Feedback control of the heat shock response by spatiotemporal regulation of Hsp70.

Cells maintain homeostasis via dynamic regulation of stress response pathways. Stress pathways transiently induce response regulons via negative feedback loops, but the extent to which individual genes provide feedback has not been comprehensively measured for any pathway. Here, we disrupted induction of each gene in the Saccharomyces cerevisiae heat shock response (HSR) and quantified cell growth and HSR dynamics following heat shock. The screen revealed a core feedback loop governing expression of the chaperone Hsp70 reinforced by an auxiliary feedback loop controlling Hsp70 subcellular localization. Mathematical modeling and live imaging demonstrated that multiple HSR targets converge to promote Hsp70 nuclear localization via its release from cytosolic condensates. Following ethanol stress, a distinct set of factors similarly converged on Hsp70, suggesting that nonredundant subsets of the HSR regulon confer feedback under different conditions. Flexible spatiotemporal feedback loops may broadly organize stress response regulons and expand their adaptive capacity.

Nonlinear Changes in Facial Affect and Posttraumatic Growth: Assessment of Ecological Momentary Assessment Video Data.

Posttraumatic Growth (PTG), characterized by newfound meaning, perspective, and purpose for trauma survivors, remains enigmatic in its nature. This state is thought to arise from the dynamic interplay of biopsychosocial factors; however, the nature of this interplay is unclear. This study aimed to investigate the intricate relationship between PTG and facial affect dynamics, shedding light on the complex interplay of biopsychosocial factors that underpin this transformative process. We conducted a comprehensive investigation involving 19 wildfire survivors who provided daily self-reported PTG ratings alongside smartphone videos analyzed using Automated Facial Affect Recognition (AFAR) software. Our findings revealed compelling evidence of self-organization within facial affect, as indicated by notably high mean R2 and shape parameter values (i.e., nonlinear indices indicative of structural integrity and flexibility). Further regression analyses unveiled a significant interaction between the degree of facial affect 'burstiness' and coping self-efficacy (CSE) on PTG. This interaction suggested that PTG development was a nuanced process intricately linked to the coherence of emotion patterns exhibited by individuals. These insights illuminate the multifaceted dynamics at play in the emergence of PTG and contribute to a broader understanding of its biopsychosocial foundations.

Adaptive preservation of orphan ribosomal proteins in chaperone-dispersed condensates.

Ribosome biogenesis is among the most resource-intensive cellular processes, with ribosomal proteins accounting for up to half of all newly synthesized proteins in eukaryotic cells. During stress, cells shut down ribosome biogenesis in part by halting rRNA synthesis, potentially leading to massive accumulation of aggregation-prone 'orphan' ribosomal proteins (oRPs). Here we show that, during heat shock in yeast and human cells, oRPs accumulate as reversible peri-nucleolar condensates recognized by the Hsp70 co-chaperone Sis1/DnaJB6. oRP condensates are liquid-like in cell-free lysate but solidify upon depletion of Sis1 or inhibition of Hsp70. When cells recover from heat shock, oRP condensates disperse in a Sis1- and Hsp70-dependent manner, and the oRP constituents are incorporated into functional ribosomes in the cytosol, enabling cells to efficiently resume growth. Preserving biomolecules in reversible condensates-like mRNAs in cytosolic stress granules and oRPs at the nucleolar periphery-may be a primary function of the Hsp70 chaperone system.

Biopsychosocial Resilience through a Complex Adaptive Systems Lens: A Narrative Review of Nonlinear Modeling Approaches.

Human resilience is often considered as static traits using a reductionist approach. More recent work has demonstrated it to be a dynamic and emergent property of complex systems. This narrative review explores human resilience through a self-organizing framework with a specific emphasis on the application of nonlinear modeling approaches. Four classes of approaches are examined: univariate dynamics, bivariate coupling, topological modeling, and network modeling. Univariate dynamics capture the temporal structure and flexibility within a single time series, while bivariate coupling approaches quantify the interaction dynamics and coordination between two time series. Topological modeling identifies bifurcations and attractor dynamics as signals of critical transitions relative to emergence and system stability. Network modeling represents system structure with a focus on connectivity, flexibility, and system integrity. Applying a complex systems framework, this review provides insights into data modeling opportunities for characterizing important features of a system's capacity to bounce back and recover from stress. These characteristics are connected to meta-flexibility, which characterizes a system's adaptive responsiveness to stressors, including post-traumatic growth, and the relation between meta-flexibility and metastability is discussed. Overall, this review provides a foundation of tools for researchers interested in under-standing human resilience through a complex systems framework.

Blue light receptor CRY1 regulates HSFA1d nuclear localization to promote plant thermotolerance.

Temperature increases as light intensity rises, but whether light signals can be directly linked to high temperature response in plants is unclear. Here, we find that light pre-treatment enables plants to survive better under high temperature, designated as light-induced thermotolerance (LIT). With short-term light treatment, plants induce light-signaling pathway genes and heat shock genes. Blue light photoreceptor cryptochrome 1 (CRY1) is required for LIT. We also find that CRY1 physically interacts with the heat shock transcription factor A1d (HsfA1d) and that HsfA1d is involved in thermotolerance under light treatment. Furthermore, CRY1 promotes HsfA1d nuclear localization through importin alpha 1 (IMPα1). Consistent with this, CRY1 shares more than half of the chromatin binding sites with HsfA1d. Mutation of CRY1 (cry1-304) diminishes a large number of HsfA1d binding sites that are shared with CRY1. We present a model where, by coupling light sensing to high-temperature stress, CRY1 confers thermotolerance in plants via HsfA1d.

Multicenter matched-pair study comparing BACT/ALERT® MP reagent systems for the recovery of mycobacteria from specimens other than blood.

The BACT/ALERT® MP Reagent System is a broth culture medium for optimal detection and recovery of mycobacteria from clinical samples. The MP formulation was recently modified to improve detection and recovery times. A multicenter prospective matched pair study design was conducted to validate the performance of improved MP (MP-I) versus current MP (MP-C) bottles utilizing nonsterile and normally sterile samples, except blood, from patients suspected of having mycobacterial infections. A total of 1488 clinical samples were collected to obtain 212 mycobacteria samples by either or both MP culture bottles. MP-I and MP-C sensitivities were 86.6% and 81.4%, respectively, but the difference was not significant (P = 0.163) while specificities were 96.8% and 93.8%, respectively, and that difference was significant (P = 0.002). Overall recovery was 94.34% for MP-I and 88.68% for MP-C (recovery was 100% for both bottles with 52 seeded samples). Overall performance of MP-I was better than MP-C for sensitivity, specificity, and recovery.

Transcriptional regulation of Sis1 promotes fitness but not feedback in the heat shock response.

The heat shock response (HSR) controls expression of molecular chaperones to maintain protein homeostasis. Previously, we proposed a feedback loop model of the HSR in which heat-denatured proteins sequester the chaperone Hsp70 to activate the HSR, and subsequent induction of Hsp70 deactivates the HSR (Krakowiak et al., 2018; Zheng et al., 2016). However, recent work has implicated newly synthesized proteins (NSPs) - rather than unfolded mature proteins - and the Hsp70 co-chaperone Sis1 in HSR regulation, yet their contributions to HSR dynamics have not been determined. Here, we generate a new mathematical model that incorporates NSPs and Sis1 into the HSR activation mechanism, and we perform genetic decoupling and pulse-labeling experiments to demonstrate that Sis1 induction is dispensable for HSR deactivation. Rather than providing negative feedback to the HSR, transcriptional regulation of Sis1 by Hsf1 promotes fitness by coordinating stress granules and carbon metabolism. These results support an overall model in which NSPs signal the HSR by sequestering Sis1 and Hsp70, while induction of Hsp70 - but not Sis1 - attenuates the response.

Multicenter Clinical Performance Evaluation of Omadacycline Susceptibility Testing of Enterobacterales on VITEK 2 Systems.

We present the first performance evaluation results for omadacycline on the VITEK 2 and VITEK 2 Compact Systems (bioMérieux, Inc.). The trial was conducted at four external sites and one internal site. All sites were in the United States, geographically dispersed as follows: Indianapolis, IN; Schaumburg, IL; Wilsonville, OR; Cleveland, OH; and Hazelwood, MO. In this multisite study, omadacycline was tested against 858 Enterobacterales on the VITEK 2 antimicrobial susceptibility test (AST) Gram-negative (GN) card, and the results were compared to the Clinical and Laboratory Standards Institute broth microdilution (BMD) reference method. The results were analyzed and are presented as essential agreement (EA), category agreement (CA), minor error (mE) rates, major error (ME) rates, and very major error (VME) rates following the US Food and Drug Administration (FDA) and International Standards Organization (ISO) performance criteria requirements. Omadacycline has susceptibility testing interpretive criteria (breakpoints) established by the FDA only; nevertheless, the analysis was also performed using the ISO acceptance criteria to satisfy the registration needs of countries outside the United States. The analysis following FDA criteria (including only Klebsiella pneumoniae and Enterobacter cloacae) showed the following performance: EA = 97.9% (410/419), CA = 94.3% (395/419), VME = 2% (1/51), with no ME present. The performance following ISO criteria (including all Enterobacterales tested) after error resolutions was EA = 98.1% (842/858) and CA = 96.9% (831/858). No ME or VME were observed. The VITEK 2 test met the ISO and FDA criteria of ≥ 95% reproducibility, and ≥ 95% quality control (QC) results within acceptable ranges for QC organisms. In June 2022, the omadacycline VITEK 2 test received FDA 510(k) clearance (K213931) FDA as a diagnostic device to be used in the treatment of acute bacterial skin and skin-structure infections caused by E. cloacae and K. pneumoniae, and for treatment of community-acquired bacterial pneumonia caused by K. pneumoniae. The new VITEK 2 AST-GN omadacycline test provides an alternative to the BMD reference method testing and increases the range of automated diagnostic tools available for determining omadacycline MICs in Enterobacterales.

Inducible transcriptional condensates drive 3D genome reorganization in the heat shock response.

Mammalian developmental and disease-associated genes concentrate large quantities of the transcriptional machinery by forming membrane-less compartments known as transcriptional condensates. However, it is unknown whether these structures are evolutionarily conserved or involved in 3D genome reorganization. Here, we identify inducible transcriptional condensates in the yeast heat shock response (HSR). HSR condensates are biophysically dynamic spatiotemporal clusters of the sequence-specific transcription factor heat shock factor 1 (Hsf1) with Mediator and RNA Pol II. Uniquely, HSR condensates drive the coalescence of multiple Hsf1 target genes, even those located on different chromosomes. Binding of the chaperone Hsp70 to a site on Hsf1 represses clustering, whereas an intrinsically disordered region on Hsf1 promotes condensate formation and intergenic interactions. Mutation of both Hsf1 determinants reprograms HSR condensates to become constitutively active without intergenic coalescence, which comes at a fitness cost. These results suggest that transcriptional condensates are ancient and flexible compartments of eukaryotic gene control.

Stability and flexibility in psychotherapy process predict outcome.

Ten good outcome and ten poor outcome psychotherapy cases were compared to investigate whether or not the temporal stability and flexibility of their process variables can predict their outcomes. Each participant was monitored daily using the Therapy Process Questionnaire (TPQ), which has 43 items and seven sub-scales, and responses over time were analyzed in terms of correlation robustness and correlation variability across the TPQ sub-scales. "Correlation robustness" and "correlation variability" are two basic characteristics of any correlation matrix: the first is calculated as the sum of the absolute values of Pearson correlation coefficients, the second as the standard deviation of Pearson correlation coefficients. The results demonstrated that the patients within the poor outcome group had lower values on both variables, suggesting lower stability and flexibility. Furthermore, a higher number of cycles of increase and decrease in correlation robustness and variability of the TPQ sub-scales was observed within good outcome psychotherapies, suggesting that, these cycles can be considered as process-markers of good-outcomes. These results provide support for the validity of these quantitative process-parameters, correlation robustness and variability, in predicting psychotherapeutic outcomes. Moreover, the results lend support to the common clinical experience of alternating periods of flexibility and integration being beneficial to good psychotherapeutic processes.

Multicenter Clinical Evaluation of Vitek 2 Meropenem-Vaborbactam for Susceptibility Testing of Enterobacterales and Pseudomonas aeruginosa.

The carbapenem/beta-lactamase inhibitor meropenem-vaborbactam (MEV) used to treat complicated urinary tract infections and pyelonephritis in adults was approved in 2017 by the U.S. Food and Drug Administration (FDA). Here, we evaluated Vitek 2 MEV (bioMérieux, Durham, NC) compared to the reference broth microdilution (BMD) method. Of 449 Enterobacterales isolates analyzed per FDA/CLSI breakpoints, the overall performance was 98.2% essential agreement (EA), 98.7% category agreement (CA), and 0% very major errors (VME) or major errors (ME). For 438 FDA intended-for-use Enterobacterales isolates, performance was 98.2% EA, 98.6% CA, and 0% VME or ME. Evaluable EA was 81.0%, but with only 42 on-scale evaluable results. Individual species demonstrated EA and CA rates of ≥90% without any VME or ME. When evaluated using European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints, overall Vitek 2 MEV performance for Enterobacterales and Pseudomonas aeruginosa demonstrated 97.3% EA, 99.2% CA, 2.3% VME, and 0.6% ME (after error resolution: 97.3% EA, 99.4% CA, 2.2% VME, and 0.4% ME) compared to the reference BMD method. Performance for P. aeruginosa included 92.2% EA, 97.4% CA, 0% VME, and 3.0% ME (after error resolution: 92.2% EA, 98.7% CA, 0% VME, and 1.5% ME). Performance for Enterobacterales included 98.2% EA, 99.6% CA, 3.0% VME, and 0.2% ME. Evaluable EA was 80.6% but was based on only 67 evaluable results. These findings support Vitek 2 MEV as an accurate automated system for MEV susceptibility testing of Enterobacterales and P. aeruginosa and could be an alternate solution to the manual-labor-intensive reference BMD method.

Burstiness and Stochasticity in the Malleability of Physical Activity.

This study examined whether patterns of self-organization in physical activity (PA) predicted long-term success in a yearlong PA intervention. Increased moderate to vigorous PA (MVPA) was targeted in insufficiently active adults (N = 512) via goal setting and financial reinforcement. The degree to which inverse power law distributions, which are reflective of self-organization, summarized (a) daily MVPA and (b) time elapsed between meeting daily goals (goal attainment interresponse times) was calculated. Goal attainment interresponse times were also used to calculate burstiness, the degree to which meeting daily goals clustered in time. Inverse power laws accurately summarized interresponse times, but not daily MVPA. For participants with higher levels of MVPA early in the study, burstiness in reaching goals was associated with long-term resistance to intervention, while stochasticity in meeting goals predicted receptiveness to intervention. These results suggest that burstiness may measure self-organizing resistance to change, while PA stochasticity could be a precondition for behavioral malleability.

Primordial super-enhancers: heat shock-induced chromatin organization in yeast.

Specialized mechanisms ensure proper expression of critically important genes such as those specifying cell identity or conferring protection from environmental stress. Investigations of the heat shock response have been critical in elucidating basic concepts of transcriptional control. Recent studies demonstrate that in response to thermal stress, heat shock-responsive genes associate with high levels of transcriptional activators and coactivators and those in yeast intensely interact across and between chromosomes, coalescing into condensates. In mammalian cells, cell identity genes that are regulated by super-enhancers (SEs) are also densely occupied by transcriptional machinery that form phase-separated condensates. We suggest that the stress-remodeled yeast nucleome bears functional and structural resemblance to mammalian SEs, and will reveal fundamental mechanisms of gene control by transcriptional condensates.

Multicenter evaluation of the VITEK MS matrix-assisted laser desorption/ionization-time of flight mass spectrometry system for identification of bacteria, including Brucella, and yeasts.

The use of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry has proven to be rapid and accurate for the majority of clinical isolates. Some gaps remain concerning rare, emerging, or highly pathogenic species, showing the need to continuously expand the databases. In this multicenter study, we evaluated the accuracy of the VITEK MS v3.2 database in identifying 1172 unique isolates compared to identification by DNA sequence analysis. A total of 93.6% of the isolates were identified to species or group/complex level. A remaining 5.2% of the isolates were identified to the genus level. Forty tests gave a result of no identification (0.9%) and 12 tests (0.3%) gave a discordant identification compared to the reference identification. VITEK MS is also the first MALDI-TOF MS system that is able to delineate the four members of the Acinetobacter baumannii complex at species level without any specific protocol or special analysis method. These findings demonstrate that the VITEK MS v3.2 database is highly accurate for the identification of bacteria and fungi encountered in the clinical laboratory as well as emerging species like Candida auris and the highly pathogenic Brucella species.

Subcellular localization of the J-protein Sis1 regulates the heat shock response.

Cells exposed to heat shock induce a conserved gene expression program, the heat shock response (HSR), encoding protein homeostasis (proteostasis) factors. Heat shock also triggers proteostasis factors to form subcellular quality control bodies, but the relationship between these spatial structures and the HSR is unclear. Here we show that localization of the J-protein Sis1, a cofactor for the chaperone Hsp70, controls HSR activation in yeast. Under nonstress conditions, Sis1 is concentrated in the nucleoplasm, where it promotes Hsp70 binding to the transcription factor Hsf1, repressing the HSR. Upon heat shock, Sis1 forms an interconnected network with other proteostasis factors that spans the nucleolus and the surface of the endoplasmic reticulum. We propose that localization of Sis1 to this network directs Hsp70 activity away from Hsf1 in the nucleoplasm, leaving Hsf1 free to induce the HSR. In this manner, Sis1 couples HSR activation to the spatial organization of the proteostasis network.

Persistent Activation of mRNA Translation by Transient Hsp90 Inhibition.

The heat shock protein 90 (Hsp90) chaperone functions as a protein-folding buffer and plays a role promoting the evolution of new heritable traits. To better understand how Hsp90 can affect mRNA translation, we screen more than 1,600 factors involved in mRNA regulation for physical interactions with Hsp90 in human cells. The mRNA binding protein CPEB2 strongly binds Hsp90 via its prion domain. In a yeast model, transient inhibition of Hsp90 results in persistent activation of a CPEB translation reporter even in the absence of exogenous CPEB that persists for 30 generations after the inhibitor is removed. Ribosomal profiling reveals that some endogenous yeast mRNAs, including HAC1, show a persistent change in translation efficiency following transient Hsp90 inhibition. Thus, transient loss of Hsp90 function can promote a nongenetic inheritance of a translational state affecting specific mRNAs, introducing a mechanism by which Hsp90 can promote phenotypic variation.

Regulation of Hsf1 and the Heat Shock Response.

The heat shock response (HSR) is characterized by the induction of molecular chaperones following a sudden increase in temperature. In eukaryotes, the HSR comprises the set of genes controlled by the transcription factor Hsf1. The HSR is induced by defects in co-translational protein folding, ribosome biogenesis, organellar targeting of nascent proteins, and protein degradation by the ubiquitin proteasome system. Upon heat shock, these processes may be endogenous sources of polypeptide ligands that activate the HSR. Mechanistically, these ligands are thought to titrate the chaperone Hsp70 away from Hsf1, releasing Hsf1 to induce the full arsenal of cellular chaperones to restore protein homeostasis. In metazoans, this cell-autonomous feedback loop is modulated by the microenvironment and neuronal cues to enable tissue-level and organism-wide coordination.

Multi-kinase control of environmental stress responsive transcription.

Cells respond to changes in environmental conditions by activating signal transduction pathways and gene expression programs. Here we present a dataset to explore the relationship between environmental stresses, kinases, and global gene expression in yeast. We subjected 28 drug-sensitive kinase mutants to 10 environmental conditions in the presence of inhibitor and performed mRNA deep sequencing. With these data, we reconstructed canonical stress pathways and identified examples of crosstalk among pathways. The data also implicated numerous kinases in novel environment-specific roles. However, rather than regulating dedicated sets of target genes, individual kinases tuned the magnitude of induction of the environmental stress response (ESR)-a gene expression signature shared across the set of perturbations-in environment-specific ways. This suggests that the ESR integrates inputs from multiple sensory kinases to modulate gene expression and growth control. As an example, we provide experimental evidence that the high osmolarity glycerol pathway is an upstream negative regulator of protein kinase A, a known inhibitor of the ESR. These results elaborate the central axis of cellular stress response signaling.

Strategies for Engineering and Rewiring Kinase Regulation.

Eukaryotic protein kinases (EPKs) catalyze the transfer of a phosphate group onto another protein in response to appropriate regulatory cues. In doing so, they provide a primary means for cellular information transfer. Consequently, EPKs play crucial roles in cell differentiation and cell-cycle progression, and kinase dysregulation is associated with numerous disease phenotypes including cancer. Nonnative cues for synthetically regulating kinases are thus much sought after, both for dissecting cell signaling pathways and for pharmaceutical development. In recent years advances in protein engineering and sequence analysis have led to new approaches for manipulating kinase activity, localization, and in some instances specificity. These tools have revealed fundamental principles of intracellular signaling and suggest paths forward for the design of therapeutic allosteric kinase regulators.