Transcriptional analysis of an immune-responsive serine protease from Indian malarial vector, Anopheles culicifacies

<p>Abstract</p> <p>Background</p> <p>The main vector for transmission of malaria in India is the <it>Anopheles culicifacies </it>mosquito species, a naturally selected subgroup of which is completely refractory (R) to transmission of the malaria parasite, <it>Plasmodium vivax</it>;</p> <p>Results</p> <p>Here, we report the molecular characterization of a serine protease (<it>acsp30</it>)-encoding gene from <it>A. culicifacies</it>, which was expressed in high abundance in the refractory strain compared to the susceptible (S) strain. The transcriptional upregulation of <it>acsp30 </it>upon <it>Plasmodium </it>challenge in the refractory strain coincided with ookinete invasion of mosquito midgut. Gene organization and primary sequence of <it>acsp30 </it>were identical in the R and S strains suggesting a divergent regulatory status of <it>acsp30 </it>in these strains. To examine this further, the upstream regulatory sequences of <it>acsp30 </it>were isolated, cloned and evaluated for the presence of promoter activity. The 702 bp upstream region of <it>acsp30 </it>from the two strains revealed sequence divergence. The promoter activity measured by luciferase-based reporter assay was shown to be 1.5-fold higher in the R strain than in the S. Gel shift experiments demonstrated a differential recruitment of nuclear proteins to upstream sequences of <it>acsp30 </it>as well as a difference in the composition of nuclear proteins in the two strains, both of which might contribute to the relative abundance of <it>acsp30 </it>in the R strain;</p> <p>Conclusion</p> <p>The specific upregulation of <it>acsp30 </it>in the R strain only in response to <it>Plasmodium </it>infection is suggestive of its role in contributing the refractory phenotype to the <it>A. culicifacies </it>mosquito population.</p

Comparative Study of the Effect of Early Versus Late Initiation of Epidural Analgesia on Labour

Background: Epidural analgesia also known as regional analgesia has been established as a safe and an effective method of pain relief during labor. It was thought that epidurals may possibly interfere with labor and consequently increase the rate of cesarean deliveries or instrumental deliveries or other adverse effect. . A more recent review concluded that epidural analgesia is not associated with such a risk. But, the timing of placement of epidural analgesia has been a controversial issue and how early laboring women can benefit from epidural analgesia is still debated. Hence this comparative study determines the effect of early versus late initiation of epidural analgesia on labor.Objective: To compare the effect of early versus late initiation of epidural analgesia on the duration of labour and the mode of delivery.Methodology: A randomized trial in which 100 term women in early labor at less than3 cm of cervical dilatation were assigned to either immediate initiation of epidural analgesia at first request (50 women) or delay of epidural until the cervix was dilated to at least 4 cm (50 women).Results: At initiation of the epidural, the mean cervical dilatation was 3.1 cm in the early epidural group and 4.4 cm in the late group (P value 0.0000). The mean duration from initiation to full dilatation was significantly shorter in the early compared to the late epidural group: 5.57 hours and 6.3hours respectively amongst primigravida (P = 0.0001) and 3.04 hours and 4.07 hours respectively amongst multigravida. The rates of cesarean section were not significantly different between the groups i.e. 6% and 6% in both early and late groups (P = 0.82) which was not significant. When questioned after delivery regarding their next labor, the women indicated a preference for early epidural.Conclusion: Epidural analgesia in the early labour, following the first request for epidural at cervical dilation of 2-3 cm does not prolong the progression of labor and does not increase the rate of Cesarean deliveries , instrumental vaginal deliveries , and other adverse effects in laboring women compared with the delayed analgesia at the cervical dilation of 4.0 cm or more. Furthermore, it was associated with shorter duration of the first stage of labor and was clearly preferred by the women

Interaction of Gene-Cloned and Insect Cell-Expressed Aminopeptidase N of Spodoptera litura with Insecticidal Crystal Protein Cry1C

Insecticidal toxins produced by Bacillus thuringiensis interact with specific receptors located in the midguts of susceptible larvae, and the interaction is followed by a series of biochemical events that lead to the death of the insect. In order to elucidate the mechanism of action of B. thuringiensis toxins, receptor protein-encoding genes from many insect species have been cloned and characterized. In this paper we report the cloning, expression, and characterization of Cry toxin-interacting aminopeptidase N (APN) isolated from the midgut of a polyphagous pest, Spodoptera litura. The S. litura APN cDNA was expressed in the Sf21 insect cell line by using a baculovirus expression system. Immunofluorescence staining of the cells revealed that the expressed APN was located at the surface of Sf21 cells. Treatment of Sf21 cells expressing S. litura APN with phosphatidylinositol-specific phospholipase C demonstrated that the APN was anchored in the membrane by a glycosylphosphatidylinositol moiety. Interaction of the expressed receptor with different Cry toxins was examined by immunofluorescence toxin binding studies and ligand blot and immunoprecipitation analyses. By these experiments we showed that the bioactive toxin, Cry1C, binds to the recombinant APN, while the nonbioactive toxin, Cry1Ac, showed no interaction

siRNA-directed silencing of transgene expressed in cultured insect cells

RNA interference (RNAi) has emerged as a powerful tool to rapidly analyze gene functions in a wide variety of eukaryotic organisms as well as in cultured cell lines. We demonstrate here that RNAi can be applied to study the function of a transgene expressed in an insect cell line (Spodoptera frugiperda, Sf21). The aminopeptidase N gene (apn) targeted for silencing in the present study was isolated from the midgut of Spodoptera litura larvae and expressed in Sf21 cells using baculovirus expression system. The recombinant APN protein expressed at the surface of Sf21 cells was shown to interact with insecticidal crystal protein, Cry1C, by in vitro experiments. The exogenous addition/transfection of APN dsRNA or siRNA in the cultured cells resulted in partial/complete inhibition of expression of apn leading to the loss of toxin binding to the transgene expressing cells. These experiments highlighted the usefulness of RNAi as a tool to study the function of an expressed transgene in insect cell line and to study the specificity of receptor-ligand interaction

How viruses Evade host responses ?

Humans have evolved a highly complex defence system to combat a wide variety of pathogens. Among the pathogens, developing a remedy for most of the viral infections still remains a great challenge as viruses evolve very rapidly as well as produce a variety of proteins. These features provide viruses with strategies to evade almost any hurdle imposed by the host defences to block their replication. It is important to understand the mechanisms of virus-host interactions, as this information can prove useful in symptomatic treatment of viral infections and aid in the design of antiviral vaccines. Further, since viruses rely almost entirely on the host cell machinery for their propagation, these studies educate us about our own cells and the basic mechanisms of gene expression and immunity

CBC parameters and morphological alterations in peripheral blood cells in COVID-19 patients: Their significance and correlation with clinical course

Background:SARS-CoV2&nbsp; infection&nbsp; induces inflammatory responses and acute lung injury in human beings.CBC and its derivatives are important investigative tools in its prognosis. However very few studies highlight the importance of Peripheral blood cell morphology in this disease.Aim:To Analyze significance of the CBC, &nbsp;derived parameters and peripheral blood cells morphology in Covid-19 patients,and to study their correlation with its Clinical Status of the patients at admission and at subsequent assessment during hospital stay. Material and methods: A Single center retrospective study of laboratory-confirmed 26 COVID-19 patients admitted at SSPH &amp; PGTI, from April to July 2020., CBC, its derived parameters and Romanowsky stained peripheral blood&nbsp; smears were analysed at two points of time during clinical course. Data was tabulated and analyzed using SPSS22 software.Results:On admission, 42.3% cases were mildly symptomatic and 19.2% were moderate to severely symptomatic requiring oxygen/ventilatory support. No significant statistical findings noted in CBC and its derived parameters at the&nbsp; time of admission . Follow-up, revealed a significant change in WBC and platelet count. (p=0.002). 7 cases showed persistent changes in CBC parameters and blood cell morphology and 3 out of 7 had moderate to severe clinical course. Bizarre atypical mononuclear cells , 2-3 times the size of RBC with dense homogenous chromatin, nuclear&nbsp; membrane irregularity and deep blue cytoplasm(?covicytes) were the most significant morphological finding. Conclusion: It is evident from our studies that a relevant number of cases having moderate to severe symptoms showed persistence of morphological changes and alteration in CBC parameters

Recombinantly expressed isoenzymic aminopeptidases from Helicoverpa armigera (American cotton bollworm) midgut display differential interaction with closely related Bacillus thuringiensis insecticidal proteins.

Several investigators have independently identified membrane-associated aminopeptidases in the midgut of insect larvae as the initial interacting ligand to the insecticidal crystal proteins of Bacillus thuringiensis. Though several isoenzymes of aminopeptidases have been identified from the midgut of an insect and their corresponding cDNA cloned, only one of the isoform has been expressed heterologously and studied for its binding to Cry toxins. Here we report the cloning and expression of two aminopeptidases N from Helicoverpa armigera (American cotton bollworm) (HaAPNs). The full-length cDNA of H. armigera APN1 (haapn1) is 3205 bp in size and encodes a 1000-amino-acid protein, while H. armigera APN2 (haapn2) is 3116 bp in size and corresponds to a 1012-amino-acid protein. Structurally these proteins show sequence similarity to other insect aminopeptidases and possess characteristic aminopeptidase motifs. Both the genes have been expressed in Trichoplusia ni (cabbage looper) cells using a baculovirus expression vector. The expressed aminopeptidases are membrane-associated, catalytically active and glycosylated. Ligand-blot analysis of both these aminopeptidases with bioactive Cry1Aa, Cry1Ab and Cry1Ac proteins displayed differential interaction. All the three toxins bound to HaAPN1, whereas only Cry1Ac interacted with HaAPN2. This is the first report demonstrating differential Cry-toxin-binding abilities of two different aminopeptidases from a susceptible insect

Transcriptional analysis of an immune-responsive serine protease from Indian malarial vector, -0

<p><b>Copyright information:</b></p><p>Taken from "Transcriptional analysis of an immune-responsive serine protease from Indian malarial vector, "</p><p>http://www.biomedcentral.com/1471-2199/8/33</p><p>BMC Molecular Biology 2007;8():33-33.</p><p>Published online 15 May 2007</p><p>PMCID:PMC1876469.</p><p></p> marked by 'Δ'. The putative 17 amino acid signal peptide is underlined with a solid line and the putative 19 amino acid propeptide is underlined with a dotted line. '' indicates the cleavage site of signal peptide and '▼' shows the activation site. The conserved cysteines are shown in bold and larger font. . The tree was constructed by the neighbour-joining method with the Best Tree mode. A value of 0.1 corresponds to a difference of 10% between two sequences. GenBank™ accession numbers are as follows; predicted serine protease (agCP8348), AAAB01008980.1; serine protease (AgSp24D), : immune-responsive chymotrypsin-like serine protease-related protein (ISPR1), : EASTER-like serine protease (Sp14D1), : serine protease (CULQU), : serine protease K17/F1R1 (CHRBE), Q9GSM5; serine protease (DROYA),