Nucleocapsid Protein of Newcastle Disease Virus as an Antigenic Carrier

Newcastle disease virus (NDV) is an economically important avian virus that causes loss to the poultry industry. It has a wide host range infecting 27 of the 50 orders of birds. Generally, the virus consists of six structural proteins: nucleocapsid (NP), phosphoprotein (P), matrix (M), fusion (F), haemagglutinin-neuramidase (HN) and large (L). The NP protein resembles the classical herringbone morphology when observed under electron microscope. However, the morphology changed into individual ring-like particles when the myc epitope and six histidine residues were fused to the C-terminal end of the protein. Further investigation showed that the C-terminus of this protein derivative is exposed on the surface of the ring-like particles. In this project, several chimeric proteins have been constructed in which the antigenic regions of the HN or F protein of NDV strain AF2240, myc epitope and six histidine residues were linked to the C-tenninus of the NP protein. The chimeric proteins were expressed efficiently in Escherichia coli as detected by Western blot analysis. Electron microscopic analysis on these proteins revealed that they assembled into ring-like particles. These chimeric NP proteins exhibited antigenicity of the myc epitope suggesting that the foreign sequences were exposed on the surface of the particles. Chickens vaccinated with the chimeric particles exhibited an immune response against NDY. However, no protection was observed when the vaccinated chickens were challenged by the virus

Enhanced secretory production of hemolysin-mediated cyclodextrin-glucanotransferase in Escherichia coli by random mutagenesis of the ABC transporter system.

The hemolysin transport system was found to mediate the release of cyclodextrin glucanotransferase (CGTase) into the extracellular medium when it was fused to the C-terminal 61 amino acids of HlyA (HlyAs(61)). To produce an improved-secretion variant, the hly components (hlyAs, hlyB and hlyD) were engineered by directed evolution using error-prone PCR. Hly mutants were screened on solid LB-starch plate for halo zone larger than the parent strain. Through screening of about 1 × 10(4) Escherichia coli BL21(DE3) transformants, we succeeded in isolating five mutants that showed a 35-217% increase in the secretion level of CGTase-HlyAs(61) relative to the wild-type strain. The mutation sites of each mutant were located at HlyB, primarily along the transmembrane domain, implying that the corresponding region was important for the improved secretion of the target protein. In this study we describe the finding of novel site(s) of HlyB responsible for enhancing secretion of CGTase in E. coli

Global, regional, and national burden of colorectal cancer and its risk factors, 1990–2019: a systematic analysis for the Global Burden of Disease Study 2019

Funding: F Carvalho and E Fernandes acknowledge support from Fundação para a Ciência e a Tecnologia, I.P. (FCT), in the scope of the project UIDP/04378/2020 and UIDB/04378/2020 of the Research Unit on Applied Molecular Biosciences UCIBIO and the project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy i4HB; FCT/MCTES through the project UIDB/50006/2020. J Conde acknowledges the European Research Council Starting Grant (ERC-StG-2019-848325). V M Costa acknowledges the grant SFRH/BHD/110001/2015, received by Portuguese national funds through Fundação para a Ciência e Tecnologia (FCT), IP, under the Norma Transitória DL57/2016/CP1334/CT0006.proofepub_ahead_of_prin

Diabetes mortality and trends before 25 years of age : an analysis of the Global Burden of Disease Study 2019

Background: Diabetes, particularly type 1 diabetes, at younger ages can be a largely preventable cause of death with the correct health care and services. We aimed to evaluate diabetes mortality and trends at ages younger than 25 years globally using data from the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2019. Methods: We used estimates of GBD 2019 to calculate international diabetes mortality at ages younger than 25 years in 1990 and 2019. Data sources for causes of death were obtained from vital registration systems, verbal autopsies, and other surveillance systems for 1990–2019. We estimated death rates for each location using the GBD Cause of Death Ensemble model. We analysed the association of age-standardised death rates per 100 000 population with the Socio-demographic Index (SDI) and a measure of universal health coverage (UHC) and described the variability within SDI quintiles. We present estimates with their 95% uncertainty intervals. Findings: In 2019, 16 300 (95% uncertainty interval 14 200 to 18 900) global deaths due to diabetes (type 1 and 2 combined) occurred in people younger than 25 years and 73·7% (68·3 to 77·4) were classified as due to type 1 diabetes. The age-standardised death rate was 0·50 (0·44 to 0·58) per 100 000 population, and 15 900 (97·5%) of these deaths occurred in low to high-middle SDI countries. The rate was 0·13 (0·12 to 0·14) per 100 000 population in the high SDI quintile, 0·60 (0·51 to 0·70) per 100 000 population in the low-middle SDI quintile, and 0·71 (0·60 to 0·86) per 100 000 population in the low SDI quintile. Within SDI quintiles, we observed large variability in rates across countries, in part explained by the extent of UHC (r2=0·62). From 1990 to 2019, age-standardised death rates decreased globally by 17·0% (−28·4 to −2·9) for all diabetes, and by 21·0% (–33·0 to −5·9) when considering only type 1 diabetes. However, the low SDI quintile had the lowest decline for both all diabetes (−13·6% [–28·4 to 3·4]) and for type 1 diabetes (−13·6% [–29·3 to 8·9]). Interpretation: Decreasing diabetes mortality at ages younger than 25 years remains an important challenge, especially in low and low-middle SDI countries. Inadequate diagnosis and treatment of diabetes is likely to be major contributor to these early deaths, highlighting the urgent need to provide better access to insulin and basic diabetes education and care. This mortality metric, derived from readily available and frequently updated GBD data, can help to monitor preventable diabetes-related deaths over time globally, aligned with the UN's Sustainable Development Targets, and serve as an indicator of the adequacy of basic diabetes care for type 1 and type 2 diabetes across nations. Funding: Bill & Melinda Gates Foundation.publishedVersionPeer reviewe

Global, regional, and national burden of disorders affecting the nervous system, 1990–2021: a systematic analysis for the Global Burden of Disease Study 2021

BackgroundDisorders affecting the nervous system are diverse and include neurodevelopmental disorders, late-life neurodegeneration, and newly emergent conditions, such as cognitive impairment following COVID-19. Previous publications from the Global Burden of Disease, Injuries, and Risk Factor Study estimated the burden of 15 neurological conditions in 2015 and 2016, but these analyses did not include neurodevelopmental disorders, as defined by the International Classification of Diseases (ICD)-11, or a subset of cases of congenital, neonatal, and infectious conditions that cause neurological damage. Here, we estimate nervous system health loss caused by 37 unique conditions and their associated risk factors globally, regionally, and nationally from 1990 to 2021.MethodsWe estimated mortality, prevalence, years lived with disability (YLDs), years of life lost (YLLs), and disability-adjusted life-years (DALYs), with corresponding 95% uncertainty intervals (UIs), by age and sex in 204 countries and territories, from 1990 to 2021. We included morbidity and deaths due to neurological conditions, for which health loss is directly due to damage to the CNS or peripheral nervous system. We also isolated neurological health loss from conditions for which nervous system morbidity is a consequence, but not the primary feature, including a subset of congenital conditions (ie, chromosomal anomalies and congenital birth defects), neonatal conditions (ie, jaundice, preterm birth, and sepsis), infectious diseases (ie, COVID-19, cystic echinococcosis, malaria, syphilis, and Zika virus disease), and diabetic neuropathy. By conducting a sequela-level analysis of the health outcomes for these conditions, only cases where nervous system damage occurred were included, and YLDs were recalculated to isolate the non-fatal burden directly attributable to nervous system health loss. A comorbidity correction was used to calculate total prevalence of all conditions that affect the nervous system combined.FindingsGlobally, the 37 conditions affecting the nervous system were collectively ranked as the leading group cause of DALYs in 2021 (443 million, 95% UI 378–521), affecting 3·40 billion (3·20–3·62) individuals (43·1%, 40·5–45·9 of the global population); global DALY counts attributed to these conditions increased by 18·2% (8·7–26·7) between 1990 and 2021. Age-standardised rates of deaths per 100 000 people attributed to these conditions decreased from 1990 to 2021 by 33·6% (27·6–38·8), and age-standardised rates of DALYs attributed to these conditions decreased by 27·0% (21·5–32·4). Age-standardised prevalence was almost stable, with a change of 1·5% (0·7–2·4). The ten conditions with the highest age-standardised DALYs in 2021 were stroke, neonatal encephalopathy, migraine, Alzheimer's disease and other dementias, diabetic neuropathy, meningitis, epilepsy, neurological complications due to preterm birth, autism spectrum disorder, and nervous system cancer.InterpretationAs the leading cause of overall disease burden in the world, with increasing global DALY counts, effective prevention, treatment, and rehabilitation strategies for disorders affecting the nervous system are needed

Detection of toluene degradation in bacteria isolated from oil contaminated soils

Toluene (C7H8) a hydrocarbon in crude oil, is a common contaminant in soil and groundwater. In this study, the ability to degrade toluene was investigated from twelve bacteria isolates which were isolated from soil contaminated with oil. Out of 12 bacterial isolates tested, most of Pseudomonas sp. showed the capability to grow in 1 mM of toluene compared with other isolates on the third day of incubation. Based on enzyme assays towards toluene monooxygenase, Pseudomonas aeruginosa UKMP-14T and Bacillus cereus UKMP-6G were shown to have the highest ability to degrade toluene. The toluene monoxygenase activity was analysed by using two calorimetric methods, Horseradish peroxidase (HRP) and indole-indigo. Both of the methods measured the production of catechol by the enzymatic reaction of toluene monooxygenase. In the HRP assay, the highest enzyme activity was 0.274 U/mL, exhibited by Pseudomonas aeruginosa UKMP-14T. However, for indole-indigo assay, Bacillus cereus UKMP-6G produced the highest enzyme activity of 0.291 U/ml. Results from both experiments showed that Pseudomonas aeruginosa UKMP-14T and Bacillus cereus UKMP-6G were able to degrade toluene

Effects of point mutations at positions 79, 85 and 91 of the nipah virus leader sequence to its minigenome expression

Nipah virus has been identified as the causative agent responsible for an outbreak of fatal human viral encephalitis in Malaysia and Singapore in 1998 to 1999. In vitro replication assays with Nipah virus minigenome carrying CAT gene (chloramphenicol acetyltransferase) has been developed without the use of infectious virus to allow further study of the replication of Nipah virus in vitro. It has been reported that the viral RNA replication and transcription activity of paramyxoviruses are controlled by essential sequences located in the terminal regions of viral genomic and antigenomic RNAs. In this study, single base substitution was carried out on the Nipah virus minigenome separately at the three guanine residues (G) located in positions 79, 85 and 91 of the leader promoter within the 5’ non-translated region (NTR) of the nucleocapsid gene (N) mRNA region. The guanine residues of these positions were substituted with the cytosine (C) or adenine (A) residue, respectively by using the overlapping PCR-mediated mutagenesis method. The resultant mutants containing the desired point mutation were confirmed by sequencing. The mutants were analyzed to determine the effect of substitution mutation on the viral transcription activity of the minigenome. It was found that the substitution of G at positions 79 and 85 decreased the efficiency of transcription of Nipah virus minigenome while the substitution of G at position 91 did not. Our findings also showed that the effect of transition mutation gave more impact than the transversion mutation in term of suppression effect upon the transcription activity of minigenome

Cloning of Aspergillus Niger BglA and expression of recombinant β-glucosidase in methylotrophic yeast Pichia Pastoris

Full length cDNA of bglA gene encoding Aspergillus niger ATCC10574 β-glucosidase was isolated and sequenced. The cDNA has a length of 2583 bp which encodes a polypeptide of 860 amino acid residues with predicted pI value of 4.6 and molecular weight of 93 kDa. Amino acid analysis of BGLA from four different isolates of A. niger, isolates ATCC10574, ATCC1015, B1 and CBS513.88, detected a total of 29 amino acids differences. The degree of differences varies between different variants, from 0.46% up to 2.9%. Around 34% of these differences were located in β-glucosidase two conserved domains, the glycosyl hydrolase family 3 N-terminal and the C-terminal domains. Both of the domains are important for the catalytic activity of the enzyme and these differences might contribute to different biophysical and biochemical enzyme properties. Heterologous expression of BGLA in methylotrophic yeast, Pichia pastoris has been carried out using methanol as inducer resulting in the production of recombinant protein with molecular weight around 90 kDa. β-glucosidase activity was detected from the culture filtrate using UVstimulated fluorescence of cleaved fluorescence substrate, 4-methylumbelliferyl-β-D-glucopyranoside (MUGlc). The specific activity of the crude recombinant enzyme for cellobiose hydrolysis was 18 U/mg