth, bone disease, and molecular profiling of whole myelomatous bone

Background

Induction of osteolytic bone lesions in multiple myeloma is caused by an uncoupling of osteoclastic bone resorption and osteoblastic bone formation. Current management of myeloma bone disease is limited to the use of antiresorptive agents such as bisphosphonates.

Methodology/Principal Findings

We tested the effects of daily administered parathyroid hormone (PTH) on bone disease and myeloma growth, and we investigated molecular mechanisms by analyzing gene expression profiles of unique myeloma cell lines and primary myeloma cells engrafted in SCID-rab and SCID-hu mouse models. PTH resulted in increased bone mineral density of myelomatous bones and reduced tumor burden, which reflected the dependence of primary myeloma cells on the bone marrow microenvironment. Treatment with PTH also increased bone mineral density of uninvolved murine bones in myelomatous hosts and bone mineral density of implanted human bones in nonmyelomatous hosts. In myelomatous bone, PTH markedly increased the number of osteoblasts and bone-formation parameters, and the number of osteoclasts was unaffected or moderately reduced. Pretreatment with PTH before injecting myeloma cells increased bone mineral density of the implanted bone and delayed tumor progression. Human global gene expression profiling of myelomatous bones from SCID-hu mice treated with PTH or saline revealed activation of multiple distinct pathways involved in bone formation and coupling; involvement of Wnt signaling was prominent. Treatment with PTH also downregulated markers typically expressed by osteoclasts and myeloma cells, and altered expression of genes that control oxidative stress and inflammation. PTH receptors were not expressed by myeloma cells, and PTH had no effect on myeloma cell growth in vitro.

Conclusions/Significance

We conclude that PTH-induced bone formation in myelomatous bones is mediated by activation of multiple signaling pathways involved in osteoblastogenesis and attenuated bone resorption and myeloma growth; mechanisms involve increased osteoblast production of anti-myeloma factors and minimized myeloma induction of inflammatory conditions.     

Proteome analysis of soybean roots subjected to short-term drought stress

Drought is one of the most important constraints on the growth and productivity of many crops, including soybeans. However, as a primary sensing organ, the plant root response to drought has not been well documented at the proteomic level. In the present study, we carried out a proteome analysis in combination with physiological analyses of soybean roots subjected to severe but recoverable drought stress at the seedling stage. Drought stress resulted in the increased accumulation of reactive oxygen species and subsequent lipid peroxidation. The proline content increased in drought-stressed plants and then decreased during the period of recovery. The high-resolution proteome map demonstrated significant variations in about 45 protein spots detected on Comassie briliant blue-stained 2-DE gels. Of these, 28 proteins were identified by mass spectrometry; the levels of 5 protein spots were increased, 21 were decreased and 2 spots were newly detected under drought condition. When the stress was terminated by watering the plants for 4 days, in most cases, the protein levels tended towards the control level. The proteins identified in this study are involved in a variety of cellular functions, including carbohydrate and nitrogen metabolism, cell wall modification, signal transduction, cell defense and programmed cell death, and they contribute to the molecular mechanism of drought tolerance in soybean plants. Analysis of protein expression patterns revealed that proteins associated with osmotic adjustment, defense signaling and programmed cell death play important roles for soybean plant drought adaptation. The identification of these proteins provides new insight that may lead to a better understanding of the molecular basis of the drought stress responses.     

Virus elimination and pathogen-free plantlets regeneration in Cucurbita pepo L.

An efficient in vitro protocol was established for developing pathogen-free plantlets in Cucurbita pepo through meristem culture. Meristems of about 0.3–0.5 mm in size were isolated from shoot tips of 25–30 day old in vitro grown plants. For primary establishment of isolated apical meristem, MS liquid medium supplemented with 2.0 mgl KIN and 0.5 mg/l GA₃ was found to be most effective in both cultivars. MS semisolid medium containing 2.0 mg/l BAP were found to be most effective for shoot development from primarily established meristem in both cultivars. A good number of shoots were not concomitant with good rooting. The best root induction was found in media having 1.0 mg/l IBA in cv. Bulum. It was found that cv. Bulum was better than cv. Rumbo in all stages of meristem culture. The presence of virus in plantlets was achieved by DAS-ELISA test, where 68–81% plantlets have been proved to be virus free among the studied viruses. Healthy growth and vigour was observed in meristem derived plants over their source plants after cultivation under natural conditions.     

Fluoroquinolone Resistance Mechanisms of Shigella flexneri Isolated in Bangladesh

Objective

To investigate the prevalence and mechanisms of fluoroquinolone resistance in Shigella species isolated in Bangladesh and to compare with similar strains isolated in China.

Methods

A total of 3789 Shigella isolates collected from Clinical Microbiology Laboratory of icddr,b, during 2004–2010 were analyzed for antibiotic susceptibility. Analysis of plasmids, plasmid-mediated quinolone-resistance genes, PFGE, and sequencing of genes of the quinolone-resistance-determining regions (QRDR) were conducted in representative strains isolated in Bangladesh and compared with strains isolated in Zhengding, China. In addition, the role of efflux-pump was studied by using the efflux-pump inhibitor carbonyl cyanide-m-chlorophenylhydrazone (CCCP).

Results

Resistance to ciprofloxacin in Shigella species increased from 0% in 2004 to 44% in 2010 and S. flexneri was the predominant species. Of Shigella spp, ciprofloxacin resistant (Cip^R) strains were mostly found among S. flexneri (8.3%), followed by S. sonnei (1.5%). Within S. flexneri (n = 2181), 14.5% were resistance to ciprofloxacin of which serotype 2a was predominant (96%). MIC of ciprofloxacin, norfloxacin, and ofloxacin were 6–32 mg/L, 8–32 mg/L, and 8–24 mg/L, respectively in S. flexneri 2a isolates. Sequencing of QRDR genes of resistant isolates showed double mutations in gyrA gene (Ser⁸³Leu, Asp⁸⁷Asn/Gly) and single mutation in parC gene (Ser⁸⁰Ile). A difference in amino acid substitution at position 87 was found between strains isolated in Bangladesh (Asp⁸⁷Asn) and China (Asp⁸⁷Gly) except for one. A novel mutation at position 211 (His→Tyr) in gyrA gene was detected only in the Bangladeshi strains. Susceptibility to ciprofloxacin was increased by the presence of CCCP indicating the involvement of energy dependent active efflux pumps. A single PFGE type was found in isolates from Bangladesh and China suggesting their genetic relatedness.

Conclusions

Emergence of fluoroquinolone resistance in Shigella undermines a major challenge in current treatment strategies which needs to be followed up by using empirical therapeutic strategies.     

Insight into the biochemical characterization of phytocystatin from Glycine max and its interaction with Cd+2 and Ni+2

Phytocystatins are cysteine proteinase inhibitors ubiquitously present in plants and animals. They are known to carry out various significant physiological functions and also maintain the balance of protease‐antiprotease activity. In the present disquisition, a phytocystatin after preliminary treatment has been isolated and purified to homogeneity from soybean (Glycine max) by a simple two‐step stratagem using ammonium sulfate fractionation and gel filtration chromatography performed on Sephacryl S‐100‐HR. Soybean phytocystatin (SBPC) was purified with a fold purification of 635 and percent yield of 77.6%. A single band was observed on native gel electrophoresis confirming the homogeneity of the purified SBPC. The molecular weight of SBPC was found to be 19.05 kDa as determined by SDS‐PAGE. The SBPC was found to be devoid of carbohydrate moieties and sulfhydryl group content. The binding stoichiometry of SBPC‐papain interaction was determined by isothermal calorimetry suggesting 1:1 complex, and the value of binding constant (K) was found to be 2.78 × 10⁵ M^?1 The affinity of binding (K_d) value obtained through ITC was 3.59 × 10^?6 M. The purified SBPC was found to be stable in the pH range of 3 to 7 and is thermostable up to 50°C. The UV‐visible and fluorescence studies showed significant changes in the conformation upon the formation of the SBPC‐papain complex. Furthermore, fluorescence spectroscopy, ANS binding, and caseinolytic activity assay were conducted out to explore the effect of metal ions on SBPC which showed that there was a loss in the inhibitory activity along with conformational changes of SBPC upon complex formation with Cd⁺² and Ni⁺².     

Xenon reduces activation of transient receptor potential vanilloid type 1 (TRPV1) in rat dorsal root ganglion cells and in human TRPV1-expressing HEK293 cells

AimsXenon provides effective analgesia in several pain states at sub-anaesthetic doses. Our aim was to examine whether xenon may mediate its analgesic effect, in part, through reducing the activity of transient receptor potential vanilloid type 1 (TRPV1), a receptor known to be involved in certain inflammatory pain conditions.Main methodsWe studied the effect of xenon on capsaicin-evoked cobalt uptake in rat cultured primary sensory neurons and in human TRPV1 (hTRPV1)-expressing human embryonic kidney 293 (HEK293) cells. We also examined xenon's effect on the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in the rat spinal dorsal horn evoked by hind-paw injection of capsaicin.Key findingsXenon (75%) reduced the number of primary sensory neurons responding to the TRPV1 agonist, capsaicin (100 nM–1 μM) by ~ 25% to ~ 50%. Xenon reduced the number of heterologously-expressed hTRPV1 activated by 300 nM capsaicin by ~ 50%. Xenon (80%) reduced by ~ 40% the number of phosporylated ERK1/2-expressing neurons in rat spinal dorsal horn resulting from hind-paw capsaicin injection.SignificanceXenon substantially reduces the activity of TRPV1 in response to noxious stimulation by the specific TRPV1 agonist, capsaicin, suggesting a possible role for xenon as an adjunct analgesic where hTRPV1 is an active contributor to the excitation of primary afferents which initiates the pain sensation.     

Identification of Gy4 nulls and development of multiplex PCR-based co-dominant marker for Gy4 and α’ subunit of β-conglycinin in soybean

Alpha prime (α’) subunit of β-conglycinin and Gy4 subunit of glycinin are two important subunits of soybean storage protein which have negative effects on food processing, total amino acid content, and hypersensitivity reactions. It has been possible to reduce or remove some of these problems from soybean by screening or developing mutant lines. The objective of this study was to establish a simple, cheap DNA marker for Gy4 and α’ subunit for use in non-seed destructive, marker-assisted selection (MAS) that can identify these two mutants at the same time in a unique PCR reaction. To achieve this objective, we identified eight of Gy4 mutants from diverse soybean accessions from the USDA Soybean Germplasm Collection and described a multiplex PCR based co-dominant DNA marker for Gy4 subunit of glycinin. Then we crossed one of these Gy4 mutants with Keburi (α’ mutant) for development of double mutant variety and established a multiplex PCR based, co-dominant DNA marker for screening Gy4 and α’ mutants. Thus, using this newly developed marker to identify Gy4 and α’ mutants in breeding programs we could save our time, labor, and resources.     

Length–weight and length–length relationships of five Mystus species from the Ganges and Rupsha rivers,Bangladesh

This study focused on the length–weight and length–length relationships of five Mystus species from Bangladesh. A sum of 398 individuals (Mystus bleekeri = 47, Mystus cavasius = 171, Mystus gulio = 59, Mystus tengra = 65, and Mystus vittatus = 56) was collected from the Ganges and Rupsha rivers, Bangladesh. Fishes were caught by gill net (mesh sizes: 2.0–4.0 cm) and cast net (mesh sizes: 1.5–3.0 cm) from July 2014 to June 2015. Total length (TL), fork length (FL) and standard length (SL) were measured to 0.1 cm, while whole body weight (W) was taken to the nearest 0.1 g for each individual. The TL ranged from 6.0–13.5 cm for M. bleekeri, 5.0–15.0 cm for M. cavasius, 7.4–17.2 cm for M. gulio, 4.6–11.6 cm for M. tengra and 5.5–12.3 cm for M. vittatus. The W varied from 3.0–18.2 g for M. bleekeri, 1.3–30.4 g for M. cavasius, 6.1–62.2 g for M. gulio, 1.7–15.1 g for M. tengra and 2.7–19.2 g for M. vittatus. All LWRs were highly significant (p < .001), with all r² values ≥.950. The LLRs were also highly significant (p < .001), with all r² ≥.980. This study provides information on LWRs and LLRs for M. gulio and M. tengra for the first time. The results of this study can be very effective for stock assessment of Mystus species in the Ganges and Rupsha rivers as well as in the surrounding ecosystems.     

Impairment of reproduction in the red flour beetle, Tribolium castaneum (Herbst) (Col., Tenebrionidae) due to larval feeding on triflumuron-treated diet

The larvae of Tribolium castaneum (Herbst) of different ages were exposed to various concentrations viz., 0.0001, 0.001 and 0.01 ppm of triflumuron up to pupation. The adults were allowed to feed on untreated diet. The preoviposition period, fecundity, egg viability and incubation period of the eggs laid were studied. Triflumuron significantly lengthened the preoviposition and incubation periods (P < 0.001). Fecundity and egg viability were also reduced (P < 0.001) due to the treatment. Both the strains were affected by the treatment.