Bioanalytical method validation and application to a phase 1, double-blind, randomized pharmacokinetic trial of a standardized Centella asiatica (L.) Urban water extract product in healthy older adults.

Introduction: Centella asiatica is an herbaceous plant reputed in Eastern medicine to improve memory. Preclinical studies have shown that C. asiatica aqueous extract (CAW) improves neuronal health, reduces oxidative stress, and positively impacts learning and cognition. This study aimed to develop and validate bioanalytical methods for detecting known bioactive compounds from C. asiatica in human biological matrices and apply them to a human pharmacokinetic trial in healthy older adults. Methods: High performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was used for detecting triterpenes and caffeoylquinic acids from C. asiatica, or their metabolites, in human plasma and urine. Validation parameters including linearity, precision, accuracy, recovery and thermal stability were evaluated. The method was applied to a Phase I, randomized, double-blind, crossover trial of two doses (2 or 4 g) of a standardized C. asiatica water extract product (CAP) in eight healthy older adults. Pharmacokinetic parameters were measured over a 12-h post administration period and acute safety was assessed. Results: The method satisfied US Food & Drug Administration criteria for linearity and recovery of the analytes of interest in human plasma and urine. The method also satisfied criteria for precision and accuracy at medium and high concentrations. Single administration of 2 and 4 g of CAP was well tolerated and safe in healthy older adults. The parent triterpene glycosides, asiaticoside and madecassoside, were not detected in plasma and in minimal amounts in urinary excretion analyses, while the aglycones, asiatic acid and madecassic acid, showed readily detectable pharmacokinetic profiles. Similarly, the di-caffeoylquinic acids and mono-caffeoylquinic acids were detected in low quantities, while their putative metabolites showed readily detectable pharmacokinetic profiles and urinary excretion. Discussion: This method was able to identify and calculate the concentration of triterpenes and caffeoylquinic acids from C. asiatica, or their metabolites, in human plasma and urine. The oral absorption of these key compounds from CAP, and its acute safety in healthy older adults, support the use of this C. asiatica product in future clinical trials.

Pharmacokinetics and Pharmacodynamics of Key Components of a Standardized Centella asiatica Product in Cognitively Impaired Older Adults: A Phase 1, Double-Blind, Randomized Clinical Trial.

Centella asiatica is reputed in Eastern medicine to improve cognitive function in humans. Preclinical studies have demonstrated that aqueous extracts of C. asiatica improve cognition in mouse models of aging and Alzheimer's disease (AD) through the modulation of mitochondrial biogenesis and nuclear factor-erythroid-2-related factor 2 (Nrf2)-dependent antioxidant response genes. This randomized, double-blind, crossover Phase I trial explored the oral bioavailability and pharmacokinetics of key compounds from two doses (2 g and 4 g) of a standardized C. asiatica aqueous extract product (CAP), over 10 h, in four mildly demented older adults on cholinesterase inhibitor therapy. The analysis focused on triterpenes (TTs) and caffeoylquinic acids (CQAs), which are known to contribute to C. asiatica's neurological activity. The acute safety of CAP and the effects on NRF2 gene expression in peripheral blood mononuclear cells were evaluated. Single administration of 2 g or 4 g of CAP was safe and well-tolerated. The TT aglycones, asiatic acid and madecassic acid, were identified in plasma and urine, while the parent glycosides, asiaticoside and madecassoside, although abundant in CAP, were absent in plasma and had limited renal excretion. Similarly, mono- and di-CQAs showed delayed absorption and limited presence in plasma or urine, while the putative metabolites of these compounds showed detectable plasma pharmacokinetic profiles and urinary excretion. CAP elicited a temporal change in NRF2 gene expression, mirroring the TT aglycone's pharmacokinetic curve in a paradoxical dose-dependent manner. The oral bioavailability of active compounds or their metabolites, NRF2 target engagement, and the acute safety and tolerability of CAP support the validity of using CAP in future clinical studies.

Unintentional Epinephrine Auto-Injector Maxillofacial Injury in a Pediatric Patient.

CASE PRESENTATION: A four-year-old female patient presented to the emergency department with an epinephrine auto-injector that had unintentionally discharged into her mandible. There was difficulty removing the auto-injector at bedside. Images we acquired noted needle curvature not present in an off-the-shelf model. She was sedated, and the auto-injector was removed by retracing the angle of discharge, with care taken not to inject epinephrine into the patient. DISCUSSION: Epinephrine auto-injector accidental discharges are an unusual injury pattern, but the incidence of such events is increasing in the United States. The emergency clinician should be cognizant of complicating factors with discharges, such as bent needles. Here we discuss a case of discharge into the maxillofacial region (lower jaw), with approaches to treatment.

Comparison of Internal Jugular Vein Cross-Section Area During a Russian Tilt-Table Protocol and Microgravity.

BACKGROUND: To date, we lack U.S. data on the effects of the long-used Russian tilt-table training protocol known as the Russian pre-launch tilt-table training protocol on internal jugular vein cross sectional area (IJV-CSA) in microgravity.CASE REPORT: A case study of a single healthy male astronaut volunteer was used for this study. The right IJV-CSA was measured using real time ultrasound at set times throughout the Russian pre-launch tilt-table training protocol, a method of physiological preparation for microgravity using tilt-table training. In microgravity, the subjects right IJV-CSA was measured again for comparison. The mean difference from in-flight right IJV-CSA for pre-tilt (0) was 0.438 cm², for 15 was 0.887 cm², for 30 was 0.864 cm², for 50 was 1.15 cm², and for post-tilt (0) the difference was 0.305 cm².DISCUSSION: The cross-sectional areas of the subjects right IJV-CSA were significantly different between in-flight values and several angles of the Russian tilt-table protocol, except for the 0 measurement. In summary, this case-study represents the first time IJV-CSA has been compared between various angles of a tilt-table training protocol and microgravity in the same astronaut subject. The findings support prior cohort studies studying the same principles. Further investigation is merited; both to better describe the relationship between the cardiovascular effects of tilt-table simulations of microgravity and their correlating in-flight values, and to evaluate and study the Russian tilt-table protocol effects on cardiovascular physiology from a training and preparation perspective.David J, Scheuring RA, Morgan A, Olsen C, Sargsyan A, Grishin A. Comparison of internal jugular vein cross-section area during a Russian tilt-table protocol and microgravity. Aerosp Med Hum Perform. 2021; 92(3):207211.

Qualitative analysis of the Meals, Mindfulness, & Moving Forward (M3 ) lifestyle programme: Cultivating a 'safe space' to start on a 'new path' for youth with early episode psychosis.

AIM: The Meals, Mindfulness, & Moving Forward (M3 ) programme included nutrition education, hands-on cooking classes, mindfulness meditation practice, physical activities and facilitated group sharing. M3 was designed as a supplement to standard care for youths (age 15-25 years) with first-episode psychosis (FEP) who were clients of coordinated specialty care teams. M3 's primary aim was feasibility by demonstrating high programme attendance; secondary aims included cardiometabolic measures. Data collection included quantitative and qualitative outcomes. The aim of the qualitative study was to understand participants' and study partners' experiences during the programme and to understand programme elements that were helpful for young people to sustain healthy lifestyle choices 6 weeks post-programme. METHODS: During the last programme session, we conducted two focus groups, one with participants (n = 13) and one with their study partners (n = 11); 6 weeks post-intervention, individual semi-structured interviews were conducted with 11 participants. All interviews were audio recorded and transcribed; grounded theory methods guided thematic analysis. RESULTS: Main themes from the focus groups included appreciation for a 'non-stigmatizing' environment providing participants and study partners with a sense of 'dignity' that enabled a 'new path'. Six weeks post-intervention, participants reported continued use of mindfulness practice to stay grounded and assist with making healthful lifestyle changes. However, many were unsure of how to sustain these changes long-term. CONCLUSION: The results suggest that young people with FEP value a non-stigmatizing space that allows for social engagement and facilitates healthy behaviours. Short-term, M3 participants reported behaviour change but wanted on-going support to sustain healthy behaviours.

An evidence-based approach to nonoperative management of traumatic hemorrhagic shock in the emergency department.

The management of traumatic hemorrhagic shock has evolved, with increasing emphasis on damage control resuscitation principles. Despite these advances, hemorrhage is still the leading preventable cause of death in trauma. This issue provides evidence-based recommendations for the assessment and treatment of traumatic hemorrhagic shock. Hemostatic techniques as well as correction of hemorrhagic hypovolemia and traumatic coagulopathy are presented. The safety and efficacy of practices such as resuscitative endovascular balloon occlusion of the aorta (REBOA), viscoelastic clot testing, and whole blood resuscitation are also reviewed.

Oxidized Products of Omega-6 and Omega-3 Long Chain Fatty Acids Are Associated with Increased White Matter Hyperintensity and Poorer Executive Function Performance in a Cohort of Cognitively Normal Hypertensive Older Adults.

BACKGROUND: Cerebrovascular disease is a common cause of dementia in older adults, and potentially preventable with early intervention. Oxylipins are produced from the oxidation of long-chain polyunsaturated fatty acids (PUFA) possessing potent vascular effects. Oxylipins generated from the cytochrome P450 pathway are enzymatically converted to diols by soluble epoxide hydrolase (sEH); sEH products have been associated with small vessel ischemic disease. Little is known about oxylipins' impact on markers of dementia risk. OBJECTIVE: An exploratory examination of the association between omega-6 and omega-3 derived oxylipins, brain MRI, and cognition. METHODS: Thirty-seven non-demented participants with controlled hypertension (mean age 65.6 years) were enrolled in a dementia prevention study investigating fish oil and lipoic acid on preserving cognitive function. Baseline associations between plasma oxylipins, white matter hyperintensity (WMH), and Trails-B were examined using linear regression. P450-derived diol/epoxide ratio was an indirect measure of sEH activity. RESULTS: Omega-6 derived 9-HODE was associated with increased WMH (p = 0.017) and reduced grey matter volume (p = 0.02). Omega-6 P450-derived diol/epoxide ratio 9,10-DiHOME/9,10-EpOME was associated with increased WMH (p = 0.035) and poorer performance on Trails-B (p = 0.05); ratio14,15-DHET/14,15-EET was associated with increased WMH (p = 0.045). Omega-3 P450-derived diol/epoxide ratio 19,20-DiHDPE/19,20-EpDPE was associated with increased WMH (p = 0.04) and poorer performance on Trails-B (p = 0.04). Arachidonic acid was associated with better performance on Trails-B (p = 0.012); Omega-3 derived 16,17-EpDPE was associated with decreased WMH (p = 0.005). CONCLUSIONS: With the exception of arachidonic acid, it was specific oxylipin products, not their parent PUFAs, that were associated with unfavorable and favorable MRI and cognitive markers of dementia risk.

Loss of TXNIP enhances peritoneal metastasis and can be abrogated by dual TORC1/2 inhibition.

Peritoneal metastasis (PM) is a debilitating consequence of multiple cancers. As cancer cells lose tonic signaling related to attachment dependence, critical morphologic shifts result in alteration of the transcriptome. Identifying key genes associated with this transformation may lead to targeted therapies for this devastating complication. TC71, CHLA9, PANC1, YOU and HEYA8 cell lines were grown as tumor spheroids in polyHEMA coated plates. Temporal profiling of transcriptomic alterations over 72 hrs was used to develop a comprehensive PM model. We identified transcriptomic outliers using Gaussian mixtures model clustering to identify drivers of spheroid formation. Outliers were validated in The Cancer Genome Atlas (TCGA) and an ovarian tissue microarray (TMA) and by modulation in ovarian cancer models in vitro and in peritoneal xenograft models. Outlier analysis of PM genes identified the gene TXNIP and the TORC signaling as central to PM. Ovarian cancer spheroids isolated from patient ascites had significantly higher TXNIP than their attached counterparts (p = 0.047). TXNIP levels predicted progression-free (log-rank p = 0.026) survival in stage 1/2 ovarian cancer and overall survival (log rank p = 0.047) in stage 3/4 ovarian cancer. In vitro, TXNIP silencing was associated with increased mTOR signaling and enhanced spheroid development which could be overcome by TAK228, a TORC1/2 inhibitor. Similarly, in vivo peritoneal xenograft models of carcinomatosis were prevented by TAK228. PM is driven by TXNIP-associated TORC1/2 signaling. This work provides the first evidence that TORC1/2 inhibition may prevent PM.

Systematic Review of Live Tissue Versus Simulation Education for Prehospital Trauma Providers.

BACKGROUND: Advanced simulation capabilities have provided medical educators novel approaches for learners. Simulation has successfully replaced many aspects of medical education that previously used animal live-tissue training (LTT) for physician education. However, prehospital trauma providers, such as combat medics, currently used LTT to prepare for patient care. This use of LTT has sparked a debate about the optimal educational modality for this unique learner population. At this time, there is no clear evidence-based recommendation available to recommend either LTT or simulation as a superior modality. METHODS: The authors performed a systematic review of observational studies and randomized control trials (RCTs) to examine the use of LTT versus simulation in the trauma education of prehospital providers. The authors judged studies for inclusion and data abstraction independently and in duplicate, while also assessing quality and risk of bias. Since the literature demonstrated a heterogeneous background, no meta-analysis was performed. RESULTS: 12 studies met inclusion criteria: seven RCTs, four prospective cohorts, one cross-sectional study. Two of seven RCTs were presented as abstracts only. Ten of 12 studies were performed in a military setting, whereas two occurred in a civilian setting. Four studies used swine, two used goats, one used swine and goats, one used canines, and four did not specify the animal type. The authors used the Cochrane Collaboration tool to assess RCTs and found a considerable risk of bias. They used the Newcastle-Ottawa score to assess prospective cohorts (mean score of 5.75 ± 0.5, range 1-9), and the cross-sectional study (score 4, range 1-9). CONCLUSION: The existing literature provides limited, low-to-moderate quality outcome data. Evidence does not exist at this time to recommend either LTT or simulation as a superior educational modality for prehospital trauma care providers.

Association between early life adversity and inflammation during infancy.

Early life adversity is associated with adult elevations of inflammatory markers such as circulating levels of C-reactive protein (CRP). Few studies have measured inflammation or its association with psychosocial stress during infancy. Existing evidence suggests that early adversity can embed itself into young children's biology with implications for lifelong development. This study examined the association between psychosocial stress and salivary CRP in infants. Early adversity in the form of socioeconomic disadvantage and maternal psychosocial stress were measured when infants were 17 months old. Resting state saliva samples were collected to assess CRP (pg/ml) levels via enzyme immunoassay. Results revealed that both socioeconomic disadvantage and maternal psychosocial stress were independently associated with higher infant CRP levels. These results raise questions about timing of exposure to adversity, and about the potentially lasting effects on inflammatory processes when such exposure occurs early in development.

Increased levels of inflammation among infants with disorganized histories of attachment.

Early life adversity is associated with adult elevations of inflammatory markers such as circulating levels of C-reactive protein (CRP). Few studies have examined inflammation levels during infancy nor the associations between sources of adversity and concurrent inflammation early in life. Existing evidence suggests that early adversity in the form of compromised caregiving relationships can embed itself into young children's biology with implications for lifelong development. This study examined the association between infants' histories of attachment with their mothers and salivary concentrations of CRP, all of which were assessed when infants were 17 months of age. Results show that infants with disorganized attachments histories and those exhibiting disorganized and avoidant regulatory behaviors when faced with an attachment stressor were all associated with significantly elevated levels of salivary CRP. These results suggest that exposure to significant interpersonal adversity very early in life may engender a proinflamotry phenotype with life-long implications for health.

Autophagy Induction Results in Enhanced Anoikis Resistance in Models of Peritoneal Disease.

Peritoneal carcinomatosis and peritoneal sarcomatosis is a potential complication of nearly all solid tumors and results in profoundly increased morbidity and mortality. Despite the ubiquity of peritoneal carcinomatosis/peritoneal sarcomatosis, there are no clinically relevant targeted therapies for either its treatment or prevention. To identify potential therapies, we developed in vitro models of peritoneal carcinomatosis/peritoneal sarcomatosis using tumor cell lines and patient-derived spheroids (PDS) that recapitulate anoikis resistance and spheroid proliferation across multiple cancer types. Epithelial- and mesenchymal-derived cancer cell lines (YOU, PANC1, HEYA8, CHLA10, and TC71) were used to generate spheroids and establish growth characteristics. Differential gene expression analyses of these spheroids to matched adherent cells revealed a consensus spheroid signature. This spheroid signature discriminates primary tumor specimens from tumor cells found in ascites of ovarian cancer patients and in our PDS models. Key in this gene expression signature is BNIP3 and BNIP3L, known regulators of autophagy and apoptosis. Elevated BNIP3 mRNA expression is associated with poor survival in ovarian cancer patients and elevated BNIP3 protein, as measured by IHC, and is also associated with higher grade tumors and shorter survival. Pharmacologic induction of autophagy with rapamycin significantly increased spheroid formation and survival while decreasing the induction of apoptosis. In contrast, the autophagy inhibitor hydroxychloroquine abrogated spheroid formation with a clear increase in apoptosis. Modulation of BNIP3 and the critical autophagy gene Beclin-1 (BECN1) also caused a significant decrease in spheroid formation. Combined, these data demonstrate how modulation of BNIP3-related autophagy, in PDS and in vitro spheroid models, alters the survival and morphology of spheroids. IMPLICATIONS: Development of BNIP3/BNIP3L-targeting agents or autophagy-targeting agents may reduce morbidity and mortality associated with peritoneal carcinomatosis and sarcomatosis. Mol Cancer Res; 15(1); 26-34. ©2016 AACR.

Analysis of Layperson Tourniquet Application Using a Novel Color-Coded Device.

OBJECTIVE: To determine whether a color-coded tourniquet designed for public use increases successful tourniquet application by laypeople. METHODS: This was a randomized study conducted on April 25, 2015. The study occurred during the Maryland Day activity at the University of Maryland in College Park, Maryland. Investigators recruited participants with posters displayed at major crosswalks around a central testing area. A total of 157 volunteers aged 18 years or older and without prior military service or medical training were enrolled. A participant stood in front of a waist-down mannequin with an isolated leg injury while an investigator read aloud a mass causality scenario. The investigator then asked the participant to apply a tourniquet to the mannequin's leg. All participants received a 4-step illustrated just-in-time (JiT) instruction card designed to facilitate layperson tourniquet application. Test participants received a color-coded tourniquet designed for layperson use with instructions printed on the device. Control participants received a black Combat Application Tourniquet (C-A-T; Composite Resources, Rock Hill, SC). Participants were randomized in a 1:1 ratio in blocks of 50. The primary outcome was the proportion of successful tourniquet applications by those who received color-coded tourniquets compared to those who received black tourniquets. Secondary outcomes included validation of previous data analyzing layperson success with tourniquet application, time for successful placement, reasons for failed applications, and participant self-willingness and comfort using tourniquets. We also analyzed demographic data on the study population and inter-rater reliability regarding the assessment of successful tourniquet application. RESULTS: Participants supplied with color-coded tourniquets successfully placed the device 51.38% of the time, compared to 44.71% of the time for controls using a black tourniquet (risk ratio: 1.15; 95% confidence interval: 0.83-1.59; P=0.404). Participants' self-reported willingness to use a tourniquet rose from 40.8% before the study to 80.3% after the study (P<0.05). CONCLUSIONS: The color-coded device did not significantly increase laypeople's proportion of successful tourniquet applications when compared with a standard black device. However, this study reproduced pilot study data showing that laypeople can successfully apply tourniquets about half the time if provided JiT instructions. Age, sex, race, income, and highest level of education were not found to impact one's ability to properly apply a tourniquet. Laypeople's willingness to apply tourniquets doubled to 80% after brief exposure to the device. These results affirm the feasibility of engaging laypeople as immediate lifesavers of trauma victims and justify further efforts to boost rates of proper application.

Characterization of the effects of heat stress on the DNA-intercalating dye EvaGreen for potential use with the joint biological agent identification and diagnostic system.

Although advances in real-time polymerase chain reaction (PCR) technology and equipment have facilitated field research, only a limited selection of reagents do not require cold storage. This study explored the temperature stability of the commercially available DNA-intercalating dye EvaGreen after exposure to a spectrum of temperatures for 176 days by analyzing quantification cycle (Cq) and end fluorescence levels during amplification of the invA gene of Salmonella typhimurium. To further characterize potential dye stability, the effects of small differences in dye volume were examined and dye samples were subjected to an Air Force deployment to the Middle East. Significant differences in Cq and end fluorescence were found; however, the magnitude of mean Cq differences was less than one cycle and the magnitude of mean fluorescence differences was less than that attributable to a difference of 0.25 µL of dye per 25 µL reaction. Liquid EvaGreen dye may thus be stable at temperatures as high as 65°C for up to 6 months for use in real-time PCR. These results warrant further investigation by using liquid EvaGreen dye to adapt traditional lab-based real-time PCR assays for Joint Biological Agent Identification and Diagnostic System use and testing the assays in the field.

Transglutaminase-2 mediates calcium-regulated crosslinking of the Y-box 1 (YB-1) translation-regulatory protein in TGFß1-activated myofibroblasts.

Myofibroblast differentiation is required for wound healing and accompanied by activation of smooth muscle α-actin (SMαA) gene expression. The stress-response protein, Y-box binding protein-1 (YB-1) binds SMαA mRNA and regulates its translational activity. Activation of SMαA gene expression in human pulmonary myofibroblasts by TGFß1 was associated with formation of denaturation-resistant YB-1 oligomers with selective affinity for a known translation-silencer sequence in SMαA mRNA. We have determined that YB-1 is a substrate for the protein-crosslinking enzyme transglutaminase 2 (TG2) that catalyzes calcium-dependent formation of covalent γ-glutamyl-isopeptide linkages in response to reactive oxygen signaling. TG2 transamidation reactions using intact cells, cell lysates, and recombinant YB-1 revealed covalent crosslinking of the 50 kDa YB-1 polypeptide into protein oligomers that were distributed during SDS-PAGE over a 75-250 kDa size range. In vitro YB-1 transamidation required nanomolar levels of calcium and was enhanced by the presence of SMαA mRNA. In human pulmonary fibroblasts, YB-1 crosslinking was inhibited by (a) anti-oxidant cystamine, (b) the reactive-oxygen antagonist, diphenyleneiodonium, (c) competitive inhibition of TG2 transamidation using the aminyl-surrogate substrate, monodansylcadaverine, and (d) transfection with small-interfering RNA specific for human TG2 mRNA. YB-1 crosslinking was partially reversible as a function of oligomer-substrate availability and TG2 enzyme concentration. Intracellular calcium accumulation and peroxidative stress in injury-activated myofibroblasts may govern SMαA mRNA translational activity during wound healing via TG2-mediated crosslinking of the YB-1 mRNA-binding protein.

Y-box binding protein-1 implicated in translational control of fetal myocardial gene expression after cardiac transplant.

Peri-transplant surgical trauma and ischemia/reperfusion injury in accepted murine heterotopic heart grafts has been associated with myofibroblast differentiation, cardiac fibrosis and biomechanical-stress activation of the fetal myocardial smooth muscle α-actin (SMαA) gene. The wound-healing agonists, transforming growth factor ß1 and thrombin, are known to coordinate SMαA mRNA transcription and translation in activated myofibroblasts by altering the subcellular localization and mRNA-binding affinity of the Y-box binding protein-1 (YB-1) cold-shock domain (CSD) protein that governs a variety of cellular responses to metabolic stress. YB-1 accumulated in polyribosome-enriched regions of the sarcoplasm proximal to cardiac intercalated discs in accepted heart grafts. YB-1 binding to a purine-rich motif in exon 3 of SMαA mRNA that regulates translational efficiency increased substantially in perfusion-isolated, rod-shaped adult rat cardiomyocytes during phenotypic de-differentiation in the presence of serum-derived growth factors. Cardiomyocyte de-differentiation was accompanied by the loss of a 60 kDa YB-1 variant that was highly expressed in both adult myocardium and freshly isolated myocytes and replacement with the 50 kDa form of YB-1 (p50) typically expressed in myofibroblasts that demonstrated sequence-specific interaction with SMαA mRNA. Accumulation of p50 YB-1 in reprogrammed, de-differentiated myocytes was associated with a 10-fold increase in SMαA protein expression. Endomyocardial biopsies collected from patients up to 14 years after heart transplant showed variable yet coordinately elevated expression of SMαA and p50 YB-1 protein and demonstrable p50 YB-1:SMαA mRNA interaction. The p60 YB-1 variant in human heart graft samples, but neither mouse p60 nor mouse or human p50, reacted with an antibody specific for the phosphoserine 102 modification in the YB-1 CSD. Modulation of YB-1 subcellular compartmentalization and mRNA-binding activity may be linked with reprogramming of contractile protein gene expression in ventricular cardiomyocytes that could contribute to maladaptive remodeling in accepted, long-term heart grafts.

pH induced swelling of PVP microgel particles--a first order phase transition?

The pH-induced swelling of poly(2-vinylpyridine) microgel particles was studied using dynamic light scattering. The increase in particle diameter with decreasing pH was modeled using a well-established thermodynamic model for microgel swelling. The Maxwell construction was applied to extend the model and yield a prediction for a pH-responsive microgel across the entire pH range. The model predicts a first order phase transition for polymer-solvent combinations with a Flory interaction parameter, χ, greater than a critical value χ(c). The modified theory compared favorably to the dynamic light scattering data for the hydrodynamic diameter of microgel particles based on 2-vinylpyridine at different pH values. In particular, the swelling transition is both predicted theoretically and observed experimentally to occur at a pH lower than the pK(a) of the polymer.

Comparison of immune responses and protective efficacy of intranasal prime-boost immunization regimens using adenovirus-based and CpG/HH2 adjuvanted-subunit vaccines against genital Chlamydia muridarum infection.

An efficacious Chlamydia vaccine is urgently needed to control Chlamydia infections. Heterologous prime-boost vaccination regimens are emerging as a promising strategy for preventing intracellular viral and bacterial infections. However, it remains to be determined if this regimen would be a feasible and effective approach for Chlamydia infection. In this study, we examined the immune response and the protective efficacy induced by various vaccination regimens using a recombinant adenovirus vector expressing the Chlamydia antigen CPAF (AdCPAF) and recombinant CPAF (rCPAF) subunit vaccines formulated with CpG oligodeoxynucleotides and/or a synthetic immunomodulatory peptide HH2 as adjuvants. A single dose of AdCPAF stimulated potent antibody production but weak cellular immune responses in mice. A booster rCPAF vaccine formulated with both CpG and HH2, but not CpG alone or HH2 alone, showed robust adjuvant effects on induction of Th1-biased cellular immune responses in mice primed with AdCPAF. In contrast, a homologous regimen using rCPAF/CpG/HH2 subunit vaccine for both priming and boosting induced a weak antibody response, but potent cellular immunity with a mixed Th1/Th17 profile. Despite the disparities observed in humoral and cellular immune responses, both the heterologous and homologous prime-boost regimens conferred significant immune protection against genital Chlamydia muridarum challenge in C3H/HeN and BALB/c mice.

Serum response factor neutralizes Pur alpha- and Pur beta-mediated repression of the fetal vascular smooth muscle alpha-actin gene in stressed adult cardiomyocytes.

Mouse hearts subjected to repeated transplant surgery and ischemia-reperfusion injury develop substantial interstitial and perivascular fibrosis that was spatially associated with dysfunctional activation of fetal smooth muscle alpha-actin (SM alpha A) gene expression in graft ventricular cardiomyocytes. Compared with cardiac fibroblasts in which nuclear levels of the Sp1 and Smad 2/3 transcriptional-activating proteins increased markedly after transplant injury, the most abundant SM alpha A gene-activating protein in cardiomyocyte nuclei was serum response factor (SRF). Additionally, cardiac intercalated discs in heart grafts contained substantial deposits of Pur alpha, an mRNA-binding protein and known negative modulator of SRF-activated SM alpha A gene transcription. Activation of fetal SM alpha A gene expression in perfusion-isolated adult cardiomyocytes was linked to elevated binding of a novel protein complex consisting of SRF and Pur alpha to a purine-rich DNA element in the SM alpha A promoter called SPUR, previously shown to be required for induction of SM alpha A gene transcription in injury-activated myofibroblasts. Increased SRF binding to SPUR DNA plus one of two nearby CArG box consensus elements was observed in SM alpha A-positive cardiomyocytes in parallel with enhanced Pur alpha:SPUR protein:protein interaction. The data suggest that de novo activation of the normally silent SM alpha A gene in reprogrammed adult cardiomyocytes is linked to elevated interaction of SRF with fetal-specific CArG and injury-activated SPUR elements in the SM alpha A promoter as well as the appearance of novel Pur alpha protein complexes in both the nuclear and cytosolic compartments of these cells.

Induction of vascular smooth muscle alpha-actin gene transcription in transforming growth factor beta1-activated myofibroblasts mediated by dynamic interplay between the Pur repressor proteins and Sp1/Smad coactivators.

The mouse vascular smooth muscle alpha-actin (SMA) gene enhancer is activated in fibroblasts by transforming growth factor beta1 (TGFbeta1), a potent mediator of myofibroblast differentiation and wound healing. The SMA enhancer contains tandem sites for the Sp1 transcriptional activator protein and Puralpha and beta repressor proteins. We have examined dynamic interplay between these divergent proteins to identify checkpoints for possible control of myofibroblast differentiation during chronic inflammatory disease. A novel element in the SMA enhancer named SPUR was responsible for both basal and TGFbeta1-dependent transcriptional activation in fibroblasts and capable of binding Sp1 and Pur proteins. A novel Sp1:Pur:SPUR complex was dissociated when SMA enhancer activity was increased by TGFbeta1 or Smad protein overexpression. Physical association of Pur proteins with Smad2/3 was observed as was binding of Smads to an upstream enhancer region that undergoes DNA duplex unwinding in TGFbeta1-activated myofibroblasts. Purbeta repression of the SMA enhancer could not be relieved by TGFbeta1, whereas repression mediated by Puralpha was partially rescued by TGFbeta1 or overexpression of Smad proteins. Interplay between Pur repressor isoforms and Sp1 and Smad coactivators may regulate SMA enhancer output in TGFbeta1-activated myofibroblasts during episodes of wound repair and tissue remodeling.