Subclinical atherosclerosis and accelerated epigenetic age mediated by inflammation: a multi-omics study.

AIMS: Epigenetic age is emerging as a personalized and accurate predictor of biological age. The aim of this article is to assess the association of subclinical atherosclerosis with accelerated epigenetic age and to investigate the underlying mechanisms mediating this association. METHODS AND RESULTS: Whole blood methylomics, transcriptomics, and plasma proteomics were obtained for 391 participants of the Progression of Early Subclinical Atherosclerosis study. Epigenetic age was calculated from methylomics data for each participant. Its divergence from chronological age is termed epigenetic age acceleration. Subclinical atherosclerosis burden was estimated by multi-territory 2D/3D vascular ultrasound and by coronary artery calcification. In healthy individuals, the presence, extension, and progression of subclinical atherosclerosis were associated with a significant acceleration of the Grim epigenetic age, a predictor of health and lifespan, regardless of traditional cardiovascular risk factors. Individuals with an accelerated Grim epigenetic age were characterized by an increased systemic inflammation and associated with a score of low-grade, chronic inflammation. Mediation analysis using transcriptomics and proteomics data revealed key pro-inflammatory pathways (IL6, Inflammasome, and IL10) and genes (IL1B, OSM, TLR5, and CD14) mediating the association between subclinical atherosclerosis and epigenetic age acceleration. CONCLUSION: The presence, extension, and progression of subclinical atherosclerosis in middle-aged asymptomatic individuals are associated with an acceleration in the Grim epigenetic age. Mediation analysis using transcriptomics and proteomics data suggests a key role of systemic inflammation in this association, reinforcing the relevance of interventions on inflammation to prevent cardiovascular disease.

Aging-Associated miR-217 Aggravates Atherosclerosis and Promotes Cardiovascular Dysfunction.

OBJECTIVE: microRNAs are master regulators of gene expression with essential roles in virtually all biological processes. miR-217 has been associated with aging and cellular senescence, but its role in vascular disease is not understood. Approach and Results: We have used an inducible endothelium-specific knock-in mouse model to address the role of miR-217 in vascular function and atherosclerosis. miR-217 reduced NO production and promoted endothelial dysfunction, increased blood pressure, and exacerbated atherosclerosis in proatherogenic apoE-/- mice. Moreover, increased endothelial miR-217 expression led to the development of coronary artery disease and altered left ventricular heart function, inducing diastolic and systolic dysfunction. Conversely, inhibition of endogenous vascular miR-217 in apoE-/- mice improved vascular contractility and diminished atherosclerosis. Transcriptome analysis revealed that miR-217 regulates an endothelial signaling hub and downregulates a network of eNOS (endothelial NO synthase) activators, including VEGF (vascular endothelial growth factor) and apelin receptor pathways, resulting in diminished eNOS expression. Further analysis revealed that human plasma miR-217 is a biomarker of vascular aging and cardiovascular risk. CONCLUSIONS: Our results highlight the therapeutic potential of miR-217 inhibitors in aging-related cardiovascular disease.

Cycle length of nonsustanied ventricular tachycardias among ICD patients: implications on subsequent appropriate therapies.

BACKGROUND: ICD patients with episodes of nonsustained ventricular tachycardias (NSVT) are at risk of appropriate therapies. However, the relationship between the cycle length (CL) of such NSVTs and the subsequent incidence of appropriate interventions is unknown. METHODS: 416 ICD patients with LVEF < 45% were studied. ICD programming was standardized. NSVT was defined as any VT of 5 or more beats at ≥ 150 bpm occurred in the first 6 months after implantation that terminated spontaneously and was not preceded by any appropriate therapy. The mean follow-up was 41 ± 27 months. RESULTS: We analyzed 2201 NSVTs (mean CL = 323 ms) that occurred in 250 patients; 111 of such episodes were fast (CL ≤ 300 ms). Secondary prevention (HR = 1.7; p < 0.001), number of NSVT episodes (HR = 1.05; 95% CI 1.04-1.07; p < 0.001) and beta-blocker treatment (HR = 0.7; p = 0.04) were independent predictors of appropriate interventions; however, the mean CL of NSVTs was not (p = 0.6). There was a correlation between the mean CL of NSVTs and the CL of the first monomorphic VT: r = 0.88; p < 0.001. This correlation was especially robust in individuals with > 5 NSVTs (r = 0.97; p < 0.001), with an agreement between both values greater than 95%. Patients with any fast NSVT experienced a higher incidence of VF episodes (26%) compared to those without NVSTs (3%) or with only slow NSVTs (7%); p < 0.001. CONCLUSIONS: Unlike the burden, the CL of NSVTs is not a predictor of subsequent appropriate interventions. However, there is a close relationship between the CL of NSVTs and that of arrhythmias that will later lead to appropriate therapies.

Correlation between protein concentration and bacterial cell size can reveal mechanisms of gene expression.

Classically, gene expression is modeled as a chemical process with reaction rates dependent on the concentration of the reactants (typically, DNA loci, plasmids, RNA, enzymes, etc). Other variables like cell size are in general ignored. Size dynamics can become an important variable due to the low number of many of these reactants, imperfectly symmetric cell partitioning and molecule segregation. In this work we measure the correlation between size and protein concentration by observing the gene expression of the RpOD gene from a low-copy plasmid in Escherichia coli during balanced growth in different media. A positive correlation was found, and we used it to examine possible models of cell size dynamics and plasmid replication. We implemented a previously developed model describing the full gene expression process including transcription, translation, loci replication, cell division and molecule segregation. By comparing with the observed correlation, we determine that the transcription rate must be proportional to the size times the number of plasmids. We discuss how fluctuations in plasmid segregation, due to the low copy number, can impose limits in this correlation.

Genetic diversity of Fusarium pseudocircinatum in the central western region of Mexico: the case of big-leaf mahogany malformation disease.

Fusarium pseudocircinatum is the main causal agent of big-leaf mahogany malformation disease (BLMMD) of mahogany (Swietenia macrophylla) in Mexico. Although, BLMMD is the most important disease for this high-value timber species, there is a lack of information on the genetic variation present in geographically diverse isolates of F. pseudocircinatum. The objective of this study was to determine the genetic diversity of populations of F. pseudocircinatum causing BLMMD in the central western region of Mexico. A total of 611 big-leaf mahogany trees were inspected at eight sites in four states (Colima, Guerrero, Jalisco and Michoacán); of these, 42.7% showed malformation symptoms similar to those of BLMMD. Of 374 Fusarium isolates that were recovered, 277 were identified as F. pseudocircinatum, 56 were F. mexicanum, and 41 were Fusarium spp. An ISSR analysis of the F. pseudocircinatum isolates generated 51 bands of which 38 were polymorphic (76.8%) with a mean of 17 bands per primer. A total of 87 multilocus genotypes (MLGs) were identified. Nei's genetic diversity analysis showed that the isolates had a high genetic diversity average (0.147), with values ranging from 0.070 to 0.365 depending of the geographical location. An analysis of molecular variance revealed that the variation within the populations was low (27.36%), while the variation within MLGs was significant (72.64%), indicating genetic flow. Overall, the genetic variability of F. pseudocircinatum populations was high and the MLGs from Colima (Colima) and Gabriel Zamora (Michoacán) were placed centrally, which possibly is evidence of ancestry and indicates its dispersion routes in the central western region of Mexico.

The Effectiveness of an Oral Sensorimotor Stimulation Protocol for the Early Achievement of Exclusive Oral Feeding in Premature Infants. A Randomized, Controlled Trial.

Aims: This study is aimed to investigate the effectiveness of an oral stimulation protocol in preterm infants compared to usual care, to reduce the time for achieving safe full oral feeding.Methods: 47 preterm infants (25- 30 weeks of gestational age) were randomized into two groups. Babies of the EG (n = 24) received a 10-minute oral stimulation protocol while the CG (n = 23) received the standard care. The primary outcome were the days from the initiation of the intervention until the achievement of full oral feeding. Secondary outcomes were: days from the first day the intervention started until achieving a first oral intake of 30% in the first 5 minutes, days from the first day the intervention started until achieving a first oral intake of 100%, and days of hospitalization. A parametric survival model with Gaussian distribution was used.Results: The EG achieved full oral feeding 8.3 days before the CG (p = 0.013). EG also achieved the first oral intake of 30% in the first five minutes, 6.03 days before (p = 0.019) and of 100%, 5.88 days before (p = 0.040). EG also spent 6.9 days less hospitalized than CG (p = 0.028).Conclusion: Oral stimulation in preterm infants significantly shortens the time to achieve full oral feeding and reduces the length of hospitalization.

Healthcare personnel in 2016-2019 prospective cohort infrequently got vaccinated, worked while ill, and frequently used antibiotics rather than antivirals against viral influenza illnesses.

Background: Uncertainty about risk of illness and the value of influenza vaccines negatively affects vaccine uptake among persons targeted for influenza vaccination. Methods: During 2016-2019, we followed a cohort of healthcare personnel (HCP) targeted for free-of-charge influenza vaccination in five Lima hospitals to quantify risk of influenza, workplace presenteeism (coming to work despite illness), and absenteeism (taking time off from work because of illness). The HCP who developed acute respiratory illnesses (ARI) (≥1 of acute cough, runny nose, body aches, or feverishness) were tested for influenza using reverse-transcription polymerase chain reaction (rt-PCR). Findings: The cohort (2968 HCP) contributed 950,888 person-days. Only 36 (6%) of 605 HCP who participated every year were vaccinated. The HCP had 5750 ARI and 147 rt-PCR-confirmed influenza illnesses. The weighted incidence of laboratory-confirmed influenza was 10.0/100 person-years; 37% used antibiotics, and 0.7% used antivirals to treat these illnesses. The HCP with laboratory-confirmed influenza were present at work while ill for a cumulative 1187 hours. Interpretation: HCP were frequently ill and often worked rather than stayed at home while ill. Our findings suggest the need for continuing medical education about the risk of influenza and benefits of vaccination and stay-at-home-while-ill policies.

Plasmodium falciparum genetic diversity maintained and amplified over 5 years of a low transmission endemic in the Peruvian Amazon.

Plasmodium falciparum entered into the Peruvian Amazon in 1994, sparking an epidemic between 1995 and 1998. Since 2000, there has been sustained low P. falciparum transmission. The Malaria Immunology and Genetics in the Amazon project has longitudinally followed members of the community of Zungarococha (N = 1,945, 4 villages) with active household and health center-based visits each year since 2003. We examined parasite population structure and traced the parasite genetic diversity temporally and spatially. We genotyped infections over 5 years (2003-2007) using 14 microsatellite (MS) markers scattered across ten different chromosomes. Despite low transmission, there was considerable genetic diversity, which we compared with other geographic regions. We detected 182 different haplotypes from 302 parasites in 217 infections. Structure v2.2 identified five clusters (subpopulations) of phylogenetically related clones. To consider genetic diversity on a more detailed level, we defined haplotype families (hapfams) by grouping haplotypes with three or less loci differences. We identified 34 different hapfams identified. The F(st) statistic and heterozygosity analysis showed the five clusters were maintained in each village throughout this time. A minimum spanning network (MSN), stratified by the year of detection, showed that haplotypes within hapfams had allele differences and haplotypes within a cluster definition were more separated in the later years (2006-2007). We modeled hapfam detection and loss, accounting for sample size and stochastic fluctuations in frequencies overtime. Principle component analysis of genetic variation revealed patterns of genetic structure with time rather than village. The population structure, genetic diversity, appearance/disappearance of the different haplotypes from 2003 to 2007 provides a genome-wide "real-time" perspective of P. falciparum parasites in a low transmission region.

Intrapericardial Administration of Secretomes from Menstrual Blood-Derived Mesenchymal Stromal Cells: Effects on Immune-Related Genes in a Porcine Model of Myocardial Infarction.

Acute myocardial infarction (AMI) is a manifestation of ischemic heart disease where the immune system plays an important role in the re-establishment of homeostasis. We hypothesize that the anti-inflammatory activity of secretomes from menstrual blood-derived mesenchymal stromal cells (S-MenSCs) and IFNγ/TNFα-primed MenSCs (S-MenSCs*) may be considered a therapeutic option for the treatment of AMI. To assess this hypothesis, we have evaluated the effect of S-MenSCs and S-MenSCs* on cardiac function parameters and the involvement of immune-related genes using a porcine model of AMI. Twelve pigs were randomly divided into three biogroups: AMI/Placebo, AMI/S-MenSCs, and AMI/S-MenSCs*. AMI models were generated using a closed chest coronary occlusion-reperfusion procedure and, after 72 h, the different treatments were intrapericardially administered. Cardiac function parameters were monitored by magnetic resonance imaging before and 7 days post-therapy. Transcriptomic analyses in the infarcted tissue identified 571 transcripts associated with the Gene Ontology term Immune response, of which 57 were differentially expressed when different biogroups were compared. Moreover, a prediction of the interactions between differentially expressed genes (DEGs) and miRNAs from secretomes revealed that some DEGs in the infarction area, such as STAT3, IGFR1, or BCL6 could be targeted by previously identified miRNAs in secretomes from MenSCs. In conclusion, the intrapericardial administration of secretome early after infarction has a significant impact on the expression of immune-related genes in the infarcted myocardium. This confirms the immunomodulatory potential of intrapericardially delivered secretomes and opens new therapeutic perspectives in myocardial infarction treatment.

The Clinical outcomes, healthcare resource utilization, and related costs (COHERENT) model. Application in heart failure patients.

INTRODUCTION AND OBJECTIVES: Composite endpoints are widely used but have several limitations. The Clinical outcomes, healthcare resource utilization and related costs (COHERENT) model is a new approach for visually displaying and comparing composite endpoints including all their components (incidence, timing, duration) and related costs. We aimed to assess the validity of the COHERENT model in a patient cohort. METHODS: A color graphic system displaying the percentage of patients in each clinical situation (vital status and location: at home, emergency department [ED] or hospital) and related costs at each time point during follow-up was created based on a list of mutually exclusive clinical situations coded in a hierarchical fashion. The system was tested in a cohort of 1126 patients with acute heart failure from 25 hospitals. The system calculated and displayed the time spent in each clinical situation and health care resource utilization-related costs over 30 days. RESULTS: The model illustrated the times spent over 30 days (2.12% in ED, 23.6% in index hospitalization, 2.7% in readmissions, 65.5% alive at home, and 6.02% dead), showing significant differences between patient groups, hospitals, and health care systems. The tool calculated and displayed the daily and cumulative health care-related costs over time (total, €4 895 070; mean, €144.91 per patient/d). CONCLUSIONS: The COHERENT model is a new, easy-to-interpret, visual display of composite endpoints, enabling comparisons between patient groups and cohorts, including related costs. The model may constitute a useful new approach for clinical trials or observational studies, and a tool for benchmarking, and value-based health care implementation.

Evaluation of respiratory anatomical-functional sequelae in patients who recovered from COVID-19.

INTRODUCTION: Coronavirus disease 19 (COVID-19) has been a global public health emergency, with 209.89 million cases of infection with SARS-CoV-2 recorded, resulting in 4,401,675 deaths. After recuperation, it is probable that COVID-19 patients have sequelae of the disease. This study aimed to evaluate the respiratory anatomical-functional sequelae in Mexican patients who recovered from COVID-19. METHODOLOGY: This study included twenty-four patients who recovered from COVID-19 and eight non-infected patients (controls). Participants were screened for SARS-CoV-2 and the presence of IgM/IgG antibodies. Pulmonary function and lung anatomical abnormalities were evaluated by spirometry and computerized tomography. RESULTS: A total of 45.8% of the patients had pulmonary function with obstructive patterns: 70.8% of recovered cases had COVID-19 Reporting and Data System (CO-RADS) 1, 20.8% CO-RADS 3 and 16.7% CO-RADS 4. A total of 35.3% of patients with CO-RADS 1 also showed bilateral nodal growth; 70.8% of patients tested positive for IgG and 8.4% for IgG/IgM, and 20.8% tested negative for both antibodies. CONCLUSIONS: There were respiratory anatomical and functional sequelae in Mexican patients who recovered from COVID-19, with a high occurrence of pulmonary obstructive patterns in the study population. These observations indicate the importance of the routine evaluation of sequelae in Mexican patients who recovered from COVID-19 and the need for strict follow-up to improve the quality of life of these patients.

Comparable diagnostic accuracy of SARS-CoV-2 Spike RBD and N-specific IgG tests to determine pre-vaccination nation-wide baseline seroprevalence in Mexico.

A major challenge for developing countries during the COVID-19 pandemic is affordable and adequate monitoring of disease progression and population exposure as the primary source relevant epidemiological indicators. Serology testing enables assessing population exposure and to guide vaccination strategies but requires rigorous accuracy validation before population-wide implementation. We adapted a two-step ELISA protocol as a single-step protocol for detection of IgG against the Receptor Binding Domain (RBD) of SARS-CoV-2 spike protein and compared its diagnostic accuracy with a commercial immunoassay anti-nucleoprotein IgG. Both methods yielded adequate and comparable diagnostic accuracy after 3 weeks post-symptom onset and were implemented in a nation-wide population based serological survey during August-November 2020. Anti-RBD National seroprevalence was 23.6%, 1.3% lower, but not significantly, than for anti-N. Double positive seroprevalence was 19.7%. Anti-N single-positive seroprevalence was 3.72% and anti-RBD single-positive seroprevalence was 1.98%. Discrepancies in the positivity to either single marker may be due to different kinetics of each antibody marker as well as the heterogeneity of the sampling time in regards to local epidemic waves. Baseline single positivity prevalence will be useful to assess the serological impact of vaccination and natural infection in further serosurveillance efforts.

Phylogenomics of Salvia L. subgenus Calosphace (Lamiaceae).

The evolutionary relationships of Salvia have been difficult to estimate. In this study, we used the Next Generation Sequencing method Hyb-Seq to evaluate relationships among 90 Lamiaceae samples, including representatives of Mentheae, Ocimeae, Salvia subgenera Audibertia, Leonia, Salvia, and 69 species of subgenus Calosphace, representing 32 of Epling's sections. A bait set was designed in MarkerMiner using available transcriptome data to enrich 119 variable nuclear loci. Nuclear and chloroplast loci were assembled with hybphylomaker (HPM), followed by coalescent approach analyses for nuclear data (ASTRAL, BEAST) and a concatenated Maximum Likelihood analysis of chloroplast loci. The HPM assembly had an average of 1,314,368 mapped reads for the sample and 527 putative exons. Phylogenetic inferences resolved strongly supported relationships for the deep-level nodes, agreeing with previous hypotheses which assumed that subgenus Audibertia is sister to subgenus Calosphace. Within subgenus Calosphace, we recovered eight monophyletic sections sensu Epling, Cardinalis, Hastatae, Incarnatae, and Uricae in all the analyses (nDNA and cpDNA), Biflorae, Lavanduloideae, and Sigmoideae in nuclear analyses (ASTRAL, BEAST) and Curtiflorae in ASTRAL trees. Network analysis supports deep node relationships, some of the main clades, and recovers reticulation within the core Calosphace. The chloroplast phylogeny resolved deep nodes and four monophyletic Calosphace sections. Placement of S. axillaris is distinct in nuclear evidence and chloroplast, as sister to the rest of the S. subg. Calosphace in chloroplast and a clade with "Hastatae clade" sister to the rest of the subgenus in nuclear evidence. We also tested the monophyly of S. hispanica, S. polystachia, S. purpurea, and S. tiliifolia, including two samples of each, and found that S. hispanica and S. purpurea are monophyletic. Our baits can be used in future studies of Lamiaceae phylogeny to estimate relationships between genera and among species. In this study, we presented a Hyb-Seq phylogeny for complex, recently diverged Salvia, which could be implemented in other Lamiaceae.

Timing of Ablation and Prognosis of Patients With Electrical Storm and Scar-Related Left Ventricular Dysfunction.

Although radiofrequency catheter ablation (RFCA) is indicated in electrical storm (ES) refractory to antiarrhythmic drugs, its most appropriate timing has not been determined. Our objective is to analyse the impact of the timing of RFCA on 30-day mortality in patients with ES and previous scar-related systolic dysfunction. In this multi-centre study, we analysed 104 patients (age: 72 ± 10, left ventricular ejection fraction: 30 ± 6%) attended consecutively due to an ES caused by monomorphic ventricular tachycardia. Sixty-four subjects were treated with RFCA (mean time from admission = 83 ± 67 hours) and 40 were not. Upon admission 25 (24%) individuals had severe heart failure. Mortality rate at 30 days was 24 (23%) patients. RFCA was associated with a reduction of 30-day mortality (hazard ratio = 0.2; p = 0.008). After showing a positive correlation between the time of the RFCA (hours) and survival at 30 days (C-statistic = 0.77; p <0.001), we found that only subjects ablated >48 hours after admission had lower mortality at 30 days than those treated conservatively: 38% (no RFCA) versus 30% (RFCA ≤48 hours) versus 7% (RFCA >48 hours) (adjusted hazard ratio for RFCA >48 hours vs others = 0.2; p = 0.007). Among the patients ablated, those who were non-inducible had lower 30-day mortality: 8% versus 29% (p = 0.03). Extracorporeal membrane oxygenation was associated with a higher rate of non-inducibility in RFCA >48 hours (100% vs 76%; p = 0.03), but not in RFCA ≤48 hours (60% vs 60%; p = 1). In conclusion, among high-risk patients with ES, RFCA performed >48 hours after admission is associated with a reduction in 30-day mortality. In such subjects, the probability of successful RFCA increases when performed under extracorporeal membrane oxygenation support.

Unraveling the Molecular Signature of Extracellular Vesicles From Endometrial-Derived Mesenchymal Stem Cells: Potential Modulatory Effects and Therapeutic Applications.

Endometrial-derived Mesenchymal Stem Cells (endMSCs) are involved in the regeneration and remodeling of human endometrium, being considered one of the most promising candidates for stem cell-based therapies. Their therapeutic effects have been found to be mediated by extracellular vesicles (EV-endMSCs) with pro-angiogenic, anti-apoptotic, and immunomodulatory effects. Based on that, the main goal of this study was to characterize the proteome and microRNAome of these EV-endMSCs by proteomics and transcriptomics approaches. Additionally, we hypothesized that inflammatory priming of endMSCs may contribute to modify the therapeutic potential of these vesicles. High-throughput proteomics revealed that 617 proteins were functionally annotated as Extracellular exosome (GO:0070062), corresponding to the 70% of the EV-endMSC proteome. Bioinformatics analyses allowed us to identify that these proteins were involved in adaptive/innate immune response, complement activation, antigen processing/presentation, negative regulation of apoptosis, and different signaling pathways, among others. Of note, multiplexed quantitative proteomics and Systems Biology analyses showed that IFNγ priming significantly modulated the protein profile of these vesicles. As expected, proteins involved in antigen processing and presentation were significantly increased. Interestingly, immunomodulatory proteins, such as CSF1, ERAP1, or PYCARD were modified. Regarding miRNAs expression profile in EV-endMSCs, Next-Generation Sequencing (NGS) showed that the preferred site of microRNAome targeting was the nucleus (n = 371 microTargets), significantly affecting signal transduction (GO:0007165), cell proliferation (GO:0008283), and apoptotic processes (GO:0006915), among others. Interestingly, NGS analyses highlighted that several miRNAs, such as hsa-miR-150-5p or hsa-miR-196b-5p, were differentially expressed in IFNγ-primed EV-endMSCs. These miRNAs have a functional involvement in glucocorticoid receptor signaling, IL-6/8/12 signaling, and in the role of macrophages. In summary, these results allowed us to understand the complexity of the molecular networks in EV-endMSCs and their potential effects on target cells. To our knowledge, this is the first comprehensive study based on proteomic and genomic approaches to unravel the therapeutic potential of these extracellular vesicles, that may be used as immunomodulatory effectors in the treatment of inflammatory conditions.

Single-cell microscopy of suspension cultures using a microfluidics-assisted cell screening platform.

Studies that rely on fluorescence imaging of nonadherent cells that are cultured in suspension, such as Escherichia coli, are often hampered by trade-offs that must be made between data throughput and imaging resolution. We developed a platform for microfluidics-assisted cell screening (MACS) that overcomes this trade-off by temporarily immobilizing suspension cells within a microfluidics chip. This enables high-throughput and automated single-cell microscopy for a wide range of cell types and sizes. As cells can be rapidly sampled directly from a suspension culture, MACS bypasses the need for sample preparation, and therefore allows measurements without perturbing the native cell physiology. The setup can also be integrated with complex growth chambers, and can be used to enrich or sort the imaged cells. Furthermore, MACS facilitates the visualization of individual cytoplasmic fluorescent proteins (FPs) in E. coli, allowing low-abundance proteins to be counted using standard total internal reflection fluorescence (TIRF) microscopy. Finally, MACS can be used to impart mechanical pressure for assessing the structural integrity of individual cells and their response to mechanical perturbations, or to make cells take up chemicals that otherwise would not pass through the membrane. This protocol describes the assembly of electronic control circuitry, the construction of liquid-handling components and the creation of the MACS microfluidics chip. The operation of MACS is described, and automation software is provided to integrate MACS control with image acquisition. Finally, we provide instructions for extending MACS using an external growth chamber (1 d) and for how to sort rare cells of interest.

Mechanical slowing-down of cytoplasmic diffusion allows in vivo counting of proteins in individual cells.

Many key regulatory proteins in bacteria are present in too low numbers to be detected with conventional methods, which poses a particular challenge for single-cell analyses because such proteins can contribute greatly to phenotypic heterogeneity. Here we develop a microfluidics-based platform that enables single-molecule counting of low-abundance proteins by mechanically slowing-down their diffusion within the cytoplasm of live Escherichia coli (E. coli) cells. Our technique also allows for automated microscopy at high throughput with minimal perturbation to native physiology, as well as viable enrichment/retrieval. We illustrate the method by analysing the control of the master regulator of the E. coli stress response, RpoS, by its adapter protein, SprE (RssB). Quantification of SprE numbers shows that though SprE is necessary for RpoS degradation, it is expressed at levels as low as 3-4 molecules per average cell cycle, and fluctuations in SprE are approximately Poisson distributed during exponential phase with no sign of bursting.

Cost-effectiveness Analysis of Viscosupplementation versus Conventional Supportive Therapy for Knee Osteoarthritis in Colombia.

BACKGROUND: Treatment goals for knee osteoarthritis (OA) include preservation of mobility, control of pain, and delaying total knee replacement (TKR). OBJECTIVE: To estimate the cost-effectiveness of viscosupplementation (hylan G-F 20) alone compared with conventional supportive therapy (CST) in the treatment of knee OA in Colombia. METHODS: Microsimulation in patients with knee OA, modeling of clinical outcomes (disease progression, symptom improvement, TKR), and estimation of associated costs were performed (drugs, diagnostic tests, procedures, and hospitalizations). The probabilities for disease progression and clinical events were correlated with patients' characteristics. Clinical outcome information was obtained from the literature. The costs were drawn from institutional databases from health maintenance organizations and the Colombian standard tariffs handbook (ISS 2001. Agreement No. 256 of 2001. Tariffs for the health promoter Social Security EPS-ISS. Social Insurance Board of Directors. December 19, 2001). Sensitivity analyses were performed for costs and transition probabilities. RESULTS: Monte-Carlo simulation for 1000 patients with knee OA showed that viscosupplementation with hylan G-F 20 delayed the occurrence of TKR by 3 years compared with CST. Western Ontario and McMaster Universities Arthritis Index scores indicate improvement in symptoms and function with hylan G-F 20. The incremental cost-effectiveness ratio for viscosupplementation is dominant, with reduction of US $576 in treatment cost in favor of hylan G-F 20, with more cost-effectiveness per quality-adjusted life-year during the first 10 years of treatment compared with CST. CONCLUSIONS: The results of mathematical simulation indicate that in comparison to conventional support therapies, viscosupplementation with hylan G-F 20 improved disease symptoms, joint function, and quality of life, reduced direct treatment costs, delayed TKR by 3 years, and was cost-effective in Colombia.

The human HDL proteome displays high inter-individual variability and is altered dynamically in response to angioplasty-induced atheroma plaque rupture.

Recent findings support potential roles for HDL in cardiovascular pathophysiology not related to lipid metabolism. We address whether HDL proteome is dynamically altered in atheroma plaque rupture. We used immunoaffinity purification of HDL samples from coronary artery disease patients before and after percutaneous transluminal coronary angioplasty (PTCA), a model of atheroma plaque disruption. Samples were analyzed by quantitative proteomics using stable isotope labeling and results were subjected to statistical analysis of protein variance using a novel algorithm. We observed high protein variability in HDL composition between individuals, indicating that HDL protein composition is highly patient-specific. However, intra-individual protein variances remained at low levels, confirming the reproducibility of the method used for HDL isolation and protein quantification. A systems biology analysis of HDL protein alterations induced by PTCA revealed an increase in two protein clusters that included several apolipoproteins, fibrinogen-like protein 1 and other intracellular proteins, and a decrease in antithrombin-III, annexin A1 and several immunoglobulins. Our results support the concept of HDL as dynamic platforms that donate and receive a variety of molecules and provide an improved methodology to use HDL proteome for the systematic analysis of differences among individuals and the search for cardiovascular biomarkers. Biological significance The HDL proteome is an interesting model of clinical relevance and has been previously described to be dynamically altered in response to pathophysiological conditions and cardiovascular diseases. Our study suggests that interindividual variability of HDL proteome is higher than previously thought and provided the detection of a set of proteins that changed their abundance in response to plaque rupture, supporting the concept of HDL as dynamic platforms that donate and receive a variety of molecules.