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Background: Immunization is the most economic and most efficient solution to prevent children from infectious diseases. Despite launch of several programmes by the government, parents attitude and knowledge towards vaccination is what matters most. Objective:(i) To study immunization related details of children in general population of UHTC. (ii) To counsel mothers regarding immunization. Methodology: A questionnaire based cross sectional study was conducted for 2 month duration (August and September 2020) among 100 mothers who visited general outdoor of a UHTC in Udaipur city.Data was entered in MS Excel and analysed through SPSS. Result: Majority of the mothers were of the age group 21-25 years (56%),educated upto secondary school (49%), were housewives / unemployed (46%) . The source of information for the maximum participants were health workers. Majority chose government set ups for vaccination of their children (91%).Among all, 69% were fully immunized,30% were partially immunized and only 1% were not immunized at all. An overall 38% drop-out rate was seen. The most common reason of non-vaccination was found to be fear of side effects among the parents (37%).Conclusion: Active and comprehensive involvement of sociologists, behavioural scientists and health personnel is very important to produce effective changes in the attitudes and practices regarding immunization of children.

BACKGROUND: Amid the ongoing COVID-19 pandemic, we aimed to study its epidemiological and clinical characterstics OBJECTIVE: (1)To study epidemiological profile of first 100 covid cases admitted at DCH MB Hospital. (2)To study progression of disease among these covid-19 cases for 28 days.METHODOLOGY:This is observational cross sectional study of first hundred COVID-19 cases admitted at DCH, MB Hospital,Udaipur and followed up for 28 days.Data obtained from Case Investigation Form and indoor records.RESULT:Majority were males(60%)Mean age was 35.88 years in males and 35.99 years in females.History of contact present in 62% and travel in 13%.41% were symptomatic and 14% had comorbidity.Recovery had no significant relation with gender,symptoms or comorbidity.Progression was good and CFR was 1%.CONCLUSION:Recovery and progression was good among the patients,need to avoid travel and contact with others was noticed and people with comorbidities required to be more alert.

This communication discusses the discovery of novel reverse tricyclic pyridones as inhibitors of Janus kinase 2 (JAK2). By using a kinase cross screening approach coupled with molecular modeling, a unique inhibitor–water interaction was discovered to impart excellent broad kinase selectivity. Improvements in intrinsic potency were achieved by utilizing a rapid library approach, while targeted structural changes to lower lipophilicity led to improved rat pharmacokinetics. This multi-pronged approach led to the identification of 31, which demonstrated encouraging rat pharmacokinetics, in vivo potency, and excellent off-target kinase selectivity.

The poly-(ADP-ribose) polymerase (PARP) inhibitor, MK-4827, is a novel potent, orally bioavailable PARP-1 and PARP-2 inhibitor currently in phase I clinical trials for cancer treatment. No preclinical data currently exist on the combination of MK-4827 with radiotherapy. The current study examined combined treatment efficacy of MK-4827 and fractionated radiotherapy using a variety of human tumor xenografts of differing p53 status: Calu-6 (p53 null), A549 (p53 wild-type [wt]) and H-460 (p53 wt) lung cancers and triple negative MDA-MB-231 human breast carcinoma. To mimic clinical application of radiotherapy, fractionated radiation (2 Gy per fraction) schedules given once or twice daily for 1 to 2 weeks combined with MK-4827, 50 mg/kg once daily or 25 mg/kg twice daily, were used. MK-4827 was found to be highly and similarly effective in both radiation schedules but maximum radiation enhancement was observed when MK-4827 was given at a dose of 50 mg/kg once daily (EF = 2.2). MK-4827 radiosensitized all four tumors studied regardless of their p53 status. MK-4827 reduced PAR levels in tumors by 1 h after administration which persisted for up to 24 h. This long period of PARP inhibition potentially adds to the flexibility of design of future clinical trials. Thus, MK-4827 shows high potential to improve the efficacy of radiotherapy.

Polycythemia vera (PV) is a myeloproliferative disorder characterized by increased red cell mass and splenomegaly in the absence of secondary causes [Tefferi A., Spivak J.L., Polycythemia vera: scientific advances and current practice. Semin Hematol 2005;42(4):206-20.]. Recently, several laboratories have discovered that the vast majority of patients with PV carry a single, activating mutation (V617F) in the pseudokinase domain of Janus kinase 2 (Jak2) [Zhao R, Xing S, Li Z, Fu X, Li Q, Krantz SB, et al., Identification of an acquired JAK2 mutation in polycythemia vera. J Biol Chem 2005;280(24):22788-92; James C, Ugo V, Le Couedic JP, Staerk J, Delhommeau F, Lacout C, et al., A unique clonal JAK2 mutation leading to constitutive signalling causes polycythemia vera. Nature 2005;434(7037):1144-8; Kralovics R, Passamonti F, Buser AS, Teo SS, Tiedt R, Passweg JR, et al., A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Engl J Med 2005;352(17):1779-90; Levine RL, Wadleigh M, Cools J, Ebert BL, Wernig G, Huntly BJ, et al., Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer Cell 2005;7(4):387-97.]. This discovery has spurred interest in developing therapies for PV via inhibition of Jak2. We induced polycythemia in mice by administering high dose recombinant erythropoietin (Epo) and determined that administration recapitulates almost all of the major and minor diagnostic features of human PV. We then tested a selective, small molecule inhibitor of Jak2 (Jak2i) and showed that this treatment prevents polycythemia. This prevention of polycythemia was accompanied by lower hematocrits, reduced spleen sizes and reductions in Stat5 phosphorylation (pStat5). Surprisingly, Epo rapidly (

Cyclin-dependent kinases (CDKs) mediate proliferation and neuronal development, while aberrant CDK activity is associated with cancer and neurodegeneration. Consequently, pharmacologic inhibitors, such as 2,6,9-trisubstituted purines, which potently inhibit CDKs 1, 2, and 5, were developed to combat these pathologies. One agent, R -roscovitine (CYC202), has advanced to clinical trials as a potential cancer therapy. In primary neuronal cultures, these agents have been used to delineate the physiologic and pathologic functions of CDKs, and associated signaling pathways. Herein we demonstrate that three 2,6,9-trisubstituted purines: olomoucine, roscovitine, and purvalanol, used at concentrations ascribed by others to potently inhibit CDKs 1, 2, and 5, are powerful triggers of death in maturing cerebellar granule neurons, assessed by loss of mitochondrial reductive capacity and differential staining with fluorescent indicators of living/dead neurons. Based on several criteria, including delayed time course and establishment of an irreversible commitment point of death, pyknotic cell and nuclear morphology, and caspase-3 cleavage, the death process is apoptotic. However, pharmacological and biochemical data indicate that apoptosis is independent of CDK 1, 2, or 5 inhibition. This is based on the pattern of changes in c-jun mRNA, c-Jun protein, and Ca 2+ /cAMP response element binding protein (CREB) phosphorylation, and also, the ineffectiveness of structurally distinct CDK 1, 2, and 5 inhibitors butyrolactone-1 and PNU112445A to induce apoptosis. Collectively, our results, and those of others, indicate that the CDK regulation of transcription (CDKs 7 and 9) should be examined as a target of these agents, and as an indirect mediator of neuronal fate.

CaMKIV is enriched in neuronal nuclei and mediates Ca2+-dependent survival via transcription factor phosphorylation. Cultured cerebellar granule neurons were used to examine whether distinct modes of Ca2+ signaling differentially modulate CaMKIV expression and function. For long-term survival, these neurons require 25 mm KCl or NMDA, which stimulates Ca2+ entry through voltage-sensitive Ca2+ channels or NMDA receptors (NRs). Lower levels of Ca2+ entry through NRs support survival of a neuronal subpopulation grown in 5 mm KCl media. Several effects were demonstrated: (i) sustained exposure to 25 mm KCl or 140 µm NMDA produced CaMKIV down-regulation, compared to 5 mm KCl cultures; (ii) CaMKIV down-regulation was attenuated by nifedipine, APV and CaM kinase inhibitors, indicating that it is Ca2+ dependent and reversible; (iii) down-regulation was both selective for nuclear substrates and calpain-mediated; (iv) proteolysis was exacerbated by leptomycin B, a nuclear export inhibitor. Although CaMKIV proteolysis by trophic agents seems paradoxical in light of evidence supporting its critical role in survival, the CaMKIV/CREB signal transduction pathway was preserved, as assessed by CaM kinase-mediated CREB phosphorylation, and the ability of CaM kinase inhibitors to interfere with KCl-mediated survival. We hypothesize that limited calpain-mediated proteolysis of CaMKIV is a negative feedback response to the sustained activation of a Ca2+ and CaMKIV signaling pathway by these agents.

Protein kinase C (PKC) isozymes constitute a family of at least 12 structurally related serine-threonine kinases that are differentially regulated and localized, and are presumed to mediate distinct intracellular functions. To explore their roles in intact cells, investigators are developing cell-permeable, isoform-selective inhibitors. 2,2',3,3',4,4'-Hexahydroxy-1, 1'-biphenyl-6,6'-dimethanol dimethyl ether (HBDDE) is reported to be a selective inhibitor of PKC alpha and gamma with IC(50) values of 43 and 50 microM, respectively, using an in vitro assay. However, data examining the potency and selectivity of HBDDE in intact cells are lacking. Employing rodent cerebellar granule neurons as a model system, we investigated the effects of HBDDE using cell survival as a functional end-point. HBDDE induced an apoptotic form of cell death that was dependent upon protein synthesis and included activation of a terminal executioner of apoptosis, caspase 3. The concentration of HBDDE required for half-maximal cell death was less than 10 microM ( approximately 5-fold less than the reported IC(50) values for PKC alpha and gamma in vitro). Furthermore, HBDDE induced apoptosis even after phorbol-ester-mediated down-regulation of PKC alpha and gamma, indicating that this effect is independent of these isoforms. Consistent with this, 2-[1-(3-dimethylaminopropyl) indol-3-yl]-3-(indol-3-yl)-maleimide (GF 109203X), a general inhibitor of all classical and some novel PKCs, did not interfere with survival. Thus, HBDDE should not be used as an isoform-selective inhibitor of PKC alpha or gamma in intact cells. Nevertheless, identification of its target in granule neurons will provide valuable information about survival pathways.

Multiple isoforms of type II Ca(2+)-calmodulin-dependent kinase (CaM KII) are composed of two major neuron-specific subunits, designated alpha and beta, and two less well-characterized subunits that are also expressed in non-neuronal tissues, designated delta and gamma. Regulated expression of these 4 gene products, and several variants produced by alternative splicing, shows temporal and regional specificity and influences intracellular targeting. We used immunoblotting and RT-PCR to analyze subunit and variant expression and distribution in cultured cerebellar astrocytes and neurons, and whole cerebellar cortex from rodent brain. The data indicate that: (i) astrocytes express a single splice variant of delta, namely delta(2); (ii) like neurons, astrocytes express two forms of CaM KII gamma; gamma(B) and gamma(A); (iii) these CaM KII variants are enriched in the supernate fraction in astrocytes, and the particulate fraction in neurons; (iv) unlike neurons, astrocytes do not express detectable levels of alpha or beta subunits or their respective splice variants. The results indicate that neurons and astrocytes express distinct CaM KII subunits and variants that localize to distinct subcellular compartments and, by inference, exert distinct cellular functions.

A series of carboxamide-substituted thiophenes demonstrating inhibition of JAK2 is described. Development of this chemical series began with the bioisosteric replacement of a urea substituent by a pyridyl ring. Issues of chemical and metabolic stability were solved using the results of both in vitro and in vivo studies, ultimately delivering compounds such as 24 and 25 that performed well in an acute PK/PD model measuring p-STAT5 inhibition.

Aberrant kinase activation resulting from mutation, amplification, or translocation can drive growth and survival in a subset of human cancer. FGFR2 is amplified in breast and gastric cancer, and we report here the first characterization of FGFR2 gene amplification in colorectal cancer in the NCI-H716 colorectal cancer cell line. FGFR2 is highly expressed and activated in NCI-H716 cells, and FGFR selective small molecule inhibitors or FGFR2 shRNA strongly inhibited cell viability in vitro, indicating "addiction" of NCI-H716 cells to FGFR2. NCI-H716 growth in a xenograft model was also inhibited by an FGFR small molecule inhibitor. FGFR2 was required for activation of multiple downstream signaling proteins including AKT, ERK, S6RP and NFKB. Inhibition of downstream kinases such as AKT or ERK alone had modest effects on proliferation, whereas combined inhibition of AKT and ERK signaling resulted in a loss of viability similar to FGFR2 inhibition. We identified elevated FGFR2 expression in a small subset of primary colorectal cancer, however FGFR2 amplification was not observed. Although FGFR2 amplification is not common in primary colon cancer or lymph node and liver metastases, other subsets of colorectal cancer such as ascites, from which the NCI-H716 cell line was derived, have yet to be tested. These results suggest that emerging FGFR inhibitor therapeutics may have efficacy in a subset of colon cancer driven by FGFR2 amplification.

This paper describes the discovery and design of a novel class of JAK2 inhibitors. Furthermore, we detail the optimization of a screening hit using ligand binding efficiency and log D. These efforts led to the identification of compound 41, which demonstrates in vivo activity in our study.

NMDA receptors (NMDAR) contribute to neuronal development throughout the CNS. However, their mode(s) of activation preceding synaptic maturation is unclear, as they are not co-localized with alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors (AMPARs) which normally provide sufficient depolarization to relieve voltage-dependent blockade by Mg(2+). We used cerebellar granule neurons (CGNs) cultured at a near-physiological KCl concentration to examine maturation-dependent changes in NMDAR responses. In contrast, most studies use KCl-supplemented medium to promote survival. At 2-4 days in vitro CGNs: (i) express developmental markers resembling the in vivo migratory phenotype; (ii) maintain a basal amount of calcium responsive element-binding protein phosphorylation that requires NMDARs and calcium/calmodulin-dependent kinases, but not AMPARs; (iii) exhibit NMDA-mediated Ca(2+) influx not effectively blocked by ambient Mg(2+) (0.75 mM) or AMPARs; (iv) maintain a more depolarized resting membrane potential and increased resistance compared to synaptically-connected CGNs. Moreover, migrating CGNs in explant cultures demonstrate NMDA-mediated Ca(2+) influx not effectively blocked by 0.75 mM Mg(2+), and NMDAR but not AMPAR antagonists slow migration. These data suggest the biophysical properties of immature CGNs render NMDARs less sensitive to Mg(2+) blockade, enhancing the likelihood of activation in the absence of AMPAR depolarization.

J. Neurochem. (2010) 114, 191–202. Abstract NMDA receptors (NMDAR) contribute to neuronal development throughout the CNS. However, their mode(s) of activation preceding synaptic maturation is unclear, as they are not co-localized with alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors (AMPARs) which normally provide sufficient depolarization to relieve voltage-dependent blockade by Mg2+. We used cerebellar granule neurons (CGNs) cultured at a near-physiological KCl concentration to examine maturation-dependent changes in NMDAR responses. In contrast, most studies use KCl-supplemented medium to promote survival. At 2–4 days in vitro CGNs: (i) express developmental markers resembling the in vivo migratory phenotype; (ii) maintain a basal amount of calcium responsive element-binding protein phosphorylation that requires NMDARs and calcium/calmodulin-dependent kinases, but not AMPARs; (iii) exhibit NMDA-mediated Ca2+ influx not effectively blocked by ambient Mg2+ (0.75 mM) or AMPARs; (iv) maintain a more depolarized resting membrane potential and increased resistance compared to synaptically-connected CGNs. Moreover, migrating CGNs in explant cultures demonstrate NMDA-mediated Ca2+ influx not effectively blocked by 0.75 mM Mg2+, and NMDAR but not AMPAR antagonists slow migration. These data suggest the biophysical properties of immature CGNs render NMDARs less sensitive to Mg2+ blockade, enhancing the likelihood of activation in the absence of AMPAR depolarization.

c-Met is a receptor tyrosine kinase (RTK) with a critical role in many fundamental cellular processes, including cell proliferation and differentiation. Deregulated c-Met signaling has been implicated in both the initiation and progression of human cancers and therefore represents an attractive target for anticancer therapy. Monitoring the phosphorylation status of relevant tyrosine residues provides an important method of assessing c-Met kinase activity. This report describes a novel assay to monitor c-Met phosphorylation in cells using Amplified Luminescent Proximity Homogeneous Assay (AlphaScreen) technology. Using AlphaScreen, the authors were able to detect both global and site-specific phosphorylation of c-Met in transformed cell lines. Data obtained from the AlphaScreen assay were compared to data obtained from a high-content imaging (HCI) method developed in parallel to monitor c-Met phosphorylation at the single cell level. The AlphaScreen assay was miniaturized to a 384-well format with acceptable signal-to-background ratio (S/B) and Z' statistics and was employed to measure c-Met kinase activity in situ after treatment with potent c-Met-specific kinase inhibitors. The authors discuss the utility of quantifying endogenous cellular c-Met phosphorylation in lead optimization and how the modular design of the AlphaScreen assay allows its adaptation to measure cellular activity of other kinases.

CaMKIV is enriched in neuronal nuclei and mediates Ca2+-dependent survival via transcription factor phosphorylation. Cultured cerebellar granule neurons were used to examine whether distinct modes of Ca2+ signaling differentially modulate CaMKIV expression and function. For long-term survival, these neurons require 25 mm KCl or NMDA, which stimulates Ca2+ entry through voltage-sensitive Ca2+ channels or NMDA receptors (NRs). Lower levels of Ca2+ entry through NRs support survival of a neuronal subpopulation grown in 5 mm KCl media. Several effects were demonstrated: (i) sustained exposure to 25 mM KCl or 140 microM NMDA produced CaMKIV down-regulation, compared to 5 mM KCl cultures; (ii) CaMKIV down-regulation was attenuated by nifedipine, APV and CaM kinase inhibitors, indicating that it is Ca2+ dependent and reversible; (iii) down-regulation was both selective for nuclear substrates and calpain-mediated; (iv) proteolysis was exacerbated by leptomycin B, a nuclear export inhibitor. Although CaMKIV proteolysis by trophic agents seems paradoxical in light of evidence supporting its critical role in survival, the CaMKIV/CREB signal transduction pathway was preserved, as assessed by CaM kinase-mediated CREB phosphorylation, and the ability of CaM kinase inhibitors to interfere with KCl-mediated survival. We hypothesize that limited calpain-mediated proteolysis of CaMKIV is a negative feedback response to the sustained activation of a Ca2+ and CaMKIV signaling pathway by these agents.

The poly-(ADP-ribose) polymerase (PARP) inhibitor, MK-4827, is a novel potent, orally bioavailable PARP-1 and PARP-2 inhibitor currently in phase I clinical trials for cancer treatment. No preclinical data currently exist on the combination of MK-4827 with radiotherapy (XRT). We examined whether MK-4827 enhances radiosensitivity of a variety of human tumor xenografts of differing p53 status. Human lung cancer xenografts, Calu-6 (p53 null), A549 (p53 wild-type [wt]) and H-460 (p53 wt) and triple negative MDA-MB-231 human breast carcinoma xenografts were used. Tumor xenografts growing in the hind leg of nude mice were exposed to fractionated XRT at 8 mm (diameter). Fractionated XRT (2 Gy per fraction) was delivered for 7 (twice daily, 6 h in between) or 14 (once daily) consecutive days, total dose 28 Gy. MK-4827 was given by gavage once (50 mg/kg, 1 h before first XRT of the day) or twice daily (25 mg/kg, 1 h before each XRT of the day) starting when tumors were 6 mm diameter. Mice received (a) no treatment, (b) MK-4827, (c) XRT or (d) MK-4827 plus XRT. Tumor growth delay (TGD, days for tumors to grow from 8.0 mm to 12.0 mm) was the endpoint. Radiation enhancement factor (EF) was then calculated. MK-4827 alone had no effect on tumor growth for any of the tumors types. For p53 mutant Calu-6 xenografts TGD for untreated tumors was 13.2 ± 0.5 and for twice daily XRT was 30.9 ± 2.4 days. Combination of XRT for 7 days with MK-4827, 25mg/kg twice daily for 21 consecutive days, further extended TGD to 39.7 ± 2.0 days (EF 1.4) and 47.8 ± 4.0 days (EF 2.03) for MK-4827, 50mg/kg once daily. Maximum radiation enhancement was observed when MK-4827 was given at a dose of 50 mg/kg once daily. For XRT and MK-4827 given once daily, MK-4827 was continued until 2 days after completion of XRT, MK-4827 was found to be similarly effective and the EF was 1.7. For p53 wt H460and A459 xenografts, twice daily XRT and once daily MK-4827 was used. MK-4827 was continued until 2 days after completion of XRT. TGD for untreated H460 and A459 xenografts was 7.8 ± 0.5 and 33.2 ± 1.5 days respectively; whereas it was 15.6 ± 0.5 and 53.2 ± 3.1 days respectively for irradiated tumors. TGDs were further extended to 26.0 ± 3.5 days for H460 (EF 2.2) and 64.1 ± 4.2 days (EF 1.9) for A549 xenografts in MK-4827 plus XRT groups. Similarly, using MDA-MB-231 human breast carcinoma xenografts, the potency of combination treatment was greater than XRT alone showing essentially no local tumor regrowth until the occurrence of metastases. When MK-4827 was given as a single dose of 50 mg/kg and tumors were assayed for PAR, MK-4827 reduced PAR levels in tumors by 1 h after administration which persisted for up to 24 h. In conclusion, MK-4827 strongly enhanced the effect of radiation on a variety of human tumor xenografts irrespective of p53 status. The long lasting PARP inhibition (1-24h) potentially adds flexibility to future clinical trial design. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1461. doi:1538-7445.AM2012-1461

FGFR2 amplification has been reported in subsets of gastric cancer and breast cancer. We describe FGFR2 amplification in a cell line derived from a colorectal carcinoma arising in a young patient with ulcerative colitis. The cell line has features of endocrine differentiation, representing a rare (< 1%) subset of colorectal cancer. FGFR2 amplification in this cell line results in an overexpressed and highly activated FGFR2 kinase. The FGFR selective inhibitor PD173074 blocked FGFR2 phosphorylation and inhibited Erk, Akt, and S6 ribosomal protein phosphorylation. FGFR2 inhibition resulted in profound cell death as measured by robust accumulation of cells in subG1 by flow cytometry and PARP cleavage by western analysis. A structurally unrelated FGFR2 inhibitor, MK2461, also caused cell death and inhibited downstream signaling pathways. RTK arrays revealed coactivation of EGFR family kinases, and a functional role for these kinases is being investigated. Interestingly, an FGFR2 isoform in this cell line migrated at 160kD, in contrast to the 140kD isoform observed in SNU16 and other cell lines. However, C-terminal FGFR2 antibodies did not react with the 160kD FGFR2 isoform, suggesting that it has a C-terminal deletion similar to the FGFR2 isoform in OCUM2M and KatoIII cell lines. We are currently defining the frequency of FGFR2 overexpression and activation in colon cancer tissue microarrays. The strong growth inhibition and induction of cell death observed in vitro suggests that colorectal cancer tumor cells harboring FGFR2 amplification / activation may show similar response to FGFR inhibitor therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 284.