HILPDA promotes NASH-driven HCC development by restraining intracellular fatty acid flux in hypoxia.

BACKGROUND & AIMS: The prevalence of non-alcoholic steatohepatitis (NASH)-driven hepatocellular carcinoma (HCC) is rising rapidly, yet its underlying mechanisms remain unclear. Herein, we aim to determine the role of hypoxia-inducible lipid droplet associated protein (HILPDA)/hypoxia-inducible gene 2 (HIG2), a selective inhibitor of intracellular lipolysis, in NASH-driven HCC. METHODS: The clinical significance of HILPDA was assessed in human NASH-driven HCC specimens by immunohistochemistry and transcriptomics analyses. The oncogenic effect of HILPDA was assessed in human HCC cells and in 3D epithelial spheroids upon exposure to free fatty acids and either normoxia or hypoxia. Lipidomics profiling of wild-type and HILPDA knockout HCC cells was assessed via shotgun and targeted approaches. Wild-type (Hilpdafl/fl) and hepatocyte-specific Hilpda knockout (HilpdaΔHep) mice were fed a Western diet and high sugar in drinking water while receiving carbon tetrachloride to induce NASH-driven HCC. RESULTS: In patients with NASH-driven HCC, upregulated HILPDA expression is strongly associated with poor survival. In oxygen-deprived and lipid-loaded culture conditions, HILPDA promotes viability of human hepatoma cells and growth of 3D epithelial spheroids. Lack of HILPDA triggered flux of polyunsaturated fatty acids to membrane phospholipids and of saturated fatty acids to ceramide synthesis, exacerbating lipid peroxidation and apoptosis in hypoxia. The apoptosis induced by HILPDA deficiency was reversed by pharmacological inhibition of ceramide synthesis. In our experimental mouse model of NASH-driven HCC, HilpdaΔHep exhibited reduced hepatic steatosis and tumorigenesis but increased oxidative stress in the liver. Single-cell analysis supports a dual role of hepatic HILPDA in protecting HCC cells and facilitating the establishment of a pro-tumorigenic immune microenvironment in NASH. CONCLUSIONS: Hepatic HILPDA is a pivotal oncometabolic factor in the NASH liver microenvironment and represents a potential novel therapeutic target. IMPACT AND IMPLICATIONS: Non-alcoholic steatohepatitis (NASH, chronic metabolic liver disease caused by buildup of fat, inflammation and damage in the liver) is emerging as the leading risk factor and the fastest growing cause of hepatocellular carcinoma (HCC), the most common form of liver cancer. While curative therapeutic options exist for HCC, it frequently presents at a late stage when such options are no longer effective and only systemic therapies are available. However, systemic therapies are still associated with poor efficacy and some side effects. In addition, no approved drugs are available for NASH. Therefore, understanding the underlying metabolic alterations occurring during NASH-driven HCC is key to identifying new cancer treatments that target the unique metabolic needs of cancer cells.

Identification of motifs and mechanisms for lipid droplet targeting of the lipolytic inhibitors G0S2 and HIG2.

G0S2 and HIG2 are two selective inhibitors of ATGL (also known as PNPLA2), the key enzyme for intracellular lipolysis. Whereas G0S2 regulates triglyceride (TG) mobilization in adipocytes and hepatocytes, HIG2 functions to enhance intracellular TG accumulation under hypoxic conditions. A homologous hydrophobic domain (HD) is shared by G0S2 and HIG2 (also known as HILPDA) for binding to ATGL. However, the determinants of their lipid droplet (LD) localization are unknown. Here, we study how G0S2 and HIG2 are targeted to LDs, and identify both ATGL-independent and -dependent mechanisms. Structural prediction and studies in cells reveal that ATGL-independent localization of G0S2 to both the endoplasmic reticulum (ER) and LDs is mediated by a hairpin structure consisting of two hydrophobic sequences. Positively charged residues in the hinge region play a crucial role in sorting G0S2, which initially localizes to ER, to LDs. Interestingly, the role of these positive charges becomes dispensable when ATGL is co-expressed. In comparison, HIG2, which lacks a similar hairpin structure, is dependent on ATGL for its full LD targeting. Thus, our studies identify specific structural features and mechanisms for mediating accumulation of these two ATGL inhibitors on LDs.

Defining Disc Biopsy Timing in Relation to Blood Culture Results for Inpatients with Suspected Discitis-Osteomyelitis.

PURPOSE: To determine optimal timing of biopsy for suspected discitis-osteomyelitis (DOM) with respect to preliminary blood culture results and the effect of biopsy timing on hospital length of stay (LOS). MATERIALS AND METHODS: This retrospective study reviewed disc/vertebral biopsies for suspected DOM performed between 2010 and 2018. A total of 107 disc/vertebral biopsies were performed on 96 inpatients (mean ± SD age 57.9 ± 14.5 years, 68 men/28 women) for suspected DOM, and 100 cases of DOM were clinically confirmed and treated. Descriptive and regression statistics were performed with LOS as the primary outcome. RESULTS: Of disc biopsies in clinically confirmed cases, 68% were positive; 20% of all biopsies had preliminary positive blood cultures after 2 hospital days. There was no difference in LOS between cases with biopsy performed ≤ 2 days after blood culture and cases with biopsy performed > 2 days after blood culture (P = .40). Regression analysis showed no association between positive biopsy results and sepsis, white blood cell (WBC) count, erythrocyte sedimentation rate (ESR), or C-reactive protein (CRP). Biopsy yield was not significantly decreased in patients previously taking antibiotics (P = .09). CONCLUSIONS: Waiting 2 days for preliminary blood culture results could avoid disc/vertebral biopsy in 20% of patients and does not significantly impact hospital LOS. Additionally, clinical factors (sepsis, WBC count, CRP, and ESR) do not have predictive value for positive disc biopsy results.

Hypoxia, hypoxia-inducible gene 2 (HIG2)/HILPDA, and intracellular lipolysis in cancer.

Tumor tissues are chronically exposed to hypoxia owing to aberrant vascularity. Hypoxia induces metabolic alterations in cancer, thereby promoting aggressive malignancy and metastasis. While previous efforts largely focused on adaptive responses in glucose and glutamine metabolism, recent studies have begun to yield important insight into the hypoxic regulation of lipid metabolic reprogramming in cancer. Emerging evidence points to lipid droplet (LD) accumulation as a hallmark of hypoxic cancer cells. One critical underlying mechanism involves the inhibition of adipose triglyceride lipase (ATGL)-mediated intracellular lipolysis by a small protein encoded by hypoxia-inducible gene 2 (HIG2), also known as hypoxia inducible lipid droplet associated (HILPDA). In this review we summarize and discuss recent key findings on hypoxia-dependent regulation of metabolic adaptations especially lipolysis in cancer. We also pose several questions and hypotheses pertaining to the metabolic impact of lipolytic regulation in cancer under hypoxia and during hypoxia-reoxygenation transition.

Differences between ecological niches in northern and southern populations of Angolan black and white colobus monkeys (Colobus angolensis palliatus and Colobus angolensis sharpei) throughout Kenya and Tanzania.

Ecological niche models can be useful for clarifying relationships between environmental factors and a species' geographic distribution. In this study, we use presence-only data and environmental layers to create an ecological niche model to better understand the distribution of the East African Angolan black and white colobus monkey, Colobus angolensis palliatus, and to assess whether the model supports considering the population as two separate subspecies, Colobus angolensis sharpei and C. a. palliatus. We found the range of the predicted distribution for suitable habitat of C. a. palliatus as currently classified to be only 12.4% of that shown in the International Union for Conservation of Nature Red List range map and to be fragmented. As C. angolensis is considered a "Least Concern" species, this difference suggests that generalized maps may lead to understating the species' extinction risk. When presence points were divided into two previously proposed subspecies -C. a. palliatus (Kenya and Northern Tanzania) and C. a. sharpei (Southern Tanzania)-we found significant environmental differences between the distributions. The most important ecological variable for C. a. palliatus was predominantly precipitation of the driest month (69.1%) whereas for C. a. sharpei annual precipitation (44.8%) and land cover (normalized difference vegetation index, 16.4%) were the most important. When comparing suitable ranges for the separate distributions, we found only a 1.2% geographical overlap. These differences are consistent with previous subspecies delineations of C. a. palliatus and C. a. sharpei based upon morphology, pelage, and genetics. Our study suggests that extirpation of C. a. palliatus in suitable habitat areas and occurrence of this subspecies in anthropogenic environments, warrant further consideration for conservation actions.

Imaging of Acute Low Back Pain.

Acute low back pain, defined as less than 6 weeks in duration, does not require imaging in the absence of "red flags" that may indicate a cause, such as fracture, infection, or malignancy. When imaging is indicated, it is important to rule out a host of abnormalities that may be responsible for the pain and any associated symptoms. A common mnemonic VINDICATE can help ensure a thorough consideration of the possible causes.

Effects of implementing evidence-based appropriateness guidelines for epidural steroid injection in chronic low back pain: the EAGER (Esi Appropriateness GuidElines pRotocol) study.

Objective: Chronic low back pain is very common and often treated with epidural steroid injections (ESIs). As ESI referrals had been rapidly increasing at our Veterans' Administration hospital, we were concerned that they were supplanting more comprehensive care. The objective was to determine how referral patterns and multidisciplinary care might change with the implementation of evidence-based guidelines. Methods: In this retrospective observational study, multidisciplinary evidence-based guidelines were implemented in 2014 (EAGER: Esi Appropriateness GuidElines pRotocol) as part of the ordering process for an ESI. Time series analysis was performed to assess the primary outcome of subspecialty referral pattern, that is, the number of patients receiving referrals to ancillary services which might serve to provide a more comprehensive approach to their back pain. Secondary outcomes included patient-level changes (ie, body mass index, number of injections, opioid use), which were compared before and after protocol implementation. Results: Comparing preimplementation and postimplementation protocol periods, referrals to physical medicine/rehabilitation increased 11.7% (p=0.003) per year and integrative health increased 2.1% (p<0.001) per year among the 2294 individual patients who received ESI through the neurointerventional radiology service. Of 100 randomly selected patients for patient-level analysis, the median body mass index decreased from 31.57 to 30.22 (p=<0.001) and the mean number of injections decreased from 1.76 to 0.73 (p<0.001). The percentage of patients using oral opioid analgesics decreased from 72% to 49% (p=<0.001). Conclusion: Implementation of evidence-based guidelines for ESI referral helps guide patients into a more comprehensive care pathway for chronic low back pain and is correlated with patient-level changes such as decreased body mass index and decreased opioid usage.

Release of plasmid DNA-encoding IL-10 from PLGA microparticles facilitates long-term reversal of neuropathic pain following a single intrathecal administration.

PURPOSE: Interleukin-10 (IL-10) is an anti-inflammatory molecule that has achieved interest as a therapeutic for neuropathic pain. In this work, the potential of plasmid DNA-encoding IL-10 (pDNA-IL-10) slowly released from biodegradable microparticles to provide long-term pain relief in an animal model of neuropathic pain was investigated. METHODS: PLGA microparticles encapsulating pDNA-IL-10 were developed and assessed both in vitro and in vivo. RESULTS: In vitro, pDNA containing microparticles activated macrophages, enhanced the production of nitric oxide, and increased the production of IL-10 protein relative to levels achieved with unencapsulated pDNA-IL-10. In vivo, intrathecally administered microparticles embedded in meningeal tissue, induced phagocytic cell recruitment to the cerebrospinal fluid, and relieved neuropathic pain for greater than 74 days following a single intrathecal administration, a feat not achieved with unencapsulated pDNA. Therapeutic effects of microparticle-delivered pDNA-IL-10 were blocked in the presence of IL-10-neutralizing antibody, and elevated levels of plasmid-derived IL-10 were detected in tissues for a prolonged time period post-injection (>28 days), demonstrating that therapeutic effects are dependent on IL-10 protein production. CONCLUSIONS: These studies demonstrate that microparticle encapsulation significantly enhances the potency of intrathecally administered pDNA, which may be extended to treat other disorders that require intrathecal gene therapy.