Detection of mpox virus in wastewater provides forewarning of clinical cases in Canadian cities.

Wastewater-based surveillance (WBS) has shown to be an effective tool in monitoring the spread of SARS-CoV-2 and has helped guide public health actions. Consequently, WBS has expanded to now include the monitoring of mpox virus (MPXV) to contribute to its mitigation efforts. In this study, we demonstrate a unique sample processing and a molecular diagnostic strategy for MPXV detection that can inform on the epidemiological situation of mpox outbreaks through WBS. We conducted WBS for MPXV in 22 Canadian wastewater treatment plants (WWTPs) for 14 weeks. Three MPXV qPCR assays were assessed in this study for the detection of MPXV which include the G2R assays (G2R_WA and G2R_G) developed by the Centers for Disease Control and Prevention (CDC) in 2010, and an in-house-developed assay that we have termed G2R_NML. The G2R_NML assay was designed using reference genomes from the 2022 MPXV outbreak and provides a larger qPCR amplicon size to facilitate Sanger sequencing. Results show that all three assays have similar limits of detection and are able to detect the presence of MPXV in wastewater. The G2R_NML assay produced a significantly greater number of Sanger sequence-confirmed MPXV results compared to the CDC G2R assays. Detection of MPXV was possible where provincial surveillance indicated overall low caseloads, and in some sites forewarning of up to several weeks was observed. Overall, this study proposes that WBS of MPXV provides additional information to help fill knowledge gaps in clinical case-surveillance and is potentially an essential component to the management of mpox.

Emergence and spread of SARS-CoV-2 variants of concern in Canada: a retrospective analysis from clinical and wastewater data.

BACKGROUND: The spread of SARS-CoV-2 has been studied at unprecedented levels worldwide. In jurisdictions where molecular analysis was performed on large scales, the emergence and competition of numerous SARS-CoV-2lineages have been observed in near real-time. Lineage identification, traditionally performed from clinical samples, can also be determined by sampling wastewater from sewersheds serving populations of interest. Variants of concern (VOCs) and SARS-CoV-2 lineages associated with increased transmissibility and/or severity are of particular interest. METHOD: Here, we consider clinical and wastewater data sources to assess the emergence and spread of VOCs in Canada retrospectively. RESULTS: We show that, overall, wastewater-based VOC identification provides similar insights to the surveillance based on clinical samples. Based on clinical data, we observed synchrony in VOC introduction as well as similar emergence speeds across most Canadian provinces despite the large geographical size of the country and differences in provincial public health measures. CONCLUSION: In particular, it took approximately four months for VOC Alpha and Delta to contribute to half of the incidence. In contrast, VOC Omicron achieved the same contribution in less than one month. This study provides significant benchmarks to enhance planning for future VOCs, and to some extent for future pandemics caused by other pathogens, by quantifying the rate of SARS-CoV-2 VOCs invasion in Canada.

Preparing for Mpox Resurgence: Surveillance Lessons From Outbreaks in Toronto, Canada.

BACKGROUND: With many global jurisdictions, Toronto, Canada, experienced an mpox outbreak in spring/summer 2022. Cases declined following implementation of a large vaccination campaign. A surge in early 2023 led to speculation that asymptomatic and/or undetected local transmission was occurring in the city. METHODS: Mpox cases and positive laboratory results are reported to Toronto Public Health. Epidemic curves and descriptive risk factor summaries for the 2022 and 2023 outbreaks were generated. First- and second-dose vaccination was monitored. Mpox virus wastewater surveillance and whole genome sequencing were conducted to generate hypotheses about the source of the 2023 resurgence. RESULTS: An overall 515 cases were reported in spring/summer 2022 and 17 in the 2022-2023 resurgence. Wastewater data correlated with the timing of cases. Whole genome sequencing showed that 2022-2023 cases were distinct from 2022 cases and closer to sequences from another country, suggesting a new importation as a source. At the start of the resurgence, approximately 16% of first-dose vaccine recipients had completed their second dose. CONCLUSIONS: This investigation demonstrates the importance of ongoing surveillance and preparedness for mpox outbreaks. Undetected local transmission was not a likely source of the 2022-2023 resurgence. Ongoing preexposure vaccine promotion remains important to mitigate disease burden.

Assessment of rapid wastewater surveillance for determination of communicable disease spread in municipalities.

Wastewater surveillance (WS) helps to improve the understanding of the spread of communicable diseases in communities. WS can assist public health decision-makers in the design and implementation of timely mitigation measures. There is an increased need to use reliable, cost-effective, simple, and rapid WS systems, given traditional analytical (or 'gold-standard') programs are instrument/time-intensive, and dependent on highly skilled personnel. This study investigated the application of the portable GeneXpert platform for WS of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus (IAV), influenza B virus (IBV), and respiratory syncytial virus (RSV). The GeneXpert system with the Xpert Xpress-SARS-CoV-2/Flu/RSV test kit uses reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to analyze wastewater samples. From September 2022 through January 2023, wastewater samples were collected from the influents of municipal wastewater treatment plants (MWTPs) of Saskatoon, Prince Albert, and North Battleford in the province of Saskatchewan, Canada. Both raw and concentrated wastewater samples were subjected to the GeneXpert analysis. Results showed that the Saskatoon wastewater viral loads were significantly correlated to Saskatchewan's influenza and COVID-19 clinical cases, with a lead time of 10 days for IAV and a lag time of 4 days for SARS-CoV-2. Additionally, the GeneXpert analysis of the three cities' wastewater samples showed that the raw WS could capture the dynamics of SARS-CoV-2 and IAV due to their correlation with concentrated WS. Interestingly, IBV loads were not detected in any wastewater samples, while the Saskatoon and Prince Albert wastewater samples collected following the 2023 holiday season (end of December and beginning of January) were positive for RSV. This study indicates that the GeneXpert has excellent potential for use in the development of an early warning system for transmissible disease in municipalities and limited-resource communities while simultaneously providing stakeholders with an efficient WS methodology.

Coupling wastewater-based epidemiological surveillance and modelling of SARS-COV-2/COVID-19: Practical applications at the Public Health Agency of Canada.

Wastewater-based surveillance (WBS) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) offers a complementary tool for clinical surveillance to detect and monitor coronavirus disease 2019 (COVID-19). Since both symptomatic and asymptomatic individuals infected with SARS-CoV-2 can shed the virus through the fecal route, WBS has the potential to measure community prevalence of COVID-19 without restrictions from healthcare-seeking behaviours and clinical testing capacity. During the Omicron wave, the limited capacity of clinical testing to identify COVID-19 cases in many jurisdictions highlighted the utility of WBS to estimate disease prevalence and inform public health strategies; however, there is a plethora of in-sewage, environmental and laboratory factors that can influence WBS outcomes. The implementation of WBS, therefore, requires a comprehensive framework to outline a pipeline that accounts for these complex and nuanced factors. This article reviews the framework of the national WBS conducted at the Public Health Agency of Canada to present WBS methods used in Canada to track and monitor SARS-CoV-2. In particular, we focus on five Canadian cities-Vancouver, Edmonton, Toronto, Montréal and Halifax-whose wastewater signals are analyzed by a mathematical model to provide case forecasts and reproduction number estimates. The goal of this work is to share our insights on approaches to implement WBS. Importantly, the national WBS system has implications beyond COVID-19, as a similar framework can be applied to monitor other infectious disease pathogens or antimicrobial resistance in the community.

Tafazzin deficiency attenuates anti-cluster of differentiation 40 and interleukin-4 activation of mouse B lymphocytes.

Barth syndrome (BTHS) is a rare X-linked genetic disease caused by mutations in TAFAZZIN. The tafazzin (Taz) protein is a cardiolipin remodeling enzyme required for maintaining mitochondrial function. Patients with BTHS exhibit impaired mitochondrial respiratory chain and metabolic function and are susceptible to serious infections. B lymphocytes (B cells) play a vital role in humoral immunity required to eradicate circulating antigens from pathogens. Intact mitochondrial respiration is required for proper B-cell function. We investigated whether Taz deficiency in mouse B cells altered their response to activation by anti-cluster of differentiation 40 (anti-CD40) + interleukin-4 (IL-4). B cells were isolated from 3-4-month-old wild type (WT) or tafazzin knockdown (TazKD) mice and were stimulated with anti-CD40 + IL-4 for 24 h and cellular bioenergetics, surface marker expression, proliferation, antibody production, and proteasome and immunoproteasome activities determined. TazKD B cells exhibited reduced mRNA expression of Taz, lowered levels of cardiolipin, and impairment in both oxidative phosphorylation and glycolysis compared to WT B cells. In addition, anti-CD40 + IL-4 stimulated TazKD B cells expressed lower levels of the immunogenic surface markers, cluster of differentiation 86 (CD86) and cluster of differentiation 69 (CD69), exhibited a lower proliferation rate, reduced production of immunoglobulin M and immunoglobulin G, and reduced proteasome and immunoproteasome proteolytic activities compared to WT B cells stimulated with anti-CD40 + IL-4. The results indicate that Taz is required to support T-cell-dependent signaling activation of mouse B cells.

Statistical framework to support the epidemiological interpretation of SARS-CoV-2 concentration in municipal wastewater.

The ribonucleic acid (RNA) of the severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) is detectable in municipal wastewater as infected individuals can shed the virus in their feces. Viral concentration in wastewater can inform the severity of the COVID-19 pandemic but observations can be noisy and sparse and hence hamper the epidemiological interpretation. Motivated by a Canadian nationwide wastewater surveillance data set, unlike previous studies, we propose a novel Bayesian statistical framework based on the theories of functional data analysis to tackle the challenges embedded in the longitudinal wastewater monitoring data. By employing this framework to analyze the large-scale data set from the nationwide wastewater surveillance program covering 15 sampling sites across Canada, we successfully detect the true trends of viral concentration out of noisy and sparsely observed viral concentrations, and accurately forecast the future trajectory of viral concentrations in wastewater. Along with the excellent performance assessment using simulated data, this study shows that the proposed novel framework is a useful statistical tool and has a significant potential in supporting the epidemiological interpretation of noisy viral concentration measurements from wastewater samples in a real-life setting.

Tafazzin deficiency impairs mitochondrial metabolism and function of lipopolysaccharide activated B lymphocytes in mice.

B lymphocytes are responsible for humoral immunity and play a key role in the immune response. Optimal mitochondrial function is required to support B cell activity during activation. We examined how deficiency of tafazzin, a cardiolipin remodeling enzyme required for mitochondrial function, alters the metabolic activity of B cells and their response to activation by lipopolysaccharide in mice. B cells were isolated from 3-month-old wild type or tafazzin knockdown mice and incubated for up to 72 h with lipopolysaccharide and cell proliferation, expression of cell surface markers, secretion of antibodies and chemokines, proteasome and immunoproteasome activities, and metabolic function determined. In addition, proteomic analysis was performed to identify altered levels of proteins involved in survival, immunogenic, proteasomal and mitochondrial processes. Compared to wild type lipopolysaccharide activated B cells, lipopolysaccharide activated tafazzin knockdown B cells exhibited significantly reduced proliferation, lowered expression of cluster of differentiation 86 and cluster of differentiation 69 surface markers, reduced secretion of immunoglobulin M antibody, reduced secretion of keratinocytes-derived chemokine and macrophage-inflammatory protein-2, reduced proteasome and immunoproteasome activities, and reduced mitochondrial respiration and glycolysis. Proteomic analysis revealed significant alterations in key protein targets that regulate cell survival, immunogenicity, proteasomal processing and mitochondrial function consistent with the findings of the above functional studies. The results indicate that the cardiolipin transacylase enzyme tafazzin plays a key role in regulating mouse B cell function and metabolic activity during activation through modulation of mitochondrial function.

Generation of False-Positive SARS-CoV-2 Antigen Results with Testing Conditions outside Manufacturer Recommendations: A Scientific Approach to Pandemic Misinformation.

Antigen-based rapid diagnostics tests (Ag-RDTs) are useful tools for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection. However, misleading demonstrations of the Abbott Panbio coronavirus disease 2019 (COVID-19) Ag-RDT on social media claimed that SARS-CoV-2 antigen could be detected in municipal water and food products. To offer a scientific rebuttal to pandemic misinformation and disinformation, this study explored the impact of using the Panbio SARS-CoV-2 assay with conditions falling outside manufacturer recommendations. Using Panbio, various water and food products, laboratory buffers, and SARS-CoV-2-negative clinical specimens were tested with and without manufacturer buffer. Additional experiments were conducted to assess the role of each Panbio buffer component (tricine, NaCl, pH, and Tween 20) as well as the impact of temperature (4°C, 20°C, and 45°C) and humidity (90%) on assay performance. Direct sample testing (without the kit buffer) resulted in false-positive signals resembling those obtained with SARS-CoV-2 positive controls tested under proper conditions. The likely explanation of these artifacts is nonspecific interactions between the SARS-CoV-2-specific conjugated and capture antibodies, as proteinase K treatment abrogated this phenomenon, and thermal shift assays showed pH-induced conformational changes under conditions promoting artifact formation. Omitting, altering, and reverse engineering the kit buffer all supported the importance of maintaining buffering capacity, ionic strength, and pH for accurate kit function. Interestingly, the Panbio assay could tolerate some extremes of temperature and humidity outside manufacturer claims. Our data support strict adherence to manufacturer instructions to avoid false-positive SARS-CoV-2 Ag-RDT reactions, otherwise resulting in anxiety, overuse of public health resources, and dissemination of misinformation. IMPORTANCE With the Panbio severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen test being deployed in over 120 countries worldwide, understanding conditions required for its ideal performance is critical. Recently on social media, this kit was shown to generate false positives when manufacturer recommendations were not followed. While erroneous results from improper use of a test may not be surprising to some health care professionals, understanding why false positives occur can help reduce the propagation of misinformation and provide a scientific rebuttal for these aberrant findings. This study demonstrated that the kit buffer's pH, ionic strength, and buffering capacity were critical components to ensure proper kit function and avoid generation of false-positive results. Typically, false positives arise from cross-reacting or interfering substances; however, this study demonstrated a mechanism where false positives were generated under conditions favoring nonspecific interactions between the two antibodies designed for SARS-CoV-2 antigen detection. Following the manufacturer instructions is critical for accurate test results.

Reduced protein kinase C delta association with a higher molecular weight complex in mitochondria of Barth Syndrome lymphoblasts.

The protein kinase C delta (PKCδ) signalosome exists as a high molecular weight complex in mitochondria and controls mitochondrial oxidative phosphorylation. Barth Syndrome (BTHS) is a rare X-linked genetic disease in which mitochondrial oxidative phosphorylation is impaired due to a mutation in the gene TAFAZZIN which results in reduction in the phospholipid cardiolipin and an accumulation of monolysocardiolipin. Here we examined if PKCδ association with a higher molecular weight complex was altered in mitochondria of BTHS lymphoblasts. Immunoblot analysis of blue native-polyacrylamide gel electrophoresis mitochondrial fractions revealed that PKCδ associated with a higher molecular weight complex in control lymphoblasts but this was markedly reduced in BTHS patient B lymphoblasts in spite of an increase in PKCδ protein expression. We hypothesize that the lack of PKCδ within this higher molecular weight complex may contribute to defective mitochondrial PKCδ signaling and thus to the bioenergetic defects observed in BTHS.

The Phosphoenolpyruvate Carboxykinase Is a Key Metabolic Enzyme and Critical Virulence Factor of Leishmania major.

There is currently no effective vaccine against leishmaniasis because of the lack of sufficient knowledge about the Ags that stimulate host-protective and long-lasting T cell-mediated immunity. We previously identified Leishmania phosphoenolpyruvate carboxykinase (PEPCK, a gluconeogenic enzyme) as an immunodominant Ag that is expressed by both the insect (promastigote) and mammalian (amastigote) stages of the parasite. In this study, we investigated the role of PEPCK in metabolism, virulence, and immunopathogenicity of Leishmania major We show that targeted loss of PEPCK results in impaired proliferation of L. major in axenic culture and bone marrow-derived macrophages. Furthermore, the deficiency of PEPCK results in highly attenuated pathology in vivo. BALB/c mice infected with PEPCK-deficient parasites failed to develop any cutaneous lesions despite harboring parasites at the cutaneous site of infection. This was associated with a dramatic reduction in the frequency of cytokine (IFN-γ, IL-4, and IL-10)-producing CD4+ T cells in spleens and lymph nodes draining the infection site. Cells from mice infected with PEPCK-deficient parasites also produced significantly low levels of these cytokines into the culture supernatant following in vitro restimulation with soluble Leishmania Ag. PEPCK-deficient parasites exhibited significantly greater extracellular acidification rate, increased proton leak, and decreased ATP-coupling efficiency and oxygen consumption rates in comparison with their wild-type and addback counterparts. Taken together, these results show that PEPCK is a critical metabolic enzyme for Leishmania, and its deletion results in altered metabolic activity and attenuation of virulence.

Cardiolipin deficiency elevates susceptibility to a lipotoxic hypertrophic cardiomyopathy.

Cardiolipin (CL) is a unique tetra-acyl phospholipid localized to the inner mitochondrial membrane and essential for normal respiratory function. It has been previously reported that the failing human heart and several rodent models of cardiac pathology have a selective loss of CL. A rare genetic disease, Barth syndrome (BTHS), is similarly characterized by a cardiomyopathy due to reduced levels of cardiolipin. A mouse model of cardiolipin deficiency was recently developed by knocking-down the cardiolipin biosynthetic enzyme tafazzin (TAZ KD). These mice develop an age-dependent cardiomyopathy due to mitochondrial dysfunction. Since reduced mitochondrial capacity in the heart may promote the accumulation of lipids, we examined whether cardiolipin deficiency in the TAZ KD mice promotes the development of a lipotoxic cardiomyopathy. In addition, we investigated whether treatment with resveratrol, a small cardioprotective nutraceutical, attenuated the aberrant lipid accumulation and associated cardiomyopathy. Mice deficient in tafazzin and the wildtype littermate controls were fed a low-fat diet, or a high-fat diet with or without resveratrol for 16 weeks. In the absence of obesity, TAZ KD mice developed a hypertrophic cardiomyopathy characterized by reduced left-ventricle (LV) volume (~36%) and 30-50% increases in isovolumetric contraction (IVCT) and relaxation times (IVRT). The progression of cardiac hypertrophy with tafazzin-deficiency was associated with several underlying pathological processes including altered mitochondrial complex I mediated respiration, elevated oxidative damage (~50% increase in reactive oxygen species, ROS), the accumulation of triglyceride (~250%) as well as lipids associated with lipotoxicity (diacylglyceride ~70%, free-cholesterol ~44%, ceramide N:16-35%) compared to the low-fat fed controls. Treatment of TAZ KD mice with resveratrol maintained normal LV volumes and preserved systolic function of the heart. The beneficial effect of resveratrol on cardiac function was accompanied by a significant improvement in mitochondrial respiration, ROS production and oxidative damage to the myocardium. Resveratrol treatment also attenuated the development of cardiac steatosis in tafazzin-deficient mice through reduced de novo fatty acid synthesis. These results indicate for the first time that cardiolipin deficiency promotes the development of a hypertrophic lipotoxic cardiomyopathy. Furthermore, we determined that dietary resveratrol attenuates the cardiomyopathy by reducing ROS, cardiac steatosis and maintaining mitochondrial function.

Mitochondrial Respiration Correlates with Prognostic Markers in Chronic Lymphocytic Leukemia and Is Normalized by Ibrutinib Treatment.

Mitochondrial bioenergetics profiling, a measure of oxygen consumption rates, correlates with prognostic markers and can be used to assess response to therapy in chronic lymphocytic leukemia (CLL) cells. In this study, we measured mitochondrial respiration rates in primary CLL cells using respirometry to evaluate mitochondrial function. We found significant increases in mitochondrial respiration rates in CLL versus control B lymphocytes. We also observed amongst CLL patients that advanced age, female sex, zeta-chain-associated protein of 70 kD (ZAP-70+), cluster of differentiation 38 (CD38+), and elevated ß2-microglobulin (ß2-M) predicted increased maximal respiration rates. ZAP-70+ CLL cells exhibited significantly higher bioenergetics than B lymphocytes or ZAP-70- CLL cells and were more sensitive to the uncoupler, carbonyl cyanide-p-trifluoro-methoxyphenylhydrazone (FCCP). Univariable and multivariable linear regression analysis demonstrated that ZAP-70+ predicted increased maximal respiration. ZAP-70+ is a surrogate for B cell receptor (BCR) activation and can be targeted by ibrutinib, which is a clinically approved Bruton's tyrosine kinase (BTK) inhibitor. Therefore, we evaluated the oxygen consumption rates (OCR) of CLL cells and plasma chemokine (C-C motif) ligands 3 and 4 (CCL3/CCL4) levels from ibrutinib-treated patients and demonstrated decreased OCR similar to control B lymphocytes, suggesting that ibrutinib treatment resets the mitochondrial bioenergetics, while diminished CCL3/CCL4 levels indicate the down regulation of the BCR signaling pathway in CLL. Our data support evaluation of mitochondrial respiration as a preclinical tool for the response assessment of CLL cells.

Transcriptional Modulation by Idelalisib Synergizes with Bendamustine in Chronic Lymphocytic Leukemia.

: The phosphatidyl-inositol 3 kinase (PI3K) δ inhibitor, idelalisib (IDE), is a potent inhibitor of the B-cell receptor pathway and a novel and highly effective agent for the treatment of chronic lymphocytic leukemia (CLL). We evaluated the activities of IDE in comparison to bendamusine (BEN), a commonly used alkylating agent, in primary CLL cells ex vivo. In contrast to BEN, IDE was cytotoxic to cells from extensively-treated patients, including those with a deletion (del)17p. Cross-resistance was not observed between BEN and IDE, confirming their different modes of cytotoxicity. Marked synergy was seen between BEN and IDE, even in cases that were resistant to BEN or IDE individually, and those with deletion (del) 17p. CD40L/interleukin 4 (IL4) co-treatment mimicking the CLL microenvironment increased resistance to IDE, but synergy was retained. PI3Kδ-deficient murine splenic B cells were more resistant to IDE and showed reduced synergy with BEN, thus confirming the importance of functional PI3Kδ protein. Although IDE was observed to induce γH2AX, IDE did not enhance activation of the DNA damage response nor DNA repair activity. Interestingly, IDE decreased global RNA synthesis and was antagonistic with 5,6-Dichlorobenzimidazole 1-b-D-ribofuranoside (DRB), an inhibitor of transcription. These findings add to the increasingly complex cellular effects of IDE, and B cell receptor (BCR) inhibitors in general, in CLL.

TAPP Adaptors Control B Cell Metabolism by Modulating the Phosphatidylinositol 3-Kinase Signaling Pathway: A Novel Regulatory Circuit Preventing Autoimmunity.

Class I PI3K enzymes play critical roles in B cell activation by phosphorylating plasma membrane lipids to generate two distinct phosphoinositide (PI) products, PI(3,4,5)P3 and PI(3,4)P2. These PIs each bind distinct but overlapping sets of intracellular proteins that control cell survival, cytoskeletal reorganization, and metabolic activity. The tandem PH domain containing proteins (TAPPs) bind with high specificity to PI(3,4)P2, and their genetic uncoupling from PI(3,4)P2 in TAPP knock in (KI) mice was previously found to cause chronic B cell activation, abnormal germinal centers (GCs), and autoimmunity. In this article, we find that TAPPs provide feedback regulation affecting PI3K signaling and metabolic activation of B cells. Upon activation, TAPP KI B cells show enhanced metabolic activity associated with increased extracellular acidification rate, increased expression of glucose transporter GLUT1, and increased glucose uptake. TAPP KI B cells show markedly increased activation of the PI3K-regulated kinases Akt, GSK3ß, and p70-S6K. Conversely, overexpression of the C-terminal TAPP PH domains in B cells can inhibit Akt phosphorylation by a mechanism requiring the TAPP PI(3,4)P2-binding pocket. Inhibition of the PI3K pathway in TAPP KI B cells reduced GLUT1 expression and glucose uptake, whereas inhibition of Akt alone was not sufficient to normalize these responses. TAPP KI GC B cells also show increased GLUT1 and glucose uptake, and treatment with the inhibitor of glycolysis 2-deoxy-D-glucose reduced chronic GC responses and autoantibody production within these mice. Our findings show that TAPP-PI(3,4)P2 interaction controls activation of glycolysis and highlights the significance of this pathway for B cell activation, GC responses, and autoimmunity.

Expression of human monolysocardiolipin acyltransferase-1 improves mitochondrial function in Barth syndrome lymphoblasts.

The mitochondrial polyglycerophospholipid cardiolipin (CL) is remodeled to obtain specific fatty acyl chains. This is predominantly accomplished by the transacylase enzyme tafazzin (TAZ). Barth syndrome (BTHS) patients with TAZ gene mutations exhibit impaired TAZ activity and loss in mitochondrial respiratory function. Previous studies identified monolysocardiolipin acyltransferase-1 (MLCL AT-1) as a mitochondrial enzyme capable of remodeling CL with fatty acid. In this study, we analyzed what relationship, if any, exists between TAZ and MLCL AT-1 with regard to CL remodeling and whether transfection of BTHS lymphoblasts with an MLCL AT-1 expression construct improves mitochondrial respiratory function. In healthy lymphoblasts, reduction in TAZ expression through TAZ RNAi transfection resulted in a compensatory increase in MLCL AT-1 mRNA, protein, and enzyme activity, but CL mass was unaltered. In contrast, BTHS lymphoblasts exhibited decreased TAZ gene and protein expression but in addition decreased MLCL AT-1 expression and CL mass. Transfection of BTHS lymphoblasts with MLCL AT-1 expression construct increased CL, improved mitochondrial basal respiration and protein leak, and decreased the proportion of cells producing superoxide but did not restore CL molecular species composition to control levels. In addition, BTHS lymphoblasts exhibited higher rates of glycolysis compared with healthy controls to compensate for reduced mitochondrial respiratory function. Mitochondrial supercomplex assembly was significantly impaired in BTHS lymphoblasts, and transfection of BTHS lymphoblasts with MLCL AT-1 expression construct did not restore supercomplex assembly. The results suggest that expression of MLCL AT-1 depends on functional TAZ in healthy cells. In addition, transfection of BTHS lymphoblasts with an MLCL AT-1 expression construct compensates, but not completely, for loss of mitochondrial respiratory function.

EWS-FLI1 confers exquisite sensitivity to NAMPT inhibition in Ewing sarcoma cells.

Ewing sarcoma (EwS) is the second most common bone cancer in children and adolescents with a high metastatic potential. EwS development is driven by a specific chromosomal translocation resulting in the generation of a chimeric EWS-ETS transcription factor, most frequently EWS-FLI1.Nicotinamide adenine dinucleotide (NAD) is a key metabolite of energy metabolism involved in cellular redox reactions, DNA repair, and in the maintenance of genomic stability. This study describes targeting nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme of NAD synthesis, by FK866 in EwS cells. Here we report that blocking NAMPT leads to exhaustive NAD depletion in EwS cells, followed by a metabolic collapse and cell death. Using conditional EWS-FLI1 knockdown by doxycycline-inducible shRNA revealed that EWS-FLI1 depletion significantly reduces the sensitivity of EwS cells to NAMPT inhibition. Consistent with this finding, a comparison of 7 EwS cell lines of different genotypes with 5 Non-EwS cell lines and mesenchymal stem cells revealed significantly higher FK866 sensitivity of EWS-ETS positive EwS cells, with IC50 values mostly below 1nM.Taken together, our data reveal evidence of an important role of the NAMPT-mediated NAD salvage pathway in the energy homeostasis of EwS cells and suggest NAMPT inhibition as a potential new treatment approach for Ewing sarcoma.

Glucose Uptake and Triacylglycerol Synthesis Are Increased in Barth Syndrome Lymphoblasts.

Barth syndrome (BTHS) is an X-linked genetic disease resulting in loss of cardiolipin (Ptd2Gro). Patients may be predisposed to hypoglycemia and exhibit increases in whole-body glucose disposal rates and a higher fat mass percentage. We examined the reasons for this in BTHS lymphoblasts. BTHS lymphoblasts exhibited a 60% increase (p < 0.004) in 2-[1,2-3H(N)]deoxy-D-glucose uptake, a 40% increase (p < 0.01) in glucose transporter-3 protein expression, an increase in phosphorylated-adenosine monophosphate kinase (AMPK) and a 58% increase (p < 0.001) in the phosphorylated-AMPK/AMPK ratio compared to controls. In addition, BTHS lymphoblasts exhibited a 90% (p < 0.001) increase in D-[U-14C]glucose incorporated into 1,2,3-triacyl-sn-glycerol (TAG) and a 29% increase (p < 0.025) in 1,2-diacyl-sn-glycerol acyltransferase-2 activity compared to controls. Thus, BTHS lymphoblasts exhibit increased glucose transport and increased glucose utilization for TAG synthesis. These results may, in part, explain why BTHS patients exhibit an increase in whole-body glucose disposal rates, may be predisposed to hypoglycemia and exhibit a higher fat mass percentage.

Impaired Cardiolipin Biosynthesis Prevents Hepatic Steatosis and Diet-Induced Obesity.

Mitochondria are the nexus of energy metabolism, and consequently their dysfunction has been implicated in the development of metabolic complications and progression to insulin resistance and type 2 diabetes. The unique tetra-acyl phospholipid cardiolipin (CL) is located in the inner mitochondrial membrane, where it maintains mitochondrial integrity. Here we show that knockdown of Tafazzin (TAZ kd), a CL transacylase, in mice results in protection against the development of obesity, insulin resistance, and hepatic steatosis. We determined that hypermetabolism protected TAZ kd mice from weight gain. Unexpectedly, the large reduction of CL in the heart and skeletal muscle of TAZ kd mice was not mirrored in the liver. As a result, TAZ kd mice exhibited normal hepatic mitochondrial supercomplex formation and elevated hepatic fatty acid oxidation. Collectively, these studies identify a key role for hepatic CL remodeling in regulating susceptibility to insulin resistance and as a novel therapeutic target for diet-induced obesity.