Identification and stratification of systemic lupus erythematosus patients into two transcriptionally distinct clusters based on IFN-I signature.

OBJECTIVE: Despite the significant advancement in the understanding of the pathophysiology of systemic lupus erythematosus (SLE) variable clinical response to newer therapies remain a major concern, especially for patients with lupus nephritis and neuropsychiatric systemic lupus erythematosus (NPSLE). We performed this study with an objective to comprehensively characterize Indian SLE patients with renal and neuropsychiatric manifestation with respect to their gene signature, cytokine profile and immune cell phenotypes. METHODS: We characterized 68 Indian SLE subjects with diverse clinical profiles and disease activity and tried to identify differentially expressed genes and enriched pathways. To understand the temporal profile, same patients were followed at 6 and 12-months intervals. Additionally, auto-antibody profile, levels of various chemokines, cytokines and the proportion of different immune cells and their activation status were captured in these subjects. RESULTS: Multiple IFN-related pathways were enriched with significant increase in IFN-I gene signature in SLE patients as compared to normal healthy volunteers (NHV). We identified two transcriptionally distinct clusters within the same cohort of SLE patients with differential immune cell activation status, auto-antibody as well as plasma chemokines and cytokines profile. CONCLUSIONS: Identification of two distinct clusters of patients based on IFN-I signature provided new insights into the heterogeneity of underlying disease pathogenesis of Indian SLE cohort. Importantly, patient within those clusters retain their distinct expression dynamics of IFN-I signature over the time course of one year despite change in disease activity. This study will guide clinicians and researchers while designing future clinical trials on Indian SLE cohort.

Impact of early versus late tracheostomy on patient outcomes in a tertiary care multispeciality ICU.

BACKGROUND AND AIMS: Tracheostomy is a commonly performed procedure in critically ill patients because patients requiring chronic mechanical ventilation (MV) are rising by as much as 5.5% per year. The controversy on likely benefits of early versus late tracheostomy is ongoing. We aimed to study the impact of early versus late tracheostomy on patient outcomes. MATERIAL AND METHODS: A retrospective observational study was performed in intensive care unit (ICU) patients who underwent tracheostomy in a 31-bedded multispeciality ICU of a 350-bedded tertiary care hospital, over a period of 1 year. Data collected included the age, sex, APACHE II score, indication for tracheostomy, timing of procedure, whether surgical or percutaneous, any complication, MV days, ICU stay, and patient outcome. Patients were divided into two groups for statistical comparison: early ≤7 days and late >7 days of MV. RESULTS: A total of 102 patients underwent tracheostomy over the study period, of which 19 were excluded because of inadequate data and exclusion criteria. Of the 83 study patients, 60 had percutaneous, while 23 had surgical tracheostomy. About 51 (61.45%) had early, while 32 (38.55%) had late tracheostomy. On statistical analysis, there was a significant difference in MV days (5 vs 12.5 days, P = 0.002), ICU stay (10 vs 16 days, P = 0.004), mortality (21.6% vs 43.8%, P = 0.032), and decannulation rate (29.41% vs 6.25%, P = 0.009). No difference was observed in hospital stay or complication rates. CONCLUSION: Early tracheostomy is associated with both morbidity and mortality benefits. Patients requiring MV should be given an option of early tracheostomy.

Mild Encephalopathy/Encephalitis with Reversible Splenial Lesion in a Patient with Salmonella typhi Infection: An Unusual Presentation with Excellent Prognosis.

Mild encephalopathy/encephalitis with reversible splenial lesion (MERS) is an uncommon clinicoradiological entity reported mainly in East Asian population. Mild encephalopathy/encephalitis with reversible splenial lesion is characterized by neuropsychiatric manifestations, magnetic resonance imaging (MRI) findings of the reversible lesions in the splenium of corpus callosum, and good clinical outcomes. These transient splenial lesions are not specific to a particular condition and have been described mainly in children in various situations including epilepsy or peri-ictal state, antiepileptic drug use, and infectious agents such as influenza virus, Mycoplasma pneumoniae, Legionella pneumophila, and O-157 Escherichia coli. Mild encephalopathy/encephalitis with reversible splenial lesion is an uncommon complication of Salmonella infection and has been described earlier in a child who made excellent clinical recovery. We report a case of Salmonella typhi encephalopathy in a young adult who presented with reversible transient splenial lesions on MRI. The patient recovered without neurological sequelae. Awareness of these lesions is important as these are uncommon findings on MRI and carry excellent prognosis. HOW TO CITE THIS ARTICLE: Chopra P, Bhatia RS, Chopra R. Mild Encephalopathy/Encephalitis with Reversible Splenial Lesion in a Patient with Salmonella typhi Infection: An Unusual Presentation with Excellent Prognosis. Indian J Crit Care Med 2019;23(12):584-586.

Biochemical characterization of an S-adenosyl-l-methionine-dependent methyltransferase (Rv0469) of Mycobacterium tuberculosis.

Tuberculostearic acid (l0-methylstearic acid, TSA) is a major constituent of mycobacterial membrane phospholipids, and its biosynthesis involves the direct methylation of oleic acid esterified as a component of phospholipids. The methyltransferases of mycobacteria were long proposed to be involved in the synthesis of methyl-branched short-chain fatty acids, but direct experimental evidence is still lacking. In this study, we identified the methyltransferase encoded by umaA in Mycobacterium tuberculosis H37Rv as a novel S-adenosyl-l-methionine (SAM)-dependent methyltransferase capable of catalyzing the conversion of olefinic double bond of phospholipid-linked oleic acid to biologically essential TSA. Therefore, UmaA, catalyzing such modifications, offer a viable target for chemotherapeutic intervention.

Perioperative Management of Spontaneous Intracranial Hemorrhage in a Patient With Hemophilia A in a Resource Limited Country.

Intracranial hemorrhage (ICH) is a serious complication of hemophilia A with high morbidity and mortality. The management of such cases is complicated by nonspecific and often delayed presentation, increased frequency of rebleeding, low awareness regarding clotting factor replacement, and debate regarding the efficacy of surgical interventions. We report a case of an 18-year-old male patient with hemophilia A, who first presented to the emergency department in India in a comatose state. Neuroimaging revealed subdural hematoma with midline shift and uncal herniation. The patient was successfully managed with perioperative cryoprecipitate and factor VIII replacement, tiered intracranial pressure lowering strategies, and early decompressive craniectomy with clot evacuation. In India, there are no standardized guidelines for screening and routine care for hereditary diseases like hemophilia. In a resource-deficient country, management was complicated by the limited availability of factor VIII in the emergent setting, as well as the inability to obtain serial factor levels in the postoperative period. We hope that this article helps to guide the management of ICH and hemophilia in resource-limited countries.

Development of a cell death-based method for the screening of nuclear factor-kappaB inhibitors.

Nuclear factor kappa B (NF-kappaB) plays a significant role in immunity and inflammation and represents a first choice as pharmacological target for anti-inflammatory therapy. However, research in this field has been hampered by the fact that no convenient assay suitable for large-scale screening procedures is available. The present study provides a cell death-based assay method for screening of nuclear factor-kappaB inhibitors. In this study, we observed that four distinct pharmacologic inhibitors of NF-kappaB, pyrrolidine dithiocarbamate (PDTC), N-tosyl-L-lysyl chloromethyl ketone (TPCK), genistein and BAY11-7082, resulted in the cell death of murine macrophages, J774A.1. DNA-binding experiments showed that lethal doses were consistent with those required for NF-kappaB inhibition. DNA fragmentation analysis showed that cell death is apoptotic in nature. Further studies suggested that NF-kappaB inhibitors induced apoptosis is independent of the involvement of other markers of cell death such as caspases and p38 MAP (Mitogen activated protein) kinase. From this study, we conclude that NF-kappaB activation may represent an important survival mechanism in macrophages. This study also provides a new cell-based screening method, as any compound that will inhibit NF-kappaB activity will result in the death of macrophages.

An improved zinc cocktail-mediated fluorescence polarization-based kinase assay for high-throughput screening of kinase inhibitors.

During the past few years, high-throughput screening (HTS) has provided a useful resource to researchers involved in the development of kinase inhibitors as a novel therapeutic modality. However, with all the choices among kinase assays, there is not yet a one-size-fits-all assay. Therefore, selection of a specific kinase assay is a daunting task. HTS assays should be homogeneous, cost effective, use nonradioactive reagents, generic and not time consuming. Here, we report an improved method of assaying protein kinase activity using a zinc cocktail in a fluorescence polarization-(FP) based format. Assay conditions were standardized manually and validated in a HTS format using a liquid handler. We validated this assay for both serine/threonine and tyrosine (receptor/nonreceptor) kinases. The results obtained in the HTS assay system were comparable to the commercially available fluorescence-based assay. We suggest that the reported assay is a cost-effective alternative to the IMAP-based generic kinase assay.

Cloning and characterization of GTP-binding proteins of Mycobacterium tuberculosis H(37)Rv.

GTP-binding proteins (G-proteins) are highly conserved signaling molecules that participate in cellular signaling and bacterial pathogenesis by regulating the activity of cognate GTPases. However, the exact role of G-proteins in the pathogenesis of Mycobacterium tuberculosis is poorly understood. The complete genome sequence of M. tuberculosis H(37)Rv, suggests the presence of several homologs of bacterial G-proteins. In the present study, three G-proteins, Era, Obg and LepA of M. tuberculosis H(37)Rv were cloned and expressed in Escherichia coli. Purified proteins showed GTP-binding and hydrolyzing activities. A point mutation in the conserved GTP-binding motif, AspXXGly (Asp to Ala) in Era (Asp-258) and Obg (Asp-212) proteins resulted in the loss of the associated activities, confirming that known key residues in well-established G-proteins are also conserved in mycobacterial homologs. This study confirms that Era, Obg and LepA of M. tuberculosis H(37)Rv possess GTPase activity and provide a platform to understand the physiological significance of these proteins in associated pathogenesis.

Nucleoside diphosphate kinase from Mycobacterium tuberculosis cleaves single strand DNA within the human c-myc promoter in an enzyme-catalyzed reaction.

The reason for secretion of nucleoside diphosphate kinase (NdK), an enzyme involved in maintaining the cellular pool of nucleoside triphosphates in both prokaryotes and eukaryotes, by Mycobacterium tuberculosis is intriguing. We recently observed that NdK from M.tuberculosis (mNdK) localizes within nuclei of HeLa and COS-1 cells and also nicks chromosomal DNA in situ (A. K. Saini, K. Maithal, P. Chand, S. Chowdhury, R. Vohra, A. Goyal, G. P. Dubey, P. Chopra, R. Chandra, A. K. Tyagi, Y. Singh and V. Tandon (2004) J. Biol. Chem., 279, 50142-50149). In the current study, using a molecular beacon approach, we demonstrate that the mNdK catalyzes the cleavage of single strand DNA. It displays Michaelis-Menten kinetics with a kcat/K(M) of 9.65 (+/-0.88) x 10(6) M(-1) s(-1). High affinity (K(d) approximately K(M) of approximately 66 nM) and sequence-specific binding to the sense strand of the nuclease hypersensitive region in the c-myc promoter was observed. This is the first study demonstrating that the cleavage reaction is also enzyme-catalyzed in addition to the enzymatic kinase activity of multifunctional NdK. Using our approach, we demonstrate that GDP competitively inhibits the nuclease activity with a K(I) of approximately 1.9 mM. Recent evidence implicates mNdK as a potent virulence factor in tuberculosis owing to its DNase-like activity. In this context, our results demonstrate a molecular mechanism that could be the basis for assessing in situ DNA damage by secretory mNdK.

Nucleoside diphosphate kinase of Mycobacterium tuberculosis acts as GTPase-activating protein for Rho-GTPases.

Several bacterial pathogens secrete proteins into the host cells that act as GTPase-activating proteins (GAPs) for Rho-GTPases and convert GTP-bound active form to GDP-bound inactive form. However, no such effector molecule has been identified in Mycobacterium tuberculosis. In this study, we show that culture supernatant of M. tuberculosis H(37)Rv harbors a protein that stimulates the conversion of GTP-bound Rho-GTPases to the GDP-bound form. Nucleoside diphosphate kinase (Ndk) was identified as this culture supernatant protein that stimulated in vitro GTP hydrolysis by members of Rho-GTPases. The histidine-117 mutant of Ndk, which is impaired for autophosphorylation and nucleotide-binding activities, shows GAP activity. These results suggest that Ndk of M. tuberculosis functions as a Rho-GAP to downregulate Rho-GTPases, and this activity may aid in pathogenesis of the bacteria.

PknH, a transmembrane Hank's type serine/threonine kinase from Mycobacterium tuberculosis is differentially expressed under stress conditions.

Serine/threonine protein kinases (STPKs) represent a burgeoning concept in prokaryotic signaling and have been implicated in a range of control mechanisms. This paper describes the enzymatic and molecular characterization of PknH, a mycobacterial STPK. After cloning and expression as a Glutathione-S-transferase fusion protein in E. coli, PknH was found to phosphorylate itself and exogenous substrates like myelin basic protein and histone. The kinase activity of PknH was inhibited by the kinase inhibitors staurosporine and H-7. The results confirmed that PknH is a transmembrane protein and is restricted to members of the Mycobacterium tuberculosis complex. In addition, transcriptional analysis of pknH in M. tuberculosis under various stress conditions revealed that exposure to low pH and heat shock decreased the level of pknH transcription significantly. This is the first report describing differential expression of a mycobacterial kinase in response to stress conditions which can indicate its ability to regulate cellular events promoting bacterial adaptation to environmental change.

New drug targets for Mycobacterium tuberculosis.

In spite of the availability of effective chemotherapy and Bacille-Calmette-Guerin (BCG) vaccine, tuberculosis remains a leading infectious killer world-wide. Many factors such as, human immunodeficiency virus (HIV) co-infection, drug resistance, lack of patient compliance with chemotherapy, delay in diagnosis, variable efficacy of BCG vaccine and various other factors contribute to the mortality due to tuberculosis. In spite of the new advances in understanding the biology of Mycobacterium tuberculosis, and availability of functional genomic tools, such as microarray and proteomics, in combination with modern approaches, no new drug has been developed in the past 30 yr. Therefore, there is an urgent need to identify new drug targets in mycobacteria and eventually, develop new drugs. The release of the complete genome sequence of M. tuberculosis has facilitated a more rational, and directional approach to search for new drug targets. In general, gene products involved in mycobacterial metabolism, persistence, transcription, cell wall synthesis and virulence would be possible targets for the development of new drugs. The exploitation of host cell signaling pathways for the benefit of the pathogen is a phenomenon that deserves to be looked into with a new perspective in the current scenario to combat M. tuberculosis. Reversible phosphorylation and dephosphorylation, which are carried out by specific protein kinases and phosphatases have been shown to modify the host proteins and help in the establishment of disease by several pathogenic bacteria. In this review, we discuss some possible drug targets for M. tuberculosis.

Emerging therapeutic interventions for idiopathic pulmonary fibrosis.

INTRODUCTION: Idiopathic pulmonary fibrosis (IPF) is a devastating and relentlessly progressive lung disorder. Previously, it was thought to be a chronic inflammatory disease; however, it is now considered to be an epithelial-fibroblastic disease. In accordance with this paradigm change, efforts toward the development of novel therapeutic targets for IPF have acquired a new direction. Currently available therapies are largely ineffective in reversing the lung damage, and lung transplantation is the only effective treatment for end-stage disease. Limitations in advancement of IPF therapeutics are due to a poor understanding of its pathogenesis, unavailability of reliable animal models and slow disease progression. Recent research on IPF has resulted in the identification of a plethora of novel targets that are in various stages of development and offers hope that in the near future that there will be better therapeutic options available for the treatment of IPF. AREAS COVERED: This review discusses existing therapies and highlights some of the recent, novel therapeutics being explored in the current clinical landscape for the treatment of this chronic, disabling disorder. The review also discusses the pathogenic rationale behind current therapies. EXPERT OPINION: Targeting one fibrotic signaling pathway at a time may not have any significant effect on the control of IPF. It is therefore recommended that future IPF management focuses on targeting multiple pro-fibrotic pathways associated with its complex pathogenesis.

Hepatocyte growth factor is an attractive target for the treatment of pulmonary fibrosis.

INTRODUCTION: Pulmonary fibrosis (PF) is a progressive fatal disorder and is characterized by alveolar epithelial injury, myofibroblast proliferation, and extracellular matrix remodeling, resulting in irreversible distortion of lung's architecture. Available therapies are associated with side effects and show restricted efficacy. Therefore, there is an urgent need to find a therapeutic solution to PF. Therapeutic strategies interfering myofibroblast expansion, apoptosis of epithelial and endothelial cells might be beneficial for treatment of PF. Hepatocyte growth factor (HGF), a pleiotropic growth factor, plays an important role in lung development, inflammation, repair, and regeneration. In animal model of PF, administration of recombinant HGF protein or ectopic HGF expression ameliorates fibrosis. AREAS COVERED: The focus of this review is to highlight HGF as a promising therapeutic approach for the treatment of PF. The review discusses the currently available treatment option for PF as well as highlights the possible beneficial effect of HGF as a drug target. EXPERT OPINION: HGF with its anti-fibrotic effect provides a promising new therapeutic approach by protecting lung from fibrotic remodeling and also promoting normal regeneration of lung. The development of HGF mimetics may provide a potential attractive therapy for treatment of this devastating and complex disease.

Chirality and anaesthetic drugs: A review and an update.

Many molecules can exist as right-handed and left-handed forms that are non-superimposable mirror images of each other. They are known as enantiomers or substances of opposite shape. Such compounds are also said to be chiral (Greek chiros meaning 'hand'). Such chiral molecules are of great relevance to anaesthetic theory and practice. This review summarizes the basic concepts, pharmacokinetic and pharmacodynamic aspects of chirality, and some specific examples of their application in anaesthesia, along with recent advances to elucidate the anaesthetic mechanisms. Chirality is relevant to anaesthesia, simply because more than half of the synthetic agents used in anaesthesia practice are chiral drugs. Almost all these synthetic chiral drugs are administered as racemic mixture, rather than as single pure enantiomers. These mixtures are not drug formulations containing two or more therapeutic substances, but combination of isomeric substances, with the therapeutic activity residing mainly in one of the enantiomer. The other enantiomer can have undesirable properties, have different therapeutic activities or be pharmacologically inert. Specific examples of application of chirality in anaesthetic drugs include inhalational general anaesthetics (e.g. isoflurane), intravenous anaesthetics (e.g. etomidate, thiopentone), neuromuscular blocking agents (e.g. cisatracurium), local anaesthetics (e.g. ropivacaine and levobupivacaine) and other agents (e.g. levosimendan, dexmedetomidine, L-cysteine). In the recent advances, chirality study has not only helped new drug development as mentioned above, but has also contributed in a more profound way to the understanding of the mechanism of anaesthesia and anaesthetic drugs.

Polo-like kinase inhibitors: an emerging opportunity for cancer therapeutics.

IMPORTANCE OF THE FIELD: The Polo-like kinase (Plk) family has emerged as an important regulator in cell cycle progression. Plks belong to a family of serine/threonine kinases and exist in four isoforms Plk1- 4. However, only one of these isoforms, Plk1, is shown to be involved in the activation of Cdc2, chromosome segregation, centrosome maturation, bipolar spindle formation and execution of cytokinesis. The activity of Plk1 is elevated in tissues and cells with a high mitotic index. In patients, Plk1 is overexpressed in tumors including those derived from lung, breast, colon, pancreas, prostate and ovary. Plk1 depletion is associated with the decrease in cell viability and induction of apoptosis in various cancerous cells. Several Plk1 inhibitors are in different phases of clinical development for anticancer therapy. AREAS COVERED IN THIS REVIEW: The focus of present review is to highlight Plk1 as a promising therapeutic approach for the treatment of cancer. The review discusses the role of Plk1 in cancer and the current status of Plk1 inhibitors, as well as highlighting the possible beneficial effect of inhibition of Plk1 as compared to other mitotic targets. WHAT THE READER WILL GAIN: Readers will get a comprehensive overview of Plk1 as a novel anticancer drug target. This review will also update readers about the progress made in the field of Plk1 inhibitors. TAKE HOME MESSAGE: The current literature about Plk1 inhibitors and knockout studies favor Plk1 inhibition as a potential antitumor therapy.

Human spleen tyrosine kinase (Syk) recombinant expression systems for high-throughput assays.

Spleen tyrosine kinase (Syk) is an important non-receptor tyrosine kinase and its aberrant regulation is associated with a variety of allergic disorders and autoimmune diseases. To identify small molecule inhibitors of Syk in high-throughput assays, recombinant Syk protein is needed in bulk quantity. We studied the expression of recombinant human Syk in three heterologous systems: E. coli, baculovirus expression vector system (BEVS), and the cellular slime mold Dictyostelium discoideum (Dd). Syk activity was higher in the BEVS as compared to the Dd expression host, whereas in E. coli, no activity was observed under our assay conditions. Purified Syk kinase domain protein from BEVS showed concentration dependent inhibition with OXSI-2, a known Syk inhibitor. Molecular modeling and docking studies were performed to understand the binding mode and critical interactions of the inhibitor with catalytic domain of Syk. The BEVS generated Syk kinase domain showed stability upon multiple freeze-thaw cycles and exhibited significantly higher levels of tyrosine phosphorylation at pTyr(525)/Tyr(526) in the Syk activation loop. Based on our data, we conclude that BEVS is the ideal host to produce an active and stable enzyme, which can be successfully employed for screening of Syk inhibitors in a high-throughput system.

Pharmacological profile of AW-814141, a novel, potent, selective and orally active inhibitor of p38 MAP kinase.

The p38 mitogen activated protein kinase (MAPK) is a key signaling molecule that plays a crucial role in the progression of various inflammatory diseases such as rheumatoid arthritis (RA), asthma and chronic obstructive pulmonary disease. The objective of the present study was to evaluate the anti-inflammatory activity of a p38 MAPK inhibitor, AW-814141. AW-814141 inhibited enzymatic activity of recombinant p38-alpha and beta isoforms with IC(50) value of 100nM and 158nM, respectively. AW-814141 also inhibited the release of tumor necrosis factor (TNF)-alpha by lipopolysaccharide (LPS) treated human peripheral blood mononuclear cells with an IC(50) value of 212nM and demonstrated selectivity against a panel of few kinases. Oral administration of AW-814141 (10mpk) in LPS-injected mice resulted in a significant reduction in TNF-alpha production in the circulation. In a carrageenan-induced rat paw edema model and collagen-induced arthritis model (CIA), AW-814141 dose dependently inhibited paw swelling. In different in vivo efficacy models, efficacy of AW-814141 was found to be better as compared to the reference compounds (Vx-745 and BIRB-796). This study demonstrated that AW-814141 is a novel p38 MAPK inhibitor and it displays promising in vitro and in vivo anti-inflammatory activities and can be used for the treatment of rheumatoid arthritis.

Patients with symptomatic primary hyperparathyroidism: an anaesthetic challenge.

SUMMARY: Primary hyperparathyroidism is a disease characterized by hypercalcaemia attributable to autonomous overproduction of parathormone. Many patients with primary hyperparathyroidism are asymptomatic. Osteoporosis and nephrolithiasis are some of the major sequelae seen in the symptomatic patients. Parathyroidectomy is the only curative therapy. However anaesthetic management of such patients may be problematic with associated cardiac arrhythmias and skeletal muscle weakness. Low serum albumin and alteration in the acid base status in the perioperative period can affect the serum calcium level and thus adds to the existing problem. We present the successful anaesthetic management of a patient with primary hyperparathyroidism who initially presented with pathological fractures, and discuss the anaesthetic issues involved.