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Abstract Colorectal cancer (CRC) is a devastating disease, ranking as the second leading cause of cancer-related deaths worldwide. Immune checkpoint inhibitors (ICIs) have emerged as promising treatments; however, their efficacy is largely restricted to a subgroup of microsatellite instable (MSI) CRCs. In contrast, microsatellite stable (MSS) CRCs, which account for the majority of cases, exhibit variable and generally weaker response to ICIs, with only a subset demonstrating exceptional responsiveness. Identifying novel cancer-specific tissue (CST) markers predictive of immunotherapy response is crucial for refining patient selection and overcoming treatment resistance. In this study, we developed clinically relevant CRC organoids and autologous immune system interaction platforms to model ICI response. We conducted a comprehensive molecular characterization of both responder and non-responder models, identifying CST markers that predict ICI response. Validation of these findings was performed using an independent cohort of patient specimens through multiplex immunofluorescence. Furthermore, we demonstrated that knocking out a key gene from the identified predictive signature in resistant organoids restored immune sensitivity and induced T-cell-mediated apoptosis. Overall, our results provide novel insights into the mechanisms underlying immunotherapy resistance and suggest new markers for enhancing patient selection. These findings may pave the way for new therapeutic options in MSS patients, potentially broadening the cohort of individuals eligible for immunotherapy.

Abstract Aims Risk factors for asymptomatic echocardiographic abnormalities that predict symptomatic heart failure (HF) may provide insight into early mechanisms of HF pathogenesis. We examined risk factors associated with asymptomatic echocardiographic structural, systolic, and diastolic abnormalities, separately and in combination, and interactions between risk factors, in the prospective community‐based SCReening Evaluation of the Evolution of New HF (SCREEN‐HF) Study cohort of 3190 participants at increased risk of cardiovascular disease. Methods and results Inclusion criteria were age ≥ 60 years with one or more of hypertension, diabetes, ischaemic heart disease, valvular heart disease, abnormal heart rhythm, cerebrovascular disease, or renal impairment. Exclusion criteria were known HF, ejection fraction mild valve abnormality. Structural, systolic, and diastolic echocardiographic abnormalities were defined according to the Atherosclerosis Risk in Communities study criteria, and risk factors for asymptomatic structural, systolic, and diastolic abnormalities were identified using logistic regression analysis. In multivariable analysis, increased body mass index (BMI), non‐steroidal anti‐inflammatory drug therapy, and alcohol intake were risk factors for isolated structural abnormality, whereas male gender, increased heart rate, atrial fibrillation (AF), angiotensin‐converting enzyme inhibitor therapy, and obstructive sleep apnoea were associated with a lower risk. Moreover, male gender, smoking, increased systolic blood pressure, and physical inactivity were risk factors for isolated systolic abnormality, whereas increased pulse pressure and antihypertensive therapy were associated with a lower risk. Furthermore, increased age, blood pressure, amino‐terminal pro‐B‐type natriuretic peptide level, and warfarin therapy (associated with AF) were risk factors for isolated diastolic abnormality, whereas increased heart rate and triglyceride level (associated with BMI) were associated with a lower risk. The association of increased heart rate with lower risk of structural and diastolic abnormalities was independent of β‐blocker therapy. Interactions between risk factors differed for structural, systolic, and diastolic abnormalities. Conclusions The different risk factors for asymptomatic structural, systolic, and diastolic abnormalities that predict symptomatic HF, and the interactions between risk factors, illustrate how these structural, systolic, and diastolic abnormalities represent unique trajectories that lead to symptomatic HF. Improved understanding of these trajectories may assist in the design of HF prevention strategies.

Background: Despite well-established efficacy for patients with a cardiovascular diagnosis or event, exercise-based cardiac rehabilitation program participation and completion has remained alarmingly low due to both system-level barriers and patient-level factors. Patient mental health, particularly depression, is now recognized as significantly associated with reduced enrollment, participation, attendance, and completion of a cardiac rehabilitation program. More recently, anxiety sensitivity has emerged as an independent construct, related to but distinct from both depression and anxiety. Anxiety sensitivity has been reported to be adversely associated with participation in exercise and, thus, may be important for patients in cardiac rehabilitation. Accordingly, the objective of this study was to conduct a scoping review to summarize the evidence for associations between anxiety sensitivity and cardiovascular disease risk factors, exercise, and clinical outcomes in cardiac rehabilitation. Methods: A formal scoping review, following PRISMA-ScR guidelines, was undertaken. Searches of MEDLINE, Web of Science, CINAHL, PSYCINFO, and Scopus databases were conducted, supplemented by hand searches; studies published through December of 2020 were included. The initial screening was based on titles and abstracts and the second stage of screening was based on full text examination. Results: The final search results included 28 studies. Studies reported statistically significant associations between anxiety sensitivity and exercise, cardiovascular disease, and participation in cardiac rehabilitation. Many studies, however, were conducted in non-clinical, community-based populations; there were few studies conducted in cardiovascular disease and cardiac rehabilitation clinical patient populations. Additionally, significant gaps remain in our understanding of the sex-based differences in the complex relationships between anxiety sensitivity, exercise and cardiac rehabilitation. Conclusion: More research is needed to understand specific associations between anxiety sensitivity and clinical outcomes among clinical cardiovascular disease patients and participants in cardiac rehabilitation programs. Treatment of anxiety sensitivity to optimize clinical outcomes in cardiac rehabilitation programs should be investigated in future studies.

Background: Mediated by outcomes such as improved exercise capacity, cardiac rehabilitation (CR) reduces morbidity and mortality. For accuracy, an individual CR patient's change must be measured reliably, an issue not typically considered in practice. Drawing from psychometric theory, we calculated reliable change indices (RCIs), to measure individual CR patients’ true clinical change, apart from that from error and test practice/exposure, in exercise capacity, anxiety, and depression. Methods: Indirectly calculated exercise capacity (peak metabolic equivalents [METs]) and psychological symptoms were each measured twice, 1 week apart, by administering treadmill tests or the Hospital Anxiety and Depression Scale (HADS) to separate samples of 35 (mean age: 59.0 years; 6 women) and 96 (mean age: 64.4 years; 32 women) CR patients, respectively. Using test-retest reliability and mean difference scores from these samples to estimate error and practice/exposure effects, we calculated RCIs for a separate cohort (n = 2066; mean age: 62.0 years; 533 women) who completed 6-month CR, and compared change distributions (worsened/unchanged/improved) based on critical RCIs, mean and percent changes, cut-off scores, and standard deviations. Results: Practice/exposure effects were nonsignificant, except the mean HADS anxiety score decreased significantly (P ≤ 0.013; d = 0.17, small effect). Test-retest reliabilities were high (METs r = 0.934; HADS anxiety score r = 0.912; HADS depression score r = 0.90; P < 0.001). Among 2066 CR patients, RCI distributions differed (P < 0.001) from those of most other change criteria. Conclusions: Change ascertainment depends on criterion choice. A Canadian Cardiovascular Society CR quality indicator of increase by 0.5 MET may be too small to assess individuals’ functional capacity change. RCIs offer a pragmatic approach to benchmarking reliable change frequency, and pending further validation, could be used for feedback to individual patients. Résumé: Contexte: Grâce à des résultats tels que l'amélioration de la capacité d'exercice, la réadaptation cardiaque (RC) réduit la morbidité et la mortalité. Pour être précis, le changement de RC d'un patient doit être mesuré de manière fiable, un aspect qui n'est généralement pas pris en compte dans la pratique. En nous inspirant de la théorie psychométrique, nous avons déterminé des indices de changement fiables afin de mesurer le véritable changement clinique individuel des patients en RC en lien avec la capacité d'entraînement, l'anxiété et la dépression et indépendamment des erreurs et de la pratique du test/l'exposition au test. Méthodologie: La capacité d'entraînement calculée indirectement (l’équivalent métabolique maximum [MET]) et les symptômes psychologiques ont été mesurés deux fois, à une semaine d'intervalle, par le biais de tests sur tapis roulant ou de l’échelle de l’évaluation de l'anxiété et de la dépression en milieu hospitalier (HADS), auxquels ont respectivement été soumis des échantillons de 35 personnes en RC (âge moyen : 59,0 ans; 6 femmes) et de 96 personnes en RC (âge moyen : 64,4 ans; 32 femmes). En recourant à la fiabilité test-retest et aux écarts moyens dans les scores de ces échantillons pour faire une approximation des effets d'erreur et de pratique/exposition, nous avons déterminé des indices de changement fiables pour une cohorte distincte (n = 2 066; âge moyen : 62,0 ans; 533 femmes) qui a terminé une RC de six mois, et comparé la répartition des changements (aggravé/inchangé/amélioré) sur la base des indices de changement fiables critiques, des changements moyens et en pourcentage, des scores-seuils et des écarts types. Résultats: Les effets de la pratique/exposition étaient peu significatifs, sauf une diminution du score moyen de l'anxiété sur l’échelle HADS (p ≤ 0,013; d = 0,17, petit effet). La fiabilité test-retest était élevée (MET, r = 0,934; score de l'anxiété sur l’échelle HADS, r = 0,912; score de la dépression sur l’échelle HADS, r = 0,90; p < 0,001). Parmi les 2 066 patients en RC, la répartition des indices de changement fiables différait (p < 0,001) de celle de la plupart des autres critères de changement. Conclusions: Le constat du changement dépend du choix du critère. Une augmentation de 0,5 MET selon l'indicateur de qualité en RC de la Société canadienne de cardiologie est peut-être trop faible pour évaluer le changement de capacité fonctionnelle des individus. Les indices de changement fiables offrent une approche pragmatique pour comparer la fréquence du changement fiable et, en attendant une vérification plus poussée, ils pourraient être utilisés comme rétroaction fournie individuellement aux patients.

Abstract Background Given the age-related decline in glomerular filtration rate (GFR) in healthy individuals, we examined the association of all-cause death or cardiovascular event with the Kidney age - Chronological age Difference (KCD) score, whereby an individual’s kidney age is estimated from their estimated GFR (eGFR) and the age-dependent eGFR decline reported for healthy living potential kidney donors. Methods We examined the association between death or cardiovascular event and KCD score, age-dependent stepped eGFR criteria (eGFRstep), and eGFR

Abstract Aim Heart failure (HF) incidence increases markedly with age. We examined age‐associated longitudinal change in cardiac structure and function, and their prediction by age and cardiovascular disease (CVD) risk factors, in a community‐based cohort aged ≥60 years at increased CVD risk but without HF. Methods and results CVD risk factors were recorded in 3065 participants who underwent a baseline echocardiographic examination, of whom 2358 attended a follow‐up examination 3.8 [median, inter‐quartile range (IQR) 3.5, 4.2] years later. Median age was 71 (IQR 67, 76) years and 55% of participants were male. Age was associated with longitudinal increase in left ventricular (LV) mass index (LVMI); decrease in LV volumes; increase in LV ejection fraction; decrease in mitral annular systolic velocity; decrease in diastolic function (decreased mitral early diastolic annular velocity (e′); and increase in left atrial volume index, mitral peak early diastolic flow velocity (E)/e′ ratio, and tricuspid regurgitant velocity (TRVmax) in men and women, except for TRVmax in men). In multivariable analysis, longitudinal increase in LVMI was explained by CVD risk factors alone, whereas age, together with CVD risk factors, independently predicted longitudinal change in all other echocardiographic parameters. CVD risk factors were differentially associated with longitudinal change in different echocardiographic parameters. Conclusions Whereas the increase in LVMI with age was explained by CVD risk factors alone, age, together with risk factors, independently predicted longitudinal change in all other echocardiographic parameters, providing evidence for age‐specific mechanisms of change in cardiac structure and function as people age. Age‐associated change in LVMI, LV volumes, and diastolic function resembled what might be expected for the evolution of HF with preserved ejection fraction. Given the differential association of different CVD risk factors with longitudinal change in different echocardiographic parameters, therapies aimed at attenuation of age‐associated change in cardiac structure and function, and HF evolution, will likely need to address multiple CVD risk factors.

Abstract Aims We sought to determine which echocardiographic markers of left ventricular (LV) remodeling and diastolic dysfunction can contribute as incremental and independent prognostic information in addition to current clinical risk markers of ischemic LV systolic dysfunction in the Surgical Treatment for Ischemic Heart Failure (STICH) trial. Methods and results The cohort consisted of 1511 of 2136 patients in STICH for whom baseline transmitral Doppler (E/A ratio) could be measured by an echocardiographic core laboratory blinded to treatment and outcomes, and prognostic value of echocardiographic variables was determined by a Cox regression model. E/A ratio was the most significant predictor of mortality amongst diastolic variables with lowest mortality for E/A closest 0.8, although mortality was consistently low for E/A 0.6 to 1.0. Mortality increased for E/A 1.0 up to approximately 2.3, beyond which there was no further increase in risk. Larger LV end-systolic volume index (LVESVI) and E/A 1.0 had incremental negative effects on mortality when added to a clinical multivariable model, where creatinine, LVESVI, age, and E/A ratio accounted for 74% of the prognostic information for predicting risk. LVESVI and E/A ratio were stronger predictors of prognosis than New York Heart Association functional class, anemia, diabetes, history of atrial fibrillation, and stroke. Conclusions Echocardiographic markers of advanced LV remodeling and diastolic dysfunction added incremental prognostic value to current clinical risk markers. LVESVI and E/A ratio outperformed other markers and should be considered as standard in assessing risks in ischemic heart failure. E/A closest to 0.8 was the most optimal filling pattern.

Abstract Aims We investigated whether addition of diastolic dysfunction (DD) and longitudinal strain (LS) to Stage B heart failure (SBHF) criteria (structural or systolic abnormality) improves prediction of symptomatic HF in participants of the SCReening Evaluation of the Evolution of New Heart Failure study, a self‐selected population at increased cardiovascular disease risk recruited from members of a health insurance fund in Melbourne and Shepparton, Australia. Both American Society of Echocardiography and European Association of Cardiovascular Imaging (ASE/EACVI) criteria and age‐specific Atherosclerosis Risk in Communities (ARIC) study criteria, for SBHF and DD, and ARIC criteria for abnormal LS, were examined. Methods and results Inclusion criteria were age ≥60 years with one or more of self‐reported ischaemic or other heart disease, irregular or rapid heart rhythm, cerebrovascular disease, renal impairment, or treatment for hypertension or diabetes for ≥2 years. Exclusion criteria were known HF, or ejection fraction mild valve abnormality detected on previous echocardiography or other imaging. Echocardiography was performed in 3190 participants who were followed for a median of 3.9 (interquartile range: 3.4, 4.5) years after echocardiography. Symptomatic HF was diagnosed in 139 participants at a median of 3.1 (interquartile range: 2.1, 3.9) years after echocardiography. ARIC structural, systolic, and diastolic abnormalities predicted HF in univariate and multivariable proportional hazards analyses, whereas ASE/EACVI structural and systolic, but not diastolic, abnormalities predicted HF. ARIC and ASE/EACVI SBHF criteria predicted HF with sensitivities of 81% and 55%, specificities of 39% and 76%, and C statistics of 0.60 (95% confidence interval: 0.57, 0.64) and 0.66 (0.61, 0.71), respectively. Adding ARIC DD to SBHF increased sensitivity to 94% with specificity of 24% and C statistic of 0.59 (0.57, 0.61), whereas addition of ASE/EACVI DD to SBHF increased sensitivity to 97% but reduced specificity to 9% and the C statistic to 0.52 (0.50, 0.54, P

Abstract Background Burkholderia pseudomallei is a human pathogen causing severe infections in tropical and subtropical regions and is classified as a bio-threat agent. B. thailandensis strain E264 has been proposed as less pathogenic surrogate for understanding the interactions of B. pseudomallei with host cells. Results We show that, unlike B. thailandensis strain E264, the pattern of growth of B. thailandensis strain E555 in macrophages is similar to that of B. pseudomallei. We have genome sequenced B. thailandensis strain E555 and using the annotated sequence identified genes and proteins up-regulated during infection. Changes in gene expression identified more of the known B. pseudomallei virulence factors than changes in protein levels and used together we identified 16% of the currently known B. pseudomallei virulence factors. These findings demonstrate the utility of B. thailandensis strain E555 to study virulence of B. pseudomallei. Conclusions A weakness of studies using B. thailandensis as a surrogate for B. pseudomallei is that the strains used replicate at a slower rate in infected cells. We show that the pattern of growth of B. thailandensis strain E555 in macrophages closely mirrors that of B. pseudomallei. Using this infection model we have shown that virulence factors of B. pseudomallei can be identified as genes or proteins whose expression is elevated on the infection of macrophages. This finding confirms the utility of B. thailandensis strain E555 as a surrogate for B. pseudomallei and this strain should be used for future studies on virulence mechanisms.

Objective: To develop a bank of text messages for a lifestyle-based self-management intervention for people with low back pain (LBP). Design: Iterative development process. Setting: Community and primary care. Participants: Fifteen researchers, clinicians, and consumer representatives participated in the concept and initial content development phase. Twelve experts (researchers and clinicians) and 12 consumers participated in the experts and consumers review phase. Full study sample of participants was N=39. Interventions: Not applicable. Main Outcome Measures: We first conducted two 2-hour workshops to identify important domains for people with LBP, sources of content, appropriate volume, and timing of the messages. The messages were then drafted by a team of writers. Second, we invited expert researchers and clinicians to review and score the messages using a 5-item psychometric scale according to (1) the appropriateness of the content and (2) the likelihood of clinical effectiveness and to provide written feedback. Messages scoring ≤8 out of 10 points would be modified accordingly. Consumers were invited to review the messages and score them using a 5-item psychometric scale according to the utility of the content, the understanding of the content, and language acceptability and to provide feedback. Messages scoring ≤12 out of 15 points would be improved. Results: Exercise, education, mood, sleep, use of care, and medication domains were identified and 82 domain-specific evidence-based messages were written. Messages received a mean score of 8.3 out of 10 points by experts. Twenty-nine messages were modified accordingly. The mean score of the messages based on consumers feedback was of 12.5 out of 15 points. Thirty-six messages were improved. Conclusions: We developed a bank of text messages for an evidence-based self-management intervention using a theory-based, iterative, codesign process with researchers, consumers, and clinicians. This article provides scientific support for future development of text message interventions within the pain field.

Abstract Background The reasons for reduced exercise capacity in diabetes mellitus (DM) remains incompletely understood, although diastolic dysfunction and diabetic cardiomyopathy are often favored explanations. However, there is a paucity of literature detailing cardiac function and reserve during incremental exercise to evaluate its significance and contribution. We sought to determine associations between comprehensive measures of cardiac function during exercise and maximal oxygen consumption ($$\dot{V}O_{2}$$ V˙O2 peak), with the hypothesis that the reduction in exercise capacity and cardiac function would be associated with co-morbidities and sedentary behavior rather than diabetes itself. Methods This case–control study involved 60 subjects [20 with type 1 DM (T1DM), 20 T2DM, and 10 healthy controls age/sex-matched to each diabetes subtype] performing cardiopulmonary exercise testing and bicycle ergometer echocardiography studies. Measures of biventricular function were assessed during incremental exercise to maximal intensity. Results T2DM subjects were middle-aged (52 ± 11 years) with a mean T2DM diagnosis of 12 ± 7 years and modest glycemic control (HbA1c 57 ± 12 mmol/mol). T1DM participants were younger (35 ± 8 years), with a 19 ± 10 year history of T1DM and suboptimal glycemic control (HbA1c 65 ± 16 mmol/mol). Participants with T2DM were heavier than their controls (body mass index 29.3 ± 3.4 kg/m2 vs. 24.7 ± 2.9, P = 0.001), performed less exercise (10 ± 12 vs. 28 ± 30 MET hours/week, P = 0.031) and had lower exercise capacity ($$\dot{V}O_{2}$$ V˙O2 peak = 26 ± 6 vs. 38 ± 8 ml/min/kg, P

Murine dendritic cells, when pulsed with heat-killed Burkholderia pseudomallei and used to immunise naïve mice, have previously been shown to induce protective immunity in vivo. We have now demonstrated the in vitro priming of naïve human T cells against heat-killed B. pseudomallei, by co-culture with syngeneic B. pseudomallei-pulsed dendritic cells. Additionally, we have enriched the DC fraction such that a study of the differential response induced by pulsed DCs of either myeloid or plasmacytoid lineage in syngeneic human T cells was achievable. Whilst both mDCs and pDCs were activated by pulsing, the mDCs contributed the major response to B. pseudomallei with the expression of the migration marker CCR7 and a significantly greater secretion of the proinflammatory TNFα and IL1β. When these DC factions were combined and used to prime syngeneic T cells, a significant proliferation was observed in the CD4+ fraction. Here, we have achieved human T cell priming in vitro with unadjuvanted B. pseudomallei, the causative organism of melioidosis, for which there is currently no approved vaccine. We propose that the approach we have taken could be used to screen for the human cellular response to candidate vaccines and formulations, in order to enhance the cell-mediated immunity required to protect against this intracellular pathogen and potentially more broadly against other, difficult-to-treat intracellular pathogens. To date, the polysaccharide capsule of B. pseudomallei, fused to a standard carrier protein, e.g., Crm, looks a likely vaccine candidate. Dendritic cells (DCs), providing, as they do, the first line of defence to infection, process and present microbial products to the immune system to direct downstream immune responses. Here, we have sought to use DCs ex vivo to identify immunogenic products from heat-killed B. pseudomallei. Using practical volumes of fresh human donor blood, we show that heat-killed B. pseudomallei activated and stimulated the expression of pro-inflammatory cytokines TNF-α, IL-1β and IL-6 from both myeloid and plasmacytoid DCs. Furthermore, B. pseudomallei-pulsed DCs cultured with naïve syngeneic T cells ex vivo, induced the activation and proliferation of the CD4+ T-cell population, which was identified by cell surface marker staining using flow cytometry. Thus, both DC subsets are important for driving primary T helper cell responses to B. pseudomallei in healthy individuals and have the potential to be used to identify immunogenic components of B. pseudomallei for future therapies and vaccines.

Historically, disease progression in animal models of Q fever has been carried out using PCR to monitor the presence of Coxiella burnetii DNA in the host. However, the colonization and dissemination of other bacterial infections in animal models are tracked using viable counts, enabling an accurate assessment of viable bacterial load within tissues. Following recent advances in the culture methods, it has become possible to do the same with C. burnetii. Here we compare and contrast the different information gained by using PCR or viable counts to study this disease. Viable bacteria were cleared from organs much faster than previously reported when assessed by bacterial DNA, but weight loss and clinical signs improved while animals were still heavily infected.

The concept of oxidative stress, the factors responsible for oxidative stress and on those substances in foods that may have in vivo effects in terms of decreasing the adverse effects of reactive oxygen species (ROS) and reactive nitrogen species (RNS) on normal physiological function are reviewed. Methods of analysis of dietary redox reactive compounds and the role of dietary antioxidants in preventing disease are discussed. Literature related to the role of consumption of dietary bioactives (antioxidants) on the prevention of adverse health outcomes, with clinical trials in human subjects as the primary focus is reviewed.

There is a requirement for an efficacious vaccine to protect people against infection from Francisella tularensis, the etiological agent of tularemia. The lipopolysaccharide (LPS) of F. tularensis is suboptimally protective against a parenteral lethal challenge in mice. To develop a more efficacious subunit vaccine, we have used a novel biosynthetic technique of protein glycan coupling technology (PGCT) that exploits bacterial N-linked glycosylation to recombinantly conjugate F. tularensis O-antigen glycans to the immunogenic carrier protein Pseudomonas aeruginosa exoprotein A (ExoA). Previously, we demonstrated that an ExoA glycoconjugate with two glycosylation sequons was capable of providing significant protection to mice against a challenge with a low-virulence strain of F. tularensis. Here, we have generated a more heavily glycosylated conjugate vaccine and evaluated its efficacy in a Fischer 344 rat model of tularemia. We demonstrate that this glycoconjugate vaccine protected rats against disease and the lethality of an inhalational challenge with F. tularensis Schu S4. Our data highlights the potential of this biosynthetic approach for the creation of next-generation tularemia subunit vaccines.

Red radish anthocyanins are relatively stable due to the acylation of anthocyanins with organic and phenolic acid moieties. The objective of this study was to create and identify a novel ethyl-bridged acylated anthocyanin with a stable violet color from red radishes in the presence of acetaldehyde and catechin. After incubation at ambient temperature for one week at 21 °C the reaction mixture with acetaldehyde and catechin turned from bright red to a vivid purple color. The newly formed compounds were tentatively identified with liquid chromatography-electrospray ionization-mass spectrometry. Color stability (CIE L*C*h) was monitored over six months at 21 °C and anthocyanin stability was evaluated by HPLC. Next, radishes were fermented by Zymomonas mobilis, a high acetaldehyde producing bacteria. Natural sources of catechin were used to replace the catechin standard, but the same purple color did not appear with the addition of cocoa powder or fava beans. The ethyliden-bridged radish anthocyanins possess excellent color stability and could serve as a natural food colorant.

The effect of feeding cranberry pomace (CBP) on selected metabolic parameters associated with high fructose feeding (58% by weight) was investigated in growing Sprague–Dawley rats. Compared to a positive control (modified AIN93 diet), high fructose feeding increased fasting plasma insulin, cholesterol, and triacylglycerols (TAG), post-prandial plasma TAG as well as homeostatic assessment models of insulin resistance and β-cell function, but not weight gain, diet intake and efficiency, abdominal fat, oral glucose tolerance, and fasting and post-prandial plasma glucose and cholesterol levels. Inclusion of CBP was effective in minimizing or ameliorating some of the metabolic anomalies, such as increased fasting plasma insulin, cholesterol, and TAG level as well as decreasing insulin resistance, especially when extruded CBP was fed at 3% of the diet. Feeding HF diets was only partially effective in augmenting some of the metabolic factors associated with high fructose feeding, including triacylglycerolemia and insulin resistance, but not weight gain or abdominal obesity. Overall, CBP was effective in minimizing or ameliorating the metabolic anomalies observed in the current study, more so with 3% extruded CBP.

Burkholderia pseudomallei is the causative agent of the severe tropical disease melioidosis, which commonly presents as sepsis. The B. pseudomallei K96243 genome encodes eleven predicted autotransporters, a diverse family of secreted and outer membrane proteins often associated with virulence. In a systematic study of these autotransporters, we constructed insertion mutants in each gene predicted to encode an autotransporter and assessed them for three pathogenesis-associated phenotypes: virulence in the BALB/c intra-peritoneal mouse melioidosis model, net intracellular replication in J774.2 murine macrophage-like cells and survival in 45% (v/v) normal human serum. From the complete repertoire of eleven autotransporter mutants, we identified eight mutants which exhibited an increase in median lethal dose of 1 to 2-log10 compared to the isogenic parent strain (bcaA, boaA, boaB, bpaA, bpaC, bpaE, bpaF and bimA). Four mutants, all demonstrating attenuation for virulence, exhibited reduced net intracellular replication in J774.2 macrophage-like cells (bimA, boaB, bpaC and bpaE). A single mutant (bpaC) was identified that exhibited significantly reduced serum survival compared to wild-type. The bpaC mutant, which demonstrated attenuation for virulence and net intracellular replication, was sensitive to complement-mediated killing via the classical and/or lectin pathway. Serum resistance was rescued by in trans complementation. Subsequently, we expressed recombinant proteins of the passenger domain of four predicted autotransporters representing each of the phenotypic groups identified: those attenuated for virulence (BcaA), those attenuated for virulence and net intracellular replication (BpaE), the BpaC mutant with defects in virulence, net intracellular replication and serum resistance and those displaying wild-type phenotypes (BatA). Only BcaA and BpaE elicited a strong IFN-γ response in a restimulation assay using whole blood from seropositive donors and were recognised by seropositive human sera from the endemic area. To conclude, several predicted autotransporters contribute to B. pseudomallei virulence and BpaC may do so by conferring resistance against complement-mediated killing.

Aim. We examined endocannabinoids (ECs) in relation to bariatric surgery and the association between plasma ECs and markers of insulin resistance. Methods. A study of 20 participants undergoing bariatric surgery. Fasting and 2-hour plasma glucose, lipids, insulin, and C-peptide were recorded preoperatively and 6 months postoperatively with plasma ECs (AEA, 2-AG) and endocannabinoid-related lipids (PEA, OEA). Results. Gender-specific analysis showed differences in AEA, OEA, and PEA preoperatively with reductions in AEA and PEA in females postoperatively. Preoperatively, AEA was correlated with 2-hour glucose (r=0.55, P=0.01), HOMA-IR (r=0.61, P=0.009), and HOMA %S (r=-0.71, P=0.002). OEA was correlated with weight (r=0.49, P=0.03), waist circumference (r=0.52, P=0.02), fasting insulin (r=0.49, P=0.04), and HOMA-IR (r=0.48, P=0.05). PEA was correlated with fasting insulin (r=0.49, P=0.04). 2-AG had a negative correlation with fasting glucose (r=-0.59, P=0.04). Conclusion. Gender differences exist in circulating ECs in obese subjects. Females show changes in AEA and PEA after bariatric surgery. Specific correlations exist between different ECs and markers of obesity and insulin and glucose homeostasis.

Pro-inflammatory cytokines have been noted to increase following exercise but their relationship to exercise-induced cardiac dysfunction has not previously been investigated. We sought to evaluate whether exercise-induced cardiac dysfunction was associated with increases in cytokines, particularly the pro-inflammatory cytokines IL-1β, IL-12p70 and TNFα, which have been most implicated in cardiac pathology.40 well-trained endurance athletes underwent evaluation prior to and immediately following one of four endurance sporting events ranging from 3 to 11 hours duration. Cytokines (IL-1β, IL-6, IL-8, IL-10, IL-12p70 and TNFα) were analyzed by flow cytometry from serum samples collected within 50 minutes of race completion. Cardiac troponin (cTnI) and B-type natriuretic peptide were combined with an echocardiographic assessment of cardiac function, and a composite of cTnI > 0.04 μg/L, BNP increase > 10 ng/L and a decrease in right ventricular ejection (RVEF) > 10% were prospectively defined as evidence of myocardial dysfunction.Relative to baseline, IL-6 IL-8 and IL-10 increased 8.5-, 2.9-, and 7.1-fold, respectively, P

The Gram-negative bacterium Burkholderia pseudomallei causes melioidosis and is a CDC category B bioterrorism agent. Toll-like receptor (TLR)-2 impairs host defense during pulmonary B.pseudomallei infection while TLR4 only has limited impact. We investigated the role of TLRs in B.pseudomallei-lipopolysaccharide (LPS) induced inflammation. Purified B.pseudomallei-LPS activated only TLR2-transfected-HEK-cells during short stimulation but both HEK-TLR2 and HEK-TLR4-cells after 24 h. In human blood, an additive effect of TLR2 on TLR4-mediated signalling induced by B.pseudomallei-LPS was observed. In contrast, murine peritoneal macrophages recognized B.pseudomallei-LPS solely through TLR4. Intranasal inoculation of B.pseudomallei-LPS showed that both TLR4-knockout(-/-) and TLR2x4-/-, but not TLR2-/- mice, displayed diminished cytokine responses and neutrophil influx compared to wild-type controls. These data suggest that B.pseudomallei-LPS signalling occurs solely through murine TLR4, while in human models TLR2 plays an additional role, highlighting important differences between specificity of human and murine models that may have important consequences for B.pseudomallei-LPS sensing by TLRs and subsequent susceptibility to melioidosis.

Lipophilic cationic technetium-99m-complexes are widely used for myocardial perfusion imaging (MPI). However, inherent uncertainties in the supply chain of molybdenum-99, the parent isotope required for manufacturing 99Mo/99mTc generators, intensifies the need for discovery of novel MPI agents incorporating alternative radionuclides. Recently, germanium/gallium (Ge/Ga) generators capable of producing high quality 68Ga, an isotope with excellent emission characteristics for clinical PET imaging, have emerged. Herein, we report a novel 68Ga-complex identified through mechanism-based cell screening that holds promise as a generator-produced radiopharmaceutical for PET MPI.

[This corrects the article DOI: 10.1371/journal.pone.0079461.].

Melioidosis is a severe infectious disease caused by Burkholderia pseudomallei. It is refractory to antibiotic treatment and there is currently no licensed vaccine. In this report we detail the construction and protective efficacy of a polysaccharide-protein conjugate composed of B. pseudomallei lipopolysaccharide and the Hc fragment of tetanus toxin. Immunisation of mice with the lipopolysaccharide-conjugate led to significantly reduced bacterial burdens in the spleen 48 hours after challenge and afforded significant protection against a lethal challenge with B. pseudomallei. The conjugate generated significantly higher levels of antigen-specific IgG1 and IgG2a than in lipopolysaccharide-immunised mice. Immunisation with the conjugate also demonstrated a bias towards Th1 type responses, evidenced by high levels of IgG2a. In contrast, immunisation with unconjugated lipopolysaccharide evoked almost no IgG2a demonstrating a bias towards Th2 type responses. This study demonstrates the effectiveness of this approach in the development of an efficacious and protective vaccine against melioidosis.

The autotransporters are a large and diverse family of bacterial secreted and outer membrane proteins, which are present in many Gram-negative bacterial pathogens and play a role in numerous environmental and virulence-associated interactions. As part of a larger systematic study on the autotransporters of Burkholderia pseudomallei, the causative agent of the severe tropical disease melioidosis, we have constructed an insertion mutant in the bpss1439 gene encoding an unstudied predicted trimeric autotransporter adhesin. The bpss1439 mutant demonstrated a significant reduction in biofilm formation at 48 hours in comparison to its parent 10276 wild-type strain. This phenotype was complemented to wild-type levels by the introduction of a full-length copy of the bpss1439 gene in trans. Examination of the wild-type and bpss1439 mutant strains under biofilm-inducing conditions by microscopy after 48 hours confirmed that the bpss1439 mutant produced less biofilm compared to wild-type. Additionally, it was observed that this phenotype was due to low levels of bacterial adhesion to the abiotic surface as well as reduced microcolony formation. In a murine melioidosis model, the bpss1439 mutant strain demonstrated a moderate attenuation for virulence compared to the wild-type strain. This attenuation was abrogated by in trans complementation, suggesting that bpss1439 plays a subtle role in the pathogenesis of B. pseudomallei. Taken together, these studies indicate that BPSS1439 is a novel predicted autotransporter involved in biofilm formation of B. pseudomallei; hence, this factor was named BbfA, Burkholderia biofilm factor A.

BACKGROUND: Obesity is associated with diastolic dysfunction, lower maximal myocardial blood flow, impaired myocardial metabolism and increased risk of heart failure. We examined the association between obesity, left ventricular filling pressure and myocardial structure. METHODS: We performed histological analysis of non-ischemic myocardium from 57 patients (46 men and 11 women) undergoing coronary artery bypass graft surgery who did not have previous cardiac surgery, myocardial infarction, heart failure, atrial fibrillation or loop diuretic therapy. RESULTS: Non-obese (body mass index, BMI, ≤ 30 kg/m(2), n=33) and obese patients (BMI >30 kg/m(2), n=24) did not differ with respect to myocardial total, interstitial or perivascular fibrosis, arteriolar dimensions, or cardiomyocyte width. Obese patients had lower capillary length density (1145 ± 239, mean ± SD, vs. 1371 ± 333 mm/mm(3), P=0.007) and higher diffusion radius (16.9 ± 1.5 vs. 15.6 ± 2.0 μm, P=0.012), in comparison with non-obese patients. However, the diffusion radius/cardiomyocyte width ratio of obese patients (0.73 ± 0.11 μm/μm) was not significantly different from that of non-obese patients (0.71 ± 0.11 μm/μm), suggesting that differences in cardiomyocyte width explained in part the differences in capillary length density and diffusion radius between non-obese and obese patients. Increased BMI was associated with increased pulmonary capillary wedge pressure (PCWP, P

Glycoconjugate-based vaccines have proved to be effective at producing long-lasting protection against numerous pathogens. Here, we describe the application of bacterial protein glycan coupling technology (PGCT) to generate a novel recombinant glycoconjugate vaccine. We demonstrate the conjugation of the Francisella tularensis O-antigen to the Pseudomonas aeruginosa carrier protein exotoxin A using the Campylobacter jejuni PglB oligosaccharyltransferase. The resultant recombinant F. tularensis glycoconjugate vaccine is expressed in Escherichia coli where yields of 3 mg l−1 of culture were routinely produced in a single-step purification process. Vaccination of BALB/c mice with the purified glycoconjugate boosted IgG levels and significantly increased the time to death upon subsequent challenge with F. tularensis subsp. holarctica. PGCT allows different polysaccharide and protein combinations to be produced recombinantly and could be easily applicable for the production of diverse glycoconjugate vaccines.

BACKGROUND: Heart failure is associated with abnormalities of myocardial structure, and plasma levels of the advanced glycation end-product (AGE) N(ε)-(carboxymethyl)lysine (CML) correlate with the severity and prognosis of heart failure. Aging is associated with diastolic dysfunction and increased risk of heart failure, and we investigated the hypothesis that diastolic dysfunction of aging humans is associated with altered myocardial structure and plasma AGE levels. METHODS: We performed histological analysis of non-ischemic left ventricular myocardial biopsies and measured plasma levels of the AGEs CML and low molecular weight fluorophores (LMWFs) in 26 men undergoing coronary artery bypass graft surgery who had transthoracic echocardiography before surgery. None had previous cardiac surgery, myocardial infarction, atrial fibrillation, or heart failure. RESULTS: The patients were aged 43-78 years and increasing age was associated with echocardiographic indices of diastolic dysfunction, with higher mitral Doppler flow velocity A wave (r = 0.50, P = 0.02), lower mitral E/A wave ratio (r = 0.64, P = 0.001), longer mitral valve deceleration time (r = 0.42, P = 0.03) and lower early diastolic peak velocity of the mitral septal annulus, e' (r = 0.55, P = 0.008). However, neither mitral E/A ratio nor mitral septal e' was correlated with myocardial total, interstitial or perivascular fibrosis (picrosirius red), immunostaining for collagens I and III, CML, and receptor for AGEs (RAGE), cardiomyocyte width, capillary length density, diffusion radius or arteriolar dimensions. Plasma AGE levels were not associated with age. However, plasma CML levels were associated with E/A ratio (r = 0.44, P = 0.04) and e' (r = 0.51, P = 0.02) and LMWF levels were associated with E/A ratio (r = 0.49, P = 0.02). Moreover, the mitral E/A ratio remained correlated with plasma LMWF levels in all patients (P = 0.04) and the mitral septal e' remained correlated with plasma CML levels in non-diabetic patients (P = 0.007) when age was a covariate. CONCLUSIONS: Diastolic dysfunction of aging was independent of myocardial structure but was associated with plasma AGE levels.

Bioluminescence imaging (BLI) of luciferase reporters in small animal models offers an attractive approach to monitor regulation of gene expression, signal transduction, and protein-protein interactions, as well as following tumor progression, cell engraftment, infectious pathogens, and target-specific drug action. Conventional BLI can be repeated within the same animal after bolus reinjections of a bioluminescent substrate. However, intervals between image acquisitions are governed by substrate pharmacokinetics and excretion, therefore restricting temporal resolution of reinjection protocols to the order of hours, limiting analyses of processes in vivo with short time constants. To eliminate these constraints, we examined use of implanted micro-osmotic pumps for continuous, long-term delivery of bioluminescent substrates. Pump-assisted d -luciferin delivery enabled BLI for ⩾ 7 days from a variety of luciferase reporters. Pumps allowed direct repetitive imaging at < 5-minute intervals of the pharmacodynamics of proteasome- and IKK-inhibiting drugs in mice bearing tumors stably expressing ubiquitin-firefly luciferase or IκBα-firefly luciferase fusion reporters. Circadian oscillations in the olfactory bulbs of transgenic rats expressing firefly luciferase under the control of the period1 promoter also were temporally resolved over the course of several days. We conclude that implanted pumps provide reliable, prolonged substrate delivery for high temporal resolution BLI, traversing complications of repetitive substrate injections.

The use of real-time reporters has accelerated our understanding of gene expression in vivo. This study examined the feasibility of a luciferase-based reporter to image spatiotemporal changes in fetal gene expression in utero. We chose to monitor Period1 ( Per1 ) because it is expressed broadly in the body and plays a role in circadian rhythmicity. Using rats carrying a Per1::luc transgene, we repetitively imaged fetuses in utero throughout gestation. We found that bioluminescence was specific to transgenic pups, increased dramatically on embryonic day 10 (10 days after successful mating), and continued to increase logarithmically until birth. Diurnal fluctuations in Per1 expression were apparent several days prior to birth. These results demonstrate the feasibility of in utero imaging of mammalian gene expression, tracking of fetal gene expression from the same litter, and early detection of mammalian clock gene expression. We conclude that luciferase-based reporters can provide a sensitive, noninvasive measure of in utero gene expression.

Bioluminescence, positron emission tomography (PET), and fluorescence modalities are currently available for noninvasive imaging in vivo, each with its own merits. To exploit the combined strengths of each and facilitate multimodality imaging, we engineered a dual-reporter construct in which firefly luciferase (FLuc) and a 12–amino acid nonstructural linker were fused in frame to the N-terminus of a mutant herpes simplex virus thymidine kinase (mNLS-SR39TK) kinetically enhanced for positron emission tomography (PET). Furthermore, a triple-reporter construct was developed in which monster green fluorescent protein (MGFP), a recently available enhanced fluorescent protein, was introduced into the fusion vector downstream of an internal ribosome entry site (IRES) to allow analysis by fluorescence microscopy or flow cytometry without compromising the specific activities of the upstream fusion components. FLuc bioluminescence was measured with a cooled charge-coupled device camera and mNLS-SR39TK activity by 9-[4-[ 18 F]fluoro-3-(hydroxymethyl) butyl guanine ( 18 F-FHBG) microPET or 3 H-penciclovir net accumulation. Importantly, HeLa cells transiently transfected with the FLuc-mNLS-SR39TK-IRES-MGFP triple reporter retained the same specific activities of the FLuc-mNLS-SR39TK heteroenzyme and the individual unfused enzymes with no change in protein half-lives. The presence of the IRES-MGFP modestly decreased upstream heteroprotein expression. In living mice, somatic gene transfer of a ubiquitin promoter-driven FLuc-mNLS-SR39TK-IRES-MGFP plasmid showed a > 1,000-fold increase in liver photon flux and a > 2-fold increase in liver retention of 18 F-FHBG by microPET compared with mice treated with control plasmid. Multifocal hepatocellular fluorescence was readily observed by standard confocal microscopy. This second-generation triple reporter incorporating enhanced components enables bioluminescence, PET, and fluorescence imaging of cells and living animals.

The best characterized mechanism of multidrug resistance (MDR) in cancer involves the MDR1 efflux transporter P-glycoprotein (Pgp). The positron-emitting radiotracer hexakis (2-methoxyisobutylisonitrile)- 94m Tc ( 94m Tc-MIBI) was synthesized and validated in cell transport studies as a substrate for MDR1 Pgp. In vivo small-scale PET imaging and biodistribution studies of mdr1a/1b (−/−) gene deleted and wild-type mice demonstrated the use of 94m Tc-MIBI to detect Pgp function. The reversal effect of a Pgp modulator was shown in tissue distribution studies of KB 3–1 (Pgp-) and KB 8–5 (Pgp+) tumor-bearing nude mice. The current 94m Tc-MIBI experiments parallel previous studies employing 99m Tc-MIBI, showing essentially identical performance of the two technetium radiotracers and providing biological validation of 94m Tc-MIBI for PET imaging of multidrug resistance.

Background: In patients with preserved left ventricular ejection fraction (LVEF), small ventricular size has been associated with reduced functional capacity, but its impact on clinical outcomes is unclear. Objectives: The goal of this study was to determine the relationship between small heart size and premature mortality within a large multicenter adult patient cohort with transthoracic echocardiographic examinations. Methods: We divided 366,484 individuals with LVEF ≥50% (including a subset of 279,442 individuals with high-normal LVEF ≥60%) by sex and increasing quartiles for LV end-diastolic volume (LVEDV), LVEDV indexed to body surface area (LVEDVi), and LV end-diastolic diameter to assess associations with 5-year mortality through linkage with the National Death Index. Results: During approximately 2 million person-years of follow-up, 65,241 deaths occurred. Increasing LV chamber size was associated with reduced odds of 5-year all-cause mortality, particularly for higher LVEF. As compared with the larger quartiles, the smallest cardiac size quartiles were associated with higher 5-year all-cause mortality, even after adjusting for age. The smallest LVEDVi quartile was associated with a 14% to 18% higher odds of 5-year all-cause mortality, with a greater effect with high-normal LVEF. There was a U-shaped relationship between LV chamber size and all-cause mortality. For cardiovascular-related mortality, females in the smallest LVEDVi quartile had a 17% increased odds of mortality, which increased to 30% in those with LVEF ≥60%. In men, there was no significant association between smallest cardiac size and cardiovascular-related mortality. Conclusions: In individuals with normal LVEF, small ventricular size is associated with increased mortality, particularly among females and those with higher LVEF.