Dynamin-dependent entry of Chlamydia trachomatis is sequentially regulated by the effectors TarP and TmeA.

Chlamydia invasion of epithelial cells is a pathogen-driven process involving two functionally distinct effectors - TarP and TmeA. They collaborate to promote robust actin dynamics at sites of entry. Here, we extend studies on the molecular mechanism of invasion by implicating the host GTPase dynamin 2 (Dyn2) in the completion of pathogen uptake. Importantly, Dyn2 function is modulated by TarP and TmeA at the levels of recruitment and activation through oligomerization, respectively. TarP-dependent recruitment requires phosphatidylinositol 3-kinase and the small GTPase Rac1, while TmeA has a post-recruitment role related to Dyn2 oligomerization. This is based on the rescue of invasion duration and efficiency in the absence of TmeA by the Dyn2 oligomer-stabilizing small molecule activator Ryngo 1-23. Notably, Dyn2 also regulated turnover of TarP- and TmeA-associated actin networks, with disrupted Dyn2 function resulting in aberrant turnover dynamics, thus establishing the interdependent functional relationship between Dyn2 and the effectors TarP and TmeA.

Metabolic model guided CRISPRi identifies a central role for phosphoglycerate mutase in Chlamydia trachomatis persistence.

Upon nutrient starvation, Chlamydia trachomatis serovar L2 (CTL) shifts from its normal growth to a non-replicating form, termed persistence. It is unclear if persistence reflects an adaptive response or a lack thereof. To understand this, transcriptomics data were collected for CTL grown under nutrient-replete and nutrient-starved conditions. Applying K-means clustering on transcriptomics data revealed a global transcriptomic rewiring of CTL under stress conditions in the absence of any canonical global stress regulator. This is consistent with previous data that suggested that CTL's stress response is due to a lack of an adaptive response mechanism. To investigate the impact of this on CTL metabolism, we reconstructed a genome-scale metabolic model of CTL (iCTL278) and contextualized it with the collected transcriptomics data. Using the metabolic bottleneck analysis on contextualized iCTL278, we observed that phosphoglycerate mutase (pgm) regulates the entry of CTL to the persistence state. Our data indicate that pgm has the highest thermodynamics driving force and lowest enzymatic cost. Furthermore, CRISPRi-driven knockdown of pgm in the presence or absence of tryptophan revealed the importance of this gene in modulating persistence. Hence, this work, for the first time, introduces thermodynamics and enzyme cost as tools to gain a deeper understanding on CTL persistence. IMPORTANCE: This study uses a metabolic model to investigate factors that contribute to the persistence of Chlamydia trachomatis serovar L2 (CTL) under tryptophan and iron starvation conditions. As CTL lacks many canonical transcriptional regulators, the model was used to assess two prevailing hypotheses on persistence-that the chlamydial response to nutrient starvation represents a passive response due to the lack of regulators or that it is an active response by the bacterium. K-means clustering of stress-induced transcriptomics data revealed striking evidence in favor of the lack of adaptive (i.e., a passive) response. To find the metabolic signature of this, metabolic modeling pin-pointed pgm as a potential regulator of persistence. Thermodynamic driving force, enzyme cost, and CRISPRi knockdown of pgm supported this finding. Overall, this work introduces thermodynamic driving force and enzyme cost as a tool to understand chlamydial persistence, demonstrating how systems biology-guided CRISPRi can unravel complex bacterial phenomena.

Exploiting Nonclassical Motion of a Trapped Ion Crystal for Quantum-Enhanced Metrology of Global and Differential Spin Rotations.

We theoretically investigate prospects for the creation of nonclassical spin states in trapped ion arrays by coupling to a squeezed state of the collective motion of the ions. The correlations of the generated spin states can be tailored for quantum-enhanced sensing of global or differential rotations of subensembles of the spins by working with specific vibrational modes of the ion array. We propose a pair of protocols to utilize the generated states and demonstrate their viability even for small systems, while assessing limitations imposed by spin-motion entanglement and technical noise. Our work suggests new opportunities for the preparation of many-body states with tailored correlations for quantum-enhanced metrology in spin-boson systems.

Multispecies mass mortality in the Beagle Channel associated with paralytic shellfish toxins.

The Beagle Channel is a Subantarctic semi-estuarine environment at the southern tip of South America, where intoxication events associated with harmful algal blooms have been reported since 1886, including a world record in toxicity due to Alexandrium catenella in 1992. Toxic algae affect public health and ecosystem services, particularly mussel aquaculture and fisheries management. During the austral summer of 2022, an intense bloom of A. catenella (5 × 104 cells L-1) occurred in the Beagle Channel, leading to the second most toxic event in the area, with mussel toxicity reaching 197,266 µg STXeq kg-1. This event was synchronous with the mortality of marine organisms from different trophic levels and terrestrial fauna, i.e., two Fuegian red foxes and a southern caracara. Stomach content and liver samples from dead kelp gulls (Larus dominicanus), Magellanic penguins (Spheniscus magellanicus), papua penguins (Pygoscelis papua), and imperial cormorants (Leucocarbo atriceps), presented variable paralytic shellfish toxins (PST) levels (up to 3427 µg STXeq kg-1) as measured by high performance liquid chromatography (HPLC), suggesting that deaths were associated with high PST toxicity level. The different toxin profiles found in phytoplankton, zooplankton, squat lobsters (Grimothea gregaria), Fuegian sprat (Sprattus fuegensis), and seabirds evidenced possible toxin transformation along the food web and the possible transfer vectors. The unexpected detection of PST in terrestrial fauna (up to 2707 µg STXeq kg-1) suggested intoxication by scavenging on squat lobsters, which had high toxicity (26,663 µg STXeq kg-1). PST trace levels were also detected in a liver sample of a dead false killer whale (Pseudorca crassidens), an oceanic odontocete stranded on the coast during the bloom. Overall, our results denote the exceptional nature of the toxic, multispecies mortality event and that toxins may propagate to several levels of the food web in this Subantarctic environment.

The asymptotic distribution of the permutation entropy.

Ordinal patterns serve as a robust symbolic transformation technique, enabling the unveiling of latent dynamics within time series data. This methodology involves constructing histograms of patterns, followed by the calculation of both entropy and statistical complexity-an avenue yet to be fully understood in terms of its statistical properties. While asymptotic results can be derived by assuming a multinomial distribution for histogram proportions, the challenge emerges from the non-independence present in the sequence of ordinal patterns. Consequently, the direct application of the multinomial assumption is questionable. This study focuses on the computation of the asymptotic distribution of permutation entropy, considering the inherent patterns' correlation structure. Furthermore, the research delves into a comparative analysis, pitting this distribution against the entropy derived from a multinomial law. We present simulation algorithms for sampling time series with prescribed histograms of patterns and transition probabilities between them. Through this analysis, we better understand the intricacies of ordinal patterns and their statistical attributes.

Dynamin-dependent entry of Chlamydia trachomatis is sequentially regulated by the effectors TarP and TmeA.

Chlamydia invasion of epithelial cells is a pathogen-driven process involving two functionally distinct effectors - TarP and TmeA. They collaborate to promote robust actin dynamics at sites of entry. Here, we extend studies on the molecular mechanism of invasion by implicating the host GTPase dynamin 2 (Dyn2) in the completion of pathogen uptake. Importantly, Dyn2 function is modulated by TarP and TmeA at the levels of recruitment and activation through oligomerization, respectively. TarP-dependent recruitment requires phosphatidylinositol 3-kinase and the small GTPase Rac1, while TmeA has a post-recruitment role related to Dyn2 oligomerization. This is based on the rescue of invasion duration and efficiency in the absence of TmeA by the Dyn2 oligomer-stabilizing small molecule activator Ryngo 1-23. Notably, Dyn2 also regulated turnover of TarP- and TmeA-associated actin networks, with disrupted Dyn2 function resulting in aberrant turnover dynamics, thus establishing the interdependent functional relationship between Dyn2 and the effectors TarP and TmeA.

Men who have sex with men experience low anxiety and few barriers to performing anal self or companion examinations: a qualitative study of the Prevent Anal Cancer Palpation Study.

While rare in the larger population, anal cancer incidence is significantly higher in groups such as sexual minority men and people living with HIV. This qualitative analysis examined participants' experiences and perceptions of barriers to anal self-examination and anal companion examination through interviews completed as a part of a larger clinical trial. Interviews were conducted online with participants (n = 131) within a week of their baseline appointment between January 2020 and October 2021. Content analysis denoted participants' thoughts and perceptions about anal self-examination and anal companion examinations. Of the 131 cisgender men interviewed (mean age 49.9 years, SD 12.7), 92.4% identified as gay, 54.9% identified as white, 22.1% identified as Black, 19.9% identified as Latino, and 44.3% of participants were living with HIV. Participants did not report feelings of excessive anxiety when an abnormality was detected. However, three salient themes emerged as to why participants may not perform an anal self-examination or anal companion examination: (1) physical limitation(s), (2) potential sexualisation of the examination, and (3) level of comfort discussing anal health. Future work must continue to explore methods that not only decrease stigma surrounding anal health but also bolster feelings of accessibility to perform self and couple examinations.

The role of infected epithelial cells in Chlamydia-associated fibrosis.

Ocular, genital, and anogenital infection by the obligate intracellular pathogen Chlamydia trachomatis have been consistently associated with scar-forming sequelae. In cases of chronic or repeated infection of the female genital tract, infection-associated fibrosis of the fallopian tubes can result in ectopic pregnancy or infertility. In light of this urgent concern to public health, the underlying mechanism of C. trachomatis-associated scarring is a topic of ongoing study. Fibrosis is understood to be an outcome of persistent injury and/or dysregulated wound healing, in which an aberrantly activated myofibroblast population mediates hypertrophic remodeling of the basement membrane via deposition of collagens and other components of the extracellular matrix, as well as induction of epithelial cell proliferation via growth factor signaling. Initial study of infection-associated immune cell recruitment and pro-inflammatory signaling have suggested the cellular paradigm of chlamydial pathogenesis, wherein inflammation-associated tissue damage and fibrosis are the indirect result of an immune response to the pathogen initiated by host epithelial cells. However, recent work has revealed more direct routes by which C. trachomatis may induce scarring, such as infection-associated induction of growth factor signaling and pro-fibrotic remodeling of the extracellular matrix. Additionally, C. trachomatis infection has been shown to induce an epithelial-to-mesenchymal transition in host epithelial cells, prompting transdifferentiation into a myofibroblast-like phenotype. In this review, we summarize the field's current understanding of Chlamydia-associated fibrosis, reviewing key new findings and identifying opportunities for further research.

DJ-1 Oncogene as a Potential Diagnostic and Prognostic Biomarker for Head and Neck Cancer.

Background Current methods used to diagnose and prognosticate oropharyngeal cancer have contributed to unfavorable patient survival rates that have not significantly improved for the last several decades. Precision medicine oncology relies on molecular diagnostics and biomarkers to supplement existing methods of detecting and prognosticating cancers. This study evaluated the expression of DJ-1, an oncogene that is implicated in the pathogenesis of oral squamous cell carcinoma (OSCC), the most common type of head and neck cancer, to determine its utility as a diagnostic and prognostic biomarker. Methodology Immunohistochemistry (IHC) was performed on 13 normal oral mucosa tissue samples and 143 OSCC tissue samples of varying histopathological grades. Computer-assisted image analysis was performed using the Aperio ImageScope software from Leica Biosystems (Buffalo Grove, IL), which utilizes an algorithm of positive pixel counting for the quantification of immunoreactivity and the percentage of positive cell staining, generating a histo-score (H-score). The comparisons of the average H-scores of the different groups were made using a two-tailed T-test with P ≤ 0.05 set as the level of significance. Results The study found a significant increase in DJ-1 expression in oral squamous cell carcinoma tissue samples in comparison to the normal oral mucosa tissue samples. Additionally, the study documented a significant upregulation in DJ-1 expression in the OSCC tissue samples with high histopathological grades compared to the OSCC tissue samples with low histopathological grades. Conclusions DJ-1 expression patterns were able to reliably differentiate between oral squamous cell carcinoma and the normal counterpart tissues of the oral mucosa, thereby highlighting its role as a potential diagnostic biomarker. Moreover, DJ-1 expression significantly correlates with the OSCC histological grade, which serves as an indicator of the differentiation status and a predictor of the biological behavior of malignant neoplasms, adding to DJ-1's potential utility as a prognostic biomarker for this common type of head and neck cancer.

Chlamydia trachomatis induces the transcriptional activity of host YAP in a Hippo-independent fashion.

Introduction: The obligate intracellular pathogen Chlamydia trachomatis is the causative agent of the most common bacterial sexually transmitted disease worldwide. While the host response to infection by this pathogen has been well characterized, it remains unclear to what extent host gene expression during infection is the product of Chlamydia-directed modulation of host transcription factors. Methods: To identify transcription factors potentially modulated by Chlamydia during infection, we infected immortalized endocervical epithelial cells (End1/E6E7) with the anogenital C. trachomatis serovar L2, harvesting polyadenylated RNA for bulk RNA-sequencing. Subsequent experiments elucidating the mechanism of infection-mediated YAP activation assayed YAP target gene expression via qRT-PCR, YAP nuclear translocation via quantitative immunofluorescence, and YAP phosphorylation via Western blotting. Results: RNA sequencing of Chlamydia-infected endocervical epithelial cells revealed gene expression consistent with activity of YAP, a transcriptional coactivator implicated in cell proliferation, wound healing, and fibrosis. After confirming induction of YAP target genes during infection, we observed an infection-dependent increase in YAP nuclear translocation sensitive to inhibition of bacterial protein synthesis. While Hippo-mediated phosphoinhibition of YAP at S127 was unaffected by C. trachomatis infection, Hippo-independent phosphorylation at Y357 was increased. Infection did not enhance nuclear translocation of Y357F mutant YAP, illustrating a requirement for phosphorylation at this residue. Pharmacological inhibition of host Src-family kinase activity attenuated YAP Y357 phosphorylation, but not nuclear translocation - which was instead sensitive to inhibition of Abl. Discussion: Our results define a transcriptome-altering mechanism of pathogen-directed YAP activation that bypasses canonical inhibition by the Hippo kinase cascade, with a potential link to chlamydial fibrosis and other advanced disease sequelae. Additional study is required to determine the specific role of infection-associated Y357 phosphorylation and Abl activity in chlamydial induction of YAP.

Characterization of DJ-1, PTEN, and p-Akt as Prognostic Biomarkers in the Progression of Oral Squamous Cell Carcinoma.

Objective Oral cancer has a five-year survival rate of 68%, and the methods used to assess it still rely heavily on morphology. Protein biomarkers can potentially increase the predictive power of histopathological evaluation. This study aims to examine the expression of three closely linked proteins implicated in the pathogenesis of oral squamous cell carcinoma (OSCC); protein deglycase (DJ-1), an oncogene, phosphatase, and tensin homolog (PTEN), a tumor suppressor gene, and phosphorylated protein kinase B (p-Akt), the activated form of a vital serine/threonine kinase, which is involved in the oncogenesis of several human malignancies, throughout the tumor progression steps to establish their potential as prognostic biomarkers. Study design Western blot analysis was carried out using four different cell lines representing the successive steps of OSCC progression, including normal oral keratinocytes, dysplastic oral keratinocytes, locally invasive OSCC, and metastatic OSCC.  Results DJ-1 expression was found to be upregulated gradually throughout the successive steps of OSCC progression from normal to dysplastic to locally invasive to metastatic OSCC. PTEN expression showed an overall opposite trend. Interestingly, a significant downregulation of p-Akt was seen in the locally invasive OSCC cells, although it was followed by a significant increase in p-Akt expression in the metastatic OSCC cell line, which is consistent with the role of p-Akt in the motility and migration of cancer cells. Conclusion This study documented trends in expression patterns of three important signaling molecules, DJ-1, PTEN, and p-Akt, in normal, premalignant, and malignant oral keratinocytes. The oncogenic DJ-1 and tumor suppressor PTEN were expressed in a manner consistent with their respective roles in tumorigenesis, while p-Akt only showed a significant upregulation in the metastatic OSCC cells. Overall, all three proteins exhibited unique trends throughout the progressive stages of OSCC tumor progression, thereby adding to their potential utility as prognostic biomarkers for oral cancer patients.

Reliability of Surgical Apgar Score (SAS) in predicting immediate post-operative extubation and intensive care unit admission after major abdominal surgeries: A single center three-year retrospective study

Background@#Surgical Apgar Score (SAS) is a simple, inexpensive, and readily available ten-point scoring system using patient's parameters which include surgical blood loss, lowest recorded mean arterial pressure (MAP) and lowest intraoperative heart rate in predicting 30-day post-operative morbidities. This study determined the reliability of SAS in predicting immediate post-operative extubation and immediate intensive care unit (ICU) admission among patients who underwent major abdominal surgeries in a tertiary hospital in Iloilo City.@*Methods@#A descriptive retrospective cross- sectional study conducted in a tertiary hospital in Iloilo City included patients aged 19 and above who underwent major abdominal surgery from January 1, 2017 to December 31, 2019, and met the study's inclusion criteria. Purposive sampling was utilized. Demographics, clinical data, intraoperative data, management as well as treatment course, post- operative course and patient outcome were extracted, and data collected were utilized for data processing and analysis. Frequency count, mean and standard deviation were utilized for descriptive statistics; T-test and One-way Analysis of Variance (ANOVA) were utilized to determine statistical difference among groups. Logistic regression analysis was employed to assess association between SAS and immediate extubation and post- op ICU admission. Statistical Package of the Social Sciences (SPSS) software version 23.0 was utilized for statistical computations. A probability level of p<0.05 was utilized to determine statistical significance.@*Results@#The study consisted of 221 patients predominantly female 64.3 % (n=142) with the mean age of 55.80 17.53. Mean SAS was 6.79 $ 1.3 with a total of 13 (5.9%) patients who were classified as high risk (SAS 0-4), 152 (68.8%) patients as medium risk (SAS 5-7) and 56 (25.3%) patients as low risk with SAS 8-10. On logistic regression analysis, mean arterial pressure (MAP), lowest heart rate and estimated blood loss were significantly associated with decision to do immediate post-operative extubation and immediate ICU admission (p<0.001). Those with higher MAP were 1.19 times more likely to be extubated (OR 1.199, CI: 1.078-1.334, p<0.001) and higher estimated blood loss more likely to be admitted in the ICU (OR 1.006, CI: 1.004-1.009, p<0.001). Lastly, those with higher heart rates were 1.2 times more likely to be admitted in the ICU post-operatively. Low-Risk SAS (Score of 8-10) is predictive of immediate post-operative extubation with 97.7% sensitivity and 75.6% specificity. High Risk SAS (score of 0-4) is predictive of immediate post-operative ICU admission with a sensitivity of 76.1% and 98.3% specificity.@*Conclusion@#SAS is a reliable and valid predictive tool in determining immediate post- operative extubation and ICU admission among patients undergoing major abdominal surgeries. Multicentric, longitudinal and prospective studies are further required to confirm results.Keywords: Surgical Apgar Score (SAS), extubation, intensive care, critical care, abdominal surgery

Machine Learning and Metabolic Model Guided CRISPRi Reveals a Central Role for Phosphoglycerate Mutase in Chlamydia trachomatis Persistence.

Upon nutrient starvation, Chlamydia trachomatis serovar L2 (CTL) shifts from its normal growth to a non-replicating form, termed persistence. It is unclear if persistence is an adaptive response or lack of it. To understand that transcriptomics data were collected for nutrient-sufficient and nutrient-starved CTL. Applying machine learning approaches on transcriptomics data revealed a global transcriptomic rewiring of CTL under stress conditions without having any global stress regulator. This indicated that CTL's stress response is due to lack of an adaptive response mechanism. To investigate the impact of this on CTL metabolism, we reconstructed a genome-scale metabolic model of CTL (iCTL278) and contextualized it with the collected transcriptomics data. Using the metabolic bottleneck analysis on contextualized iCTL278, we observed phosphoglycerate mutase (pgm) regulates the entry of CTL to the persistence. Later, pgm was found to have the highest thermodynamics driving force and lowest enzymatic cost. Furthermore, CRISPRi-driven knockdown of pgm and tryptophan starvation experiments revealed the importance of this gene in inducing persistence. Hence, this work, for the first time, introduced thermodynamics and enzyme-cost as tools to gain deeper understanding on CTL persistence.

Statistical properties of the entropy from ordinal patterns.

The ultimate purpose of the statistical analysis of ordinal patterns is to characterize the distribution of the features they induce. In particular, knowing the joint distribution of the pair entropy-statistical complexity for a large class of time series models would allow statistical tests that are unavailable to date. Working in this direction, we characterize the asymptotic distribution of the empirical Shannon's entropy for any model under which the true normalized entropy is neither zero nor one. We obtain the asymptotic distribution from the central limit theorem (assuming large time series), the multivariate delta method, and a third-order correction of its mean value. We discuss the applicability of other results (exact, first-, and second-order corrections) regarding their accuracy and numerical stability. Within a general framework for building test statistics about Shannon's entropy, we present a bilateral test that verifies if there is enough evidence to reject the hypothesis that two signals produce ordinal patterns with the same Shannon's entropy. We applied this bilateral test to the daily maximum temperature time series from three cities (Dublin, Edinburgh, and Miami) and obtained sensible results.