A Qualitative Study on the Experiences of Preclinical Students in Learning Clinical and Communication Skills at a Simulation Centre.

Introduction: Simulation centres (SC) and its learning resources are now firmly established as part of medical education. In SC, medical students obtain both knowledge and skills based on a combination of theory and practice using provided resources. This study aims to explore medical students' use of SC learning resources to learn clinical and communication skills based on Kolb's experiential learning cycle. This is based on the research question 'How are the SC resources useful in supporting preclinical medical students' clinical and communication skills learning?' The findings of the study can make a case for further enhancement of SC design and resources for medical students in the preclinical phase. Methods: A qualitative study involving 20 preclinical medical students with learning experiences in SC was conducted between December 2019 and 2020 at a medical school in Malaysia. Semi-structured interview questions were developed based on Kolb's learning cycle. The data were thematically analysed using the six phases of Braun and Clarke's thematic analysis. Results: Three main themes were identified based on preclinical medical students' experiences in SC; they were 'preparation for authentic clinical experience', 'accessibility of multiple resources for learning and support' and 'opportunities to learn and improve'. Conclusions: The SC's resources have a significant and positive role in supporting preclinical medical students learn clinical and communication skills. The SC resources prepared them for authentic clinical experiences with a patient-centred care approach and self-directed learning opportunities. Social support from peers, peer tutors and academics emerged as a key finding and resource of the SC as they help preclinical students learn and improve.

Corrigendum: Immune Response in Myocardial Injury: In Situ Hybridization and Immunohistochemistry Techniques for SARS-CoV-2 Detection in COVID-19 Autopsies.

[This corrects the article DOI: 10.3389/fmolb.2021.658932.].

An exploratory study on the challenges faced and coping strategies used by preclinical medical students during the COVID-19 crisis.

PURPOSE: The impact of the coronavirus disease 2019 crisis on medical education includes reduced clinical training, a significant loss of learning time and a probable decline in confidence of being a doctor. These recent changes will have significant effect on the well-being of medical students and interventional support needs to be given early. This study explores the challenges faced and coping strategies used by preclinical medical students during the crisis. METHODS: A qualitative study involving 13 preclinical medical students was conducted between August and September 2020 at a medical school in Malaysia. An in-depth individual interview via Microsoft Teams (Microsoft Corp.) with semi-structured questions was conducted. The recorded interview data were thematically analyzed using the six phases of Braun and Clarke's Thematic Analysis. RESULTS: The challenges faced were identified under three themes: psychosocial impact of lockdown, significant lifestyle changes, and impact on professional progression. Meanwhile, four themes emerged in coping strategies that include behavioral strategies, re-appraisal of the uncertainties of situation, active coping mechanisms, and regulation of emotion with coping reserve. There are indications that personality traits determine strategies to cope with challenges faced during the crisis which may either lead to resilience building or experiencing burnout. CONCLUSION: The findings of the study highlighted the urgent need to develop early preventive and intervention strategies to address the mental health of medical students to mitigate stress and promote positive well-being in times of crisis.

Discovery of novel pyridyl carboxamides as potent CCR5 antagonists and optimization of their pharmacokinetic profile in rats.

A novel series of pyridyl carboxamide-based CCR5 inhibitors was designed, synthesized, and demonstrated to be highly potent against HIV-1 infection in both HOS and PBL assays. Attempts to evaluate this series of compounds in a rat PK model revealed its instability in rat plasma. A hypothesis for this liability was proposed, and strategies to overcome this issue were pursued, leading to discovery of highly potent 40 and 41, which featured dramatically improved rat PK profiles.

Immune Response in Myocardial Injury: In Situ Hybridization and Immunohistochemistry Techniques for SARS-CoV-2 Detection in COVID-19 Autopsies.

Coronavirus disease-19 (COVID-19) is caused by the newly discovered coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While the lung remains the primary target site of COVID-19 injury, damage to myocardium, and other organs also contribute to the morbidity and mortality of this disease. There is also increasing demand to visualize viral components within tissue specimens. Here we discuss the cardiac autopsy findings of 12 intensive care unit (ICU) naïve and PCR-positive COVID-19 cases using a combination of histological, Immunohistochemical/immunofluorescent and molecular techniques. We performed SARS-CoV-2 qRT-PCR on fresh tissue from all cases; RNA-ISH and IHC for SARS-CoV-2 were performed on selected cases using FFPE tissue from heart. Eight of these patients also had positive post-mortem serology for SARS-CoV-2. Histopathologic changes in the coronary vessels and inflammation of the myocardium as well as in the endocardium were documented which support the reports of a cardiac component to the viral infection. As in the pulmonary reports, widespread platelet and fibrin thrombi were also identified in the cardiac tissue. In keeping with vaccine-induced activation of virus-specific CD4+ and CD8+ T cells, and release of cytokines such as interferon-gamma (IFNγ), we observed similar immune cellular distribution and cytokines in these patients. Immunohistochemical and immunofluorescent localisation for the viral Spike (S-protein) protein and the nucleocapsid protein (NP) were performed; presence of these aggregates may possibly contribute to cardiac ischemia and even remodelling.

Mechanism of NS2B-mediated activation of NS3pro in dengue virus: molecular dynamics simulations and bioassays.

The NS2B cofactor is critical for proteolytic activation of the flavivirus NS3 protease. To elucidate the mechanism involved in NS2B-mediated activation of NS3 protease, molecular dynamic simulation, principal component analysis, molecular docking, mutagenesis, and bioassay studies were carried out on both the dengue virus NS3pro and NS2B-NS3pro systems. The results revealed that the NS2B-NS3pro complex is more rigid than NS3pro alone due to its robust hydrogen bond and hydrophobic interaction networks within the complex. These potent networks lead to remodeling of the secondary and tertiary structures of the protease that facilitates cleavage sequence recognition and binding of substrates. The cofactor is also essential for proper domain motion that contributes to substrate binding. Hence, the NS2B cofactor plays a dual role in enzyme activation by facilitating the refolding of the NS3pro domain as well as being directly involved in substrate binding/interactions. Kinetic analyses indicated for the first time that Glu92 and Asp50 in NS2B and Gln27, Gln35, and Arg54 in NS3pro may provide secondary interaction points for substrate binding. These new insights on the mechanistic contributions of the NS2B cofactor to NS3 activation may be utilized to refine current computer-based search strategies to raise the quality of candidate molecules identified as potent inhibitors against flaviviruses.

Design of N-Benzoxaborole Benzofuran GSK8175-Optimization of Human Pharmacokinetics Inspired by Metabolites of a Failed Clinical HCV Inhibitor.

We previously described the discovery of GSK5852 (1), a non-nucleoside polymerase (NS5B) inhibitor of hepatitis C virus (HCV), in which an N-benzyl boronic acid was essential for potent antiviral activity. Unfortunately, facile benzylic oxidation resulted in a short plasma half-life (5 h) in human volunteers, and a backup program was initiated to remove metabolic liabilities associated with 1. Herein, we describe second-generation NS5B inhibitors including GSK8175 (49), a sulfonamide- N-benzoxaborole analog with low in vivo clearance across preclinical species and broad-spectrum activity against HCV replicons. An X-ray structure of NS5B protein cocrystallized with 49 revealed unique protein-inhibitor interactions mediated by an extensive network of ordered water molecules and the first evidence of boronate complex formation within the binding pocket. In clinical studies, 49 displayed a 60-63 h half-life and a robust decrease in viral RNA levels in HCV-infected patients, thereby validating our hypothesis that reducing benzylic oxidation would improve human pharmacokinetics and lower efficacious doses relative to 1.

Signature Optical Cues: Emerging Technologies for Monitoring Plant Health.

Optical technologies can be developed as practical tools for monitoring plant health by providing unique spectral signatures that can be related to specific plant stresses. Signatures from thermal and fluorescence imaging have been used successfully to track pathogen invasion before visual symptoms are observed. Another approach for noninvasive plant health monitoring involves elucidating the manner with which light interacts with the plant leaf and being able to identify changes in spectral characteristics in response to specific stresses. To achieve this, an important step is to understand the biochemical and anatomical features governing leaf reflectance, transmission and absorption. Many studies have opened up possibilities that subtle changes in leaf reflectance spectra can be analyzed in a plethora of ways for discriminating nutrient and water stress, but with limited success. There has also been interest in developing transgenic phytosensors to elucidate plant status in relation to environmental conditions. This approach involves unambiguous signal creation whereby genetic modification to generate reporter plants has resulted in distinct optical signals emitted in response to specific stressors. Most of these studies are limited to laboratory or controlled greenhouse environments at leaf level. The practical translation of spectral cues for application under field conditions at canopy and regional levels by remote aerial sensing remains a challenge. The movement towards technology development is well exemplified by the Controlled Ecological Life Support System under development by NASA which brings together technologies for monitoring plant status concomitantly with instrumentation for environmental monitoring and feedback control.

An SRLLR motif downstream of the scissile bond enhances enterokinase cleavage efficiency.

In a previous paper, we reported more efficient enterokinase cleavage at a C-terminal non-target LKGDR(201) site compared with an internally sited canonical recognition site, DDDDK(156). When this non-target site was placed internally to replace DDDDK(156) between the thioredoxin moiety and mouse NT-proCNP(1-50), this site was poorly processed leading us to conclude that efficient processing at LKGDR(201) in the first instance was due to its accessibility at the C-terminus of the fusion protein. Subsequently, we reasoned that treatment of thioredoxin-fused NT-proCNP(1-81) would allow us to retrieve full-length NT-proCNP(1-81) without undue processing at the LKGDR(201) site since this non-target site would now be located internally about 36 residues away from the C-terminus and hence not be hydrolyzed efficiently. Surprisingly, ESI-MS data showed that the LKGDR site in thioredoxin-fused human NT-proCNP(1-81) was still very efficiently cleaved and revealed a new but slow hydrolysis site with the sequence RVDTK/SRAAW to yield a peptide consistent with NT-proCNP(58-81). The evidence obtained from these experiments led us to postulate that efficient cleavage at the non-target LKGDR(201) site was not merely influenced by steric constraints but also by the sequence context downstream of the scissile bond. Hence, we constructed variants of thioredoxin-mouse NT-proCNP(1-50) where SRLLR residues (i.e. those immediately downstream from the LKGDR(201) site in NT-proCNP(1-50)) were systematically added one at a time downstream of the internal DDDDK(156) site. To evaluate the relative effects of site accessibility and downstream sequence context on the efficiency of enterokinase cleavage, we have also replaced the native LKGDR(201) sequence with DDDDK(201). Our results showed that incremental addition of SRLLR residues led to a steady increase in the rate of hydrolysis at DDDDK(156). Further variants comprising DDDDK(156)SS, DDDDK(156)SD and DDDDK(156)RR showed that the minimal critical determinants for enhanced enterokinase cleavage are serine in the P1' position followed by a serine or a basic residue, lysine or arginine, in the P2' position. Our data provided conclusive evidence that the influence of downstream sequences on recombinant light chain enterokinase activity was greater than accessibility of the target site at the terminus region of the protein. We further showed that the catalytic efficiency of the native holoenzyme was influenced primarily by residues on the N-terminal side of the scissile bond while being neutral to residues on the C-terminal side. Finally, we found that cleavage of all nine fusion proteins reflects accurate hydrolysis at the DDDDK(156) and DDDDK(201) sites when recombinant light chain enterokinase was used while non-specific processing at secondary sites were observed when these fusion proteins were treated with the native holoenzyme.

SARS-CoV virus-host interactions and comparative etiologies of acute respiratory distress syndrome as determined by transcriptional and cytokine profiling of formalin-fixed paraffin-embedded tissues.

These studies attempt to understand more fully the host response and pathogenesis associated with severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) by monitoring gene expression using formalin-fixed paraffin-embedded (FFPE) pulmonary autopsy tissues. These tissues were from patients in different hospitals in Singapore who were diagnosed with various microbial infections, including SARS-CoV, that caused acute respiratory distress syndrome (ARDS). Global expression patterns showed limited correlation between end-stage ARDS and the initiating pathogen, but when focusing on a subset of genes implicated in pulmonary pathogenesis, molecular signatures of pulmonary disease were obtained and appeared to be influenced by preexisting pulmonary complications and also bacterial components of infection. Many factors detected during pulmonary damage and repair, such as extracellular matrix (ECM) components, transforming growth factor (TGF) enhancers, acute-phase proteins, and antioxidants, were included in the molecular profiles of these ARDS lung tissues. In addition, differential expression of cytokines within these pulmonary tissues were observed, including notable genes involved in the interferon (IFN) pathway, such as Stat1, IFN regulatory factor-1 (IRF-1), interleukin-6 (IL-6), IL-8, and IL-18, that are often characterized as elevated in ARDS patients.

Synthesis of (+)-bullatacin via the highly diastereoselective [3+2] annulation reaction of a racemic aldehyde and a nonracemic allylsilane.

[reaction: see text] A total synthesis of (+)-bullatacin has been accomplished via a diastereoselective [3+2] annulation reaction of the highly enantiomerically enriched allylsilane 3 and racemic aldehyde 4, which provides the key bis-tetrahydrofuran fragment 15 with > or = 20:1 ds.

Lung pathology of severe acute respiratory syndrome (SARS): a study of 8 autopsy cases from Singapore.

Severe acute respiratory syndrome (SARS) is an infectious condition caused by the SARS-associated coronavirus (SARS-CoV), a new member in the family Coronaviridae. To evaluate the lung pathology in this life-threatening respiratory illness, we studied postmortem lung sections from 8 patients who died from SARS during the spring 2003 Singapore outbreak. The predominant pattern of lung injury in all 8 cases was diffuse alveolar damage. The histology varied according to the duration of illness. Cases of 10 or fewer days' duration demonstrated acute-phase diffuse alveolar damage (DAD), airspace edema, and bronchiolar fibrin. Cases of more than 10 days' duration exhibited organizing-phase DAD, type II pneumocyte hyperplasia, squamous metaplasia, multinucleated giant cells, and acute bronchopneumonia. In acute-phase DAD, pancytokeratin staining was positive in hyaline membranes along alveolar walls and highlighted the absence of pneumocytes. Multinucleated cells were shown to be both type II pneumocytes and macrophages by pancytokeratin, thyroid transcription factor-1, and CD68 staining. SARS-CoV RNA was identified by reverse transcriptase-polymerase chain reaction in 7 of 8 cases in fresh autopsy tissue and in 8 of 8 cases in formalin-fixed, paraffin-embedded lung tissue, including the 1 negative case in fresh tissue. Understanding the pathology of DAD in SARS patients may provide the basis for therapeutic strategies. Further studies of the pathogenesis of SARS may reveal new insight into the mechanisms of DAD.

Concerning the origin of the high beta-selectivity of glycosidation reactions of 2-deoxy-2-iodo-glucopyranosyl trichloroacetimidates.

[structure: see text] Studies on the glycosidation reactions of conformationally constrained glycosyl imidates 8a and 8b were performed to evaluate the possible involvement of "conformationally inverted" oxonium ion intermediates in glycosidation reactions with 2-deoxy-2-iodo-glucopyranosyl donors. The mechanistic implications of this study are discussed, and intermediates 23 and 24 are invoked to rationalize the observed beta-selectivities.

Invasive middle ear cholesterol granuloma involving the basal turn of the cochlea with profound sensorineural hearing loss.

STUDY DESIGN: A retrospective surgical case review. SETTING: A tertiary-care, university teaching hospital. PATIENTS: The authors describe a unique case operated on for a middle ear cholesterol granuloma, which had invaded the cochlea and vestibule causing profound sensorineural deafness. INTERVENTIONS: Extended radical mastoidectomy and labyrinthectomy with musculofascial seal. RESULTS: Successful postoperative outcome with no recurrence seen after 2 years of follow up. CONCLUSION: To the authors' knowledge, this is the first reported case of a primary middle ear cholesterol granuloma with direct invasion into the cochlea. Such invasion of the otic capsule by cholesterol granulomas is rare and presents a diagnostic challenge to the attending otologist and radiologist. Salient points of the case history, pathogenesis, imaging studies, histopathology, and management are presented with a review of the current literature.

A His6-SUMO-eXact tag for producing human prepro-urocortin 2 in Escherichia coli for raising monoclonal antibodies.

This is a first report of recombinant production of human prepro-Urocortin 2 in Escherichia coli by N-terminal fusion with a triple His6-SUMO-eXact tag and its subsequent use as an antigen for the production and screening of very high affinity monoclonal antibodies. The rationale for this combinatorial construct is that the His tag allows first step protein purification of insoluble and soluble proteins, the SUMO tag enhances protein expression level and solubility, while the eXact tag facilitates anion-triggered on-column cleavage of the triple tag to recover pure native proteins in a simple two-step protein purification procedure. Compared with an eXact fusion alone, the presence of the SUMO moiety enhanced overall expression levels by 4 to 10 fold but not the solubility of the highly basic prepro-Urocortin 2. Insoluble SUMO-eXact-preproUCN2 was purified in milligram quantities by denaturing IMAC and solubilized in native phosphate buffer by on-column refolding or step-wise dialysis. Only a small fraction of this solubilized protein was able to bind onto the eXact™ affinity column and cleaved by NaF treatment. To test whether binding and cleavage failure was due to improperly refolded SUMO-eXact-preproUCN2 or to the presence of N- and C-terminal sequences flanking the eXact moiety, we created a SUMO-eXact-thioredoxin construct which was overexpressed mainly in the soluble form. This protein bound to and was cleaved efficiently on the eXact™ column to yield native thioredoxin. Solubilized SUMO-eXact-preproUCN2 was used successfully to generate two high affinity mouse monoclonal antibodies (KD~10⁻¹° and 10⁻¹¹ M) specific to the pro-region of Urocortin 2.

Hepatitis C replication inhibitors that target the viral NS4B protein.

We describe the preclinical development and in vivo efficacy of a novel chemical series that inhibits hepatitis C virus replication via direct interaction with the viral nonstructural protein 4B (NS4B). Significant potency improvements were realized through isosteric modifications to our initial lead 1a. The temptation to improve antiviral activity while compromising physicochemical properties was tempered by the judicial use of ligand efficiency indices during lead optimization. In this manner, compound 1a was transformed into (+)-28a which possessed an improved antiviral profile with no increase in molecular weight and only a modest elevation in lipophilicity. Additionally, we employed a chimeric "humanized" mouse model of HCV infection to demonstrate for the first time that a small molecule with high in vitro affinity for NS4B can inhibit viral replication in vivo. This successful proof-of-concept study suggests that drugs targeting NS4B may represent a viable treatment option for curing HCV infection.

Breast cancer stem cells: an update.

Breast cancer is a significant cause of morbidity and mortality in women with a high incidence of recurrence or treatment failure. Growing evidence suggests that cancer stem cells (CSCs) most likely contribute to tumour progression, spread and therapy failure. However, despite extensive research and the tremendous clinical potential of such cells in possible therapeutic management, the real nature of CSCs remains an enigma. In this review, we discuss the fundamental properties and molecular target of CSCs and focus on recent advances regarding the identification of CSC markers with emphasis on breast cancer and the underlying molecular mechanism of CSC phenotypes. We also discuss experimental evidence of targeting molecular pathways in order to modulate breast CSC behaviour in tumourigenesis and the controversies associated with it that potentially weaken the CSC model in breast cancer and other cancers as well.

Rational design of potent non-nucleoside inhibitors of HIV-1 reverse transcriptase.

A new series of non-nucleoside reverse transcriptase inhibitors based on an imidazole-amide biarylether scaffold has been identified and shown to possess potent antiviral activity against HIV-1, including the NNRTI-resistant Y188L mutated virus. X-ray crystallography of inhibitors bound to reverse transcriptase, including a structure of the Y188L RT protein, was used extensively to help identify and optimize the key hydrogen-bonding motif. This led directly to the design of compound 43 that exhibits remarkable antiviral activity (EC50<1 nM) against a wide range of NNRTI-resistant viruses and a favorable pharmacokinetic profile across multiple species.

Imidazo[1,2-a]pyridines That Directly Interact with Hepatitis C NS4B: Initial Preclinical Characterization.

A series of imidazo[1,2-a]pyridines which directly bind to HCV Non-Structural Protein 4B (NS4B) is described. This series demonstrates potent in vitro inhibition of HCV replication (EC50 < 10 nM), direct binding to purified NS4B protein (IC50 < 20 nM), and an HCV resistance pattern associated with NS4B (H94N/R, V105L/M, F98L) that are unique among reported HCV clinical assets, suggestive of the potential for additive or synergistic combination with other small molecule inhibitors of HCV replication.