A roadmap toward implementing health technology assessment in Egypt.

Background: The Egyptian healthcare system is currently in the early phase of health technology assessment (HTA) implementation. The aim of this study is to propose an implementation roadmap based on the national healthcare system status. Methods: A survey was conducted among Egyptian healthcare sector decision-makers to assess the current and future (preferred) HTA implementation status in Egypt based on a widely used international scorecard methodology. Subsequently, interviews were conducted with experts representing middle- and top-tier management in the Egyptian healthcare system to interpret the survey results and recommend specific actions. Results: Experts recommended more capacity-building programs for HTA and health economics. Additionally, they proposed establishing HTA units in separate healthcare authorities and merging them into a single central HTA unit in the long term. Regarding the scope of implementation, experts recommended commencing with the assessment of innovative pharmaceuticals, and thereafter, expanding the scope to cover all health technologies in the long term. Additionally, they recommended using innovative tools such as "multi-criteria decision analysis (MCDA)" for tendering, and "managed entry agreements" for reimbursement decisions. Local burden of diseases and costing studies were also recommended to facilitate the implementation of HTA. Conclusion: Experts agreed that several actions are required for successful HTA implementation in Egypt, including coordination between HTA bodies, application of an explicit MCDA framework, and strengthening of local evidence generation. To implement these actions, investment in technical capacity-building is indispensable. Most experts favored using multiple and soft cost-effectiveness thresholds. Efforts should be made to publish HTA submission guidelines and timelines of the processes.

Draft genome sequence of a prodigiosin-hyperproducing Serratia marcescens strain isolated from Cairo, Egypt.

Serratia marcescens is a Gram-negative bacterium with both environmental and host-associated strains. Pigmentation is reportedly inversely correlated with infection frequency, and prodigiosin is one of Serratia pigments that has medical and industrial applications. Here, we report the draft genome sequence of prodigiosin-hyperproducing Serratia marcescens strain N2, isolated from Cairo, Egypt. The sequence is assembled into 142 contigs, with a combined size of 5,570,793 bp. The assembled genome carries typical S. marcescens genes, with potential prodigiosin-biosynthesizing genes detected.

Nanoparticles of ZnO/Berberine complex contract COVID-19 and respiratory co-bacterial infection in addition to elimination of hydroxychloroquine toxicity.

Purpose: A novel coronavirus (COVID-19) that has not been previously identified in humans and has no specific treatment has recently spread. Treatment trials using antiviral and immune-modulating drugs such as hydroxychloroquine (HCQ) were used to control this viral outbreak however several side effects have emerged. Berberine (BER) is an alkaloid that has been reported to reveal some pharmacological properties including antioxidant and antimicrobial activities. Additionally, Zinc oxide nanoparticles (ZnO-NPs) possess potent antioxidant and anti-inflammatory properties. Therefore, this study was undertaken to estimate the efficiency of both BER and synthetic ZnO/BER complex as an anti-COVID-19 therapy. Methods: First, the ZnO/BER complex was prepared by the facile mixing method. Then in vitro studies on the two compounds were conducted including VeroE6 toxicity, anti-COVID-19 activity, determination of inhibitory activity towards papain-like proteinase (PL pro) and spike protein- and receptor- binding domain (RBD) as well as assessment of drug toxicity on RBCs. Results: The results showed that ZnO/BER complex acts as an anti-COVID-19 by inhibiting spike protein binding with angiotensin-converting enzyme II (ACE II), PL pro activity, spike protein and E protein levels, and expression of both E-gene and RNA dependent RNA polymerase (RdRp) at a concentration lower than that of BER or ZnO-NPs alone. Furthermore, ZnO/BER complex had antioxidant and antimicrobial properties where it prevents the auto oxidation of 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and the culture of lower respiratory system bacteria that affected Covid 19 patients. The ZnO/BER complex prevented as well the HCQ cytotoxic effect on both RBC and WBC (in vitro) and hepatotoxicity, nephrotoxicity and anemia that occurred after HCQ long administration in vivo. Conclusion: The ZnO/BER complex can be accounted as promising anti-COVID 19 candidate because it inhibited the virus entry, replication, and assembly. Furthermore, it could be used to treat a second bacterial infection that took place in hospitalized COVID 19 patients. Moreover, ZnO/BER complex was found to eliminate the toxicity of long-term administration of HCQ in vivo.

Safety and immunogenicity evaluation of inactivated whole-virus-SARS-COV-2 as emerging vaccine development in Egypt.

BACKGROUND: Current worldwide pandemic coronavirus disease 2019 (COVID-19) with high numbers of mortality rates and huge economic problems require an urgent demand for safe and effective vaccine development. Inactivated SARS-CoV2 vaccine with alum. Hydroxide can play an important role in reducing the impacts of the COVID-19 pandemic. In this study, vaccine efficacy was evaluated through the detection of the neutralizing antibodies that protect mice from challenge with SARS-CoV 2 3 weeks after the second dose. We conclude that the vaccine described here has safety and desirable properties, and our data support further development and plans for clinical trials. METHODS: Characterized SARS-COV-2 strain, severe acute respiratory syndrome coronavirus 2 isolates (SARS-CoV-2/human/EGY/Egy-SERVAC/2020) with accession numbers; MT981440; MT981439; MT981441; MT974071; MT974069; and MW250352 at GenBank were isolated from Egyptian patients SARS-CoV-2-positive. Development of inactivated vaccine was carried out in a BSL-3 facilities and the immunogenicity was determined in mice at two doses (55 and 100 µg per dose). RESULTS: The distinct cytopathic effect induced by SARS-COV-2 propagation on Vero cell monolayers and the viral particles were identified as Coronaviridae by transmission electron microscopy and RT-PCR on infected cells cultures. Immunogenicity of the developed vaccine indicated the high antigen-binding and neutralizing antibody titers, regardless of the dose concentration, with excellent safety profiles and no deaths or clinical symptoms in mice groups. The efficacy of the inactivated vaccine formulation was tested by the wild virus challenge of the vaccinated mice and viral replication detection in lung tissues. CONCLUSIONS: Vaccinated mice recorded complete protection from challenge infection via inhibition of SARS-COV-2 replication in the lung tissues of mice following virus challenge, regardless of the level of serum neutralizing antibodies. This finding will support future trials for the evaluation of an applicable SARS-CoV-2 vaccine candidate.

Investigating the antimicrobial, antioxidant and cytotoxic activities of the biological synthesized glutathione selenium nano-incorporation.

Aqueous glutathione selenium nano-incorporation (GSH-SeN-Inco) was prepared by gamma radiation in presence of microbial glutathione (GSH) and selenium dioxide. The novel prepared GSH-SeN-Inco are validated by UV-vis spectroscopy, TEM (17.5 nm), DLS, XRD, EDX and FTIR spectrum reveals the presence of GSH moiety that coating the selenium nanoparticles (SeNPs) forming GSH-SeN-Inco. The XRD analysis verified the presence of metallic SeNPs. The nucleation and radiolysis mechanism of GSH-SeN-Inco formation are also discussed. The size GSH-SeN-Inco (17.5 nm) is affected by certain factors such as concentration of GSH, selenium dioxide, and absorbed dose of gamma radiation. The present study explored the positive role of GSH-SeN-Inco as an antitumor activity against HepG-2 and MCF-7, with IC50 at a concentration of 1.00 and 0.9 mM, respectively. The GSH-SeN-Inco show significant scavenging activity at 33%. The GSH-SeN-Inco shows antimicrobial potential against Gram-negative and Gram-positive bacteria with significant MIC especially Escherichia coli ATCC 25922 at 5.20 µg/ml.

Purification and characterization of bacteriocins-like inhibitory substances from food isolated Enterococcus faecalis OS13 with activity against nosocomial enterococci.

Nosocomial infections caused by enterococci are an ongoing global threat. Thus, finding therapeutic agents for the treatment of such infections are crucial. Some Enterococcus faecalis strains are able to produce antimicrobial peptides called bacteriocins. We analyzed 65 E. faecalis isolates from 43 food samples and 22 clinical samples in Egypt for 17 common bacteriocin-encoding genes of Enterococcus spp. These genes were absent in 11 isolates that showed antimicrobial activity putatively due to bacteriocins (three from food, including isolate OS13, and eight from clinical isolates). The food-isolated E. faecalis OS13 produced bacteriocin-like inhibitory substances (BLIS) named enterocin OS13, which comprised two peptides (enterocin OS13α OS13ß) that inhibited the growth of antibiotic-resistant nosocomial E. faecalis and E. faecium isolates. The molecular weights of enterocin OS13α and OS13ß were determined as 8079 Da and 7859 Da, respectively, and both were heat-labile. Enterocin OS13α was sensitive to proteinase K, while enterocin OS13ß was resistant. Characterization of E. faecalis OS13 isolate revealed that it belonged to sequence type 116. It was non-hemolytic, bile salt hydrolase-negative, gelatinase-positive, and sensitive to ampicillin, penicillin, vancomycin, erythromycin, kanamycin, and gentamicin. In conclusion, BLIS as enterocin OS13α and OS13ß represent antimicrobial agents with activities against antibiotic-resistant enterococcal isolates.

In-Vitro Inactivation of Sabin-Polioviruses for Development of Safe and Effective Polio Vaccine.

After years of global collaboration; we are steps away from a polio-free world. However, the currently conventional inactivated polio vaccine (cIPV) is suboptimal for the post eradication era. cIPV production cost and biosafety hazards hinder its availability and coverage of the global demands. Production of IPV from the attenuated Sabin strains (sIPV) was an ideal solution and scientists work extensively to perfect a safe, effective and affordable sIPV. This study investigated the ability of hydrogen peroxide (H2O2), ascorbic acid (AA) and epigallocatechin-3-gallate (EGCG) as alternatives for Formaldehyde (HCHO) to inactivate Sabin-polioviruses strains for sIPV production. Sabin-polioviruses vaccine strains were individually treated with AA, EGCG or H2O2 and were compared to HCHO. This was investigated by determination of the inactivation kinetics on HEP2C cells, testing of D-antigen preservation by ELISA and the immune response in Wistar rats of the four vaccine preparations. H2O2, AA and EGCG were able to inactivate polioviruses within 24 h while HCHO required 96 h. Significant high D-antigen levels were observed using AA, EGCG and H2O2 compared to HCHO. Rat sera tested for neutralizing antibodies showed comparable results. These findings support the idea of using these inactivating agents as safe and time- saving alternatives for HCHO to produce sIPV.

Basil Essential Oil and Its Nanoemulsion Mitigate Non Alcoholic Steatohepatitis in Rat Model with Special Reference to Gut Microbiota.

The present research evaluated the protective effect of basil essential oil nanoemulsion (BNO) and its parent basil essential oil (BO) towards steatohepatitis. Chemical composition of BO was assessed followed by formulation into different BNOs using the low energy spontaneous emulsification technique. An ideal formula of BNO was selected among the others based on its ultra-fine particle size (15.42 nm) and physical stability at 25-37°C, which was then tested in steatohepatitis rat model along with BO. Rats were divided into four groups, the first was fed on balanced diet (C), and the other groups were maintained on high fructose saturated fat diet deficient in choline to induce steatohepatitis, one of such groups served as control steatohepatitis (SC), the other groups received daily oral dose of BO and BNO, respectively. Microbiota (Firmicutes and Bacteroidetes) were counted in colon content and their ratio (F/B) was calculated. Liver fat, plasma lipid profile, plama interlukin-6, plasma lipopolysaccharides and plasma and colon content of lipocaline were assessed with histopathological examination of liver and colon. Results showed that the major volatile components of BO were linalool (60.9 %), eugenol (5.1 %) and eucalyptol (9.5%). SC group exhibited significant increase in liver lipids, plasma triglycerides, total cholesterol (TC), low density lipoprotein cholesterol and significant reduction in high density lipoprotein-cholesterol (HDL-C) compared to C group. Significant increase in plasma TC/HDL-C, interlukin-6, and lipocaline and F/B ratio and lipocaline in colon content were demonstrated in SC group without changes in plasma lipopolysaccharides compared to C. Histopathology of SC group showed liver fatty degeneration and fibroblasts activation while the colon demonstrated erosion and mucosal epithelium detachment. Treatment with either BNO or BO showed improvement compared to SC group. BNO was superior in reducing F/B ratio, liver lipids and histopathological changes. BO was more efficient in reducing TC, triglycerides and low density lipoprotein cholesterol. It is concluded that BO and BNO reduced the progression of nonalcoholic steatohepatitis in rat model. Gut microbiota in relation to steatohepatitis and related new therapies needs further investigations.

Complete genome sequence and comparative analysis of two potential probiotics Bacillus subtilis isolated from honey and honeybee microbiomes.

BACKGROUND: We have previously isolated Bacillus subtilis HMNig-2 and MENO2 strains, from honey and the honeybee gut microbiome respectively, and demonstrated these strains to produce levansucrase with potential probiotics characteristics. Here we report their complete genome sequence and comparative analysis with other and other B. subtilis strains. RESULTS: The complete genome sequences of Bacillus subtilis HMNig-2 and MENO2 were de novo assembled from MiSeq paired-end sequence reads and annotated using the RAST tool. Whole-genome alignments were performed to identify functional differences associated with their potential use as probiotics. CONCLUSIONS: The comparative analysis and the availability of the genome sequence of these two strains will provide comprehensive analysis about the diversity of these valuable Bacillus strains and the possible impact of the environment on bacterial evolution. SIGNIFICANCE AND IMPACT OF STUDY: We introduce complete genome sequence of two new B. subtilis strains HMNig-2 and MENO2 with probiotic potential and as cell factories for the production of levan and other valuable components for pharmaceutical and industrial applications.

Acquisition of Colistin Resistance Links Cell Membrane Thickness Alteration with a Point Mutation in the lpxD Gene in Acinetobacter baumannii.

Acinetobacter baumannii is one of the most common causes of nosocomial infections in intensive care units. Its ability to acquire diverse mechanisms of resistance limits the therapeutic choices for its treatment. This especially concerns colistin, which has been reused recently as a last-resort drug against A. baumannii. Here, we explored the impact of gaining colistin resistance on the susceptibility of A. baumannii to other antibiotics and linked colistin resistance acquisition to a gene mutation in A. baumannii. The susceptibility of 95 A. baumannii isolates revealed that 89 isolates were multi-drug resistance (MDR), and nine isolates were resistant to colistin. Subsequently, three isolates, i.e., MS48, MS50, and MS64, exhibited different resistance patterns when colistin resistance was induced and gained resistance to almost all tested antibiotics. Upon TEM examination, morphological alterations were reported for all induced isolates and a colistin-resistant clinical isolate (MS34Col-R) compared to the parental sensitive strains. Finally, genetic alterations in PmrB and LpxACD were assessed, and a point mutation in LpxD was identified in the MS64Col-R and MS34Col-R mutants, corresponding to Lys117Glu substitution in the lipid-binding domain. Our findings shed light on the implications of using colistin in the treatment of A. baumannii, especially at sub-minimum inhibitory concentrations concentrations, since cross-resistance to other classes of antibiotics may emerge, beside the rapid acquisition of resistance against colistin itself due to distinct genetic events.

Emergence of colistin resistance in multidrug-resistant Klebsiella pneumoniae and Escherichia coli strains isolated from cancer patients.

BACKGROUND: Colistin resistance is mainly driven by alterations in the Gram-negative outer membrane lipopolysaccharides and is caused, in most cases, by mutations in mgrB gene. However, the recent emergence of plasmid-encoded colistin resistance among Enterobacteriaceae strains represents a serious threat to global public health. In this paper we have investigated the rates of colistin resistance and the underlying mechanisms in 450 Klebsiella pneumoniae and Escherichia coli isolates obtained from cancer patients in Egypt. METHODS: Colistin susceptibility and minimum inhibitory concentrations were determined according to the European Committee on Antimicrobial Susceptibility Testing, by broth microdilution, and by E-test. The mcr-1, mcr-2 and mgrB genes were detected by PCR and then sequenced. Clonal diversity in colistin-resistant K. pneumoniae was evaluated by multilocus sequence typing. RESULTS: Forty (8.8%) colistin-resistant isolates, including 22 K. pneumoniae and 18 E. coli, were isolated over 18 months. Of these, 50% were carbapenem-resistant, out of which nine were blaOXA-48 and seven blaNDM-1 positive. The mechanisms of colistin resistance could be revealed only in three of the 40 resistant strains, being represented by mcr-1 in one blaNDM-1-positive E. coli strain and in one K. pneumoniae ST11 and by mgrB mutations, detected in one K. pneumoniae isolate. None of the studied isolates harbored mcr-2. CONCLUSIONS: Our results demonstrate a high frequency of colistin resistance in enterobacterial strains isolated from cancer patients, but a low prevalence of the most well known resistance mechanisms.

Skin tissue responses to transient heating with memory-dependent derivative.

In this work, the new concept of "memory dependent derivative" in the Pennes' bio-heat transfer process of skin tissues is employed to investigate the one-dimensional problem of a skin tissue under sinusoidal heat flux conditions. Laplace transform technique is utilized to solve the problem. We investigate, numerically, the bio-heat transfer equation with memory-dependent derivative to find the effect on the tissue temperature of the kernel function and the time-delay parameter which are characteristic of memory dependent derivative heat transfer. Correlations are made with the results obtained in the case of the absence of memory-dependent derivative parameters. The effects of the time-delay on the temperature distribution in skin tissue for different forms of kernel functions are examined.

Hospital Microbiome Variations As Analyzed by High-Throughput Sequencing.

Hospital-acquired infections remain a serious threat to human life and are becoming a top public health issue. As the latest advances in sequencing technologies have allowed the unbiased identification of bacterial communities, we aimed to implement emerging omics technologies to characterize a hospital's microbiome at the center of Cairo, Egypt. To this end, we screened surfaces and inanimate objects in the hospital, focusing on bed sheets and door knobs, with additional screening for resistant microbes and resistance genes. While bacterial load and community composition were not dramatically different between door knobs of hospital units with different hygiene levels, the bacterial communities on door knob samples were richer and more diverse than those detected on bed sheets. Bacteria detected on door knobs were a mix of those associated with dust/particulate matter/debris (e.g., Bacillus, Geobacillus, Aeribacillus) and skin-associated bacteria (e.g., Staphylococcus, Corynebacterium). The latter were among the core genera shared by all analyzed samples. Conversely, bacteria that were more abundant in bed sheets were not associated with a particular source (e.g., Pseudomonas and Nitrobacter). Resistance screening indicated an expansion of a mobile beta-lactamase-encoding gene (blaTEM), reflecting its current global spread. This study is a first step toward more comprehensive screening of hospital surfaces and correlating their microbiome with hospital outbreaks or chronic infections. We conclude that, as hospitals are unique built environments, these findings can inform future infection control strategies in hospitals and health care-related built environments, and attest to the importance of the emerging hospital microbiome research field.

Combination Therapy of Mesenchymal Stromal Cells and Interleukin-4 Attenuates Rheumatoid Arthritis in a Collagen-Induced Murine Model.

Rheumatoid arthritis (RA) is a disease of the joints that causes decreased quality of life. Mesenchymal stromal cells (MSCs) have immunosuppressive properties, with possible use in the treatment of RA. Similarly, interleukin (IL)-4 has been shown as a potential RA treatment. However, their combination has not been explored before. Therefore, this study aimed to investigate the effect of a combination therapy of MSCs and IL-4 in the treatment of RA, using a murine collagen-induced arthritis (CIA) model. Arthritis was induced in Balb/c mice by two intradermal injections of type II collagen (CII), at days 0 and 21. CIA mice were randomly assigned to four groups; group I received an intravenous injection of mouse bone marrow-derived MSCs, while group II received an intraperitoneal injection of IL-4. Group III received a combined treatment of MSC and IL-4, while group IV served as a CIA diseased control group receiving phosphate buffer saline (PBS). A fifth group of healthy mice served as the normal healthy control. To assess changes induced by different treatments, levels of RA-associated inflammatory cytokines and biomarkers were measured in the serum, knee joints, and synovial tissue, using ELISA and Real Time-qPCR. Histopathological features of knee joints were analyzed for all groups. Results showed that combined MSC and IL-4 treatment alleviated signs of synovitis in CIA mice, reverting to the values of healthy controls. This was evident by the decrease in the levels of rheumatic factor (RF), C-reactive protein (CRP) and anti-nuclear antibodies (ANA) by 64, 80, and 71%, respectively, compared to the diseased group. Moreover, tumor necrosis factor-alpha (TNF- α) and monocyte chemoattractant protein-1 (MCP-1) levels decreased by 63 and 68%, respectively. Similarly, our gene expression data showed improvement in mice receiving combined therapy compared to other groups receiving single treatment, where cartilage oligomeric matrix protein (Comp), tissue inhibitor metalloproteinase-1 (Timp1), matrix metalloproteinase1 (Mmp-1), and IL-1 receptor (Il-1r) gene expression levels decreased by 75, 70, and 78%, respectively. Collectively, treatment with a combined therapy of MSC and IL-4 might have an efficient therapeutic effect on arthritis. Thus, further studies are needed to assess the potential of different MSC populations in conjugation with IL-4 in the treatment of experimental arthritis.

Preclinical study of safety and immunogenicity of combined rubella and human papillomavirus vaccines: Towards enhancing vaccination uptake rates in developing countries.

Rubella vaccine was not part of national immunization programs (NIP) in several countries in the Middle East and North Africa (MENA), South-East Asia (SEA), and South Africa regions until the year 2000. Therefore, immunization coverage of females older than 20 years old in these countries has been the focus of national campaigns for rubella elimination in developing countries. Vaccines against human papillomavirus (HPV) are not part of NIPs in developing countries. To enhance the advantages of rubella-directed immunization campaigns and to increase HPV vaccine uptake in developing countries, this study aimed to test the stability, potency, efficacy and safety of a combined rubella and HPV vaccine. Female BALB/c mice were immunized subcutaneously with proposed combined HPV16/HPV18 VLP and rubella vaccine at weeks (W) 0, 3 then with HPV vaccine at W 7. Immunized mice developed antigen-specific antibodies against rubella and HPV significantly higher than mice immunized with rubella or HPV vaccine alone. The combined vaccine induced significantly higher splenocyte proliferation than control groups. In addition, pro-inflammatory cytokines IL-4, IL-6, IL-2, and IFNγ levels were significantly higher in mice immunized with the combined vaccine than control groups. Overall, the combined vaccine was safe and immunogenic offering antibody protection as well as eliciting a cellular immune response against rubella and HPV viruses in a single vaccine. This combined vaccine can be of great value to females above 20 years old in the SEA, MENA and South Africa regions offering coverage to rubella vaccine and a potential increase in HPV vaccine uptake rates after appropriate clinical testing.

Novel blaCTX-M variants and genotype-phenotype correlations among clinical isolates of extended spectrum beta lactamase-producing Escherichia coli.

The rapid emergence of multiresistant microbial pathogens, dubbed superbugs, is a serious threat to human health. Extended spectrum beta lactamase (ESBL)-producing Escherichia coli is a superbug causing worldwide outbreaks, necessitating timely and accurate tracking of resistant strains. Accordingly, this study was designed to investigate the spread of ESBL-producing Escherichia coli isolates, to analyze the effect of different genotypic and phenotypic factors on in vitro resistance patterns, and to assess the diagnostic value of commonly used ESBL genetic markers. For that purpose, we cultured 250 clinical isolates and screened their susceptibility to beta-lactam antibiotics. Among 12 antibiotics screened, only imipenem seems to have remained resilient. We subsequently analyzed the ESBL phenotype of Escherichia coli isolates and examined potential associations between their resistance phenotypes and patient-related factors. ESBL genotyping of 198 multiresistant isolates indicated that 179 contained at least one blaCTX-M gene. As we statistically dissected the data, we found associations between overall resistance and body site / type of disease. Additionally, we confirmed the diagnostic value of testing both blaCTX-M-1 and blaCTX-M-15 in providing better prediction of overall resistance. Finally, on sequencing the amplification products of detected blaCTX-M genes, we discovered two novel variants, which we named blaCTX-M-14.2 and blaCTX-M-15.2.

A Validation Study of the Limulus Amebocyte Lysate Test as an End-Product Endotoxin Test for Polyvalent Horse Snake Antivenom.

The only definitive management of snake envenoming is the use of snake antivenom. Endotoxin contamination is a serious threat to the safe use of parenteral drugs. A greater understanding of the nature of limulus amebocyte lysate (LAL) test interference and use of permissible dilutions has minimized enhancement problems. Common interference issues include suboptimal pH, enzyme or protein modification, and nonspecific LAL activation. This study aimed at determining the interference factors associated with validating the antivenom sera preparations to avoid false-positive results when testing snake antivenom serum samples by the LAL method. Phase I (preliminary screening/interference assay) was performed to determine a compatible test dilution, which was then used in Phase II (inhibition-enhancement/validation study). The best approach to resolve interference issues was dilution by 1:80 (maximum valid dilution) plus a specific treatment as heat-activation at 70°C-80°C for 10 min with rehydration of LAL reagent with endotoxin-specific buffer solution.LAY ABSTRACT: Snake antivenom sera are produced by immunizing horses with repeated nonlethal doses of snake venom. Bacterial endotoxins constitute one of the major problems in the formulation of pharmaceutical products. One such method for detecting endotoxin levels is the bacterial endotoxin test (BET). However, some substances show strong interfering action with the BET that cannot be avoided by simply diluting the sample solution. In this work, the test for interfering factors was performed as two identical series of product dilutions-one spiked with 2λ and one left unspiked. The result of the interference test revealed the noninterfering dilution (NID) of the product, which was used for the actual validation. Our results showed that after treating the samples using different procedures, such as heat activation at 70-80°C for 10 min followed by centrifugation at 2000 rpm for 10 min and dilution of samples in BD100 (biodispersing agent), inhibition and enhancement up to 1:100 maximum valid dilution (MVD) were observed. Finally, to resolve this inhibition/enhancement problem, the activated sample was heated at 70-80°C for 10 min with rehydration of the Endosafe LAL reagent in an endotoxin-specific buffer solution (BG120) to block ß-d-glucans and limulus amebocyte lysate (LAL) reactive material (LAL-RM).

Comparative Structural Analysis of Different Mycobacteriophage-Derived Mycolylarabinogalactan Esterases (Lysin B).

Mycobacteriophage endolysins have emerged as a potential alternative to the current antimycobacterial agents. This study focuses on mycolylarabinogalactan hydrolase (LysB) enzymes of the α/ß-hydrolase family, which disrupt the unique mycolic acid layer of mycobacterium cell wall. Multiple sequence alignment and structural analysis studies showed LysB-D29, the only enzyme with a solved three-dimensional structure, to share several common features with esterases (lacking lid domain) and lipases (acting on long chain lipids). Sequence and structural comparisons of 30 LysB homology models showed great variation in domain organizations and total protein length with major differences in the loop-5 motif harboring the catalytic histidine residue. Docking of different p-nitrophenyl ligands (C4-C18) to LysB-3D models revealed that the differences in length and residues of loop-5 contributed towards wide diversity of active site conformations (long tunnels, deep and superficial funnels, shallow bowls, and a narrow buried cave) resembling that of lipases, cutinases, and esterases. A set of seven LysB enzymes were recombinantly produced; their activity against p-nitrophenyl esters could be related to their active site conformation and acyl binding site. LysB-D29 (long tunnel) showed the highest activity with long chain p-nitrophenyl palmitate followed by LysB-Omega (shallow bowl) and LysB-Saal (deep funnel).

Polymorphisms of base-excision repair genes and the hepatocarcinogenesis.

AIM: To determine the possible association between polymorphisms of DNA repair genes, including XRCC1 Arg194Tryp, Arg280His, and Arg399Glu, APE1 Asp148Glu, and NEIL2 Arg257Leu, and the risk of developing hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC). METHODS: A total of 264 subjects were recruited in this retrospective case-control study and were categorized into four groups: 88 control subjects (CR), 53 chronic hepatitis C patients (CHC), 36 liver cirrhotic patients (LC), and 87 HCC patients. The XRCC1 Arg194Tryp, Arg280His, and Arg399Glu polymorphisms were detected using PCR-RFLP, while real-time PCR was used to genotype APE1 Asp148Glu and NEIL2 Arg257Leu. RESULTS: Our data revealed that, compared with the healthy controls, for those subjects with the XRCC1 Arg194Trp genotype, the risk of developing CHC, LC, and HCC was increased by 6.66- (odds ratio (OR) = 6.667; 95% confidence interval (CI) = 3.244-13.701; P > 0.01), 3.85- (OR = 3.852; 95% CI = 1.797-8.256; P > 0.01), and 2.14-fold (OR = 2.14; 95% CI = 1.13-4.06; P > 0.05), respectively. There was no association between the risk of HCC development and the XRCC1 Arg280His or XRCC1 Arg399Gln genotypes. Moreover, the analysis showed a lack of association between APE1 Asp148Glu and the risk of HCC development. The analysis of clinicopathological parameters showed that the HCC patients with the XRCC1 Arg280His polymorphism were 2.9 fold more likely to have hepatic lesions in both hepatic lobes (OR: 2.9; 95% CI: 1.15-7.29). Notably, in the HCC patients, the prevalence of the APE1 polymorphism in the males was four times higher than that in the females (OR = 4; 95% CI = 1.129-14.175; P > 0.05). CONCLUSION: Our results indicate that the XRCC1 Arg194Trp polymorphism could be a risk factor for HCV-related HCC development in Egypt.