Repurposing antiviral drugs on recently emerged viral infections: A review article.

Finer understandings of drugs, for newly emerged diseases are becoming difficult nowadays. The contemporary approach is Drug Repurposing. Drug repurposing implies the exploration of surviving drugs for new restorative motive. Apart from conventional drug approaches, it is a profitable, brisk and reliable approach. The equivalent therapies for newly emerging and remerging viral infections are strenuous spot these days. The drug repurposing has helped in treating many viral reprofiling infectious diseases like CoVID-19, MERS, SARS, Influenza, Swine flu, Hanta, Zika, Ebola, Marburg, Human Adeno virus infection etc. The present review looks at describing the drug repurposing approach in various viral infections.

Therapeutic hypothermia for moderate and severe hypoxic ischaemic encephalopathy in newborns using low-cost devices - ice packs and phase changing material.

Background: Different methods have been used for therapeutic hypothermia for neonates with moderate-to-severe hypoxic ischaemic encephalopathy (HIE). As standard cooling devices are expensive, there is a need to establish the safety and efficacy of low-cost devices such as ice packs (IP) and phase changing material (PCM). Aim: To assess the efficacy and safety of therapeutic hypothermia (TH) and the clinico-laboratory profile of neonates who underwent cooling with IP or PCM. Methods: The study was retrospective. TH for moderate-to-severe HIE was initiated with IP between 2012 and 2014 and with PCM (MiraCradleTM) from September 2014. A standard protocol for inclusion and management during TH was used for all newborns. All data were collected by means of a local cooling registry. Results: Sixty-two cooled newborns (IP 29, PCM 33) were included in the study. Mean gestational age was 38.6 (1.7) weeks and mean birthweight 2920.6 g (450.7); 66.1% were inborn and 91.9% had moderate encephalopathy. Mean (SD) core temperature during cooling was 33.47°C (0.33) for PCM and 33.44°C (0.34) for IP. Adverse events observed during TH were thrombocytopenia (54.8%), coagulopathy (30.6%), shock (30.6%), skin changes (12.9%) and persistent pulmonary hypertension (8.1%). Forty-nine infants were discharged, two died and 11 were discharged against medical advice. TH was prematurely stopped in seven newborns with serious adverse events such as disseminated intravascular coagulation (DIC), gangrene and arrhythmia (IP 5, PCM 2). Conclusion: Low-cost devices are safe and effective alternatives for maintaining TH in low-resource settings with adequate monitoring. Abbreviations: DAMA, discharged against medical advice; DIC, disseminated intravascular coagulation; HELIX, Hypothermia for Encephalopathy in Low- and Middle-Income Countries Trial; HIE, hypoxic ischaemic encephalopathy; IP, ice packs; LMIC, low- and middle-income countries; NICHD, National Institute of Child Health and Human Development; PCM, phase changing; TH, therapeutic hypothermia (TH); TOBY, total body hypothermia for neonatal encephalopathy.

Umbilical cord milking in preterm neonates requiring resuscitation: A randomized controlled trial.

OBJECTIVE: To evaluate the effect of cord milking on short term morbidity and hematologic parameters at 6 weeks in preterm neonates requiring resuscitation. METHODS: This trial randomized preterm infants requiring resuscitation to milking group and no milking group. Multiple pregnancy, Rh negative mothers, hydrops, cord abnormalities were excluded. The primary outcome was hemoglobin and serum ferritin at 6 weeks of life. Secondary outcomes were common preterm morbidities and mortality. RESULTS: 60 neonates were included in the study. Infants in the milking group had higher hemoglobin (10.07 g/dl vs 8.9 g/dl; p 0.003) and higher serum ferritin level (244.8 ng/ml vs 148.5 ng/ml; p 0.04) compared to no milking group. CONCLUSIONS: In preterm neonates requiring resuscitation, umbilical cord milking results in higher hemoglobin and ferritin at 6 weeks of life. It can be a used as a placental transfusion strategy in preterm neonates requiring resuscitation with no significant adverse effects. CLINICAL TRIAL REGISTRATION: Clinical trials registry -India CTRI/2015/01/005436, www.ctri.nic.in.

Probiotics for Promoting Feed Tolerance in Very Low Birth Weight Neonates - A Randomized Controlled Trial.

OBJECTIVE: To measure the efficacy of a probiotic formulation on time to reach full enteral feeds in VLBW (very low birth weight) newborns. DESIGN: Blinded randomized control trial. SETTING: A tertiary care neonatal intensive care unit (NICU) in Southern India between August 2012 to November 2013. PARTICIPANTS: 104 newborns with a birth weight of 750-1499 g on enteral feeds. INTERVENTION: Probiotic group (n=52) received a multicomponent probiotic formulation of Lactobacillus acidophilus, Lactobacillus rhamnosus, Bifidobacterium longum and Saccharomyces boulardii once a day at a dose of 1.25×109 CFU from the time of initiation of enteral feeds till discharge and the control group (n=52) received only breast milk. OUTCOME MEASURE: Time to reach full enteral feeds (150 mL/kg/day). RESULTS: The mean (SD) time to reach full enteral feeding was 11.2 (8.3) days in probiotic vs. 12.7 (8.9) in no probiotic group; (P=0.4), and was not significantly different between the two study groups. There was a trend towards lower necrotizing enterocolitis in the probiotic group (4% vs. 12%). CONCLUSION: Probiotic supplementation does not seem to result in significant improvement of feed tolerance in VLBW newborns.

Effect of early versus delayed cord clamping on hematological status of preterm infants at 6 wk of age.

OBJECTIVE: To compare the effect of early cord clamping (ECC) vs. delayed cord clamping (DCC) on hematocrit and serum ferritin at 6 wk of life in preterm infants. METHODS: This randomized controlled trial was conducted in the delivery room and neonatal intensive care unit of a tertiary hospital. One hundred preterm infants born between 30 (0)/7 and 36 (6)/7 wk were randomized to either early or delayed cord clamping groups. Parental informed consent was obtained prior to the delivery. In the ECC group, the cord was clamped immediately after the delivery of the baby and in the DCC group; the cord was clamped beyond 2 min after the baby was delivered. Hematocrit and serum ferritin at 6 wk of life were the primary outcomes. Incidence of anemia, polycythemia and significant jaundice were the main secondary outcomes. RESULTS: The mean hematocrit (27.3 ± 3.8 % vs. 31.8 ± 3.5 %, p value 0.00) and the mean serum ferritin (136.9 ± 83.8 ng/mL vs. 178.9 ± 92.8 ng/mL, p value 0.037) at 6 wk of age were significantly higher in the infants randomized to DCC group. The hematocrit on day 1 was also significantly higher in the DCC group (50.8 ± 5.2 % vs. 58.5 ± 5.1 %, p value 0.00). The DCC group required significantly longer duration of phototherapy (55.3 ± 40.0 h vs. 36.7 ± 32.6 h, p value 0.016) and had a trend towards higher risk of polycythemia. CONCLUSIONS: Delaying the cord clamping by 2 min, significantly improves the hematocrit value at birth and this beneficial effect continues till at least 2nd mo of life.

Dental trauma in children with disabilities in India: a comparative study.

AIM: To investigate and describe the characteristics of traumatic dental injury (TDI) in children with disabilities attending special schools in Bangalore, India and to compare these with a matched group of healthy children. METHODS: The sample included 231 children with disabilities aged 6-16 years and 231 age- and sex-matched healthy children. Data were collected through clinical examinations according to the modified Ellis classification of TDI. RESULTS: All the dental injuries involved maxillary incisor teeth, and trauma was noted in 12.1 % of disabled children as compared to 6.9 % among the control group which showed statistical significance. There was no difference in the distribution of traumatic injuries between the genders and no difference in the mean age was found between the study and the control groups. Simple fractures involving little or no dentine were the most frequent type of injury. CONCLUSIONS: The data suggest that the TDI prevalence in children with disability was higher than that of non-disabled children.

Screen for inhibitors of the coupled transglycosylase-transpeptidase of peptidoglycan biosynthesis in Escherichia coli.

Class A high-molecular-weight penicillin-binding protein 1a (PBP1a) and PBP1b of Escherichia coli have both transglycosylase (TG) and transpeptidase (TP) activity. These enzymes are difficult to assay, since their substrates are difficult to prepare. We show the activity of PBP1a or PBP1b can be measured in membranes by cloning the PBP into an E. coli ponB::Spcr strain. Using this assay, we show that PBP1a is approximately 10-fold more sensitive to penicillin than PBP1b and that the 50% inhibitory concentration (IC50) of moenomycin, a TG inhibitor, is approximately 10-fold higher in the PBP transformants than in wild-type membranes; this increase in IC50 in transformants can be used to test the specificity of test compounds for inhibition of the TG. Alternatively, the coupled TG-TP activity of PBP1b can be directly measured in a two-step microplate assay. In the first step, radiolabeled lipid II, the TG substrate, was made in membranes of the E. coli ponB::Spcr strain by incubation with the peptidoglycan sugar precursors. In the second step, the TG-TP activity was assayed by adding a source of PBP1b to the membranes. The coupled TG-TP activity converts lipid II to cross-linked peptidoglycan, which was specifically captured by wheat germ agglutinin-coated scintillation proximity beads in the presence of 0.2% Sarkosyl (B. Chandrakala et al., Antimicrob. Agents Chemother. 48:30-40, 2004). The TG-TP assay was inhibited by penicillin and moenomycin as expected. Surprisingly, tunicamycin and nisin also inhibited the assay, and paper chromatography analysis revealed that both inhibited the transglycosylase. The assay can be used to screen for novel antibacterial agents.

Development of a microplate-based scintillation proximity assay for MraY using a modified substrate.

MraY is an established target for the discovery of antibacterial agents. The conventional assay for MraY uses radioactive substrate and analysis of products after paper chromatography or butanol extraction. Synthesis of radiolabeled substrate has been done in vitro using purified enzymes or by growing cells on radiolabeled precursors. The authors report a simple and rapid method to chemically radiolabel MraY substrate, UDP-MurNAc-pentapeptide. Specific activity obtained by this method was more than 100 times higher than the conventionally labeled substrate, and yields are high enough to support the requirements of high-throughput screening (HTS). The authors have developed a microplate-based homogeneous assay for MraY in which the product is captured on wheat germ agglutinin (WGA) scintillation proximity assay (SPA) beads. The assay was validated by showing inhibition by specific inhibitors of MraY but not by inhibitors of other enzymes of peptidoglycan synthesis. The assay uses wild-type membranes of Escherichia coli, giving it an advantage over recently described assays that need the protein to be overexpressed. In addition, it has an advantage over the high-throughput MraY-MurG coupled assay reported in the literature because it is MraY specific, and therefore hits obtained in this assay do not need further deconvolution. It has potential for use in HTS approaches to find novel inhibitors of MraY.

Scintillation proximity assay for inhibitors of Escherichia coli MurG and, optionally, MraY.

MurG and MraY, essential enzymes involved in the synthesis of bacterial peptidoglycan, are difficult to assay because the substrates are lipidic and hard to prepare in large quantities. Based on the use of Escherichia coli membranes lacking PBP1b, we report a high-throughput method to measure the activity of MurG and, optionally, MraY as well. In these membranes, incubation with the two peptidoglycan sugar precursors results in accumulation of lipid II rather than the peptidoglycan produced by wild-type membranes. MurG was assayed by addition of UDP-[3H]N-acetylglucosamine to membranes in which lipid I was preformed by incubation with UDP-N-acetyl-muramylpentapeptide, and the product was captured by wheat germ agglutinin scintillation proximity assay beads. In a modification of the assay, the activity of MraY was coupled to that of MurG by addition of both sugar precursors together in a single step. This allows simultaneous detection of inhibitors of either enzyme. Both assays could be performed using wild-type membranes by addition of the transglycosylase inhibitor moenomycin. Nisin and vancomycin inhibited the MurG reaction; the MraY-MurG assay was inhibited by tunicamycin as well. Inhibitors of other enzymes of peptidoglycan synthesis--penicillin G, moenomycin, and bacitracin--had no effect. Surprisingly, however, the beta-lactam cephalosporin C inhibited both the MurG and MraY-MurG assays, indicating a secondary mechanism by which this drug inhibits bacterial growth. In addition, it inhibited NADH dehydrogenase in membranes, a hitherto-unreported activity. These assays can be used to screen for novel antibacterial agents.

High-throughput screen for inhibitors of transglycosylase and/or transpeptidase activities of Escherichia coli penicillin binding protein 1b.

Penicillin binding protein (PBP) 1b of Escherichia coli has both transglycosylase and transpeptidase activities, which are attractive targets for the discovery of new antibacterial agents. A high-throughput assay that detects inhibitors of the PBPs was described previously, but it cannot distinguish them from inhibitors of the MraY, MurG, and lipid pyrophosphorylase. We report on a method that distinguishes inhibitors of both activities of the PBPs from those of the other three enzymes. Radioactive peptidoglycan was synthesized by using E. coli membranes. Following termination of the reaction the products were analyzed in three ways. Wheat germ agglutinin (WGA)-coated scintillation proximity assay (SPA) beads were added to one set, and the same beads together with a detergent were added to a second set. Type A polyethylenimine-coated WGA-coated SPA beads were added to a third set. By comparison of the results of assays run in parallel under the first two conditions, inhibitors of the transpeptidase and transglycosylase could be distinguished from inhibitors of the other enzymes, as the inhibitors of the other enzymes showed similar inhibitory concentrations (IC(50)s) under both conditions but the inhibitors of the PBPs showed insignificant inhibition in the absence of detergent. Furthermore, comparison of the results of assays run under conditions two and three enabled the distinction of transpeptidase inhibitors. Penicillin and other beta-lactams showed insignificant inhibition with type A beads compared with that shown with WGA-coated SPA beads plus detergent. However, inhibitors of the other four enzymes (tunicamycin, nisin, bacitracin, and moenomycin) showed similar IC(50)s under both conditions. We show that the main PBP being measured under these conditions is PBP 1b. This screen can be used to find novel transglycosylase or transpeptidase inhibitors.

S-adenosyl-L-methionine is required for DNA cleavage by type III restriction enzymes.

The requirement of S-adenosyl-L-methionine (AdoMet) in the cleavage reaction carried out by type III restriction-modification enzymes has been investigated. We show that DNA restriction by EcoPI restriction enzyme does not take place in the absence of exogenously added AdoMet. Interestingly, the closely related EcoP15I enzyme has endogenously bound AdoMet and therefore does not require the addition of the cofactor for DNA cleavage. By employing a variety of AdoMet analogs, which differ structurally from AdoMet, this study demonstrates that the carboxyl group and any substitution at the epsilon carbon of methionine is absolutely essential for DNA cleavage. Such analogs could bring about the necessary conformational change(s) in the enzyme, which make the enzyme proficient in DNA cleavage. Our studies, which include native polyacrylamide gel electrophoresis, molecular size exclusion chromatography, UV, fluorescence and circular dichroism spectroscopy, clearly demonstrate that the holoenzyme and apoenzyme forms of EcoP15I restriction enzyme have different conformations. Furthermore, the Res and Mod subunits of the EcoP15I restriction enzyme can be separated by gel filtration chromatography in the presence of 2 M NaCl. Reconstitution experiments, which involve mixing of the isolated subunits, result in an apoenzyme form, which is restriction proficient in the presence of AdoMet. However, mixing the Res subunit with Mod subunit deficient in AdoMet binding does not result in a functional restriction enzyme. These observations are consistent with the fact that AdoMet is required for DNA cleavage. In vivo complementation of the defective mod allele with a wild-type mod allele showed that an active restriction enzyme could be formed. Furthermore, we show that while the purified c2-134 mutant restriction enzyme is unable to cleave DNA, the c2-440 mutant enzyme is able to cleave DNA albeit poorly. Taken together, these results suggest that AdoMet binding causes conformational changes in the restriction enzyme and is necessary to bring about DNA cleavage.

Novel scintillation proximity assay for measuring membrane-associated steps of peptidoglycan biosynthesis in Escherichia coli.

We have developed a novel, high-throughput scintillation proximity assay to measure the membrane-associated steps (stages 2 and 3) of peptidoglycan synthesis in Escherichia coli. At least five enzymes are involved in these two stages, all of which are thought to be essential for the survival of the cell. The individual enzymes are difficult to assay since the substrates are lipidic and difficult to isolate in large quantities and analysis is done by paper chromatography. We have assayed all five enzymes in a single mixture by monitoring synthesis of cross-linked peptidoglycan, which is the final product of the pathway. E. coli membranes are incubated with the two sugar precursors, UDP-N-acetyl muramylpentapeptide and UDP-[(3)H]-N-acetylglucosamine. The radiolabel is incorporated into peptidoglycan, which is captured using wheat germ agglutinin-coated scintillation proximity assay beads. The assay monitors the activity of the translocase (MraY), the transferase (MurG), the lipid pyrophosphorylase, and the transglycosylase and transpeptidase activities of the penicillin-binding proteins. Vancomyin, tunicamycin, nisin, moenomycin, bacitracin, and penicillin inhibit the assay, and these inhibitors have been used to validate the assay. The search for new antimicrobial agents that act via the late stages of peptidoglycan biosynthesis can now be performed in high throughput in a microtiter plate.