HIV/AIDS and COVID-19: Shared Lessons from Two Pandemics.

The global experiences with the HIV/AIDS and COVID-19 pandemics hold important lessons for preparing for, and responding to, future outbreaks of emerging or re-emerging infectious diseases. Scores of infectious diseases have emerged or re-emerged over the past four decades, and future outbreaks are inevitable. The next emerging pathogen likely will again come from unanticipated sources and pose puzzles in terms of microbiology, transmission, natural history, pathogenesis, epidemiology, and will present challenges to developing countermeasures such as diagnostics, therapeutics, and vaccines.

Ubiquitin's Conformational Heterogeneity as Discerned by Nuclear Magnetic Resonance Spectroscopy.

Visualizing a protein's molecular motions has been a long standing topic of research in the biophysics community. Largely this has been done by exploiting nuclear magnetic resonance spectroscopy (NMR), and arguably no protein's molecular motions have been better characterized by NMR than that of ubiquitin (Ub), a 76 amino acid polypeptide essential in ubiquitination-a key regulatory system within cells. Herein, we discuss ubiquitin's conformational plasticity as visualized, at atomic resolution, by more than 35 years of NMR work.

Broccoli aptamer allows quantitative transcription regulation studies in vitro.

Quantitative transcription regulation studies in vivo and in vitro often make use of reporter proteins. Here we show that using Broccoli aptamers, quantitative study of transcription in various regulatory scenarios is possible without a translational step. To explore the method we studied several regulatory scenarios that we analyzed using thermodynamic occupancy-based models, and found excellent agreement with previous studies.

Cellular Applications of DNP Solid-State NMR - State of the Art and a Look to the Future.

Sensitivity enhanced dynamic nuclear polarization solid-state NMR is emerging as a powerful technique for probing the structural properties of conformationally homogenous and heterogenous biomolecular species irrespective of size at atomic resolution within their native environments. Herein we detail advancements that have made acquiring such data, specifically within the confines of intact bacterial and eukaryotic cell a reality and further discuss the type of structural information that can presently be garnered by the technique's exploitation. Subsequently, we discuss bottlenecks that have thus far curbed cellular DNP-ssNMR's broader adoption namely due a lack of sensitivity and spectral resolution.

Genetic switching by the Lac repressor is based on two-state Monod-Wyman-Changeux allostery.

High-resolution NMR spectroscopy enabled us to characterize allosteric transitions between various functional states of the dimeric Lac repressor. In the absence of ligands, the dimer exists in a dynamic equilibrium between DNA-bound and inducer-bound conformations. Binding of either effector shifts this equilibrium toward either bound state.

A high-field cellular DNP-supported solid-state NMR approach to study proteins with sub-cellular specificity.

Studying the structural aspects of proteins within sub-cellular compartments is of growing interest. Dynamic nuclear polarization supported solid-state NMR (DNP-ssNMR) is uniquely suited to provide such information, but critically lacks the desired sensitivity and resolution. Here we utilize SNAPol-1, a novel biradical, to conduct DNP-ssNMR at high-magnetic fields (800 MHz/527 GHz) inside HeLa cells and isolated cell nuclei electroporated with [C,N] labeled ubiquitin.

Pandemic Preparedness and Response: Lessons From COVID-19.

The global experience with COVID-19 holds important lessons for preparing for, and responding to, future emergences of pathogens with pandemic potential.

Characterizing proteins in a native bacterial environment using solid-state NMR spectroscopy.

For a long time, solid-state nuclear magnetic resonance (ssNMR) has been employed to study complex biomolecular systems at the detailed chemical, structural, or dynamic level. Recent progress in high-resolution and high-sensitivity ssNMR, in combination with innovative sample preparation and labeling schemes, offers novel opportunities to study proteins in their native setting irrespective of the molecular tumbling rate. This protocol describes biochemical preparation schemes to obtain cellular samples of both soluble as well as insoluble or membrane-associated proteins in bacteria.

Characterization of nucleosome sediments for protein interaction studies by solid-state NMR spectroscopy.

Regulation of DNA-templated processes such as gene transcription and DNA repair depend on the interaction of a wide range of proteins with the nucleosome, the fundamental building block of chromatin. Both solution and solid-state NMR spectroscopy have become an attractive approach to study the dynamics and interactions of nucleosomes, despite their high molecular weight of ~ 200 kDa. For solid-state NMR (ssNMR) studies, dilute solutions of nucleosomes are converted to a dense phase by sedimentation or precipitation.

When Small becomes Too Big: Expanding the Use of In-Cell Solid-State NMR Spectroscopy.

Solution-state NMR spectroscopy has become a powerful tool to study soluble proteins in cells, provided that they tumble sufficiently fast. In addition, cryo-electron tomography (cryo-ET) has recently displayed a tremendous potential to probe structures of large proteins and assemblies in their native cellular environments. However, challenges remain to obtain atomic-level information in native cell settings for proteins that are small, disordered, or are strongly engaged in intermolecular interactions.

Ending the Human Immunodeficiency Virus Pandemic: Optimizing the Prevention and Treatment Toolkits.

Unprecedented basic and clinical biomedical research advances over the past 4 decades have led to the development of "toolkits" of highly effective interventions for preventing and treating human immunodeficiency virus (HIV). Despite many successes in decreasing the incidence and mortality of HIV, major challenges remain in the goal of ending the HIV pandemic in the United States and globally. Overcoming these challenges will require optimization of the implementation of existing interventions for HIV prevention and treatment together with the continued development of new and innovative approaches that can be readily utilized by individuals with HIV and those at risk of infection.

DNP-Supported Solid-State NMR Spectroscopy of Proteins Inside Mammalian Cells.

Elucidating at atomic level how proteins interact and are chemically modified in cells represents a leading frontier in structural biology. We have developed a tailored solid-state NMR spectroscopic approach that allows studying protein structure inside human cells at atomic level under high-sensitivity dynamic nuclear polarization (DNP) conditions. We demonstrate the method using ubiquitin (Ub), which is critically involved in cellular functioning.

Acute Flaccid Myelitis: Something Old and Something New.

Since 2014, acute flaccid myelitis (AFM), a long-recognized condition associated with polioviruses, nonpolio enteroviruses, and various other viral and nonviral causes, has been reemerging globally in epidemic form. This unanticipated reemergence is ironic, given that polioviruses, once the major causes of AFM, are now at the very threshold of global eradication and cannot therefore explain any aspect of AFM reemergence. Instead, the new AFM epidemic has been temporally associated with reemergences of nonpolio enteroviruses such as EV-D68, until recently thought to be an obscure virus of extremely low endemicity.

Function and Interactions of ERCC1-XPF in DNA Damage Response.

Numerous proteins are involved in the multiple pathways of the DNA damage response network and play a key role to protect the genome from the wide variety of damages that can occur to DNA. An example of this is the structure-specific endonuclease ERCC1-XPF. This heterodimeric complex is in particular involved in nucleotide excision repair (NER), but also in double strand break repair and interstrand cross-link repair pathways.

iSEE: Interface structure, evolution, and energy-based machine learning predictor of binding affinity changes upon mutations.

Quantitative evaluation of binding affinity changes upon mutations is crucial for protein engineering and drug design. Machine learning-based methods are gaining increasing momentum in this field. Due to the limited number of experimental data, using a small number of sensitive predictive features is vital to the generalization and robustness of such machine learning methods.

Guest Editorial: Critical Thinking in Education and Research-Why and How?

"A responsible scientist is able to step back, critically reflect on his or her own doings, and also explain them to a wider audience. Critical thinking includes the ability to talk to people working in other areas, as well as the broader public. It has to be taught to students and fostered at the university level, and should be practiced in relation to one's own work …" Read more in the Guest Editorial.

Efficient switching of mCherry fluorescence using chemical caging.

Fluorophores with dynamic or controllable fluorescence emission have become essential tools for advanced imaging, such as superresolution imaging. These applications have driven the continuing development of photoactivatable or photoconvertible labels, including genetically encoded fluorescent proteins. These new probes work well but require the introduction of new labels that may interfere with the proper functioning of existing constructs and therefore require extensive functional characterization.

Single-stranded DNA Binding by the Helix-Hairpin-Helix Domain of XPF Protein Contributes to the Substrate Specificity of the ERCC1-XPF Protein Complex.

The nucleotide excision repair protein complex ERCC1-XPF is required for incision of DNA upstream of DNA damage. Functional studies have provided insights into the binding of ERCC1-XPF to various DNA substrates. However, because no structure for the ERCC1-XPF-DNA complex has been determined, the mechanism of substrate recognition remains elusive.

EGFR Dynamics Change during Activation in Native Membranes as Revealed by NMR.

The epidermal growth factor receptor (EGFR) represents one of the most common target proteins in anti-cancer therapy. To directly examine the structural and dynamical properties of EGFR activation by the epidermal growth factor (EGF) in native membranes, we have developed a solid-state nuclear magnetic resonance (ssNMR)-based approach supported by dynamic nuclear polarization (DNP). In contrast to previous crystallographic results, our experiments show that the ligand-free state of the extracellular domain (ECD) is highly dynamic, while the intracellular kinase domain (KD) is rigid.

Swiss Canine Cancer Registry 1955-2008: Occurrence of the Most Common Tumour Diagnoses and Influence of Age, Breed, Body Size, Sex and Neutering Status on Tumour Development.

This study is based on the Swiss Canine Cancer Registry, comprising 121,963 diagnostic records of dogs compiled between 1955 and 2008, in which 63,214 (51.83%) animals were diagnosed with tumour lesions through microscopical investigation. Adenoma/adenocarcinoma (n = 12,293, 18.

A model for the interaction of the G3-subdomain of Geobacillus stearothermophilus IF2 with the 30S ribosomal subunit.

Bacterial translation initiation factor IF2 complexed with GTP binds to the 30S ribosomal subunit, promotes ribosomal binding of fMet-tRNA, and favors the joining of the small and large ribosomal subunits yielding a 70S initiation complex ready to enter the translation elongation phase. Within the IF2 molecule subdomain G3, which is believed to play an important role in the IF2-30S interaction, is positioned between the GTP-binding G2 and the fMet-tRNA binding C-terminal subdomains. In this study the solution structure of subdomain G3 of Geobacillus stearothermophilus IF2 has been elucidated.