Improved expression and purification of highly-active 3 chymotrypsin-like protease from SARS-CoV-2.

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is the causative pathogen of coronavirus disease-19 (COVID-19). The COVID-19 pandemic has resulted in millions of deaths and widespread socio-economic damage worldwide. Therefore, numerous studies have been conducted to identify effective measures to control the spreading of the virus. Among various potential targets, the 3 chymotrypsin-like protease (3CLpro), also known as Mpro, stands out as the key protease of SARS-CoV-2, playing an essential role in virus replication and assembly, is the most prospective. In this study, we modified the commercial vector, pETM33-Nsp5-Mpro (plasmid # 156475, Addgene, USA), by inserting an autocleavage site (AVLQ) of 3CLpro and 6 × His-tag encoding sequences before and after the Nsp5-Mpro sequence, respectively. This modification enabled the expression of 3CLpro as an authentic N terminal protease (au3CLpro), which was purified to electrophoretic homogeneity by a single-step chromatography using two tandem Glutathione- and Ni-Sepharose columns. The enzyme au3CLpro demonstrated significantly higher activity (3169 RFU/min/µg protein) and catalytic efficiency (Kcat/Km of 0.007 µM-1.s-1) than that of the 3CLpro (com3CLpro) expressed from the commercial vector (pETM33-Nsp5-Mpro) with specific activity 889 RFU/min/µg and Kcat/Km of 0.0015 µM-1.s-1, respectively. Optimal conditions for au3CLpro activity included a 50 mM Tris-HCl buffer at pH 7, containing 150 mM NaCl and 0.1 mg/ml BSA at 37 °C.

Role of Coronary Calcium Score to Identify Candidates for ASCVD Prevention.

PURPOSE OF REVIEW: In this review, we describe the mechanism behind coronary artery calcification formation and detection, as well as its implication in cardiovascular disease (CVD) risk stratification, intervention, and prognosis in asymptomatic individuals. RECENT FINDINGS: Multiple cohort and population studies have shown that coronary artery calcium scoring is effective and reproducible in predicting the risk for cardiovascular disease. The updated 2018 ACC/AHA guideline has incorporated consideration of coronary artery calcification testing into cardiovascular disease risk stratification and therapy guidance. Coronary artery calcification's evidence-based role in detection, risk stratification, and ultimately its unique influence on therapeutic intervention and prognosis of cardiovascular disease in asymptomatic population is increasingly being recognized..

Variability in State-Based Recommendations for Management of Alpha Thalassemia Trait and Silent Carrier Detected on the Newborn Screen.

We conducted an inventory of state-based recommendations for follow-up of alpha thalassemia silent carrier and trait identified on newborn screen. We found wide variability in the nature and timing of these recommendations. We recommend a standardized recommendation to guide pediatricians in evidenced-based care for this population.

Expression of a novel mRNA transcript for human microsomal epoxide hydrolase (EPHX1) is regulated by short open reading frames within its 5'-untranslated region.

Microsomal epoxide hydrolase (mEH, EPHX1) is a critical xenobiotic-metabolizing enzyme, catalyzing both detoxification and bioactivation reactions that direct the disposition of chemical epoxides, including the carcinogenic metabolites of several polycyclic aromatic hydrocarbons. Recently, we discovered that a previously unrecognized and primate-specific EPHX1 transcript, termed E1-b, was actually the predominant driver of EPHX1 expression in all human tissues. In this study, we identify another human EPHX1 transcript, designated as E1-b'. Unusually, both the E1-b and E1-b' mRNA transcripts are generated from the use of a far upstream gene promoter, localized ∼18.5 kb 5'-upstream of the EPHX1 protein-coding region. Although expressed at comparatively lower levels than E1-b, the novel E1-b' transcript is readily detected in all tissues examined, with highest levels maintained in human ovary. The E1-b' mRNA possesses unusual functional features in its 5'-untranslated region, including a GC-rich leader sequence and two upstream AUGs that encode for short peptides of 26 and 17 amino acids in length, respectively. Results from in vitro transcription/translation assays and direct transfection in mammalian cells of either the E1-b' transcript or the encoded peptides demonstrated that the E1-b' upstream open reading frames (uORFs) are functional, with their presence markedly inhibiting the translation of EPHX1 protein, both in cis and in trans configurations. These unique uORF peptides exhibit no homology to any other known uORF sequences but likely function to mediate post-transcription regulation of EPHX1 and perhaps more broadly as translational regulators in human cells.

An efficient procedure for the expression and purification of HIV-1 protease from inclusion bodies.

Several studies have focused on HIV-1 protease for developing drugs for treating AIDS. Recombinant HIV-1 protease is used to screen new drugs from synthetic compounds or natural substances. However, large-scale expression and purification of this enzyme is difficult mainly because of its low expression and solubility. In this study, we constructed 9 recombinant plasmids containing a sequence encoding HIV-1 protease along with different fusion tags and examined the expression of the enzyme from these plasmids. Of the 9 plasmids, pET32a(+) plasmid containing the HIV-1 protease-encoding sequence along with sequences encoding an autocleavage site GTVSFNF at the N-terminus and TEV plus 6× His tag at the C-terminus showed the highest expression of the enzyme and was selected for further analysis. The recombinant protein was isolated from inclusion bodies by using 2 tandem Q- and Ni-Sepharose columns. SDS-PAGE of the obtained HIV-1 protease produced a single band of approximately 13 kDa. The enzyme was recovered efficiently (4 mg protein/L of cell culture) and had high specific activity of 1190 nmol min(-1) mg(-1) at an optimal pH of 4.7 and optimal temperature of 37 °C. This procedure for expressing and purifying HIV-1 protease is now being scaled up to produce the enzyme on a large scale for its application.

Rare silent ischemic ventricular septal aneurysm and rupture: A multimodality diagnostic approach.

A multimodality approach in addition to high level of clinical suspicion and thorough physical examination is important in the diagnosis and management of left ventricular aneurysm and rupture after a silent inferior infarction.

Broken and forgotten: A case of unintentionally retained foreign object.

Central venous catheter have become ubiquitous with greater than 15 million catheter days/year in the intensive care setting alone. However, the procedure carries with it several immediate and other delayed complications that can result in significant morbidity, mortality, and increased healthcare cost. We report a rare case of significantly delayed complications associated with intravascular loss of guide wire during central venous catheter placement and its impact on patient's long term management. The case highlights not only the importance of proper technique and safety precaution in performing an increasingly common procedure, but also the need for timely identification and rectification of medical errors, especially in the context of improved physician-patient communication.

Meta-analysis Comparing Multivessel Versus Culprit Coronary Arterial Revascularization for Patients With Non-ST-Segment Elevation Acute Coronary Syndromes.

We present a systematic review and meta-analysis comparing efficacy and safety outcomes between single procedure multivessel revascularization (MVR) and culprit vessel only revascularization in patients presenting with non-ST-segment-elevation acute coronary syndrome (NSTE-ACS). NSTE-ACS is the most common form of acute coronary syndrome (ACS), and multivessel disease is common. There is no consensus on the most efficacious single procedure revascularization strategy for patients undergoing percutaneous coronary intervention not meeting coronary artery bypass grafting criteria. Studies in PubMed and EMBASE databases were systematically reviewed, and 15 studies met criteria for inclusion in the meta-analysis. Baseline characteristics between the groups were similar. A random effects model was used to calculate odds ratios (OR) with 95% confidence intervals (CI). Heterogeneity of studies was assessed using Cochrane's Q and Higgins I2 tests. For short-term outcomes, patients who underwent MVR had higher rates of major adverse cardiac events (OR 1.14; 95% CI 1.01 to 1.29; p = 0.03); and stroke (OR 1.94; 95% CI 1.01 to 3.72; p = 0.05), but lower rates of urgent or emergent coronary artery bypass grafting (OR 0.35; 95% CI 0.29 to 0.43; p <0.00001). In the long-term, MVR patients had less frequent major adverse cardiac events (OR 0.76; 95% CI 0.61-0.93; p = 0.009), all-cause death (OR 0.83; 95% CI 0.71 to 0.97; p = 0.03), and repeat revascularization, (OR 0.62; 95% CI 0.42 to 0.90; p = 0.01). MVR following NSTE-ACS was associated with higher short-term risk, but long-term benefit. In conclusion, these results support the use of single procedure multivessel revascularization for NSTE-ACS patients who are suitable candidates at the time of percutaneous coronary intervention.

Leigh syndrome T8993C mitochondrial DNA mutation: Heteroplasmy and the first clinical presentation in a Vietnamese family.

Leigh syndrome is a rare inherited, heterogeneous and progressive neurometabolic disorder that is mainly caused by specific mutations in nuclear DNA (nDNA) or mitochondrial DNA (mtDNA). The present study reported a case of childhood Leigh syndrome with a point mutation at bp 8,993 in the mitochondrial ATPase6 gene. A 21­month­old male child had developed epilepsy, muscular weakness and vomiting, which was accompanied by high fever. Magnetic resonance imaging indicated typical characteristics of Leigh syndrome, including a symmetric abnormal signal in the dorsal medulla oblongata and Sylvian fissure enlargement in association with an abnormal signal in the periventricular white matter and in the putamina and caudate heads. The diagnosis was further supported with genetic tests including polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), sequencing, and quantitative PCR. The patient was found to carry a mitochondrial T8993C (m.T8993C) mutation in peripheral blood with 94.00±1.34% heteroplasmy. Eight of his relatives were also subjected to quantification of the m.T8993C mutation. The percentages of heteroplasmy in samples taken from the grandmother, mother, aunt, cousin 1, and cousin 2 were 16.33±1.67, 66.81±0.85, 71.66±3.22, 87.00±1.79, and 91.24±2.50%, respectively. The mutation was not found in samples taken from the father, the husband of the aunt, or the grandfather of the patient. The obtained data showed that the mutation was maternally inherited and accumulated through generations. Even though the heteroplasmy levels of his mother, aunt, cousin 1, and cousin 2 were relatively high (66.81­91.24%), they remained asymptomatic, indicating that the threshold at which this mutation shows effects is high. To the best of our knowledge, this is the first report of a case of Leigh syndrome in a Vietnamese individual harboring a mtDNA mutation at the 8,993 bp site, and showing a correlation between the heteroplasmy and clinical phenotype. These findings may be useful in helping to improve the clinical diagnosis and treatment of Leigh syndrome.