Cloning and catalytic profile of Hyalomma dromedarii leucine aminopeptidase.

Ticks have harmful impacts on both human and animal health and cause considerable economic losses. Leucine aminopeptidase enzymes (LAP) play important roles during tick infestation to liberate vital amino acids necessary for growth. The aim of the current study is to identify, express and characterize the LAP from the hard tick Hyalomma dromedarii and elucidate its biochemical characteristics. We cloned an open reading frame of 1560 bp encoding a protein of 519 amino acids. The LAP full-length was expressed in Escherichia coli BL21 (DE3) and purified. The recombinant enzyme (H.d rLAP- 6×His) had a predicted molecular mass of approximately 55 kDa. Purification and the enzymatic characteristics of H.d rLAP- 6×His were studied. The purified enzyme showed maximum activity at 37 °C and pH 8.0-8.5 using Leu-p-nitroanilide as a substrate. The activity of H.d rLAP- 6×His was sensitive to ß-mercaptoethanol, dl-dithiothreitol, 1,10- phenanthroline, bestatin HCl, and EDTA and completely abolished by 0.05 % SDS. In parallel, the enzymatic activity was enhanced by Ni2+, Mn2+ and Mg2+, partially inhibited by Na+, Cu2+, Ca2+ and completely inhibited by Zn2+.

Altering Escherichia coli envelope integrity by mimicking the lipoprotein RcsF.

Escherichia coli cell envelope is crucial for stress sensing and signal transduction, mediated by numerous protein-protein interactions to enable adaptation and survival. Interfering with these interactions might affect envelope integrity leading to bacterial death. The outer membrane lipoprotein (RcsF) is the stress sensor of the regulator of capsule synthesis (Rcs) phosphorelay that senses envelope threats. RcsF interacts with two essential proteins, IgaA (repressing the Rcs system) and BamA (inserting ß-barrel proteins in the outer membrane). Disturbing RcsF interactions may alter Rcs signaling and/or membrane integrity thus affecting bacterial survival. Here, we derived the sequence of a peptide mimicking RcsF (RcsFmim), based on the in silico docking of RcsF with IgaA. Expression of rcsFmim caused 3-to-4-fold activation of the Rcs system and perturbation of the outer membrane. Both effects result in decreased E. coli growth rate. We anticipate that RcsFmim present a candidate for future antibacterial peptide development.

Comparative assessment of homemade ELISA and lateral flow assay (LFA)in the rapid, specific and sensitive detection of SARS-CoV-2 anti-nucleocapsid protein in sera of Egyptian patients.

Several diagnostic measures have been employed to precisely detect the SARS-CoV-2 viral infection using viral antigens, nucleic acids, and other serological approaches. The sensitivity and specificity of the serological tests remain a challenging need. Here, we describe the detection of human anti-SARS-CoV-2 IgG and IgM antibodies qualitatively through two optimized in-house ELISA and lateral flow immunoassay. Both approaches are based on the prokaryotic expression of 50 kDa SARS-CoV-2 recombinant nucleocapsid protein. This SARS-CoV-2rN-6×His was used either to coat ELISA plates or to be conjugated to gold nanoparticles followed by colorimetric detection of bound human IgG or IgM. In the LFA, we show the optimization of nanoparticle size, protein-binding capacity, membrane treatment, and finally testing the potential capacity of using either the optimized ELISA or LFA in detecting antibodies raised against viral infection. Assessment of both methods was carried out using human sera-positive and negative SARS-CoV-2 antibodies. The ELISA and LFA tests showed 86%, 96.5% sensitivity, 92%, 93.75% specificity, 97%, 98.2% PPV, and 64%, 88.2% NPV, respectively. In conclusion, both approaches were able to successfully detect human antibodies against SARS-CoV-2 nucleocapsid protein. The importance of both protocols cannot be overstated in the detection and diagnosis of viral infections, especially in developing countries.

Gene Networks and Pathways Involved in Escherichia coli Response to Multiple Stressors.

Stress response helps microorganisms survive extreme environmental conditions and host immunity, making them more virulent or drug resistant. Although both reductionist approaches investigating specific genes and systems approaches analyzing individual stress conditions are being used, less is known about gene networks involved in multiple stress responses. Here, using a systems biology approach, we mined hundreds of transcriptomic data sets for key genes and pathways involved in the tolerance of the model microorganism Escherichia coli to multiple stressors. Specifically, we investigated the E. coli K-12 MG1655 transcriptome under five stresses: heat, cold, oxidative stress, nitrosative stress, and antibiotic treatment. Overlaps of transcriptional changes between studies of each stress factor and between different stressors were determined: energy-requiring metabolic pathways, transport, and motility are typically downregulated to conserve energy, while genes related to survival, bona fide stress response, biofilm formation, and DNA repair are mainly upregulated. The transcription of 15 genes with uncharacterized functions is higher in response to multiple stressors, which suggests they may play pivotal roles in stress response. In conclusion, using rank normalization of transcriptomic data, we identified a set of E. coli stress response genes and pathways, which could be potential targets to overcome antibiotic tolerance or multidrug resistance.

Assessment of specific human antibodies against SARS-CoV-2 receptor binding domain by rapid in-house ELISA.

BACKGROUND: The recently emerged SARS-CoV-2 caused a global pandemic since the last two years. The urgent need to control the spread of the virus and rapid application of the suitable health measures raised the importance of available, rapid, and accurate diagnostic approaches. OBJECTIVE: The purpose of this study is to describe a rapid in-house optimized ELISA based on the expression of the receptor binding domain (RBD) of the SARS-CoV-2 spike protein in a prokaryotic system. METHODS: We show the expression of the 30 kDa recombinant SARS-CoV-2 RBD-6×His in four different E. coli strains (at 28∘C using 0.25mM IPTG) including the expression strain E. coli BL21 (DE3) Rosetta Gami. SARS-CoV-2 rRBD-6×His protein was purified, refolded, and used as an antigen coat to assess antibody response in human sera against SARS-CoV-2 infection. RESULTS: The assessment was carried out using a total of 155 human sero-positive and negative SARS-CoV-2 antibodies. The ELISA showed 69.5% sensitivity, 88% specificity, 78.5% agreement, a positive predictive value (PPV) of 92.3%, and a negative predictive value of 56.5%. Moreover, the optical density (OD) values of positive samples significantly correlated with the commercial kit titers. CONCLUSIONS: Specific human antibodies against SARS-CoV-2 spike protein were detected by rapid in-house ELISA in sera of human COVID-19-infected patients. The availability of this in-house ELISA protocol would be valuable for various diagnostic and epidemiological applications, particularly in developing countries. Future studies are planned for the use of the generated SARS-CoV-2 rRBD-6×His protein in vaccine development and other diagnostic applications.

Value of Sodium-Glucose Co-Transporter 2 Inhibitor Versus Traditional Medication in Microalbuminuric Diabetic Patients.

BACKGROUND: Sodium glucose co-transporter 2 inhibitor (SGLT2i) is a new arment in the prevention and treatment of diabetic kidney disease with a potential effect on reducing and preventing Chronic Kidney Disease (CKD) progression. OBJECTIVE: To evaluate the effect of SGLT2 inhibitor in comparison to traditional medication in diabetic patients with microalbuminuria. METHODS: A total of 60 diabetic patients with microalbuminuria were divided into group I, where 30 patients were treated by traditional medications (RAAS blockers) and group II where 30 patients were treated by Dapagliflozin added to the traditional medications. All patients were followed up for 6 months and their Urine Albumin/Creatinine Ratio (UACR) and eGFR changes were monitered. RESULTS: UACR significantly declined after 6 months of treatment in group II with a p-value <0.001. There were no significant eGFR changes between both groups. Systolic blood pressure decreases in both groups, but the decrease was highly significant in group II (pvalue<0.001). Diastolic blood pressure decreases significantly in both groups (p-value<0.001). Also, bodyweight reduced significantly in group II with a p-value<0.001. CONCLUSION: Dapagliflozin, when added to traditional medications (RAAS Blockers), leads to a significant reduction in microalbuminuria with no significant eGFR changes.

Distinct domains of Escherichia coli IgaA connect envelope stress sensing and down-regulation of the Rcs phosphorelay across subcellular compartments.

In enterobacteria, the Rcs system (Regulator of capsule synthesis) monitors envelope integrity and induces a stress response when damages occur in the outer membrane or in the peptidoglycan layer. Built around a two-component system, Rcs controls gene expression via a cascade of phosphoryl transfer reactions. Being particularly complex, Rcs also involves the outer membrane lipoprotein RcsF and the inner membrane essential protein IgaA (Intracellular growth attenuator). RcsF and IgaA, which are located upstream of the phosphorelay, are required for normal Rcs functioning. Here, we establish the stress-dependent formation of a complex between RcsF and the periplasmic domain of IgaA as the molecular signal triggering Rcs. Moreover, molecular dissection of IgaA reveals that its negative regulatory role on Rcs is mostly carried by its first N-terminal cytoplasmic domain. Altogether, our results support a model in which IgaA regulates Rcs activation by playing a direct role in the transfer of signals from the cell envelope to the cytoplasm. This remarkable feature further distinguishes Rcs from other envelope stress response systems.

Effect of Topical Nepafenac on Central Foveal Thickness following Panretinal Photocoagulation in Diabetic Patients.

PURPOSE: To evaluate effectiveness of topical nepafenac in reducing macular edema following panretinal photocoagulation (PRP). DESIGN: Prospective randomized double-blinded controlled study. METHODS: Sixty eyes of 60 patients having proliferative or severe nonproliferative diabetic retinopathy had PRP. Patients were then divided into two groups: nepafenac group (30 eyes) receiving 1% topical nepafenac eye drops for 6 months and control group (30 eyes) receiving carboxymethylcellulose eye drops for 6 months. Best-corrected visual acuity (BCVA) and macular optical coherence tomography were followed up at 1, 2, 4, and 6 months after PRP. RESULTS: BCVA was significantly better in nepafenac group than in control group at all follow-ups (P < 0.01). At 6 months post-PRP, logMAR BCVA was 0.11 ± 0.04 (equivalent to 20/26 Snellen acuity) in the nepafenac group and 0.18 ± 0.08 (equivalent to 20/30 Snellen acuity) in the control group (P < 0.01). Central foveal thickness (CFT) increased in both groups from the first month after PRP. Increase in CFT was higher in control group than in nepafenac group throughout follow-up, but the difference became statistically significant only after 4 months. No significant ocular adverse events were reported with topical nepafenac. CONCLUSION: Topical nepafenac can minimize macular edema and stabilize visual acuity following PRP for diabetic patients.

Examining Delay Intervals in the Diagnosis and Treatment of Primary Open Angle Glaucoma in an Egyptian Population and Its Impact on Lifestyle.

Purpose. To examine causes as well as extent of delay in diagnosis and treatment of primary open angle glaucoma patients in a sample of Egyptians. Patients and Methods. 440 patients with primary open angle glaucoma were interviewed to evaluate delay in their diagnosis and treatment. The extent and cause of delay were investigated. The total delay interval, if any, was correlated with socioeconomic and other factors. Results. The median total delay was one year, with 50% of patients having a total delay of 1 year or less, of which 25% exhibited zero total delay. 25% of patients had a delay ranging from 1 to 3 years, and 25% had a total delay ranging from 3 to 27 years. Diagnostic delay accounted for 43.03% of cases. Longer delays were met in patients with certain socioeconomic factors. Patients with a positive family history of glaucoma displayed shorter delay periods. Conclusion. Significant delay in the diagnosis and treatment of glaucoma was found. Poor socioeconomic status seems to hinder timely diagnosis and treatment of POAG. Certain socioeconomic factors seem to correlate with the extent of delay. More effort is thus needed to subsidize the cost of investigations and treatment for glaucoma patients.

Analysis of Factors Affecting Patients' Compliance to Topical Antiglaucoma Medications in Egypt as a Developing Country Model.

Purpose. To study factors affecting patients' compliance to antiglaucoma medications in Egypt where there are unique factors as a developing country. Patients and Methods. A cross-sectional descriptive study on 440 Egyptian patients with open angle glaucoma (OAG) recruited for over two years. The patients were thoroughly interviewed about their age, education level, duration of glaucoma, difficulty in instilling the drops, medication regimens, a family history of glaucoma, knowledge of the disease, and the presence of medical insurance. Results. 236 (53.6%) were noncompliant compared to 204 (46.4%) who were compliant. Females had a tendency for higher compliance (p=0.061). Patient age above 50 years and low level of education and negative family history of glaucoma were factors significantly associated with poor compliance (p < 0.0001). Polytherapy and lack of medical insurance could be contributing factors. Conclusion. Egyptian patients have a high rate of noncompliance compared to the average in literature. Great effort is needed in educating patients about the importance of medications and the risk and the prognosis of this disease. Economic factors must also be taken into consideration in developing countries with large number of poor patients. We recommend simplifying drug regimens, incorporating electronic dose monitors, and creating reminders for follow-up visits of glaucoma patients.

Molecular cloning of Ra-sHSPI, a novel member of the HSP20 family from Rhipicephalus annulatus salivary glands.

Infestation of cattle by ticks of Rhipicephalus spp. results in severe veterinary and economical losses. Identification of novel proteins from tick salivary glands will enhance our understanding of several aspects of tick physiology and will aid in the development of anti-tick vaccines. Small heat shock proteins (HSPs) have important roles in infection and immunity, especially between invertebrate vectors and mammalian hosts while initially performing their molecular chaperone activity. Here, we report the identification of a small HSP gene from the salivary glands of Rhipicephalus annulatus ticks through immunoscreening of the corresponding cDNA expression library. The identified cDNA contained a 742bp sequence with 543bp open reading frame. It was subsequently cloned, expressed and successfully purified under both native and denaturing conditions. Sequence analysis and functional investigations showed that the protein belongs to the HSP20 family, hence the annotated name Ra-sHSPI. Indeed, recombinant Ra-sHSPI showed two typical in vitro activities of holdase chaperones, including thermal protection of bacterial cellular extracts and the recombinant HindIII at elevated temperatures. Moreover, the recombinant Ra-sHSPI showed strong immunogenic effect in animal model. These results pave the way toward further investigation of Ra-sHSPI role in ticks feeding and its potential use as protective antigen.

Identification of four novel Rhipicephalus annulatus upregulated salivary gland proteins as candidate vaccines.

The control of Rhipicephalus annulatus ticks in Egypt and other countries relies principally on the application of acaricides which have many drawbacks. Recently, cattle vaccination against ticks showed a potential unconventional approach to control ticks. As a target, salivary glands contain various proteins that may play specific roles during attachment, feeding and may modulate the immune system of the host. We have performed immunoscreening on expression normalized cDNA library to identify unique R. annulatus proteins from salivary gland (RaSal) that are particularly expressed during engorgement. We also present the cloning and sequencing of four novel cDNAs (RaSal1-4) from salivary glands that are expressed during feeding. RaSal4 shows 13 cysteine amino acid residues forming 6 potential disulfide bonds. We detected the expression level of the four genes during embryogenesis in eggs collected at 6, 12 and 18 days after oviposition. RT-PCR analysis detected these proteins at days 12 and 18 while slight amplification was detected at day 6 for only RaSal2. The expression of these salivary genes may put forward new vaccines to control tick infestations and tick-borne diseases.

Molecular cloning of a small heat shock protein (sHSPII) from the cattle tick Rhipicephalus (Boophilus) annulatus salivary gland.

Immunoscreening of a cDNA expression library of the Rhipicephalus (Boophilus) annulatus tick with purified rabbit anti-R annulatus salivary glands antigens polyclonal antibodies led to the identification of a 661bp sequence. The sequence includes an open reading frame of 543bp encoding a protein of 180 amino acids with calculated molecular weight of 20.51kDa, isoelectric point of 9.071 and with no signal sequence. Comparison of the deduced amino acids with protein data bank showed that the identified polypeptide belongs to the alpha crystallin small heat shock proteins superfamily and shows sequence similarity of 62% and 55% to Ixodes scapularis fed tick salivary gland protein and Ornithodoros parkeri alpha-crystallin protein, respectively. Accordingly, this protein was called Ra-sHSPII. The Ra-sHSPII protein was expressed in E. coli under T7 promotor of the pET-30b vector, purified under denaturation conditions and the immunogenicity and cross-reactivity of the recombinant Ra-sHSPII were evaluated. Direct ELISA showed that the Ra-sHSPII is a strong immunogen. In immunoblotting assay the anti-rRa-sHSPII antisera reacted specifically with purified rRa-sHSPII, with several proteins in R. annulatus whole tick, larval and gut protein extracts in addition to Hyalomma dromedarii and Ornithodoros moubata whole tick protein extracts, as examples of hard and soft tick species, respectively. The rRa-sHSPII protein confers thermal protection to other proteins in vitro as found in other sHSPs. E. coli cell extracts containing the protein were protected from heat-denatured precipitation when heated up to 100°C, whereas extracts from cells not expressing the protein were heat-sensitive at 60°C.