Real-world evaluation of access-driven Canadian treatment sequences in progressive prostate cancer (REACTIVATE).

INTRODUCTION: The results of the phase 3 ALSYMPCA trial showed that Radium-223 (Ra-223) improves overall survival (OS) and delays onset of first symptomatic skeletal event vs. placebo in patients with metastatic castration-resistant prostate cancer (mCRPC). The purpose of the REACTIVATE study was to inform the optimal placement of Ra-233 in the treatment sequence by evaluating clinical outcomes and healthcare resource utilization using real-world data from multiple Canadian provinces. METHODS: This retrospective cohort study analyzed patient outcomes according to Ra-223 placement using administrative databases of four Canadian provinces, encompassing 4301 patients with mCRPC who received at least two lines of life-prolonging therapy (LPT) for mCRPC. Outcomes included OS, event-free survival (EFS), and healthcare resource utilization. Each province was analyzed separately. RESULTS: OS, measured from the start of second-line LPT, differed between provinces: those in Ontario receiving second-line Ra-223 had a longer OS vs. those receiving it in third-line or later (hazard ratio [HR] 0.79, 95% confidence interval [CI] 0.66-0.95). There was no difference between lines of therapy in patients in British Columbia (HR 1.165, 95% CI, 0.894-1.518, p=0.2576), and OS was numerically worse but not statistically significant in patients receiving Ra-223 in second-line in Quebec (HR 1.44, 95% CI, 0.93-2.24). Other outcomes also varied across provinces, with second-line use of Ra-223 being associated with longer EFS and reduced healthcare utilization vs. third-line use in Ontario but not in Quebec. CONCLUSIONS: Significant heterogeneity exists in the management and outcomes of mCRPC between provinces, particularly regarding the placement of Ra-223 in the treatment sequence.

Absolute quantification of protein number and dynamics in single cells.

Quantitative characterization of protein abundance and interactions in live cells is necessary to understand and predict cellular behavior. The accurate determination of copy number for individual proteins and heterologous complexes in individual cells is critical because small changes in protein dosage, often less than two-fold, can have strong phenotypic consequences. Here, we review the merits and pitfalls of different quantitative fluorescence imaging methods for single-cell determination of protein abundance, localization, interactions, and dynamics. In particular, we discuss how scanning number and brightness (sN&B) and its variation, Raster scanning image correlation spectroscopy (RICS), exploit stochastic noise in small measurement volumes to quantify protein abundance, stoichiometry, and dynamics with high accuracy.

Efficacy, tolerability, and endometrial safety of ospemifene compared with current therapies for the treatment of vulvovaginal atrophy: a systematic literature review and network meta-analysis.

IMPORTANCE: Ospemifene is a novel selective estrogen receptor modulator developed for the treatment of moderate to severe postmenopausal vulvovaginal atrophy (VVA). OBJECTIVE: The aim of the study is to perform a systematic literature review (SLR) and network meta-analysis (NMA) to assess the efficacy and safety of ospemifene compared with other therapies used in the treatment of VVA in North America and Europe. EVIDENCE REVIEW: Electronic database searches were conducted in November 2021 in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Randomized or nonrandomized controlled trials targeting postmenopausal women with moderate to severe dyspareunia and/or vaginal dryness and involving ospemifene or at least one VVA local treatment were considered. Efficacy data included changes from baseline in superficial and parabasal cells, vaginal pH, and the most bothersome symptom of vaginal dryness or dyspareunia, as required for regulatory approval. Endometrial outcomes were endometrial thickness and histologic classifications, including endometrial polyp, hyperplasia, and cancer. For efficacy and safety outcomes, a Bayesian NMA was performed. Endometrial outcomes were compared in descriptive analyses. FINDINGS: A total of 44 controlled trials met the eligibility criteria ( N = 12,637 participants). Network meta-analysis results showed that ospemifene was not statistically different from other active therapies in most efficacy and safety results. For all treatments, including ospemifene, the posttreatment endometrial thickness values (up to 52 wk of treatment) were under the recognized clinical threshold value of 4 mm for significant risk of endometrial pathology. Specifically, for women treated with ospemifene, endometrial thickness ranged between 2.1 and 2.3 mm at baseline and 2.5 and 3.2 mm after treatment. No cases of endometrial carcinoma or hyperplasia were observed in ospemifene trials, nor polyps with atypical hyperplasia or cancer after up to 52 weeks of treatment. CONCLUSIONS AND RELEVANCE: Ospemifene is an efficacious, well-tolerated, and safe therapeutic option for postmenopausal women with moderate to severe symptoms of VVA. Efficacy and safety outcomes with ospemifene are similar to other VVA therapies in North America and Europe.

Pressure pushes tRNALys3 into excited conformational states.

Conformational dynamics play essential roles in RNA function. However, detailed structural characterization of excited states of RNA remains challenging. Here, we apply high hydrostatic pressure (HP) to populate excited conformational states of tRNALys3, and structurally characterize them using a combination of HP 2D-NMR, HP-SAXS (HP-small-angle X-ray scattering), and computational modeling. HP-NMR revealed that pressure disrupts the interactions of the imino protons of the uridine and guanosine U-A and G-C base pairs of tRNALys3. HP-SAXS profiles showed a change in shape, but no change in overall extension of the transfer RNA (tRNA) at HP. Configurations extracted from computational ensemble modeling of HP-SAXS profiles were consistent with the NMR results, exhibiting significant disruptions to the acceptor stem, the anticodon stem, and the D-stem regions at HP. We propose that initiation of reverse transcription of HIV RNA could make use of one or more of these excited states.

Protocol for production and purification of SARS-CoV-2 3CLpro.

3CLpro protease from SARS-CoV-2 is a primary target for COVID-19 antiviral drug development. Here, we present a protocol for 3CLpro production in Escherichia coli. We describe steps to purify 3CLpro, expressed as a fusion with the Saccharomyces cerevisiae SUMO protein, with yields up to 120 mg L-1 following cleavage. The protocol also provides isotope-enriched samples suitable for nuclear magnetic resonance (NMR) studies. We also present methods to characterize 3CLpro by mass spectrometry, X-ray crystallography, heteronuclear NMR, and a Förster-resonance-energy-transfer-based enzyme assay. For complete details on the use and execution of this protocol, please refer to Bafna et al.1.

High Pressure CPMG and CEST Reveal That Cavity Position Dictates Distinct Dynamic Disorder in the PP32 Repeat Protein.

Given the central role of conformational dynamics in protein function, it is essential to characterize the time scales and structures associated with these transitions. High pressure (HP) perturbation favors transitions to excited states because they typically occupy a smaller molar volume, thus facilitating characterization of conformational dynamics. Repeat proteins, with their straightforward architecture, provide good models for probing the sequence dependence of protein conformational dynamics. Investigations of chemical exchange by 15N CPMG relaxation dispersion analysis revealed that introduction of a cavity via substitution of isoleucine 7 by alanine in the N-terminal capping motif of the pp32 leucine-rich repeat protein leads to pressure-dependent conformational exchange detected on the 500 µs-2 ms CPMG time scale. Exchange amplitude decreased from the N- to C-terminus, revealing a gradient of conformational exchange across the protein. In contrast, introduction of a cavity in the central core of pp32 via the L60A mutation led to pressure-induced exchange on a slower (>2 ms) time scale detected by 15N-CEST analysis. Excited state 15N chemical shifts indicated that in the excited state detected by HP CEST, the N-terminal region is mostly unfolded, while the core retains native-like structure. These HP chemical exchange measurements reveal that cavity position dictates exchange on distinct time scales, highlighting the subtle, yet central role of sequence in determining protein conformational dynamics.

The microprotein Nrs1 rewires the G1/S transcriptional machinery during nitrogen limitation in budding yeast.

Commitment to cell division at the end of G1 phase, termed Start in the budding yeast Saccharomyces cerevisiae, is strongly influenced by nutrient availability. To identify new dominant activators of Start that might operate under different nutrient conditions, we screened a genome-wide ORF overexpression library for genes that bypass a Start arrest caused by absence of the G1 cyclin Cln3 and the transcriptional activator Bck2. We recovered a hypothetical gene YLR053c, renamed NRS1 for Nitrogen-Responsive Start regulator 1, which encodes a poorly characterized 108 amino acid microprotein. Endogenous Nrs1 was nuclear-localized, restricted to poor nitrogen conditions, induced upon TORC1 inhibition, and cell cycle-regulated with a peak at Start. NRS1 interacted genetically with SWI4 and SWI6, which encode subunits of the main G1/S transcription factor complex SBF. Correspondingly, Nrs1 physically interacted with Swi4 and Swi6 and was localized to G1/S promoter DNA. Nrs1 exhibited inherent transactivation activity, and fusion of Nrs1 to the SBF inhibitor Whi5 was sufficient to suppress other Start defects. Nrs1 appears to be a recently evolved microprotein that rewires the G1/S transcriptional machinery under poor nitrogen conditions.

Structural evolution of the ancient enzyme, dissimilatory sulfite reductase.

Dissimilatory sulfite reductase is an ancient enzyme that has linked the global sulfur and carbon biogeochemical cycles since at least 3.47 Gya. While much has been learned about the phylogenetic distribution and diversity of DsrAB across environmental gradients, far less is known about the structural changes that occurred to maintain DsrAB function as the enzyme accompanied diversification of sulfate/sulfite reducing organisms (SRO) into new environments. Analyses of available crystal structures of DsrAB from Archaeoglobus fulgidus and Desulfovibrio vulgaris, representing early and late evolving lineages, respectively, show that certain features of DsrAB are structurally conserved, including active siro-heme binding motifs. Whether such structural features are conserved among DsrAB recovered from varied environments, including hot spring environments that host representatives of the earliest evolving SRO lineage (e.g., MV2-Eury), is not known. To begin to overcome these gaps in our understanding of the evolution of DsrAB, structural models from MV2.Eury were generated and evolutionary sequence co-variance analyses were conducted on a curated DsrAB database. Phylogenetically diverse DsrAB harbor many conserved functional residues including those that ligate active siro-heme(s). However, evolutionary co-variance analysis of monomeric DsrAB subunits revealed several False Positive Evolutionary Couplings (FPEC) that correspond to residues that have co-evolved despite being too spatially distant in the monomeric structure to allow for direct contact. One set of FPECs corresponds to residues that form a structural path between the two active siro-heme moieties across the interface between heterodimers, suggesting the potential for allostery or electron transfer within the enzyme complex. Other FPECs correspond to structural loops and gaps that may have been selected to stabilize enzyme function in different environments. These structural bioinformatics results suggest that DsrAB has maintained allosteric communication pathways between subunits as SRO diversified into new environments. The observations outlined here provide a framework for future biochemical and structural analyses of DsrAB to examine potential allosteric control of this enzyme.

Biological Networks across Scales-The Theoretical and Empirical Foundations for Time-Varying Complex Networks that Connect Structure and Function across Levels of Biological Organization.

Many biological systems across scales of size and complexity exhibit a time-varying complex network structure that emerges and self-organizes as a result of interactions with the environment. Network interactions optimize some intrinsic cost functions that are unknown and involve for example energy efficiency, robustness, resilience, and frailty. A wide range of networks exist in biology, from gene regulatory networks important for organismal development, protein interaction networks that govern physiology and metabolism, and neural networks that store and convey information to networks of microbes that form microbiomes within hosts, animal contact networks that underlie social systems, and networks of populations on the landscape connected by migration. Increasing availability of extensive (big) data is amplifying our ability to quantify biological networks. Similarly, theoretical methods that describe network structure and dynamics are being developed. Beyond static networks representing snapshots of biological systems, collections of longitudinal data series can help either at defining and characterizing network dynamics over time or analyzing the dynamics constrained to networked architectures. Moreover, due to interactions with the environment and other biological systems, a biological network may not be fully observable. Also, subnetworks may emerge and disappear as a result of the need for the biological system to cope with for example invaders or new information flows. The confluence of these developments renders tractable the question of how the structure of biological networks predicts and controls network dynamics. In particular, there may be structural features that result in homeostatic networks with specific higher-order statistics (e.g., multifractal spectrum), which maintain stability over time through robustness and/or resilience to perturbation. Alternative, plastic networks may respond to perturbation by (adaptive to catastrophic) shifts in structure. Here, we explore the opportunity for discovering universal laws connecting the structure of biological networks with their function, positioning them on the spectrum of time-evolving network structure, that is, dynamics of networks, from highly stable to exquisitely sensitive to perturbation. If such general laws exist, they could transform our ability to predict the response of biological systems to perturbations-an increasingly urgent priority in the face of anthropogenic changes to the environment that affect life across the gamut of organizational scales.

Short-term efficacy of latanoprostene bunod for the treatment of open-angle glaucoma and ocular hypertension: a systematic literature review and a network meta-analysis.

BACKGROUND/AIMS: To assess the comparative efficacy of latanoprostene bunod (LBN), a novel prostaglandin analogue (PGA), to other medications for open-angle glaucoma and ocular hypertension on lowering intraocular pressure (IOP). METHODS: A systematic literature review adapted from the Li et al (Ophthalmology, 2016) study was conducted. Medline, Embase and PubMed were searched for randomised controlled trials published between 1 January 2014 and 19 March 2020. Studies had to report IOP reduction after 3 months for at least two different treatments among placebo, PGAs (bimatoprost 0.01%, bimatoprost 0.03%, latanoprost, LBN, tafluprost, unoprostone) or apraclonidine, betaxolol, brimonidine, brinzolamide, carteolol, dorzolamide, levobunolol, timolol, travoprost. A Bayesian network meta-analysis was performed to provide the relative effect in terms of mean difference (95% credible interval) of IOP reduction and ranking probabilities. Surface under the cumulative ranking curve (SUCRA) was generated. RESULTS: A total of 106 trials were included with data for 18 523 participants. LBN was significantly more effective than unoprostone (-3.45 (-4.77 to -2.12)). Although relative effect was not significative, compared with other PGAs, LBN numerically outperformed latanoprost (-0.70 (-1.83 to 0.43)) and tafluoprost (-0.41 (-1.87 to 1.07)), was similar to bimatoprost 0.01% (-0.02(-1.59 to 1.55)) and was slightly disadvantaged by bimatoprost 0.03% (-0.17 (-1.42 to 1.07)). LBN was significantly more efficient than the beta-blockers apraclonidine, betaxolol, brimonidine, brinzolamide, carteolol, dorzolamide and timolol. According to SUCRA, LBN was ranked second after bimatoprost 0.03%, followed by bimatoprost 0.01%. CONCLUSION: LBN was significantly more effective than the PGA unoprostone and most of the beta-blockers. Compared with the most widely used PGAs, LBN numerically outperformed latanoprost and travoprost and was similar to bimatoprost 0.01%.

Hidden intermediates in Mango III RNA aptamer folding revealed by pressure perturbation.

Fluorescent RNA aptamers have the potential to enable routine quantitation and localization of RNA molecules and serve as models for understanding biologically active aptamers. In recent years, several fluorescent aptamers have been selected and modified to improve their properties, revealing that small changes to the RNA or the ligands can modify significantly their fluorescent properties. Although structural biology approaches have revealed the bound, ground state of several fluorescent aptamers, characterization of low-abundance, excited states in these systems is crucial to understanding their folding pathways. Here we use pressure as an alternative variable to probe the suboptimal states of the Mango III aptamer with both fluorescence and NMR spectroscopy approaches. At moderate KCl concentrations, increasing pressure disrupted the G-quadruplex structure of the Mango III RNA and led to an intermediate with lower fluorescence. These observations indicate the existence of suboptimal RNA structural states that still bind the TO1-biotin fluorophore and moderately enhance fluorescence. At higher KCl concentration as well, the intermediate fluorescence state was populated at high pressure, but the G-quadruplex remained stable at high pressure, supporting the notion of parallel folding and/or binding pathways. These results demonstrate the usefulness of pressure for characterizing RNA folding intermediates.

Comparative Assessment of NMR Probes for the Experimental Description of Protein Folding Pathways with High-Pressure NMR.

Multidimensional NMR intrinsically provides multiple probes that can be used for deciphering the folding pathways of proteins: NH amide and CαHα groups are strategically located on the backbone of the protein, while CH3 groups, on the side-chain of methylated residues, are involved in important stabilizing interactions in the hydrophobic core. Combined with high hydrostatic pressure, these observables provide a powerful tool to explore the conformational landscapes of proteins. In the present study, we made a comparative assessment of the NH, CαHα, and CH3 groups for analyzing the unfolding pathway of ∆+PHS Staphylococcal Nuclease. These probes yield a similar description of the folding pathway, with virtually identical thermodynamic parameters for the unfolding reaction, despite some notable differences. Thus, if partial unfolding begins at identical pressure for these observables (especially in the case of backbone probes) and concerns similar regions of the molecule, the residues involved in contact losses are not necessarily the same. In addition, an unexpected slight shift toward higher pressure was observed in the sequence of the scenario of unfolding with CαHα when compared to amide groups.

Protein unfolded states populated at high and ambient pressure are similarly compact.

The relationship between the dimensions of pressure-unfolded states of proteins compared with those at ambient pressure is controversial; resolving this issue is related directly to the mechanisms of pressure denaturation. Moreover, a significant pressure dependence of the compactness of unfolded states would complicate the interpretation of folding parameters from pressure perturbation and make comparison to those obtained using alternative perturbation approaches difficult. Here, we determined the compactness of the pressure-unfolded state of a small, cooperatively folding model protein, CTL9-I98A, as a function of temperature. This protein undergoes both thermal unfolding and cold denaturation, and the temperature dependence of the compactness at atmospheric pressure is known. High-pressure small angle x-ray scattering studies, yielding the radius of gyration and high-pressure diffusion ordered spectroscopy NMR experiments, yielding the hydrodynamic radius were carried out as a function of temperature at 250 MPa, a pressure at which the protein is unfolded. The radius of gyration values obtained at any given temperature at 250 MPa were similar to those reported previously at ambient pressure, and the trends with temperature are similar as well, although the pressure-unfolded state appears to undergo more pronounced expansion at high temperature than the unfolded state at atmospheric pressure. At 250 MPa, the compaction of the unfolded chain was maximal between 25 and 30°C, and the chain expanded upon both cooling and heating. These results reveal that the pressure-unfolded state of this protein is very similar to that observed at ambient pressure, demonstrating that pressure perturbation represents a powerful approach for observing the unfolded states of proteins under otherwise near-native conditions.

Hepatitis C virus drugs that inhibit SARS-CoV-2 papain-like protease synergize with remdesivir to suppress viral replication in cell culture.

Effective control of COVID-19 requires antivirals directed against SARS-CoV-2. We assessed 10 hepatitis C virus (HCV) protease-inhibitor drugs as potential SARS-CoV-2 antivirals. There is a striking structural similarity of the substrate binding clefts of SARS-CoV-2 main protease (Mpro) and HCV NS3/4A protease. Virtual docking experiments show that these HCV drugs can potentially bind into the Mpro substrate-binding cleft. We show that seven HCV drugs inhibit both SARS-CoV-2 Mpro protease activity and SARS-CoV-2 virus replication in Vero and/or human cells. However, their Mpro inhibiting activities did not correlate with their antiviral activities. This conundrum is resolved by demonstrating that four HCV protease inhibitor drugs, simeprevir, vaniprevir, paritaprevir, and grazoprevir inhibit the SARS CoV-2 papain-like protease (PLpro). HCV drugs that inhibit PLpro synergize with the viral polymerase inhibitor remdesivir to inhibit virus replication, increasing remdesivir's antiviral activity as much as 10-fold, while those that only inhibit Mpro do not synergize with remdesivir.

The Molecular Basis for Life in Extreme Environments.

Sampling and genomic efforts over the past decade have revealed an enormous quantity and diversity of life in Earth's extreme environments. This new knowledge of life on Earth poses the challenge of understandingits molecular basis in such inhospitable conditions, given that such conditions lead to loss of structure and of function in biomolecules from mesophiles. In this review, we discuss the physicochemical properties of extreme environments. We present the state of recent progress in extreme environmental genomics. We then present an overview of our current understanding of the biomolecular adaptation to extreme conditions. As our current and future understanding of biomolecular structure-function relationships in extremophiles requires methodologies adapted to extremes of pressure, temperature, and chemical composition, advances in instrumentation for probing biophysical properties under extreme conditions are presented. Finally, we briefly discuss possible future directions in extreme biophysics.

An oligomeric switch controls the Mrr-induced SOS response in E. coli.

Mrr from Escherichia coli K12 is a type IV restriction endonuclease whose role is to recognize and cleave foreign methylated DNA. Beyond this protective role, Mrr can inflict chromosomal DNA damage that elicits the SOS response in the host cell upon heterologous expression of specific methyltransferases such as M.HhaII, or after exposure to high pressure (HP). Activation of Mrr in response to these perturbations involves an oligomeric switch that dissociates inactive homo-tetramers into active dimers. Here we used scanning number and brightness (sN&B) analysis to determine in vivo the stoichiometry of a constitutively active Mrr mutant predicted to be dimeric and examine other GFP-Mrr mutants compromised in their response to either M.HhaII activity or HP shock. We also observed in vitro the direct pressure-induced tetramer dissociation by HP fluorescence correlation spectroscopy of purified GFP-Mrr. To shed light on the linkages between subunit interactions and activity of Mrr and its variants, we built a structural model of the full-length tetramer bound to DNA. Similar to functionally related endonucleases, the conserved DNA cleavage domain would be sequestered by the DNA recognition domain in the Mrr inactive tetramer, dissociating into an enzymatically active dimer upon interaction with multiple DNA sites.

G1/S transcription factors assemble in increasing numbers of discrete clusters through G1 phase.

In budding yeast, the transcription factors SBF and MBF activate a large program of gene expression in late G1 phase that underlies commitment to cell division, termed Start. SBF/MBF are limiting with respect to target promoters in small G1 phase cells and accumulate as cells grow, raising the questions of how SBF/MBF are dynamically distributed across the G1/S regulon and how this impacts the Start transition. Super-resolution Photo-Activatable Localization Microscopy (PALM) mapping of the static positions of SBF/MBF subunits in fixed cells revealed each transcription factor was organized into discrete clusters containing approximately eight copies regardless of cell size and that the total number of clusters increased as cells grew through G1 phase. Stochastic modeling using reasonable biophysical parameters recapitulated growth-dependent SBF/MBF clustering and predicted TF dynamics that were confirmed in live cell PALM experiments. This spatio-temporal organization of SBF/MBF may help coordinate activation of G1/S regulon and the Start transition.