Structure-function study of a Ca2+-independent metacaspase involved in lateral root emergence.

Metacaspases are part of an evolutionarily broad family of multifunctional cysteine proteases, involved in disease and normal development. As the structure-function relationship of metacaspases remains poorly understood, we solved the X-ray crystal structure of an Arabidopsis thaliana type II metacaspase (AtMCA-IIf) belonging to a particular subgroup not requiring calcium ions for activation. To study metacaspase activity in plants, we developed an in vitro chemical screen to identify small molecule metacaspase inhibitors and found several hits with a minimal thioxodihydropyrimidine-dione structure, of which some are specific AtMCA-IIf inhibitors. We provide mechanistic insight into the basis of inhibition by the TDP-containing compounds through molecular docking onto the AtMCA-IIf crystal structure. Finally, a TDP-containing compound (TDP6) effectively hampered lateral root emergence in vivo, probably through inhibition of metacaspases specifically expressed in the endodermal cells overlying developing lateral root primordia. In the future, the small compound inhibitors and crystal structure of AtMCA-IIf can be used to study metacaspases in other species, such as important human pathogens, including those causing neglected diseases.

Patient and Provider Satisfaction with Asynchronous Versus Synchronous Telepsychiatry in Primary Care: A Secondary Mixed-Methods Analysis of a Randomized Controlled Trial.

Background: Asynchronous telepsychiatry (ATP) consultations are a novel form of psychiatric consultation. Studies comparing patient and provider satisfaction for ATP with that for synchronous telepsychiatry (STP) do not exist. Methods: This mixed-methods study is a secondary analysis of patients' and primary care providers' (PCPs) satisfaction from a randomized clinical trial of ATP compared with STP. Patients and their PCPs completed satisfaction surveys, and provided unstructured feedback about their experiences with either ATP or STP. Differences in patient satisfaction were assessed using mixed-effects logistic regression models, and the qualitative data were analyzed using thematic analysis with an inductive coding framework. Results: Patient satisfaction overall was high with 84% and 97% of respondents at 6 months reported being somewhat or completely satisfied with ATP and STP, respectively. Patients in the STP group were more likely to report being completely satisfied, to recommend the program to a friend, and to report being comfortable with their care compared with ATP (all p < 0.05). However, there was no difference between the patients in ATP and STP in perceived change in clinical outcomes (p = 0.51). The PCP quantitative data were small, and thus only summarized descriptively. Conclusions: Patients expressed their overall satisfaction with both STP and ATP. Patients in ATP reported more concerns about the process, likely because feedback after ATP was slower than that after STP consultations. PCPs had no apparent preference for STP or ATP, and reported implementing the psychiatrists' recommendations for both groups when such recommendations were made, which supports our previous findings. Trial Registration: ClinicalTrials.gov NCT02084979; https://clinicaltrials.gov/ct2/show/NCT02084979.

A new species of Diolcogaster (Hymenoptera: Braconidae) from Brazil of potential use in the biological control of the pest Hypercompe brasiliensis (Stoll) (Lepidoptera: Erebidae).

Species of Diolcogaster parasitize Lepidoptera pests of commercial plants. The diversity of this genus is high, but few species of Diolcogaster have been described. The description of a new Diolcogaster species provides information for the biological control using this insect. This study presents the description and key notes on the biology of a new Diolcogaster parasitoid wasp. This species was reared from a caterpillar of Hypercompe brasiliensis collected after feeding on a Gloxinia perennis plant important to floriculture. Two complementary identification analyzes were performed on Diolcogaster adult bodies. The first was the analyses of its external morphology and the second its molecular analysis (mitochondrial DNA). The morphological analysis defined the insect as a new species of Diolcogaster, named Diolcogaster joanesi sp. nov. A maximum-likelihood (ML) analysis partially confirmed the morphological analysis, placing D. joanesi within a cluster including a previously identified species (Diolcogaster choi) and seven other morphospecies. The proximity of D. joanesi to D. choi is discussed and an updated key for all New World species of the xanthaspis group is provided. Twenty-eight adult wasps were obtained (22 females and six males) out of 50 cocoons which larvae emerged from the caterpillar host. The findings contribute to the broader understanding of Diolcogaster in the Neotropics and its potential for the biological control of lepidopteran defoliators.

Intraoperative circulatory arrest secondary to high-risk pulmonary embolism. Case series and updated literature review.

BACKGROUND: Intraoperative pulmonary embolism (PE) with cardiac arrest (CA) represents a critical and potentially fatal condition. Available treatments include systemic thrombolysis, catheter-based thrombus fragmentation or aspiration, and surgical embolectomy. However, limited studies are focused on the optimal treatment choice for this critical condition. We present a case series and an updated review of the management of intraoperative CA secondary to PE. METHODS: A retrospective review of patients who developed high-risk intraoperative PE was performed between June 2012 and June 2022. For the updated review, a literature search on PubMed and Scopus was conducted which resulted in the inclusion of a total of 46 articles. RESULTS: A total of 196 174 major non-cardiac surgeries were performed between 2012 and 2022. Eight cases of intraoperative CA secondary to high-risk PE were identified. We found a mortality rate of 75%. Anticoagulation therapy was administered to one patient (12.5%), while two patients (25%) underwent thrombolysis, and one case (12.5%) underwent mechanical thrombectomy combined with thrombus aspiration. Based on the literature review and our 10-year experience, we propose an algorithm for the management of intraoperative CA caused by PE. CONCLUSION: The essential components for adequate management of intraoperative PE with CA include hemodynamic support, cardiopulmonary resuscitation, and the implementation of a primary perfusion intervention. The prompt identification of the criteria for each specific treatment modality, guided by the individual patient's characteristics, is necessary for an optimal approach.

Dead space ratio as a tool in nitric oxide weaning: a study in pulmonary hypertensive disease.

OBJECTIVES: To describe the association between successful weaning of inhaled nitric oxide and trends in dead space ratio during such weans in patients empirically initiated on nitric oxide therapy out of concern of pulmonary hypertensive crisis. PATIENTS: Children in a cardiac intensive care unit initiated on inhaled nitric oxide out of clinical concern for pulmonary hypertensive crisis retrospectively over 2 years. MEASUREMENTS AND MAIN RESULTS: Twenty-seven patients were included, and nitric oxide was successfully discontinued in 23/27. These patients exhibited decreases in dead space ratio (0.18 versus 0.11, p = 0.047) during nitric oxide weaning, and with no changes in dead space ratio between pre- and post-nitric oxide initiation (p = 0.88) and discontinuation (p = 0.63) phases. These successful patients had a median age of 10 months [4.0, 57.0] and had a pre-existent diagnosis of CHD in 6/23 and pulmonary hypertension in 2/23. Those who failed nitric oxide discontinuation trended with a higher dead space ratio at presentation (0.24 versus 0.10), were more likely to carry a prior diagnosis of pulmonary hypertension (50% versus 8.7%), and had longer mechanical ventilation days (5 versus 12). CONCLUSIONS: Patients empirically placed on nitric oxide out of concern of pulmonary hypertensive crisis and successfully weaned off showed unchanged or decreased dead space ratio throughout the initiation to discontinuation phases of nitric oxide therapy. Trends in dead space ratio may aid in determining true need for nitric oxide and facilitate effective weaning. Further studies are needed to directly compare trends between success and failure groups.

Primary Care Physician Adherence to Telepsychiatry Recommendations: Intermediate Outcomes from a Randomized Clinical Trial.

Objective:To compare clinical recommendations given by psychiatrists and the adherence to these recommendations by primary care physicians (PCP) following consultations conducted by asynchronous telepsychiatry (ATP) and synchronous telepsychiatry (STP).Materials and Methods:ATP and STP consultations were compared using intermediate data from a randomized clinical trial with adult participant enrollment between April 2014 and December 2017. In both study arms, PCPs received written recommendations from the psychiatrist after each encounter. Independent clinicians reviewed PCP documentation to measure adherence to those recommendations in the 6 months following the baseline consultation.Results:Medical records were reviewed for 645 psychiatrists' consult recommendations; 344 from 61 ATP consultations and 301 from 62 STP consultations. Of those recommendations, 191 (56%) and 173 (58%) were rated fully adherent by two independent raters for ATP and STP, respectively. In a multilevel ordinal logistic regression model adjusted for recommendation type and recommended implementation timing, there was no statistically significant difference in adherence to recommendations for ATP compared with STP (adjusted odds ratio = 0.91, 95% confidence interval = 0.51-1.62). The profiles of recommendation type were comparable between ATP and STP.Conclusions:This is the first PCP adherence study comparing two forms of telemedicine. Although we did not find evidence of a difference between ATP and STP; this study supports the feasibility and acceptability of ATP and STP for the provision of collaborative psychiatric care. Clinical Trial Identifier NCT02084979.

Clinical Outcomes of Asynchronous Versus Synchronous Telepsychiatry in Primary Care: Randomized Controlled Trial.

BACKGROUND: Asynchronous telepsychiatry (ATP; delayed-time) consultations are a novel form of psychiatric consultation in primary care settings. Longitudinal studies comparing clinical outcomes for ATP with synchronous telepsychiatry (STP) are lacking. OBJECTIVE: This study aims to determine the effectiveness of ATP in improving clinical outcomes in English- and Spanish-speaking primary care patients compared with STP, the telepsychiatry usual care method. METHODS: Overall, 36 primary care physicians from 3 primary care clinics referred a heterogeneous sample of 401 treatment-seeking adult patients with nonurgent psychiatric disorders. A total of 184 (94 ATP and 90 STP) English- and Spanish-speaking participants (36/184, 19.6% Hispanic) were enrolled and randomized, and 160 (80 ATP and 80 STP) of them completed baseline evaluations. Patients were treated by their primary care physicians using a collaborative care model in consultation with the University of California Davis Health telepsychiatrists, who consulted with patients every 6 months for up to 2 years using ATP or STP. Primary outcomes (the clinician-rated Clinical Global Impressions [CGI] scale and the Global Assessment of Functioning [GAF]) and secondary outcomes (patients' self-reported physical and mental health and depression) outcomes were assessed every 6 months. RESULTS: For clinician-rated primary outcomes, ATP did not promote greater improvement than STP at 6-month follow-up (ATP vs STP, adjusted difference in follow-up at 6 months vs baseline differences for CGI: 0.2, 95% CI -0.2 to 0.6; P=.28; and GAF: -0.6, 95% CI -3.1 to 1.9; P=.66) or 12-month follow-up (ATP vs STP, adjusted difference in follow-up at 12 months vs baseline differences for CGI: 0.4, 95% CI -0.04 to 0.8; P=.07; and GAF: -0.5, 95% CI -3.3 to 2.2; P=.70), but patients in both arms had statistically and clinically significant improvements in both outcomes. There were no significant differences in improvement from baseline between ATP and STP on any patient self-reported ratings at any follow-up (all P values were between .17 and .96). Dropout rates were higher than predicted but similar between the 2 arms. Of those with baseline visits, 46.8% (75/160) did not have a follow-up at 1 year, and 72.7% (107/147) did not have a follow-up at 2 years. No serious adverse events were associated with the intervention. CONCLUSIONS: This is the first longitudinal study to demonstrate that ATP can improve clinical outcomes in English- and Spanish-speaking primary care patients. Although we did not find evidence that ATP is superior to STP in improving clinical outcomes, it is potentially a key part of stepped mental health interventions available in primary care. ATP presents a possible solution to the workforce shortage of psychiatrists and a strategy for improving existing systems of care. TRIAL REGISTRATION: ClinicalTrials.gov NCT02084979; https://clinicaltrials.gov/ct2/show/NCT02084979.

In vivo detection of protein cysteine sulfenylation in plastids.

Protein cysteine thiols are post-translationally modified under oxidative stress conditions. Illuminated chloroplasts are one of the important sources of hydrogen peroxide (H2 O2 ) and are highly sensitive to environmental stimuli, yet a comprehensive view of the oxidation-sensitive chloroplast proteome is still missing. By targeting the sulfenic acid YAP1C-trapping technology to the plastids of light-grown Arabidopsis cells, we identified 132 putatively sulfenylated plastid proteins upon H2 O2 pulse treatment. Almost half of the sulfenylated proteins are enzymes of the amino acid metabolism. Using metabolomics, we observed a reversible decrease in the levels of the amino acids Ala, Asn, Cys, Gln, Glu, His, Ile, Leu, Lys, Phe, Ser, Thr and Val after H2 O2 treatment, which is in line with an anticipated decrease in the levels of the glycolysis and tricarboxylic acid metabolites. Through the identification of an organelle-tailored proteome, we demonstrated that the subcellular targeting of the YAP1C probe enables us to study in vivo cysteine sulfenylation at the organellar level. All in all, the identification of these oxidation events in plastids revealed that several enzymes of the amino acid metabolism rapidly undergo cysteine oxidation upon oxidative stress.

Synthesis strategies for non-symmetric, photochromic diarylethenes.

Diarylethenes (DAEs) represent an important class of photochromes with notable characteristics, like thermally irreversible photoisomerization and high fatigue resistance. Structural diversification of the DAE scaffold has enabled further refinement of photochromic properties and realization of new applications, ranging from advanced materials to tools for studying biological systems. In particular, methods for synthesizing non-symmetric DAE scaffolds, which are typically more challenging to synthesize than their symmetric counterparts, have grown over the past 20 years. These developments are surveyed in this review, with discussion of how access to these compounds has contributed to the improvement of photochromic properties and paved the way for exploring new applications of DAEs. First, non-symmetric DAE structures are classified and their uses and applications are overviewed. Subsequent sections discuss the main strategies that have been used to access non-symmetric DAEs with examples illustrating the impact of non-symmetric DAEs in the growing field of light-controlled molecular systems.

Assessment of the interaction between the flux-signaling metabolite fructose-1,6-bisphosphate and the bacterial transcription factors CggR and Cra.

Bacteria regulate cell physiology in response to extra- and intracellular cues. Recent work showed that metabolic fluxes are reported by specific metabolites, whose concentrations correlate with flux through the respective metabolic pathway. An example of a flux-signaling metabolite is fructose-1,6-bisphosphate (FBP). In turn, FBP was proposed to allosterically regulate master regulators of carbon metabolism, Cra in Escherichia coli and CggR in Bacillus subtilis. However, a number of questions on the FBP-mediated regulation of these transcription factors is still open. Here, using thermal shift assays and microscale thermophoresis we demonstrate that FBP does not bind Cra, even at millimolar physiological concentration, and with electrophoretic mobility shift assays we also did not find FBP-mediated impairment of Cra's affinity for its operator site, while fructose-1-phosphate does. Furthermore, we show for the first time that FBP binds CggR within the millimolar physiological concentration range of the metabolite, and decreases DNA-binding activity of this transcription factor. Molecular docking experiments only identified a single FBP binding site CggR. Our results provide the long thought after clarity with regards to regulation of Cra activity in E. coli and reveals that E. coli and B. subtilis use distinct cellular mechanism to transduce glycolytic flux signals into transcriptional regulation.

Modulating Polymer Dispersity with Light: Cationic Polymerization of Vinyl Ethers Using Photochromic Initiators.

Deterministic methods for tuning polymer dispersity are rare, especially for nonradical polymerizations. Reported here is the first example of photomodulating dispersity in controlled cationic polymerizations of vinyl ethers using carboxy-functionalized dithienylethene initiators. Reversible photoisomerization of these initiators induces changes in their acidities by up to an order of magnitude. Using the more acidic, ring-closed isomers as initiators results in polymers with lower dispersities. The degree of light-induced pKa change in the initiators correlates with the degree of dispersity change in polymers derived from the isomeric initiators. The polymerizations are controlled, and dynamic photoswitching of dispersity during the polymerization reaction was demonstrated. This work provides a framework for photomodulating dispersity in other controlled polymerizations and developing one-pot block copolymerization reactions in which the dispersities of component blocks can be controlled using light.

The Listeria Small RNA Rli27 Regulates a Cell Wall Protein inside Eukaryotic Cells by Targeting a Long 5'-UTR Variant.

Listeria monocytogenes is a bacterial pathogen whose genome encodes many cell wall proteins that bind covalently to peptidoglycan. Some members of this protein family have a key role in virulence, and recent studies show that some of these, such as Lmo0514, are upregulated in bacteria that colonize eukaryotic cells. The regulatory mechanisms that lead to these changes in cell wall proteins remain poorly characterized. Here we studied the regulation responsible for increased Lmo0514 protein levels in intracellular bacteria. The amount of this protein increased markedly in intracellular bacteria (>200-fold), which greatly exceeded the increase in lmo0514 transcript levels (∼6-fold). Rapid amplification of 5'-cDNA ends (RACE) assays identified two lmo0514 transcripts with 5'-untranslated regions (5'-UTR) of 28 and 234 nucleotides. The transcript containing the long 5'-UTR is upregulated by intracellular bacteria. The 234-nucleotide 5'-UTR is also the target of a small RNA (sRNA) denoted Rli27, which we identified by bioinformatics analysis as having extensive base pairing potential with the long 5'-UTR. The interaction is predicted to increase accessibility of the Shine-Dalgarno sequence occluded in the long 5'-UTR and thus to promote Lmo0514 protein production inside the eukaryotic cell. Real-time quantitative PCR showed that Rli27 is upregulated in intracellular bacteria. In vivo experiments indicated a decrease in Lmo0514 protein levels in intracellular bacteria that lacked Rli27. Wild-type Lmo0514 levels were restored by expressing the wild-type Rli27 molecule but not a mutated version unable to interact with the lmo0514 long 5'-UTR. These findings emphasize how 5'-UTR length affects regulation by defined sRNA. In addition, they demonstrate how alterations in the relative abundance of two transcripts with distinct 5'-UTR confine the action of an sRNA for a specific target to bacteria that occupy the intracellular eukaryotic niche.

Serum microRNAs as novel biomarkers for primary sclerosing cholangitis and cholangiocarcinoma.

The diagnosis of primary sclerosing cholangitis (PSC) is difficult due to the lack of sensitive and specific biomarkers, as is the early diagnosis of cholangiocarcinoma (CC), a complication of PSC. The aim of this study was to identify specific serum miRNAs as diagnostic biomarkers for PSC and CC. The levels of 667 miRNAs were evaluated in 90 human serum samples (30 PSC, 30 CC and 30 control subjects) to identify disease-associated candidate miRNAs (discovery phase). The deregulated miRNAs were validated in an independent cohort of 140 samples [40 PSC, 40 CC, 20 primary biliary cirrhosis (PBC) and 40 controls]. Receiver operating characteristic (ROC) curves were established and only miRNAs with an area under the curve (AUC) > 0·70 were considered useful as biomarkers. In the discovery phase we identified the following: 21 miRNAs expressed differentially in PSC, 33 in CC and 26 in both in comparison to control subjects as well as 24 miRNAs expressed differentially between PSC and CC. After the validation phase, miR-200c was found to be expressed differentially in PSC versus controls, whereas miR-483-5p and miR-194 showed deregulated expression in CC compared with controls. We also demonstrate a difference in the expression of miR-222 and miR-483-5p in CC versus PSC. Combination of these specific miRNAs further improved the specificity and accuracy of diagnosis. This study provides a basis for the use of miRNAs as biomarkers for the diagnosis of PSC and CC.

Highly sensitive index of sympathetic activity based on time-frequency spectral analysis of electrodermal activity.

Time-domain indices of electrodermal activity (EDA) have been used as a marker of sympathetic tone. However, they often show high variation between subjects and low consistency, which has precluded their general use as a marker of sympathetic tone. To examine whether power spectral density analysis of EDA can provide more consistent results, we recently performed a variety of sympathetic tone-evoking experiments (43). We found significant increase in the spectral power in the frequency range of 0.045 to 0.25 Hz when sympathetic tone-evoking stimuli were induced. The sympathetic tone assessed by the power spectral density of EDA was found to have lower variation and more sensitivity for certain, but not all, stimuli compared with the time-domain analysis of EDA. We surmise that this lack of sensitivity in certain sympathetic tone-inducing conditions with time-invariant spectral analysis of EDA may lie in its inability to characterize time-varying dynamics of the sympathetic tone. To overcome the disadvantages of time-domain and time-invariant power spectral indices of EDA, we developed a highly sensitive index of sympathetic tone, based on time-frequency analysis of EDA signals. Its efficacy was tested using experiments designed to elicit sympathetic dynamics. Twelve subjects underwent four tests known to elicit sympathetic tone arousal: cold pressor, tilt table, stand test, and the Stroop task. We hypothesize that a more sensitive measure of sympathetic control can be developed using time-varying spectral analysis. Variable frequency complex demodulation, a recently developed technique for time-frequency analysis, was used to obtain spectral amplitudes associated with EDA. We found that the time-varying spectral frequency band 0.08-0.24 Hz was most responsive to stimulation. Spectral power for frequencies higher than 0.24 Hz were determined to be not related to the sympathetic dynamics because they comprised less than 5% of the total power. The mean value of time-varying spectral amplitudes in the frequency band 0.08-0.24 Hz were used as the index of sympathetic tone, termed TVSymp. TVSymp was found to be overall the most sensitive to the stimuli, as evidenced by a low coefficient of variation (0.54), and higher consistency (intra-class correlation, 0.96) and sensitivity (Youden's index > 0.75), area under the receiver operating characteristic (ROC) curve (>0.8, accuracy > 0.88) compared with time-domain and time-invariant spectral indices, including heart rate variability.

A Longitudinal Investigation of Syndemic Conditions Among Young Gay, Bisexual, and Other MSM: The P18 Cohort Study.

The persistence of disparities in STI/HIV risk among a new generation of emerging adult gay, bisexual, and other men who have sex with men (YMSM) warrant holistic frameworks and new methodologies for investigating the behaviors related to STI/HIV in this group. In order to better understand the continued existence of these disparities in STI/HIV risk among YMSM, the present study evaluated the presence and persistence of syndemic conditions among YMSM by examining the co-occurrence of alcohol and drug use, unprotected sexual behavior, and mental health burden over time. Four waves of data, collected over the first 18 months of a 7 wave, 36-month prospective cohort study of YMSM (n=600) were used to examine the extent to which measurement models of drug use, unprotected sexual behavior, and mental health burden remained consistent across time using latent class modeling. Health challenges persisted across time as these YMSM emerged into young adulthood and the measurement models for the latent constructs of drug use and unprotected sexual behavior were essentially consistent across time whereas models for mental health burden varied over time. In addition to confirming the the robustness of our measurement models which capture a more holistic understandings of the health conditions of drug use, unprotected sex, and mental health burden, these findings underscore the ongoing health challenges YMSM face as they mature into young adulthood. These ongoing health challenges, which have been understood as forming a syndemic, persist over time, and add further evidence to support ongoing and vigilant comprehensive health programming for sexual minority men that move beyond a sole focus on HIV.

Color difference thresholds in dentistry.

PURPOSE: The aim of this prospective multicenter study was to determine 50:50% perceptibility threshold (PT) and 50:50% acceptability threshold (AT) of dental ceramic under simulated clinical settings. MATERIALS AND METHODS: The spectral radiance of 63 monochromatic ceramic specimens was determined using a non-contact spectroradiometer. A total of 60 specimen pairs, divided into 3 sets of 20 specimen pairs (medium to light shades, medium to dark shades, and dark shades), were selected for psychophysical experiment. The coordinating center and seven research sites obtained the Institutional Review Board (IRB) approvals prior the beginning of the experiment. Each research site had 25 observers, divided into five groups of five observers: dentists-D, dental students-S, dental auxiliaries-A, dental technicians-T, and lay persons-L. There were 35 observers per group (five observers per group at each site ×7 sites), for a total of 175 observers. Visual color comparisons were performed using a viewing booth. Takagi-Sugeno-Kang (TSK) fuzzy approximation was used for fitting the data points. The 50:50% PT and 50:50% AT were determined in CIELAB and CIEDE2000. The t-test was used to evaluate the statistical significance in thresholds differences. RESULTS: The CIELAB 50:50% PT was ΔEab = 1.2, whereas 50:50% AT was ΔEab = 2.7. Corresponding CIEDE2000 (ΔE00 ) values were 0.8 and 1.8, respectively. 50:50% PT by the observer group revealed differences among groups D, A, T, and L as compared with 50:50% PT for all observers. The 50:50% AT for all observers was statistically different than 50:50% AT in groups T and L. CONCLUSION: A 50:50% perceptibility and ATs were significantly different. The same is true for differences between two color difference formulas ΔE00 /ΔEab . Observer groups and sites showed high level of statistical difference in all thresholds. CLINICAL SIGNIFICANCE: Visual color difference thresholds can serve as a quality control tool to guide the selection of esthetic dental materials, evaluate clinical performance, and interpret visual and instrumental findings in clinical dentistry, dental research, and subsequent standardization. The importance of quality control in dentistry is reinforced by increased esthetic demands of patients and dental professionals.

Genome expression analysis of nonproliferating intracellular Salmonella enterica serovar Typhimurium unravels an acid pH-dependent PhoP-PhoQ response essential for dormancy.

Genome-wide expression analyses have provided clues on how Salmonella proliferates inside cultured macrophages and epithelial cells. However, in vivo studies show that Salmonella does not replicate massively within host cells, leaving the underlying mechanisms of such growth control largely undefined. In vitro infection models based on fibroblasts or dendritic cells reveal limited proliferation of the pathogen, but it is presently unknown whether these phenomena reflect events occurring in vivo. Fibroblasts are distinctive, since they represent a nonphagocytic cell type in which S. enterica serovar Typhimurium actively attenuates intracellular growth. Here, we show in the mouse model that S. Typhimurium restrains intracellular growth within nonphagocytic cells positioned in the intestinal lamina propria. This response requires a functional PhoP-PhoQ system and is reproduced in primary fibroblasts isolated from the mouse intestine. The fibroblast infection model was exploited to generate transcriptome data, which revealed that ∼2% (98 genes) of the S. Typhimurium genome is differentially expressed in nongrowing intracellular bacteria. Changes include metabolic reprogramming to microaerophilic conditions, induction of virulence plasmid genes, upregulation of the pathogenicity islands SPI-1 and SPI-2, and shutdown of flagella production and chemotaxis. Comparison of relative protein levels of several PhoP-PhoQ-regulated functions (PagN, PagP, and VirK) in nongrowing intracellular bacteria and extracellular bacteria exposed to diverse PhoP-PhoQ-inducing signals denoted a regulation responding to acidic pH. These data demonstrate that S. Typhimurium restrains intracellular growth in vivo and support a model in which dormant intracellular bacteria could sense vacuolar acidification to stimulate the PhoP-PhoQ system for preventing intracellular overgrowth.

Mutation of Arabidopsis SME1 and Sm core assembly improves oxidative stress resilience.

Alternative splicing is a key posttranscriptional gene regulatory process, acting in diverse adaptive and basal plant processes. Splicing of precursor-messenger RNA (pre-mRNA) is catalyzed by a dynamic ribonucleoprotein complex, designated the spliceosome. In a suppressor screen, we identified a nonsense mutation in the Smith (Sm) antigen protein SME1 to alleviate photorespiratory H2O2-dependent cell death in catalase deficient plants. Similar attenuation of cell death was observed upon chemical inhibition of the spliceosome, suggesting pre-mRNA splicing inhibition to be responsible for the observed cell death alleviation. Furthermore, the sme1-2 mutants showed increased tolerance to the reactive oxygen species inducing herbicide methyl viologen. Both an mRNA-seq and shotgun proteomic analysis in sme1-2 mutants displayed a constitutive molecular stress response, together with extensive alterations in pre-mRNA splicing of transcripts encoding metabolic enzymes and RNA binding proteins, even under unstressed conditions. Using SME1 as a bait to identify protein interactors, we provide experimental evidence for almost 50 homologs of the mammalian spliceosome-associated protein to reside in the Arabidopsis thaliana spliceosome complexes and propose roles in pre-mRNA splicing for four uncharacterized plant proteins. Furthermore, as for sme1-2, a mutant in the Sm core assembly protein ICLN resulted in a decreased sensitivity to methyl viologen. Taken together, these data show that both a perturbed Sm core composition and assembly results in the activation of a defense response and in enhanced resilience to oxidative stress.

Human G3BP1 interacts with beta-F1-ATPase mRNA and inhibits its translation.

The post-transcriptional regulation of nuclear mRNAs that encode core components of mitochondria has relevant implications in cell physiology. The mRNA that encodes the catalytic subunit of the mitochondrial H(+)-ATP synthase subunit beta (ATP5B, beta-F1-ATPase) is localized in a large ribonucleoprotein (RNP) complex (beta-F1-RNP), which is subjected to stringent translational control during development and the cell cycle, and in carcinogenesis. Because downregulation of beta-F1-ATPase is a conserved feature of most prevalent human carcinomas, we have investigated the molecular composition of the human beta-F1-RNP. By means of an improved affinity-chromatography procedure and protein sequencing we have identified nine RNA-binding proteins (RNABPs) of the beta-F1-RNP. Immunoprecipitation assays of Ras-GAP SH3 binding protein 1 (G3BP1) and fluorescent in-situ hybridization of mRNA indicate a direct interaction of the endogenous G3BP1 with mRNA of beta-F1-ATPase (beta-F1 mRNA). RNA-bridged trimolecular fluorescence complementation (TriFC) assays confirm the interaction of G3BP1 with the 3'-UTR of beta-F1 mRNA in cytoplasmic RNA-granules. Confocal and high-resolution immunoelectron-microscopy experiments suggest that the beta-F1-RNP is sorted to the periphery of mitochondria. Molecular and functional studies indicate that the interaction of G3BP1 with beta-F1 mRNA inhibits its translation at the initiation level, supporting a role for G3BP1 in the glycolytic switch that occurs in cancer.