Quantitation of mitochondrial dynamics by photolabeling of individual organelles shows that mitochondrial fusion is blocked during the Bax activation phase of apoptosis

A dynamic balance of organelle fusion and fission regulates mitochondrial morphology. During apoptosis this balance is altered, leading to an extensive fragmentation of the mitochondria. Here, we describe a novel assay of mitochondrial dynamics based on confocal imaging of cells expressing a mitochondrial matrix–targeted photoactivable green fluorescent protein that enables detection and quantification of organelle fusion in living cells. Using this assay, we visualize and quantitate mitochondrial fusion rates in healthy and apoptotic cells. During apoptosis, mitochondrial fusion is blocked independently of caspase activation. The block in mitochondrial fusion occurs within the same time range as Bax coalescence on the mitochondria and outer mitochondrial membrane permeabilization, and it may be a consequence of Bax/Bak activation during apoptosis

Structural basis for recruitment of mitochondrial fission complexes by Fis1

Mitochondrial fission controls mitochondrial shape and physiology, including mitochondrial remodeling in apoptosis. During assembly of the yeast mitochondrial fission complex, the outer membrane protein Fis1 recruits the dynamin-related GTPase Dnm1 to mitochondria. Fis1 contains a tetratricopeptide repeat (TPR) domain and interacts with Dnm1 via the molecular adaptors Mdv1 and Caf4. By using crystallographic analysis of adaptor-Fis1 complexes, we show that these adaptors use two helices to bind to both the concave and convex surfaces of the Fis1 TPR domain. Fis1 therefore contains two interaction interfaces, a binding mode that, to our knowledge, has not been observed previously for TPR domains. Genetic and biochemical studies indicate that both binding interfaces are important for binding of Mdv1 and Caf4 to Fis1 and for mitochondrial fission activity in vivo. Our results reveal how Fis1 recruits the mitochondrial fission complex and will facilitate efforts to manipulate mitochondrial fission

Autophagy machinery genes are differentially required for autophagy and Parkin-mediated mitophagy

Autophagy is a highly conserved process from yeast to mammals that recycles proteins and organelles in response to various stresses such as nutrition depletion, unfolded protein or protein aggregation formation and pathogen infections. ULK1 complex consisting of ULK1/ATG13/ATG101/FIP200 in mammals is the key component in the most upstream initiation pathway that triggers downstream PI3K complex activation (containing Vps34/Beclin1/ATG14/p150) through phosphorylation of Beclin1 to induce autophagy membrane initiation. Active Vps34 then generates PI(3)P that becomes a docking point to recruit other proteins, mainly the uniquitin‐like conjugation system such as ATG12‐ATG5 conjugates to lipidate ATG8/LC3 to complete the closure of the autophagysomes. ULK1 complex also phosphorylates the only membrane‐spanning member ATG9A to assist efficient LC3 recruitment. Genetic studies with knockout mice have suggested essential roles of each autophagy machinery genes at each step. However, recently studies suggested that each pathway seems to be independently recruited to mitochondria during Parkin‐mediated mitophagy. By generating ATG5, ATG9A, ATG13, ATG14 and FIP200 single, double, pentaKO in HeLa cells, we found that unlike in mouse, different ATG genes are differentially required for autophagy and mitophagy. While ATG5, ATG9A and FIP200 KO completely block p62 degradation during starvation, ATG13 and ATG14 KO exhibits mild inhibition. ATG9A, ATG13 and ATG14 KO also display mild inhibition of mitophagy, while ATG5 KO shows moderate block and FIP200 exhibit the strongest block of mitophagy, almost to the same extent as ATG5/9A/13/14 QKO. While ULK1/ULK2 both seem to be dispensable for autophagy from other studies, the differential requirement of ATG13, ULK1/2 and FIP200 for autophagy and mitophagy that are components of the same complex suggest a complex regulation of autophagy and mitophagy than previously believed

Human immunodeficiency virus rebound after suppression to < 400 copies/mL during initial highly active antiretroviral therapy regimens, according to prior nucleoside experience and duration of suppression

This study evaluated 1433 human immunodeficiency virus (HIV)-infected patients starting highly active antiretroviral therapy (HAART), 409 (28%) of whom had prior nucleoside experience and achieved an HIV load of <400 copies/mL by 24 weeks of therapy. Three hundred seven patients experienced virus rebound during a total of 2773.3 person-years of follow-up. There was a higher rate of virus rebound among the patients with pre-HAART nucleoside experience (relative hazard [RH], 2.86; 95% confidence interval, 2.22-3.84; P < .0001) and a decreasing rate of virus rebound with increasing duration of virus suppression (i.e., time since achieving a virus load of <400 HIV RNA copies/mL) among both the nucleoside-experienced and naive patients (P < .0001), but the difference between the groups persisted into the third year of follow-up (P = .0007). Even patients who had experienced <2 months of nucleoside therapy before beginning HAART had an increased risk of virus rebound (RH, 1.95; P = .009). It appears that only a small period of pre-HAART nucleoside therapy is sufficient to confer a disadvantage, in terms of risk of virus rebound, that persists for several years

A randomised trial of subcutaneous intermittent interleukin-2 without antiretroviral therapy in HIV-infected patients: the UK-Vanguard Study

Objective: The objective of the trial was to evaluate in a pilot setting the safety and efficacy of interleukin-2 (IL-2) therapy when used without concomitant antiretroviral therapy as a treatment for HIV infection. Design and Setting: This was a multicentre randomised three-arm trial conducted between September 1998 and March 2001 at three clinical centres in the United Kingdom. Participants: Participants were 36 antiretroviral treatment naive HIV-1-infected patients with baseline CD4 T lymphocyte counts of at least 350 cells/mm(3). Interventions: Participants were randomly assigned to receive IL-2 at 15 million international units (MIU) per day ( 12 participants) or 9 MIU/day ( 12 participants) or no treatment ( 12 participants). IL-2 was administered by twice-daily subcutaneous injections for five consecutive days every 8 wk. Outcome Measures: Primary outcome was the change from baseline CD4 T lymphocyte count at 24 wk. Safety and plasma HIV RNA levels were also monitored every 4 wk through 24 wk. The two IL-2 dose groups were combined for the primary analysis. Results: Area under curve (AUC) for change in the mean CD4 T lymphocyte count through 24 wk was 129 cells/mm(3) for those assigned IL-2 ( both dose groups combined) and 13 cells/mm(3) for control participants (95% CI for difference, 51.3 - 181.2 cells/mm(3); p = 0.0009). Compared to the control group, significant increases in CD4 cell count were observed for both IL-2 dose groups: 104.2/mm(3) ( p = 0.008) and 128.4 cells/mm(3) ( p = 0.002) for the 4.5 and 7.5 MIU dose groups, respectively. There were no significant differences between the IL-2 (0.13 log(10) copies/ ml) and control (0.09 log(10) copies/ml) groups for AUC of change in plasma HIV RNA over the 24-wk period of follow- up ( 95% CI for difference, - 0.17 to 0.26; p = 0.70). Grade 4 and dose-limiting side effects were in keeping with those previously reported for IL-2 therapy. Conclusions: In participants with HIV infection and baseline CD4 T lymphocyte counts of at least 350 cells/mm(3), intermittent subcutaneous IL-2 without concomitant antiretroviral therapy was well tolerated and produced significant increases in CD4 T lymphocyte counts and did not adversely affect plasma HIV RNA levels

Distinct roles in autophagy and importance in infectivity of the two ATG4 cysteine peptidases of leishmania major

Macroautophagy in Leishmania, which is important for the cellular remodeling required during differentiation, relies upon the hydrolytic activity of two ATG4 cysteine peptidases (ATG4.1 and ATG4.2). We have investigated the individual contributions of each ATG4 to Leishmania major by generating individual gene deletion mutants (Δatg4.1 and Δatg4.2); double mutants could not be generated, indicating that ATG4 activity is required for parasite viability. Both mutants were viable as promastigotes and infected macrophages in vitro and mice, but Δatg4.2 survived poorly irrespective of infection with promastigotes or amastigotes, whereas this was the case only when promastigotes of Δatg4.1 were used. Promastigotes of Δatg4.2 but not Δatg4.1 were more susceptible than wild type promastigotes to starvation and oxidative stresses, which correlated with increased reactive oxygen species levels and oxidatively damaged proteins in the cells as well as impaired mitochondrial function. The antioxidant N-acetylcysteine reversed this phenotype, reducing both basal and induced autophagy and restoring mitochondrial function, indicating a relationship between reactive oxygen species levels and autophagy. Deletion of ATG4.2 had a more dramatic effect upon autophagy than did deletion of ATG4.1. This phenotype is consistent with a reduced efficiency in the autophagic process in Δatg4.2, possibly due to ATG4.2 having a key role in removal of ATG8 from mature autophagosomes and thus facilitating delivery to the lysosomal network. These findings show that there is a level of functional redundancy between the two ATG4s, and that ATG4.2 appears to be the more important. Moreover, the low infectivity of Δatg4.2 demonstrates that autophagy is important for the virulence of the parasite

Choice of first-line antiretroviral therapy regimen and treatment outcomes for HIV in a middle income compared to a high income country: a cohort study

BACKGROUND: The range of combination antiretroviral therapy (cART) regimens available in many middle-income countries differs from those suggested in international HIV treatment guidelines. We compared first-line cART regimens, timing of initiation and treatment outcomes in a middle income setting (HIV Centre, Belgrade, Serbia - HCB) with a high-income country (Royal Free London Hospital, UK - RFH). METHODS: All antiretroviral-naïve HIV-positive individuals from HCB and RFH starting cART between 2003 and 2012 were included. 12-month viral load and CD4 count responses were compared, considering the first available measurement 12-24 months post-cART. The percentage that had made an antiretroviral switch for any reason, or for toxicity and the percentage that had died by 36 months (the latest time at which sufficient numbers remained under follow-up) were investigated using standard survival methods. RESULTS: 361/597 (61 %) of individuals initiating cART at HCB had a prior AIDS diagnosis, compared to 337/1763 (19 %) at RFH. Median pre-ART CD4 counts were 177 and 238 cells/mm(3) respectively (p < 0.0001). The most frequently prescribed antiretrovirals were zidovudine with lamivudine (149; 25 %) and efavirenz [329, 55 %] at HCB and emtricitabine with tenofovir (899; 51 %) and efavirenz [681, 39 %] at RFH. At HCB, a median of 2 CD4 count measurements in the first year of cART were taken, compared to 5 at RFH (p < 0.0001). Median (IQR) CD4 cell increase after 12 months was +211 (+86, +359) and +212 (+105, +318) respectively. 287 (48 %) individuals from HCB and 1452 (82 %) from RFH had an available viral load measurement, of which 271 (94 %) and 1280 (88 %) were <400 copies/mL (p < 0.0001). After 36 months, comparable percentages had made at least one antiretroviral switch (77 % HCB vs. 78 % RFH; p = 0.23). However, switches for toxicity/patient choice were more common at RFH. After 12 and 36 months of cART 3 % and 8 % of individuals died at HCB, versus 2 % and 4 % at RFH (p < 0.0001). CONCLUSION: In middle-income countries, cART is usually started at an advanced stage of HIV disease, resulting in higher mortality rates than in high income countries, supporting improved testing campaigns for early detection of HIV infection and early introduction of newer cART regimens

Pink1 and Parkin regulate Drosophila intestinal stem cell proliferation during stress and aging.

Intestinal stem cells (ISCs) maintain the midgut epithelium in Drosophila melanogaster Proper cellular turnover and tissue function rely on tightly regulated rates of ISC division and appropriate differentiation of daughter cells. However, aging and epithelial injury cause elevated ISC proliferation and decreased capacity for terminal differentiation of daughter enteroblasts (EBs). The mechanisms causing functional decline of stem cells with age remain elusive; however, recent findings suggest that stem cell metabolism plays an important role in the regulation of stem cell activity. Here, we investigate how alterations in mitochondrial homeostasis modulate stem cell behavior in vivo via RNA interference-mediated knockdown of factors involved in mitochondrial dynamics. ISC/EB-specific knockdown of the mitophagy-related genes Pink1 or Parkin suppresses the age-related loss of tissue homeostasis, despite dramatic changes in mitochondrial ultrastructure and mitochondrial damage in ISCs/EBs. Maintenance of tissue homeostasis upon reduction of Pink1 or Parkin appears to result from reduction of age- and stress-induced ISC proliferation, in part, through induction of ISC senescence. Our results indicate an uncoupling of cellular, tissue, and organismal aging through inhibition of ISC proliferation and provide insight into strategies used by stem cells to maintain tissue homeostasis despite severe damage to organelles

Adenovirus-mediated hPNPase(old-35) gene transfer as a therapeutic strategy for neuroblastoma

Current treatment options for neuroblastoma fail to eradicate the disease in the majority of high-risk patients, clearly mandating development of innovative therapeutic strategies. Gene therapy represents a promising approach for reversing the neoplastic phenotype or driving tumor cells to self-destruction. We presently studied the effects of adenovirus-mediated gene transfer of human polynucleotide phosphorylase (hPNPase(old-35)), a 3',5'-exoribonuclease with growth-inhibitory properties, in neuroblastoma cells. Transgene expression was driven by either the cytomegalovirus (CMV) promoter or by a tumor-selective promoter derived from progression elevated gene-3 (PEG-3). Our data demonstrate that efficient adenoviral transduction of neuroblastoma cells and robust transgene expression are feasible objectives, that the PEG-3 promoter is capable of selectively targeting gene expression in the majority of neuroblastoma cells, and that hPNPase(old-35) induces profound growth suppression and apoptosis of malignant neuroblastoma cells, while exerting limited effects on normal neural crest-derived melanocytes. These findings support future applications of hPNPase(old-35) for targeted gene-based therapy of neuroblastoma and suggest that combination with the PEG-3 promoter holds promise for creating a potent and selective neuroblastoma therapeutic

Estimation of the effect of SLCO1B1 polymorphisms on lopinavir plasma concentration in HIV-Infected Adults

Background—The organic anion transporting polypeptides (OATP)/SLCO family represents an important class of hepatic drug uptake transporters that mediate the sodium independent transport of a diverse range of amphipathic organic compounds, including the protease inhibitors. The SLCO1B1 521T>C (rs4149056) single nucleotide polymorphism (SNP) has been consistently associated with reduced transport activity in vivo, and we previously showed an association of this polymorphism with lopinavir plasma concentrations. The aim of this study was to develop a population pharmacokinetic (PK) model to quantify the impact of 521T>C. Methods—A population PK analysis was performed with 594 plasma samples from 375 patients receiving lopinavir/ritonavir. Non-linear mixed effects modelling was applied to explore the effects of SLCO1B1 521T>C and patient demographics. Simulations of the lopinavir concentration profile were performed with different dosing regimens considering the different alleles. Results—A one-compartment model with first-order absorption best described the data. Population clearance was 5.67 L/h with inter-patient variability of 37%. Body weight was the only demographic factor influencing clearance, which increased 0.5 L/h for every 10 kg increase. Homozygosity for the C allele was associated with a 37% lower clearance, and 14% for heterozygosity, which were statistically significant. Conclusion—These data show an association between SLCO1B1 521T>C and lopinavir clearance. The association is likely to be mediated through reduced uptake by hepatocytes leading to higher plasma concentrations of lopinavir. Further studies are now required to confirm the association and to assess the influence of other polymorphisms in the SLCO family on lopinavir PK