Targeting Apollo-NADP+ to Image NADPH Generation in Pancreatic Beta-Cell Organelles.

NADPH/NADP+ redox state supports numerous reactions related to cell growth and survival; yet the full impact is difficult to appreciate due to organelle compartmentalization of NADPH and NADP+. To study glucose-stimulated NADPH production in pancreatic beta-cell organelles, we targeted the Apollo-NADP+ sensor by first selecting the most pH-stable version of the single-color sensor. We subsequently targeted mTurquoise2-Apollo-NADP+ to various organelles and confirmed activity in the cytoplasm, mitochondrial matrix, nucleus, and peroxisome. Finally, we measured the glucose- and glutamine-stimulated NADPH responses by single- and dual-color imaging of the targeted sensors. Overall, we developed multiple organelle-targeted Apollo-NADP+ sensors to reveal the prominent role of beta-cell mitochondria in determining NADPH production in the cytoplasm, nucleus, and peroxisome.

Psychological changes in successful completers of an HIV-tailored smoking cessation program: mood, attachment and self-efficacy.

High rates of cigarette smoking is the leading contributor to the increasing risk of cardiovascular disease (CVD) among people living with HIV (PLH). Relapse rates among PLH who quit smoking are high among those receiving standard care, which may be due to several unique social and psychological challenges PLH face when they attempt to quit smoking. The purpose of the current study was to examine change in relevant psychological factors in a subgroup of participants (n = 14) who remained smoke-free at 6-months follow-up in an HIV-tailored smoking cessation counselling program (N = 50). We examined self-reported depressive symptoms, attachment style and self-efficacy across 5 time points (baseline, quite date, 4, 12 and 24 weeks). At study baseline, mean depression scores fell above the clinical cut off of 16 (M = 16.31; SD = 13.53) on the Centre for Epidemiological Studies - Depression (CES-D) scale and fell below the clinical cut off at 24 weeks post quit date (M = 13.36; SD = 10.62). Results of multi-level modeling indicated a significant linear reduction in depressive symptoms and a significant linear improvement in self-efficacy to refrain from smoking across study visits. These results suggest that positive change in mood and self-efficacy may be helpful for PLH who remain smoke-free during a quit attempt.

Role of RIPK1 in SMAC mimetics-induced apoptosis in primary human HIV-infected macrophages.

Macrophages serve as viral reservoirs due to their resistance to apoptosis and HIV-cytopathic effects. We have previously shown that inhibitor of apoptosis proteins (IAPs) confer resistance to HIV-Vpr-induced apoptosis in normal macrophages. Herein, we show that second mitochondrial activator of caspases (SMAC) mimetics (SM) induce apoptosis of monocyte-derived macrophages (MDMs) infected in vitro with a R5-tropic laboratory strain expressing heat stable antigen, chronically infected U1 cells, and ex-vivo derived MDMs from HIV-infected individuals. To understand the mechanism governing SM-induced cell death, we show that SM-induced cell death of primary HIV-infected macrophages was independent of the acquisition of M1 phenotype following HIV infection of macrophages. Instead, SM-induced cell death was found to be mediated by IAPs as downregulation of IAPs by siRNAs induced cell death of HIV-infected macrophages. Moreover, HIV infection caused receptor interacting protein kinase-1 (RIPK1) degradation which in concert with IAP1/2 downregulation following SM treatment may result in apoptosis of macrophages. Altogether, our results show that SM selectively induce apoptosis in primary human macrophages infected in vitro with HIV possibly through RIPK1. Moreover, modulation of the IAP pathways may be a potential strategy for selective killing of HIV-infected macrophages in vivo.

Leveraging multimodal microscopy to optimize deep learning models for cell segmentation.

Deep learning provides an opportunity to automatically segment and extract cellular features from high-throughput microscopy images. Many labeling strategies have been developed for this purpose, ranging from the use of fluorescent markers to label-free approaches. However, differences in the channels available to each respective training dataset make it difficult to directly compare the effectiveness of these strategies across studies. Here, we explore training models using subimage stacks composed of channels sampled from larger, "hyper-labeled," image stacks. This allows us to directly compare a variety of labeling strategies and training approaches on identical cells. This approach revealed that fluorescence-based strategies generally provide higher segmentation accuracies but were less accurate than label-free models when labeling was inconsistent. The relative strengths of label and label-free techniques could be combined through the use of merging fluorescence channels and using out-of-focus brightfield images. Beyond comparing labeling strategies, using subimage stacks for training was also found to provide a method of simulating a wide range of labeling conditions, increasing the ability of the final model to accommodate a greater range of candidate cell labeling strategies.

Breathlessness catastrophizing relates to poorer quality of life in adults with cystic fibrosis.

BACKGROUND: Cystic Fibrosis (CF) is the most common genetic disease affecting children and young adults in Canada. It is a multi-system disease, however lung disease is largely responsible for mortality. Treatment advances have resulted in increased life expectancy and a subsequent need to better understand psychosocial issues associated with quality of life in adults living with CF. Emerging research suggests that anxiety and depression are related to poorer health-related quality of life (HRQoL) in CF patients, but there is little research examining cognitive processes, such as breathlessness catastrophizing. The present study addresses this gap in the literature. METHODS: Participants in this study are based on a convenience sample of patients recruited during their regular CF clinic appointments at a tertiary care center. Forty-five adults (Mage = 30.73 years) completed measures of lung function, depression, anxiety, pain, breathlessness catastrophizing, and HRQoL at one time point. RESULTS: Results of a hierarchical multiple regression indicate that increased breathlessness catastrophizing was related to poorer HRQoL, after controlling for lung function, depression, anxiety, and pain (p < .05). Depression, pain, and breathlessness catastrophizing all emerged as significant unique predictors of HRQoL. CONCLUSIONS: Breathlessness catastrophizing is a potential target for clinical intervention and might impact HRQoL. Further research on breathlessness catastrophizing in CF is warranted including longitudinal studies to examine the mechanisms by which breathlessness catastrophizing relates to HRQoL and treatment outcomes in CF.

An HIV-tailored quit-smoking counselling pilot intervention targeting depressive symptoms plus Nicotine Replacement Therapy.

Cardiovascular disease (CVD) rates among people living with HIV/AIDS (PHAs) are high. Rates of cigarette smoking, a leading contributor to CVD among PHAs, are 40-70% (2-3 times higher than the general population). Furthermore, PHAs have high rates of depression (40-60%), a risk factor for smoking cessation relapse. The current pilot study examined the effectiveness of a specifically tailored 5-session smoking cessation counselling programme for PHAs, which addressed depression, in combination with Nicotine Replacement Therapy (NRT) in a cohort of PHA smokers (n = 50). At 6-month follow-up, 28% of participants demonstrated biochemically verified abstinence from smoking. This result compares favourably to other quit-smoking intervention studies, particularly given the high percentage of HIV+ smokers with depression. At study baseline, 52% of HIV+ smokers scored above the clinical cut-off for depression on the Centre for Epidemiological Studies - Depression (CES-D) scale. HIV+ smokers with depression at study baseline demonstrated quantitatively lower depression at 6-month follow-up with a large effect size (d = 1), though it did not reach statistical significance (p = .058). Furthermore, those with depression were no more likely to relapse than those without depression (p = .33), suggesting that our counselling programme adequately addressed this significant barrier to smoking cessation among PHAs. Our pilot study indicates the importance of tailored programmes to help PHAs quit smoking, the significance of addressing depressive symptoms, and the need for tailored counselling programmes to enhance quit rates among PHAs.

Apollo-NADP(+): a spectrally tunable family of genetically encoded sensors for NADP(+).

NADPH-dependent antioxidant pathways have a critical role in scavenging hydrogen peroxide (H2O2) produced by oxidative phosphorylation. Inadequate scavenging results in H2O2 accumulation and can cause disease. To measure NADPH/NADP(+) redox states, we explored genetically encoded sensors based on steady-state fluorescence anisotropy due to FRET (fluorescence resonance energy transfer) between homologous fluorescent proteins (homoFRET); we refer to these sensors as Apollo sensors. We created an Apollo sensor for NADP(+) (Apollo-NADP(+)) that exploits NADP(+)-dependent homodimerization of enzymatically inactive glucose-6-phosphate dehydrogenase (G6PD). This sensor is reversible, responsive to glucose-stimulated metabolism and spectrally tunable for compatibility with many other sensors. We used Apollo-NADP(+) to study beta cells responding to oxidative stress and demonstrated that NADPH is significantly depleted before H2O2 accumulation by imaging a Cerulean-tagged version of Apollo-NADP(+) with the H2O2 sensor HyPer.

HIV prevention in action on the football field: the WhizzKids United program in South Africa.

The Africaid Trust is a grassroots South African non-profit organization that engages youth in HIV prevention by harnessing the popularity of football (i.e. soccer). WhizzKids United, the organization's primary program, operates a 12-week program in elementary schools in Pietermaritzburg, South Africa, which aims to impart knowledge and life skills critical to HIV prevention. The goal of this research was to compare elementary school youth who received the program to youth who only received traditional classroom-based HIV education on health behaviors and HIV-related knowledge and stigma. A secondary objective was to evaluate HIV knowledge, sexual behaviors, attitudes towards HIV and health care seeking behaviors among South African youth in grades 9-12. Elementary students who participated in the program reported greater HIV knowledge and lower HIV stigma (p < .001) than those who had not. The majority of youth in grades 9-12 report having sexual relations (55.6%), despite low levels of HIV testing (29.9%) in this high HIV prevalence region of South Africa. The results highlight the importance of supporting community-based HIV educational initiatives that engage high-risk youth in HIV prevention and the need for youth-friendly health services.

Nef protein of human immunodeficiency virus and lipopolysaccharide induce expression of CD14 on human monocytes through differential utilization of interleukin-10.

We investigated the expression of membrane-bound CD14 (mCD14) on monocytes and soluble CD14 (sCD14) released into the culture supernatants of peripheral blood lymphocytes (PBMC) from human immunodeficiency virus (HIV)-infected individuals. Monocytes from HIV-positive individuals exhibited both enhanced mCD14 expression and sCD14 production in the PBMC culture supernatants compared to the levels of mCD14 and sCD14 in HIV-negative individuals. This enhanced mCD14 expression and sCD14 production in HIV-infected individuals may be due to the effects of cytokines, the bacterial product lipopolysaccharide (LPS), and/or the HIV regulatory antigens Tat and Nef. Interleukin-10 (IL-10), an immunoregulatory cytokine, as well as LPS enhanced mCD14 expression and the release of sCD14 in the culture supernatants. HIV-Nef, unlike Tat, enhanced mCD14 expression on monocytes but did not induce the release of sCD14 into the culture supernatants. Studies conducted to investigate the mechanism underlying HIV-Nef-induced mCD14 expression revealed that HIV-Nef upregulated mCD14 expression via a mechanism that does not involve endogenously produced IL-10. In contrast, LPS upregulated the expression of mCD14 and increased the release of sCD14 via a mechanism that involves, at least in part, endogenously produced IL-10. Furthermore, dexamethasone, an anti-inflammatory and immunosuppressive agent, inhibited HIV-Nef-induced CD14 expression in an IL-10-independent manner. In contrast, dexamethasone inhibited IL-10-dependent LPS-induced CD14 expression by interfering with IL-10-induced signals but not by blocking IL-10 production. These results suggest that HIV-Nef and IL-10 constitute biologically important modulators of CD14 expression which may influence immunobiological responses to bacterial infections in HIV disease.