Multidrug resistance-associated protein 1 (MRP-1) is a ubiquitously expressed member of the ATP-binding cassette transporter family. MRP-1 is one of the primary transporters of glutathione and glutathione conjugates. This protein also transports antiretroviral therapeutics, such as HIV-1 protease inhibitors (PI). We hypothesized that inflammatory mediators that activate macrophages would modify the expression and activity of MRP-1 in macrophages. Real time PCR assays western blots and calcein efflux assays were used to show that exposure of macrophage cell line RAW 264.7 to LPS increased expression of MRP-1 at the level of mRNA and protein, as well as at the level of functional activity. Treatment of macrophages with LPS resulted in 2-fold increases of MRP-1 expression or functional activity. LPS-mediated increases in calcein efflux were repressed by the MRP-specific inhibitor MK-571. These results suggest that the effectiveness of HIV-1 PI therapy may be compromised by the presence of opportunistic infections.
The major limiting factor for production and productivity of finger millet crop is blast disease caused by Magnaporthe grisea. Since, the genome sequence information available in finger millet crop is scarce, comparative genomics plays a very important role in identification of genes/QTLs linked to the blast resistance genes using SSR markers. In the present study, a total of 58 genic SSRs were developed for use in genetic analysis of a global collection of 190 finger millet genotypes. The 58 SSRs yielded ninety five scorable alleles and the polymorphism information content varied from 0.186 to 0.677 at an average of 0.385. The gene diversity was in the range of 0.208 to 0.726 with an average of 0.487. Association mapping for blast resistance was done using 104 SSR markers which identified four QTLs for finger blast and one QTL for neck blast resistance. The genomic marker RM262 and genic marker FMBLEST32 were linked to finger blast disease at a P value of 0.007 and explained phenotypic variance (R2) of 10% and 8% respectively. The genomic marker UGEP81 was associated to finger blast at a P value of 0.009 and explained 7.5% of R2. The QTLs for neck blast was associated with the genomic SSR marker UGEP18 at a P value of 0.01, which explained 11% of R2. Three QTLs for blast resistance were found common by using both GLM and MLM approaches. The resistant alleles were found to be present mostly in the exotic genotypes. Among the genotypes of NW Himalayan region of India, VHC3997, VHC3996 and VHC3930 were found highly resistant, which may be effectively used as parents for developing blast resistant cultivars in the NW Himalayan region of India. The markers linked to the QTLs for blast resistance in the present study can be further used for cloning of the full length gene, fine mapping and their further use in the marker assisted breeding programmes for introgression of blast resistant alleles into locally adapted cultivars.
Primary juxtrarenal leiomyosarcoma of the inferior vena cava (IVC) is a rare disease and is invariably malignant. Prognosis depends on early patient diagnosis and management. We report a case of a primary IVC tumor in a 66-year-old woman which was managed successfully by surgical resection of involved IVC along with right nephrectomy. IVC reconstruction was done using a Dacron interposition graft, and the left renal vein was anastomosed end to side to the Dacron graft.
Leiomyosarcoma; Inferior vena cava; Dacron graft
Alcohol consumption, which is highly prevalent in HIV-infected individuals, poses serious concerns in terms of rate of acquisition of HIV-1 infection, HIV-1 replication, response to highly active antiretroviral therapy (HAART) and AIDS/neuroAIDS progression. However, little is known about the mechanistic pathways by which alcohol exerts these effects, especially with respect to HIV-1 replication and the patient’s response to HAART.
In this review, the authors discuss the effects of alcohol consumption on HIV-1 pathogenesis and its effect on HAART. They also describe the role of cytochrome P450 2E1 (CYP2E1) in alcohol-mediated oxidative stress and toxicity, and the role of CYP3A4 in the metabolism of drugs used in HAART (i.e., protease inhibitors (PI) and non-nucleoside reverse transcriptase inhibitors (NNRTI)). Based on the most recent findings the authors discuss the role of CYP2E1 in alcohol-mediated oxidative stress in monocytes/macrophages and astrocytes, as well as the role of CYP3A4 in alcohol–PI interactions leading to altered metabolism of PI in these cells.
The authors propose that alcohol and PI/NNRTI interact synergistically in monocytes/macrophages and astrocytes through the CYP pathway leading to an increase in oxidative stress and a decrease in response to HAART. Ultimately, this exacerbates HIV-1 pathogenesis and neuroAIDS.
alcohol; antiretroviral; cytochrome P450; HIV-1; NeuroAIDS
Upon blue light irradiation, photoactive yellow protein (PYP) undergoes a conformational change that involves large movements at the N-terminus of the protein. We reasoned that this conformational change might be used to control other protein or peptide sequences if these were introduced as linkers connecting the N and C-termini of PYP in a circular permutant. For such a design strategy to succeed, the circularly permuted PYP (cPYP) would have to fold normally and undergo a photocycle similar to that of the wild type protein. We created a test cPYP by connecting the N- and Ctermini of wild type PYP (wtPYP) with a GGSGGSGG linker polypeptide and introducing new N- and C- termini at G115 and S114 respectively. Biophysical analysis indicated that this cPYP adopts a dark state conformation much like wtPYP and undergoes wtPYP-like photoisomerization driven by blue light. However, thermal recovery of dark state cPYP is ~10-fold faster than wtPYP, so that very bright light is required to significantly populate the light state. Targeted mutations at M121E (M100 in wtPYP numbering) were found to enhance the light sensitivity substantially by lengthening the lifetime of the light state to ~10 min. NMR, circular dichroism, and UV-Vis analysis indicated that the M121E-cPYP mutant also adopts a dark state structure like that of wtPYP although protonated and deprotonated forms of the chromophore coexist giving rise to a shoulder near 380 nm in the UV-Vis absorption spectrum. Fluorine NMR studies with fluoro-tryptophan labeled M121E-cPYP show that blue-light drives large changes in conformational dynamics and leads to solvent exposure of Trp7 (Trp119 in wtPYP numbering) consistent with substantial rearrangement of the N-terminal cap structure. M121E-cPYP thus provides a scaffold that may allow a wider range of photoswitchable protein designs by replacing the linker polypeptide with a target protein or peptide sequence.
photo-control; photoactive yellow protein; PYP; optogenetics; photoisomerization; genetically encoded; circular permutation
Tobacco smoking is highly prevalent among the HIV-1-infected population. In addition to diminished immune response, smoking has been shown to increase HIV-1 replication and decrease response to antiretroviral therapy, perhaps through drug–drug interaction. However, the mechanism by which tobacco/nicotine increases HIV-1 replication and mediates drug–drug interaction is poorly understood.
In this review, the authors discuss the effects of smoking on HIV-1 pathogenesis. Since they propose a role for the cytochrome P450 (CYP) pathway in smoking-mediated HIV-1 pathogenesis, the authors briefly converse the role of CYP enzymes in tobacco-mediated oxidative stress and toxicity. Finally, the authors focus on the role of CYP enzymes, especially CYP2A6, in tobacco/nicotine metabolism and oxidative stress in HIV-1 model systems monocytes/macrophages, lymphocytes, astrocytes and neurons, which may be responsible for HIV-1 pathogenesis.
Recent findings suggest that CYP-mediated oxidative stress is a novel pathway that may be involved in smoking-mediated HIV-1 pathogenesis, including HIV-1 replication and drug–drug interaction. Thus, CYP and CYP-associated oxidative stress pathways may be potential targets to develop novel pharmaceuticals for HIV-1-infected smokers. Since HIV-1/TB co-infections are common, future study involving interactions between antiretroviral and antituberculosis drugs that involve CYP pathways would also help treat HIV-1/TB co-infected smokers effectively.
cytochrome p450; HIV-1; nicotine; oxidative stress; smoking/tobacco
An automated Multi-Leaf Collimator (MLC) system has been developed as add-on for the cobalt-60 teletherapy machines available in India. The goal of the present computational study is to validate the MLC design using Monte Carlo (MC) modeling. The study was based on the Kirloskar-supplied Phoenix model machines that closely match the Atomic Energy of Canada Limited (AECL) theratron-80 machine. The MLC is a retrofit attachment to the collimator assembly, with 14 non-divergent leaf pairs of 40 mm thick, 7 mm wide, and 150 mm long tungsten alloy plates with rounded edges and 20 mm tongue and 2 mm groove in each leaf. In the present work, the source and collimator geometry has been investigated in detail to arrive at a model that best represents the measured dosimetric data. The authors have studied in detail the proto-I MLC built for cobalt-60. The MLC field sizes were MC simulated for 2 × 2 cm2 to 14 × 14 cm2 square fields as well as irregular fields, and the percent depth dose (PDD) and profile data were compared with ROPS† treatment planning system (TPS). In addition, measured profiles using the IMATRIXX system‡ were also compared with the MC simulations. The proto-I MLC can define radiation fields up to 14 × 14 cm2 within 3 mm accuracy. The maximum measured leakage through the leaf ends in closed condition was 3.4% and interleaf leakage observed was 7.3%. Good agreement between MC results, ROPS and IMATRIXX results has been observed. The investigation also supports the hypothesis that optical and radiation field coincidence exists for the square fields studied with the MLC. Plots of the percent depth dose (PDD) data and profile data for clinically significant irregular fields have also been presented. The MC model was also investigated to speed up the calculations to allow calculations of clinically relevant conformal beams.
†Radiation Oncology Planning System (ROPS) is supplied by Tirumala Jyothi Computer Systems described at https://sites.google.com/site/tjcsrops/
‡IMATRIXX is supplied by IBA Dosimetry described at HYPERLINK http://www.iba-dosimetry.com
BEAMnrc; Cobalt-60; MLC; Monte Carlo
The prevalence of HIV-associated neurocognitive disorders (HAND) remains high in patients infected with HIV-1. The production of pro-inflammatory cytokines by astrocytes/microglia exposed to viral proteins is thought to be one of the mechanisms leading to HIV-1- mediated neurotoxicity. In the present study we examined the effects of Nef on CCL5 induction in astrocytes. The results demonstrate that CCL5 is significantly induced in Nef-transfected SVGA astrocytes. To determine the mechanisms responsible for the increased CCL5 caused by Nef, we employed siRNA and chemical antagonists. Antagonists of NF-κB, PI3K, and p38 significantly reduced the expression levels of CCL5 induced by Nef transfection. Furthermore, specific siRNAs demonstrated that the Akt, p38MAPK, NF-κB, CEBP, and AP-1 pathways play a role in Nef-mediated CCL5 expression. The results demonstrated that the PI3K/Akt and p38 MAPK pathways, along with the transcription factors NF-κB, CEBP, and AP-1, are involved in Nef-induced CCL5 production in astrocytes.
Expression level of lens epithelial derived growth factor (LEDGF) is vital for LEDGF-mediated cell survival and cytoprotection against proapoptotic stimuli. We previously demonstrated that LEDGF is transcriptionally regulated by Sp1-responsive elements within a CpG island in the LEDGF promoter. Herein, we report on the existence of epigenetic signaling involved in the repression of LEDGF transcription in lens epithelial cells (LECs) facing UVB. UVB exposure led to histone H3 dimethylation and deacetylation at its CpG island, where a histone deacetylase/histone methylase (HDAC1/SUV39H1) complex was recruited. Exposure of LECs to UVB stress altered LEDGF protein and mRNA expression as well as promoter activity, while failing to methylate the CpG island. These events were correlated with increased reactive oxygen species (ROS) and increased cell death. LEDGF promoter activity and expression remained unaltered after 5-Aza treatment, but were relieved with tricostatin A, an inhibitor of HDACs. Expression analysis disclosed that UVB radiation altered the global expression levels of acetylated histone proteins, diminished total histone acetyltransferase (HAT) activity and increased HDAC activity and HDAC1 expression. In silico analysis of LEDGF proximal promoter and ChIP analyses disclosed HDAC1/SUV39H1 complex anchored to the -170/-10 nt promoter regions at Sp1-responsive elements and also attenuated Sp1 binding, resulting in HDAC1- and SUV39H1-dependent deacetylation and dimethylation of H3 at K9. Acetylation of H3K9 was essential for LEDGF active transcription, while enrichment of H3K9me2 at Sp1-responsive elements within CpGs (-170/-10) by UVB radiation repressed LEDGF transcription. Our study may contribute to understanding diseases associated with LEDGF aberrant expression due to specific epigenetic modifications, including blinding disorders.
LEDGF; Sp1; epigenetics; transcription; histone acetyltransferases; DNA methyltransferase; histone deacetylase
In this work, addition of OH− to one-electron oxidized thymidine (dThd) and thymine nucleotides in basic aqueous glasses is investigated. At pHs ca. 9–10 where the thymine base is largely deprotonated at N3, one-electron oxidation of the thymine base by Cl2•− at ca. 155 K results in formation of a neutral thyminyl radical, T(−H)•. Assignment to T(−H)• is confirmed by employing 15N substituted 5'-TMP. At pH ≥ ca. 11.5, formation of the 5-hydroxythymin-6-yl radical, T(5OH)•, is identified as a metastable intermediate produced by OH− addition to T(−H)• at C5 at ca. 155 K. Upon further annealing to ca. 170 K, T(5OH)• readily converts to the 6-hydroxythymin-5-yl radical, T(6OH)•. One-electron oxidation of N3-methyl-thymidine (N3-Me-dThd) by Cl2•− at ca. 155 K produces the cation radical (N3-Me-dThd•+) for which we find a pH dependent competition between deprotonation from the methyl group at C5 and addition of OH− to C5. At pH 7 the 5-methyl deprotonated species is found; however, at pH ca. 9, N3-Me-dThd•+ produces T(5OH)• that on annealing up to 180 K forms T(6OH)•. Through use of deuterium substitution at C5' and on the thymine base, i.e., specifically employing [5',5”-D,D]-5'-dThd, [5',5”-D,D]-5'-TMP, [CD3]-dThd and [CD3,6D]-dThd, we find unequivocal evidence for T(5OH)• formation and its conversion to T(6OH)•. The addition of OH− to the C5 position in T(−H)• and N3-Me-dThd•+ is governed by spin and charge localization. DFT calculations predict that the conversion of the “reducing” T(5OH)• to the “oxidizing” T(6OH)• occurs by a unimolecular OH group transfer from C5 to C6 in the thymine base. The T(5OH)• to T(6OH)• conversion is found to occur more readily for deprotonated dThd and its nucleotides than for N3-Me-dThd. In agreement, calculations predict that the deprotonated thymine base has a lower energy barrier (ca. 6 kcal/mol) for OH transfer than its corresponding N3-protonated thymine base (14 kcal/mol).
The advent of nanotechnology has reignited interest in the field of pharmaceutical science for the development of nanomedicine. Nanomedicinal formulations are nanometer-sized carrier materials designed for increasing the drug tissue bioavailability, thereby improving the treatment of systemically applied chemotherapeutic drugs. Nanomedicine is a new approach to deliver the pharmaceuticals through different routes of administration with safer and more effective therapies compared to conventional methods. To date, various kinds of nanomaterials have been developed over the years to make delivery systems more effective for the treatment of various diseases. Even though nanomaterials have significant advantages due to their unique nanoscale properties, there are still significant challenges in the improvement and development of nanoformulations with composites and other materials. Here in this review, we highlight the nanomedicinal formulations aiming to improve the balance between the efficacy and the toxicity of therapeutic interventions through different routes of administration and how to design nanomedicine for safer and more effective ways to improve the treatment quality. We also emphasize the environmental and health prospects of nanomaterials for human health care.
The association of neurofibromatosis type I with invasive male breast cancer is a rare clinical entity with only one case in literature reported in 1953. Women with NF1 are at risk of developing breast cancer and men also may be at risk but there is scarce data on the risk and association of NF1 with male breast cancer due to its rarity. Established clinical trials in male breast cancer patients are lacking and the results are extrapolated from female breast cancer patients. The treatment of male breast cancer is followed as per the guidelines of premenopausal female breast cancer and tamoxifen is the hormone treatment in them. Mendes et al suggests that silencing of NF1 gene confers resistance to tamoxifen. Our conclusions are that since NF1 is mutated or deleted in one third of sporadic breast cancers, its role as a molecular driver for treatment has to be further explored.
The aim of the present study was to increase the solubility of an anti-allergic drug loratadine by making its inclusion complex with β-cyclodextrin and to develop it’s thermally triggered mucoadhesive in situ nasal gel so as to overcome first-pass effect and consequently enhance its bioavailability. A total of eight formulations were prepared by cold method and optimized by 23 full factorial design. Independent variables (concentration of poloxamer 407, concentration of carbopol 934 P, and pure drug or its inclusion complex) were optimized in order to achieve desired gelling temperature with sufficient mucoadhesive strength and maximum permeation across experimental nasal membrane. The design was validated by extra design checkpoint formulation (F9) and Pareto charts were used to help eliminate terms that did not have a statistically significant effect. The response surface plots and possible interactions between independent variables were analyzed using Design Expert Software 8.0.2 (Stat Ease, Inc., USA). Faster drug permeation with zero-order kinetics and target flux was achieved with formulation containing drug: β-cyclodextrin complex rather than those made with free drug. The optimized formulation (F8) with a gelling temperature of 28.6 ± 0.47°C and highest mucoadhesive strength of 7,676.0 ± 0.97 dyn/cm2 displayed 97.74 ± 0.87% cumulative drug permeation at 6 h. It was stable for over 3 months and histological examination revealed no remarkable damage to the nasal tissue.
23 factorial design; in situ nasal gel; loratadine; mucoadhesion; temperature-induced gelation
Dendritic cells (DCs) and macrophages (MΦs) are well-known antigen presenting cells with an ability to produce IL-12 which indicates that they have potential of directing acquired immunity toward a Th1-biased response. The aim of this study was to examine the effect of Leishmania specific KMP-11 antigen through comparison of immune responses after presentation by DCs and MΦs to T cells in Indian patients with VL. Patients with DCS and MΦs were directed against a purified Leishmania donovani antigen (KMP-11) and phytohaemagglutinin (PHA). The cytokines (IL-12, IL-10, and TGF-β) producing abilities of the DCs and MΦs against these antigens were determined by flow cytometry. The transcription factor (NF-κB) and T-cell cytokine support (IFN-γ, IL-10), which could be significant in effector immune function, were also determined. Severe hindrance in the immune protection due to Leishmania parasites, as revealed by decreased expression of IL-12 and upregulation of IL-10 and TGF-β expression in the MΦs compared to DCs, occurred in VL patients. The production of IL-12 in response to L. donovani KMP-11 antigen was increased in DCs which was reduced in MΦs of VL patients. In contrast, the presentation of KMP-11 antigen by DCs to T-lymphocytes in VL patients significantly increased the IFN-γ produced by these immune cells, whereas the levels of IL-10 were significantly elevated after presentation of KMP-11antigen by MΦs. The VL patients were observed with severely dysfunctional MΦs in terms of NF-κB activity that could be recovered only after stimulation of DCs with L. donovani KMP-11 antigen. Immunologically the better competitiveness of KMP-11 antigen through a dendritic cell delivery system may be used to revert T-cell anergy, and control strategy can be designed accordingly against kala-azar.
Background: Acute myocardial infarction carries a high mortality among cardiac patients.The discovery of the fact that certain enzymes like CPK, LDH liberated into circulation following necrosis of the myocardial cells came as boon for physicians and patients. There has been a constant search of different parameters for the diagnosis and management of CoronaryArtery Diseases (CAD).
Aim: The present study was undertaken to investigate a possible relation between the changes in plasma free fatty acid (FFA) concentration and acute myocardial infarction.
Material and Methods: Fifty cases (25 males and 25 females) of acute myocardial infarction were selected for the present study. All the patients were in the age group of 40-70 years. For the control group fifty (25 male and 25 female) subjects of same age group were selected from patient’s relatives and friends.
Plasma free fatty acid concentration was estimated by Titrametric method of Trout et al., (1960), a modified version of Dole (1956).
Statistical Analysis: The statistical analysis of the data of the present study was done by using SPSS, version 14.0.1 was used.
Results: Our study showed a significant increase in plasma FFA in the first 24 hours of acute myocardial infarction with subsequent normalisation on the 7th day.The difference between the first and the seventh day was statistically significant.
Conclusion: The FFA were found raised in cases of acute myocardial infarction.On the basis of present study, it is worth to say that estimation of serum free fatty acid should be done routinely at the earliest opportunity in all cases of acute myocardial infarction.
Plasma free fatty acid; FFA; Acute myocardial infarction; Coronary artery disease; Marker
Objectives: To determine the minimum inhibitory concentration of ciprofloxacin among 50 blood stream isolates of Salmonella enterica.
Material and Methods: A total of 50 consecutive isolates of Salmonella enterica were tested for susceptibility to antimicrobials using the Kirby Bauer disk diffusion method. Minimum inhibitory concentrations were determined using Hi-Comb strips. All results were interpreted according to the CLSI guidelines.
Results: Of the 50 isolates 70%were Salmonella
Typhi, 4% Salmonella
paratyphi A, 2% Salmonella
paratyphi B and the remaining 10% were identified only as Salmonella species. Using the CLSI 2011 breakpoints for disc diffusion, 86% (43/50) were resistant to nalidixic acid(NA), 22% (11/50) to ciprofloxacin, 12% to azithromycin, 6% to cotrimoxazole, 4% to ampicillin and 1% to chloramphenicol. The MIC50 and MIC90 of ciprofloxacin for S.Typhi were 0.181 μg/mL and 5.06 μg/mL respectively. While the same for S. paratyphi A was 0.212μg/mL and 0.228μg/mL respectively. None of the isolates were multi drug resistant and all were susceptible to ceftriaxone. Using the CLSI 2012 revised ciprofloxacin breakpoints for disc diffusion (>31mm) & MIC (<0.06 μg/mL), 90% (45/50) of these isolates were found to be resistant.
Conclusion: MIC’s of ciprofloxacin should be reported for all salmonella isolates and should be used to guide treatment. Blindly following western guidelines for a disease which is highly endemic in the subcontinent will spell the death knell of a cheap and effective drug in our armamentarium. Therefore it will be too premature to declare that “the concept of using ciprofloxacin in typhoid fever is dead!”
Salmonella typhi; Salmonella paratyphi A; Ciprofloxacin; Resistance; Breakpoints
Clonidine, an alpha-2 adrenergic receptor agonist, has well-established role in acute perioperative pain management. However, recently it has found increasing use in chronic pain conditions as well. In this review, we systematically searched and analyzed the clinical studies from “PubMed,” “PubMed central” and “Scopus” database for use of clonidine in the chronic pain. Quantitative meta-analysis was not possible as clonidine has been used in various patient populations through different routes. However, qualitative analysis of nearly thirty clinical studies provides some evidence that clonidine administered through epidural, intrathecal and local/topical route may be effective in chronic pain conditions where neuropathy is a predominant component. It may also be effective where opioids are of limited use due to inadequate pain relief or adverse effects.
Chronic pain; clonidine; neuropathy
Neurocognitive impairments remain prevalent in HIV-1 infected individuals despite current antiretroviral therapies. It is increasingly becoming evident that astrocytes play a critical role in HIV-1 neuropathogenesis through the production of proinflammatory cytokines/chemokines. HIV-1 viral protein R (Vpr) plays an important role in neuronal dysfunction; however, its role in neuroinflammation is not well characterized. The major objective of this study was to determine the effect of Vpr in induction of proinflammatory chemokine CCL5 in astrocytes and to define the underlying mechanism(s).
SVGA astrocytes were either mock transfected or were transfected with a plasmid encoding HIV-1 Vpr, and the cells were harvested at different time intervals. The mRNA level of CCL5 expression was quantified using real-time RT-PCR, and cell culture supernatants were assayed for CCL5 protein concentration. Immunocytochemistry was performed on HIV-1 Vpr transfected astrocytes to check CCL5 expression. Various signaling mechanisms such as p38 MAPK, PI3K/Akt, NF-κB and AP-1 were explored using specific chemical inhibitors and siRNAs.
HIV-1 Vpr transfected astrocytes exhibited time-dependent induction of CCL5 as compared to mock-transfected astrocytes at both the mRNA and protein level. Immunostained images of astrocytes transfected with HIV-1 Vpr also showed much higher accumulation of CCL5 in comparison to untransfected and mock-transfected astrocytes. Pre-treatment with NF-κB (SC514) and PI3K/Akt (LY294002) inhibitor partially abrogated CCL5 mRNA and protein expression levels as opposed to untreated controls after HIV-1 Vpr transfection. Specific siRNAs against p50 and p65 subunits of NF-κB, p38δ MAPK, Akt-2 and Akt-3, and AP-1 transcription factor substantially inhibited the production of CCL5 in HIV-1 Vpr transfected astrocytes.
These results demonstrate the ability of HIV-1 Vpr to induce CCL5 in astrocytes in a time-dependent manner. Furthermore, this effect was observed to be mediated by transcription factors NF-κB and AP-1 and involved the p38-MAPK and PI3K/Akt pathway.
HIV-1; Vpr; CCL5; NF-kB; AP-1; MAPK; PI3K/Akt
The incidence of HIV-associated neurological disorders (HAND) has increased during recent years even though the highly active antiretroviral therapy (HAART) has significantly curtailed the virus replication and increased the life expectancy among HIV-1 infected individuals. These neurological deficits have been attributed to HIV proteins including HIV-1 Tat. HIV-1 Tat is known to up-regulate CCL5 expression in mouse astrocytes, but the mechanism of up-regulation is not known. The present study was undertaken with the objective of determining the mechanism(s) underlying HIV-1 Tat-mediated expression of CCL5 in astrocytes. SVGA astrocytes were transiently transfected with a plasmid encoding Tat, and expression of CCL5 was studied at the mRNA and protein levels using real time RT-PCR and multiplex cytokine bead array, respectively. HIV-1 Tat showed a time-dependent increase in the CCL5 expression with peak mRNA and protein levels, observed at 1 h and 48 h post-transfection, respectively. In order to explore the mechanism(s), pharmacological inhibitors and siRNA against different pathway(s) were used. Pre-treatment with SC514 (NF-κB inhibitor), LY294002 (PI3K inhibitor), AG490 (JAK2 inhibitor) and Janex-1 (JAK3 inhibitor) showed partial reduction of the Tat-mediated induction of CCL5 suggesting involvement of JAK, PI3K/Akt and NF-κB in CCL5 expression. These results were further confirmed by knockdown of the respective genes using siRNA. Furthermore, p38 MAPK was found to be involved since the knockdown of p38δ but not other isoforms showed partial reduction in CCL5 induction. This was further confirmed at transcriptional level that AP-1, C/EBPα and C/EBPγ were involved in CCL5 up-regulation.
A nondisintegrating, floating asymmetric membrane capsule (FAMC) was developed to achieve site-specific osmotic flow of a highly water-soluble drug, ranitidine hydrochloride (RHCl), in a controlled manner. Solubility suppression of RHCl was achieved by the common ion effect, using optimized coated sodium chloride as a formulation component. The capsular wall of FAMC was prepared by the phase inversion process wherein the polymeric membrane was precipitated on glass pins by dipping them in a solution of cellulose acetate followed by quenching. Central composite design was utilized to investigate the influence of independent variables, namely, level(s) of membrane former, pore former, and osmogen, on percent cumulative drug release (response). The release mechanism of RHCl through FAMC was confirmed as osmotic pumping. The asymmetry of the membrane was characterized by scanning electron microscopy that revealed a dense nonporous outer region of membrane supported by an inner porous region. Differential scanning calorimetry indicated no incompatibility between the drug and excipients. In vitro drug release in three biorelevant media, pH 2.5 (low fed), pH 4.5 (intermediate fed), and pH 6.5 (high fed), demonstrated pH-independent release of RHCl (P > 0.05). Floating ability for 12 h of the optimized FAMC9 was visually examined during the in vitro release studies that showed maximal drug release with zero-order kinetics (r2 = 0.9991). Thus, a novel osmotically regulated floating capsular system was developed for site-specific delivery of RHCl.
Electronic supplementary material
The online version of this article (doi:10.1208/s12249-012-9870-8) contains supplementary material, which is available to authorized users.
asymmetric membrane capsule; central composite design; floating system; osmotic delivery; ranitidine hydrochloride
Lung cancer accounts for the highest number of cancer-related deaths worldwide. Early diagnosis significantly increases the disease-free survival rate and a large amount of effort has been expended in screening trials and the development of early molecular diagnostics. However, a gold standard diagnostic strategy is not yet available. Here, based on miRNA expression profile in lung cancer and using a novel in silico reverse-transcriptomics approach, followed by analysis of the interactome; we have identified potential transcription factor (TF) markers that would facilitate diagnosis of subtype specific lung cancer. A subset of seven TF markers has been used in a microarray screen and was then validated by blood-based qPCR using stage-II and IV non-small cell lung carcinomas (NSCLC). Our results suggest that overexpression of HMGA1, E2F6, IRF1, and TFDP1 and downregulation or no expression of SUV39H1, RBL1, and HNRPD in blood is suitable for diagnosis of lung adenocarcinoma and squamous cell carcinoma sub-types of NSCLC. Here, E2F6 was, for the first time, found to be upregulated in NSCLC blood samples. The miRNA-TF-miRNA interaction based molecular mechanisms of these seven markers in NSCLC revealed that HMGA1 and TFDP1 play vital roles in lung cancer tumorigenesis. The strategy developed in this work is applicable to any other cancer or disease and can assist in the identification of potential biomarkers.
Acute gastric dilatation can have multiple etiologies which may lead to ischemia of the stomach. Without proper timely diagnosis and treatment, potentially fatal events such as gastric perforation, haemorrhage, and other serious complications can occur. Here we present a 36-year-old man who came to the casualty with pain abdomen and distension for 2 days. Clinically, abdomen was asymmetrically distended more in the left hypochondrium and epigastrium region. Straight X-ray abdomen showed opacified left hypochondrium with nonspecific gaseous distension of bowel. Exploratory laparotomy revealed dilated stomach with patchy gangrene over lesser curvature and fundic area. About 4 litres of brownish fluid along with semisolid undigested food particles was sucked out (mainly undigested pieces of meat). Limited resection of gangrenous areas and primary repair were done along with feeding jejunostomy. Necrosis of the stomach was confirmed on histopathology. The patient recovered well and was discharged on the tenth postoperative day.
Objective To examine the association of family organization with metabolic control in adolescents with type 1 diabetes through the mechanisms of family self-efficacy for diabetes and disease management. Method Data from the baseline assessment of a longitudinal RCT were used, wherein 257 adolescent–parent dyads (adolescents aged 11–14) each completed the family organization subscale of the Family Environment Scale, the self-efficacy for Diabetes Self-Management Scale, the Diabetes Behavior Rating Scale, and 2 24-hr diabetes interviews. Results Structural equation modeling showed greater family organization was associated indirectly with better disease management behaviors via greater family self-efficacy (β = .38, p < .001). Greater self-efficacy was indirectly associated with better metabolic control via better disease management both concurrently (β = −.37, p < .001) and prospectively (β = −.26, p < .001). The full model indicates more family organization is indirectly associated with better metabolic control concurrently and prospectively through greater self-efficacy and better disease management (β = −.13, p < .001). Conclusions Understanding the mechanisms by which family organization is associated with metabolic control provides insight into possible avenues of prevention/intervention for better diabetes management.
disease management; family organization; self-efficacy; type 1 diabetes
Indian truck drivers and their younger apprentice drivers are at increased risk of HIV infection. We determine network and risk practices associated with willingness to adopt HIV prevention interventions currently not being used in India: rapid HIV testing, circumcision, and preexposure prophylaxis (PrEP) in order to inform the National AIDS Control Program (NACP). Truck drivers and truck cleaners were systematically recruited to participate in a social network and risk survey in Hyderabad, Southern India. Three separate composite measures of acceptability of rapid HIV testing, circumcision, and PrEP acceptability were utilized to independently assess the relationship of these prevention interventions with risk-practices and social network characteristics. An 89% participation rate yielded 1602 truck drivers and truck cleaners with 54.2% younger than 30 years of age and 2.8% HIV infected. Twenty-five percent of respondents reported sex with female sex workers (FSW) and 5% with men (MSM). Rapid testing, circumcision, and PrEP acceptability were 97.4%, 9.1%, and 85.9%, respectively. Participants reporting prosocial network characteristics were more accepting of rapid testing (adjusted odds ratio [AORs] 3.07–6.71; p<0.05) and demonstrated variable PrEP acceptability (AORs 0.08–2.22; p<0.001). Sex with FSWs was associated with PrEP acceptability (AOR 4.27; p<0.001); sex with MSM was associated with circumcision acceptability only (AOR 2.66; p<0.01). Social network factors and risk-practices were associated with novel prevention acceptability, but not consistently across intervention type and with variable directionality. The NACP will need to consider that intervention uptake may likely be most successful when efforts are targeted to individuals with specific behavior and social network characteristics.
Dengue virus (DENV) is an important human pathogen, especially in the tropical and subtropical parts of the world, causing considerable morbidity and mortality. DENV replication occurs in the cytoplasm; however, a high proportion of nonstructural protein 5 (NS5), containing methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRp) activities, accumulates in the nuclei of infected cells. The present study investigates the impact of nuclear localization of NS5 on its known functions, including viral RNA replication and subversion of the type I interferon response. By using a mutation analysis approach, we identified the most critical residues within the αβ nuclear localization signal (αβNLS), which are essential for the nuclear accumulation of this protein. Although we observed an overall correlation between reduced nuclear accumulation of NS5 and impaired RNA replication, we identified one mutant with drastically reduced amounts of nuclear NS5 and virtually unaffected RNA replication, arguing that nuclear localization of NS5 does not correlate strictly with DENV replication, at least in cell culture. Because NS5 plays an important role in blocking interferon signaling via STAT-2 (signal transducer and activator of transcription 2) degradation, the abilities of the NLS mutants to block this pathway were investigated. All mutants were able to degrade STAT-2, with accordingly similar type I interferon resistance phenotypes. Since the NLS is contained within the RdRp domain, the MTase and RdRp activities of the mutants were determined by using recombinant full-length NS5. We found that the C-terminal region of the αβNLS is a critical functional element of the RdRp domain required for polymerase activity. These results indicate that efficient DENV RNA replication requires only minimal, if any, nuclear NS5, and they identify the αβNLS as a structural element required for proper RdRp activity.