Hepatitis B Virus Infection in Eastern Libya: Current Efforts for Overcoming Regional Barriers for Its Elimination.

Approximately 2.2% of Libyans have chronic hepatitis B (CHB) and are at the highest risk of developing end-stage disease complications. Several resource-limited countries, including Libya, may be far from achieving the WHO goal of hepatitis B elimination by 2030 as a result of several testing and linkage to care (LTC) barriers. In Libya, data about the current HBV infection situation is scarce. Therefore, our study aimed to evaluate the trends of HBV in eastern Libya, Tobruk region, and try to identify the region-specific gaps and barriers that could potentially delay the WHO goal of HBV elimination. An eighteen-year retrospective review of records of the main district medical center in the region was done to estimate the trends of HBV infection and qualitative interviews with the clinical staff of the CHB registry in the region were conducted to investigate the current status of HBV management. Out of 392,952 records, 371 (0.09%) HBV-positive were recorded and declining trends of the infection were noticed over the study period. Until late 2019, there was no linkage to care or follow-up for people with HBV infection. However, a CHB registry was established in late 2019 to manage HBV infections in the region, yet there are several barriers such as the lack of diagnostic infrastructure for liver function assessment and antiviral treatment. Despite the significant decline observed in the occurrence of HBV infection and introduction of important HBV management steps such as establishment of the CHB registry, there are still several barriers that could delay the elimination of the infection.

Bone morphogenic protein-7 (BMP-7) polymorphism: Susceptibility to cirrhosis and hepatocellular carcinoma after viral hepatitis in Egyptian patients.

Aim of the study: Bone morphogenic proteins (BMPs) have both inhibitory and stimulatory effects on growth of a tumor that depend on the type of cells, the dosage and the tumor microenvironment. We aimed to investigate the impact of the bone morphogenic protein-7 (BMP-7) single nucleotide polymorphism (SNP) rs230205 [A/G] on susceptibility to hepatocellular carcinoma (HCC) progression from liver cirrhosis after viral hepatitis infection in Egyptian patients. Material and methods: The amplification-refractory mutation system (ARMS)-polymerase chain reaction (PCR) method was used to genotype the rs230205 [A/G] SNP in 150 subjects (50 patients with post-hepatitis C or B cirrhosis, 50 HCC patients, and 50 controls). Expression level of BMP-7 protein was assessed using enzyme-linked immunosorbent assay (ELISA). Results: The results revealed insignificant changes in distribution of all genotypes/alleles of the BMP-7 rs230205 [A/G] SNP between cirrhotic patients, HCC patients and controls. The AA genotype and A allele could be considered risk factors for cirrhosis (OR = 1.75, 1.50) and HCC (OR = 2.19, 1.74), respectively. The AA genotype (95% CI: 0.45-6.79) and A allele (OR = 1.50, 95% CI: 0.77-2.93) may be viewed as cirrhosis risk factors based on group segregation. Additionally, the A allele, AG and AA genotypes and their combined ORs of 2.19 (95% CI: 0.58-8.23), 1.74 (95% CI: 0.90-3.37), and 1.70 (95% CI: 0.68-4.29) could all be risk factors for HCC. No genotype or allele could be regarded as a risk factor for progression of cirrhosis to HCC, according to OR values. Conclusions: The results showed no correlation between BMP-7 rs230205 [A/G] SNP and progression of cirrhosis to HCC. To confirm our findings, additional prospective large-scale research is required.

Association Between ABO Blood Group System and COVID-19 Severity.

OBJECTIVES: A possible association between blood group systems (ABO and Rh) and coronavirus disease 2019 (COVID-19) severity has recently been investigated by various studies with conflicting results. However, due to variations in the prevalence of the ABO and Rh blood groups in different populations, their association with COVID-19 might be varied as well. Therefore, we conducted this study on Libyan participants to further investigate this association and make population-based data available to the worldwide scientific community. METHODS: In this case-control study, ABO and Rh blood groups in 419 confirmed COVID-19 cases in Zawia, Libya, and 271 healthy controls were compared using descriptive statistics and χ 2 tests. RESULTS: Blood group A was significantly more prevalent in patients with severe COVID-19 (64/125; 51.2%) than in patients with nonsevere COVID-19 (108/294, 36.7%) (P < .034), whereas the O blood group prevalence was higher in nonsevere COVID-19 cases (131/294, 44.5%) compared with severe cases (43/125, 34.4%) (P < .001). CONCLUSIONS: The results showed a significant association between blood group A and the severity of COVID-19, whereas patients with blood group O showed a low risk of developing severe COVID-19 infection. No significant association was found between Rh and susceptibility/severity of the disease.

Hepatitis C Virus Infection in Eastern Libya: Efforts Needed to Improve HCV Testing and Linkage to Care in the Resource-Limited Setting.

Hepatitis C virus (HCV) is a significant public health problem, and the elimination of its infection by 2031 is a global goal. However, due to insufficient testing, lack of linkage to care (LTC) and treatment, Libya may be far from achieving this goal. This study aimed to explore HCV testing, the care and treatment of infected people, and to assess the burden of the infection among individuals who visited the main Medical Centre in Tobruk region, eastern Libya, for various medical and surgical conditions. A research team interviewed public health officials in Tobruk Medical Center, inspected available equipment, and obtained data available for people who were positive for antibodies to HCV (anti-HCV) as part of their routine pre-invasive procedures and pre-donation screening tests from January 2005 to April 2020. HCV antibody tests were positive for 612 cases out of 368,392 (0.17%). Of those who tested positive for anti-HCV antibodies, no one had followed up by RNA test for identifying individuals with chronic HCV infection, and there are no links to outpatient care and treatment. Our findings highlight the critical need for an up-to-date HCV diagnosis and linkage to care guidelines, which includes a follow-up RNA test for anti-HCV positive patients and early linkage to care for confirmed cases to accelerate the elimination of HCV infection from the community.

Clinical Characteristics of the First 100 Patients of COVID-19 in Tobruk, Libya: A Brief Report From Low-Resource Settings.

OBJECTIVE: This study aims to report the clinical features of a cohort of patients with suspected coronavirus disease (COVID-19) from Tobruk, Libya, and reflect upon the diagnosis challenge in low-resource settings. METHODS: A descriptive report of the first 100 patients with suspected COVID-19 who have visited the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and COVID-19 screening clinic at the National Centre for Disease Control in Tobruk, Libya. RESULTS: The most common presenting symptoms were fever (90%), cough (89%), dyspnea (85%), sore throat (79%), fatigue (78%), headache (64%), loss of smell (52%), loss of taste (53%), loss of appetite (43%), nausea and vomiting (26%), diarrhea (22%), and rhinorrhea (16%); 51% of the patients had lymphocytopenia, whereas 13% had thrombocytopenia. Bilateral infiltrates were the most common radiologic finding on chest X-ray (76%), and COVID-19 IgM and/or IgG antibodies were detected in 80% of the patients, whereas only 37% of the patients were tested positive by the reverse transcriptase polymerase chain reaction (RT-PCR). CONCLUSIONS: The disease continued its spread across the region. Fever, cough, and dyspnea were the main symptoms; 21% of the patients did not have any chest X-ray abnormalities. Initial negative results for either antibody testing or RT-PCR-testing for COVID-19 do not rule out the infection.

Expression, purification and ligand binding properties of the recombinant translation initiation factor (PeIF5B) from Pisum sativum.

Gene encoding a novel translation initiation factor PeIF5B from Pisum sativum with sequence similarity to eIF5B from H. sapiens, D. melanogaster, S. cerevisiae as well as archaeal aIF5B from M. thermoautotrophicum was earlier reported by us. We now describe the expression and purification of 96 kDa recombinant PeIF5B (rPeIF5B) protein. Using fluorescence and circular dichroism spectra analyses, we show that Mg(2+) binding does not lead to any change in PeIF5B aromatic amino acid micro-environment, whereas GTP binding induces significant changes in the local environment of the aromatic amino acids. However, the protein undergoes changes in secondary structure upon metal ion and nucleotide binding. Charged initiator tRNA binding to PeIF5B is found to be cofactor dependent. PeIF5B binds to GTP in vitro as evident from autoradiography. Based on homology modeling of the catalytic domain of PeIF5B, we could confirm the conformational changes in PeIF5B following ligand binding.

Protease inhibitors: a panacea?

With the increasing evidence of protease involvement in several diseases, novel strategies for drug development involve the use of protease inhibitors (PIs). The local balance between protease inhibitors and proteases is an important determinant of the occurrence and progression of a particular disease. Hence, enzymes and their cognate inhibitors are finding their applications as diagnostic and prognostic markers. PIs are widely implicated for their use in host defense against infection, tissue repair and matrix production, blood coagulation, cancer, and they are, therefore, the current focus as therapeutic alternatives for major diseases such as AIDS and Alzheimer's diseases. This review is a brief summary of the varied role of protein protease inhibitors in controlling the activity of aberrant enzymes in several diseases afflicting mankind today.

Characterization of LEF4 ligand binding property and its role as part of baculoviral transcription machinery.

Late expression factor 4 (LEF4) is one of the four identified subunits of Autographa californica nucleopolyhedrosis virus (AcNPV) encoded RNA polymerase that carries out transcription from viral late and very late promoters. This 464-amino acid baculovirus-encoded protein also harbors 5' mRNA capping activity that includes RNA 5' triphosphatase, nucleoside triphosphatase, and guanylyltransferase activities. Hydrolysis of 5' triphosphate RNA and free NTPs is metal ion dependent property of the protein. In the present communication, we describe the structural changes in the recombinant LEF4 protein following ligand binding. Metal ion binding causes some alteration in the conformation around aromatic amino acids whereas there is no effect on tryptophan fluorescence on GTP binding in absence and presence of metal ion. It is found that GTP and divalent cation cofactor produce some prominent changes in the secondary structure of the protein. Electrophoretic mobility shift assay (EMSA) shows that LEF4 is the probable factor that acts as anchor to dock the viral RNA polymerase on the very late polyhedrin promoter (Ppolh) facilitated by other factors.

Biophysical characterization and unfolding of LEF4 factor of RNA polymerase from AcNPV.

Late expression factor 4 (LEF4) is one of the four subunits of Autographa californica nuclear polyhedrosis virus (AcNPV) RNA polymerase. LEF4 was overexpressed in Escherichia coli and recombinant protein was subjected to structural characterization. Chemical induced unfolding of LEF4 was investigated using intrinsic fluorescence, hydrophobic dye binding, fluorescence quenching, and circular dichroism (CD) techniques. The unfolding of LEF4 was found to be a non-two state, biphasic transition. Intermediate states of LEF4 at 2M GnHCl and 4M urea shared some common structural features and hence may lie on the same pathway of protein folding. Steady-state fluorescence and far-UV CD showed that while there was considerable shift in the wavelength of emission maximum (lambda(max)), the secondary structure of LEF4 intermediates at 2M GnHCl and 4M urea remained intact. Further, temperature induced denaturation of LEF4 was monitored using far-UV CD. This study points to the structural stability of LEF4 under the influence of denaturants like urea and temperature. Although LEF4 is an interesting model protein to study protein folding intermediates, in terms of functional significance the robust nature of this protein might reflect one of the several strategies adapted by the virus to survive under very adverse environmental and physiological conditions.

Identification and characterization of functional intermediates of stem bromelain during urea and guanidine hydrochloride unfolding.

By comparing changes in enzyme activity with changes in spectral features for stem bromelain (EC.3.4.22.32) in the absence and presence of urea, Guanidine hydrochloride and ethanol; four intermediate states could be identified: two activity-enhanced state obtained in the presence of 5 M urea and 2 M GnHCl, termed X and X', respectively, and a third, similarly active state closely resembling the native protein in the presence of 8-9 M urea, termed Y. The enhanced activity of these states is due to local conformational changes accompanied by increased dynamics in the active site. Further, the enzyme does not lose its activity after substantial tertiary structure changes in 8-9 M urea (Y state), suggesting that active site containing domain is more resistant to chemical denaturation than the other structural domain. This makes stem bromelain and in general cysteine proteases an exception to the hypothesis that active site is the most labile part of enzyme.

Effect of methyl mercury induced free radical stress on nucleic acids and protein: Implications on cognitive and motor functions.

Mercury pollution and acute neurotoxicity of mercury is well known. The recent reports suggest the adverse effect of low dose mercury, though the available literature is still silent on its mechanism. This study was therefore undertaken to probe the effect of low dose methyl mercury induced heavy metal toxicity on free radical stress and its impact on behaviour of male albino rats. Male albino rats were exposed to 1 mg/kg body wt of methylmercury chloride for seven days, on day 8 they were tested for motor and memory functions. They were sacrificed later for biochemical estimations for rate of lipid peroxidation, nucleic acids, proteins in cerebrum, cerebellum and brain stem. There was an increase in the rate of lipid peroxidation showing methyl mercury induced free radical stress. The motor and memory functions demonstrated a clear decline, besides there was a lowering in the levels of nucleic acids and proteins as compared to controls. The results are important in view of recent reports that methyl mercury induced free radical stress results in early ageing and may serve as an initiating factor more specifically for neurodegenerative disorders like Alzeihemer's disease and dementias. The current findings support the notion that incorporating dietary antioxidants like curcumin, ascorbic acid and α-tocopherol in routine diet from early age may help combat the risk of developing such disorders in ensuing years.

Effect of detergents and hexafluoroisopropanol on the conformation of a non-helical and a helical plant protease inhibitor.

We have earlier reported the purification of a non-helical proteinase inhibitor from Cajanus cajan and a helical proteinase/amylase inhibitor from Phaseolus aureus. The effect of detergents, viz. sodium dodecyl sulfate (SDS), sodium deoxycholate (DOC) and 3-[(3-cholamidopropy) dimethylammonio]-1-propane sulfonate (CHAPS) and hexafluoroisopropanol on the conformation of these proteinaceous inhibitors was investigated using circular dichroism spectroscopy. The present report focuses on changes in the polypeptide backbone conformation with respect to induction of helical structure. SDS causes minimal changes in the tertiary as well as secondary structure of C. cajan proteinase inhibitor. In the presence of anionic bile salt, deoxycholate, minor changes in the far-UV CD spectrum were accompanied by loss in inhibitory activity while CHAPS did not affect the inhibitor function. As judged from the changes in circular dichroic curves ([Theta](MRW) at 208 and 222 nm), the primarily disorganized polypeptide chain of C. cajan proteinase inhibitor was converted by 3,3,3,3',3',3'-hexafluoro-2-propanol (HFIP) into helical conformation. The P. aureus inhibitor showed increased helicity in the presence of SDS ([Theta](MRW) at 208 nm) as well as sodium deoxycholate and CHAPS ([Theta](MRW) at 222 nm). Fluorescence measurements show slight alterations in the emission intensities. HFIP caused a cooperative increase in alpha-helical secondary structure in the P. aureus inhibitor.

Biochemical characterization, stability studies and N-terminal sequence of a bi-functional inhibitor from Phaseolus aureus Roxb. (Mung bean).

Herein, we report the purification and biochemical characterization of a novel bi-functional protein proteinase/amylase inhibitor from the dietary leguminous pulse Phaseolus aureus Roxb. (Vigna radiata L.) by means of acetic acid precipitation, salt fractionation, ion-exchange chromatography (DEAE-cellulose) and affinity chromatography on trypsin-sepharose column. P. aureus inhibitor is a bi-functional inhibitor since it exhibits inhibitory activity towards trypsin-like and alpha-chymotrypsin-like serine proteinases as well as against alpha-amylases. It is a helix-rich protein (Mr 13,600) containing approximately eight tyrosines, one tryptophan and two cystines. N-terminal sequence alignment reveals no homology to other proteinase inhibitors reported from Phaseolus sp. thereby confirming that it is a novel inhibitor. Inhibitory activity measurements show that the inhibitor is quite stable even at extremely high temperatures and is only slightly affected by pH changes. Circular dichroism (CD) conformational studies revealed some changes in its near- as well as far-ultraviolet spectrum at extremes of pH and temperature. Treatments with trypsin for varying time periods did not alter its proteolytic inhibitory activity but caused some reduction in its amylase inhibitory activity.

Guanidine hydrochloride denaturation of human serum albumin originates by local unfolding of some stable loops in domain III.

The effect of guanidine hydrochloride (GnHCl) on the global stability of human serum albumin (HSA) has been studied by fluorescence and circular dichroism spectroscopic measurements. The differential stability of native conformation of three HSA domains were explored by using domain-specific ligands, hemin (domain I), chloroform (domain II), bilirubin (at domain I/domain II interface) and diazepam (domain III). GnHCl induced unfolding transition curves as monitored by probes for secondary and tertiary structures were cooperative but noncoincidental. A strong ANS binding to the protein was observed around 1.8 M GnHCl, suggesting existence of intermediate states in the unfolding pathway of HSA. A gradual decrease (in the GnHCl concentration range 0.0-1.8 M) in the binding of diazepam indicates that domain III is the most labile to GnHCl denaturation. A significant increase in the binding of bilirubin up to 1.4 M GnHCl and decrease thereafter leading to complete abolishment of bilirubin binding at around 2.0 M GnHCl suggest favorable rearrangement and separation of domains I and II at 1.4 and 2.0 M GnHCl concentration, respectively. Above 1.6 M GnHCl, decrease of the binding of hemin, a ligand for domain I, chloroform, which binds in domain II and lone tryptophanyl fluorescence (Trp-214 located in domain II) indicate that at higher concentration of GnHCl domains I and II start unfolding simultaneously but the stability of domain I (7.4 Kcal/mol) is much more than domain II (4.3 Kcal/mol). A pictorial model for the unfolding of HSA domains, consistent with all these results, has been formulated, suggesting that domain III is the most labile followed by domain II while domain I is the most stable. A molten globule like state of domain III around 1.8 M GnHCl has also been identified and characterized.

Spectroscopic analysis of thermal denaturation of Cajanus cajan proteinase inhibitor at neutral and acidic pH by circular dichroism.

The conformational changes accompanying thermal denaturation under neutral, acidic and reducing conditions of Cajanus cajan proteinase inhibitor were investigated using near- and far-ultraviolet circular dichroism (CD) spectroscopy. The protein inhibitor shows a reversible N<-->D transition at neutral pH with a Tm approximately equal to 63 degrees C. The negative CD band intensities at 200 nm (far-UV) and near about 280 nm (near-UV) decrease as a result of thermal stress. The effect is more pronounced at low pH and in the presence of dithiothreitol. Only partial reversibility is observed under acidic conditions. Significant changes in the near- as well as far-ultraviolet CD spectrum are observed in the presence of dithiothreitol suggestive of the importance of disulfide linkages in maintaining the structure of C. cajan proteinase inhibitor.

Low dose mercury toxicity and human health.

Post Minamata incident there has been awareness about mercury toxicity even among the general public. Previous researches contributed a vast amount of data regarding acute mercury exposure, but gradually information about the low dose [Ninomiya, T., Ohmori, H., Hashimoto, K., Tsuruta, K., Ekino, S., 1995. Expansion of methylmercury poisoning outside minamata: an epidemiological study on chronic methylmercury poisoninig outside of Minamata. Environ. Res. 70 (1) 47-50; Lebel, J., Mergler, D., Lucotte, M., Amorim, M., Dolbec, J., Miranda, D., Arantes, G., Rheault, I., Pichet, P., 1996. Evidence of early nervous system dysfunction in Amazonian populations exposed to low-levels of methylmercury. Neurotoxicology 17 (1) 157-167] of mercury toxicity has been trickling in. With mercury contaminating rain-, ground- and sea-water no one is safe. Polluted water leads to mercury laced fish, meat and vegetable. In aquatic environments, inorganic mercury is microbiologically transformed into lipophilic organic compound 'methylmercury'. This transformation makes mercury more prone to biomagnification in food chains. Consequently, populations with traditionally high dietary intake of food originating from fresh or marine environment have highest dietary exposure to mercury. Extensive research done on locals across the globe have already established this, persons who routinely consume fish or a particular species of fish are at an increased risk of methylmercury poisoning. The easy access of the toxicant to man through multiple pathways air, water, food, cosmetic products and even vaccines increase the exposure. Foetus and children are more susceptible towards mercury toxicity. Mothers consuming diet containing mercury pass the toxicant to foetus and to infants through breast milk. Decreased performance in areas of motor function and memory has been reported among children exposed to presumably safe mercury levels. Similarly, disruption of attention, fine motor function and verbal memory was also found in adults on exposure to low mercury levels. It is an occupational hazard for dental staff, chloralkali factory workers and goldminers, etc. Mercury has been found to be a causative agent of various sorts of disorders, including neurological, nephrological, immunological, cardiac, motor, reproductive and even genetic. Recently heavy metal mediated toxicity has been linked to diseases like Alzeihemer's, Parkinson's, Autism, Lupus, Amyotrophic lateral sclerosis, etc. Besides this, it poses danger to wildlife. Therefore, it becomes imperative to spread the information regarding the threat of mercury exposure amongst the scientists and masses.

Influence of salts and alcohols on the conformation of partially folded intermediate of stem bromelain at low pH.

The effect of salts and alcohols was examined on the partially folded intermediate (PFI) state of stem bromelain reported at low pH (Haq, Rasheedi, and Khan (2002) European Journal of Biochemistry 269, 47-52) by a combination of optical methods like circular dichroism, intrinsic fluorescence and ANS binding. ESI mass spectrometry was also performed to see the effect, if any, on the overall tertiary structure of the protein. Increase in ionic strength by the addition of salts resulted in folded structures somewhat different from the native enzyme. Salt-induced intermediates are characterized by increase in helical content and a significantly reduced exposure of hydrophobic clusters relative to the state at pH 2.0. The emission wavelength maximum of intrinsic fluorescence was shifted towards that of native enzyme. ESI-MS data show decreased accessibility of ionizable/protonation sites suggestive of a folded structure. On the other hand, alcohol-induced intermediates though exhibiting increased helical content are apparently largely unfolded as observed by ESI. Thermal denaturation of a representative intermediate, each from the group of salts and alcohols examined, was also performed to check their relative stabilities. While the alcohol-induced state showed a cooperative thermal transition, the salt-induced state shows non-cooperative thermal denaturation.

Protein proteinase inhibitor genes in combat against insects, pests, and pathogens: natural and engineered phytoprotection.

The continual need to increase food production necessitates the development and application of novel biotechnologies to enable the provision of improved crop varieties in a timely and cost-effective way. A milestone in this field was the introduction of Bacillus thuringiensis (Bt) entomotoxic proteins into plants. Despite the success of this technology, there is need for development of alternative strategies of phytoprotection. Biotechnology offers sustainable solutions to the problem of pests, pathogens, and plant parasitic nematodes in the form of other insecticidal protein genes. A variety of genes, besides (Bt) toxins that are now available for genetic engineering for pest resistance are genes for vegetative insecticidal proteins, proteinase inhibitors, alpha-amylase inhibitors, and plant lectins. This review presents a comprehensive summary of research efforts that focus on the potential use and advantages of using proteinase inhibitor genes to engineer insect- and pest-resistance. Crop protection by means of PI genes is an important component of Integrated Pest Management programmes.