• Machine Learning And MRI-Based Diagnostic Models For ADHD: Are We There Yet?

      Zhang-James, Yanli; Hoogman, Martine; Franke, Barbara; Faraone, Stephen V. (Cold Spring Harbor Laboratory, 2020-10-23)
      Machine learning (ML) has been applied to develop magnetic resonance imaging (MRI)-based diagnostic classifiers for attention-deficit/hyperactivity disorder (ADHD). This systematic review examines this literature to clarify its clinical significance and to assess the implications of the various analytic methods applied. We found that, although most of studies reported the classification accuracies, they varied in choice of MRI modalities, ML models, cross-validation and testing methods, and sample sizes. We found that the accuracies of cross-validation methods inflated the performance estimation compared with those of a held-out test, compromising the model generalizability. Test accuracies have increased with publication year but were not associated with training sample sizes. Improved test accuracy over time was likely due to the use of better ML methods along with strategies to deal with data imbalances. Ultimately, large multi-modal imaging datasets, and potentially the combination with other types of data, like cognitive data and/or genetics, will be essential to achieve the goal of developing clinically useful imaging classification tools for ADHD in the future.
    • Machine-Learning Prediction of Comorbid Substance Use Disorders in ADHD Youth Using Swedish Registry Data

      Zhang-James, Yanli; Chen, Qi; Kuja-Halkola, Ralf; Lichtenstein, Paul; Larsson, Henrik; Faraone, Stephen V. (Cold Spring Harbor Laboratory, 2019-06-06)
      Background: Children with attention-deficit/hyperactivity disorder (ADHD) have a high risk for substance use disorders (SUDs). Early identification of at-risk youth would help allocate scarce resources for prevention programs. Methods: Psychiatric and somatic diagnoses, family history of these disorders, measures of socioeconomic distress, and information about birth complications were obtained from the national registers in Sweden for 19,787 children with ADHD born between 1989 and 1993. We trained (a) a cross-sectional random forest (RF) model using data available by age 17 to predict SUD diagnosis between ages 18 and 19; and (b) a longitudinal recurrent neural network (RNN) model with the Long Short-Term Memory (LSTM) architecture to predict new diagnoses at each age. Results: The area under the receiver operating characteristic curve (AUC) was 0.73(95%CI 0.70–0.76) for the random forest model (RF). Removing prior diagnosis from the predictors, the RF model was still able to achieve significant AUCs when predicting all SUD diagnoses (0.69, 95%CI 0.66–0.72) or new diagnoses (0.67, 95%CI: 0.64, 0.71) during age 18–19. For the model predicting new diagnoses, model calibration was good with a low Brier score of 0.086. Longitudinal LSTM model was able to predict later SUD risks at as early as 2 years age, 10 years before the earliest diagnosis. The average AUC from longitudinal models predicting new diagnoses 1, 2, 5 and 10 years in the future was 0.63. Conclusions: Population registry data can be used to predict at-risk comorbid SUDs in individuals with ADHD. Such predictions can be made many years prior to age of the onset, and their SUD risks can be monitored using longitudinal models over years during child development. Nevertheless, more work is needed to create prediction models based on electronic health records or linked population registers that are sufficiently accurate for use in the clinic.
    • A matter of life and death: human cytomegalovirus induction of monocyte survival and differentiation into macrophages through manipulation of the PI3K/Akt pathway

      Chan, Gary; Cojohari, Olesea (2017)
      Human cytomegalovirus (HCMV) is a ubiquitous β-herpesvirus infecting up to 80% of the US population and reaching 100% seroprevalence in many parts of the world. In mostindividuals HCMV infection is usually asymptomatic. In contrast, in immunodeficient or immunonaive people, such as transplant recipients and the developing fetus, the virus is a major cause of morbidity and mortality. During a primary infection, HCMVcan spread very effectively in the body infecting many organ types and monocytes are believed to be the principal cell type responsible for HCMV dissemination throughout the body. Monocytes, however, are naturally programmed to undergo apoptosis after 48h in the circulation and are not permissive for viral replication. Our lab has shown that in order to combat these biological hurdles, HCMV promotes survival of these short-lived cells past their 48h “viability gate”. Besides inducing survival, the virus also mediates the differentiation of monocytes into macrophages skewed towards an M1 pro-inflammatory phenotype with select M2 anti-inflammatory features, which are long-lived cells, permissive for viral replication. However, the mechanisms used by HCMV to concomitantly induce survival and macrophage differentiation -two linked but separate processes, are not fully understood. The studies in this thesis reveal that upon binding and entry, HCMV initiates a survival program in monocytes by inducing a rapid and sustained activation of the PI3K/Akt pathway, which isdifferent from that induced by myeloid growth factors. Moreover, after inducing cellular survival across the 48-h viability gate, the virus also employsthe PI3K/Akt pathway to regulate caspase 3 activation which mediatesthe atypical M1/M2 polarization. Our work suggests that virus not only makes use of the PI3K/Akt pathway, but manipulates it at multiple levels toallow for viral-specific downstream functional changes.Deciphering how the virus uniquely maneuvers signaling pathways in monocytes to drive their survival and differentiation might allow us to develop new treatments targeting HCMV-infected monocytes and preventing viral spread and disease.
    • MECHANISM OF ANT1-INDUCED HUMAN DISEASES AND STRESS SIGNALING

      Liu, Yaxin (2016)
      Adenine nucleotidetranslocase(Ant) is a mitochondrial inner membraneprotein, the primaryfunction of which is to mediate the ADP/ATP exchange acrossthe inner membrane. Missense mutations in Ant1, the skeletal muscle-and heart-specificisoform, induce human disordersincluding autosomal dominant ProgressiveExternalOpthalmoplegia(adPEO), cardiomyopathyand myopathy. Several models were proposed to interpret the pathogenesis of mutant Ant1-induced diseases, but no consensus has been reached.Our lab has previouslyfound that mutant Aac2, the homologof Ant1 in yeast, causes cell death due to the mitochondrialbiogenesis defect. In the present study, we provided biochemical evidencesupporting the idea that themutant Aac2 proteins are misfolded, which derailsthe proteostasis on the inner membrane.We found that the assemblyand stability of multipleprotein complexeson the inner membrane are affected, including those involved in mitochondrialrespirationand protein transport. In human cells, the mutant Ant1 proteinshave reduced steady-state levels and increased degradation, consistent with misfolding and increased susceptibility to protein quality control machineries. In the second part of thework, we sought to identify the cellularsignalingpathways that respond to Ant1-induced proteostatic stress on the mitochondrialinner membrane. We generatedANT1alleles that have enhanced toxicity and are able to induce proteostatic stress on the inner membrane in human. Although these mutant Ant1 proteins only mildly affectmitochondrialrespiration, they trigger robust transcriptional responses in the nucleus to remodel cellular signaling. The upregulationof the cytosolic chaperone/ubiquitin-proteasome systems, and the downregulation of mTOR signaling may serveas adaptiveresponses to mitigate mitochondrial PrecursorOver-AccumulationStress (mPOS), a novel pathway of cell death recently discovered in yeast. We also found that humancells respond to mitochondrialinner membrane stress by increasingEgr1 signaling and the alternative splicing of many genes important for cell survival/deathcontrol. Insummary, the data suggested that protein misfoldingcauses Ant1-induced pathogenesisin human diseases. Our study provided support for mitochondria-induced proteostatic stress in the cytosol (or mPOS) in human cells. We also identified several novel mitochondria-to-nucleus signaling pathways, which may help in developingtherapeutic interventionsfor the treatmentof mitochondria-induced pathologies.
    • Mechanism of gene regulation of HNF4α

      Lu, Hong; Guo, Shangdong (2018)
      Hepatocyte Nuclear Factor 4 alpha(HNF4α)is a masterregulatorthat modulatesthe liver development andfunction. The dysfunction of HNF4αcauses multiplehumandiseases, such as hepatocellular carcinoma(HCC) and maturity onset diabetes of the young1 (MODY1). Incontrast,the restoration of HNF4α can inhibit the development of liver cancer and improve the liver function simultaneously.However, HNF4αis anorphan nuclear receptor whose activating ligand remains elusive. Therefore, an alternative approach to enhance the HNF4αactivity is to up-regulate the proteinexpression.While a great progress has been made on the functional study of HNF4α,the mechanistic details regarding the gene regulationofHNF4αare still a vast knowledge gap. The present study was aimed to investigatethe mechanism of gene regulation ofHNF4αsystematically. In chapter 2 and chapter 3, we reported the strong translational inhibition of both humanand mouse HNF4αinduced by the nucleicacid secondary structuretermed “G-quadruplex”(G4)within the 5` untranslated region(UTR).By performing the deletion/mutation studies, we determined the compositionof the G4in HNF4A 5`UTR. We further speculated thatthis G4 required the adjacent cis-elements, such as the RNA-binding proteins and other secondary structures, to form a conjunction for the strong translational inhibition. We for the first time reported the RNA-G4 induced translational repression within the 5`UTR of a tumor suppressor and highlighted the significanceof the “biostability”of G4s in exerting their biological functions. In chapter 4, we conductedacomprehensivestudy that coveredthe auto-regulation, transcriptionalregulation and transactivation activity of HNF4α. By creatingvariousreporter constructs, we were able to validate the self-stimulation of HNF4αand discovered the strong correlation between HNF4αand its corresponding anti-sense RNA, HNF4A-AS1. Additionally, we identified novel HNF4αmutations such as Q277X that may affect the crosstalk of HNF4αwith other transcriptionfactors.Overall, the novel findings from our study shedlight on the gene regulation of HNF4αand providefurther insights into ourultimate goal that is to up-regulate HNF4αprotein expression/activity to treat human diseases.
    • Mechanisms of aseptic loosening in total knee arthroplasty

      Cyndari, Karen (2017)
      Introduction: Cemented Total Knee Arthroplasty (TKA) is the gold standard of care for end-stage, multi-etiologic arthritis. While the longevity of these devices may now reach or even surpass 15 years in service, a minority (~10%) will fail prematurely due to a process called aseptic loosening. Historically, this process has been attributed to an inflammatory reaction against wear debris from the TKA polyethylene (PE) insert. However, we have previously estimated supraphysiologic fluid shear stress (FSS) (exceeding 900 Pa) at the cement-bone interface of cemented joint replacements, and examined this as a possible alternative cause of increased osteoclast activity. Methods: We analyzed the cement-bone interlock of tibial and femoral components from en-bloc, postmortem-retrieved, non-revised TKAs to explore the process of loss of fixation, prior to any clinical loosening. For the tibial components, we used a novel protocol wherein whole undecalcified bone+PMMA cement segments from the proximal tibia were embedded in Spurr’s resin and thinly sectioned. Polarized light microscopy was used to identify and quantify co-located PE debris. Using a novel bioreactor developed by our lab called the Multi-Well Fluid Loading (MFL) System, we examined static, subphysiologic, physiologic, and supraphysiologic FSS on RAW 264.7 osteoclast activity and morphology, with and without PE particle treatment. Results: We found no association been the amount or presence of PE debris and the amount or location of loss of interlock in retrieved TKAs. FSS up to 17 Pa increased the ability of osteoclasts to resorb mineral, and FSS up to 4.4 Pa induced the formation of larger osteoclasts. FSS and fluid shear rate interacted together to increase the area of actin rings, while PE treatment increased the number of actin rings and TRAP production. FSS up to 4.4 Pa decreased expression of Ctsk and Il1a, but PE co-treatment abolished this effect. Conclusions: These results indicate there may be alternative factors leading to aseptic loosening apart from PE debris. We demonstrated that osteoclasts are mechanosensitive and able to adjust activity, morphology, and gene expression based on FSS. Further, PE interferes with osteoclast gene downregulation in response to FSS, indicating PE could be a potentiator of osteoclast activity or differentiation.
    • MECHANISMS OF WISKOTT ALDRICH SYNDROME PROTEIN WSP1 POSITIONING AND REGULATION AT SITES OF ENDOCYTOSIS IN S. POMBE

      Sirotkin, Vladimir; Macquarrie, Cameron Dale (2020-12-30)
      Branched actin networks nucleated by the Arp2/3 complex provide force needed to carry out endocytosis. The Arp2/3 complex is activated by Nucleation Promoting Factors (NPFs) including Wiskott-Aldrich Syndrome protein WASp. The WASp Interacting Protein WIP binds WASp, protecting it from degradation. Humans with mutations disrupting this interaction develop a serious immune disorder, Wiskott-Aldrich Syndrome. However, in the fission yeast S. pombe WASp homolog Wsp1 remains stable in the absence of WIP homolog Vrp1, providing an ideal environment to study additional WASp and WIP functions. In fission yeast, Wsp1, Vrp1, and the class-1 myosin Myo1 localize to sites of endocytosis, known as actin patches. Wsp1 and Myo1 play an important role in activating the Arp2/3 complex and initiating the actin network needed to internalize endocytic vesicles. S. pombe endocytosis is a rapid, reproducible event involving over 40 proteins. While several of these proteins are throught to regulate branched actin assembly, many still have poorly defined functions. Importantly, how WASp proteins are regulated at sites of endocytosis remains unclear. The following studies explore mechanisms of Wsp1 regulation using quantitative live cell imaging. In Chapter 2, we observed Wsp1, Vrp1, and Myo1 forming a transient complex near the membrane, positioning branched filaments in a way that optimizes force generation. In Chapter 3, we explored the role of WIP homolog Vrp1 in actin assembly and discovered the Vrp1-Wsp1 interaction is essential for Wsp1-mediated branched actin assembly. In Chapter 4, we examined how a separate module of endocytic coat proteins contributes to actin patch assembly and discovered the coat protein Sla1 inhibits Wsp1 NPF activity, after the endocytic vesicle begins to internalize. In Chapter 5, we examined how Wsp1 and Myo1 impact additional endocytic modules and discovered Wsp1 plays an important role in expediating endocytosis and Myo1 contributes to the localization of several proteins. Lastly, in Chapter 6, we observed that blocking the fastgrowing end of actin filaments does not impact actin assembly in patches. These studies provide key insight into how WASp family proteins are regulated in vivo.
    • Memory and Effector B cell Responses to Viral and Intracellular Bacterial Infection

      Gary M. Winslow; Newell, Krista (2021)
      Infection with an intracellular pathogen presents the host with the immunological challenge of intracellular access and of clearing infection without excessive damage to host tissues. This challenge was long thought to be addressed primarily by cell-mediated immunity, but is now known to include a significant humoral component. To better understand the B cell-mediated contribution to intracellular pathogen control, we investigated memory and effector B cell responses to the intracellular bacterial pathogen Ehrlichia muris, and SARS-CoV-2 infection. B cells expressing the transcription factor T-bet were identified in both mice and humans, and T-bet played an important role in directing antibody class switch recombination, but not in the generation of memory B cells during E. muris infection. T-bet expression was identified in cells resembling murine B-1 B cells, an innate-like subset of B cells important for early T cell-independent B cell responses. These results suggest that T-bet expression in B-1 B cells may contribute to their participation in the early B cell response to murine intracellular bacterial infection. Following human SARS-CoV-2 infection, we revealed that in addition to the canonical class-switched B cell memory response, the presence of a substantial pool of peripheral blood unswitched IgM+ memory B cells was correlated with reduced symptom duration and enhanced generation of antigen-specific antibody. These IgM+ memory B cells were stable, unlike the contracting plasmablast response. These studies underscore the importance of innate and unswitched B cell subsets to the functional plasticity of the humoral response and contribute to our understanding of correlates of innate protection and adaptive immunity.
    • Meta-Analysis of Alzheimer's Disease Risk with Obesity, Diabetes, and Related Disorders

      Profenno, Louis A.; Porsteinsson, Anton P.; Faraone, Stephen V. (Elsevier BV, 2010-03)
      Background: Late-onset Alzheimer’s disease (AD) is a multifactorial and heterogeneous disorder with major risk factors including advanced age, presence of an apolipoprotein E 4 (APOE4) allele, and family history of AD. Other risk factors may be obesity and diabetes and related disorders, which are highly prevalent. Methods: We reviewed longitudinal epidemiological studies of body mass, diabetes, metabolic syndrome, and glucose and insulin levels on risk for AD. We conducted meta-analyses of the results from these studies. Results: For obesity assessed by body mass index, the pooled effect size for AD was 1.59 (95% confidence interval [CI] 1.02–2.5; z 2.0; p .042), and for diabetes, the pooled effect size for AD was 1.54 (95% CI 1.33–1.79; z 5.7; p .001). Egger’s test did not find significant evidence for publication bias in the meta-analysis for obesity (t 1.4, p .21) or for diabetes (t .86, p .42). Since these disorders are highly comorbid, we conducted a meta-analysis combining all studies of obesity, diabetes, and abnormal glucose or insulin levels, which yielded a highly significant pooled effect size for AD of 1.63 (95% CI 1.39 –1.92; z 5.9; p .001). Conclusions: Obesity and diabetes significantly and independently increase risk for AD. Though the level of risk is less than that with the APOE4 allele, the high prevalence of these disorders may result in substantial increases in future incidence of AD. Physiological changes common to obesity and diabetes plausibly promote AD.
    • Meta-Analysis of the Association Between the 7-Repeat Allele of the Dopamine D4Receptor Gene and Attention Deficit Hyperactivity Disorder

      Faraone, Stephen V.; Doyle, Alysa E.; Mick, Eric; Biederman, Joseph (American Psychiatric Association Publishing, 2001-07)
      Objective: Family, twin, and adoption studies show attention deficit hyperactivity disorder (ADHD) to have a substantial genetic component. Although several studies have shown an association between ADHD and the 7-repeat allele of the dopamine D4 receptor gene (DRD4), several studies have not. Thus, the status of the ADHD-DRD4 association is uncertain. Method: Meta-analysis was applied to case-control and family-based studies of the association between ADHD and DRD4 to assess the joint evidence for the association, the influence of individual studies, and evidence for publication bias. Results: For both the case-control and family-based studies, the authors found 1) support for the association between ADHD and DRD4, 2) no evidence that this association was accounted for by any one study, and 3) no evidence for publication bias. Conclusions: Although the association between ADHD and DRD4 is small, these results suggest that it is real. Further studies are needed to clarify what variant of DRD4 (or some nearby gene) accounts for this association.
    • Metabolic Control of Autoimmunity in the Liver

      Perl, Andras; Oaks, Zachary (2016)
      Autophagy,literally meaning “self-eating,” is an integral part of cellularturnover of damaged organelles and proteins.This process is inextricably linked to mitochondrial function and turnover. Mitochondria can be degraded viaautophagy, known as mitophagy, as well as donate lipid membraneto generate autophagosomes fordigestingother organelles and proteins. On a larger scale, autophagy is essential for organ homeostasis. In the liver, autophagy ensures the turnover of damaged mitochondria that may otherwise increase oxidative stress which modifies DNA, proteins, and lipids resulting in the production of autoantigens or neoplasia. We investigated the role of autophagy and mitochondrial dysfunction prior to disease onset in mouse models of systemic lupus erythematosus (SLE). Patients and mice with SLE exhibit overexpression of transaldolase (TAL) and show predisposition to anti-phospholipid antibody production and associated liver diseases, including hepatocellular carcinoma. Wediscovered deficient mitophagy in the liver of lupus-prone mice prior to disease onset. Furthermore, these mice had increased mitochondrial respirationwith concomitant inner membrane hyperpolarization. These changes were coupled to overexpression of Rab4A, which depletes Drp1and thus inhibitsmitophagy.In addition,activation of complex I of the mechanistic target of rapamycin (mTORC1)was noted along with enhanced production of autoantibodies against mitochondrial phospholipids in lupus-prone mice. These changes were reversed by blockade of mTORC1 by rapamycin treatment in vivo. We then examined the role of TAL, a key enzyme of the pentose phosphate pathway (PPP) in mitochondrial dysfunction and oxidative stress. TAL-deficientmice showedincreased mitochondrial electron transport chain (ETC) activity and mTORC1 activation andreduced autophagy.Since inactivation of TAL caused oxidative stress via depletion of NADPH, we tested the hypothesis that aldose reductase(AR), a NADPH dependent enzymecan correct this metabolic defect without reversing the accumulation of TAL-specific substrates, sedoheptulose 7-phosphate and erythrose 4-phosphate. Moreover, deletion of AR reversed mTORC1 activation without affecting enhanced mitochondrial ETC activity or diminished autophagy. On a more global scale, predisposition to neoplasia and acetaminophen-induced liver failurewere reversed, while anti-phospholipid autoantibody production and liver fibrosis persisted in TAL/AR double-knockout mice indicatingthat the PPPmay act as a metabolic rheostat of organ-specific disease pathogenesis.
    • Metabolic Control of Autoimmunity Through Autophagy

      Choudhary, Gourav (2019)
      Metabolism plays a key role in immune cell activation and differentiation. Immune cell activation depending on their biosynthetic and bioenergetic needs leads to profound metabolic reprograming. Proinflammatory subsets of immune system cells such as effector T cells show dependency on glycolysis, whereas, regulatory T cells rely on oxidative phosphorylation. Under metabolic stress, immune cells utilize autophagy to overcome nutrient scarcity, an alternate method of recycling amino acids and other metabolic precursors. Limitation of nutrients such as amino acids activates mechanistic target of rapamycin (mTOR) in the immune cells. mTOR acts as a metabolic mediator, associated with mitochondria and metabolic needs of the immune cells. Homeostasis between mTOR activation and autophagy decides the fate and functionality of specific immune cells. The activation of mTOR is widely acknowledged in the pathogenesis of SLE, whereas, autophagy has been linked with antigen processing, presentation, and immunoregulation. In this study, we focused on Rab4A, an endosomal GTPase and Transaldolase, a rate limiting enzyme of the pentose phosphate pathway (PPP). Rab4A is over expressed in SLE T cells and facilitates lysosomal degradation of CD4 and CD3. Transaldolase is also overexpressed in T cells from SLE patients and SLE prone mice. First, we examined the role of Rab4A in a pristane-induced mouse model of SLE. Since Rab4A protects from pristane-induced alveolar lung hemorrhage, we tested the hypothesis that Rab4A will also protect from pristane-induced lupus nephritis. We found that overexpression of a constitutively active form of Rab4A limits antinuclear antibody production. Further, we found that Rab4A protects from pristane-induced renal injury by restricting immune complex depositions in the kidney. In additions, we found that Rab4A abrogates kidney-infiltration by lymphocytes and protects from podocyte injury. Furthermore, Rab4A facilitates the lysosomal mediated activation of mTOR. Possibly, the Rab4A mediated activation of mTOR in regulatory T cells leads to suppression of pristane-induced pro-inflammation signaling. In the second part, we investigated if aldose reductase (AR) deficiency can protect from Transaldolase mediated pathogenesis of liver disease. We found a coordinated regulation between AR and TAL, leading to the disease progression.
    • MITOCHONDRIAL ELECTRON TRANSPORT CHAIN ACTIVITY IN SYSTEMIC LUPUS ERYTHEMATOSUS

      Perl, Andras; Doherty, Edward (2014)
      Systemic lupus erythematosus (SLE) is an autoimmune disorder, characterized by T cell and B cell dysfunction. SLE mitochondria have been shown to be dysfunctional with increased mass, mitochondrial potential, decreased ATP, elevated reactive oxygen species (ROS) and reactive nitrogen species (RNS) concentrations, and altered Ca2+ stores. Drug treatments that target the mitochondria have shown efficacy in treating SLE. Here we have investigated electron transport chain (ETC) activity in SLE, to better understand the causes of mitochondrial dysfunction in SLE. We have found that mitochondrial complexes I and IV of the ETC have elevated respiration in SLE compared to healthy controls after both overnight resting and anti-CD3/CD28 stimulation. We have also shown that SLE complex I is resistant to NO inhibition of respiration. SLE peripheral blood lymphocytes (PBL) have increased S-nitrosylation (SNO) while immunoprecipitated complex I had decreased SNO of proteins compared to healthy controls. The drug Nacetylcysteine (NAC) was able to inhibit complex I activity in SLE, and was found to reduce the amount of complex I protein NDUFS3 after 15 minutes as measured by western blotting. These results have led us to the conclusion that SLE mitochondrial complex I is in an active form which is resistant to SNO and is driving the production of ROS and RNS that are associated with SLE. The drug NAC is able to inhibit complex I respiration which may have therapeutic efficacy by reducing the ROS and RNS stress in SLE.
    • MITOCHONDRIAL PROTEINS AS TUMOR MARKERS AND ANTI-CANCER DRUG TARGETS

      Sheikh, Saeed; Babbar, Mansi (2017)
      Cancer is a major cause of morbidity and mortality. Identification and characterization of novel biomarkers are expected to facilitate early diagnosis and improve prognosis of human malignancies. Increasing number of studies have linked tumor progression with metabolic reprogramming. However, the players involved are not fully discovered. Therefore, understanding the cancer cell plasticity may offer a successful approach for an anti-cancer strategy. In this regard, we report the functional characterization of Coiled-coil Helix Tumor and Metabolism 1 (CHTM1) and KM1 as important regulator of cancer cell metabolism.CHTM1 is localized in cytosol and mitochondrial inter-membrane spaceand regulates mitochondrial activity. Our results demonstrate that MIA40 appears to alterCHTM1 mitochondrial localizationand stability. Further, CHTM1 cysteineresiduesinvolved in CHTM1 folding modulatescellular distributionof CHTM1. Importantly, alterations in CHTM1 expression in cancer cells affect mitochondrial activity. Given thatmitochondria play an important role in cellular response to nutrient stress, we sought to analyze the role of CHTM1 in glucose/glutamine-deprived conditions. Wehave found thatCHTM1 deficiency enhancescancer cell sensitivityto glucose/glutamine starvation and metformin treatment. Additionally, increased sensitivity of CHTM1-deficient cells to metabolic stress could be in part due to inability to activate fatty acid oxidation. Further, targeting CHTM1 expression in cancer cells reduce fatty acid oxidation causing decrease in substrate availability under metabolic stress conditions. This can explain the increase in autophagy and protein catabolism in CHTM1-deficient cancer cells under metabolic stress conditions. Mechanistic studies suggest that CHTM1-mediated alterations in cancer cell metabolism under stress conditions involve modulation of PGC1 alpha-CREB-PKC signaling.We further demonstrate that under metabolic stress, CHTM1 deficiency activates p38-AIF1pathway leading to increased cell death. CHTM1 negatively regulates p38 and interacts with AIF1 alteringAIF1release frommitochondria under metabolic stress conditions.These findings are highly significant because alterations in cancer cell metabolism are linked to pathogenesis of cancer. Most importantly, multiple human malignancies associated with breast, colon and lung tissuesshow increase in CHTM1 expression. CHTM1 appears to be a high value tumor marker, that has the potential tofacilitate earlydiagnosis of human malignancies and could also serve as a target to develop novel therapeutics to manage human malignancies. In the second part of this manuscript, we report the characterization of a novel protein temporarily named as KM1. Our results indicate that KM1 is localized inthemitochondrial inner membrane and regulates mitochondrial activity. Metabolic stress-induced increased cell death is noted in KM1 knockout cancer cells, a finding consistent with the defective mitochondria in KM1-deficient cells. Our results further demonstrate that under metabolic stress KM1 regulates mitochondrial-mediated cell death. Most importantly, KM1 levels are upregulated in breast and lung cancer tissues.Collectively, our results suggest that CHTM1 and KM1 are novel proteins and are involved in regulating cancer cell metabolism.
    • Molecular Analysisof Saccharomyc escerevisiae RNA Polymerase I Core Factor Complex and its Interaction with Promoter DNA

      Knutson, Bruce; Jackobel, Ashleigh J (2020)
      Gene transcription and protein synthesis are essential molecular processes required for all living organisms. In eukaryotes, messages encoded within DNA are transcribed by three DNA-dependent RNA polymerases (Pols I-III) into ribosomal RNA (rRNA), messenger RNA (mRNA), and transfer RNA (tRNA), respectively. General transcription factors (GTFs) help recruit Pols to their appropriate gene promoters as well as facilitate template opening and transcription start site (TSS) selection. In Saccharomyces cerevisiae, the Pol I pre-initiation complex (PIC) is formed by numerous GTFs that include Upstream Activating Factor (UAF), Core Factor (CF), TATA-binding protein (TBP), and Rrn3. This unique set of GTFs engage ribosomal DNA (rDNA) through interactions with regulatory elements of the promoter known as the Upstream Activating Sequence (UAS) and the Core Element (CE). Here, we resolve the cryo-electron microscopy (cryo-EM) structure of CF bound to the rDNA promoter at 3.8Å near-atomic resolution and determine itsDNA binding preferences in which CF preferentially binds to the GC-minor groove. Briefly, our cryo-EM studies reveal that the CF-DNA interaction is mediated by two CF subunits, Rrn7 and Rrn11. We also found that the path of promoter DNA is relatively straight in the Pol I PIC, which is strikingly different from the bent promoters observed in structures of the Pols II/III PICs. We identified three states of CF engagement with promoter DNA (States 1-3) in which CF acts as a ratchet toforceDNA into the active site of the polymerase that facilitates the melting of the double-stranded DNA template in an ATP-independent manner, another unique feature of the Pol I system. Using in vitroDNA binding assays, we have identified a 12 base pair (bp) region within the CE that is necessary and sufficient for CF binding. We have also demonstrated that the human anticancer compound CX-5461 can inhibit yeast cell growth and blocks yeast CF binding to both yeast and human rDNA promoters in vitro. Furthermore, we show that the human Core Promoter Element (CPE) can functionally replace the yeast CE in a position-dependent manner. Together, these results reveal the unique molecular architecture of the Pol I PIC and suggest a conserved sequence-independent binding mechanism of CF with promoter DNA.
    • The monoamine oxidase B gene exhibits significant association to ADHD

      Li, Jun; Wang, Yufeng; Hu, Songnian; Zhou, Rulun; Yu, Xiaomin; Wang, Bing; Guan, Lili; Yang, Li; Zhang, Feng; Faraone, Stephen V. (Wiley, 2008)
      Attention deficit hyperactivity disorder (ADHD) is a common neuropsychiatric condition with strong genetic basis. Recent work in China indicated that ADHD may be linked to Xp1–2 in the Han Chinese population. The gene encoding monoamine oxidase B (MAOB), the main enzyme degrading dopamine in the human brain, is located in this region. The current study sequenced the exons and the 50 and 30 flanking regions of theMAOBgene and found four common variants including 2276C>T and 2327C>T in exon 15, rs1799836 in intron 13 and rs1040399 in 30-UTR. We assessed the association of these variants with ADHD in 548 trios collected from 468 males and 80 females probands. TDT analysis showed that alleles of each polymorphism were preferentially transmitted to probands (rs1799836, P¼3.28E-15; rs1040399, P¼1.87E-6; 2276T>C or 2327T>C, P¼2.20E-6) and haplotype-based TDT analyses also found distorted transmission. In conclusion, this study provides the strongest evidence for the involvement of MAOB gene in the etiology of ADHD to date, at least in Han Chinese population.
    • MULTI-FUNCTIONAL EFFECTOR RESPONSES ELICITED FROM IgM MEMORY STEM CELLS

      Winslow, Gary; Kenderes, Kevin (2017)
      The response of memory B cells to challenge infection is fundamental to longterm protection against pathogens. Following challenge, memory B cells can rapidly differentiate into antibody-secreting cells (ASCs) to produce a secondary antibody response. Memory B cells have also been shown to re-enter into germinal centers and undergo additional rounds of affinity maturation. Both the isotype of the B cell and the signals that generated the B cell have been proposed to modulate how memory B cells respond. Initial studies proposed BCR-intrinsic factors are responsible for the differentiation of memory cells. IgM memory cells undergo differentiation in GCs following antigen challenge, while IgG memory cells rapidly differentiate into ASCs. Other studies found no link-between BCR isotype and differentiation. We investigated the differentiation of T-bet+ CD11c+ IgM memory B cells following challenge infection. IgM memory cells differentiated into IgM-producing plasmablasts. Other IgM memory B cells entered germinal centers, underwent class switching, and became switched memory cells. Yet other donor cells were maintained as IgM memory cells. The IgM memory cells also retained their multi-lineage potential following serial transfer. The kinetics of the IgM memory response mimicked the kinetics of the primary response. Thus, IgM memory cells can differentiate into all effector B cell lineages, and undergo self-renewal, properties that are characteristic of stem cells; however, differentiation occurs with the same kinetics of the primary response. We propose that memory B cells have varying degrees of stem cell likeness. IgM memory stem cells retain the most differentiating capacity but respond to challenge similarly to naïve cells, while IgG effector memory cells are primed to rapidly differentiate into IgG ASCs.
    • The multidimensionality of schizotypy in nonpsychotic relatives of patients with schizophrenia and its applications in ordered subsets linkage analysis of schizophrenia

      Lien, Yin-Ju; Tsuang, Hui-Chun; Chiang, Abigail; Liu, Chih-Min; Hsieh, Ming H.; Hwang, Tzung-Jeng; Liu, Shi K.; Hsiao, Po-Chang; Faraone, Stephen V.; Tsuang, Ming T.; et al. (Wiley, 2009)
      This study aimed to examine the multidimensionality of schizotypy and validate the structure using ordered subset linkage analyses on information from both relatives’ schizotypy and probands’ schizophrenia symptoms. A total of 203 and 1,310 nonpsychotic first-degree relatives from simplex and multiplex schizophrenia families, respectively, were interviewed with the Diagnostic Interview for Genetic Studies, which contains a modified Structured Interview for Schizotypy. Using Mplus program with categorical factor indicators, a four-factor model (Negative Schizotypy, Positive Schizotypy, Interpersonal Sensitivity, and Social Isolation/Introversion) was extracted by exploratory factor analysis from relatives of simplex families and was confirmed in relatives of multiplex families. The validity of each factor was supported by distinct linkage findings resulting from ordered subset analysis based on different combinations of schizophrenia–schizotypy factors. Six chromosomal regions with significant increase in nonparametric linkage z score (NPL-Z) were found as follows: 15q21.1 (NPLZ ¼3.60) for Negative Schizophrenia–Negative Schizotypy, 10q22.3 (NPL-Z¼3.83) and 15q21.3 (NPL-Z¼3.36) for Negative Schizophrenia–Social Isolation/Introversion, 5q14.2 (NPL-Z¼3.20) and 11q23.3 (NPL-Z¼3.31) for Positive Schizophrenia–Positive Schizotypy, and 4q32.1 (NPL-Z¼3.31- ) for Positive Schizophrenia–Interpersonal Sensitivity. The greatest NPL-Z of 3.83 on 10q22.3 in the subset was significantly higher than the greatest one of 2.88 in the whole sample (empirical P-value¼0.04). We concluded that a consistent four-factor model of schizotypy could be derived in nonpsychotic relatives across families of patients with different genetic loadings in schizophrenia. Their differential relations to linkage signals have etiological implications and provide further evidence for their validity. 2009 Wiley-Liss, Inc.
    • NEAR-ATOMIC RESOLUTION STRUCTURE OF THE YEAST VACUOLAR (V-) ATPASE MEMBRANE SECTOR Vo IN LIPID NANODISC

      Wilkens, Stephan; Stam, Nicholas J (2020-04-10)
      Vacuolar ATPase (V-ATPase) is a large multisubunit enzyme that acidifies subcellular organelles and the extracellular space. Its activity is regulated by reversible disassembly, causing V-ATPase dissociation into soluble V1-ATPase and membrane-integral Voproton channel sectors.The goal of this thesis project was to observethe yeast Voin a physiologically relevant, auto-inhibited state, i.e. in its form dissociated from the ATPase sector, V1, in order to better visualizethe closed pore and to identify testable hypotheses on why the pore remains closed following dissociation from V1. Towards this aim we present two chapters:In Chapter 1, we detail a single-particle negative stain EM study of lipid nanodisc reconstituted Vo,which suggesteddissociated Vois halted in theso-called rotational state 3 of the holo-enzyme.We performed site directed mutagenesis and binding studies of subunits aand dto test and validate this hypothesis.In Chapter 2,we further detaillipid nanodisc reconstitutedVoin a high-resolutioncryoEM structure,confirmingour earlier identification of Voresting in rotational state 3, andproviding structural information of the sectorat the amino acid level. Through this work we proposed apossible mechanism for transmembrane proton transport in the V-ATPaseandidentifieda new subunit member of Vo, assembly factor Voa1.The studies shown here highlight the potential of lipid nanodisc reconstitution of membrane protein complexes, give insight into a conformational mismatch between autoinhibited V1and halted Vowith the implication that the mismatch may serve to prevent unintended reassembly of V-ATPase upon activity silencing, and propose a chemical basis for transmembrane proton transport in the Voproton pore.