Evaluation of critical congenital heart defects screening using pulse oximetry in the neonatal intensive care unit.

OBJECTIVE: To evaluate the implementation of early screening for critical congenital heart defects (CCHDs) in the neonatal intensive care unit (NICU) and potential exclusion of sub-populations from universal screening. STUDY DESIGN: Prospective evaluation of CCHD screening at multiple time intervals was conducted in 21 NICUs across five states (n=4556 infants). RESULTS: Of the 4120 infants with complete screens, 92% did not have prenatal CHD diagnosis or echocardiography before screening, 72% were not receiving oxygen at 24 to 48 h and 56% were born ⩾2500 g. Thirty-seven infants failed screening (0.9%); none with an unsuspected CCHD. False positive rates were low for infants not receiving oxygen (0.5%) and those screened after weaning (0.6%), yet higher among infants born at <28 weeks (3.8%). Unnecessary echocardiograms were minimal (0.2%). CONCLUSION: Given the majority of NICU infants were ⩾2500 g, not on oxygen and not preidentified for CCHD, systematic screening at 24 to 48 h may be of benefit for early detection of CCHD with minimal burden.

Evaluation of Role of Mastoid Surgery in the Management of Safe Chronic Suppurative Otitis Media.

This study evaluates the outcome of type 1 tympanoplasty with and with out mastoidectomy. The comparative study comprises of 40 patients with CSOM safe type in dry ear. All cases were operated during a period of one and a half years. 20 of these cases were selected for tympanoplasty alone (Group A) and 20 cases were selected for Tympanoplasty with cortical mastoidectomy (Group B). Patients were reviewed after 3 weeks for inspection of the operated ear. The second and third postoperative reviews were done 6 and 12 weeks respectively for clinical assessment of the operated ear with respect to graft status, ear discharge and hearing improvement. The postoperative audiograms were recorded after 3 months. Type I tympanoplasty with cortical mastoidectomy has better graft uptake (100 %) as compared to without mastoidectomy (95 %). Post-operative A-B gap closure is better in tympano-mastoidectomy (20.48 dB) than tympanoplasty (15.75 dB) with p value <0.05. Post-operative hearing gain and graft uptake were both better with tympano-mastoidectomy and tympanoplasty.

Apolipoprotein A-I in Labeo rohita: Cloning and functional characterisation reveal its broad spectrum antimicrobial property, and indicate significant role during ectoparasitic infection.

Apolipoprotein A-I (ApoA-I) is the most abundant and multifunctional high-density lipoprotein (HDL) having a major role in lipid transport and potent antimicrobial activity against a wide range of microbes. In this study, a complete CDS of 771 bp of Labeo rohita (rohu) ApoA-I (LrApoA-I) encoding a protein of 256 amino acids was amplified, cloned and sequenced. Tissue specific transcription analysis of LrApoA-I revealed its expression in a wide range of tissues, with a very high level of expression in liver and spleen. Ontogenic study of LrApoA-I expression showed presence of transcripts in milt and 3 h post-fertilization onwards in the larvae. The expression kinetics of LrApoA-I was studied upon infection with three different types of pathogens to elucidate its functional significance. Its expression was found to be up-regulated in the anterior kidney of L. rohita post-infection with Aeromonas hydrophila. Similarly following poly I:C (poly inosinic:cytidylic) stimulation, the transcript levels increased in both the anterior kidney and liver tissues. Significant up-regulation of LrApoA-I expression was observed in skin, mucous, liver and anterior kidney of the fish challenged with the ectoparasite Argulus siamensis. Immunomodulatory effect of recombinant LrApoA-I (rApoA-I) produced in Escherichia coli was demonstrated against A. hydrophila challenge in vivo. L. rohita administered with rApoA-I at a dose of 100 µg exhibited significantly higher protection (∼55%) upon challenge with A. hydrophila 12 h post-administration of the protein, in comparison to that observed in control group, along with higher level of expression of immune-related genes. The heightened expression of ApoA-I observed post-infection reflected its involvement in immune responses against a wide range of infections including bacterial, viral as well as parasitic pathogens. Our results also suggest the possibility of using rApoA-I as an immunostimulant, particularly rendering protection against A. hydrophila.

Four pro-inflammatory cytokines of rohu (Labeo rohita) during early developmental stages, their tissue distribution and expression by leucocytes upon in-vitro stimulation.

Cytokines are important components of both adaptive and innate immunity, and are required to initiate and regulate immune responses following infection. The ontogeny and tissue specific distribution of four pro-inflammatory cytokines, interleukin-6 (IL-6), tumor necrosis factor α (TNF-α), IL-8 and IL-1ß in rohu (Labeo rohita), and their responses by leucocytes from anterior-kidney/head-kidney (HKLs), spleen (SPLs) and peripheral blood (PBLs) following stimulation with concanavalin A (ConA), ConA with phorbol 12-myristate 13-acetate (ConA/PMA) and formalin-killed Aeromonas hydrophila cells (FAH) were studied. In ontogeny study, mRNA levels of IL-6 and IL-1ß were evident in unfertilized egg stages of L. rohita whereas IL-8 and TNF-α transcripts were found from 1 to 3 h post-fertilization (hpf) onwards till day 15 post-fertilization, respectively. Basal level of all four cytokines was observed in all twelve tissues (eye, brain, heart, gill, anterior kidney, posterior kidney, spleen, liver, skin, muscle, hindgut and foregut) of L. rohita juveniles. Expression levels of IL-6 and IL-8 were found to be the highest in liver and heart tissues, respectively, while TNF-α transcripts were high in anterior kidney and liver tissues. Transcripts of IL-1ß showed high expression in muscle, heart and spleen. Upon in vitro stimulation of leucocytes, there was variable up-regulation of all the four cytokines following different treatments throughout the experimental time period. Induction of cytokines was more pronounced in PBLs stimulated with FAH compared to other stimuli. However, an up-regulated IL-8 expression was evident in all the leucocytes following stimulation with FAH thus indicating IL-8 could be used as an indicator or indirect marker to monitor vaccine status or health status of L. rohita during bacterial infection.

Immune responses and protective efficacy of recombinant outer membrane protein R (rOmpR)-based vaccine of Aeromonas hydrophila with a modified adjuvant formulation in rohu (Labeo rohita).

Despite the importance and success of developing a candidate vaccine against Aeromonas hydrophila infection in fish, little is known about the molecular mechanisms of the vaccine-induced immunoprotection in Indian major carp, Labeo rohita, primarily due to lack of information on most of the immune related genes of the species. In this study, a novel candidate antigen recombinant outer membrane protein R (rOmpR) of A. hydrophila was evaluated as a vaccine candidate along with a modified adjuvant formulation. Protective efficacy of the rOmpR immunization was assessed in terms of survival against A. hydrophila challenge as well as modulation of immune response in vaccinated fish after 1, 3, 6, 12, 24, 72 h and 10 days post-injection (using immune gene expression analysis) and 10, 28, 56 and 140 days post-injection (serum immune parameter analysis). The generated immune response was compared with a formalin-killed A. hydrophila antigen preparation using mineral oil only and modified adjuvant alone. We report a variable up-regulation of the immune-related genes viz., lysozyme G, complement factor 4, immunoglobulin M, ß2-microglobulin, major histocompatibility complex I and II, and interleukin-1ß in anterior kidney and spleen tissues at early time points post-immunization in all the groups, when compared to the control fish. The vaccinated fish also showed an increase in serum natural hemolysin titer, lysozyme and myeloperoxidase activities, and antibody titer irrespective of vaccine formulations as compared to control fish on days 10, 28 and 56. However, the increase in the serum parameters was more pronounced on day 140 in rOmpR-modified adjuvant injected group, indicating the modulatory role of this new vaccine formulation. Upon challenge with live A. hydrophila on days 56 and 140 post-immunization, significantly reduced percent mortality was noted in the group immunized with modified adjuvant based rOmpR vaccine formulation. Taken together, our results suggest that rOmpR along with modified adjuvant could potentially be used as a vaccine formulation to handle A. hydrophila infection on a long-term basis.

Is it beneficial to increase the provision of thrombolysis?-- a discrete-event simulation model.

BACKGROUND: Although Thrombolysis has been licensed in the UK since 2003, it is still administered only to a small percentage of eligible patients. AIM: We consider the impact of investing the impact of thrombolysis on important acute stroke services, and the effect on quality of life. The concept is illustrated using data from the Northern Ireland Stroke Service. DESIGN: Retrospective study. METHODS: We first present results of survival analysis utilizing length of stay (LOS) for discharge destinations, based on data from the Belfast City Hospital (BCH). None of these patients actually received thrombolysis but from those who would have been eligible, we created two initial groups, the first representing a scenario where they received thrombolysis and the second comprising those who do not receive thrombolysis. On the basis of the survival analysis, we created several subgroups based on discharge destination. We then developed a discrete event simulation (DES) model, where each group is a patient pathway within the simulation. Coxian phase type distributions were used to model the group LOS. Various scenarios were explored focusing on cost-effectiveness across hospital, community and social services had thrombolysis been administered to these patients, and the possible improvement in quality of life, should the proportion of patients who are administered thrombolysis be increased. Our aim in simulating various scenarios for this historical group of patients is to assess what the cost-effectiveness of thrombolysis would have been under different scenarios; from this we can infer the likely cost-effectiveness of future policies. RESULTS: The cost of thrombolysis is offset by reduction in hospital, community rehabilitation and institutional care costs, with a corresponding improvement in quality of life. CONCLUSION: Our model suggests that provision of thrombolysis would produce moderate overall improvement to the service assuming current levels of funding.

Optimization of inclusion body solubilization and renaturation of recombinant human growth hormone from Escherichia coli.

Recombinant human growth hormone (r-hGH) was expressed in Escherichia coli as inclusion bodies. In 10 h of fed-batch fermentation, 1.6 g/L of r-hGH was produced at a cell concentration of 25 g dry cell weight/L. Inclusion bodies from the cells were isolated and purified to homogeneity. Various buffers with and without reducing agents were used to solubilize r-hGH from the inclusion bodies and the extent of solubility was compared with that of 8 M urea as well as 6 M Gdn-HCl. Hydrophobic interactions as well as ionic interactions were found to be the dominant forces responsible for the formation of r-hGH inclusion bodies during its high-level expression in E. coli. Complete solubilization of r-hGH inclusion bodies was observed in 100 mM Tris buffer at pH 12.5 containing 2 M urea. Solubilization of r-hGH inclusion bodies in the presence of low concentrations of urea helped in retaining the existing native-like secondary structures of r-hGH, thus improving the yield of bioactive protein during refolding. Solubilized r-hGH in Tris buffer containing 2 M urea was found to be less susceptible to aggregation during buffer exchange and thus was refolded by simple dilution. The r-hGH was purified by use of DEAE-Sepharose ion-exchange chromatography and the pure monomeric r-hGH was finally obtained by using size-exclusion chromatography. The overall yield of the purified monomeric r-hGH was approximately 50% of the initial inclusion body proteins and was found to be biologically active in promoting growth of rat Nb2 lymphoma cell lines.

N-terminus of mature heat-labile enterotoxin chain B is critical for its extracellular secretion in Vibrio cholerae.

The effects of addition of a few amino acids to the amino- and carboxy-terminal regions of the mature portion of the heat-labile enterotoxin chain B (LTB) of Escherichia coli on protein export, secretion and assembly were investigated. In E. coli, LTB (secretory protein) with or without the extension at the N- or C-terminus accumulated in the periplasmic fraction. For Vibrio cholerae, LTB with the extension at the C-terminus was exported to the periplasm followed by secretion to the extracellular milieu. However, LTB with the N-terminus extension was exported to the periplasm only. Our findings suggest that in the case of V. cholerae, the N-terminus of the mature LTB plays an important role in its secretion to the extracellular milieu.

Effect of the codon following the ATG start site on the expression of ovine growth hormone in Escherichia coli.

For expression of ovine growth hormone (OGH) in inclusion bodies without an affinity histidine tag at either end of the protein, three clones, differing only in the second codon following the ATG start site, were constructed. Their expression was studied by SDS-PAGE followed by immunoblotting. Clone Ala.OGH (clone 1), beginning with Met.Ala.Phe.Pro ellipsis, did not show any expression. Clone Phe.OGH (clone 3), beginning with Met.Phe.Pro ellipsis, gave very high levels of OGH expression following IPTG induction. However, in clone Gly.OGH (clone 2), in which the Ala codon was replaced with a Gly codon at the second position after the start site, a lower level of expression was obtained. Northern hybridization analysis showed that upon IPTG induction, OGH mRNA was transcribed from all three clones. These results therefore, imply that lack of expression in clone 1 and a lower level of expression in clone 2 are not due to a failure of transcription; however, they may be due to inefficient initiation of translation. The secondary structure analysis of mRNA predicts inaccessibility of different elements of the RBS in the case of Ala.OGH (clone 1). The present study highly underscores the importance of mRNA secondary structure at the start site in regulation of expression of a cloned gene in Escherichia coli, a prokaryotic expression system.

Molecular cloning and sequence analysis of bubaline lactoferrin promoter.

The proximal promoter for bubaline lactoferin-encoding gene has been isolated, cloned and sequenced. A 468 bp fragment of the 5' flanking region of the lactoferrin gene was PCR amplified and cloned into pUC18 vector. Sequence analysis of the amplified fragment revealed the presence of one TATA box, one TATA like element, two GC boxes and one motif resembling cAMP response element (CRE) in this region. Bubaline lactoferrin promoter shares 93%, 53%, 52% and 48% homology with cattle, pig, mouse and human lactoferrin 5' flanking region, respectively.

cDNA cloning and sequence analysis of bubaline growth hormone.

The cDNA for Bubalus bubalis growth hormone (GH) has been cloned and sequence determined through RT-PCR approach. The nucleotide sequence of bubaline GH cDNA was in a single reading frame coding for a protein of 191 residues comprising a putative signal sequence of 27 amino acids. Homology comparison of the sequence with other mammalian GH cDNAs showed a very high degree of evolutionary conservation. Bubaline GH sequence shared a homology of 99.5%, 99.5%, 98.6%, 87.6% and 61.9% with that of ovine, caprine, bovine, porcine and human, respectively at amino acid level.

Molecular cloning and sequence analysis of the cDNA encoding beta-lactoglobulin in Bubalus bubalis.

The cDNA for bubaline beta-lactoglobulin (beta lg) has been cloned through RT-PCR approach and sequenced. Sequence data showed a single open reading frame coding for a protein of 180 amino acids with a signal sequence of 18 amino acid residues. Comparison with other ruminant beta lg sequences revealed a high homology indicating the protein to be conserved through evolution. The degree of homology, at amino acid level, is 96.1%, 95.6%, 93.9% and 63.7% with goat, sheep, cow and pig, respectively.

Isolation and separation of HMG proteins and histones H1 and H5 and core histones by column chromatography on phosphocellulose.

A single-step method to dissociate histones as well as nonhistone chromosomal proteins from chicken erythrocyte chromatin and their separation into histones H1, H5, core histones, and high mobility group proteins by column chromatography on phosphocellulose is presented. NaCl at 2.0 M is effective in dissociating both histones and nonhistone proteins. The core histones elute as a complex. The pH is a critical factor in separating H5 from the core histones.

Expression and stable germline transmission of neomycin-resistance gene in transgenic mice.

Transgenic mice were produced to study the expression of amino-3' glycosyl phosphotransferase gene (neomycin resistance gene) in the embryonic fibroblast cells. A 1.9 Kb linear fragment of neomycin resistance gene under the control of pPGK promoter was microinjected into the pronucleus of mouse embryos. Out of 64 potential founders born, 5 were identified to be transgenic by the polymerase chain reaction (PCR) and southern hybridization. Multiple mice from first and second generation from two transgenic founders (N-10 and N-32) were analysed to determine the germline transmission. It was found to be 24.6 and 71.4% in first and second generation respectively. Results were also further confirmed by RT-PCR, sequencing and in vitro bioassays.

Ca(2+)-ATPases in the cochlear duct.

Differing levels of the Ca(2+)-ATPase enzymes that reside on the plasma membrane (PM) and on the endoplasmic reticulum (ER) were identified in individual rat cochlear tissues by the use of a semi-quantitative enzyme-linked immunosorbent assay (ELISA). Unlike other studies, a specific antibody to PM Ca(2+)-ATPase was used to detect significantly greater levels (about 2x) of PM Ca(2+)-ATPase in the stria vascularis (SV) than that in the spiral ligament (SL) and organ of Corti (OC) tissues. Similarly, levels of ER Ca(2+)-ATPase were also significantly higher in the SV than in the SL and OC tissues. The presence of ER Ca(2+)-ATPase in the tissues of the SV has not been demonstrated previously. Given the importance of Ca2+ homeostasis in the inner ear, the statistically significantly higher densities of both PM and ER Ca(2+)-ATPase measured in the SV relative to the SL and OC regions would indicate tissue-specific responses to fluctuations in systemic and local Ca2+ concentrations.

Altered calcium homeostasis in the rat cochlear duct and endogenous corticosteroid insufficiency.

Free calcium concentration (CCa2+) profiles were evaluated in perilymph, endolymph, marginal cells, spiral ligament and blood serum of adrenalectomized (ADX) rats. Free CCa2+ was significantly greater in perilymph and significantly reduced in the serum of the ADX animals as compared to sham-operated animals. In addition, higher levels of free CCa2+ were found in the spiral ligament in ADX animals. Free CCa2+ did not appear to be affected by ADX in marginal cells and endolymph. These data suggest that marked reductions in endogenous levels of corticosteroids may have a systematic effect on free CCa2+ that is detectable in blood serum as well as cochlear fluids and tissues.