Harnessing Immunomodulatory Polymers for Treatment of Autoimmunity, Allergy, and Transplant Rejection.

Autoimmunity, allergy, and transplant rejection are a collection of chronic diseases that are currently incurable, drastically decrease patient quality of life, and consume considerable health care resources. Underlying each of these diseases is a dysregulated immune system that results in the mounting of an inflammatory response against self or an innocuous antigen. As a consequence, afflicted patients are required to adhere to lifelong regimens of multiple immunomodulatory drugs to control disease and reclaim agency. Unfortunately, current immunomodulatory drugs are associated with a myriad of side effects and adverse events, such as increased risk of cancer and increased risk of serious infection, which negatively impacts patient adherence rates and quality of life. The field of immunoengineering is a new discipline that aims to harness endogenous biological pathways to thwart disease and minimize side effects using novel biomaterial-based strategies. We highlight and discuss polymeric micro/nanoparticles with inherent immunomodulatory properties that are currently under investigation in biomaterial-based therapies for treatment of autoimmunity, allergy, and transplant rejection.

APLpred: A machine learning-based tool for accurate prediction and characterization of asparagine peptide lyases using sequence-derived optimal features.

Asparagine peptide lyase (APL) is among the seven groups of proteases, also known as proteolytic enzymes, which are classified according to their catalytic residue. APLs are synthesized as precursors or propeptides that undergo self-cleavage through autoproteolytic reaction. At present, APLs are grouped into 10 families belonging to six different clans of proteases. Recognizing their critical roles in many biological processes including virus maturation, and virulence, accurate identification and characterization of APLs is indispensable. Experimental identification and characterization of APLs is laborious and time-consuming. Here, we developed APLpred, a novel support vector machine (SVM) based predictor that can predict APLs from the primary sequences. APLpred was developed using Boruta-based optimal features derived from seven encodings and subsequently trained using five machine learning algorithms. After evaluating each model on an independent dataset, we selected APLpred (an SVM-based model) due to its consistent performance during cross-validation and independent evaluation. We anticipate APLpred will be an effective tool for identifying APLs. This could aid in designing inhibitors against these enzymes and exploring their functions. The APLpred web server is freely available at https://procarb.org/APLpred/.

Efficacy of Probiotic Strains Lactobacillus sakei Probio65 and Lactobacillus plantarum Probio-093 in Management of Obesity: An In Vitro and In Vivo Analysis.

The prevalence of obesity, characterized by an excessive accumulation of adipose tissue and adipocyte hypertrophy, presents a major public health challenge. This study investigates the therapeutic potential of two probiotic strains, Lactobacillus sakei Probio65 and Lactobacillus plantarum Probio-093, in the context of obesity. Utilizing 3T3-L1 cell-derived human adipocytes, we assessed Probio65's and Probio-093's capacity to mitigate triglyceride accumulation and influence adipocytokine production in vitro. Subsequently, an in vivo trial with male C57BL/6J mice examined the effects of both probiotic strains on adipose tissue characteristics, body weight, fat mass, and obesity-related gene expression. This study employed both live and ethanol-extracted bacterial cells. The results demonstrated significant reductions in the triglyceride deposition, body weight, and adipose tissue mass in the treated groups (p < 0.05). Furthermore, both strains modulated adipokine profiles by downregulating proinflammatory markers such as PAI-1, leptin, TNF-α, STAMP2, F4/80, resistin, and MCP-1, and upregulating the insulin-sensitive transporter GLUT4 and the anti-inflammatory adiponectin (p < 0.05). Our findings suggest that Lactobacillus sakei Probio65 and Lactobacillus plantarum Probio-093 are promising agents for microbiome-targeted anti-obesity therapies, offering the effective mitigation of obesity and improvement in adipocyte function in a murine model.

Comparison of Short-Term Health Care Utilization Between Telemedicine-Delivered vs In-Person Care Visits for Heart Failure.

Background: Patients with heart failure (HF) are a medically complex population with frequent hospitalizations. Downstream health care utilization following primary care delivered by telemedicine compared to in-person is unknown. Objectives: The purpose of this study was to understand differences in return in-person visits, emergency department (ED) encounters, and hospitalizations following a telemedicine vs an in-person primary care visit for patients with HF seen for a HF-related complaint. Methods: This was an observational study of all primary care visits for HF from January 1, 2022, to December 31, 2022, in an integrated health care delivery system. We compared 7-day in-person follow-up visits, ED visits, and hospitalizations (all-cause and HF-specific) by index visit type. Results: We included 3,902 primary care visits with a primary diagnosis of HF. Most visits utilized telephone or video visits (58.4% total; 44.9% telephone, 13.5% video). After adjustment, telephone visits were associated with more in-person follow-up visits (6.14% vs 4.20%; adjusted OR: 1.08-2.21; P < 0.05) but fewer ED visits (6.12% vs 8.07%; adjusted OR: 0.55-0.97; P < 0.05) compared to in-person visits. Most hospitalized patients (74%) had an admitting diagnosis of HF. There was no difference between 7-day all-cause hospitalization following telephone or video visits compared to in-person visits. Conclusions: Most patients used telemedicine to address HF-specific primary care concerns. Telephone visits were associated with slightly higher short-term in-person primary care follow-up but lower ED utilization. Overall, downstream ED visits and hospitalizations were low. Telephone and video visits appear to offer safe alternatives to in-person care for HF-related primary care and are a promising health care delivery strategy.

Environmental Pollution and Cardiovascular Disease: Part 2 of 2: Soil, Water, and Other Forms of Pollution.

With a growing body of evidence that now links environmental pollution to adverse cardiovascular disease (CVD) outcomes, pollution has emerged as an important risk factor for CVD. There is thus an urgent need to better understand the role of pollution in CVD, key pathophysiological mechanisms, and to raise awareness among health care providers, the scientific community, the general population, and regulatory authorities about the CV impact of pollution and strategies to reduce it. This article is part 2 of a 2-part state-of-the-art review on the topic of pollution and CVD-herein we discuss major environmental pollutants and their effects on CVD, highlighting pathophysiological mechanisms, and strategies to reduce CVD risk.

Environmental Pollution and Cardiovascular Disease: Part 1 of 2: Air Pollution.

Cardiovascular disease (CVD) is the leading cause of morbidity and mortality worldwide. Over the past 50 years, there has been a substantial decline in the incidence of CVD and related mortality in high-income countries, largely due to the mitigation of modifiable risk factors such as smoking, hypertension, and diabetes. However, a significant burden of CVD remains in low- to middle-income countries, despite their lower prevalence of traditional risk factors; other environmental factors, particularly pollution, play a significant role in this attributable risk. Mounting evidence underscores a strong association between pollution and adverse health effects, including CVD. This article is part 1 of a 2-part state-of-the-art review and discusses air pollution and its adverse effects on CVD, highlighting pathophysiological mechanisms and methods to reduce air pollution and exposure to these pollutants.

Engineering antigen-presenting cells for immunotherapy of autoimmunity.

Autoimmune diseases are burdensome conditions that affect a significant fraction of the global population. The hallmark of autoimmune disease is a host's immune system being licensed to attack its tissues based on specific antigens. There are no cures for autoimmune diseases. The current clinical standard for treating autoimmune diseases is the administration of immunosuppressants, which weaken the immune system and reduce auto-inflammatory responses. However, people living with autoimmune diseases are subject to toxicity, fail to mount a sufficient immune response to protect against pathogens, and are more likely to develop infections. Therefore, there is a concerted effort to develop more effective means of targeting immunomodulatory therapies to antigen-presenting cells, which are involved in modulating the immune responses to specific antigens. In this review, we highlight approaches that are currently in development to target antigen-presenting cells and improve therapeutic outcomes in autoimmune diseases.

Emergency Department Referral of Patients With Chest Pain for Noninvasive Cardiac Testing and 2-Year Clinical Outcomes.

BACKGROUND: Noninvasive cardiac testing (NICT) has been associated with decreased long-term risks of major adverse cardiac events (MACEs) among emergency department patients at high coronary risk. It is unclear whether this association extends to patients without evidence of myocardial injury on initial ECG and cardiac troponin testing. METHODS: A retrospective cohort study was conducted of patients presenting with chest pain between 2013 and 2019 to 21 emergency departments within an integrated health care system in Northern California, excluding patients with ST-segment-elevation myocardial infarction or myocardial injury by serum troponin testing. To account for confounding by indication, we grouped patient encounters by the NICT referral rate of the initially assigned emergency physician relative to local peers within discrete time periods. The primary outcome was MACE within 2 years. Secondary outcomes were coronary revascularization and MACE, inclusive of all-cause mortality. Associations between the NICT referral group (low, intermediate, or high) and outcomes were assessed using risk-adjusted proportional hazards methods with censoring for competing events. RESULTS: Among 144 577 eligible patient encounters, the median age was 58 years (interquartile range, 48-68) and 57% were female. Thirty-day NICT referral was 13.0%, 19.9%, and 27.8% in low, intermediate, and high NICT referral groups, respectively, with a good balance of baseline covariates between groups. Compared with the low NICT referral group, there was no significant decrease in the adjusted hazard ratio of MACE within the intermediate (adjusted hazard ratio, 1.08 [95% CI, 1.02-1.14]) or high (adjusted hazard ratio, 1.05 [95% CI, 0.99-1.11]) NICT referral groups. Results were similar for MACE, inclusive of all-cause mortality, and coronary revascularization, as well as subgroup analyses stratified by estimated risk (history, electrocardiogram, age, risk factors, troponin [HEART] score: percent classified as low risk, 48.2%; moderate risk, 49.2%; and high risk, 2.7%). CONCLUSIONS: Increases in NICT referrals were not associated with changes in the hazard of MACE within 2 years following emergency department visits for chest pain without evidence of acute myocardial injury. These findings further highlight the need for evidence-based guidance regarding the appropriate use of NICT in this population.

Computational prediction of phosphorylation sites of SARS-CoV-2 infection using feature fusion and optimization strategies.

SARS-CoV-2's global spread has instigated a critical health and economic emergency, impacting countless individuals. Understanding the virus's phosphorylation sites is vital to unravel the molecular intricacies of the infection and subsequent changes in host cellular processes. Several computational methods have been proposed to identify phosphorylation sites, typically focusing on specific residue (S/T) or Y phosphorylation sites. Unfortunately, current predictive tools perform best on these specific residues and may not extend their efficacy to other residues, emphasizing the urgent need for enhanced methodologies. In this study, we developed a novel predictor that integrated all the residues (STY) phosphorylation sites information. We extracted ten different feature descriptors, primarily derived from composition, evolutionary, and position-specific information, and assessed their discriminative power through five classifiers. Our results indicated that Light Gradient Boosting (LGB) showed superior performance, and five descriptors displayed excellent discriminative capabilities. Subsequently, we identified the top two integrated features have high discriminative capability and trained with LGB to develop the final prediction model, LGB-IPs. The proposed approach shows an excellent performance on 10-fold cross-validation with an ACC, MCC, and AUC values of 0.831, 0.662, 0.907, respectively. Notably, these performances are replicated in the independent evaluation. Consequently, our approach may provide valuable insights into the phosphorylation mechanisms in SARS-CoV-2 infection for biomedical researchers.

Diet quality and cardiovascular disease risk among breast cancer survivors in the Pathways Study.

BACKGROUND: Women with breast cancer are at higher risk of cardiovascular disease (CVD) compared with women without breast cancer. Whether higher diet quality at breast cancer diagnosis lowers this risk remains unknown. We set out to determine if higher diet quality at breast cancer diagnosis was related to lower risk of CVD and CVD-related death. METHODS: This analysis included 3415 participants from the Pathway Study, a prospective cohort of women diagnosed with invasive breast cancer at Kaiser Permanente Northern California between 2005 and 2013 and followed through December 31, 2021. Scores from 5 diet quality indices consistent with healthy eating were obtained at the time of breast cancer diagnosis. Scores were categorized into ascending quartiles of concordance for each diet quality index, and multivariable adjusted hazard ratios (HRs) and 95% confidence intervals (CIs) were estimated. P values were 2-sided. RESULTS: The Dietary Approaches to Stop Hypertension diet quality index was associated with lower risk of heart failure (HR = 0.53, 95% CI = 0.33 to 0.87; Ptrend = .03), arrhythmia (HR = 0.77, 95% CI = 0.62 to 0.94; Ptrend = .008), cardiac arrest (HR = 0.77, 95% CI = 0.61 to 0.96; Ptrend = .02), valvular heart disease (HR = 0.79, 95% CI = 0.64 to 0.98; Ptrend = .046), venous thromboembolic disease (HR = 0.75, 95% CI = 0.60 to 0.93; Ptrend = .01), and CVD-related death (HR = 0.70, 95% CI = 0.50 to 0.99; Ptrend = .04), when comparing the highest with lowest quartiles. Inverse associations were also found between the healthy plant-based dietary index and heart failure (HR = 0.60, 95% CI = 0.39 to 0.94; Ptrend = .02), as well as the alternate Mediterranean dietary index and arrhythmia (HR = 0.74, 95% CI = 0.60 to 0.93; Ptrend = .02). CONCLUSION: Among newly diagnosed breast cancer patients, higher diet quality at diagnosis was associated with lower risk of CVD events and death.

UHRF1 poly-auto-ubiquitination induced by the anti-cancer drug, thymoquinone, is involved in the DNA repair machinery recruitment.

DNA methylation is one of the most important epigenetic mark involved in many physiologic cellular processes and pathologies. During mitosis, the transmission of DNA methylation patterns from a mother to the daughter cells is ensured through the action of the Ubiquitin-like, containing PHD and RING domains, 1/DNA methyltransferase 1 (UHRF1/DNMT1) tandem. UHRF1 is involved in the silencing of many tumor suppressor genes (TSGs) via mechanisms that remain largely to be deciphered. The present study investigated the role and the regulation of UHRF1 poly-ubiquitination induced by thymoquinone, a natural anti-cancer drug, known to enhance or re-activate the expression of TSGs. We found that the auto-ubiquitination of UHRF1, induced by TQ, is mediated by reactive oxygen species, and occurs following DNA damage. We demonstrated that the poly-ubiquitinated form of UHRF1 is K63-linked and can still silence the tumor suppressor gene p16INK4A/CDKN2A. We further showed that TQ-induced auto-ubiquitination is mediated via the activity of Tip60. Since this latter is known as a nuclear receptor co-factor, we investigated if the glucocorticoid receptor (GR) might be involved in the regulation of UHRF1 ubiquitination. Activation of the GR, with dexamethasone, did not influence auto-ubiquitination of UHRF1. However, we could observe that TQ induced a K48-linked poly-ubiquitination of GR, probably involved in the proteosomal degradation pathway. Mass-spectrometry analysis of FLAG-HA-tagged UHRF1 identified UHRF1 partners involved in DNA repair and showed that TQ increased their association with UHRF1, suggesting that poly-ubiquitination of UHRF1 is involved in the DNA repair process. We propose that poly-ubiquitination of UHRF1 serves as a scaffold to recruit the DNA repair machinery at DNA damage sites.

U.S. Filipino Adults Have Elevated Prevalence of Hypertension Across the Adult Lifespan: Findings From a Cross-Sectional Electronic Health Record Study.

Introduction: The prevalence of hypertension increases with age and differs by race and ethnicity. Among U.S. Asian adults, prevalence is higher for Filipino adults than for other major Asian subgroups, but whether this disparity exists across the adult lifespan is unknown. This study examined hypertension prevalence by age decade, comparing Filipino adults with South Asian, Chinese, Black, Hispanic, and White adults. Methods: This cross-sectional study used 2015-2016 electronic health record data from a Northern California integrated healthcare delivery system for 1,839,603 adults aged 30-79 years, including 128,124 Filipino adults. Hypertension was defined by diagnosis codes. Sex-specific prevalence was calculated by race and ethnicity overall and by 10-year age decade from ages 30-39 years to 70-79 years. The prevalence of hypertension among 5 racial and ethnic groups was compared within each decade (with Filipino as the reference), adjusting for age, English language, diabetes, smoking, and weight category. Results: Decade-specific prevalence of hypertension among Filipino men and women, respectively, was 9.7% and 8.5% for ages 30-39 years, 26.0% and 23.9% for ages 40-49 years, 45.9% and 44.4% for ages 50-59 years, 65.4% and 63.9% for ages 60-69 years, and 82.1% and 82.9% for ages 70-79 years. Across all age decades, hypertension prevalence among Filipino adults largely tracked with Black adults and was much higher than among South Asian, Chinese, White, and Hispanic adults. This pattern remained after adjusting for covariates, with the largest differences observed for adults aged <60 years. Conclusions: Similar to Black adults, Filipino adults have persistently higher hypertension prevalence than South Asian, Chinese, Hispanic, and White adults across the adult lifespan. These findings underscore the importance of surveillance and prevention efforts for this high-risk Asian group beginning in early adulthood.

Polygenic risk and incident coronary heart disease in a large multiethnic cohort.

Objective: Many studies support the notion that polygenic risk scores (PRS) improve risk prediction for coronary heart disease (CHD) beyond conventional risk factors. However, PRS are not yet considered risk-enhancing factor in guidelines. Our objective was to determine the predictive performance of a commercially available PRS (CARDIO inCode-Score®) compared with the Pooled Cohorts Equations (PCE) in a contemporary, multi-ethnic cohort. Methods: Participants (n = 63,070; 67 % female; 18 % non-European) without prior CHD were followed from 2007 through 12/31/2022. The association between the PRS and incident CHD was assessed using Cox regression adjusting for genetic ancestry and risk factors. Event rates were estimated by categories of PCE and by low/intermediate/high genetic risk within PCE categories; risk discrimination and net reclassification improvement (NRI) were also assessed. Results: There were 3,289 incident CHD events during 14 years of follow-up. Adjusted hazard ratio (aHR) for incident CHD per 1 SD increase in PRS was 1.18 (95 % CI:1.14-1.22), and the aHR for the upper vs lower quintile of the PRS was 1.66 (95 % CI:1.49-1.86). The association was consistent in both sexes, in European participants compared with all minority groups combined and was strongest in the first 5 years of follow-up. The increase in the C-statistic was 0.004 (0.747 vs. 0.751; p < 0.0001); the NRI was 2.4 (0.9-3.8) for the entire cohort and 9.7 (7.5-12.0) for intermediate PCE risk individuals. After incorporating high genetic risk, a further 10 percent of participants at borderline/intermediate PCE risk would be candidates for statin therapy. Conclusion: Inclusion of polygenic risk improved identification of primary prevention individuals who may benefit from more intensive risk factor modification.

Synthesis of biocomposites from microalgal peptide incorporated polycaprolactone/ κ- carrageenan nanofibers and their antibacterial and wound healing property.

Antimicrobial peptides (AMPs) are promising novel agents for targeting a wide range of pathogens. In this study, microalgal peptides derived from native microalgae were incorporated into polycaprolactone (PCL) with ƙ-Carrageenan (ƙ-C) forming nanofibers using the electrospinning method. The peptides incorporated in the nanofibers were characterized by fourier infrared spectroscopy, thermogravimetric analysis, scanning electron microscopy (SEM), and contact angle measurement. The results showed that peptides with molecular weights < 10 kDa, when loaded into nanofibers, exhibited lower wettability. The SEM analysis revealed a thin, smooth, interconnected bead-like structures. The antimicrobial activity of the electrospun nanofibers was evaluated through disc diffusion, and minimum inhibitory activity against Escherichia coli (MTTC 443), and Staphylococcus aureus (MTTC 96), resulting in zones of inhibition of 24 ± 0.5 mm and 14 ± 0.5 mm, respectively. The in vitro biocompatibility of the synthesized nanofibers was confirmed using in HEK 293 cell lines with an increased cell viability. Interestingly, the fibers also exhibited a significant wound-healing properties when used in vitro scratch assays. In conclusion, algal peptides incorporated with PCL/ ƙ-C were found to exhibit antimicrobial and biocompatible biomaterials for wound healing applications.

Identification and Characterization of HIRIP3 as a Histone H2A Chaperone.

HIRIP3 is a mammalian protein homologous to the yeast H2A.Z deposition chaperone Chz1. However, the structural basis underlying Chz's binding preference for H2A.Z over H2A, as well as the mechanism through which Chz1 modulates histone deposition or replacement, remains enigmatic. In this study, we aimed to characterize the function of HIRIP3 and to identify its interacting partners in HeLa cells. Our findings reveal that HIRIP3 is specifically associated in vivo with H2A-H2B dimers and CK2 kinase. While bacterially expressed HIRIP3 exhibited a similar binding affinity towards H2A and H2A.Z, the associated CK2 kinase showed a notable preference for H2A phosphorylation at serine 1. The recombinant HIRIP3 physically interacted with the H2A αC helix through an extended CHZ domain and played a crucial role in depositing the canonical core histones onto naked DNA. Our results demonstrate that mammalian HIRIP3 acts as an H2A histone chaperone, assisting in its selective phosphorylation by Ck2 kinase at serine 1 and facilitating its deposition onto chromatin.

Lack of leisure time physical activity and variations in cardiovascular mortality across US communities: a comprehensive county-level analysis (2011-2019).

OBJECTIVES: To investigate the associations between county-level proportions of adults not engaging in leisure-time physical activity (no LTPA) and age-adjusted cardiovascular mortality (AACVM) rates in the overall US population and across demographics. METHODS: Analysing 2900 US counties from 2011 to 2019, we used the Centers for Disease Control and Prevention (CDC) databases to obtain annual AACVM rates. No LTPA data were sourced from the CDC's Behavioural Risk Factor Surveillance System survey and county-specific rates were calculated using a validated multilevel regression and poststratification modelling approach. Multiple regression models assessed associations with county characteristics such as socioeconomic, environmental, clinical and healthcare access factors. Poisson generalised linear mixed models were employed to calculate incidence rate ratios (IRR) and additional yearly deaths (AYD) per 100 000 persons. RESULTS: Of 309.9 million residents in 2900 counties in 2011, 7.38 million (2.4%) cardiovascular deaths occurred by 2019. County attributes such as socioeconomic, environmental and clinical factors accounted for up to 65% (adjusted R2=0.65) of variance in no LTPA rates. No LTPA rates associated with higher AACVM across demographics, notably among middle-aged adults (standardised IRR: 1.06; 95% CI (1.04 to 1.07)), particularly women (1.09; 95% CI (1.07 to 1.12)). The highest AYDs were among elderly non-Hispanic black individuals (AYD=68/100 000). CONCLUSIONS: Our study reveals a robust association between the high prevalence of no LTPA and elevated AACVM rates beyond other social determinants. The most at-risk groups were middle-aged women and elderly non-Hispanic black individuals. Further, county-level characteristics accounted for substantial variance in community LTPA rates. These results emphasise the need for targeted public health measures to boost physical activity, especially in high-risk communities, to reduce AACVM.

TV Viewing From Young Adulthood to Middle Age and Cardiovascular Disease Risk.

INTRODUCTION: Few studies have longitudinally examined TV viewing trajectories and cardiovascular disease risk factors. The objective of this study was to determine the association between level and annualized changes in young adult TV viewing and the incidence of cardiovascular disease risk factors from young adulthood to middle age. METHODS: In 2023, prospective community-based cohort data of 4,318 Coronary Artery Risk Development in Young Adults study participants (1990-1991 to 2015-2016) were analyzed. Individualized daily TV viewing trajectories for each participant were developed using linear mixed models. RESULTS: Every additional hour of TV viewing at age 23 years was associated with higher odds of incident hypertension (AOR=1.16; 95% CI=1.11, 1.22), diabetes (AOR=1.19; 95% CI=1.11, 1.28), high triglycerides (AOR=1.17; 95% CI=1.08, 1.26), dyslipidemia (AOR=1.10; 95% CI=1.03, 1.16), and obesity (AOR=1.12; 95% CI=1.06, 1.17). In addition, each hourly increase in daily TV viewing was associated with higher annual odds of incident hypertension (AOR=1.26; 95% CI=1.16, 1.37), low high-density lipoprotein cholesterol (AOR=1.15; 95% CI=1.03, 1.30), high triglycerides (AOR=1.32; 95% CI=1.15, 1.51), dyslipidemia (AOR=1.22; 95% CI=1.11, 1.34), and obesity (AOR=1.17; 95% CI=1.07, 1.27) over the follow-up period. CONCLUSIONS: In this prospective cohort study, higher TV viewing in young adulthood and annual increases in TV viewing were associated with incident hypertension, high triglycerides, and obesity. Young adulthood as well as behaviors across midlife may be important time periods to promote healthful TV viewing behavior patterns.

A Multi-Fluorophore Staining Scheme for Identification and Quantification of Vomocytosis.

Vomocytosis is a process by which fungal pathogens, for instance, Cryptococcus neoformans (CN), escape from the digestive phagolysosome of phagocytic cells after ingestion. Interestingly, this expulsion leaves both the pathogen and phagocyte unharmed, and is believed to be an important mechanism by which CNs disseminate throughout infected hosts. This phenomenon was discovered in 2006, and research to date has relied almost entirely on quantification via manual counting of vomocytosis events in time-lapse microscopy videos. This archaic method has the significant disadvantages of requiring excessive labor in manual analysis, limited throughput capabilities, and low accuracy due to subjectivity. Here, we present an alternative method to measure vomocytosis rates using a multi-fluorophore reporter system comprised of two in situ staining steps during infection and a flow cytometry readout. This approach overcomes the limitations of conventional time lapse microscopy methods, with key advantages of high throughput capability, simple procedural steps, and accurate objective readouts. This study rigorously characterizes this vomocytosis reporter system in CN-infected MΦ and DC cultures via fluorescence microscopy, confocal microscopy, and flow cytometry. Here, this fluorescent tool is used to observe differences in expulsion rates after phagosome-modifying drug treatments and additionally utilized to distinguish differences in biochemical compositions among fluorescence-activated cell sorted fungal populations via Raman spectroscopy. Furthermore, this reporter scheme is demonstrated to be adaptable for use in measuring potential biomaterial particle expulsion events. Ultimately, the fluorescent reporter system presented here provides a universal tool for vomocytosis rate measurement of phagocytosed material. This facile approach opens the door to previously unfeasible types of vomocytosis-related studies such as high throughput treatment mechanistic screening and downstream characterization of expelled material.

An Image Processing Algorithm for Facile and Reproducible Quantification of Vomocytosis.

Vomocytosis is a process that occurs when internalized fungal pathogens escape from phagocytes without compromising the viability of the pathogen and the host cell. Manual quantification of time-lapse microscopy videos is currently used as the standard to study pathogen behavior and vomocytosis incidence. However, human-driven quantification of vomocytosis (and the closely related phenomenon, exocytosis) is incredibly burdensome, especially when a large volume of cells and interactions needs to be analyzed. In this study, we designed a MATLAB algorithm that measures the extent of colocalization between the phagocyte and fungal cell (Cryptococcus neoformans; CN) and rapidly reports the occurrence of vomocytosis in a high throughput manner. Our code processes multichannel, time-lapse microscopy videos of cocultured CN and immune cells that have each been fluorescently stained with unique dyes and provides quantitative readouts of the spatiotemporally dynamic process that is vomocytosis. This study also explored metrics, such as the rate of change of pathogen colocalization with the host cell, that could potentially be used to predict vomocytosis occurrence based on the quantitative data collected. Ultimately, the algorithm quantifies vomocytosis events and reduces the time for video analysis from over 1 h to just 10 min, a reduction in labor of 83%, while simultaneously minimizing human error. This tool significantly minimizes the vomocytosis analysis pipeline, accelerates our ability to elucidate unstudied aspects of this phenomenon, and expedites our ability to characterize CN strains for the study of their epidemiology and virulence.

MBD4 loss results in global reactivation of promoters and retroelements with low methylated CpG density.

BACKGROUND: Inherited defects in the base-excision repair gene MBD4 predispose individuals to adenomatous polyposis and colorectal cancer, which is characterized by an accumulation of C > T transitions resulting from spontaneous deamination of 5'-methylcytosine. METHODS: Here, we have investigated the potential role of MBD4 in regulating DNA methylation levels using genome-wide transcriptome and methylome analyses. Additionally, we have elucidated its function through a series of in vitro experiments. RESULTS: Here we show that the protein MBD4 is required for DNA methylation maintenance and G/T mismatch repair. Transcriptome and methylome analyses reveal a genome-wide hypomethylation of promoters, gene bodies and repetitive elements in the absence of MBD4 in vivo. Methylation mark loss is accompanied by a broad transcriptional derepression phenotype affecting promoters and retroelements with low methylated CpG density. MBD4 in vivo forms a complex with the mismatch repair proteins (MMR), which exhibits high bi-functional glycosylase/AP-lyase endonuclease specific activity towards methylated DNA substrates containing a G/T mismatch. Experiments using recombinant proteins reveal that the association of MBD4 with the MMR protein MLH1 is required for this activity. CONCLUSIONS: Our data identify MBD4 as an enzyme specifically designed to repair deaminated 5-methylcytosines and underscores its critical role in safeguarding against methylation damage. Furthermore, it illustrates how MBD4 functions in normal and pathological conditions.