Characterization and regulation of salt upregulated cyclophilin from a halotolerant strain of Penicillium oxalicum.

Penicillium species are an industrially important group of fungi. Cyclophilins are ubiquitous proteins and several members of this family exhibit peptidyl-prolyl cis-trans isomerase (PPIase) activity. We had earlier demonstrated that the salt-induced PPIase activity in a halotolerant strain of P. oxalicum was associated with enhanced expression of a cyclophilin gene, PoxCYP18. Cloning and characterization of PoxCYP18 revealed that its cDNA consists of 522 bp encoding a protein of 173 amino acid residues, with predicted molecular mass and pI values of 18.91 kDa and 8.87, respectively. The recombinant PoxCYP18 can catalyze cis-trans isomerization of peptidyl-prolyl bond with a catalytic efficiency of 1.46 × 107 M-1 s-1 and is inhibited specifically only by cyclosporin A, with an inhibition constant of 5.04 ± 1.13 nM. PoxCYP18 consists of two cysteine residues at positions - 45 and - 170, and loses its activity under oxidizing conditions. Substitution of these residues alone or together by site-directed mutagenesis revealed that the PPIase activity of PoxCYP18 is regulated through a redox mechanism involving the formation of disulfide linkages. Heterologous expression of PoxCYP18 conferred enhanced tolerance to salt stress in transgenic E. coli cells, implying that this protein imparts protection to cellular processes against salt-induced damage.

Temozolomide-fatty acid conjugates for glioblastoma multiforme: In vitro and in vivo evaluation.

Glioblastoma multiforme (GBM) is the deadliest brain tumor with a poor prognosis and limited therapeutic options. Temozolomide (TMZ) is the first-line chemotherapeutic agent used for the treatment of GBM; however, it suffers from several limitations, including short half-life, rapid metabolism, <1% brain bioavailability, methyl guanine methyl transferase (MGMT) based chemoresistance, and hematological toxicities. Several approaches have been adopted to overcome these limitations, particularly by using nanotechnology-based systems, but its physicochemical properties make TMZ challenging to load into these nanocarriers. In the current research, we conjugated TMZ with different fatty acids, i.e., linoleic acid (LA), oleic acid (OA), and palmitic acid (PA), to obtain TMZ-fatty acid conjugates, which are comparatively hydrophobic, less prone to degradation and potent. These conjugates were thoroughly characterized using 1H NMR spectroscopy, high-resolution mass spectrometry (HR-MS), and reverse phase-high performance liquid chromatography (RP-HPLC). The synthesized conjugates, namely Temozolomide-oleic acid (TOA,6R1), Temozolomide-linoleic acid (TLA, 6R2), and Temozolomide-palmitic acid (TPA, 6R3), showed an IC50 of 101.4, 67.97, and 672.04 µM, respectively in C6 cells and 428.257, 366.43 and 413.69 µM, respectively in U87-MG cells. On the other hand, the free TMZ showed an IC50 of >1000 µM and 564.23 µM in C6 and U87-MG, respectively. Further, the in vivo efficacy of the TMZ-fatty acid conjugates was evaluated in the C6-induced orthotropic rat glioblastoma model, wherein the TMZ-fatty acid conjugate showed improved survival rate (1.6 folds) and overall health of the animals. Collectively, the conjugation of fatty acids with TMZ improves its anticancer potential against glioblastoma multiforme (GBM).

Polymeric and small molecule-conjugates of temozolomide as improved therapeutic agents for glioblastoma multiforme.

Temozolomide (TMZ), an imidazotetrazine, is a second-generation DNA alkylating agent used as a first-line treatment of glioblastoma multiforme (GBM). It was approved by FDA in 2005 and declared a blockbuster drug in 2008. Although TMZ has shown 100% oral bioavailability and crosses the blood-brain barrier effectively, however it suffers from limitations such as a short half-life (∼1.8 h), rapid metabolism, and lesser accumulation in the brain (∼10-20%). Additionally, development of chemoresistance has been associated with its use. Since it is a potential chemotherapeutic agent with an unmet medical need, advanced delivery strategies have been explored to overcome the associated limitations of TMZ. Nanocarriers including liposomes, solid lipid nanoparticles (SLNs), nanostructure lipid carriers (NLCs), and polymeric nanoparticles have demonstrated their ability to improve its circulation time, stability, tissue-specific accumulation, sustained release, and cellular uptake. Because of the appreciable water solubility of TMZ (∼5 mg/mL), the physical loading of TMZ in these nanocarriers is always challenging. Alternatively, the conjugation approach, wherein TMZ has been conjugated to polymers or small molecules, has been explored with improved outcomes in vitro and in vivo. This review emphasized the practical evidence of the conjugation strategy to improve the therapeutic potential of TMZ in the treatment of glioblastoma multiforme.

A real world multi center study on efficacy and safety of natalizumab in Indian patients with multiple sclerosis.

BACKGROUND: Natalizumab (NTZ) is increasingly being used in Indian multiple sclerosis (MS) patients. There are no reports on its safety and efficacy, especially with respect to the occurrence of progressive multifocal leukoencephalopathy (PML). OBJECTIVES: To describe the patient characteristics, treatment outcomes, and adverse events, especially the occurrence of PML in NTZ-treated patients. METHODS: A multicentre ambispective study was conducted across 18 centres, from Jan 2012 to Dec 2021. Patients at and above the age of 18 years treated with NTZ were included. Descriptive and comparative statistics were applied to analyze data. RESULTS: During the study period of 9 years, 116 patients were treated with NTZ. Mean age of the cohort was 35.6 ± 9.7 years; 83/116 (71.6%) were females. Relapse rate for the entire cohort in the year before NTZ was 3.1 ± 1.51 while one year after was 0.20±0.57 (p = 0.001; CI 2.45 -3.35). EDSS of the entire cohort in the year before NTZ was 4.5 ± 1.94 and one year after was 3.8 ± 2.7 (p = 0.013; CI 0.16-1.36). At last follow up (38.3 ± 22.78 months) there were no cases of PML identified. CONCLUSIONS: Natalizumab is highly effective and safe in Indian MS patients, with no cases of PML identified at last follow up.

Folate targeted hybrid lipo-polymeric nanoplexes containing docetaxel and miRNA-34a for breast cancer treatment.

In spite of established evidence of the synergistic combination of hydrophobic anticancer molecule and microRNA for breast cancer treatment, their in vivo delivery has not been realized owing to their instability in the biological milieu and varied physicochemical properties. The present work reports folate targeted hybrid lipo-polymeric nanoplexes for co-delivering DTX and miR-34a. These nanoplexes exhibited a mean size of 129.3 nm with complexation efficiency at an 8:1 N/P ratio. The obtained nanoplexes demonstrated higher entrapment efficiency of DTX (94.8%) with a sustained release profile up to 85% till 48 h. Further, an improved transfection efficiency in MDA-MB-231 and 4T1 breast cancer cells was observed with uptake primarily through lipid-raft and clathrin-mediated endocytosis. Further, nanoplexes showed improved cytotoxicity (~3.5-5 folds), apoptosis (~1.6-2.0 folds), and change in expression of apoptotic genes (~4-7 folds) compared to the free treatment group in breast cancer cells. In vivo systemic administration of FA-functionalized DTX and FAM-siRNA-loaded nanoplexes showed an improved area under the curve (AUC) as well as circulation half-life compared to free DTX and naked FAM-labelled siRNA. Acute toxicity studies of the cationic polymer showed no toxicity at a dose equivalent to 10 mg/kg based on the hematological, biochemical, and histopathological examination.

RNA Interference Nanotherapeutics for Treatment of Glioblastoma Multiforme.

Nucleic acid therapeutics for RNA interference (RNAi) are gaining attention in the treatment and management of several kinds of the so-called "undruggable" tumors via targeting specific molecular pathways or oncogenes. Synthetic ribonucleic acid (RNAs) oligonucleotides like siRNA, miRNA, shRNA, and lncRNA have shown potential as novel therapeutics. However, the delivery of such oligonucleotides is significantly hampered by their physiochemical (such as hydrophilicity, negative charge, and instability) and biopharmaceutical features (in vivo serum stability, fast renal clearance, interaction with extracellular proteins, and hindrance in cellular internalization) that markedly reduce their biological activity. Recently, several nanocarriers have evolved as suitable non-viral vectors for oligonucleotide delivery, which are known to either complex or conjugate with these oligonucleotides efficiently and also overcome the extracellular and intracellular barriers, thereby allowing access to the tumoral micro-environment for the better and desired outcome in glioblastoma multiforme (GBM). This Review focuses on the up-to-date advancements in the field of RNAi nanotherapeutics utilized for GBM treatment.

Role of Cysteine Residues in Regulation of Peptidyl-prolyl cis-trans Isomerase Activity of Wheat Cyclophilin TaCYPA-1.

BACKGROUND: The wheat cyclophilin, TaCYPA-1, shows peptidyl-prolyl cis-trans isomerase (PPIase) activity. However, the significance of different cysteine residues in regulation of PPIase activity of this protein has not been investigated. OBJECTIVES: The main objective of this study was to analyze the role of different disulphide linkages in redox mechanisms that modulate the PPIase activity of TaCYPA-1. METHOD: Site-directed mutants of TaCYPA-1 were generated by substituting cysteine residues at positions -40 and -122 with serine and -126 with proline. The recombinant proteins were expressed in Escherichia coli and purified. The effect of Cu2+ and N-ethylmaleimide was studied on PPIase activity of the purified recombinant cyclophilins for analyzing the role of different cysteine residues in the modulation of enzyme activity. The changes in secondary structure of TaCYPA-1 and its mutants were analysed by recording far UV CD spectra. The effect of different cysteine substitutions on thermotolerance of E. coli was studied by monitoring the cell growth at 47 °C after 2 h, 3 h and 5 h of heat stress. RESULTS: The catalytic efficiencies (Kcat/Km) of TaCYPA-1C40S (0.37 X 106 M-1 s-1) and TaCYPA- 1C122S (0.31 X 106 M-1 s-1) were significantly lower as compared to the native TaCYPA-1 (1.33 X 106 M-1 s-1), whereas Kcat/Km of the double mutant TaCYPA-1C40S/C122S was significantly higher (2.36 X 106 M-1 s-1). Compared to the wild-type TaCYPA-1, the different mutants also showed differential sensitivity to Cu2+. Furthermore, the results of this study also revealed that despite lacking PPIase activity, the mutant TaCYPA-1C126P was able to confer partial protection against heat stress. CONCLUSION: This study revealed that mutation of different cysteine residues in TaCYPA-1 results in differential effect on PPIase activity. It was also observed that of the two pairs of cysteine residues i.e. Cys40/Cys168 and Cys122/Cys126, the latter appears to play major role in redox regulation of TaCYPA-1. The mutant TaCYPA-1C126P, though lacking PPIase activity, was able to impart partial tolerance to heat stress in E. coli, suggesting other functions besides cis to trans isomerisation. This study, therefore, provides new insights into the regulation of plant cyclophilins.

Expression of a cyclophilin OsCyp2-P isolated from a salt-tolerant landrace of rice in tobacco alleviates stress via ion homeostasis and limiting ROS accumulation.

Cyclophilins are a set of ubiquitous proteins present in all subcellular compartments, involved in a wide variety of cellular processes. Comparative bioinformatics analysis of the rice and Arabidopsis genomes led us to identify novel putative cyclophilin gene family members in both the genomes not reported previously. We grouped cyclophilin members with similar molecular weight and subtypes together in the phylogenetic tree which indicated their co-evolution in rice and Arabidopsis. We also characterized a rice cyclophilin gene, OsCyp2-P (Os02g0121300), isolated from a salinity-tolerant landrace, Pokkali. Publicly available massively parallel signature sequencing (MPSS) and microarray data, besides our quantitative real time PCR (qRT-PCR) data suggest that transcript abundance of OsCyp2-P is regulated under different stress conditions in a developmental and organ specific manner. Ectopic expression of OsCyp2-P imparted multiple abiotic stress tolerance to transgenic tobacco plants as evidenced by higher root length, shoot length, chlorophyll content, and K(+)/Na(+) ratio under stress conditions. Transgenic plants also showed reduced lipid peroxidase content, electrolyte leakage, and superoxide content under stress conditions suggesting better ion homeostasis than WT plants. Localization studies confirmed that OsCyp2-P is localized in both cytosol and nucleus, indicating its possible interaction with several other proteins. The overall results suggest the explicit role of OsCyp2-P in bestowing multiple abiotic stress tolerance at the whole plant level. OsCyp2-P operates via reactive oxygen species (ROS) scavenging and ion homeostasis and thus is a promising candidate gene for enhancing multiple abiotic stress tolerance in crop plants.

The calmodulin-like protein CML43 functions as a salicylic-acid-inducible root-specific Ca(2+) sensor in Arabidopsis.

Many signalling pathways in plants are regulated by the second messenger calcium (Ca(2+)). In the standard model, Ca(2+)-sensor proteins, such as CaM (calmodulin), detect Ca(2+) signals and subsequently regulate downstream targets to advance the signal transduction cascade. In addition to CaM, plants possess many CMLs (CaM-like proteins) that are predicted to function as Ca(2+) sensors, but which remain largely uncharacterized. In the present study, we examined the biochemical properties, subcellular localization and tissue-specific distribution of Arabidopsis CML43. Our data indicate that CML43 displays characteristics typical of Ca(2+) sensors, including high-affinity Ca(2+) binding, conformational changes upon Ca(2+) binding that expose hydrophobic regions and stabilization of structure in the presence of Mg(2+) or Ca(2+). In vivo localization analysis demonstrates that CML43 resides in cytosolic and nuclear compartments. Transgenic plants expressing a CML43:GUS (ß-glucoronidase) promoter reporter gene revealed that CML43 promoter activity is restricted almost exclusively to root tips under normal growth conditions. GUS reporter activity in these transgenic plants was strongly increased when exposed to the defence compound SA (salicylic acid). Furthermore, immunoblot analysis revealed that the CML43 protein accumulates following treatment with SA. Collectively, our findings suggest that CML43 functions as a Ca(2+) sensor in root tips during both normal growth and plant immune response.

Heterologous expression of a salinity and developmentally regulated rice cyclophilin gene (OsCyp2) in E. coli and S. cerevisiae confers tolerance towards multiple abiotic stresses.

Cyclophilin 2 (OsCyp2) is a cytosolic member of immunophilin family from rice. We have isolated its full length cDNA (1,056 bp) with an open reading frame of 519 bp encoding a polypeptide of 172 amino acids and an estimated pI of 8.61. Peptidyl prolyl cis-trans isomerase activity of the protein was determined using N-succinyl-ala-ala-pro-phe-p-nitroanilidine as peptide substrate. It has a catalytic efficiency (K (cat)/K (m)) of 4.5 x 10(6)/(mol/l)/s, which is comparable to known cyclophilins from plants. Its activity is specifically inhibited by cyclosporin A, a macrolide drug inhibitor of cyclophilins. Transcript analysis showed it to be a developmentally and differentially regulated gene; showing changes in abundance at seedling, tillering and heading stage under non-stress and salinity stress conditions. Expression of OsCyp2 enhances the ability of Escherichia coli to survive under diverse abiotic stresses viz. salinity, high temperature, osmotic stress (mannitol) and oxidative stress (H(2)O(2)). OsCyp2 was able to complement the yeast mutant lacking native Cyp2 and also improved the growth of wild type yeast under above-mentioned stress conditions. Based on these results, we propose that OsCyp2 may serve as a 'suitable candidate' for raising transgenic plants for enhanced multiple abiotic stress tolerance.

Unexplained neurological events during bathing in young people: Possible association with the use of gas geysers.

Here, we report sudden, unexplained neurological collapse in 14 young people while bathing with hot water associated with the use of liquefied petroleum gas (LPG)-based water heaters (gas geysers) in ill-ventilated bathrooms. None of the patients reported any circumstantial evidence of seizures or prior epilepsy. One patient developed cortical blindness and demonstrated posterior leucoencephalopathy on imaging studies. The remaining patients made rapid and excellent recovery without any residual neurological sequelae. In these cases, the results of all routine investigations, i.e., serum chemistry, brain imaging (computed tomography in 2 and magnetic resonance imaging in 10) and electroencephalography were normal. The clinical clustering of these cases in winter months with similar presentations of reversible encephalopathy probably indicates an inhalational toxin exposure. Therefore, we postulate a hypothesis that harmful emissions consisting of carbon monoxide (CO), hydrocarbon gases (HC) and nitrogen oxides (NOx), produced by incomplete combustion of LPG might be responsible for the cellular injury and subsequent transient neurological deficits. Physicians should be aware of this entity in order to avoid misdiagnosis of this condition as seizures, and a public awareness should also be created regarding the proper use of these devices.

Fungal granuloma of the brain caused by Cladosporium bantianum--a case report and review of literature.

Involvement of the brain by neurotropic, dematiaceous fungi namely Cladosporium bantianum is extremely rare. The disease is very resistant to treatment and prone for frequent relapses despite treatment with amphotericin B and flucytosine, the drugs of choice for the infection. Surgery is often required for resection of the fungal granuloma. Isolation of the fungus from the tissue specimens and its culture, showing dark colored fungal colonies clinches the diagnosis. Animal inoculation studies can provide insights to the portal of entry of the organism. We hereby report a case of fungal granuloma of the brain due to C. bantianum, which responded favorably to intensive antifungal treatment alone, with relevant review of literature.