Application of machine learning algorithms to predict 30-day hospital readmission following cement augmentation for osteoporotic vertebral compression fractures.

Objective: Osteoporosis is a common skeletal disease that greatly increases the risk of pathologic fractures and accounts for approximately 700,000 vertebral compression fractures (VCFs) annually in the United States. Cement augmentation procedures such as balloon kyphoplasty (KP) and percutaneous vertebroplasty (VP) have demonstrated efficacy in the treatment of VCFs, however, some studies report rates of readmission as high as 10.8% following such procedures. The purpose of this study was to employ Machine Learning (ML) algorithms to predict 30-day hospital readmission following cement augmentation procedures for the treatment of VCFs using the American College of Surgeons National Surgical Quality Improvement Program (ACS-NSQIP) database. Methods: ACS-NSQIP was queried to identify patients undergoing either KP or VP from 2011 to 2014. Three ML algorithms were constructed and tasked with predicting post-operative readmissions within this cohort of patients. Results: Postoperative pneumonia, ASA Class 2 designation, age, partially-dependent functional status, and a history of smoking were independently identified as highly predictive of readmission by all ML algorithms. Among these variables postoperative pneumonia (p < 0.01), ASA Class 2 designation (p < 0.01), age (p = 0.002), and partially-dependent functional status (p < 0.01) were found to be statistically significant. Predictions were generated with an average AUC value of 0.757 and an average accuracy of 80.5%. Conclusions: Postoperative pneumonia, ASA Class 2 designation, partially-dependent functional status, and age are perioperative variables associated with 30-day readmission following cement augmentation procedures. The use of ML allows for quantification of the relative contributions of these variables toward producing readmission.

Use of real-time monitors to evaluate the potential exposure of secondhand electronic cigarette particulate matter inside vehicles.

Electronic cigarette (ECIG) use continues to be highly prevalent, especially among youth and young adults. Potential exposure from secondhand ECIG particulate matter (PM) places bystanders in danger of inhaling harmful substances, especially in confined spaces. This study was conducted to measure the potential exposure from secondhand ECIG PM exposure in vehicles, with participants completing a 30-min ECIG use session in their own vehicle with their preferred ECIG device. Sessions included a 5-min, 10-puff directed bout (30-s interpuff interval), followed by a 25-min ad libitum bout in which participants could take as many puffs as desired. Real-time PM1, PM2.5, and PM10 (the 50% efficiency mass cut-off of that passes through a size-selective inlet at 1 µm, 2.5 µm, and 10 µm aerodynamic diameters, respectively) measurements were captured during the sessions using portable PM monitors (MiniWRAS, pDR, SidePak, and GeoAir2 low-cost monitors). A total of 56 participants with valid measurements were included in the study, with a total of 13 unique ECIG device brands, including Vuse Alto, Box Air Bar, ElfBar, Esco Bar, Aegis Legend, Hyde Edge, JUUL, Kang Onee Stick, Kang Onee Stick Plus, Nord X, Nord 2, Nord 3, and Vaporesso. During the 5-min directed bout, the highest real-time PM2.5 mean concentrations were 175 µg/m3 for the MiniWRAS, 1050 µg/m3 for pDR and 3314 µg/m3 for SidePak. The filter measurements were not detectable in most experiments, except for two participants, with one taking 205 puffs and the other taking 285 puffs, approximately 10 times the mean (30) puffs of all participants. The evaluation of GeoAir2 with the MiniWRAS showed a wide range of Pearson correlation coefficient (r) values, ranging from -0.03 to 1.00, for the 13 ECIG brands. The mass median diameter (0.31 µm-3.42 µm) and geometric standard deviation (2.47-8.21) were different based on the participants for the same ECIG brand.

Secondhand electronic cigarette aerosol in vehicles impacts indoor air quality.

BACKGROUND: Electronic cigarette (ECIG) use in vehicles represents a public health concern due to the potential for exposure to high concentrations of particulate matter (PM) and other toxicants. This study examined the impact of ECIG use on air quality in vehicles. METHODS: People who reported current ECIG use (n=60; mean age=20.5, SD=2.3) completed a brief survey and a 30-min ECIG use session in their own vehicle. Using a protocol similar to clinical laboratory studies involving tobacco use, participants took 10 directed puffs (i.e., a directed bout with one puff every 30s for 5min) followed by a 25-min ad libitum period in which participants took as many puffs as desired. PM 2.5µm in diameter or smaller (PM2.5) were measured using aerosol monitors set up to sample air from the breathing zone of the passenger seat and total puffs were recorded. The association between peak PM2.5 concentration and puff count was examined. RESULTS: Participants took a median 18 total puffs during the sessions. Median PM2.5 concentrations increased from 4.78µg/m3 at baseline to 107.40µg/m3 after the directed bout. Median peak PM2.5 concentration was 464.48µg/m3 and ranged from 9.56µg/m3 to 143,503.91µg/m3 (IQR=132.72-1604.68). After removing two extreme outliers for puff count and PM2.5 concentrations, puff count was significantly correlated with peak PM2.5 concentration during the ad libitum bout (r=0.32, p=0.015). CONCLUSIONS: ECIG use in vehicles impacts air quality negatively and may pose health risks to those present in vehicles when ECIG use is occurring.

Anatomic Assessment of L1-S1 Neuroforaminal Dimensions Using Computed Tomography.

BACKGROUND: Although the radiographic parameters for diagnosing central lumbar canal stenosis are well described, parameters for the diagnosis of neuroforaminal stenosis (NFS) are less well defined. Previous studies have used magnetic resonance imaging (MRI) and radiography to describe neuroforaminal dimensions (NFDs). Those methods, however, have limitations that may substantially distort measurements. Existing literature on the use of computed tomography (CT) to investigate normal NFDs is limited. METHODS: This anatomic assessment evaluated CT imaging of 300 female and 300 male subjects between 18 and 35 years of age to determine normal NFDs, specifically the sagittal anteroposterior width, axial anteroposterior width, craniocaudal height, and area. Statistical analyses were performed to assess differences in NFDs according to variables including sex, age, height, weight, body mass index, and ethnicity. RESULTS: Overall, mean NFDs were 9.08 mm for sagittal anteroposterior width, 8.93 mm for axial anteroposterior width, 17.46 mm for craniocaudal height, and 134.78 mm 2 for area (n = 6,000 measurements each). Male subjects had larger NFDs than females at multiple levels. Both Caucasian and Asian subjects had larger NFDs than African-American subjects at multiple levels. There were no associations between foraminal dimensions and anthropometric factors. CONCLUSIONS: This study describes CT-based L1-S1 NFDs in young, healthy patients who presented with reasons other than back pain or pathology affecting the neuroforamen. Dimensions were influenced by sex and ethnicity but were not influenced by anthropometric factors. LEVEL OF EVIDENCE: Diagnostic Level III . See Instructions for Authors for a complete description of levels of evidence.

Rehabilitation of a lateral ankle reconstruction in a male professional football player - A narrative case report.

OBJECTIVES: Lateral ankle sprains involving the ATFL and CFL are common injuries in football with a high recurrence rate. There is a lack of research to guide post-operative rehabilitation of football players following lateral ligament ankle reconstructive surgery. This narrative case report discusses the management of a lateral ligament reconstruction in a male professional football player. METHODS: A 25-year-old professional footballer underwent a lateral ankle reconstruction following recurrent lateral ankle sprains leading to an unstable ankle. RESULTS: Following 11-weeks of rehabilitation the player was cleared to return to full-contact training. The player competed in his first competitive match 13-weeks post-injury and completed a 6-month full-training block, without episodes of pain or instability. CONCLUSION: This case report illustrates the rehabilitation process of a football player following a lateral ankle ligament reconstruction within a timeframe expected in elite sport.

Electronic cigarette users' reactions and responses to a hypothetical ban of flavoured electronic cigarette liquids.

BackgroundRegulations have been proposed to limit e-cigarette flavours, but limited research has examined potential impacts of such policies. This study examined adult e-cigarette users' reactions to a hypothetical e-cigarette flavour ban. METHODS: In 2019, a convenience sample of current e-cigarette users in the USA (n=81, 53.1% women, mean age=37.6, 59.3% dual users of cigarettes) completed an online concept mapping study. Participants provided statements describing anticipated reactions to a hypothetical policy in which only tobacco, menthol or unflavoured e-cigarettes were available for purchase. Seventy-one unique statements were generated. Participants sorted statements into thematic groups and rated statements on how likely they would be to have each reaction. Multidimensional scaling was used to identify thematic clusters of statements. RESULTS: Twelve clusters were identified: negative reaction, take action against flavour limitation, youth prevention effectiveness perception, tolerance, acceptance, willingness to try new flavours, maintain vaping, reduce vaping, new flavours as vaping cessation transition, alternative sources for banned flavours, do-it-yourself mixing behaviours and alternative tobacco products. The highest rated cluster (negative reaction) described being angry or upset that flavours were banned, while the lowest rated clusters related to quitting/reducing e-cigarette use or switching to other tobacco products. Non-tobacco or non-menthol/mint flavoured e-cigarette users had higher ratings for clusters describing negative sentiment for the hypothetical policy. CONCLUSIONS: Some e-cigarette users may dislike an e-cigarette flavour ban; however, some e-cigarette users would likely be willing to use tobacco, menthol or unflavoured e-cigarette liquids with lower likelihood of quitting vaping or switching to other tobacco products.

Individual haplotyping of whale sharks from seawater environmental DNA.

Population genetic data can provide valuable information on the demography of a species. For rare and elusive marine megafauna, samples for generating the data are traditionally obtained from tissue biopsies, which can be logistically difficult and expensive to collect and require invasive sampling techniques. Analysis of environmental DNA (eDNA) offers an alternative, minimally invasive approach to provide important genetic information. Although eDNA approaches have been studied extensively for species detection and biodiversity monitoring in metabarcoding studies, the potential for the technique to address population-level questions remains largely unexplored. Here, we applied "eDNA haplotyping" to obtain estimates of the intraspecific genetic diversity of a whale shark (Rhincodon typus) aggregation at Ningaloo reef, Australia. Over 2 weeks, we collected seawater samples directly behind individual sharks prior to taking a tissue biopsy sample from the same animal. Our data showed a 100% match between mtDNA sequences recovered in the eDNA and tissue sample for all 28 individuals sampled. In the seawater samples, >97% of all reads were assigned to six dominant haplotypes, and a clear dominant signal (~99% of sample reads) was recovered in each sample. Our study demonstrates accurate individual-level haplotyping from seawater eDNA. When DNA from one individual clearly dominates each eDNA sample, it provides many of the same opportunities for population genetic analyses as a tissue sample, potentially removing the need for tissue sampling. Our results show that eDNA approaches for population-level analyses have the potential to supply critical demographic data for the conservation and management of marine megafauna.

The Contextual Essentiality of Mitochondrial Genes in Cancer.

Mitochondria are key organelles in eukaryotic evolution that perform crucial roles as metabolic and cellular signaling hubs. Mitochondrial function and dysfunction are associated with a range of diseases, including cancer. Mitochondria support cancer cell proliferation through biosynthetic reactions and their role in signaling, and can also promote tumorigenesis via processes such as the production of reactive oxygen species (ROS). The advent of (nuclear) genome-wide CRISPR-Cas9 deletion screens has provided gene-level resolution of the requirement of nuclear-encoded mitochondrial genes (NEMGs) for cancer cell viability (essentiality). More recently, it has become apparent that the essentiality of NEMGs is highly dependent on the cancer cell context. In particular, key tumor microenvironmental factors such as hypoxia, and changes in nutrient (e.g., glucose) availability, significantly influence the essentiality of NEMGs. In this mini-review we will discuss recent advances in our understanding of the contribution of NEMGs to cancer from CRISPR-Cas9 deletion screens, and discuss emerging concepts surrounding the context-dependent nature of mitochondrial gene essentiality.

Corrigendum: VHL-Mediated Regulation of CHCHD4 and Mitochondrial Function.

[This corrects the article DOI: 10.3389/fonc.2018.00388.].

Genome-wide CRISPR/Cas9 deletion screen defines mitochondrial gene essentiality and identifies routes for tumour cell viability in hypoxia.

Mitochondria are typically essential for the viability of eukaryotic cells, and utilize oxygen and nutrients (e.g. glucose) to perform key metabolic functions that maintain energetic homeostasis and support proliferation. Here we provide a comprehensive functional annotation of mitochondrial genes that are essential for the viability of a large panel (625) of tumour cell lines. We perform genome-wide CRISPR/Cas9 deletion screening in normoxia-glucose, hypoxia-glucose and normoxia-galactose conditions, and identify both unique and overlapping genes whose loss influences tumour cell viability under these different metabolic conditions. We discover that loss of certain oxidative phosphorylation (OXPHOS) genes (e.g. SDHC) improves tumour cell growth in hypoxia-glucose, but reduces growth in normoxia, indicating a metabolic switch in OXPHOS gene function. Moreover, compared to normoxia-glucose, loss of genes involved in energy-consuming processes that are energetically demanding, such as translation and actin polymerization, improve cell viability under both hypoxia-glucose and normoxia-galactose. Collectively, our study defines mitochondrial gene essentiality in tumour cells, highlighting that essentiality is dependent on the metabolic environment, and identifies routes for regulating tumour cell viability in hypoxia.

The Role of the Dental Community in Oropharyngeal Cancer Prevention Through HPV Vaccine Advocacy.

As rates of human papillomavirus (HPV)-related oropharyngeal cancer (OPC) continue to rise, the dental community's role in primary prevention efforts related to HPV vaccination will become increasingly important. The aim of this study was to assess knowledge, beliefs, practices, and perceived barriers regarding HPV and HPV vaccine advocacy within the dental community. A sample of 266 dentists and dental hygienists completed an online survey, and responses were then analyzed using frequencies of responses, t tests, chi-square tests, and Spearman's correlations. Ninety percent of providers believe it is important to play an "active role" in their patients' general medical care, yet only 50% believe it is their responsibility to recommend the HPV vaccine. Only 50% feel knowledgeable enough to recommend the vaccine. 78.6% of providers rarely discuss HPV vaccination with their age-appropriate patients, and 82% rarely recommend the vaccine. The two most strongly agreed-upon barriers were "parent concerns about the safety or efficacy of the vaccine" (71.6%) and "parent opposition to HPV vaccination for philosophical or religious reasons" (72.6%). Dentists were more knowledgeable about HPV vaccination and more likely to recommend the vaccine than hygienists. Higher levels of HPV-related knowledge correlated positively with beliefs and practices that support HPV vaccine advocacy. We have identified multiple opportunities for intervention aimed at increasing vaccine advocacy among oral health providers. These include clarification of role from dental professional organizations, alleviation of concerns related to perceived parental objection to the vaccine, and educational interventions targeting knowledge deficits among oral health providers.

CHCHD4 regulates tumour proliferation and EMT-related phenotypes, through respiratory chain-mediated metabolism.

BACKGROUND: Mitochondrial oxidative phosphorylation (OXPHOS) via the respiratory chain is required for the maintenance of tumour cell proliferation and regulation of epithelial to mesenchymal transition (EMT)-related phenotypes through mechanisms that are not fully understood. The essential mitochondrial import protein coiled-coil helix coiled-coil helix domain-containing protein 4 (CHCHD4) controls respiratory chain complex activity and oxygen consumption, and regulates the growth of tumours in vivo. In this study, we interrogate the importance of CHCHD4-regulated mitochondrial metabolism for tumour cell proliferation and EMT-related phenotypes, and elucidate key pathways involved. RESULTS: Using in silico analyses of 967 tumour cell lines, and tumours from different cancer patient cohorts, we show that CHCHD4 expression positively correlates with OXPHOS and proliferative pathways including the mTORC1 signalling pathway. We show that CHCHD4 expression significantly correlates with the doubling time of a range of tumour cell lines, and that CHCHD4-mediated tumour cell growth and mTORC1 signalling is coupled to respiratory chain complex I (CI) activity. Using global metabolomics analysis, we show that CHCHD4 regulates amino acid metabolism, and that CHCHD4-mediated tumour cell growth is dependent on glutamine. We show that CHCHD4-mediated tumour cell growth is linked to CI-regulated mTORC1 signalling and amino acid metabolism. Finally, we show that CHCHD4 expression in tumours is inversely correlated with EMT-related gene expression, and that increased CHCHD4 expression in tumour cells modulates EMT-related phenotypes. CONCLUSIONS: CHCHD4 drives tumour cell growth and activates mTORC1 signalling through its control of respiratory chain mediated metabolism and complex I biology, and also regulates EMT-related phenotypes of tumour cells.

CHCHD4 confers metabolic vulnerabilities to tumour cells through its control of the mitochondrial respiratory chain.

Background: Tumour cells rely on glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) to survive. Thus, mitochondrial OXPHOS has become an increasingly attractive area for therapeutic exploitation in cancer. However, mitochondria are required for intracellular oxygenation and normal physiological processes, and it remains unclear which mitochondrial molecular mechanisms might provide therapeutic benefit. Previously, we discovered that coiled-coil-helix-coiled-coil-helix domain-containing protein 4 (CHCHD4) is critical for regulating intracellular oxygenation and required for the cellular response to hypoxia (low oxygenation) in tumour cells through molecular mechanisms that we do not yet fully understand. Overexpression of CHCHD4 in human cancers correlates with increased tumour progression and poor patient survival. Results: Here, we show that elevated CHCHD4 expression provides a proliferative and metabolic advantage to tumour cells in normoxia and hypoxia. Using stable isotope labelling with amino acids in cell culture (SILAC) and analysis of the whole mitochondrial proteome, we show that CHCHD4 dynamically affects the expression of a broad range of mitochondrial respiratory chain subunits from complex I-V, including multiple subunits of complex I (CI) required for complex assembly that are essential for cell survival. We found that loss of CHCHD4 protects tumour cells from respiratory chain inhibition at CI, while elevated CHCHD4 expression in tumour cells leads to significantly increased sensitivity to CI inhibition, in part through the production of mitochondrial reactive oxygen species (ROS). Conclusions: Our study highlights an important role for CHCHD4 in regulating tumour cell metabolism and reveals that CHCHD4 confers metabolic vulnerabilities to tumour cells through its control of the mitochondrial respiratory chain and CI biology.

Corrigendum: CHCHD4 Regulates Intracellular Oxygenation and Perinuclear Distribution of Mitochondria.

[This corrects the article DOI: 10.3389/fonc.2017.00071.].

Exploring the molecular interface between hypoxia-inducible factor signalling and mitochondria.

Oxygen is required for the survival of the majority of eukaryotic organisms, as it is important for many cellular processes. Eukaryotic cells utilize oxygen for the production of biochemical energy in the form of adenosine triphosphate (ATP) generated from the catabolism of carbon-rich fuels such as glucose, lipids and glutamine. The intracellular sites of oxygen consumption-coupled ATP production are the mitochondria, double-membraned organelles that provide a dynamic and multifaceted role in cell signalling and metabolism. Highly evolutionarily conserved molecular mechanisms exist to sense and respond to changes in cellular oxygen levels. The primary transcriptional regulators of the response to decreased oxygen levels (hypoxia) are the hypoxia-inducible factors (HIFs), which play important roles in both physiological and pathophysiological contexts. In this review we explore the relationship between HIF-regulated signalling pathways and the mitochondria, including the regulation of mitochondrial metabolism, biogenesis and distribution.

VHL-Mediated Regulation of CHCHD4 and Mitochondrial Function.

Dysregulated mitochondrial function is associated with the pathology of a wide range of diseases including renal disease and cancer. Thus, investigating regulators of mitochondrial function is of particular interest. Previous work has shown that the von Hippel-Lindau tumor suppressor protein (pVHL) regulates mitochondrial biogenesis and respiratory chain function. pVHL is best known as an E3-ubiquitin ligase for the α-subunit of the hypoxia inducible factor (HIF) family of dimeric transcription factors. In normoxia, pVHL recognizes and binds hydroxylated HIF-α (HIF-1α and HIF-2α), targeting it for ubiquitination and proteasomal degradation. In this way, HIF transcriptional activity is tightly controlled at the level of HIF-α protein stability. At least 80% of clear cell renal carcinomas exhibit inactivation of the VHL gene, which leads to HIF-α protein stabilization and constitutive HIF activation. Constitutive HIF activation in renal carcinoma drives tumor progression and metastasis. Reconstitution of wild-type VHL protein (pVHL) in pVHL-defective renal carcinoma cells not only suppresses HIF activation and tumor growth, but also enhances mitochondrial respiratory chain function via mechanisms that are not fully elucidated. Here, we show that pVHL regulates mitochondrial function when re-expressed in pVHL-defective 786O and RCC10 renal carcinoma cells distinct from its regulation of HIF-α. Expression of CHCHD4, a key component of the disulphide relay system (DRS) involved in mitochondrial protein import within the intermembrane space (IMS) was elevated by pVHL re-expression alongside enhanced expression of respiratory chain subunits of complex I (NDUFB10) and complex IV (mtCO-2 and COX IV). These changes correlated with increased oxygen consumption rate (OCR) and dynamic changes in glucose and glutamine metabolism. Knockdown of HIF-2α also led to increased OCR, and elevated expression of CHCHD4, NDUFB10, and COXIV in 786O cells. Expression of pVHL mutant proteins (R200W, N78S, D126N, and S183L) that constitutively stabilize HIF-α but differentially promote glycolytic metabolism, were also found to differentially promote the pVHL-mediated mitochondrial phenotype. Parallel changes in mitochondrial morphology and the mitochondrial network were observed. Our study reveals a new role for pVHL in regulating CHCHD4 and mitochondrial function in renal carcinoma cells.

CHCHD4 Regulates Intracellular Oxygenation and Perinuclear Distribution of Mitochondria.

Hypoxia is a characteristic of the tumor microenvironment and is known to contribute to tumor progression and treatment resistance. Hypoxia-inducible factor (HIF) dimeric transcription factors control the cellular response to reduced oxygenation by regulating the expression of genes involved in metabolic adaptation, cell motility, and survival. Alterations in mitochondrial metabolism are not only a downstream consequence of HIF-signaling but mitochondria reciprocally regulate HIF signaling through multiple means, including oxygen consumption, metabolic intermediates, and reactive oxygen species generation. CHCHD4 is a redox-sensitive mitochondrial protein, which we previously identified and showed to be a novel regulator of HIF and hypoxia responses in tumors. Elevated expression of CHCHD4 in human tumors correlates with the hypoxia gene signature, disease progression, and poor patient survival. Here, we show that either long-term (72 h) exposure to hypoxia (1% O2) or elevated expression of CHCHD4 in tumor cells in normoxia leads to perinuclear accumulation of mitochondria, which is dependent on the expression of HIF-1α. Furthermore, we show that CHCHD4 is required for perinuclear localization of mitochondria and HIF activation in response to long-term hypoxia. Mutation of the functionally important highly conserved cysteines within the Cys-Pro-Cys motif of CHCHD4 or inhibition of complex IV activity (by sodium azide) redistributes mitochondria from the perinuclear region toward the periphery of the cell and blocks HIF activation. Finally, we show that CHCHD4-mediated perinuclear localization of mitochondria is associated with increased intracellular hypoxia within the perinuclear region and constitutive basal HIF activation in normoxia. Our study demonstrates that the intracellular distribution of the mitochondrial network is an important feature of the cellular response to hypoxia, contributing to hypoxic signaling via HIF activation and regulated by way of the cross talk between CHCHD4 and HIF-1α.

Plasma metabolomics reveals a diagnostic metabolic fingerprint for mitochondrial aconitase (ACO2) deficiency.

Mitochondrial respiratory chain dysfunction has been identified in a number of neurodegenerative disorders. Infantile cerebellar-retinal degeneration associated with mutations in the mitochondrial aconitase 2 gene (ACO2) has been recently described as a neurodegenerative disease of autosomal recessive inheritance. To date there is no biomarker for ACO2 deficiency and diagnosis relies on genetic analysis. Here we report global metabolic profiling in eight patients with ACO2 deficiency. Using an LC-MS-based metabolomics platform we have identified several metabolites with affected plasma concentrations including the tricarboxylic acid cycle metabolites cis-aconitate, isocitrate and alpha-ketoglutarate, as well as phosphoenolpyruvate and hydroxybutyrate. Taken together we report a diagnostic metabolic fingerprint for mitochondrial aconitase 2 deficiency.

Serine 162, an essential residue for the mitochondrial localization, stability and anti-apoptotic function of Mcl-1.

Mcl-1 is an anti-apoptotic member of the Bcl-2 family that plays a key role in normal development, but also in pathologies such as cancer. It has some unusual properties compared to other anti-apoptotic members of the Bcl-2 family, and its expression and function are dynamically regulated by a variety of post-transcriptional and post-translational processes. Of note, Mcl-1 protein has a very short half life, and its stability and function may be regulated by reversible phosphorylation. There is also evidence to suggest that it may be localized to different subcellular compartments. The aim of this work was to determine whether residues within the PEST region of Mcl-1 that may undergo reversible phosphorylation, also regulate its subcellular distribution. We show that EGFP:Mcl-1 localizes mainly to the mitochondria of HeLa cells, with some additional cytoplasmic and nuclear localization. The mutations, S64A, S64E, S121A, S159A, T163A and T163E did not significantly affect the localization of Mcl-1. However, mutation of Ser162 to the phospho-null residue, Alanine resulted in an essentially nuclear localization, with some cytoplasmic but no mitochondrial localization. This mutant Mcl-1 protein, S162A, showed significantly decreased stability and it decreased the ability to protect against Bak-induced apoptosis. These data identify a new molecular determinant of Mcl-1 function, localization and stability that may be important for understanding the role of this protein in disease.

Human CHCHD4 mitochondrial proteins regulate cellular oxygen consumption rate and metabolism and provide a critical role in hypoxia signaling and tumor progression.

Increased expression of the regulatory subunit of HIFs (HIF-1α or HIF-2α) is associated with metabolic adaptation, angiogenesis, and tumor progression. Understanding how HIFs are regulated is of intense interest. Intriguingly, the molecular mechanisms that link mitochondrial function with the HIF-regulated response to hypoxia remain to be unraveled. Here we describe what we believe to be novel functions of the human gene CHCHD4 in this context. We found that CHCHD4 encodes 2 alternatively spliced, differentially expressed isoforms (CHCHD4.1 and CHCHD4.2). CHCHD4.1 is identical to MIA40, the homolog of yeast Mia40, a key component of the mitochondrial disulfide relay system that regulates electron transfer to cytochrome c. Further analysis revealed that CHCHD4 proteins contain an evolutionarily conserved coiled-coil-helix-coiled-coil-helix (CHCH) domain important for mitochondrial localization. Modulation of CHCHD4 protein expression in tumor cells regulated cellular oxygen consumption rate and metabolism. Targeting CHCHD4 expression blocked HIF-1α induction and function in hypoxia and resulted in inhibition of tumor growth and angiogenesis in vivo. Overexpression of CHCHD4 proteins in tumor cells enhanced HIF-1α protein stabilization in hypoxic conditions, an effect insensitive to antioxidant treatment. In human cancers, increased CHCHD4 expression was found to correlate with the hypoxia gene expression signature, increasing tumor grade, and reduced patient survival. Thus, our study identifies a mitochondrial mechanism that is critical for regulating the hypoxic response in tumors.