Ultralight ultrafast enzymes

Inorganic materials depleted of heavy stable isotopes are known to deviate strongly in some physico-chemical properties from their isotopically natural (native) counterparts; however, in biotechnology such effects have not been investigated yet. Here we explored for the first time the effect of simultaneous depletion of the heavy carbon, hydrogen, oxygen and nitrogen isotopes on the bacterium E. coli and the enzymes expressed in it. Bacteria showed faster growth, with proteins exhibiting higher thermal stability, while for recombinant enzymes expressed in ultralight media, faster kinetics was discovered. At room temperature, luciferase, thioredoxin and dihydrofolate reductase showed a 40-250% increase in activity compared to the native counterparts. The efficiency of ultralight Pfu DNA polymerase in polymerase chain reaction was also significantly higher than that of the normal enzyme. At 10 °C, the advantage factor of ultralight enzymes typically increased by 50%, which points towards the reduction in structural entropy as the main factor explaining the kinetic effect of heavy isotope depletion. Ultralight enzymes may find an application where extreme reaction rates are required.

Subacute Combined Degeneration Associated with Methylenetetrahydrofolate Reductase (MTHFR) C677T Polymorphism

Subacute combined degeneration (SCD) is a metabolic disease caused by deficiency of vitamin B12. Rarely, it could be associated with genetic problem. An old male presented with progressive both hands weakness. Laboratory study showed deficiency of vitamin B12, but the cause was not clear. We performed a genetic study and methylenetetrahydrofolate reductase (MTHFR) C677T homozygous polymorphism with 30% of normal enzyme activity was confirmed. This case suggests SCD may occur in association with a genetic problem with MTHFR C677T polymorphism.

Elevated Pancreatic Enzymes in ICU Patients With COVID-19 in Wuhan, China: A Retrospective Study

Background: Pancreatic enzyme elevation has been reported in patients with COVID-19 during the pandemic. However, with the shortage of medical resources and information, several challenges are faced in the examination and treatment of this condition in COVID-19 patients. There is little information on whether such condition is caused by pancreatic injury, and if this is a warning sign of life threatening complications like multiple organ failure in patients. The objective of this study is to explore the relationship between elevated pancreatic enzymes and the underlying risk factors during the management of COVID-19 patients.Method: A total of 55 COVID-19 patients admitted to the intensive care unit (ICU) of Wuhan Jinyintan hospital from January 1 to March 30, 2020 were enrolled in this study. All participants underwent transabdominal ultrasound imaging to assess their pancreas.Results: Out of the 55 patients, three patients had pancreatitis, 29 (52.7%) with elevated pancreatic enzymes, and 23 (41.8%) without. The most common symptoms of patients with COVID-19 were fever and cough. There was no statistical difference in most baseline characteristics except myalgia on admission. Compared with those having normal enzyme levels, patients with elevated pancreatic enzymes had higher rates of mortality (79.3 vs. 52.2%; P = 0.038), and lower rates of discharge (20.7 vs. 47.8%; P = 0.038). Patients with elevated enzymes had higher incidence of mechanical ventilation (P = 0.004) and kidney injury (P = 0.042) than patients without elevated pancreatic enzymes. The results of multivariable logistic analysis showed that the odds ratio were 10.202 (P = 0.002) for mechanical ventilation and 7.673 (P = 0.014) for kidney injury with the elevated enzymes vs. the normal conditions.Conclusions: The findings show that the incidences of pancreatic enzymes elevation are not low in critical COVID-19 patients and only a few of them progressed to acute pancreatitis (AP). Increased pancreatic enzymes levels is associated with poor prognosis in COVID-19 patients. In addition, the kidney injury and oxygenation degradation are associated with the pancreatic enzymes elevation in COVID-19 patients.

PREVALENCE AND MOLECULAR CHARACTERIZATION OF GLUCOSE-6-PHOSPHATE DEHYDROGENASE (G6PD) DEFICIENCY IN FEMALES FROM PREVIOUSLY MALARIA ENDEMIC REGIONS IN NORTHEASTERN THAILAND AND IDENTIFICATION OF A NOVEL G6PD VARIANT

Introduction: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common X-linked enzymopathy, highly prevalent in areas where malaria is or has been endemic. Prevalence of G6PD deficiency and characterization of G6PD variants in females from previously malaria endemic areas of northeast Thailand remain unstudied. Methods: Prevalence of G6PD deficiency was determined by a fluorescent spot test (FST) and multiplex allele specific (AS)- and restriction fragment length polymorphic (RFLP)-PCR developed for detection of common G6PD variants in the Thai population. Results: Prevalence of G6PD deficiency in female samples (n = 355) was 18% by FST and 27% by PCR-based genotyping. The most common variant was G6PD Viangchan (54%), followed by G6PD Canton (11%) and G6PD Union (11%); in addition, a novel heterozygous variant, G6PD Khon Kaen (c.305T>C, p.F102S located in the coenzyme-binding domain), was identified. The majority (75%) of G6PD activities of heterozygotes were within the intermediate deficiency range (30-80% of median normal enzyme activity). Conclusion: High prevalence of G6PD deficiency was present in females from northeast Thailand, the majority being due to heterozygosity of G6PD variants. The findings will have a bearing on an inclusion of primaquine in antimalarial-based policies for malaria elimination in populations with high prevalence in G6PD deficiency.

Linking Enzyme Upregulation to Autophagic Failure: A Potential Biomarker for GM1 Gangliosidosis

With an increasing aging population, neurodegenerative diseases are having an increased impact on society. Typically, these diseases are diagnosed significantly past symptom onset, decreasing the possibility of effective treatment. A non-invasive biomarker and specific target are needed to diagnose and treat the disease before late-stage symptoms. GM1 Gangliosidosis is a lysosomal storage disease where lysosomal enzyme β-galactosidase is missing. As a result, GM1 ganglioside is not broken down and accumulates in the cell, ultimately leading to cell death. One of the main aspects of GM1 Gangliosidosis, and other neurodegenerative diseases, is impaired autophagy: reduced fusion of autophagosomes and lysosomes to degrade cellular waste.In this paper, we show that healthy cells (NSV3) have approximately 13 times more co-localization of lysosomes and autophagosomes than GM1 Gangliosidosis-diseased cells (GM1SV3), as demonstrated via immunofluorescence. GM1SV3 fold normal enzyme activity of β-galactosidase was downregulated while mannosidase, and hexosaminidase A were both upregulated. When inducing impaired autophagy in NSV3 via starvation, co-localization gradually decreases with increased starvation time. Most notably, after 48-hour starvation, healthy cells (NSV3) showed no significant difference in co-localization compared to GM1SV3. NSV3 under starvation conditions showed a significant increase between time starved and fold normal enzyme activity, with a positive correlation being observed. Activities of mannosidase, and hexosaminidase A of starved NSV3 closely resemble, and surpass, GM1SV3 after 12-hour starvation.These observations have the potential to expand the conversation regarding impaired autophagy as a potential biomarker for disease progression and diagnostics and as a treatment target.

AB1014 CLINICAL AND LABORATORY PHENOTYPES IN PATIENTS DIAGNOSED WITH SARCOIDOSIS IN A VETERAN’S HOSPITAL

Background:Sarcoidosis is a disease which affect any organ or tissue in the body, but is predisposed to lung involvement in up to 90% of cases. Its cause is not yet established, although a multicausal model with genetic, environmental and infectious factors is proposed.Objectives:Determine the clinical, paraclinical characteristics and survival of patients diagnosed with Sarcoidosis treated at the Central Military Hospital.Methods:Historical cohort study of patients who were diagnosed with Sarcoidosis at the Central Military Hospital in the last 5 years. A review of medical records, laboratory results and radiological studies was performed and verification of survival status free of mortality was documented by independent outcomes group. A descriptive analysis was done using standard methods and non-parametric survival was calculated using the Kaplan Meyer method with confidence interval in STATA 15.Results:Twenty patients with confirmatory biopsy of sarcoidosis. Average age 44 ± 15.3 years, 55% (11/20) of the patients were men. Dyspnea was the most frequent reason for consultation 50% (10/20), S. Löfgren 10% (2/20), the lung was the most frequent location 65% (13/20), mild hypercalcemia and normal enzyme converting angiotensin levels were found. 60% of patients had grade 1 commitment on the chest radiograph. 60% (12/20) of the patients received immunosuppressive treatment. The overall survival at 5 years was 92% (95% CI 57-99).Conclusion:Sarcoidosis is a disease of young patients with a slight predominance in men. Pulmonary involvement is the most frequent, followed by mediastinal nodal involvement. Most patients received steroid treatment at some time in their life. The long-term survival of the disease is more than 90%, which indicates its benign course.References:[1]Iannuzzi MC, Rybicki BA, Teirstein AS. Sarcoidosis.N Engl J Med. 2007 Nov 22;357(21):2153-65.[2]Arkema EV, Grunewald J, Kullberg S, Eklund A, Askling J. Sarcoidosis incidence and prevalence: a nationwide register-based assessment in Sweden. Eur Respir J 2016; 48: 1690–1699.Acknowledgments:Not aplyDisclosure of Interests:None declared

Identification and Functional Characterization of IDS Gene Mutations Underlying Taiwanese Hunter Syndrome (Mucopolysaccharidosis Type II)

Hunter syndrome (mucopolysaccharidosis II; MPS II) is caused by a defect of the iduronate-2-sulfatase (IDS) gene. Few studies have reported integrated mutation data of Taiwanese MPS II phenotypes. In this study, we summarized genotype and phenotype correlations of confirmed MPS II patients and asymptomatic MPS II infants in Taiwan. Regular polymerase chain reaction and DNA sequencing were used to identify genetic abnormalities of 191 cases, including 51 unrelated patients with confirmed MPS II and 140 asymptomatic infants. IDS activity was analyzed in individual novel IDS variants using in vitro expression studies. Nineteen novel mutations were identified, in which the percentages of IDS activity of the novel missense mutations c.137A>C, c.311A>T, c.454A>C, c.797C>G, c.817C>T, c.998C>T, c.1106C>G, c.1400C>T, c.1402C>T, and c.1403G>A were significantly decreased (p < 0.001), c.254C>T and c.1025A>G were moderately decreased (p < 0.01), and c.851C>T was slightly decreased (p < 0.05) comparing with normal enzyme activity. The activities of the other six missense mutations were reduced but were insignificant. The results of genomic studies and their phenotypes were highly correlated. A greater understanding of the positive correlations may help to prevent the irreversible manifestations of Hunter syndrome, particularly in infants suspected of having asymptomatic MPS II. In addition, urinary glycosaminoglycan assay is important to diagnose Hunter syndrome since gene mutations are not definitive (could be non-pathogenic).

Characterization of Protein-Lipid Interactions in Biosystems Processes

Lipids correlate with membrane characteristics and functionalities as macromolecular constituentts in all cellular processes. Numerous aspects of lipid modulation of protein activity and structure are not completely understood and, thus a holistic systematic investigation activities will be pertinent. Protein-lipid interactions are the resultant impacts of membrane proteins on lipid physical states or vice versa. Encompassing research needs to be associated with strategies to elucidate whether proteins contain binding sites which are lipid specific, and that the protein-lipid complexes are ostensibly long-lived, on the time order necessary for the turnover of a normal enzyme. Biological membranes have since been determined as essential ingredients in an expansive array of cellular processes, such as photosynthesis, cell defence, signaling transduction, communication and motility. Therefore, they constitute multiple targets in both basic and applied research. Protein-lipid interactions are becoming increasingly relevant to the morphological characterization of membrane proteins as related to their functionalities. Excepting for simplified models, certain protein-lipid interactions specifically constitute remarkable challenges which require optimum experimental paradigm and design.