The Chlamydia trachomatis type III-secreted effector protein CteG induces centrosome amplification through interactions with centrin-2.

The centrosome is the main microtubule organizing center of the cell and is crucial for mitotic spindle assembly, chromosome segregation, and cell division. Centrosome duplication is tightly controlled, yet several pathogens, most notably oncogenic viruses, perturb this process leading to increased centrosome numbers. Infection by the obligate intracellular bacterium Chlamydia trachomatis (C.t.) correlates with blocked cytokinesis, supernumerary centrosomes, and multipolar spindles; however, the mechanisms behind how C.t. induces these cellular abnormalities remain largely unknown. Here we show that the secreted effector protein, CteG, binds to centrin-2 (CETN2), a key structural component of centrosomes and regulator of centriole duplication. Our data indicate that both CteG and CETN2 are necessary for infection-induced centrosome amplification, in a manner that requires the C-terminus of CteG. Strikingly, CteG is important for in vivo infection and growth in primary cervical cells but is dispensable for growth in immortalized cells, highlighting the importance of this effector protein to chlamydial infection. These findings begin to provide mechanistic insight into how C.t. induces cellular abnormalities during infection, but also indicate that obligate intracellular bacteria may contribute to cellular transformation events. Centrosome amplification mediated by CteG-CETN2 interactions may explain why chlamydial infection leads to an increased risk of cervical or ovarian cancer.

Antiactivators prevent self-sensing in Pseudomonas aeruginosa quorum sensing.

Quorum sensing is described as a widespread cell density-dependent signaling mechanism in bacteria. Groups of cells coordinate gene expression by secreting and responding to diffusible signal molecules. Theory, however, predicts that individual cells may short-circuit this mechanism by directly responding to the signals they produce irrespective of cell density. In this study, we characterize this self-sensing effect in the acyl-homoserine lactone quorum sensing system of Pseudomonas aeruginosa. We show that antiactivators, a set of proteins known to affect signal sensitivity, function to prevent self-sensing. Measuring quorum-sensing gene expression in individual cells at very low densities, we find that successive deletion of antiactivator genes qteE and qslA produces a bimodal response pattern, in which increasing proportions of constitutively induced cells coexist with uninduced cells. Comparing responses of signal-proficient and -deficient cells in cocultures, we find that signal-proficient cells show a much higher response in the antiactivator mutant background but not in the wild-type background. Our results experimentally demonstrate the antiactivator-dependent transition from group- to self-sensing in the quorum-sensing circuitry of P. aeruginosa. Taken together, these findings extend our understanding of the functional capacity of quorum sensing. They highlight the functional significance of antiactivators in the maintenance of group-level signaling and experimentally prove long-standing theoretical predictions.

The fitness benefit of pyoverdine cross-feeding by Pseudomonas protegens Pf-5.

Under iron-limiting conditions, fluorescent pseudomonads acquire iron from the environment by secreting strain-specific, iron-chelating siderophores termed pyoverdines (PVD). The rhizosphere bacterium Pseudomonas protegens Pf-5 produces its own PVD but also can cross-feed on PVDs produced by other species. Previous work has found that Pf-5 continues to produce its own PVD when allowed to cross-feed, raising questions about the benefit of heterologous PVD utilisation. Here, we investigate this question using a defined, unidirectional P. protegens Pf-5/Pseudomonas aeruginosa PAO1 cross-feeding model. Quantifying the production of PVD in the presence of heterologous PVD produced by PAO1, we show that cross-feeding Pf-5 strains reduce the production of their own PVD, while non-cross-feeding Pf-5 strains increase the production of PVD. Measuring the fitness of cross-feeding and non-cross-feeding Pf-5 strains in triple coculture with PAO1, we find that cross-feeding provides a fitness benefit to Pf-5 when the availability of heterologous PVD is high. We conclude that cross-feeding can reduce the costs of self-PVD production and may thus aid in the colonisation of iron-limited environments that contain compatible siderophores produced by other resident microbes. Taken together, these results expand our understanding of the mechanisms of interspecific competition for iron in microbial communities.

Identification and Preliminary Characterization of Novel Type III Secreted Effector Proteins in Chlamydia trachomatis.

Chlamydia trachomatis is an obligate intracellular pathogen that replicates in a host-derived vacuole termed the inclusion. Central to pathogenesis is a type III secretion system that translocates effector proteins into the host cell, which are predicted to play major roles in host cell invasion, nutrient acquisition, and immune evasion. However, until recently, the genetic intractability of C. trachomatis hindered identification and characterization of these important virulence factors. Here, we sought to expand the repertoire of identified effector proteins and confirm they are secreted during C. trachomatis infection. Utilizing bioinformatics, we identified 18 candidate substrates that had not been previously assessed for secretion, of which we show four to be secreted, using Yersinia pseudotuberculosis as a surrogate host. Using adenylate cyclase (CyaA), BlaM, and glycogen synthase kinase (GSK) secretion assays, we identified nine novel substrates that were secreted in at least one assay. Interestingly, only three of the substrates, shown to be translocated by C. trachomatis, were similarly secreted by Y. pseudotuberculosis. Using large-scale screens to determine subcellular localization and identify effectors that perturb crucial host cell processes, we identified one novel substrate, CT392, that is toxic when heterologously expressed in Saccharomyces cerevisiae. Toxicity required both the N- and C-terminal regions of the protein. Additionally, we show that these newly described substrates traffic to distinct host cell compartments, including vesicles and the cytoplasm. Collectively, our study expands the known repertoire of C. trachomatis secreted factors and highlights the importance of testing for secretion in the native host using multiple secretion assays when possible.

Clostridium muellerianum sp. nov., a carbon monoxide-oxidizing acetogen isolated from old hay.

An acid/alcohol-producing, Gram-stain-positive, obligately anaerobic, rod-shaped, non-motile, non-spore forming acetogen, designated as strain P21T, was isolated from old hay after enrichment with CO as the substrate. Spores not observed even after prolonged incubation (30 days). Phylogenetic analysis of the 16S rRNA gene sequence of strain P21T showed it was closely related to Clostridium carboxidivorans DSM 15243T (97.9%), Clostridium scatologenes DSM 757T (97.7 %) and Clostridium drakei DSM 12750T (97.7 %). The genome is 5.6 Mb and the G+C content is 29.4 mol%. Average nucleotide identity between strain P21T, C. carboxidivorans, C. scatologenes and C. drakei was 87.1, 86.4, 86.4 %, respectively. Strain P21T grew on CO:CO2, H2:CO2, l-arabinose, ribose, xylose, fructose, galactose, glucose, lactose, mannose, cellobiose, sucrose, cellulose, starch, pyruvate, choline, glutamate, histidine, serine, threonine and casamino acids. End products of metabolism were acetate, butyrate, caproate, ethanol and hexanol. Dominant cellular fatty acids (>10 %) were C16 : 0 (41.5 %), C16 : 1 ω7c/C16 : 1 ω6c (10.0 %), and a summed feature containing cyclo C17 : 1/C18 : 0 (17.3 %). Based on the phenotypic, chemotaxonomic, phylogenetic and phylogenomic analyses, strain P21T represents a new species in the genus Clostridium, for which the name Clostridium muellerianum sp. nov. is proposed. The type strain is P21T (=DSM 111390T=NCIMB 15261T).

UCP2 Overexpression Redirects Glucose into Anabolic Metabolic Pathways.

Uncoupling protein 2 (UCP2) is often upregulated in cancer cells. The UCP2 upregulation is positively correlated with enhanced proliferation, tumorigenesis, and metabolic alterations, thus suggesting that UCP2 upregulation can play a key role in sensing metabolic changes to promote tumorigenesis. To determine the global metabolic impact of UCP2 upregulation, 13 C6 glucose as a source molecule is used to "trace" the metabolic fate of carbon atoms derived from glucose. UCP2 overexpression in skin epidermal cells enhances the incorporation of 13 C label to pyruvate, tricarboxylic acid cycle intermediates, nucleotides, and amino acids, suggesting that UCP2 upregulation reprograms cellular metabolism toward macromolecule synthesis. To the best of our knowledge, this is the first study to bring to light the overall metabolic differences caused by UCP2 upregulation.

The neutral condition in conflict tasks: On the violation of the midpoint assumption in reaction time trends.

Although the relation between congruent and incongruent conditions in conflict tasks has been the primary focus of cognitive control studies, the neutral condition is often set as a baseline directly between the two conditions. However, empirical evidence suggests that the average neutral reaction time (RT) is not placed evenly between the two opposing conditions. This article set out to establish two things: First, to reinforce the informative nature of the neutral condition and second, to highlight how it can be useful for modelling. We explored how RT in the neutral condition of conflict tasks (Stroop, Flanker, and Simon Tasks) deviated from the predictions of current diffusion models. Current diffusion models of conflict tasks predict a neutral RT that is the average of the congruent and incongruent RT, called the midpoint assumption. To investigate this, we first conducted a cursory limited search that recorded the average RT's of conflict tasks with neutral conditions. Upon finding evidence of a midpoint assumption violation which showed a larger disparity between average neutral and incongruent RT, we tested the previously mentioned conflict tasks with two different sets of stimuli to establish the robustness of the effect. The midpoint assumption violation is sometimes inconsistent with the prediction of diffusion models of conflict processing (e.g., the Diffusion Model of Conflict), suggesting possible elaborations of such models.

A 3-Year Study Investigating the Utility of Patient-Reported Outcomes Measurement Information System-29 for Long-Term Follow-Up in Adult Spinal Deformity.

BACKGROUND: Adult spinal deformity (ASD) significantly impacts the quality of life due to three-dimensional spinal abnormalities. Patient-reported outcome measures, such as the Patient-Reported Outcomes Measurement Information System (PROMIS-29), play a crucial role in assessing postoperative outcomes. This study aims to investigate trends in PROMIS-29 scores over 36 months in patients undergoing long-segment thoracolumbar fusion for ASD and provide insights into its long-term utility. METHODS: A retrospective study including 163 ASD patients undergoing long-segment thoracolumbar fusion was conducted. PROMIS-29 scores were collected at baseline and at postoperative (0-), 3-, 6-, 12-, 18-, 24-, 30-, and 36-month follow-ups. Statistical analyses was performed to assess significant score changes from baseline and in consecutive recordings. RESULTS: Significant improvements in all PROMIS-29 categories were observed at 36 months, with the greatest changes in pain intensity (-35.19%, P < 0.001), physical function (+29.13%, P < 0.001), and pain interference (-28.8%, P < 0.001). Between the 0 and 3 month mark, the greatest significant changes were recorded in pain intensity (-26.5%, P < 0.001), physical function (+24.3%, P < 0.001), and anxiety (-16.9%, P < 0.018). However, scores plateaued after the 3-month mark, with zero categories showing significant changes with subsequent consecutive recordings. CONCLUSIONS: PROMIS-29 scores demonstrated notable improvements in ASD patients particularly in pain intensity, pain interference, and physical function. However, scores plateaued beyond the 3-month mark, suggesting PROMIS-29's limited sensitivity to nuanced changes in long-term patient recovery. Future investigations exploring optimal combinations of patient reported outcome measures for comprehensive short- and long-term outcome assessments in ASD surgery would be beneficial.

Basilar Impression: A Systematic Review and Meta-Analysis of Clinical Features, Operative Strategies and Outcomes.

OBJECTIVE: Basilar Impression (BI) is a rare yet debilitating abnormality of the craniovertebral junction (CVJ), known to cause life-threatening medullary brainstem compression. Our study analyzes surgical approaches for BI and related outcomes. METHODS: A systematic review was conducted using PubMed, Google Scholar, and Web of Science electronic databases according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines to critically assess primary articles examining BI. RESULTS: We analyzed 87 patients from 65 articles, mostly female (55.17%) with a mean age of 46.31 ± 17.94 years, commonly presenting with motor (59.77%) and sensory deficits (55.17%). Commonly employed procedures included posterior occipitocervical fusion (24.14%), anterior decompression (20.69%), and combined anterior decompression with posterior fusion (21.84%). Patients who underwent anterior approaches were found to be older (55.38 ± 17.67 vs. 45.49 ± 18.78 years, p < 0.05) and had a longer duration from symptom onset to surgery (57.39 ± 64.33 vs. 26.02 ± 29.60 months, p < 0.05) compared to posterior approaches. Our analysis revealed a significant association between a longer duration from symptom onset to surgery and an increased likelihood of undergoing odontoidectomy and decompression (OR: 1.02, 95% CI: 1.00-1.03, p < 0.05). Furthermore, after adjusting for all other covariates, a history of rheumatoid arthritis and the use of a posterior approach were significantly associated with an elevated risk of postoperative complications (p < 0.05). CONCLUSIONS: The treatment approach to complex CVJ disease should be tailored to the surgeon's experience and the nature of the compressive pathology.

Global mapping of the Chlamydia trachomatis conventional secreted effector - host interactome reveals CebN interacts with nucleoporins and Rae1 to impede STAT1 nuclear translocation.

Chlamydia trachomatis (C.t.), the leading cause of bacterial sexually transmitted infections, employs a type III secretion system (T3SS) to translocate two classes of effectors, inclusion membrane proteins and conventional T3SS (cT3SS) effectors, into the host cell to counter host defense mechanisms. Here we employed three assays to directly evaluate secretion during infection, validating secretion for 23 cT3SS effectors. As bioinformatic analyses have been largely unrevealing, we conducted affinity purification-mass spectrometry to identify host targets and gain insights into the functions of these effectors, identifying high confidence interacting partners for 21 cT3SS effectors. We demonstrate that CebN localizes to the nuclear envelope in infected and bystander cells where it interacts with multiple nucleoporins and Rae1, blocking STAT1 nuclear import following IFN-γ stimulation. By building a cT3SS effector-host interactome, we have identified novel pathways that are targeted during bacterial infection and have begun to address how C.t. effectors combat cell autonomous immunity.

Conserved chromatin and repetitive patterns reveal slow genome evolution in frogs.

Frogs are an ecologically diverse and phylogenetically ancient group of anuran amphibians that include important vertebrate cell and developmental model systems, notably the genus Xenopus. Here we report a high-quality reference genome sequence for the western clawed frog, Xenopus tropicalis, along with draft chromosome-scale sequences of three distantly related emerging model frog species, Eleutherodactylus coqui, Engystomops pustulosus, and Hymenochirus boettgeri. Frog chromosomes have remained remarkably stable since the Mesozoic Era, with limited Robertsonian (i.e., arm-preserving) translocations and end-to-end fusions found among the smaller chromosomes. Conservation of synteny includes conservation of centromere locations, marked by centromeric tandem repeats associated with Cenp-a binding surrounded by pericentromeric LINE/L1 elements. This work explores the structure of chromosomes across frogs, using a dense meiotic linkage map for X. tropicalis and chromatin conformation capture (Hi-C) data for all species. Abundant satellite repeats occupy the unusually long (~20 megabase) terminal regions of each chromosome that coincide with high rates of recombination. Both embryonic and differentiated cells show reproducible associations of centromeric chromatin and of telomeres, reflecting a Rabl-like configuration. Our comparative analyses reveal 13 conserved ancestral anuran chromosomes from which contemporary frog genomes were constructed.

Parameters, architecture and emergent properties of the Pseudomonas aeruginosa LasI/LasR quorum-sensing circuit.

Quorum sensing is a widespread process in bacteria that controls collective behaviours in response to cell density. Populations of cells coordinate gene expression through the perception of self-produced chemical signals. Although this process is well-characterized genetically and biochemically, quantitative information about network properties, including induction dynamics and steady-state behaviour, is scarce. Here we integrate experiments with mathematical modelling to quantitatively analyse the LasI/LasR quorum sensing pathway in the opportunistic pathogen Pseudomonas aeruginosa. We determine key kinetic parameters of the pathway and, using the parametrized model, show that quorum sensing behaves as a bistable hysteretic switch, with stable on and off states. We investigate the significance of feedback architecture and find that positive feedback on signal production is critical for induction dynamics and bistability, whereas positive feedback on receptor expression and negative feedback on signal production play a minor role. Taken together, our data-based modelling approach reveals fundamental and emergent properties of a bacterial quorum sensing circuit, and provides evidence that native quorum sensing can indeed function as the gene expression switch it is commonly perceived to be.

Examining the context, logistics, and outcomes of food prescription programs: A scoping review.

BACKGROUND: Obesity and associated metabolic conditions are endemic. Finding new strategies to mitigate the impact on wellbeing and healthcare systems is critical. Food prescription programs (FPPs) have been promoted as one route to address this problem in a way that simultaneously addresses the socio-cultural context of obesity. Yet, little is known about the standard practices and logistics of using food prescription programs as an effective intervention. OBJECTIVES: To 1) identify the context in which food prescription programs are used; 2) identify implementation logistics of food prescription program; and 3) understand the scope of food prescription program outcomes. METHODS: A scoping review was conducted from October 2019 to May 2020 using Google Scholar, EBSCOhost, and AcademicOne Search to identify research articles focused on the implementation of prescription food programs in the US. Updates to articles were made in May of 2021 and May of 2022 to ensure the most up-to-date sample for analysis. There was no publication date restriction for article inclusion. RESULTS: A total of 213 articles were identified for abstract review via the search strategy, and 30 articles were included for analysis following article exclusion. Overall, there was little consistency among included articles regarding the target population, participant recruitment, delivery, and evaluation of the food prescription programs implemented. Most food prescription programs studied were associated with farmers markets, lasted less than 6 months, and utilized produce consumption and biometric data as primary outcomes measures. CONCLUSION: Significant gaps in the literature concerning the long-term effectiveness, impact on health behaviors, screening of eligible participants, and logistics for implementation were identified. Future research should focus on addressing these shortcomings in the current literature to improve the implementation, sustainability, and scaling of food prescription programs.

Treatment of Leptomeningeal Carcinomatosis Following Treatment of Cerebellar Metastasis of HER2+ (Human Epidermal Growth Factor Receptor 2 Positive) Breast Cancer: Case Report and Review of Literature.

Leptomeningeal carcinomatosis (LC) after metastasis of breast cancer is a rare occurrence with potentially devastating complications. Treatment options are limited, and there is a lack of literature on this topic. We report the case of a 38-year-old woman with estrogen/progesterone receptor negative (ER/PR-), human epidermal growth factor receptor 2 positive (HER2+) invasive ductal carcinoma of the left breast who underwent bilateral mastectomies with axillary lymph node dissection and chemotherapy treatment. The patient returned 11 months later with persistent headaches. Imaging and resection found cerebellar metastasis of the breast carcinoma. The brain metastasis was treated with further chemotherapy and stereotactic radiosurgery. Follow-up imaging showed the development of small lesions outside the radiation site. Metabolic studies were performed to determine if the new lesions were due to tumor recurrence or radiation necrosis, but the studies were inconclusive as to the etiology of these lesions. The patient later developed LC that was successfully treated with full resolution of the disease using intrathecal trastuzumab. There are currently no consensuses on treatment guidelines for treating LC. Here, we demonstrate successful treatment of LC from an ER/PR-, HER2+ breast carcinoma with intrathecal trastuzumab.

Inexpensive Apparatus for High-Quality Imaging of Microbial Growth on Agar Plates.

The ability to capture images of results or processes is an important tool in the biologist's tool kit. In microbiology, capturing high-quality images of microbial growth on agar plates is difficult due to the reflective surface of the plates and limitations in common photography techniques. Equipment is available to overcome these challenges, but acquisition costs are high. We have developed and tested an inexpensive and efficient apparatus for high-quality imaging of microbial colonies. The imaging box, as we have named the apparatus, is designed to eliminate glare and reduce reflections on the surface of the plate while providing uniform, diffuse light from all sides. The imaging box was used to capture hundreds of images in research and teaching lab settings.

Cooperation and Cheating through a Secreted Aminopeptidase in the Pseudomonas aeruginosa RpoS Response.

The global stress response controlled by the alternative sigma factor RpoS protects enteric bacteria from a variety of environmental stressors. The role of RpoS in other, nonenteric bacteria, such as the opportunistic pathogen Pseudomonas aeruginosa, is less well understood. Here, we employed experimental social evolution to reveal that cooperative behavior via secreted public goods is an important function in the RpoS response of P. aeruginosa Using whole-genome sequencing, we identified rpoS loss-of-function mutants among isolates evolved in a protein growth medium that requires extracellular proteolysis. We found that rpoS mutants comprise up to 25% of the evolved population and that they behave as social cheaters, with low fitness in isolation but high fitness in mixed culture with the cooperating wild type. We conclude that rpoS mutants cheat because they exploit an RpoS-controlled public good produced by the wild type, the secreted aminopeptidase PaAP, and because they do not carry the metabolic costs of expressing PaAP and many other gene products in the large RpoS regulon. Our results suggest that PaAP is an integral part of a proteolytic sequence in P. aeruginosa that permits the utilization of protein as a nutrient source. Our work broadens the scope of stress response functions in bacteria.IMPORTANCE Bacterial stress responses are generally considered protective measures taken by individual cells. Enabled by an experimental evolution approach, we describe a contrasting property, collective nutrient acquisition, in the RpoS-dependent stress response of the opportunistic human pathogen P. aeruginosa Specifically, we identify the secreted P. aeruginosa aminopeptidase (PaAP) as an essential RpoS-controlled function in extracellular proteolysis. As a secreted "public good," PaAP permits cheating by rpoS mutants that save the metabolic costs of expressing RpoS-controlled genes dispensable under the given growth conditions. Proteolytic enzymes are important virulence factors in P. aeruginosa pathogenesis and constitute a potential target for antimicrobial therapy. More broadly, our work contributes to recent findings in higher organisms that stress affects not only individual fitness and competitiveness but also cooperative behavior.

Public goods and cheating in microbes.

Communication and cooperation are not restricted to complex, higher organisms. Microbes, too, perform a variety of collective, multicellular behaviors, including biofilm formation, quorum sensing, nutrient acquisition, and dispersal. The products of these microbial cooperative behaviors are generally referred to as public goods. Here we describe the nature of microbial public goods, the associated problem of cheating, and ways in which microbes maintain public goods in the face of cheating. We highlight work in a growing field at the interface of microbiology, evolution, and ecology that combines multiple approaches in experimental evolution, genetics, and mathematical modeling.

Bacterial Cheaters Evade Punishment by Cyanide.

In all domains of life, mechanisms exist that protect cooperating groups from exploitation by cheaters. Recent observations with the bacterium Pseudomonas aeruginosa have suggested a paradigmatic cheater control mechanism in which cooperator cells punish or "police" cheater cells by cyanide poisoning. These cheater cells are deficient in a pleiotropic quorum-sensing regulator that controls the production of cooperative secretions including cyanide, and presumably also cyanide resistance. In this study, we directly tested and refuted the cyanide policing model. Contrary to the hypothesis, cheater fitness was unaffected by the presence of cyanide. Cheater mutants grew equally well in co-cultures with either cyanide-proficient or cyanide-deficient cooperators, and they were as resistant to exogenous cyanide as wild-type cells. We show that these behaviors are the result of quorum-sensing-independent and cyanide-responsive resistance gene regulation. Our results highlight the role of genetic architecture in the evolution of cooperative behavior.

Ternary Deep Eutectic Solvent Behavior of Water and Urea?Choline Chloride Mixtures.

Deep eutectic solvents (DESs) have potential as designer media whose physical, chemical, electrochemical, and spectroscopic properties are easily and broadly tuned. Due to the ubiquity of water and the hygroscopic nature of many DES components, it is important to understand how water affects DESs both on the nanoscale and in the bulk. Here, we present a study of the physical properties of DES/water mixtures. We found that water appears to form a ternary DES when mixed with 2:1 urea/ChCl.

Proton-Coupled Electron Transfer and Substituent Effects in Catechol-Based Deep Eutectic Solvents: Gross and Fine Tuning of Redox Activity.

The 1,2-diol moiety in a variety of substituted catechols allows formation of room temperature ionic melts in a 2:1 ratio with choline chloride or choline dihydrogen citrate. These deep eutectic solvents were 4.3-6.6 M in redox active catechols. Substituents on 3- and 4-substituted catechols shift both E° and pKa such that Hammett parameters predict the observed Ep for oxidation in square wave voltammetry. The proton acceptor for the proton-coupled oxidation shifts the observed Ep more strongly than the substituents within the substituents and acceptors reported here. The shift is predicted well by the pKa of the conjugate acid of the proton acceptor, i.e., water in aqueous solutions or chloride or dihydrogen citrate in the DESs in this study. Together, the substituent and the proton acceptor allow gross and fine-tuning of the oxidation potential for catechol over 750 mV, the first demonstration of control of the thermodynamics of proton-coupled electron transfer in deep eutectic solvents. Changing the substituents on the HBD affords fine control in tens of millivolts, while changing the base strength of the anion of the organic salt affords gross control across hundreds of millivolts.