Measurements of Protein Folding/Misfolding using Pulsed Electron Spin Resonance

We describe a new electron spin resonance (ESR) method that measures interspin distances at physiological conditions. The method is used to examine the melting of a polyalanine peptide, which is of considerable current interest in the field of protein folding. One end of the peptide is tagged with a Cu(II) ion and the other with a nitroxide spin label. The rapidly relaxing electron spin, Cu(II), enhances the electron spin flip rate of the nitroxide. This change in the relaxation rate depends on the interspin distance, r. As the peptide unfolds, the interspin distance changes. The ESR determined interspin distance decreases from the folded to unfolded state whereas the MD simulation shows an increased distance of the peptide in the PPII geometry. Therefore, the ESR results indicate that the polyalanine peptide does not melt into an extended PPII structure in the unfolded state. The ESR results also show that the change in interspin distances is clearly in concordance with the change in helicity and demonstrates an important application of the pulsed ESR method to monitor unfolding transition at physiological temperatures in biomolecules.In the second part of this research, the local environment of the Cu(II) ion in amyloid-¥â peptide (A¥â) is investigated by electron spin echo envelope modulation ESR spectroscopy. The aggregation of A¥â is implicated in the pathogenesis of Alzheimer¡¯s disease. Distinct differences in coordination of Cu(II) to A¥â are observed as the Cu(II) concentration increases. The overall morphology of A¥â aggregates, shown by transmission electron microscopy and atomic force microscopy images, also depends on the Cu(II) concentration. At low concentration of Cu(II), A¥â fibrils are observed and Cu(II) is coordinated by N-terminal histidine residues. As the concentration of Cu(II) increases, the images suggest a significant increase in the proportion of granular amorphous aggregates, and ESR spectra indicate a second copper-binding site that exists in a proton-rich environment. The results strongly suggest that the misfolding mechanism of A¥â depends on the Cu(II) concentration. This research significantly enhances our understanding of the misfolding mechanism in A¥â, and elucidates the relationship between the microscopic Cu(II)-A¥â interaction and macroscopic structure

SARS-CoV-2 aberrantly elevates mitochondrial bioenergetics to induce robust virus propagation

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a 'highly transmissible respiratory pathogen, leading to severe multi-organ damage. However, knowledge regarding SARS-CoV-2-induced cellular alterations is limited. In this study, we report that SARS-CoV-2 aberrantly elevates mitochondrial bioenergetics and activates the EGFR-mediated cell survival signal cascade during the early stage of viral infection. SARS-CoV-2 causes an increase in mitochondrial transmembrane potential via the SARS-CoV-2 RNA-nucleocapsid cluster, thereby abnormally promoting mitochondrial elongation and the OXPHOS process, followed by enhancing ATP production. Furthermore, SARS-CoV-2 activates the EGFR signal cascade and subsequently induces mitochondrial EGFR trafficking, contributing to abnormal OXPHOS process and viral propagation. Approved EGFR inhibitors remarkably reduce SARS-CoV-2 propagation, among which vandetanib exhibits the highest antiviral efficacy. Treatment of SARS-CoV-2-infected cells with vandetanib decreases SARS-CoV-2-induced EGFR trafficking to the mitochondria and restores SARS-CoV-2-induced aberrant elevation in OXPHOS process and ATP generation, thereby resulting in the reduction of SARS-CoV-2 propagation. Furthermore, oral administration of vandetanib to SARS-CoV-2-infected hACE2 transgenic mice reduces SARS-CoV-2 propagation in lung tissue and mitigates SARS-CoV-2-induced lung inflammation. Vandetanib also exhibits potent antiviral activity against various SARS-CoV-2 variants of concern, including alpha, beta, delta and omicron, in in vitro cell culture experiments. Taken together, our findings provide novel insight into SARS-CoV-2-induced alterations in mitochondrial dynamics and EGFR trafficking during the early stage of viral infection and their roles in robust SARS-CoV-2 propagation, suggesting that EGFR is an attractive host target for combating COVID-19

Issues in Developing and Evaluating a Culturally Tailored Internet Cancer Support Group

The purpose of this paper is to explore practical issues in developing and implementing a culturally tailored Internet Cancer Support Group for a group of ethnic minority cancer patients—Asian American cancer patients. Throughout the research process of the original study testing the Internet cancer support group, research team made written records of practical issues and plausible rationales for the issues. Weekly group discussion among research team members was conducted, and the discussion records were evaluated and analyzed using a content analysis (with individual words as the unit of analysis). The codes from the analysis process were categorized into idea themes, through which the issues were extracted. The issues included those in: (a) difficulties in using multiple languages; (b) collaboration with the IT department and technical challenges (c) difficulties in recruitment; (d) difficulties in retention; (e) optimal timing; and (f) characteristics of the users. Based on the findings, we suggested researchers to plan a workable translation process, check technical needs in advance, use multiple strategies to recruit and retain research participants, plan the right time for data collection, and consider characteristics of the users in the study design

Internet Recruitment of Asian American Breast Cancer Survivors

The purpose of this paper is to identify practical issues in Internet recruitment of racial/ethnic minorities by analyzing an Internet intervention study conducted with Asian American breast cancer survivors, and to propose directions for recruitment of racial/ethnic minorities for future Internet research. Six practical issues were identified: (a) a relatively fewer number of Internet communities/groups; (b) hindrances in establishing authenticity; (c) difficulties in gaining entrée from the webmasters or website owners of Internet communities/groups; (d) the necessity of racially/ethnically matched research team members; (e) flexibility required in recruitment strategies; and (f) strategies to overcome the low response rate

Dysregulated protocadherin-pathway activity as an intrinsic defect in induced pluripotent stem cell-derived cortical interneurons from subjects with schizophrenia.

We generated cortical interneurons (cINs) from induced pluripotent stem cells derived from 14 healthy controls and 14 subjects with schizophrenia. Both healthy control cINs and schizophrenia cINs were authentic, fired spontaneously, received functional excitatory inputs from host neurons, and induced GABA-mediated inhibition in host neurons in vivo. However, schizophrenia cINs had dysregulated expression of protocadherin genes, which lie within documented schizophrenia loci. Mice lacking protocadherin-α showed defective arborization and synaptic density of prefrontal cortex cINs and behavioral abnormalities. Schizophrenia cINs similarly showed defects in synaptic density and arborization that were reversed by inhibitors of protein kinase C, a downstream kinase in the protocadherin pathway. These findings reveal an intrinsic abnormality in schizophrenia cINs in the absence of any circuit-driven pathology. They also demonstrate the utility of homogenous and functional populations of a relevant neuronal subtype for probing pathogenesis mechanisms during development

Direct Visualization of HIV-1 with Correlative Live-Cell Microscopy and Cryo-Electron Tomography

SummaryCryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-native state and therefore has the potential to help elucidate early events of HIV-1 infection in host cells. However, structural details of infecting HIV-1 have not been observed, due to technological challenges in working with rare and dynamic HIV-1 particles in human cells. Here, we report structural analysis of HIV-1 and host-cell interactions by means of a correlative high-speed 3D live-cell-imaging and cryoET method. Using this method, we showed under near-native conditions that intact hyperstable mutant HIV-1 cores are released into the cytoplasm of host cells. We further obtained direct evidence to suggest that a hyperstable mutant capsid, E45A, showed delayed capsid disassembly compared to the wild-type capsid. Together, these results demonstrate the advantages of our correlative live-cell and cryoET approach for imaging dynamic processes, such as viral infection