Impact of daily high dose oral vitamin D therapy on the inflammatory markers in patients with COVID 19 disease.

COVID 19 is known to cause immune dysregulation and vitamin D is a known immunomodulator. This study aims to objectively investigate the impact of Pulse D therapy in reducing the inflammatory markers of COVID-19. Consented COVID-19 patients with hypovitaminosis D were evaluated for inflammatory markers (N/L ratio, CRP, LDH, IL6, Ferritin) along with vitamin D on 0th day and 9th/11th day as per their respective BMI category. Subjects were randomised into VD and NVD groups. VD group received Pulse D therapy (targeted daily supplementation of 60,000 IUs of vitamin D for 8 or 10 days depending upon their BMI) in addition to the standard treatment. NVD group received standard treatment alone. Differences in the variables between the two groups were analysed for statistical significance. Eighty seven out of one hundred and thirty subjects have completed the study (VD:44, NVD:43). Vitamin D level has increased from 16 ± 6 ng/ml to 89 ± 32 ng/ml after Pulse D therapy in VD group and highly significant (p < 0.01) reduction of all the measured inflammatory markers was noted. Reduction of markers in NVD group was insignificant (p > 0.05). The difference in the reduction of markers between the groups (NVD vs VD) was highly significant (p < 0.01). Therapeutic improvement in vitamin D to 80-100 ng/ml has significantly reduced the inflammatory markers associated with COVID-19 without any side effects. Hence, adjunctive Pulse D therapy can be added safely to the existing treatment protocols of COVID-19 for improved outcomes.

P21-activated kinase 1 (Pak1) signaling influences therapeutic outcome in pancreatic cancer.

BACKGROUND: Therapeutic resistance to gemcitabine in pancreatic ductal adenocarcinoma (PDAC) is attributed to various cellular mechanisms and signaling molecules that influence as a single factor or in combination. DESIGN: In this study, utilizing in vitro p21-activated kinase 1 (Pak1) overexpression and knockdown cell line models along with in vivo athymic mouse tumor xenograft models and clinical samples, we demonstrate that Pak1 is a crucial signaling kinase in gemcitabine resistance. RESULTS: Pak1 kindles resistance via modulation of epithelial-mesenchymal transition and activation of pancreatic stellate cells. Our results from gemcitabine-resistant and -sensitive cell line models showed that elevated Pak1 kinase activity is required to confer gemcitabine resistance. This was substantiated by elevated levels of phosphorylated Pak1 and ribonucleotide reductase M1 levels in the majority of human PDAC tumors when compared with normal. Delineation of the signaling pathway revealed that Pak1 confers resistance to gemcitabine by preventing DNA damage, inhibiting apoptosis and regulating survival signals via NF-κB. Furthermore, we found that Pak1 is an upstream interacting substrate of transforming growth factor ß-activated kinase 1-a molecule implicated in gemcitabine resistance. Molecular mechanistic studies revealed that gemcitabine docks with the active site of Pak1; furthermore, gemcitabine treatment induces Pak1 kinase activity both in vivo and in cell-free system. Finally, results from athymic mouse tumor models illustrated that Pak1 inhibition by IPA-3 enhances the cytotoxicity of gemcitabine and brings about pancreatic tumor regression. CONCLUSION: To our knowledge, this is the first study illustrating the mechanistic role of Pak1 in causing gemcitabine resistance via multiple signaling crosstalks, and hence Pak1-specific inhibitors will prove to be a better adjuvant with existing chemotherapy modality for PDAC.

Pyridoxal 5' phosphate protects islets against streptozotocin-induced beta-cell dysfunction--in vitro and in vivo.

Administration of pyridoxal 5' phosphate (PLP) has demonstrated beneficial effects in the management of diabetes, albeit the mechanism(s) are not clearly understood. The present study addressed the islet-cell function(s) in streptozotocin (STZ)-induced diabetic mice both in vitro and in vivo. Primary islet cells primed with or without PLP (5 mmol/L) were treated with STZ (2 mmol/L) and were measured for cell viability, insulin secretion, free radicals and mRNA of Insulin and Pdx1. The specificity of PLP's response on insulin secretion was assessed with amino oxy acetic acid (AOAA)-PLP inhibitor. In vivo, the STZ (200 mg/kg b.w)-treated diabetic mice received 10 mmol/L PLP intraperitoneally a day before (PLP + STZ) or after (STZ + PLP) with three more doses once every 48 h. On 7, 14 and 21 d of STZ treatment, physiological parameters, islet morphology, insulin:glucagon, insulin:HSP104, and mRNA of Insulin, Glut2, Pdx1 and Reg1 were determined. In vitro, PLP protected islets against STZ-induced changes in viability, insulin secretion, prevented increase in free radical levels and normalized mRNA of Insulin and Pdx1. Further, AOAA inhibited PLP-induced insulin secretion in islets. In vivo, PLP treatment normalized STZ-induced changes in physiological parameters, circulating levels of PLP and insulin. Also, islet morphology, insulin:glucagon, insulin:HSP104 and mRNA levels of Insulin, Pdx1 and Glut2 were restored by 21 d. Although PLP treatment (pre- and post-STZ) prevented development of frank diabetes, STZ + PLP mice showed transient hyperglycemia, and increased mRNA for Reg1. The data suggest the cytoprotective vis-à-vis insulinotrophic effects of PLP against STZ-induced beta-cell dysfunction and underline its prophylactic use in the management of diabetes.

Antimicrobial properties of the stem bark of Saraca indica (Caesalpiniaceae).

Chloroform, methanol, aqueous and ethanolic extracts of the stem bark of Saraca indica were investigated for their antibacterial and antifungal activity against standard strains of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Bacillus cereus, Klebsiella pneumoniae, Proteus mirabilis, Salmonella typhimurium and Streptococcus pneumoniae and the fungi: Candida albicans and Cryptococcus albidus. Methanolic and aqueous extract exhibited antimicrobial activity with MIC ranging from 0.5-2% and 1-3% respectively. Methanolic extract exhibited the strongest activity against both bacteria and fungi.

Susceptibilities of Escherichia coli and Staphylococcus aureus to Aloe barbadensis.

The in vitro susceptibilities of Escherichia coli and Staphylococcus aureus were evaluated and the two organisms were susceptible to the inner gel of aloe barbadensis, though it was more effective against Staphylococcus aureus than Escherichia coli. The reduction for Aloe Vera (AV) needed to suppress the growth of the gram-positive bacterium was attributed to the structural differences between the two organisms.

Erratum: A neo-clerodane diterpene from Teucrium tomentosum. Corrigendum.

The chemical name of the title compound in the paper by Devi, Malathi, Rajan, Aravind, Krishnakumari & Ravikumar [Acta Cryst. (2004), E60, o117-o119] is corrected and the structural diagram is updated.[This corrects the article DOI: 10.1107/S1600536803028745.].

Immuolocalization of nestin in pancreatic tissue of mice at different ages.

AIM: To localize nestin positive cells (NPC) in pancreatic tissue of mice of different ages. METHODS: Paraffin sections of 6-8 mum of fixed pancreatic samples were mounted on poly-L-lysine coated slides and used for Immunolocalization using appropriate primary antibodies (Nestin, Insulin, Glucagon), followed by addition of a fluorescently labeled secondary antibody. The antigen-antibody localization was captured using a confocal microscope (Leica SP 5 series). RESULTS: In 3-6 d pups, the NPC were localized towards the periphery of the endocrine portion, as evident from immunolocalization of insulin and glucagon, while NPC were absent in the acinar portion. At 2 wk, NPC were localized in both the exocrine and endocrine portions. Interestingly, in 4-wk-old mice NPC were seen only in the endocrine portion, towards the periphery, and were colocalised with the glucagon positive cells. In the pancreas of 8- wk-old mice, the NPC were predominantly localized in the central region of the islet clusters, where immunostaining for insulin was at a maximum. CONCLUSION: We report for the first time the immunolocalization of NPC in the pancreas of mice of different ages (3 d to 8 wk) with reference to insulin and glucagon positive cells. The heterogeneous localization of the NPC observed may be of functional and developmental significance and suggest(s) that mice pancreatic tissue can be a potential source of progenitor cells. NPC from the pancreas can be isolated, proliferated and programmed to differentiate into insulin secreting cells under the appropriate microenvironment.

Group I introns and GNRA tetraloops: remnants of 'The RNA world'?

GNRA tetraloops, found in high frequency in natural RNAs, make loop-receptor interactions, stabilizing the tertiary structure of Group I introns, a class of small RNAs. Analyzing 230 Group I introns, to study the distribution and sequence pattern of the GNRA tetraloops, we suggest that these features reflect the ancestral nature of these catalytic molecules, in a prebiotic RNA world. The adenosine rich GNRA tetraloops would have interacted with each other through long range RNA-RNA interactions to form higher order structures forming potential sites that render the propensity for the short RNAs to bind to metal ions from the prebiotic pool, aiding them to act as metalloenzymes.

Probing RNA-antibiotic interactions: a FTIR study.

The crystal structure determination of antibiotic binding sites on the 30S ribosomal subunit and the increasing demand for developing RNA-based drugs has prompted us to study the direct binding of spectinomycin, vancomycin and bleomycin with yeast total RNA using Fourier transform infrared (FTIR) spectroscopy. We report that the OH of spectinomycin and the peptide group of vancomycin can bind to the bases of RNA, which might depend on Mg2+ concentration. Bleomycin on the other hand does not show such a drastic effect on yeast total RNA. This study might help in developing innovative strategies utilizing RNA molecules to perform a variety of essential biological functions.

Analysis of structure, function, and evolutionary origin of the ob gene product--leptin.

Leptin, the ob gene product, is a 167 amino acid polypeptide known to play a key role in regulating the fat stores of the body and is found in all eukaryotes, including mammals, aves, and also in invertebrates. To gain insight into the structure-function relation and origin of leptin, we have analyzed the amino acid sequence of leptin from 23 species by computing the frequency of occurrence of amino acids, their secondary structure, sequence homology, et cetera. Extensive conservation is observed within the leptin sequences of all the species, suggesting an evolutionary relatedness among them. It is interesting to note that human leptin shares a very high degree of homology with gorilla, chimpanzee, and orangutan indicative of a common function of leptin in them. Analysis of the codon bias in leptin from 11 species reveals that sminthopsis shows highest variation compared to human while less variation is observed in chimpanzee and orangutan, possibly reflecting the closeness in their evolution. Thus, understanding leptin's three-dimensional structure along with primary and secondary structure might enable us to understand the functional role played by this multifaceted adipocyte derived protein.

Possible inhibition of group I intron RNA by resveratrol and genistein.

The increasing incidence of pathogens and drug resistance has become major threat in the current arena. Hence, there is a need for the development of alternative therapeutic target to combat increase in resistance problem other than the cell membrane. Besides DNA, recently RNA has been recognized as a central target site for drug design. Group I intron RNA is a unique class of RNA molecule that undergoes self-catalytic activity due to its unique folded structure that catalyze number of cellular reactions. Recently, in vitro studies have shown that the folded structure of group I intron RNA could be a potential target site for therapeutic agents. Its presence in human pathogen like Candida albicans and absence in humans, suggests that the intron could act as an alternative therapeutic target. Therefore, our interest has been to explore the RNA binding activity of dietary compounds resveratrol and genistein. The binding efficacy of resveratrol and genistein (P/D ratio's - 11.76, 4.71, 2.35, 1.17, 0.58) to group I intron RNA transcript and circular-intervening sequences (C-IVS) of Tetrahymena thermophila and the binding efficacy of resveratrol and genistein (P/D ratio's - 2.35, 1.17, 0.58, 0.29) to 25S rRNA of C. albicans is measured by quantification of the RNA using densitometric method. This suggests that these natural compounds might bind with intron RNA and acts as an potential target and modulates the cellular process during therapeutic intervention.

The isomeric compounds nimbolide and isonimbolide.

Nimbolide [systematic name: (4alpha,5alpha,6alpha,7alpha,15beta,17alpha)-7,15:21,23-diepoxy-6-hydroxy-4,8-dimethyl-1-oxo-18,24-dinor-11,12-secochola-2,13,20,22-tetraene-4,11-dicarboxylic acid gamma-lactone methyl ester], C27H30O7, was isolated from the leaves of Azadirachta indica, and its isomer, isonimbolide [systematic name: (4alpha,5alpha,6alpha,7alpha,15alpha)-7,15:21,23-diepoxy-6-hydroxy-4,8-dimethyl-1-oxo-18,24-dinor-11,12-secochola-2,16,20,22-tetraene-4,11-dicarboxylic acid gamma-lactone methyl ester], was prepared from a novel rearrangement reaction of nimbolide, using boron trifluoride etherate and tetrabutylammonium bromide. The reaction conditions are probably responsible for the ether cleavage, double-bond rearrangement and reformation of the ether linkage. As a result, there are conformational changes in two cyclopentane rings and the side-chain -CH2COOMe group. In isonimbolide, an R(4)(4)(24) hydrogen-bond motif is observed.

Antifeedant neo-clerodanes from Teucrium tomentosum Heyne. (Labiatae).

From the acetone extract of Teucrium tomentosum, a new antifeedant neo-clerodane diterpenoid teuctosin (1) was isolated along with teuflin (2), teucrin-H(2) (3), 6beta-hydroxyteuscordin (4), 6beta-acetylteuscordin (5) and montanin-D (6). The structure of the new compound was elucidated comprehensively using 1D and 2D NMR methods and confirmed by X-ray crystallography. All the compounds showed effective antifeedancy against Plutella xylostella and Spodoptera litura at 10 mug/cm(2) of leaf area.

Samaderin B and C from Samadera indica.

Samaderin B, or (1R,2S,5R,5aR,7aS,11S,11aS,11bR,14S)-1,7,7a,11,11a,11b-hexahydro-1,11-dihydroxy-8,11a,14-trimethyl-2H-5a,2,5-(methanoxymetheno)naphth[1,2-d]oxepine-4,6,10(5H)-trione, C(19)H(22)O(7), and samaderin C, or (1R,2S,5R,5aR,7aS,10S,11S,11aS,11bR,14S)-7,7a,10,11,11a,11b-hexahydro-1,10,11-trihydroxy-8,11a,14-trimethyl-2H-5a,2,5-(methanoxymetheno)naphth[1,2-d]oxepine-4,6(1H,5H)-dione, C(19)H(24)O(7), were isolated from the seed kernels of Samadera indica and were shown to exhibit antifeedant activity against Spodoptera litura third-instar larvae. The replacement of the carbonyl group in samaderin B by a hydroxy group in samaderin C causes conformational changes at the substitution site, but the overall conformation is not affected; however, the compounds pack differently in the crystal lattice.

A new clerodane diterpene with antifeedant activity from Teucrium tomentosum.

A new diterpene, viz. 3beta-acetoxy-4alpha,18:15,16-diepoxy-6beta,12-dihydroxyneocleroda-13(16),14-dien-19,20-olide, C(22)H(28)O(8), exhibiting antifeedant activity against Spodoptera litura was isolated from the aerial parts of Teucrium tomentosum and its structure is reported. One of the two fused rings has a distorted-chair conformation and the other has a chair conformation. The molecules in the crystal are stabilized via O--H...O and C--H...O hydrogen bonds.

De-intercalation of ethidium bromide and acridine orange by xanthine derivatives and their modulatory effect on anticancer agents: a study of DNA-directed toxicity enlightened by time correlated single photon counting.

Time Correlated Single Photon Counting (TCSPC) was used for the first time to analyze the effect/changes in the mode of intercalation of ethidium bromide (EtBr) and acridine orange (AO) to calf thymus DNA brought about due to interaction of naturally occurring methylxanthines such as theophylline (X1), theobromine (X2) and caffeine (X3). UV absorption and fluorescence studies were also carried to observe the behaviour of these xanthines on the modulation of the binding mode of anticancer agents (cisplatin, novantrone, and actinomycin D) and certain intercalating dyes (EtBr and AO) to DNA. In TCSPC analysis we found that when the concentration of the drugs (X1, X2 and X3) increased from 0.025 mM to 2 mM i.e. P/D 2.4 to P/D 0.03 reduction in intercalation of EtBr and AO was observed, suggesting that xanthine derivatives could play very important role in reducing the DNA-directed toxicity in a dose dependent manner. In TCSPC, the amplitude of smaller lifetime component A(1) and higher lifetime component A(2) are attributed to free and intercalated dye concentration and their variation could indicate the process of intercalation or reduced intercalation of EtBr and AO by xanthine derivatives. We found that at the maximum drug concentration the smaller lifetime component A(1) was increased by 7-8% and 17-37% in EtBr and AO intercalated complex respectively. Also the changes in lifetime and fluorescence decay profile were observed for the DNA-intercalated dyes before and after treatment with xanthines. Especially, at maximum P/D 0.03 the lifetime of DNA-intercalated EtBr and AO reduced by 1-2 ns. The present analysis reveals that xanthines are able to interact with free dyes and also with intercalated dyes, suggesting that when they interact with free dyes they might inhibit the further intercalation of dye molecules to DNA and the interaction with intercalated dyes might lead to displacement of the dyes resulting in de-intercalation. The results obtained from UV and fluorescence spectroscopy also support the present investigation of probable interaction of xanthines with the DNA damaging agents in modulating/reducing the DNA-directed toxicity.

RNA binding efficacy of theophylline, theobromine and caffeine.

The binding of naturally occurring methylxanthines such as theophylline, theobromine and caffeine to nucleic acids are reckoned to be pivotal as they are able to modulate the cellular activities. We explore the interaction of yeast RNA binding efficacy of the above xanthine derivatives by using UV absorption differential spectroscopy and Fourier Transform Infrared (FTIR) spectroscopy. Both the analyses show discrimination in their binding affinity to RNA. The differential UV-spectrum at P/D 3.3 reveals the greater RNA binding activity for theophylline (85 +/- 5%), whereas moderate and comparatively less binding activity for theobromine (45 +/- 5%) and caffeine (30 +/- 5%) and the binding activity was found to depend on concentration of the drugs. In FTIR analysis we observed changes in the amino group (NH) of RNA complexed by drugs, where the NH band is found to become very broad, indicating hydrogen bonding (H-bonding) with theophylline (3343.4 cm(-1)), theobromine (3379.8 cm(-1)) and caffeine (3343 cm(-1)) as compared to the free RNA (3341.6 cm(-1)). Furthermore in RNA-theophylline complex, it is observed that the carbonyl (C=O) vibration frequency (nu(C=O)) of both drug (nu(C=O)=1718, 1666 cm(-1)) as well as RNA (nu(C=O)=1699, 1658 cm(-1)) disappeared and a new vibration band appeared around 1703 cm(-1), indicating that the C=O and NH groups of drug and RNA are effectively involved in H-bonding. Whereas in RNA-theobromine and RNA-caffeine complexes, we found very little changes in C=O frequency and only broadening of the NH band of RNA due to complexation is observed in these groups. The changes in the vibrations of G-C/A-U bands and other bending frequencies are discussed. Thus the discrimination in the binding affinity of methylxanthines with RNA molecule shows that strong RNA binding drugs like theophylline can selectively be delivered to RNA targets of microbial pathogens having the mechanism of RNA catalysis.