Increased Mosquito Midgut Infection by Dengue Virus Recruitment of Plasmin Is Blocked by an Endogenous Kazal-type Inhibitor.

Dengue symptoms include alteration of blood coagulation and fibrinolysis, causing severe hemorrhage and death. Here, we demonstrate that higher concentration of plasmin, the human fibrinolytic factor, in blood meal enhances dengue virus (DENV) infection in mosquito midgut and dissemination in mosquitoes. We also show that mosquitoes express a plasmin-selective Kazal-type inhibitor (AaTI) in the midgut to inhibit plasmin proteolysis and revert the enhanced infection. Using bio-layer interferometry, we show that DENV, plasmin, and AaTI interact to form a tripartite complex. Eventually, plasmin increases midgut internalization of dextran molecules and this is reverted by AaTI. Our study demonstrates that (1) DENV recruits plasmin to increase local proteolytic activity in the midgut, thus degrading the glycocalyx and enhancing DENV internalization and (2) AaTI can act as a transmission-blocking agent by inhibiting plasmin proteolysis. Our results indicate that dengue pathogenesis enhances DENV fitness by increasing its infectivity to mosquitoes.

Engineering of serine protease for improved thermostability and catalytic activity using rational design.

The study involves the isolation and characterization of a serine peptidase, named SP, from Pseudomonas aeruginosa. In addition to basic characterization, the protein was engineered, by site-directed mutagenesis of selected non-catalytic residues, to increase its thermal stability and catalytic activity. Among the eight-point mutations, predicted by FireProt, two mutants, A29G and V336I, yielded a positive impact. The Tm of A29G and V336I showed an increase by 5 °C and also a substantial increase in residual activity of the enzyme at elevated temperature. Moreover, the catalytic activity of A29G and V336I also showed an increase of 1.4-fold activity, compared to the wild-type (WT). Moreover, molecular docking simulations also predicted better substrate affinity of the mutants. We have also performed molecular dynamics (MD) simulations at 315 and 345 K, and the MD data at 345 K demonstrates improved thermostability for the mutants, compared to the WT. Our findings not only contribute to a better understanding of the structure-stability-activity relationship of SP but also highlights, that modification of non-catalytic residues could also promote favourable catalytic behaviour.

Identifying Protein Interactions with Histone Peptides Using Bio-layer Interferometry.

Histone post-translational modifications (PTMs) regulate numerous cellular processes, including gene transcription, cell division, and DNA damage repair. Most histone PTMs affect the recruitment or exclusion of reader proteins from chromatin. Here, we present a protocol to measure affinity and interaction kinetics between histone peptides and the recombinant protein using Bio-layer interferometry.

Prolyl isomerization of the CENP-A N-terminus regulates centromeric integrity in fission yeast.

Centromeric identity and chromosome segregation are determined by the precise centromeric targeting of CENP-A, the centromere-specific histone H3 variant. The significance of the amino-terminal domain (NTD) of CENP-A in this process remains unclear. Here, we assessed the functional significance of each residue within the NTD of CENP-A from Schizosaccharomyces pombe (SpCENP-A) and identified a proline-rich 'GRANT' (Genomic stability-Regulating site within CENP-A N-Terminus) motif that is important for CENP-A function. Through sequential mutagenesis, we show that GRANT proline residues are essential for coordinating SpCENP-A centromeric targeting. GRANT proline-15 (P15), in particular, undergoes cis-trans isomerization to regulate chromosome segregation fidelity, which appears to be carried out by two FK506-binding protein (FKBP) family prolyl cis-trans isomerases. Using proteomics analysis, we further identified the SpCENP-A-localizing chaperone Sim3 as a SpCENP-A NTD interacting protein that is dependent on GRANT proline residues. Ectopic expression of sim3+ complemented the chromosome segregation defect arising from the loss of these proline residues. Overall, cis-trans proline isomerization is a post-translational modification of the SpCENP-A NTD that confers precise propagation of centromeric integrity in fission yeast, presumably via targeting SpCENP-A to the centromere.

Regulation of transcriptional silencing and chromodomain protein localization at centromeric heterochromatin by histone H3 tyrosine 41 phosphorylation in fission yeast.

Heterochromatin silencing is critical for genomic integrity and cell survival. It is orchestrated by chromodomain (CD)-containing proteins that bind to methylated histone H3 lysine 9 (H3K9me), a hallmark of heterochromatin. Here, we show that phosphorylation of tyrosine 41 (H3Y41p)-a novel histone H3 modification-participates in the regulation of heterochromatin in fission yeast. We show that a loss-of-function mutant of H3Y41 can suppress heterochromatin de-silencing in the centromere and subtelomere repeat regions, suggesting a de-silencing role for H3Y41p on heterochromatin. Furthermore, we show both in vitro and in vivo that H3Y41p differentially regulates two CD-containing proteins without the change in the level of H3K9 methylation: it promotes the binding of Chp1 to histone H3 and the exclusion of Swi6. H3Y41p is preferentially enriched on centromeric heterochromatin during M- to early S phase, which coincides with the localization switch of Swi6/Chp1. The loss-of-function H3Y41 mutant could suppress the hypersensitivity of the RNAi mutants towards hydroxyurea (HU), which arrests replication in S phase. Overall, we describe H3Y41p as a novel histone modification that differentially regulates heterochromatin silencing in fission yeast via the binding of CD-containing proteins.

Use of personal digital assistants to detect healthcare-associated infections in a neonatal intensive care unit in Egypt.

INTRODUCTION: Personal digital assistants (PDAs) used in electronic laboratory-based surveillance are a promising alternative to conventional surveillance to detect healthcare-associated infections (HAIs). The aim of the study was to monitor, detect, and analyze HAIs using PDAs in a neonatal intensive care unit (NICU). METHODOLOGY: In this descriptive study, 1,053 neonates admitted to the NICU in the obstetrics and gynecology ward at the Cairo University hospital were included and evaluated for HAIs by collecting data using PDAs programmed by Naval Medical Research Unit 3, Cairo, with the definitions for HAIs provided by the National Healthcare Safety Network of the Centers for Disease Control and Prevention. Case records were reviewed three times a week over 19 months, from March 2012 to September 2013. RESULTS: Of 124 suspected episodes of infection recorded in PDAs, 89 confirmed episodes of infection were identified. HAI and NICU infection rates were 7.4 and 2.72/1,000 patient-days, respectively. Primary bloodstream infection was detected in 81 episodes and pneumonia in 8 episodes. The majority of infections (62%) were acquired in the ward before NICU admission. Klebsiella spp. was isolated most frequently (42%), followed by coagulase-negative Staphylococci (31%). CONCLUSIONS: This study is the first to report the use of PDAs in surveillance to detect HAIs in the NICU in our hospital. The majority of infections were acquired at the obstetric care department, indicating the importance of implementing rigorous prevention and control programs and a more detailed surveillance to identify other risk factors for infections.

Screening for Chlamydia trachomatis in Egyptian women with unexplained infertility, comparing real-time PCR techniques to standard serology tests: case control study.

BACKGROUND: To study the prevalence of Chlamydia infection in women with primary and secondary unexplained infertility using ELISA technique for antibody detection and real time, fully automated PCR for antigen detection and to explore its association with circulating antisperm antibodies (ASA). METHODS: A total of 50 women with unexplained infertility enrolled in this case control study and a control group of 44 infertile women with a known cause of infertility. Endocervical specimens were collected for Chlamydia antigen detection using PCR and serum samples for antibodies detection. Circulating anti-sperm antibodies were detected using sperm antibody Latex Agglutination tests. RESULTS: The overall prevalence of Chlamydial infection in unexplained infertility cases as detected by both ELISA and PCR was 40 % (20/50). The prevalence of current Chlamydial genital infection as detected by real-time PCR was only 6.0 % (3/50); two of which were also IgM positive. Prevalence of ASA was 6.0 % (3/50); all were sero-negative for anti-C.trachomatis IgM and were PCR negative. CONCLUSION: The incidence of Chlamydial infection in Egyptian patients with unexplained infertility is relatively high. In the setting of fertility investigations; screening for anti. C.trachomatis antibodies using ELISA, and treatment of positive cases should be considered. The presence of circulating ASA does not correlate with the presence of old or current Chlamydia infection in women with unexplained infertility.