Recent Advances in Phytochemical-Based Topical Applications for the Management of Eczema: A Review.

Eczema (atopic dermatitis, AD) is a skin disease characterized by skin barrier dysfunction due to various factors, including genetics, immune system abnormalities, and environmental triggers. Application of emollients and topical drugs such as corticosteroids and calcineurin inhibitors form the mainstay of treatments for this challenging condition. This review aims to summarize the recent advances made in phytochemical-based topical applications to treat AD and the different carriers that are being used. In this review, the clinical efficacy of several plant extracts and bioactive phytochemical compounds in treating AD are discussed. The anti-atopic effects of the herbs are evident through improvements in the Scoring Atopic Dermatitis (SCORAD) index, reduced epidermal thickness, decreased transepidermal water loss, and alleviated itching and dryness in individuals affected by AD as well as in AD mouse models. Histopathological studies and serum analyses conducted in AD mouse models demonstrated a reduction in key inflammatory factors, including thymic stromal lymphopoietin (TSLP), serum immunoglobulin E (IgE), and interleukins (IL). Additionally, there was an observed upregulation of the filaggrin (FLG) gene, which regulates the proteins constituting the stratum corneum, the outermost layer of the epidermis. Carriers play a crucial role in topical drug applications, influencing dose delivery, retention, and bioavailability. This discussion delves into the efficacy of various nanocarriers, including liposomes, ethosomes, nanoemulsions, micelles, nanocrystals, solid-lipid nanoparticles, and polymeric nanoparticles. Consequently, the potential long-term side effects such as atrophy, eruptions, lymphoma, pain, and allergic reactions that are associated with current topical treatments, including emollients, topical corticosteroids, topical calcineurin inhibitors, and crisaborole, can potentially be mitigated through the use of phytochemical-based natural topical treatments.

NAD+ depletion enhances reovirus-induced oncolysis in multiple myeloma.

Cancer cell energy metabolism plays an important role in dictating the efficacy of oncolysis by oncolytic viruses. To understand the role of multiple myeloma metabolism in reovirus oncolysis, we performed semi-targeted mass spectrometry-based metabolomics on 12 multiple myeloma cell lines and revealed a negative correlation between NAD+ levels and susceptibility to oncolysis. Likewise, a negative correlation was observed between the activity of the rate-limiting NAD+ synthesis enzyme NAMPT and oncolysis. Indeed, depletion of NAD+ levels by pharmacological inhibition of NAMPT using FK866 sensitized several myeloma cell lines to reovirus-induced killing. The myelomas that were most sensitive to this combination therapy expressed a functional p53 and had a metabolic and transcriptomic profile favoring mitochondrial metabolism over glycolysis, with the highest synergistic effect in KMS12 cells. Mechanistically, U-13C-labeled glucose flux, extracellular flux analysis, multiplex proteomics, and cell death assays revealed that the reovirus + FK866 combination caused mitochondrial dysfunction and energy depletion, leading to enhanced autophagic cell death in KMS12 cells. Finally, the combination of reovirus and NAD+ depletion achieved greater antitumor effects in KMS12 tumors in vivo and patient-derived CD138+ multiple myeloma cells. These findings identify NAD+ depletion as a potential combinatorial strategy to enhance the efficacy of oncolytic virus-based therapies in multiple myeloma.

After virus exposure, early bystander naïve CD8 T cell activation relies on NAD+ salvage metabolism.

CD8 T cells play a central role in antiviral immunity. Type I interferons are among the earliest responders after virus exposure and can cause extensive reprogramming and antigen-independent bystander activation of CD8 T cells. Although bystander activation of pre-existing memory CD8 T cells is known to play an important role in host defense and immunopathology, its impact on naïve CD8 T cells remains underappreciated. Here we report that exposure to reovirus, both in vitro or in vivo, promotes bystander activation of naïve CD8 T cells within 24 hours and that this distinct subtype of CD8 T cell displays an innate, antiviral, type I interferon sensitized signature. The induction of bystander naïve CD8 T cells is STAT1 dependent and regulated through nicotinamide phosphoribosyl transferase (NAMPT)-mediated enzymatic actions within NAD+ salvage metabolic biosynthesis. These findings identify a novel aspect of CD8 T cell activation following virus infection with implications for human health and physiology.

What respiratory physicians should know about narcolepsy and other hypersomnias.

Narcolepsy and related central disorders of hypersomnolence may present to the sleep clinic with excessive daytime sleepiness. A strong clinical suspicion and awareness of the diagnostic clues, such as cataplexy, are essential to avoid unnecessary diagnostic delay. This review provides an overview of the epidemiology, pathophysiology, clinical features, diagnostic criteria and management of narcolepsy and related disorders, including idiopathic hypersomnia, Kleine-Levin syndrome (recurrent episodic hypersomnia) and secondary central disorders of hypersomnolence.

Emergency medical admissions and COVID-19: impact on 30-day mortality and hospital length of stay.

BACKGROUND: The COVID-19 pandemic has put considerable strain on healthcare systems. AIM: To investigate the effect of the COVID-19 pandemic on 30-day in-hospital mortality, length of stay (LOS) and resource utilization in acute medical care. METHODS: We compared emergency medical admissions to a single secondary care centre during 2020 to the preceding 18 years (2002-2019). We investigated 30-day in-hospital mortality with a multiple variable logistic regression model. Utilization of procedures/services was related to LOS with zero truncated Poisson regression. RESULTS: There were 132,715 admissions in 67,185 patients over the 19-year study. There was a linear reduction in 30-day in-hospital mortality over time; over the most recent 5 years (2016-2020), there was a relative risk reduction of 36%, from 7.9 to 4.3% with a number needed to treat of 27.7. Emergency medical admissions increased 18.8% to 10,452 in 2020 with COVID-19 admissions representing 3.5%. 18.6% of COVID-19 cases required ICU admission with a median stay of 10.1 days (IQR 3.8, 16.0). COVID-19 was a significant univariate predictor of 30-day in-hospital mortality, 18.5% (95%CI: 13.9, 23.1) vs. 3.0% (95%CI: 2.7, 3.4)-OR 7.3 (95%CI: 5.3, 10.1). ICU admission was the dominant outcome predictor-OR 12.4 (95%CI: 7.7, 20.1). COVID-19 mortality in the last third of 2020 improved-OR 0.64 (95%CI: 0.47, 0.86). Hospital LOS and resource utilization were increased. CONCLUSION: A diagnosis of COVID-19 was associated with significantly increased mortality and LOS but represented only 3.5% of admissions and did not attenuate the established temporal decline in overall in-hospital mortality.

DMG26: A Targeted Sequencing Panel for Mutation Profiling to Address Gaps in the Prognostication of Multiple Myeloma.

Multiple myeloma presents with numerous primary genomic lesions that broadly dichotomize cases into hyperdiploidy or IgH translocated. Clinically, these large alterations are assessed by fluorescence in situ hybridization (FISH) for risk stratification at diagnosis. Secondary focal events, including indels and single-nucleotide variants, are also reported; however, their clinical correlates are poorly described, and FISH has insufficient resolution to assess many of them. This study examined the exonic sequences of 26 genes reported to be mutated in >1% of patients with myeloma using a custom panel. These exons were sequenced to approximately 1000 times in a cohort of 76 patients from Atlantic Canada with detailed clinical correlates and in four multiple myeloma cell lines. Across the 76 patients, 255 mutations and 33 focal copy number variations were identified. High-severity mutations and mutations predicted by FATHMM-XF to be pathogenic identified patients with significantly reduced progression-free survival. These mutations were mutually exclusive from the Revised International Staging System high-risk FISH markers and were independent of all biochemical parameters of the Revised International Staging System. Applying our panel to patients classified by FISH to be standard risk successfully reclassified patients into high- and standard-risk groups. Furthermore, three patients in our cohort each had two high-risk markers; two of these patients developed plasma cell leukemia, a rare and severe clinical sequela of multiple myeloma.

Neuronal mitochondrial calcium uniporter deficiency exacerbates axonal injury and suppresses remyelination in mice subjected to experimental autoimmune encephalomyelitis.

High-capacity mitochondrial calcium (Ca2+) uptake by the mitochondrial Ca2+ uniporter (MCU) is strategically positioned to support the survival and remyelination of axons in multiple sclerosis (MS) by undocking mitochondria, buffering Ca2+ and elevating adenosine triphosphate (ATP) synthesis at metabolically stressed sites. Respiratory chain deficits in MS are proposed to metabolically compromise axon survival and remyelination by suppressing MCU activity. In support of this hypothesis, clinical scores, mitochondrial dysfunction, myelin loss, axon damage and inflammation were elevated while remyelination was blocked in neuronal MCU deficient (Thy1-MCU Def) mice relative to Thy1 controls subjected to experimental autoimmune encephalomyelitis (EAE). At the first sign of walking deficits, mitochondria in EAE/Thy1 axons showed signs of activation. By contrast, cytoskeletal damage, fragmented mitochondria and large autophagosomes were seen in EAE/Thy1-MCU Def axons. As EAE severity increased, EAE/Thy1 axons were filled with massively swollen mitochondria with damaged cristae while EAE/Thy1-MCU Def axons were riddled with late autophagosomes. ATP concentrations and mitochondrial gene expression were suppressed while calpain activity, autophagy-related gene mRNA levels and autophagosome marker (LC3) co-localization in Thy1-expressing neurons were elevated in the spinal cords of EAE/Thy1-MCU Def compared to EAE/Thy1 mice. These findings suggest that MCU inhibition contributes to axonal damage that drives MS progression.

Targeted Metabolic Reprogramming to Improve the Efficacy of Oncolytic Virus Therapy.

Oncolytic viruses (OVs) represent a promising new class of cancer therapeutics and cause antitumor effects by two major mechanisms: (1) directly killing cancer cells in a process known as oncolysis, or (2) initiating a powerful antitumor immune response. Interestingly, energy metabolism, within either cancer cells or immune cells, plays a pivotal role in defining the outcome of OV-mediated antitumor effects. Following therapeutic administration, OVs must hijack host cell metabolic pathways to acquire building blocks such as nucleotides, lipids, and amino acids for the process of replication that is necessary for oncolysis. Additionally, OV-stimulated antitumor immune responses are highly dependent on the metabolic state within the tumor microenvironment. Thus, metabolic reprogramming strategies bear the potential to enhance the efficacy of both OV-mediated oncolysis and antitumor immune responses.

Targeting NAD+ Synthesis to Potentiate CD38-Based Immunotherapy of Multiple Myeloma.

Antibodies targeting CD38, a NAD+-degrading enzyme, have emerged as a promising immunotherapy against multiple myeloma (MM). Currently, the mechanisms by which anti-CD38 antibodies establish their therapeutic effects are poorly understood. Here, we advocate for the depletion of NAD+ to enhance the efficacy of anti-CD38-based immunotherapies in MM.

Quantitative Proteome Responses to Oncolytic Reovirus in GM-CSF- and M-CSF-Differentiated Bone Marrow-Derived Cells.

The efficacy of oncolytic viruses (OVs), such as reovirus, is dictated by host immune responses, including those mediated by the pro- versus anti-inflammatory macrophages. As such, a detailed understanding of the interaction between reovirus and different macrophage types is critical for therapeutic efficacy. To explore reovirus-macrophage interactions, we performed tandem mass tag (TMT)-based quantitative temporal proteomics on mouse bone marrow-derived macrophages (BMMs) generated with two cytokines, macrophage colony stimulating factor (M-CSF) and granulocytic-macrophage colony stimulating factor (GM-CSF), representing anti- and proinflammatory macrophages, respectively. We quantified 6863 proteins across five time points in duplicate, comparing M-CSF (M-BMM) and GM-CSF (GM-BMM) in response to OV. We find that GM-BMMs have lower expression of key intrinsic proteins that facilitate an antiviral immune response, express higher levels of reovirus receptor protein JAM-A, and are more susceptible to oncolytic reovirus infection compared to M-BMMs. Interestingly, although M-BMMs are less susceptible to reovirus infection and subsequent cell death, they initiate an antireovirus adaptive T cell immune response comparable to that of GM-BMMs. Taken together, these data describe distinct proteome differences between these two macrophage populations in terms of their ability to mount antiviral immune responses.

Pressure modification or humidification for improving usage of continuous positive airway pressure machines in adults with obstructive sleep apnoea.

BACKGROUND: Obstructive sleep apnoea (OSA) is the repetitive closure of the upper airway during sleep. This results in disturbed sleep and excessive daytime sleepiness. It is a risk factor for long-term cardiovascular morbidity. Continuous positive airway pressure (CPAP) machines can be applied during sleep. They deliver air pressure by a nasal or oronasal mask to prevent the airway from closing, reducing sleep disturbance and improving sleep quality. Some people find them difficult to tolerate because of high pressure levels and other symptoms such as a dry mouth. Switching to machines that vary the level of air pressure required to reduce sleep disturbance could increase comfort and promote more regular use. Humidification devices humidify the air that is delivered to the upper airway through the CPAP circuit. Humidification may reduce dryness of the throat and mouth and thus improve CPAP tolerability. This updated Cochrane Review looks at modifying the delivery of positive pressure and humidification on machine usage and other clinical outcomes in OSA. OBJECTIVES: To determine the effects of positive pressure modification or humidification on increasing CPAP machine usage in adults with OSA. SEARCH METHODS: We searched Cochrane Airways Specialised Register and clinical trials registries on 15 October 2018. SELECTION CRITERIA: Randomised parallel group or cross-over trials in adults with OSA. We included studies that compared automatically adjusting CPAP (auto-CPAP), bilevel positive airway pressure (bi-PAP), CPAP with expiratory pressure relief (CPAPexp), heated humidification plus fixed CPAP, automatically adjusting CPAP with expiratory pressure relief, Bi-PAP with expiratory pressure relief, auto bi-PAP and CPAPexp with wakefulness detection with fixed pressure setting. DATA COLLECTION AND ANALYSIS: We used standard methods expected by Cochrane. We assessed the certainty of evidence using GRADE for the outcomes of machine usage, symptoms (measured by the Epworth Sleepiness Scale (ESS)), Apnoea Hypopnoea Index (AHI), quality of life measured by Functional Outcomes of Sleep Questionnaire (FOSQ), blood pressure, withdrawals and adverse events (e.g. nasal blockage or mask intolerance). The main comparison of interest in the review is auto-CPAP versus fixed CPAP. MAIN RESULTS: We included 64 studies (3922 participants, 75% male). The main comparison of auto-CPAP with fixed CPAP is based on 36 studies with 2135 participants from Europe, USA, Hong Kong and Australia. The majority of studies recruited participants who were recently diagnosed with OSA and had not used CPAP previously. They had excessive sleepiness (ESS: 13), severe sleep disturbance (AHI ranged from 22 to 59), and average body mass index (BMI) of 35 kg/m2. Interventions were delivered at home and the duration of most studies was 12 weeks or less. We judged that studies at high or unclear risk of bias likely influenced the effect of auto-CPAP on machine usage, symptoms, quality of life and tolerability, but not for other outcomes. Primary outcome Compared with average usage of about five hours per night with fixed CPAP, people probably use auto-CPAP for 13 minutes longer per night at about six weeks (mean difference (MD) 0.21 hours/night, 95% confidence interval (CI) 0.11 to 0.31; 31 studies, 1452 participants; moderate-certainty evidence). We do not have enough data to determine whether auto-CPAP increases the number of people who use machines for more than four hours per night compared with fixed CPAP (odds ratio (OR) 1.16, 95% CI 0.75 to 1.81; 2 studies, 346 participants; low-certainty evidence). Secondary outcomes Auto-CPAP probably reduces daytime sleepiness compared with fixed CPAP at about six weeks by a small amount (MD -0.44 ESS units, 95% CI -0.72 to -0.16; 25 studies, 1285 participants; moderate-certainty evidence). AHI is slightly higher with auto-CPAP than with fixed CPAP (MD 0.48 events per hour, 95% CI 0.16 to 0.80; 26 studies, 1256 participants; high-certainty evidence), although it fell with both machine types from baseline values in the studies. Ten per cent of people in auto-CPAP and 11% in the fixed CPAP arms withdrew from the studies (OR 0.90, 95% CI 0.64 to 1.27; moderate-certainty evidence). Auto-CPAP and fixed CPAP may have similar effects on quality of life, as measured by the FOSQ but more evidence is needed to be confident in this result (MD 0.12, 95% CI -0.21 to 0.46; 3 studies, 352 participants; low-certainty evidence). Two studies (353 participants) provided data on clinic-measured blood pressure. Auto-CPAP may be slightly less effective at reducing diastolic blood pressure compared to fixed CPAP (MD 2.92 mmHg, 95% CI 1.06 to 4.77 mmHg; low-certainty evidence). The two modalities of CPAP probably do not differ in their effects on systolic blood pressure (MD 1.87 mmHg, 95% CI -1.08 to 4.82; moderate-certainty evidence). Nine studies (574 participants) provided information on adverse events such as nasal blockage, dry mouth, tolerance of treatment pressure and mask leak. They used different scales to capture these outcomes and due to variation in the direction and size of effect between the studies, the comparative effects on tolerability outcomes are uncertain (very low-certainty evidence).  The evidence base for other interventions is smaller, and does not provide sufficient information to determine whether there are important differences between pressure modification strategies and fixed CPAP on machine usage outcomes, symptoms and quality of life. As with the evidence for the auto-CPAP, adverse events are measured disparately. AUTHORS' CONCLUSIONS: In adults with moderate to severe sleep apnoea starting positive airway pressure therapy, auto-CPAP probably increases machine usage by about 13 minutes per night. The effects on daytime sleepiness scores with auto-CPAP are not clinically meaningful. AHI values are slightly lower with fixed CPAP. Use of validated quality of life instruments in the studies to date has been limited, although where they have been used the effect sizes have not exceeded proposed clinically important differences. The adoption of a standardised approach to measuring tolerability would help decision-makers to balance benefits with harms from the different treatment options available. The evidence available for other pressure modification strategies does not provide a reliable basis on which to draw firm conclusions. Future studies should look at the effects of pressure modification devices and humidification in people who have already used CPAP but are unable to persist with treatment.

Inhibition of Pyruvate Dehydrogenase Kinase Enhances the Antitumor Efficacy of Oncolytic Reovirus.

Oncolytic viruses (OV) such as reovirus preferentially infect and kill cancer cells. Thus, the mechanisms that dictate the susceptibility of cancer cells to OV-induced cytotoxicity hold the key to their success in clinics. Here, we investigated whether cancer cell metabolism defines its susceptibility to OV and if OV-induced metabolic perturbations can be therapeutically targeted. Using mass spectrometry-based metabolomics and extracellular flux analysis on a panel of cancer cell lines with varying degrees of susceptibility to reovirus, we found that OV-induced changes in central energy metabolism, pyruvate metabolism, and oxidative stress correlate with their susceptibility to reovirus. In particular, reovirus infection accentuated Warburg-like metabolic perturbations in cell lines relatively resistant to oncolysis. These metabolic changes were facilitated by oxidative stress-induced inhibitory phosphorylation of pyruvate dehydrogenase (PDH) that impaired the routing of pyruvate into the tricarboxylic acid cycle and established a metabolic state unsupportive of OV replication. From the therapeutic perspective, reactivation of PDH in cancer cells that were weakly sensitive for reovirus, either through PDH kinase (PDK) inhibitors dichloroacetate and AZD7545 or short hairpin RNA-specific depletion of PDK1, enhanced the efficacy of reovirus-induced oncolysis in vitro and in vivo. These findings identify targeted metabolic reprogramming as a possible combination strategy to enhance the antitumor effects of OV in clinics. SIGNIFICANCE: This study proposes targeted metabolic reprogramming as a valid combinatorial strategy to enhance the translational efficacy of oncolytic virus-based cancer therapies.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/15/3824/F1.large.jpg.

Regulation of Cancer and Cancer-Related Genes via NAD.

SIGNIFICANCE: NAD+ is an essential redox cofactor in cellular metabolism and has emerged as an important regulator of a wide spectrum of disease conditions, most notably, cancers. As such, various strategies targeting NAD+ synthesis in cancers are in clinical trials. Recent Advances: Being a substrate required for the activity of various enzyme families, especially sirtuins and poly(adenosine diphosphate [ADP]-ribose) polymerases, NAD+-mediated signaling plays an important role in gene expression, calcium release, cell cycle progression, DNA repair, and cell proliferation. Many strategies exploring the potential of interfering with NAD+ metabolism to sensitize cancer cells to achieve anticancer benefits are highly promising, and are being pursued. CRITICAL ISSUES: With the multifaceted roles of NAD+ in cancer, it is important to understand how cellular processes are reliant on NAD+. This review summarizes how NAD+ metabolism regulates various pathophysiological processes in cancer, and how this knowledge can be exploited to devise effective anticancer therapies in clinical settings. FUTURE DIRECTIONS: In line with the redundant pathways that facilitate NAD+ metabolism, further studies should comprehensively understand the roles of the various NAD+-synthesizing as well as NAD+-utilizing biomolecules to understand its true potential in cancer treatment.

TAp73 Modifies Metabolism and Positively Regulates Growth of Cancer Stem-Like Cells in a Redox-Sensitive Manner.

PURPOSE: Stem-like cancer cells, with characteristic self-renewal abilities, remain highly refractory to various clinical interventions. As such, stemness-inhibiting entities, such as tumor suppressor p53, are therapeutically pursued for their anticancer activities. Interestingly, similar implications for tumor suppressor TAp73 in regulating stemness features within stem-like cancer cells remain unknown.Experimental Design: This study utilizes various in vitro molecular biology techniques, including immunoblotting, qRT-PCR, and mass spectrometry-based proteomics, and metabolomics approaches to study the role of TAp73 in human and murine embryonal carcinoma stem-like cells (ECSLC) as well as human breast cancer stem-like cells (BCSLC). These findings were confirmed using patient-derived brain tumor-initiating cells (BTIC) and in vivo xenograft models. RESULTS: TAp73 inhibition decreases the expression of stem cell transcription factors Oct4, Nanog, and Sox-2, as well as tumorsphere formation capacity in ECSLCs. In vivo, TAp73-deficient ECSLCs and BCSLCs demonstrate decreased tumorigenic potential when xenografted in mice. Mechanistically, TAp73 modifies the proline regulatory axis through regulation of enzymes GLS, OAT, and PYCR1 involved in the interconversion of proline-glutamine-ornithine. Further, TAp73 deficiency exacerbates glutamine dependency, enhances accumulation of reactive oxygen species through reduced superoxide dismutase 1 (SOD1) expression, and promotes differentiation by arresting cell cycle and elevating autophagy. Most importantly, the knockdown of TAp73 in CD133HI BTICs, separated from three different glioblastoma patients, strongly decreases the expression of prosurvival factors Sox-2, BMI-1, and SOD1, and profoundly decreases their self-renewal capacity as evidenced through their reduced tumorsphere formation ability. CONCLUSIONS: Collectively, we reveal a clinically relevant aspect of cancer cell growth and stemness regulation through TAp73-mediated redox-sensitive metabolic reprogramming.

RTN4 Knockdown Dysregulates the AKT Pathway, Destabilizes the Cytoskeleton, and Enhances Paclitaxel-Induced Cytotoxicity in Cancers.

Reticulon-4 (RTN4), commonly known as a neurite outgrowth inhibitor (Nogo), is emerging as an important player in human cancers. Clinically, we found lower RTN4 expression in patient-derived tumors was associated with significantly better survival in lung, breast, cervical, and renal cancer patients. To identify the role of RTN4 in cancer biology, we performed mass spectrometry-based quantitative proteomic analysis on cancer cells following RTN4 knockdown and found its link with pro-survival as well as cytoskeleton-related processes. Subsequent mechanistic investigations revealed that RTN4 regulates lipid homeostasis, AKT signaling, and cytoskeleton modulation. In particular, downregulation of RTN4 reduced sphingomyelin synthesis and impaired plasma membrane localization of AKT, wherein AKT phosphorylation, involved in many cancers, was significantly reduced without any comparable effect on AKT-related upstream kinases, in a sphingolipid-dependent manner. Furthermore, knockdown of RTN4 retarded proliferation of cancer cells in vitro as well as tumor xenografts in mice. Finally, RTN4 knockdown affected tubulin stability and promoted higher cytotoxic effects with chemotherapeutic paclitaxel in cancer cells both in vitro and in vivo. In summary, RTN4 is involved in carcinogenesis and represents a molecular candidate that may be targeted to achieve desired antitumor effects in clinics.

The NAD+ Salvage Pathway Supports PHGDH-Driven Serine Biosynthesis.

NAD+ is a key metabolic redox cofactor that is regenerated from nicotinamide through the NAD+ salvage pathway. Here, we find that inhibiting the NAD+ salvage pathway depletes serine biosynthesis from glucose by impeding the NAD+-dependent protein, 3-phosphoglycerate dehydrogenase (PHGDH). Importantly, we find that PHGDHhigh breast cancer cell lines are exquisitely sensitive to inhibition of the NAD+ salvage pathway. Further, we find that PHGDH protein levels and those of the rate-limiting enzyme of NAD+ salvage, NAMPT, correlate in ER-negative, basal-like breast cancers. Although NAD+ salvage pathway inhibitors are actively being pursued in cancer treatment, their efficacy has been poor, and our findings suggest that they may be effective for PHGDH-dependent cancers.

TRPM2 channel-mediated regulation of autophagy maintains mitochondrial function and promotes gastric cancer cell survival via the JNK-signaling pathway.

A lack of effective treatment is one of the main factors contributing to gastric cancer-related death. Discovering effective targets and understanding their underlying anti-cancer mechanism are key to achieving the best response to treatment and to limiting side effects. Although recent studies have shown that the cation channel transient receptor potential melastatin-2 (TRPM2) is crucial for cancer cell survival, the exact mechanism remains unclear, limiting its therapeutic potential. Here, using molecular and functional assays, we investigated the role of TRPM2 in survival of gastric cancer cells. Our results indicated that TRPM2 knockdown in AGS and MKN-45 cells decreases cell proliferation and enhances apoptosis. We also observed that the TRPM2 knockdown impairs mitochondrial metabolism, indicated by a decrease in basal and maximal mitochondrial oxygen consumption rates and ATP production. These mitochondrial defects coincided with a decrease in autophagy and mitophagy, indicated by reduced levels of autophagy- and mitophagy-associated proteins (i.e. ATGs, LC3A/B II, and BNIP3). Moreover, we found that TRPM2 modulates autophagy through a c-Jun N-terminal kinase (JNK)-dependent and mechanistic target of rapamycin-independent pathway. We conclude that in the absence of TRPM2, down-regulation of the JNK-signaling pathway impairs autophagy, ultimately causing the accumulation of damaged mitochondria and death of gastric cancer cells. Of note, by inhibiting cell proliferation and promoting apoptosis, the TRPM2 down-regulation enhanced the efficacy of paclitaxel and doxorubicin in gastric cancer cells. Collectively, we provide compelling evidence that TRPM2 inhibition may benefit therapeutic approaches for managing gastric cancer.

Surfen, a proteoglycan binding agent, reduces inflammation but inhibits remyelination in murine models of Multiple Sclerosis.

Proteoglycans are promising therapeutic targets in Multiple Sclerosis (MS), because they regulate many aspects of the immune response. This was studied using surfen, an agent that binds both heparan sulphate proteoglycans (HSPGs) and chondroitin sulphate proteoglycans (CSPGs). Initial cell culture work on bone marrow derived macrophages (BMDMs) found that surfen reduced concentrations of the chemokines CCL2, CCL4 and CCL5, with reduced messenger (m)RNA expression for Tumor Necrosis Factor, IL-6, IL-1ß and inducible nitric oxide synthase. These data were further explored using Experimental Autoimmune Encephalomyelitis (EAE) in mice. Surfen reduced clinical signs during EAE when administered from disease onset, and reduced infiltration by CD4 positive T cells and macrophages into the central nervous system. These mice also showed reduced mRNA expression for the chemokines CCL3 and CCL5, with reduced concentrations of CCL2, CCL3 and CCL5. During EAE, surfen treatment induced a persistent increase in Interleukin (IL)-4 concentrations which may enhance T helper 2 responses. During EAE, surfen treatment reduced mRNA expression for HSPGs (NDST1, agrin, syndecan-4, perlecan, serglycin, syndecan-1) and the CSPG versican. By contrast, surfen increased mRNA expression for the CSPG aggrecan, with no effect on neurocan. During EAE, significant positive correlations were found between mRNA expression and clinical score for syndecan-4, serglycin and syndecan-1 and a significant negative correlation for aggrecan. These correlations were absent in surfen treated mice. Repair in the later stages of MS involves remyelination, which was modeled by injecting lysolecithin (lysophosphatidylcholine, LPC) into mouse corpus callosum to create regions of demyelination. When surfen was injected 2 days after LPC, it delayed remyelination of the lesions, but had no effect when injected 7 days after LPC. The delayed remyelination was associated with local increases in CSPG expression. Therefore surfen suppresses inflammation but inhibits remyelination in these models. A mechanism in common may be increased CSPG expression.

Quantitative Temporal in Vivo Proteomics Deciphers the Transition of Virus-Driven Myeloid Cells into M2 Macrophages.

Myeloid cells play a central role in the context of viral eradication, yet precisely how these cells differentiate throughout the course of acute infections is poorly understood. In this study, we have developed a novel quantitative temporal in vivo proteomics (QTiPs) platform to capture proteomic signatures of temporally transitioning virus-driven myeloid cells directly in situ, thus taking into consideration host-virus interactions throughout the course of an infection. QTiPs, in combination with phenotypic, functional, and metabolic analyses, elucidated a pivotal role for inflammatory CD11b+, Ly6G-, Ly6Chigh-low cells in antiviral immune response and viral clearance. Most importantly, the time-resolved QTiPs data set showed the transition of CD11b+, Ly6G-, Ly6Chigh-low cells into M2-like macrophages, which displayed increased antigen-presentation capacities and bioenergetic demands late in infection. We elucidated the pivotal role of myeloid cells in virus clearance and show how these cells phenotypically, functionally, and metabolically undergo a timely transition from inflammatory to M2-like macrophages in vivo. With respect to the growing appreciation for in vivo examination of viral-host interactions and for the role of myeloid cells, this study elucidates the use of quantitative proteomics to reveal the role and response of distinct immune cell populations throughout the course of virus infection.

Measurement of Mitochondrial Cholesterol Import Using a Mitochondria-Targeted CYP11A1 Fusion Construct.

All animal membranes require cholesterol as an essential regulator of biophysical properties and function, but the levels of cholesterol vary widely among different subcellular compartments. Mitochondria, and in particular the inner mitochondrial membrane, have the lowest levels of cholesterol in the cell. Nevertheless, mitochondria need cholesterol for membrane maintenance and biogenesis, as well as oxysterol, steroid, and hepatic bile acid production. Alterations in mitochondrial cholesterol have been associated with a range of pathological conditions, including cancer, hepatosteatosis, cardiac ischemia, Alzheimer's, and Niemann-Pick Type C Disease. The mechanisms of mitochondrial cholesterol import are not fully elucidated yet, and may vary in different cell types and environmental conditions. Measuring cholesterol trafficking to the mitochondrial membranes is technically challenging because of its low abundance; for example, traditional pulse-chase experiments with isotope-labeled cholesterol are not feasible. Here, we describe improvements to a method first developed by the Miller group at the University of California to measure cholesterol trafficking to the inner mitochondrial membrane (IMM) through the conversion of cholesterol to pregnenolone. This method uses a mitochondria-targeted, ectopically expressed fusion construct of CYP11A1, ferredoxin reductase and ferredoxin. Pregnenolone is formed exclusively from cholesterol at the IMM, and can be analyzed with high sensitivity and specificity through ELISA or radioimmunoassay of the medium/buffer to reflect mitochondrial cholesterol import. This assay can be used to investigate the effects of genetic or pharmacological interventions on mitochondrial cholesterol import in cultured cells or isolated mitochondria.