Acetone production in solventogenic <Emphasis Type="Italic">Clostridium</Emphasis> species: new insights from non-enzymatic decarboxylation of acetoacetate

Development of a butanologenic strain with high selectivity for butanol production is often proposed as a possible route for improving the economics of biobutanol production by solventogenic Clostridium species. The acetoacetate decarboxylase (aadc) gene encoding acetoacetate decarboxylase (AADC), which catalyzes the decarboxylation of acetoacetate into acetone and CO₂, was successfully disrupted by homologous recombination in solventogenic Clostridium beijerinckii NCIMB 8052 to generate an aadc ⁻ mutant. Our fermentation studies revealed that this mutant produces a maximum acetone concentration of 3 g/L (in P2 medium), a value comparable to that produced by wild-type C. beijerinckii 8052. Therefore, we postulated that AADC-catalyzed decarboxylation of acetoacetate is not the sole means for acetone generation. Our subsequent finding that non-enzymatic decarboxylation of acetoacetate in vitro, under conditions similar to in vivo acetone–butanol–ethanol (ABE) fermentation, produces 1.3 to 5.2 g/L acetone between pH 6.5 and 4 helps rationalize why various knock-out and knock-down strategies designed to disrupt aadc in solventogenic Clostridium species did not eliminate acetone production during ABE fermentation. Based on these results, we discuss alternatives to enhance selectivity for butanol production.     

Effect of Dehydrozingerone, a half analog of curcumin on dexamethasone-delayed wound healing in albino rats

Oxidative stress is triggered by the wound which results in the production of reactive oxygen species (ROS), thereby delaying normal wound repair. Therefore, it is important to reduce the level of ROS to improve healing. A known antioxidant, dehydrozingerone (DHZ) was synthesized and selected for the study. The authors aimed to investigate the wound healing action of topical (100 mg/wound) and systemic (100 mg/kg, p. o.). DHZ on different wound models in normal and dexamethasone (DEX)-suppressed healing. Topical DHZ showed a significant (P < 0.05) rise in tensile strength when compared to control in normal healing. Significant (P < 0.05) wound closure was observed from 3 to 9 days in DHZ oral and gel treated groups. There was a significant (P < 0.05) rise in hydroxyproline content with the DHZ treated groups when compared to control. Systemic DHZ exhibited a significant (P < 0.05) increase in lysyl oxidase (LO) levels of 3.73 ± 0.15 nmol of H(2)O(2) when compared to control. In DEX-suppressed healing, showed good pro-healing activity with respect to the parameters mentioned above. DHZ treatment exhibited a parabolic dose response of ROS inhibition with a plateau effect at 75 μM. There was a steady and constant increase in the % NO inhibition with increasing doses of DHZ. Oral DHZ is effective in accelerating the healing process in both normal and dexamethasone-suppressed wounds. Our study suggests that DHZ (half analog of curcumin) supplementation reduces the steroid-induced delay in wound healing.     

Chronic olanzapine treatment decreases arachidonic acid turnover and prostaglandin E₂ concentration in rat brain

The atypical antipsychotic, olanzapine (OLZ), is used to treat bipolar disorder, but its therapeutic mechanism of action is not clear. Arachidonic acid (AA, 20:4n-6) plays a critical role in brain signaling and an up-regulated AA metabolic cascade was reported in postmortem brains from bipolar disorder patients. In this study, we tested whether, similar to the action of the mood stabilizers lithium, carbamazepine and valproate, chronic OLZ treatment would reduce AA turnover in rat brain. We administered OLZ (6 mg/kg/day) or vehicle i.p. to male rats once daily for 21 days. A washout group received 21 days of OLZ followed by vehicle on day 22. Two hours after the last injection, [1-1?C]AA was infused intravenously for 5 min, and timed arterial blood samples were taken. After the rat was killed at 5 min, its brain was microwaved, removed and analyzed. Chronic OLZ decreased plasma unesterified AA concentration, AA incorporation rates and AA turnover in brain phospholipids. These effects were absent after washout. Consistent with reduced AA turnover, OLZ decreased brain cyclooxygenase activity and the brain concentration of the proinflammatory AA-derived metabolite, prostaglandin E?, In view of up-regulated brain AA metabolic markers in bipolar disorder, the abilities of OLZ and the mood stabilizers to commonly decrease prostaglandin E?, and AA turnover in rat brain phospholipids, albeit by different mechanisms, may be related to their efficacy against the disease.     

The L7Ae protein binds to two kink-turns in the Pyrococcus furiosus RNase P RNA

The RNA-binding protein L7Ae, known for its role in translation (as part of ribosomes) and RNA modification (as part of sn/oRNPs), has also been identified as a subunit of archaeal RNase P, a ribonucleoprotein complex that employs an RNA catalyst for the Mg²⁺-dependent 5′ maturation of tRNAs. To better understand the assembly and catalysis of archaeal RNase P, we used a site-specific hydroxyl radical-mediated footprinting strategy to pinpoint the binding sites of Pyrococcus furiosus (Pfu) L7Ae on its cognate RNase P RNA (RPR). L7Ae derivatives with single-Cys substitutions at residues in the predicted RNA-binding interface (K42C/C71V, R46C/C71V, V95C/C71V) were modified with an iron complex of EDTA-2-aminoethyl 2-pyridyl disulfide. Upon addition of hydrogen peroxide and ascorbate, these L7Ae-tethered nucleases were expected to cleave the RPR at nucleotides proximal to the EDTA-Fe–modified residues. Indeed, footprinting experiments with an enzyme assembled with the Pfu RPR and five protein cofactors (POP5, RPP21, RPP29, RPP30 and L7Ae–EDTA-Fe) revealed specific RNA cleavages, localizing the binding sites of L7Ae to the RPR''s catalytic and specificity domains. These results support the presence of two kink-turns, the structural motifs recognized by L7Ae, in distinct functional domains of the RPR and suggest testable mechanisms by which L7Ae contributes to RNase P catalysis.     

Protective Effect of Boerhaavia diffusa L. against Mitochondrial Dysfunction in Angiotensin II Induced Hypertrophy in H9c2 Cardiomyoblast Cells

Mitochondrial dysfunction plays a critical role in the development of cardiac hypertrophy and heart failure. So mitochondria are emerging as one of the important druggable targets in the management of cardiac hypertrophy and other associated complications. In the present study, effects of ethanolic extract of Boerhaavia diffusa (BDE), a green leafy vegetable against mitochondrial dysfunction in angiotensin II (Ang II) induced hypertrophy in H9c2 cardiomyoblasts was evaluated. H9c2 cells challenged with Ang II exhibited pathological hypertrophic responses and mitochondrial dysfunction which was evident from increment in cell volume (49.09±1.13%), protein content (55.17±1.19%), LDH leakage (58.74±1.87%), increased intracellular ROS production (26.25±0.91%), mitochondrial superoxide generation (65.06±2.27%), alteration in mitochondrial transmembrane potential (ΔΨm), opening of mitochondrial permeability transition pore (mPTP) and mitochondrial swelling. In addition, activities of mitochondrial respiratory chain complexes (I-IV), aconitase, NADPH oxidase, thioredoxin reductase, oxygen consumption rate and calcium homeostasis were evaluated. Treatment with BDE significantly prevented the generation of intracellular ROS and mitochondrial superoxide radicals and protected the mitochondria by preventing dissipation of ΔΨm, opening of mPTP, mitochondrial swelling and enhanced the activities of respiratory chain complexes and oxygen consumption rate in H9c2 cells. Activities of aconitase and thioredoxin reductase which was lowered (33.77±0.68% & 45.81±0.71% respectively) due to hypertrophy, were increased in BDE treated cells (P≤0.05). Moreover, BDE also reduced the intracellular calcium overload in Ang II treated cells. Overall results revealed the protective effects of B. diffusa against mitochondrial dysfunction in hypertrophy in H9c2 cells and the present findings may shed new light on the therapeutic potential of B. diffusa in addition to its nutraceutical potentials.     

Antidiabetic Property of Symplocos cochinchinensis Is Mediated by Inhibition of Alpha Glucosidase and Enhanced Insulin Sensitivity

The study is designed to find out the biochemical basis of antidiabetic property of Symplocos cochinchinensis (SC), the main ingredient of ‘Nisakathakadi’ an Ayurvedic decoction for diabetes. Since diabetes is a multifactorial disease, ethanolic extract of the bark (SCE) and its fractions (hexane, dichloromethane, ethyl acetate and 90% ethanol) were evaluated by in vitro methods against multiple targets relevant to diabetes such as the alpha glucosidase inhibition, glucose uptake, adipogenic potential, oxidative stress, pancreatic beta cell proliferation, inhibition of protein glycation, protein tyrosine phosphatase-1B (PTP-1B) and dipeptidyl peptidase-IV (DPP-IV). Among the extracts, SCE exhibited comparatively better activity like alpha glucosidase inhibition (IC₅₀ value-82.07±2.10 µg/mL), insulin dependent glucose uptake (3 fold increase) in L6 myotubes, pancreatic beta cell regeneration in RIN-m5F (3.5 fold increase) and reduced triglyceride accumulation (22% decrease) in 3T3L1 cells, protection from hyperglycemia induced generation of reactive oxygen species in HepG2 cells (59.57% decrease) with moderate antiglycation and PTP-1B inhibition. Chemical characterization by HPLC revealed the superiority of SCE over other extracts due to presence and quantity of bioactives (beta-sitosterol, phloretin 2′glucoside, oleanolic acid) in addition to minerals like magnesium, calcium, potassium, sodium, zinc and manganese. So SCE has been subjected to oral sucrose tolerance test to evaluate its antihyperglycemic property in mild diabetic and diabetic animal models. SCE showed significant antihyperglycemic activity in in vivo diabetic models. We conclude that SC mediates the antidiabetic activity mainly via alpha glucosidase inhibition, improved insulin sensitivity, with moderate antiglycation and antioxidant activity.     

Neuropathological Responses to Chronic NMDA in Rats Are Worsened by Dietary n-3 PUFA Deprivation but Are Not Ameliorated by Fish Oil Supplementation

Background

Dietary long-chain n-3 polyunsaturated fatty acid (PUFA) supplementation may be beneficial for chronic brain illnesses, but the issue is not agreed on. We examined effects of dietary n-3 PUFA deprivation or supplementation, compared with an n-3 PUFA adequate diet (containing alpha-linolenic acid [18:3 n-3] but not docosahexaenoic acid [DHA, 22:6n-3]), on brain markers of lipid metabolism and excitotoxicity, in rats treated chronically with NMDA or saline.

Methods

Male rats after weaning were maintained on one of three diets for 15 weeks. After 12 weeks, each diet group was injected i.p. daily with saline (1 ml/kg) or a subconvulsive dose of NMDA (25 mg/kg) for 3 additional weeks. Then, brain fatty acid concentrations and various markers of excitotoxicity and fatty acid metabolism were measured.

Results

Compared to the diet-adequate group, brain DHA concentration was reduced, while n-6 docosapentaenoic acid (DPA, 22:5n-6) concentration was increased in the n-3 deficient group; arachidonic acid (AA, 20:4n-6) concentration was unchanged. These concentrations were unaffected by fish oil supplementation. Chronic NMDA increased brain cPLA₂ activity in each of the three groups, but n-3 PUFA deprivation or fish oil did not change cPLA₂ activity or protein compared with the adequate group. sPLA₂ expression was unchanged in the three conditions, whereas iPLA₂ expression was reduced by deprivation but not changed by supplementation. BDNF protein was reduced by NMDA in N-3 PUFA deficient rats, but protein levels of IL-1β, NGF, and GFAP did not differ between groups.

Conclusions

N-3 PUFA deprivation significantly worsened several pathological NMDA-induced changes produced in diet adequate rats, whereas n-3 PUFA supplementation did not affect NMDA induced changes. Supplementation may not be critical for this measured neuropathology once the diet has an adequate n-3 PUFA content.     

Evidence of Experimental Vertical Transmission of Emerging Novel ECSA Genotype of Chikungunya Virus in Aedes aegypti

Background

Chikungunya virus (CHIKV) has emerged as one of the most important arboviruses of public health significance in the past decade. The virus is mainly maintained through human-mosquito-human cycle. Other routes of transmission and the mechanism of maintenance of the virus in nature are not clearly known. Vertical transmission may be a mechanism of sustaining the virus during inter-epidemic periods. Laboratory experiments were conducted to determine whether Aedes aegypti, a principal vector, is capable of vertically transmitting CHIKV or not.

Methodology/Principal Findings

Female Ae. aegypti were orally infected with a novel ECSA genotype of CHIKV in the 2^nd gonotrophic cycle. On day 10 post infection, a non-infectious blood meal was provided to obtain another cycle of eggs. Larvae and adults developed from the eggs obtained following both infectious and non-infectious blood meal were tested for the presence of CHIKV specific RNA through real time RT-PCR. The results revealed that the larvae and adults developed from eggs derived from the infectious blood meal (2^nd gonotrophic cycle) were negative for CHIKV RNA. However, the larvae and adults developed after subsequent non-infectious blood meal (3^rd gonotrophic cycle) were positive with minimum filial infection rates of 28.2 (1∶35.5) and 20.2 (1∶49.5) respectively.

Conclusion/Significance

This study is the first to confirm experimental vertical transmission of emerging novel ECSA genotype of CHIKV in Ae. aegypti from India, indicating the possibilities of occurrence of this phenomenon in nature. This evidence may have important consequence for survival of CHIKV during adverse climatic conditions and inter-epidemic periods.     

The Interplay of Angle Strain and Aromaticity: Molecular and Electronic Structures of [0n]Paracyclophanes

The belt-like polyphenylenes, [0_n]paracyclophanes, (n = 5 and 6), have been investigated using semi-empirical, ab initio and DFT methods. The molecular structure, rotational barrier on twisting a single phenyl ring and the aromatic character within each ring as well as in the whole molecule have been evaluated. [0₅]Paracyclophane is predicted to have a quinonoid structure. In contrast, the equatorial pentaphenyl fragment found in C₇₀ as well as the hexagons of the less strained [0₆]paracyclophane have benzenoid character. Approximate band structures have been derived for larger cycles of [0_n] paracyclophanes.Electronic Supplementary Material available.     

Elucidation of structure–function relationships in Methanocaldococcus jannaschii RNase P,a multi-subunit catalytic ribonucleoprotein

RNase P is a ribonucleoprotein (RNP) that catalyzes removal of the 5′ leader from precursor tRNAs in all domains of life. A recent cryo-EM study of Methanocaldococcus jannaschii (Mja) RNase P produced a model at 4.6-Å resolution in a dimeric configuration, with each holoenzyme monomer containing one RNase P RNA (RPR) and one copy each of five RNase P proteins (RPPs; POP5, RPP30, RPP21, RPP29, L7Ae). Here, we used native mass spectrometry (MS), mass photometry (MP), and biochemical experiments that (i) validate the oligomeric state of the Mja RNase P holoenzyme in vitro, (ii) find a different stoichiometry for each holoenzyme monomer with up to two copies of L7Ae, and (iii) assess whether both L7Ae copies are necessary for optimal cleavage activity. By mutating all kink-turns in the RPR, we made the discovery that abolishing the canonical L7Ae–RPR interactions was not detrimental for RNase P assembly and function due to the redundancy provided by protein–protein interactions between L7Ae and other RPPs. Our results provide new insights into the architecture and evolution of RNase P, and highlight the utility of native MS and MP in integrated structural biology approaches that seek to augment the information obtained from low/medium-resolution cryo-EM models.