A CHO stable pool production platform for rapid clinical development of trimeric SARS-CoV-2 spike subunit vaccine antigens.

Protein expression from stably transfected Chinese hamster ovary (CHO) clones is an established but time-consuming method for manufacturing therapeutic recombinant proteins. The use of faster, alternative approaches, such as non-clonal stable pools, has been restricted due to lower productivity and longstanding regulatory guidelines. Recently, the performance of stable pools has improved dramatically, making them a viable option for quickly producing drug substance for GLP-toxicology and early-phase clinical trials in scenarios such as pandemics that demand rapid production timelines. Compared to stable CHO clones which can take several months to generate and characterize, stable pool development can be completed in only a few weeks. Here, we compared the productivity and product quality of trimeric SARS-CoV-2 spike protein ectodomains produced from stable CHO pools or clones. Using a set of biophysical and biochemical assays we show that product quality is very similar and that CHO pools demonstrate sufficient productivity to generate vaccine candidates for early clinical trials. Based on these data, we propose that regulatory guidelines should be updated to permit production of early clinical trial material from CHO pools to enable more rapid and cost-effective clinical evaluation of potentially life-saving vaccines.

The Distinctive Permutated Domain Structure of Periplasmic α-Amylase (MalS) from Glycoside Hydrolase Family 13 Subfamily 19.

Periplasmic α-amylase MalS (EC. 3.2.1.1), which belongs to glycoside hydrolase (GH) family 13 subfamily 19, is an integral component of the maltose utilization pathway in Escherichia coli K12 and used among Ecnterobacteriaceae for the effective utilization of maltodextrin. We present the crystal structure of MalS from E. coli and reveal that it has unique structural features of circularly permutated domains and a possible CBM69. The conventional C-domain of amylase consists of amino acids 120-180 (N-terminal) and 646-676 (C-terminal) in MalS, and the whole domain architecture shows the complete circular permutation of C-A-B-A-C in domain order. Regarding substrate interaction, the enzyme has a 6-glucosyl unit pocket binding it to the non-reducing end of the cleavage site. Our study found that residues D385 and F367 play important roles in the preference of MalS for maltohexaose as an initial product. At the active site of MalS, ß-CD binds more weakly than the linear substrate, possibly due to the positioning of A402. MalS has two Ca2+ binding sites that contribute significantly to the thermostability of the enzyme. Intriguingly, the study found that MalS exhibits a high binding affinity for polysaccharides such as glycogen and amylopectin. The N domain, of which the electron density map was not observed, was predicted to be CBM69 by AlphaFold2 and might have a binding site for the polysaccharides. Structural analysis of MalS provides new insight into the structure-evolution relationship in GH13 subfamily 19 enzymes and a molecular basis for understanding the details of catalytic function and substrate binding of MalS.

What are tutors' perceptions of an online tutoring project-Digital Learning Companion-During the COVID-19 pandemic? A case study in Taiwan.

The COVID-19 outbreak has had serious impact on remote education and service-learning implementation in Taiwan. To alleviate these impacts, the Digital Learning Companion, an online tutoring project, was proposed to bridge the digital divide and learning gap among remote children, while offering university students an online service-learning environment. This project recruited international students as tutors for local children. To explore tutors' perceptions of this project during the COVID-19 pandemic, qualitative research, particularly a case study, was conducted. Adopting purposive sampling, 15 participants were chosen for interviews at the end of the project, and 10 reflective videos were used to reveal further information to supplement the interview results. Content analysis was employed to analyse the data. The findings implied that using JoinNet and tutoring journals significantly facilitated the tutoring process, which led to tutors' remarkable development in skills, social relationships, multicultural experience, altruism, social responsibility, self-efficacy, and affective values. However, they encountered some challenges, such as technical problems, communication barrier, lack of tutee information, and short tutoring duration. The solutions to these challenges and insightful suggestions for the project development are pointed out. The results of this study contribute to tutors' cognitive, social, and motivational development, and support the online service-learning-integrated curriculum, which can become a reference for further studies regarding online service-learning implementation to bridge the research gap.

Knowledge, attitudes, and practices of university students regarding COVID-19: a cross-sectional study in Vietnam.

OBJECTIVE: This cross-sectional study investigated the knowledge, attitudes, and practices (KAP) of Vietnamese university students regarding COVID-19. METHODS: A validated questionnaire (Cronbach's alpha = 0.71) was used to survey 1,025 students. A convenience sampling method was used for recruiting students from April to May 2022. The Wilcoxon rank-sum test and the Kruskal-Wallis rank-sum test/Dunn test for multiple comparisons were employed to compare students' KAP scores between two groups and among three groups or more, respectively. Factors associated with students' COVID-19 KAP scores were determined via univariate and multivariate linear regression models. Variables in the multivariate linear regression models were chosen using the Bayesian Model Averaging method in R software version 4.2.0. RESULTS: A majority of students had good knowledge (75.61%), positive attitudes (98.24%), and good practices toward COVID-19 (94.93%). Regarding the COVID-19 knowledge, the proportions of students who knew that mosquito bites and exposure to/eating wild animals would not lead to COVID-19 infection were not high (47.22 and 34.34%, respectively). More importantly, 70.34% of students thought that vitamins and minerals could help prevent or cure COVID-19. Antibiotics were the first choice for COVID-19 treatment of 438 students (42.73%). Nearly half of students (48.0%) bought antibiotics to keep at home in case of COVID-19 infection. The average KAP scores of medical students (19.97 ± 3.99, 45.10 ± 3.94, 9.72 ± 1.78) and females (18.67 ± 4.44, 44.79 ± 3.79, 9.36 ± 1.84) were significantly higher than those of non-medical students (16.48 ± 4.37, 43.33 ± 4.03, 8.68 ± 1.87) and males (17.01 ± 4.55, 42.79 ± 4.39, 8.77 ± 1.97), respectively (p < 0.001). Older students were more likely to have good knowledge and practices than the younger ones (p < 0.001). In addition, students using websites of the World Health Organization/the Ministry of Health and scientific articles to seek COVID-19 information were significantly associated with higher KAP scores when compared with those not using these sources (p < 0.001, p < 0.001, and p = 0.00139, respectively). CONCLUSION: Students' KAP scores significantly varied by age, sex, major, and sources of COVID-19 information. Although many students had sufficient knowledge, positive attitudes, and good preventive practices toward COVID-19, additional education and training strategies are paramount, especially for non-medical students and males.

Focused Attention on Positive Aspects of Dementia Care in Culturally and Linguistically Diverse Communities: Qualitative Insights from Australia.

OBJECTIVES: Dementia care scholarship focuses on care challenges and less on positive aspects of care, especially among culturally and linguistically diverse (CALD) carers outside the United States. This article investigates positive aspects of dementia care across eight CALD groups in Australia. METHODS: We analyzed interviews of 112 family carers using a four-domain framework covering: a sense of personal growth, feelings of mutuality, increases in family cohesion, and a sense of personal accomplishment. RESULTS: Positive associations with care are derived from past relationships, feelings of mutual obligation, valuing changed relationships and enjoying spending time with the person with dementia. Positive aspects of care were not associated with increased family cohesion except in Vietnamese and Arab families; neither was use of ethno-specific residential aged care, except for Greek and Italian families. Religion and spirituality as a coping and comforting mechanism was inconsistently expressed. CONCLUSIONS: The study reveals the multi-dimensional nature of care, what resonates, and diverges across CALD populations. Knowing which parts of the framework apply and which do not is useful for interventions seeking to enhance positive aspects of care. CLINICAL IMPLICATIONS: Migrant populations are varied and dynamic, and practitioners should be mindful of differences within and between ethnic minority groups.

Increased Access to and Use of Endovascular Therapy Following Implementation of a 2-Tiered Regional Stroke System.

Background and Purpose- We quantified population access to endovascular-capable centers, timing, and rates of thrombectomy in Los Angeles County before and after implementing 2-tiered routing in a regional stroke system of care. Methods- In 2018, the Los Angeles County Emergency Medical Services Agency implemented transport of patients with suspected large vessel occlusions identified by Los Angeles Motor Scale ≥4 directly to designated endovascular-capable centers. We calculated population access to a designated endovascular-capable center within 30 minutes comparing 2016, before 2-tiered system planning began, to 2018 after implementation. We analyzed data from stroke centers in the region from 1 year before and after implementation to delineate changes in rates and speed of administration of tPA (tissue-type plasminogen activator) and thrombectomy and frequency of interfacility transfer. Results- With implementation of the 2-tier system, certified endovascular-capable hospitals increased from 4 to 19 centers, and within 30-minute access to endovascular care for the public in Los Angeles County, from 40% in 2016 to 93% in 2018. Comparing Emergency Medical Services-transported stroke patients in the first post-implementation year (N=3303) with those transported in the last pre-implementation year (N=3008), age, sex, and presenting deficit severity were similar. The frequency of thrombolytic therapy increased from 23.8% to 26.9% (odds ratio, 1.2 [95% CI, 1.05-1.3]; P=0.006), and median first medical contact by paramedic-to-needle time decreased by 3 minutes ([95% CI, 0-5] P=0.03). The frequency of thrombectomy increased from 6.8% to 15.1% (odds ratio, 2.4 [95% CI, 2.0-2.9]; P<0.0001), although first medical contact-to-puncture time did not change significantly, median decrease of 8 minutes ([95% CI, -4 to 20] P=0.2). The frequency of interfacility transfers declined from 3.2% to 1.0% (odds ratio, 0.3 [95% CI, 0.2-0.5]; P<0.0001). Conclusions- After implementation of 2-tiered stroke routing in the most populous US county, thrombectomy access increased to 93% of the population, and the frequency of thrombectomy more than doubled, whereas interfacility transfers declined.

Adjunctive Dexamethasone in HIV-Associated Cryptococcal Meningitis.

BACKGROUND: Cryptococcal meningitis associated with human immunodeficiency virus (HIV) infection causes more than 600,000 deaths each year worldwide. Treatment has changed little in 20 years, and there are no imminent new anticryptococcal agents. The use of adjuvant glucocorticoids reduces mortality among patients with other forms of meningitis in some populations, but their use is untested in patients with cryptococcal meningitis. METHODS: In this double-blind, randomized, placebo-controlled trial, we recruited adult patients with HIV-associated cryptococcal meningitis in Vietnam, Thailand, Indonesia, Laos, Uganda, and Malawi. All the patients received either dexamethasone or placebo for 6 weeks, along with combination antifungal therapy with amphotericin B and fluconazole. RESULTS: The trial was stopped for safety reasons after the enrollment of 451 patients. Mortality was 47% in the dexamethasone group and 41% in the placebo group by 10 weeks (hazard ratio in the dexamethasone group, 1.11; 95% confidence interval [CI], 0.84 to 1.47; P=0.45) and 57% and 49%, respectively, by 6 months (hazard ratio, 1.18; 95% CI, 0.91 to 1.53; P=0.20). The percentage of patients with disability at 10 weeks was higher in the dexamethasone group than in the placebo group, with 13% versus 25% having a prespecified good outcome (odds ratio, 0.42; 95% CI, 0.25 to 0.69; P<0.001). Clinical adverse events were more common in the dexamethasone group than in the placebo group (667 vs. 494 events, P=0.01), with more patients in the dexamethasone group having grade 3 or 4 infection (48 vs. 25 patients, P=0.003), renal events (22 vs. 7, P=0.004), and cardiac events (8 vs. 0, P=0.004). Fungal clearance in cerebrospinal fluid was slower in the dexamethasone group. Results were consistent across Asian and African sites. CONCLUSIONS: Dexamethasone did not reduce mortality among patients with HIV-associated cryptococcal meningitis and was associated with more adverse events and disability than was placebo. (Funded by the United Kingdom Department for International Development and others through the Joint Global Health Trials program; Current Controlled Trials number, ISRCTN59144167.).

Knowledge, attitudes, and practices among university students in relation to dengue fever: A multi-center study across Vietnamese regions.

OBJECTIVE: Dengue fever (DF) is a serious illness worldwide that can spread rapidly and become a dangerous epidemic. Vietnam is an endemic country affected by the health and economic burden of dengue. This study was conducted to assess the knowledge, attitudes, and practices related to DF among university students across Vietnam. METHODS: A student-based cross-sectional study was performed with a structured questionnaire in Vietnam between July and September 2018. A Chi-square test and Fisher's exact test were used to compare different issues between two student groups, including medical and non-medical student, and P-values of <0.05 were considered significant. RESULTS: A total of 1,542 students from universities in Vietnam responded to the survey, which was reflective of 315 medical students learning in public schools, 689 medical students learning in private schools, and 538 non-medical students. The majority of participants had good knowledge on the transmission of DF, good attitudes toward DF, and good practices to prevent the disease. Medical students had better knowledge of the signs and symptoms than non-medical students. However, both student groups' knowledge of signs and symptoms was low. CONCLUSIONS: Quality of education is a key determinant of the knowledge of the disease, as well as of attitudes and practices. This study supported the government's implementation of intervention projects and DF prevention campaigns and its positive participation in the community.

Engineering the fragment crystallizable (Fc) region of human IgG1 multimers and monomers to fine-tune interactions with sialic acid-dependent receptors.

Multimeric fragment crystallizable (Fc) regions and Fc-fusion proteins are actively being explored as biomimetic replacements for IVIG therapy, which is deployed to manage many diseases and conditions but is expensive and not always efficient. The Fc region of human IgG1 (IgG1-Fc) can be engineered into multimeric structures (hexa-Fcs) that bind their cognate receptors with high avidity. The critical influence of the unique N-linked glycan attached at Asn-297 on the structure and function of IgG1-Fc is well documented; however, whether the N-linked glycan has a similarly critical role in multimeric, avidly binding Fcs, is unknown. Hexa-Fc contains two N-linked sites at Asn-77 (equivalent to Asn-297 in the Fc of IgG1) and Asn-236 (equivalent to Asn-563 in the tail piece of IgM). We report here that glycosylation at Asn-297 is critical for interactions with Fc receptors and complement and that glycosylation at Asn-563 is essential for controlling multimerization. We also found that introduction of an additional fully occupied N-linked glycosylation site at the N terminus at position 1 (equivalent to Asp-221 in the Fc of IgG1) dramatically enhances overall sialic acid content of the Fc multimers. Furthermore, replacement of Cys-575 in the IgM tail piece of multimers resulted in monomers with enhanced sialic acid content and differential receptor-binding profiles. Thus insertion of additional N-linked glycans into either the hinge or tail piece of monomers or multimers leads to molecules with enhanced sialylation that may be suitable for managing inflammation or blocking pathogen invasion.

Immune recruitment or suppression by glycan engineering of endogenous and therapeutic antibodies.

Human serum IgG contains multiple glycoforms which exhibit a range of binding properties to effector molecules such as cellular Fc receptors. Emerging knowledge of how the Fc glycans contribute to the antibody structure and effector functions has opened new avenues for the exploitation of defined antibody glycoforms in the treatment of diseases. Here, we review the structure and activity of antibody glycoforms and highlight developments in antibody glycoengineering by both the manipulation of the cellular glycosylation machinery and by chemoenzymatic synthesis. We discuss wide ranging applications of antibody glycoengineering in the treatment of cancer, autoimmunity and inflammation. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc.

A continuous coupled spectrophotometric assay for debranching enzyme activity using reducing end-specific α-glucosidase.

A novel continuous spectrophotometric assay to measure the activity of the debranching enzyme and α-amylase has been developed. The assay mixture comprises the debranching enzyme (GlgX from Escherichia coli) or α-amylase (PPA from porcine pancreas), a reducing end-specific α-glucosidase (MalZ), maltodextrin-branched ß-cyclodextrin (Glcn-ß-CD) as the substrate, and the glucose oxidase/peroxidase system (GOPOD). Due to its high reducing end specificity, the branch chains of the substrates are not hydrolyzed by MalZ. After hydrolysis by GlgX or PPA, the released maltodextrins are immediately hydrolyzed into glucose from the reducing end by MalZ, whose concentration is continuously measured by GOPOD at 510 nm in a thermostat spectrophotometer. The kinetic constants determined for GlgX (Km = 0.66 ± 0.02 mM and kcat = 76.7 ± 1.5 s(-1)) are within a reasonable range compared with those measured using high-performance anion-exchange chromatography (HPAEC). The assay procedure is convenient and sensitive, and it requires lower concentrations of enzymes and substrate compared with dinitrosalicylic acid (DNS) and HPAEC analysis.

Solution Equilibrium Study of Divalent Metal Ions with Phenylpropanoid Derivatives and Acetylcysteine Ligands.

Solution equilibrium of divalent metal ions (M=Mn2+, Co2+, Ni2+, Cu2+ and Zn2+) with caffeic acid (ligand C) or dihydrocaffeic acid (ligand D) in binary system, and with acetylcysteine (ligand N) in ternary system were investigated at condition similar to human physiological temperature of 310.15 K and ionic strength of 0.15 mol·dm-3 NaCl. Potentiometry technique was used for the determination of formation constant (log ß) assisted by spectrophotometry technique. The results indicated the formation of [ML], [MLH], [ML2], [ML2H] in binary species and [MLN], [MNLH], [MNLH2] in ternary species, where L represents ligands C or D. It was found that ligand D formed more stable complexes than that of ligand C, which were affected by the presence of double bond in the carboxylate moiety of ligand C. The speciation diagrams were simulated by HySS and discussed briefly, additionally the tendency of ternary complexes was evaluated from parameters Δ log KM and log X.

Reaction kinetics of substrate transglycosylation catalyzed by TreX of Sulfolobus solfataricus and effects on glycogen breakdown.

We studied the activity of a debranching enzyme (TreX) from Sulfolobus solfataricus on glycogen-mimic substrates, branched maltotetraosyl-ß-cyclodextrin (Glc4-ß-CD), and natural glycogen to better understand substrate transglycosylation and the effect thereof on glycogen debranching in microorganisms. The validation test of Glc4-ß-CD as a glycogen mimic substrate showed that it followed the breakdown process of the well-known yeast and rat liver extract. TreX catalyzed both hydrolysis of α-1,6-glycosidic linkages and transglycosylation at relatively high (>0.5 mM) substrate concentrations. TreX transferred maltotetraosyl moieties from the donor substrate to acceptor molecules, resulting in the formation of two positional isomers of dimaltotetraosyl-α-1,6-ß-cyclodextrin [(Glc4)2-ß-CD]; these were 6(1),6(3)- and 6(1),6(4)-dimaltotetraosyl-α-1,6-ß-CD. Use of a modified Michaelis-Menten equation to study substrate transglycosylation revealed that the kcat and Km values for transglycosylation were 1.78 × 10(3) s(-1) and 3.30 mM, respectively, whereas the values for hydrolysis were 2.57 × 10(3) s(-1) and 0.206 mM, respectively. Also, enzyme catalytic efficiency (the kcat/Km ratio) increased as the degree of polymerization of branch chains rose. In the model reaction system of Escherichia coli, glucose-1-phosphate production from glycogen by the glycogen phosphorylase was elevated ∼1.45-fold in the presence of TreX compared to that produced in the absence of TreX. The results suggest that outward shifting of glycogen branch chains via transglycosylation increases the number of exposed chains susceptible to phosphorylase action. We developed a model of the glycogen breakdown process featuring both hydrolysis and transglycosylation catalyzed by the debranching enzyme.

Introducing transglycosylation activity in Bacillus licheniformis α-amylase by replacement of His235 with Glu.

To understand the role of His and Glu in the catalytic activity of Bacillus licheniformis α-amylase (BLA), His235 was replaced with Glu. The mutant enzyme, H235E, was characterized in terms of its mode of action using labeled and unlabeled maltooctaose (Glc8). H235E predominantly produced maltotridecaose (Glc13) from Glc8, exhibiting high substrate transglycosylation activity, with Km=0.38mM and kcat/Km=20.58mM(-1)s(-1) for hydrolysis, and Km2=18.38mM and kcat2/Km2=2.57mM(-1)s(-1) for transglycosylation, while the wild-type BLA exhibited high hydrolysis activity exclusively. Glu235-located on a wide open groove near subsite +1-is likely involved in transglycosylation via formation of an α-1,4-glycosidic linkage and may recognize and stabilize the non-reducing end glucose of the acceptor molecule.

Direct costs of hypertensive patients admitted to hospital in Vietnam- a bottom-up micro-costing analysis.

BACKGROUND: There is an economic burden associated with hypertension both worldwide and in Vietnam. In Vietnam, patients with uncontrolled high blood pressure are hospitalized for further diagnosis and initiation of treatment. Because there is no evidence on costs of inpatient care for hypertensive patients available yet to inform policy makers, health insurance and hospitals, this study aims to quantify direct costs of inpatient care for these patients in Vietnam. METHODS: A retrospective study was conducted in a hospital in Vietnam. Direct costs were analyzed from the health-care provider's perspective. Hospital-based costing was performed using both bottom-up and micro-costing methods. Patients with sole essential or primary hypertension (ICD-code I10) and those comorbid with sphingolipid metabolism or other lipid storage disorders (ICD-code E75) were selected. Costs were quantified based on financial and other records of the hospital. Total cost per patient resulted from an aggregation of laboratory test costs, drug costs, inpatient-days' costs and other remaining costs, including appropriate allocation of overheads. Both mean and medians, as well as interquartile ranges (IQRs) were calculated. In addition to a base-case analysis, specific scenarios were analyzed. RESULTS: 230 patients were included in the study (147 cases with I10 code only and 83 cases with I10 combined with E75). Median length of hospital stay was 6 days. Median total direct costs per patient were US$65 (IQR: 37 -95). Total costs per patient were higher in the combined hypertensive and lipid population than in the sole hypertensive population at US$78 and US$53, respectively. In all scenarios, hospital inpatient days' costs were identified as the major cost driver in the total costs. CONCLUSIONS: Costs of hospitalization of hypertensive patients is relatively high compared to annual medication treatment at a community health station for hypertension as well as to the total health expenditure per capita in Vietnam. Given that untreated/undetected hypertension likely leads to more expensive treatments of complications, these findings may justify investments by the Vietnamese health-care sector to control high blood pressure in order to save downstream health care budgets.

Mechanism of action of three different glycogen branching enzymes and their effect on bread quality.

Bread staling adversely affects the quality of bread, but starch modification by enzymes can counteract this phenomenon. Glycogen branching enzymes (GBEs) used in this study were isolated from Deinococcus geothermalis (DgGBE), Escherichia coli (EcGBE), and Vibrio vulnificus (VvGBE). These enzymes were characterized and applied for starch dough modification to determine their role in improving bread quality. First, the branching patterns, activity on amylose and amylopectin, and thermostability of the GBEs were determined and compared. EcGBE and DgGBE exhibited better thermostable characteristics than VvGBE, and all GBEs exhibited preferential catalysis of amylopectin over amylose but different degrees. VvGBE and DgGBE produced a large number of short branches. Three GBEs degraded the starch granules and generated soluble polysaccharides. Moreover, the maltose was increased in the starch slurry but most significantly in the DgGBE treatment. Degradation of the starch granules by GBEs enhanced the maltose generation of internal amylases. When used in the bread-making process, DgGBE and VvGBE increased the dough and bread volume by 9 % and 17 %, respectively. The crumb firmness and retrogradation of the bread were decreased and delayed significantly more in the DgGBE bread. Consequently, this study can contribute to understanding the detailed roles of GBEs in the baking process.