An unexpected journey for BNIP3.

Mitophagy is a cellular process that enables the selective degradation of damaged, dysfunctional, or superfluous mitochondria. During mitophagy, specific proteins recognize and tag mitochondria for degradation. These tagged mitochondria are engulfed by specialized structures called phagophores that then mature into autophagosomes/mitophagosomes. Mitophagosomes subsequently transport their mitochondrial cargo to lysosomes, where the mitochondria are broken down and recycled. While the PINK1-PRKN-dependent mitophagy pathway is well understood, mitophagy can also occur independently of this pathway. BNIP3 and BNIP3L/NIX, paralogous membrane proteins on the outer mitochondrial membrane (OMM), serve as ubiquitin-independent mitophagy receptors. Historically, BNIP3 regulation was thought to be primarily transcriptional through HIF1A (hypoxia inducible factor 1 subunit alpha). However, recent work has revealed a significant post-translational dimension, highlighting the strong role of the ubiquitin-proteasome system (UPS) in BNIP3 regulation. With these emerging concepts in mind, we aimed to develop a unified understanding of how steady-state levels of BNIP3 are established and maintained and how this regulation governs underlying cell physiology.

The ER membrane protein complex restricts mitophagy by controlling BNIP3 turnover.

Lysosomal degradation of autophagy receptors is a common proxy for selective autophagy. However, we find that two established mitophagy receptors, BNIP3 and BNIP3L/NIX, are constitutively delivered to lysosomes in an autophagy-independent manner. This alternative lysosomal delivery of BNIP3 accounts for nearly all its lysosome-mediated degradation, even upon mitophagy induction. To identify how BNIP3, a tail-anchored protein in the outer mitochondrial membrane, is delivered to lysosomes, we performed a genome-wide CRISPR screen for factors influencing BNIP3 flux. This screen revealed both known modifiers of BNIP3 stability as well as a pronounced reliance on endolysosomal components, including the ER membrane protein complex (EMC). Importantly, the endolysosomal system and the ubiquitin-proteosome system regulated BNIP3 independently. Perturbation of either mechanism is sufficient to modulate BNIP3-associated mitophagy and affect underlying cellular physiology. More broadly, these findings extend recent models for tail-anchored protein quality control and install endosomal trafficking and lysosomal degradation in the canon of pathways that tightly regulate endogenous tail-anchored protein localization.

Phosphoprotein phosphatase activity positively regulates oligomeric pyrin to trigger inflammasome assembly in phagocytes.

IMPORTANCE: Pyrin, a unique cytosolic receptor, initiates inflammatory responses against RhoA-inactivating bacterial toxins and effectors like Yersinia's YopE and YopT. Understanding pyrin regulation is crucial due to its association with dysregulated inflammatory responses, including Familial Mediterranean Fever (FMF), linked to pyrin gene mutations. FMF mutations historically acted as a defense mechanism against plague. Negative regulation of pyrin through PKN phosphorylation is well established, with Yersinia using the YopM effector to promote pyrin phosphorylation and counteract its activity. This study highlights the importance of phosphoprotein phosphatase activity in positively regulating pyrin inflammasome assembly in phagocytic cells of humans and mice. Oligomeric murine pyrin has S205 phosphorylated before inflammasome assembly, and this study implicates the dephosphorylation of murine pyrin S205 by two catalytic subunits of PP2A in macrophages. These findings offer insights for investigating the regulation of oligomeric pyrin and the balance of kinase and phosphatase activity in pyrin-associated infectious and autoinflammatory diseases.

The ER membrane protein complex governs lysosomal turnover of a mitochondrial tail-anchored protein, BNIP3, to restrict mitophagy.

Lysosomal degradation of autophagy receptors is a common proxy for selective autophagy. However, we find that two established mitophagy receptors, BNIP3 and BNIP3L/NIX, violate this assumption. Rather, BNIP3 and NIX are constitutively delivered to lysosomes in an autophagy-independent manner. This alternative lysosomal delivery of BNIP3 accounts for nearly all of its lysosome-mediated degradation, even upon mitophagy induction. To identify how BNIP3, a tail-anchored protein in the outer mitochondrial membrane, is delivered to lysosomes, we performed a genome-wide CRISPR screen for factors influencing BNIP3 flux. By this approach, we revealed both known modifiers of BNIP3 stability as well as a pronounced reliance on endolysosomal components, including the ER membrane protein complex (EMC). Importantly, the endolysosomal system regulates BNIP3 alongside, but independent of, the ubiquitin-proteosome system (UPS). Perturbation of either mechanism is sufficient to modulate BNIP3-associated mitophagy and affect underlying cellular physiology. In short, while BNIP3 can be cleared by parallel and partially compensatory quality control pathways, non-autophagic lysosomal degradation of BNIP3 is a strong post-translational modifier of BNIP3 function. More broadly, these data reveal an unanticipated connection between mitophagy and TA protein quality control, wherein the endolysosomal system provides a critical axis for regulating cellular metabolism. Moreover, these findings extend recent models for tail-anchored protein quality control and install endosomal trafficking and lysosomal degradation in the canon of pathways that ensure tight regulation of endogenous TA protein localization.

Evaluation of functional status among patients undergoing maintenance treatments for opioid use disorders.

BACKGROUND: Methadone and buprenorphine are the most prevalent types of opioid maintenance programs in Andalusia. The main objective is comparing the functional status of patients with pharmacological opioid maintenance treatments according to different socio-demographic characteristic, health and disabilities domains and sexual difficulties. METHODS: A total of 593 patients from the Andalusia community, 329 were undergoing methadone treatment and 264 were undergoing buprenorphine treatment. The patients were interviewed by socio-demographic and opioid-related variables, assessed by functioning, disability and health domains (WHODAS 2.0.) and for sexual problems (PRSexDQ-SALSEX). RESULTS: We found significant differences in the socio-demographic and the opioid-related variables as the onset of opioid use, being on previous maintenance programs, opioid intravenous use, the length of previous maintenance programs, polydrug use and elevated seroprevalence rates (HCV and HIV) between the methadone group and the buprenorphine group. Regarding health and disability domains there were differences in the Understanding and communication domain, Getting around domain, Participation in society domain and in the WHODAS 2.0. simple and complex score, favoring buprenorphine-treated patients. The methadone group referred elevated sexual impairments compared with the buprenorphine group. Opioid-related variables as seroprevalence rates, other previous lifetime maintenance program, the daily opioid dosage and the daily alcohol use are the most discriminative variables between both groups. Participation in society variables and sexual problems were the most important clinical variables in distinguishing the methadone group from the buprenorphine group regarding their functional status. CONCLUSIONS: The methadone group showed higher prevalence in opioid dependence-related variables, elevated disabilities in participation in society activities and sexual problems compared with the buprenorphine group. This study shows the importance of carry out a functional evaluation in the healthcare follow-up, especially in those areas related with social activity and with sexual problems.

Receptor-mediated clustering of FIP200 bypasses the role of LC3 lipidation in autophagy.

Autophagosome formation requires multiple autophagy-related (ATG) factors. However, we find that a subset of autophagy substrates remains robustly targeted to the lysosome in the absence of several core ATGs, including the LC3 lipidation machinery. To address this unexpected result, we performed genome-wide CRISPR screens identifying genes required for NBR1 flux in ATG7KO cells. We find that ATG7-independent autophagy still requires canonical ATG factors including FIP200. However, in the absence of LC3 lipidation, additional factors are required including TAX1BP1 and TBK1. TAX1BP1's ability to cluster FIP200 around NBR1 cargo and induce local autophagosome formation enforces cargo specificity and replaces the requirement for lipidated LC3. In support of this model, we define a ubiquitin-independent mode of TAX1BP1 recruitment to NBR1 puncta, highlighting that TAX1BP1 recruitment and clustering, rather than ubiquitin binding per se, is critical for function. Collectively, our data provide a mechanistic basis for reports of selective autophagy in cells lacking the lipidation machinery, wherein receptor-mediated clustering of upstream autophagy factors drives continued autophagosome formation.

Enantioselective PCCP Brønsted acid-catalyzed aza-Piancatelli rearrangement.

An enantioselective aza-Piancatelli rearrangement has been developed using a chiral Brønsted acid based on pentacarboxycyclopentadiene (PCCP). This reaction provides rapid access to valuable chiral 4-amino-2-cyclopentenone building blocks from readily available starting material and is operationally simple.

Impact of Nutritional Intervention on Length of Hospital Stay and Mortality among Hospitalized Patients with Malnutrition: A Clinical Randomized Controlled Trial.

OBJECTIVE: The objective of this study was to evaluate the impact of a nutritional intervention on hospital stay and mortality among hospitalized patients with malnutrition. METHODS: Hospitalized patients with a diagnosis of malnutrition were enrolled and randomly allocated to either an intervention or control group. Participants in the intervention group received an individualized nutrition plan according to energy and protein (1.0-1.5 g/kg) intake requirements as well as dietary advice based on face-to-face interviews with patients and their caregivers or family members. Individuals in the control group received standard nutritional management according to the Hospital Nutrition Department. Nutritional status and disease severity were assessed using nutritional risk screening. Length of hospital stay was defined by the number of days of hospitalization from hospital admission to medical discharge. Reference to another service or death were criteria for study withdrawal. To evaluate mortality, individuals were followed up for 6 months after hospital discharge. Hospital stay and mortality were the intention-to-treat analysis. RESULTS: A total of 55 patients with an average age of 57.1 ± 20.7 years were included into intervention (n = 28) and control (n = 27) groups, respectively. At basal condition, nutritional status, measured by nutritional risk screening score, was similar between the study groups (4.1 ± 0.8 vs 4.2 ± 1.2, p = 0.6). The average hospital stay was lower in the intervention group compared to the control group (6.4 ± 3.0 vs 8.4 ± 4.0 days, p = 0.03). Finally, the mortality rate at 6 months of follow-up was similar in both groups (hazard ratio [HR] = 0.85; 95% confidence interval [CI], 0.17-4.21). CONCLUSIONS: Results of this study suggest that, in hospitalized patients with malnutrition, nutritional intervention and dietary advice decrease hospital stay but not mortality.

Visual inspection after acetic acid (VIA) is highly heterogeneous in primary cervical screening in Amazonian Peru.

BACKGROUND: Conventional cytology (Pap) and visual inspection after the application of acetic acid (VIA) are currently used in primary screening in Peru. Studies suggest that the quality of VIA is highly variable. Over 36 000 women were screened with Pap and VIA in the TATI (Tamizaje y Tratamiento Inmediato de Lesiones Cervico-uterinas) project conducted in Amazonian Peru. Within a nested study to compare several screening techniques (C-TATI), a total of 5435 women were additionally screened with liquid-based cytology (LBC) and high-risk human papillomavirus testing (HR-HPV). We investigate the variation of positivity rates of VIA, Pap, LBC and HR-HPV in C-TATI and of VIA in the full TATI intervention. METHODS: At the screening visit, midwives collected three cervical samples for Pap, LBC and HC2 before performing VIA. The dispersion factor "D" (D = Pearson chi-square value/degrees-of-freedom) was used to measure the variability of tests results. Within C-TATI, the variability of positivity rates of VIA, Pap, LBC and HR-HPV was also graphically assessed with box- and scatter plots by midwife and month of screening. Funnel plots and smoothed scatter plots were used to correlate the variation of VIA by the number of examinations performed by each midwife over the full TATI intervention. RESULTS: Consistently over TATI, VIA results were highly variable, independently of the examiner, the time when the test was performed and the number of tests the examiner performed (D>6, p-values<0.001). In C-TATI, VIA results varied the most while those of HR-HPV varied the least (Ds>25, p-values<0.001 for VIA, Ds<1.6, p-values>0.05 for HR-HPV). No evidence for correlation between the number of VIAs done per midwife and the variability of VIA results was observed. CONCLUSION: The lack of over-dispersion for HR-HPV detection suggests that the variable VIA results do not reflect true variation in underlying disease, but a lack of consistency in human judgement.

Development of a proof of concept immunochromatographic lateral flow assay for point of care diagnosis of Mycobacterium tuberculosis.

BACKGROUND: Despite major public health initiatives and the existence of efficacious treatment regimes, tuberculosis (TB) remains a threat, particularly in resource-limited settings. A significant part of the problem is the difficulty of rapidly identifying infected individuals, and as a result, there has been renewed interest in developing better diagnostics for infection or disease caused by Mycobacterium tuberculosis. Many of the existing tools, however, have limitations such as poor sensitivity or specificity, or the need for well-equipped laboratories to function effectively. Serodiagnostic approaches in particular have long drawn attention, due to their potential utility in large field studies, particularly in resource-poor settings. Unfortunately none of the serodiagnostic approaches have so far proven useful under field conditions. RESULTS: We screened a large panel of antigens with serodiagnostic potential by ELISA and selected a subpanel that was strongly and broadly recognised by TB patients, but not by controls. These antigens were then formulated into a simple immuno-chromatographic lateral flow assay format, suitable for field use, and tested against panels of plasma and blood samples from individuals with different clinical status (confirmed TB patients, household contacts, and apparently healthy community controls), recruited from Ethiopia (a highly TB-endemic country) and Turkey (a TB meso-endemic country). While specificity was good (97-100% in non TB-endemic controls), the sensitivity was not as high as expected (46-54% in pulmonary TB, 25-29% in extra-pulmonary TB). CONCLUSIONS: Though below the level of sensitivity the consortium had set for commercial development, the assay specifically identified M. tuberculosis-infected individuals, and provides a valuable proof of concept.

Effectiveness of cervical cancer screening using visual inspection with acetic acid in Peru.

OBJECTIVE: To evaluate the effectiveness of screening using visual inspection with acetic acid (VIA). METHODS: In a low-resource area of Peru in 2005-2008, a randomly selected sample of women who had previously screened negative by VIA and Pap (intervention group), and a group of eligible women previously unscreened by VIA (comparison group) were screened by VIA. The outcome measures were histologically confirmed cervical intraepithelial neoplasia (CIN) 2-3 and invasive cervical cancer. RESULTS: There were 4252 women in the intervention group and 4392 in the comparison group. Histologically confirmed CIN 2 or worse was diagnosed in 31 (0.7%) and 115 (2.6%) women, and invasive cancer was diagnosed in 4 women (0.09%) and 43 women (1.00%), in the intervention and comparison groups, respectively. The adjusted odds ratio was 4.2 (95% confidence interval [CI], 2.7-6.4) for CIN 2 or worse, and 13.9 (95% CI, 4.9-39.6) for invasive cervical cancer in the comparison group. CONCLUSION: A lower prevalence of CIN 2-3 and invasive cervical cancer was seen in women previously screened by VIA, as compared with women not previously screened by VIA, implying that a single VIA screening can lower the population risk for cervical cancer.

Comparison of tinzaparin and acenocoumarol for the secondary prevention of venous thromboembolism: a multicentre, randomized study.

The objective of the present study was to evaluate the efficacy, safety and healthcare resource utilization of long-term treatment with tinzaparin in symptomatic patients with acute pulmonary embolism as compared to standard therapy. In this open-label trial, 102 patients with objectively confirmed pulmonary embolism were randomized to receive, after initial treatment with tinzaparin, either tinzaparin (175 IU/kg/day) or international normalized ratio-adjusted acenocoumarol for 6 months. Clinical endpoints were assessed during the 6 months of treatment. A pharmacoeconomic analysis was carried out to evaluate the cost of the long-term treatment with tinzaparin in comparison with the standard one. In an intention-to-treat analysis, one of 52 patients developed recurrent venous thromboembolism in the tinzaparin group compared with none of the 50 patients in the acenocoumarol group. One patient in each group had a major haemorrhagic complication. Six patients in the acenocoumarol group had minor bleeding compared with none in the tinzaparin group (P = 0.027). Median hospital length of stay was shorter in the tinzaparin group compared to the acenocoumarol group (7 versus 9 days; P = 0.014). When all the direct and indirect cost components were combined for the entire population, we found a slight, nonstatistically significant (mean difference €345; 95% CI 1382-2071; P = 0.69) reduction in total cost with tinzaparin. Symptomatic acute pulmonary embolism treatment with full therapeutic doses of tinzaparin for 6 months is a feasible alternative to conventional treatment with vitamin K antagonists.

Formate adsorption onto thin films of rutile TiO2 nanorods and nanowires.

We evaluate the applicability of silicon prisms to infrared (IR) spectroscopic investigations of the oxide/electrolyte interface in the attenuated total reflection (ATR) configuration. Using formic acid as a probe adsorbate, a comparison is done between a rutile nanowire film supported on Si and a rutile nanorod film supported on ZnSe. The nanowires were 2 nm in diameter and were deposited directly on Si by chemical bath deposition, whereas the nanorods were deposited on ZnSe by solution casting of an aqueous dispersion prepared from a commercial powder. The lower penetration depth of the evanescent wave for silicon was compensated by a higher internal surface area of the corresponding nanowire film. Advantageously, much higher solution concentrations can be used in the case of the Si prism without a significant contribution of solution species to the IR spectrum. Furthermore, the high chemical stability of Si opens up the possibility of performing experiments in highly acidic aqueous solutions. Upon formate adsorption at pH 3.5, a pair of intense IR bands was observed in the wavenumber range where antisymmetric v(as)(COO) vibrations are expected, namely at 1537 and 1592 cm(-1) on nanorod films and at 1544 and 1586 cm(-1) on nanowire films. The relative band intensities are different for nanorod and nanowire films. While the bands at 1537/1544 cm(-1) are assigned to formate adsorbed on the (110) face, forming the bridging bidentate (mu) structure, those at 1592/1586 cm(-1) are tentatively attributed to formate adsorbed at low coordination adsorption sites at the nanocrystal edges. Bands corresponding to the carboxylate symmetric stretching and the HCO deformation were also observed.