Persistence of Smoking-Induced Dysregulation of MiRNA Expression in the Small Airway Epithelium Despite Smoking Cessation

Even after quitting smoking, the risk of the development of chronic obstructive pulmonary disease (COPD) and lung cancer remains significantly higher compared to healthy nonsmokers. Based on the knowledge that COPD and most lung cancers start in the small airway epithelium (SAE), we hypothesized that smoking modulates miRNA expression in the SAE linked to the pathogenesis of smoking-induced airway disease, and that some of these changes persist after smoking cessation. SAE was collected from 10^th to 12^th order bronchi using fiberoptic bronchoscopy. Affymetrix miRNA 2.0 arrays were used to assess miRNA expression in the SAE from 9 healthy nonsmokers and 10 healthy smokers, before and after they quit smoking for 3 months. Smoking status was determined by urine nicotine and cotinine measurement. There were significant differences in the expression of 34 miRNAs between healthy smokers and healthy nonsmokers (p<0.01, fold-change >1.5), with functions associated with lung development, airway epithelium differentiation, inflammation and cancer. After quitting smoking for 3 months, 12 out of the 34 miRNAs did not return to normal levels, with Wnt/β-catenin signaling pathway being the top identified enriched pathway of the target genes of the persistent dysregulated miRNAs. In the context that many of these persistent smoking-dependent miRNAs are associated with differentiation, inflammatory diseases or lung cancer, it is likely that persistent smoking-related changes in SAE miRNAs play a role in the subsequent development of these disorders.     

Microbial 1-butanol production: Identification of non-native production routes and in silico engineering interventions

The potential of engineering microorganisms with non-native pathways for the synthesis of long-chain alcohols has been identified as a promising route to biofuels. We describe computationally derived predictions for assembling pathways for the production of biofuel candidate molecules and subsequent metabolic engineering modifications that optimize product yield. A graph-based algorithm illustrates that, by culling information from BRENDA and KEGG databases, all possible pathways that link the target product with metabolites present in the production host are identified. Subsequently, we apply our recent OptForce procedure to pinpoint reaction modifications that force the imposed product yield in Escherichia coli. We demonstrate this procedure by suggesting new pathways and genetic interventions for the overproduction of 1-butanol using the metabolic model for Escherichia coli. The graph-based search method recapitulates all recent discoveries based on the 2-ketovaline intermediate and hydroxybutyryl-CoA but also pinpointes one novel pathway through thiobutanoate intermediate that to the best of our knowledge has not been explored before.     

ACE2 X-ray structures reveal a large hinge-bending motion important for inhibitor binding and catalysis

The angiotensin-converting enzyme (ACE)-related carboxypeptidase, ACE2, is a type I integral membrane protein of 805 amino acids that contains one HEXXH + E zinc-binding consensus sequence. ACE2 has been implicated in the regulation of heart function and also as a functional receptor for the coronavirus that causes the severe acute respiratory syndrome (SARS). To gain further insights into this enzyme, the first crystal structures of the native and inhibitor-bound forms of the ACE2 extracellular domains were solved to 2.2- and 3.0-A resolution, respectively. Comparison of these structures revealed a large inhibitor-dependent hinge-bending movement of one catalytic subdomain relative to the other ( approximately 16 degrees ) that brings important residues into position for catalysis. The potent inhibitor MLN-4760 ((S,S)-2-[1-carboxy-2-[3-(3,5-dichlorobenzyl)-3H-imidazol4-yl]-ethylamino]-4-methylpentanoic acid) makes key binding interactions within the active site and offers insights regarding the action of residues involved in catalysis and substrate specificity. A few active site residue substitutions in ACE2 relative to ACE appear to eliminate the S(2)' substrate-binding subsite and account for the observed reactivity change from the peptidyl dipeptidase activity of ACE to the carboxypeptidase activity of ACE2.     

Comparative effectiveness of different insecticides for organic management of blueberry maggot (Diptera: Tephritidae)

Laboratory and field assays using insecticides for organic pest management were conducted on the blueberry maggot, Rhagoletis mendax Curran. Topical exposure of flies to spinosad (Entrust), pyrethrum (PyGanic 1.4 EC), azadirachtin (Aza-Direct), and phosmet (Imidan 70-W) resulted in significantly higher mortality compared with the water control after 2 and 24 h. After 24 h, there were no significant differences in fly mortality among treatments of Entrust, PyGanic, or Imidan, whereas fly mortality to Aza-Direct was significantly lower. Another laboratory assay evaluated mortality of flies after residual exposure to these insecticides on leaves, after 24 and 48 h. In this assay, there were no significant differences in fly mortality after 48 h among treatments of PyGanic, Aza-Direct, and the water control, whereas significantly higher fly mortality resulted from exposure to Entrust and Imidan. A repellency assay found no measurable effects of Aza-Direct. Large-scale field trials found no treatment effect for number of adults of the blueberry maggot captured in sticky traps; however, there were significantly lower levels of fruit-infesting larvae in treated plots compared with the untreated control. Spinosad bait (GF-120 NF Naturalyte Fruit Fly Bait), Entrust, and PyGanic were not different from imidacloprid (Provado 1.6 F). However, there was a significantly higher infestation in the plot treated with azadirachtin (Agroneem) compared with Provado. Overall, the insecticides evaluated in these trials showed good ability to control blueberry maggot, suggesting that they can be incorporated in a blueberry maggot management program under organic standards.     

Infection of the intermediate mite host with Wolbachia-depleted Litomosoides sigmodontis microfilariae: impaired L1 to L3 development and subsequent sex-ratio distortion in adult worms

The rodent filaria Litomosoides sigmodontis harbour Wolbachia, endosymbionts essential for worm embryogenesis, larval development and adult survival. To study the effect of tetracycline, which depletes Wolbachia, on the development of microfilariae (L1s, MF) to L3 in the intermediate host Ornithonyssus bacoti, and to observe the development of Wolbachia-depleted L3s in Mongolian gerbils (Meriones unguiculatus); microfilaremic gerbils were treated orally with tetracycline for 6 weeks (primary infected (1°) Tet) or untreated (1° Con). Treatment resulted in a significant reduction of Wolbachia per MF in 1° Tet gerbils. Naïve mites then fed on the 1° Tet and 1° Con gerbils in the week after treatment ended, when MF levels were not significantly different, and used to infect new gerbils (secondary infected (2°) Tet, 2° Con) via natural infection. The infection rate from dissected mites was 9% and 54% (1° Tet and 1° Con, respectively). After 3 months, worms were isolated from 2° gerbils. Significantly fewer female worms developed in 2° Tet gerbils. In contrast, there was no difference in the number of male worms that developed in 2° gerbils, resulting in a male biased sex-ratio. Although 2° Tet male worms had fewer Wolbachia than 2° Con males, development was not impaired. Female worms that developed from Wolbachia-depleted MF had Wolbachia levels equivalent to worms from 2° Con animals. Thus, tetracycline pre-treatment selected for female worms with high numbers of Wolbachia, whereas male worms had median Wolbachia levels significantly lower than 2° Con males. Therefore, female worms require a higher threshold of Wolbachia for their development. The worms analysed were only exposed to tetracycline as MF, ruling out direct effects of tetracycline during larval development in the mites or 2° gerbils, suggesting that the depletion of Wolbachia in MF was the cause of impaired larval development.     

The Apo-structure of the Low Molecular Weight Protein-tyrosine Phosphatase A (MptpA) from Mycobacterium tuberculosis Allows for Better Target-specific Drug Development

Protein-tyrosine phosphatases (PTPs) and protein-tyrosine kinases co-regulate cellular processes. In pathogenic bacteria, they are frequently exploited to act as key virulence factors for human diseases. Mycobacterium tuberculosis, the causative organism of tuberculosis, secretes a low molecular weight PTP (LMW-PTP), MptpA, which is required for its survival upon infection of host macrophages. Although there is otherwise no sequence similarity of LMW-PTPs to other classes of PTPs, the phosphate binding loop (P-loop) CX₅R and the loop containing a critical aspartic acid residue (D-loop), required for the catalytic activity, are well conserved. In most high molecular weight PTPs, ligand binding to the P-loop triggers a large conformational reorientation of the D-loop, in which it moves ∼10 Å, from an “open” to a “closed” conformation. Until now, there have been no ligand-free structures of LMW-PTPs described, and hence the dynamics of the D-loop have remained largely unknown for these PTPs. Here, we present a high resolution solution NMR structure of the free form of the MptpA LMW-PTP. In the absence of ligand and phosphate ions, the D-loop adopts an open conformation. Furthermore, we characterized the binding site of phosphate, a competitive inhibitor of LMW-PTPs, on MptpA and elucidated the involvement of both the P- and D-loop in phosphate binding. Notably, in LMW-PTPs, the phosphorylation status of two well conserved tyrosine residues, typically located in the D-loop, regulates the enzyme activity. PtkA, the kinase complementary to MptpA, phosphorylates these two tyrosine residues in MptpA. We characterized the MptpA-PtkA interaction by NMR spectroscopy to show that both the P- and D-loop form part of the binding interface.     

Cloning, expression, and homology modeling of GroEL protein from Leptospira interrogans serovar autumnalis strain N2

Leptospirosis is an infectious bacterial disease caused by Leptospira species. In this study, we cloned and sequenced the gene encoding the immunodominant protein GroEL from L. interrogans serovar Autumnalis strain N2, which was isolated from the urine of a patient during an outbreak of leptospirosis in Chennai, India. This groEL gene encodes a protein of 60 kDa with a high degree of homology (99% similarity) to those of other leptospiral serovars. Recombinant GroEL was overexpressed in Escherichia coli. Immunoblot analysis indicated that the sera from confirmed leptospirosis patients showed strong reactivity with the recombinant GroEL while no reactivity was observed with the sera from seronegative control patient. In addition, the 3D structure of GroEL was constructed using chaperonin complex cpn60 from Thermus thermophilus as template and validated. The results indicated a Z-score of ?8.35, which is in good agreement with the expected value for a protein. The superposition of the Cα traces of cpn60 structure and predicted structure of leptospiral GroEL indicates good agreement of secondary structure elements with an RMSD value of 1.5 Å. Further study is necessary to evaluate GroEL for serological diagnosis of leptospirosis and for its potential as a vaccine component.     

Patterns of Sole‐Carbon‐Source Utilization by Fast‐Growing Coastal Sand Dune Rhizobia of the Southwest Coast of India

Coastal sand dunes harbor a variety of free living and symbiotic microorganisms, which are adapted to stress conditions such as temperature, salinity and pH. The current paper explores the ability of sole‐source‐carbon utilization by symbiotic rhizobia isolated from tropical coastal sand dune wild legumes. Fast‐growing rhizobia isolated from five legume plant species (Canavalia cathartica, Canavalia maritima, Crotalaria retusa, Crotalaria verrucosa, and Derris triflorum) grown on the two coastal sand dunes of the southwest coast of India (Someshwara, S1–S5; Padubidri, P1–P5) were assessed for sole‐carbon‐source utilization patterns based on BIOLOG GN2 microplate technique. All the isolates showed high extents of utilization of the different carbon sources within 24 h of incubation at 30 °C. Cluster analysis based on quantitative and qualitative utilization of a sole carbon source revealed two distinct clusters. Cluster I consists of four isolates (S1, P2, P4, and P5), of which P2 and P5 showed greater similarities. The cluster II encompasses six isolates (S2–S5, P1 and P3), among them S3 and S5 showed high similarities. Based on the utilization of six guilds by the isolates S1, S2, P1, and P2 (polymers, carbohydrates, carboxylic acids, amides and amines, amino acids and miscellaneous), S2 and P1 exhibited high functional diversity. Principal component analysis revealed a close catabolic potential between the isolates S5 and P1; S1 and P3; S3, P2, and P5. The symbiotic rhizobia of the southwest coastal sand dune wild legumes of India studied might serve as novel inoculants to achieve legume production through nitrogen fixation under the varied conditions of tropical soils. These rhizobia were able to utilize a wide range of carbon sources, possessed functional diversity, withstood extreme conditions (temperature, salinity, pH), exhibited non‐host specificity and induced high biomass in edible legumes.     

Diterpenoids from Jatropha multifida

Chemical investigation on the stems of Jatropha multifida yielded two diterpenoids, multifolone and (4E)-jatrogrossidentadione acetate along with five known diterpenoids, a flavone and a coumarino-lignan. The structures of the compounds were settled by detailed analysis of their 1D and 2D NMR spectra. The X-ray crystallographic analysis of (4E)-jatrogrossidentadione acetate was also accomplished.     

Cell-permeant NAADP: a novel chemical tool enabling the study of Ca2+ signalling in intact cells

NAADP (nicotinic acid adenine dinucleotide phosphate) is a recently discovered second messenger, and as such, we have much yet to learn about its functions in health and disease. A bottleneck in this basic research is due to NAADP, like all second messengers, being charged to prevent it from leaking out of cells. This makes for effective biology, but imposes difficulties in experiments, as it must be injected, loaded via liposomes, or electroporated, techniques that are highly technically demanding and are possible only in certain single cell preparations. For the better understood second messenger inositol 1,4,5-trisphosphate, great success has been obtained with cell-permeant derivatives where the charged groups are masked through esterification. We now report NAADP-AM as a cell-permeant analogue of NAADP that is taken up into cells and induces NAADP-mediated Ca(2+) signalling. NAADP-AM is a powerful chemical tool that will be of enormous biological utility in a wide range of systems and will greatly facilitate research into the role of NAADP in health and disease.