A cytotoxic type-2 ribosome inactivating protein (from leafless mistletoe) lacking sugar binding activity

Articulatin-D, a 66 kDa ribosome inactivating protein (RIP) comprised of 29 kDa A-chain linked to 35 kDa B-chain, is purified from leafless mistletoe (Viscum articulatum) parasitic on Dalbergia sp. from Western Ghats (India). N-terminal sequence and LC-MS/MS analyses of A- and B-chain confirmed that articulatin-D is a type-2 RIP having high homology with other mistletoe lectins. Translation inhibition and diagnostic N-glycosidase activity of articulatin-D illustrate the presence of catalytically active A-chain. Its inability to: (i) bind to acid treated Sepharose CL-6B column, (ii) agglutinate trypsin-treated and untreated RBCs of human (A, B, O, AB), mice, rat, rabbit, buffalo, porcine, pigeon, cock, fish, sheep and goat even with 10 mg/ml of purified articulatin-D, (iii) show change in circular dichroism spectra after addition of sugar to the native protein, (iv) bind to different sugars (galactose, lactose, gal-NAc, rhamnose, arabinose, fucose and mannose) immobilized on Sepharose 4B matrix, and (v) show change in enthalpy during titration with galactose confirm that the B-chain of articulatin-D lacks sugar binding activity. Despite this, articulatin-D is highly toxic as characterized with low IC₅₀ against different cancer cell lines (Jurkat: 0.31 ± 0.02 nM, MOLT-4: 0.51 ± 0.03 nM, U-937: 0.64 ± 0.07 nM, HL-60: 0.79 ± 0.11 nM, Raji: 1.45 ± 0.09 nM). Toxicity of RIPs has been ascribed to the absence/presence of B-chain with sugar binding activity. Identification of articulatin-D, the first cytotoxic RIP with B-chain lacking sugar binding activity opens new vistas in understanding cytotoxic action of RIPs.     

Functionally diverse rhizobacteria of Saccharum munja (a native wild grass) colonizing abandoned morrum mine in Aravalli hills (Delhi)

Characterization of the rhizobacteria of native grasses naturally colonizing abandoned mine sites may help in identification of microbial inoculants for ecological-restoration programmes. Eighty one strains of Saccharum munja rhizobacteria isolated from an abandoned mine located on Aravalli mountain and 50 from bulk-region were identified using 16S rRNA sequence analyses. Based on chemical- and biological-assays they were categorized into ecologically diverse functional groups (siderophore-, IAA-, ACC-deaminase-, HCN-, polyphosphate-producers; phosphate-solubilizer; antagonistic). Eight genera, 25 species from rhizosphere and 2 genera, 5 species from bulk-region were dominated by Bacillus spp. (B. barbaricus, B. cereus, B. firmus, B. flexus, B. foraminis, B. licheniformis, B. megaterium, B. pumilus, B. subtilis, B. thuringiensis) and Paenibacillus spp. (P. alvei, P. apiarius, P. lautus, P. lentimorbus, P. polymyxa, P. popillae). Siderophore-producers were common in rhizosphere and bulk soil, whereas IAA-producers, N₂-fixers and FePO₄-solubilizers dominated rhizosphere samples. During the reproductive phase (winter) of S. munja, siderophore-, ACC-deaminase- and polyP-producers were predominant; however dominance of HCN-producers in summer might be associated with termite-infestation. In vivo ability of selected rhizobacteria (B. megaterium BOSm201, B. subtilis BGSm253, B. pumilus BGSm157, P. alvei BGSm255, P. putida BOSm217, P. aeruginosa BGSm 306) to enhance seed-germination and seedling-growth of S. munja in mine-spoil suggest their significance in natural colonization and potential for ecological-restoration of Bhatti mine.     

Medicago truncatula mtpt4 mutants reveal a role for nitrogen in the regulation of arbuscule degeneration in arbuscular mycorrhizal symbiosis

Plants acquire essential mineral nutrients such as phosphorus (P) and nitrogen (N) directly from the soil, but the majority of the vascular plants also gain access to these mineral nutrients through endosymbiotic associations with arbuscular mycorrhizal (AM) fungi. In AM symbiosis, the fungi deliver P and N to the root through branched hyphae called arbuscules. Previously we identified MtPT4, a Medicago truncatula phosphate transporter located in the periarbuscular membrane that is essential for symbiotic phosphate transport and for maintenance of the symbiosis. In mtpt4 mutants arbuscule degeneration occurs prematurely and symbiosis fails. Here, we show that premature arbuscule degeneration occurs in mtpt4 mutants even when the fungus has access to carbon from a nurse plant. Thus, carbon limitation is unlikely to be the primary cause of fungal death. Surprisingly, premature arbuscule degeneration is suppressed if mtpt4 mutants are deprived of nitrogen. In mtpt4 mutants with a low N status, arbuscule lifespan does not differ from that of the wild type, colonization of the mtpt4 root system occurs as in the wild type and the fungus completes its life cycle. Sulphur is another essential macronutrient delivered to the plant by the AM fungus; however, suppression of premature arbuscule degeneration does not occur in sulphur-deprived mtpt4 plants. The mtpt4 arbuscule phenotype is strongly correlated with shoot N levels. Analyses of an mtpt4-2 sunn-1 double mutant indicates that SUNN, required for N-mediated autoregulation of nodulation, is not involved. Together, the data reveal an unexpected role for N in the regulation of arbuscule lifespan in AM symbiosis.     

Variations in Outer-membrane Characteristics of Two Stem-nodulating Bacteria of <Emphasis Type="Italic">Sesbania rostrata</Emphasis> and its Role in Tolerance Towards Diverse Stress

Outer-membrane characteristics may determine the survivability of rhizobia under diverse abiotic and biotic stresses. Therefore, the role of lipopolysaccharides (LPS) and membrane proteins of two stem-nodulating bacteria of Sesbania rostrata (Azorhizobium caulinodans ORS571 and Rhizobium sp. WE7) in determining tolerance towards abiotic and biotic stresses (hydrophobics and phages) was investigated. Outer-membrane characteristics (LPS and membrane–protein profiles) of ORS571, WE7 and thirteen standard strains were distinct. ORS571 and WE7 also showed susceptibility towards morphologically distinct phages, i.e., ACSR16 (short-tailed) and WESR29 (long-tailed), respectively. ORS571 and WE7 were tolerant to hydrophobic compounds (triton X-100, rifampicin, crystal violet and deoxycholate). To ascertain the role of outer membrane characteristics in stress tolerance, phage-resistant transconjugant mutants of ORS571 (ORS571-M8 and ORS571-M20) and WE7 (WE7-M9) were developed. LPS- and membrane–protein profiles of mutants differed from that of respective wild types (ORS571 and WE7). In in vitro assay, phages got adsorbed onto purified LPS-membrane protein fractions of wild types. Phages did not adsorb onto membrane fraction of mutants and standard strains. Mutant with reduced expression of LPS (ORS571-M20 and WE7-M9) showed reduced tolerance towards hydrophobics. However, the tolerance was unaffected in mutant (ORS571-M8) where expression of LPS was not reduced but pattern was different. The tolerance level of mutants towards hydrophobics varied with the expression of LPS, whereas the specificity towards phages is correlated with the specific LPS pattern.     

Synthesis, molecular modeling and bio-evaluation of cycloalkyl fused 2-aminopyrimidines as antitubercular and antidiabetic agents

An economical and efficient one step synthesis of a series of 8-(arylidene)-4-(aryl)-5,6,7,8-tetrahydro-quinazolin-2-ylamines and 9-(arylidene)-4-(aryl)-6,7,8,9-tetrahydro-5H-cycloheptapyrimidin-2-ylamines by the reaction of bis-benzylidene cycloalkanones and guanidine hydrochloride in presence of NaH has been developed. All the synthesized compounds were evaluated against Mycobacterium tuberculosis H₃₇Rv strain and the α-glucosidase and glycogen phosphorylase enzymes. Few of the compounds have shown interesting in vitro activity with MIC up to 3.12 μg/mL against M. tuberculosis and very good inhibition of α-glucosidase and glycogen phosphorylase enzymes. The most potent non toxic compound 40 exhibited about 58% ex vivo activity at MIC of 3.12 μg/mL. The present study opens a new gate to synthesize antitubercular agents for diabetic TB patients. In silico docking studies indicate that mycobacterial dihydrofolate reductase is the possible target of these compounds.     

Promises and pitfalls of using high‐throughput sequencing for diet analysis

The application of high‐throughput sequencing‐based approaches to DNA extracted from environmental samples such as gut contents and faeces has become a popular tool for studying dietary habits of animals. Due to the high resolution and prey detection capacity they provide, both metabarcoding and shotgun sequencing are increasingly used to address ecological questions grounded in dietary relationships. Despite their great promise in this context, recent research has unveiled how a wealth of biological (related to the study system) and technical (related to the methodology) factors can distort the signal of taxonomic composition and diversity. Here, we review these studies in the light of high‐throughput sequencing‐based assessment of trophic interactions. We address how the study design can account for distortion factors, and how acknowledging limitations and biases inherent to sequencing‐based diet analyses are essential for obtaining reliable results, thus drawing appropriate conclusions. Furthermore, we suggest strategies to minimize the effect of distortion factors, measures to increase reproducibility, replicability and comparability of studies, and options to scale up DNA sequencing‐based diet analyses. In doing so, we aim to aid end‐users in designing reliable diet studies by informing them about the complexity and limitations of DNA sequencing‐based diet analyses, and encourage researchers to create and improve tools that will eventually drive this field to its maturity.     

Signaling Domain of Sonic Hedgehog as Cannibalistic Calcium-Regulated Zinc-Peptidase

Sonic Hedgehog (Shh) is a representative of the evolutionary closely related class of Hedgehog proteins that have essential signaling functions in animal development. The N-terminal domain (ShhN) is also assigned to the group of LAS proteins (LAS = Lysostaphin type enzymes, D-Ala-D-Ala metalloproteases, Sonic Hedgehog), of which all members harbor a structurally well-defined center; however, it is remarkable that ShhN so far is the only LAS member without proven peptidase activity. Another unique feature of ShhN in the LAS group is a double- center close to the zinc. We have studied the effect of these calcium ions on ShhN structure, dynamics, and interactions. We find that the presence of calcium has a marked impact on ShhN properties, with the two calcium ions having different effects. The more strongly bound calcium ion significantly stabilizes the overall structure. Surprisingly, the binding of the second calcium ion switches the putative catalytic center from a state similar to LAS enzymes to a state that probably is catalytically inactive. We describe in detail the mechanics of the switch, including the effect on substrate co-ordinating residues and on the putative catalytic water molecule. The properties of the putative substrate binding site suggest that ShhN could degrade other ShhN molecules, e.g. by cleavage at highly conserved glycines in ShhN. To test experimentally the stability of ShhN against autodegradation, we compare two ShhN mutants in vitro: (1) a ShhN mutant unable to bind calcium but with putative catalytic center intact, and thus, according to our hypothesis, a constitutively active peptidase, and (2) a mutant carrying additionally mutation E177A, i.e., with the putative catalytically active residue knocked out. The in vitro results are consistent with ShhN being a cannibalistic zinc-peptidase. These experiments also reveal that the peptidase activity depends on .     

Diagnostic Yield of Universal Urine Toxicology Screening in an Unselected Cohort of Stroke Patients

Background

Illicit drug use increases the risk of cerebrovascular events by a variety of mechanisms. A recent report suggested that universal urine toxicology (UTox) screening of patients with stroke may be warranted. We aimed to evaluate the diagnostic yield of urine drug screening among unselected patients admitted with acute stroke or transient ischemic attack (TIA).

Methods

Using a single-center prospective study design, we evaluated consecutive patients with acute ischemic stroke, TIA, intracerebral hemorrhage (ICH), or subarachnoid hemorrhage (SAH) over one year. Urine samples were collected within 48 hours of admission and analyzed for common classes of abused drugs. Prevalence of positive UTox screening was determined. We evaluated whether baseline demographics and clinical factors were associated with UTox results.

Results

Of 483 eligible patients (acute ischemic stroke 66.4%; TIA 18.8%; ICH 7.7%; SAH 7.0%), 414 (85.7%) completed UTox screening. The mean (standard deviation) age was 65.1 (15.6) years, 52.7% were male, and 64.3% were Caucasian. Twenty-two (4.6%) patients had positive screening—cannabinoids were detected in 13 cases (3.1%), cocaine in 5 cases (1.2%), amphetamines in 1 case, and phencyclidine in 1 case. The highest yield (14.1%) was observed in patients < 60 years old with history of tobacco use while it was < 5% in the remaining subgroups (p<0.01).

Conclusions

Consistent with current guidelines, a selective approach to UTox screening should be pursued in acute stroke evaluation. The highest diagnostic yield is likely to be for cannabinoids and cocaine testing in younger patients with a history of concurrent tobacco use.     

Mitochondrial genome variations in advanced stage endometriosis: a study in South Indian population

Background

Endometriosis is a chronic gynecological benign disease that shares several features similar to malignancy. Mitochondrial DNA (mtDNA) mutations have been reported in all most all types of tumors. However, it is not known as to whether mtDNA mutations are associated with endometriosis.

Methodology

We sequenced the entire mitochondrial genome of analogous ectopic and eutopic endometrial tissues along with blood samples from 32 advanced stage endometriosis patients to analyze the role of somatic and germ-line mtDNA variations in pathogenesis of endometriosis. All ectopic tissues were screened for tumor-specific mtDNA deletions and microsatellite instability (MSI). We also performed mtDNA haplogrouping in 128 patients and 90 controls to identify its possible association with endometriosis risk.

Principal Findings

We identified 51 somatic (novel: 31; reported: 20) and 583 germ-line mtDNA variations (novel: 53; reported: 530) in endometriosis patients. The A13603G, a novel missense mutation which leads to a substitution from serine to glycine at the codon 423 of ND5 gene showed 100% incidence in ectopic tissues. Interestingly, eutopic endometrium and peripheral leukocytes of all the patients showed heteroplasmy (A/G; 40–80%) at this locus, while their ectopic endometrium showed homoplasmic mutant allele (G/G). Superimposition of native and mutant structures of ND5 generated by homology modeling revealed no structural differences. Tumor-specific deletions and MSI were not observed in any of the ectopic tissues. Haplogrouping analysis showed a significant association between haplogroup M5 and endometriosis risk (P: 0.00069) after bonferroni correction.

Conclusions

Our findings substantiate the rationale for exploring the mitochondrial genome as a biomarker for the diagnosis of endometriosis.