Direct structural analysis of a single acyl carrier protein domain in fatty acid synthase from the fungus Saccharomyces cerevisiae.

Acyl carrier protein (ACP) is the work horse of polyketide (PKS) and fatty acid synthases (FAS) and acts as a substrate shuttling domain in these mega enzymes. In fungi, FAS forms a 2.6 MDa symmetric assembly with six identical copies of FAS1 and FAS2 polypeptides. However, ACP spatial distribution is not restricted by symmetry owing to the long and flexible loops that tether the shuttling domain to its corresponding FAS2 polypeptide. This symmetry breaking has hampered experimental investigation of substrate shuttling route in fungal FAS. Here, we develop a protein engineering and expression method to isolate asymmetric fungal FAS proteins containing odd numbers of ACP domains. Electron cryomicroscopy (cryoEM) observation of the engineered complex reveals a non-uniform distribution of the substrate shuttling domain relative to its corresponding FAS2 polypeptide at 2.9 Å resolution. This work lays the methodological foundation for experimental study of ACP shuttling route in fungi.

Human Alzheimer's disease reactive astrocytes exhibit a loss of homeostastic gene expression.

Astrocytes are one of the brain's major cell types and are responsible for maintaining neuronal homeostasis via regulating the extracellular environment, providing metabolic support, and modulating synaptic activity. In neurodegenerative diseases, such as Alzheimer's disease, astrocytes can take on a hypertrophic appearance. These reactive astrocytes are canonically associated with increases in cytoskeletal proteins, such as glial fibrillary acidic protein and vimentin. However, the molecular alterations that characterize astrocytes in human disease tissues have not been extensively studied with single cell resolution. Using single nucleus RNA sequencing data from normal, pathologic aging, and Alzheimer's disease brains, we identified the transcriptomic changes associated with reactive astrocytes. Deep learning-based clustering algorithms denoised expression data for 17,012 genes and clustered 15,529 astrocyte nuclei, identifying protoplasmic, gray matter and fibrous, white matter astrocyte clusters. RNA trajectory analyses revealed a spectrum of reactivity within protoplasmic astrocytes characterized by a modest increase of reactive genes and a marked decrease in homeostatic genes. Amyloid but not tau pathology correlated with astrocyte reactivity. To identify reactivity-associated genes, linear regressions of gene expression versus reactivity were used to identify the top 52 upregulated and 144 downregulated genes. Gene Ontology analysis revealed that upregulated genes were associated with cellular growth, responses to metal ions, inflammation, and proteostasis. Downregulated genes were involved in cellular interactions, neuronal development, ERBB signaling, and synapse regulation. Transcription factors were significantly enriched among the downregulated genes. Using co-immunofluorescence staining of Alzheimer's disease brain tissues, we confirmed pathologic downregulation of ERBB4 and transcription factor NFIA in reactive astrocytes. Our findings reveal that protoplasmic, gray matter astrocytes in Alzheimer's disease exist within a spectrum of reactivity that is marked by a strong loss of normal function.

Atomic model for core modifying region of human fatty acid synthase in complex with Denifanstat.

Fatty acid synthase (FASN) catalyzes the de novo synthesis of palmitate, a 16-carbon chain fatty acid that is the primary precursor of lipid metabolism and an important intracellular signaling molecule. FASN is an attractive drug target in diabetes, cancer, fatty liver diseases, and viral infections. Here, we develop an engineered full-length human FASN (hFASN) that enables isolation of the condensing and modifying regions of the protein post-translation. The engineered protein enables electron cryo-microscopy (cryoEM) structure determination of the core modifying region of hFASN to 2.7 Å resolution. Examination of the dehydratase dimer within this region reveals that unlike its close homolog, porcine FASN, the catalytic cavity is close-ended and is accessible only through one opening in the vicinity of the active site. The core modifying region exhibits two major global conformational variabilities that describe long-range bending and twisting motions of the complex in solution. Finally, we solved the structure of this region bound to an anti-cancer drug, Denifanstat (i.e., TVB-2640), demonstrating the utility of our approach as a platform for structure guided design of future hFASN small molecule inhibitors.

Targeting STAT5 Signaling Overcomes Resistance to IDH Inhibitors in Acute Myeloid Leukemia through Suppression of Stemness.

Mutant isocitrate dehydrogenase 1 (IDH1) and IDH2 block the differentiation of acute myeloid leukemia (AML) cells through production of R-2-hydroxyglutarate (R-2-HG). IDH inhibitors can induce differentiation of AML cells by lowering R-2-HG but have limited clinical efficacy as single agents. Here, we performed a genome-wide CRISPR knockout screen in an Idh1-mutated hematopoietic progenitor cell line to identify genes that increased the differentiation response to ivosidenib, an IDH1 inhibitor. The screen identified C-type lectin member 5a (Clec5a), which encodes a spleen tyrosine kinase (SYK)-coupled surface receptor, as one of the top hits. Knockout of Clec5a and Syk rendered cells more sensitive to ivosidenib-induced differentiation through a reduction in STAT5-dependent expression of stemness-related genes, including genes in the homeobox (HOX) family. Importantly, direct inhibition of STAT5 activity was sufficient to increase the differentiation response to IDH inhibitors in primary human IDH1- and IDH2-mutated AML cells, including those harboring mutations in receptor tyrosine kinase (RTK) and MAPK genes that have been linked to drug resistance. In patient-derived xenograft models of IDH1-mutated AML, combination treatment with ivosidenib and the STAT5 inhibitor pimozide was superior to each agent alone in inducing differentiation in leukemic cells without compromising normal hematopoiesis. These findings demonstrate that STAT5 is a critical mediator of resistance to IDH inhibitors and provide the rationale for combining STAT5 and IDH inhibitors in the treatment of IDH-mutated AML. SIGNIFICANCE: A CRISPR knockout screen identifies a mechanism of resistance to IDH inhibitors in AML involving activated STAT5 signaling, suggesting a potential strategy to improve the clinical efficacy of IDH inhibitors.

SARS-CoV-2 impairs interferon production via NSP2-induced repression of mRNA translation.

Viruses evade the innate immune response by suppressing the production or activity of cytokines such as type I interferons (IFNs). Here we report the discovery of a mechanism by which the SARS-CoV-2 virus coopts an intrinsic cellular machinery to suppress the production of the key immunostimulatory cytokine IFN-ß. We reveal that the SARS-CoV-2 encoded nonstructural protein 2 (NSP2) directly interacts with the cellular GIGYF2 protein. This interaction enhances the binding of GIGYF2 to the mRNA cap-binding protein 4EHP, thereby repressing the translation of the Ifnb1 mRNA. Depletion of GIGYF2 or 4EHP significantly enhances IFN-ß production, which inhibits SARS-CoV-2 replication. Our findings reveal a target for rescuing the antiviral innate immune response to SARS-CoV-2 and other RNA viruses.

Structural Characterization of Endogenous Tuberous Sclerosis Protein Complex Revealed Potential Polymeric Assembly.

Tuberous sclerosis protein complex (pTSC) nucleates a proteinaceous signaling hub that integrates information about the internal and external energy status of the cell in the regulation of growth and energy consumption. Biochemical and cryo-electron microscopy studies of recombinant pTSC have revealed its structure and stoichiometry and hinted at the possibility that the complex may form large oligomers. Here, we have partially purified endogenous pTSC from fasted mammalian brains of rat and pig by leveraging a recombinant antigen binding fragment (Fab) specific for the TSC2 subunit of pTSC. We demonstrate Fab-dependent purification of pTSC from membrane-solubilized fractions of the brain homogenates. Negative stain electron microscopy of the samples purified from pig brain demonstrates rod-shaped protein particles with a width of 10 nm, a variable length as small as 40 nm, and a high degree of conformational flexibility. Larger filaments are evident with a similar 10 nm width and a ≤1 µm length in linear and weblike organizations prepared from pig brain. Immunogold labeling experiments demonstrate linear aggregates of pTSC purified from mammalian brains. These observations suggest polymerization of endogenous pTSC into filamentous superstructures.

Apolipoprotein E Gene Revisited: Contribution of Rare Variants to Alzheimer's Disease Susceptibility in Southern Chinese.

BACKGROUND: APOE ε4 is the best-known risk factor for late-onset alzheimer's disease (AD). Population studies have demonstrated a relatively low prevalence of APOE ε4 among Chinese population, implying additional risk factors that are Chinese-specific may exist. Apart from - alleles, genetic variation profile along the full-length APOE has rarely been investigated. OBJECTIVE: In this study, we filled this gap by comprehensively determining all genetic variations in APOE and investigated their potential associations with late-onset AD and mild cognitive impairment (MCI) in southern Chinese. METHODS: Two hundred and fifty-seven southern Chinese participants were recruited, of whom 69 were AD patients, 83 had MCI, and 105 were normal controls. Full-length APOE from promoter to 3'UTR regions were sequenced. Genetic variants were identified and compared among the three groups. RESULTS: While APOE ε4 was more significantly found in AD patients, the prevalence of APOE ε4 in southern Chinese AD patients was the lowest when compared to other areas of China and nearby regions, as well as other countries worldwide. We further identified 13 rare non-singleton variants in APOE. Significantly more AD patients carried any of the rare non-singleton variants than MCI and normal subjects. Such difference was observed in the non-carriers of ε4-allele only. Among the identified rare variants, the potential functional impact was predicted for rs532314089, rs553874843, rs533904656 and rs370594287. CONCLUSION: Our study suggests an ethnic difference in genetic risk composition of AD in southern Chinese. Rare variants on APOE are a potential candidate for AD risk stratification biomarker in addition to APOE-ε4.

ADNC-RS, a clinical-genetic risk score, predicts Alzheimer's pathology in autopsy-confirmed Parkinson's disease and Dementia with Lewy bodies.

Growing evidence suggests overlap between Alzheimer's disease (AD) and Parkinson's disease (PD) pathophysiology in a subset of patients. Indeed, 50-80% of autopsy cases with a primary clinicopathological diagnosis of Lewy body disease (LBD)-most commonly manifesting during life as PD-have concomitant amyloid-beta and tau pathology, the defining pathologies of AD. Here we evaluated common genetic variants in genome-wide association with AD as predictors of concomitant AD pathology in the brains of people with a primary clinicopathological diagnosis of PD or Dementia with Lewy Bodies (DLB), diseases both characterized by neuronal Lewy bodies. In the first stage of our study, 127 consecutive autopsy-confirmed cases of PD or DLB from a single center were assessed for AD neuropathological change (ADNC), and these same cases were genotyped at 20 single nucleotide polymorphisms (SNPs) found by genome-wide association study to associate with risk for AD. In these 127 training set individuals, we developed a logistic regression model predicting the presence of ADNC, using backward stepwise regression for model selection and tenfold cross-validation to estimate performance. The best-fit model generated a risk score for ADNC (ADNC-RS) based on age at disease onset and genotype at three SNPs (APOE, BIN1, and SORL1 loci), with an area under the receiver operating curve (AUC) of 0.751 in our training set. In the replication stage of our study, we assessed model performance in a separate test set of the next 81 individuals genotyped in our center. In the test set, the AUC was 0.781, and individuals with ADNC-RS in the top quintile had four-fold increased likelihood of having AD pathology at autopsy compared with those in each of the lowest two quintiles. Finally, in the validation stage of our study, we applied our ADNC-RS model to 70 LBD individuals from 20 Alzheimer's Disease Research Centers (ADRC) whose autopsy and genetic data were available in the National Alzheimer's Coordinating Center (NACC) database. In this validation set, the AUC was 0.754. Thus, in patients with autopsy-confirmed PD or DLB, a simple model incorporating three AD-risk SNPs and age at disease onset substantially enriches for concomitant AD pathology at autopsy, with implications for identifying LBD patients in which targeting amyloid-beta or tau is a therapeutic strategy.

Translational control in the tumor microenvironment promotes lung metastasis: Phosphorylation of eIF4E in neutrophils.

The translation of mRNAs into proteins serves as a critical regulatory event in gene expression. In the context of cancer, deregulated translation is a hallmark of transformation, promoting the proliferation, survival, and metastatic capabilities of cancer cells. The best-studied factor involved in the translational control of cancer is the eukaryotic translation initiation factor 4E (eIF4E). We and others have shown that eIF4E availability and phosphorylation promote metastasis in mouse models of breast cancer by selectively augmenting the translation of mRNAs involved in invasion and metastasis. However, the impact of translational control in cell types within the tumor microenvironment (TME) is unknown. Here, we demonstrate that regulatory events affecting translation in cells of the TME impact cancer progression. Mice bearing a mutation in the phosphorylation site of eIF4E (S209A) in cells comprising the TME are resistant to the formation of lung metastases in a syngeneic mammary tumor model. This is associated with reduced survival of prometastatic neutrophils due to decreased expression of the antiapoptotic proteins BCL2 and MCL1. Furthermore, we demonstrate that pharmacological inhibition of eIF4E phosphorylation prevents metastatic progression in vivo, supporting the development of phosphorylation inhibitors for clinical use.

Neuropsychological Profile and Dementia Symptom Recognition in Help-Seekers in a Community Early-Detection Program in Hong Kong.

OBJECTIVES: To examine the neuropsychological and clinical profile of help-seekers in an early-detection community dementia program and to explore any relationship between profiles and time to seek help. DESIGN: Cross-sectional. SETTING: Early-detection community dementia program. PARTICIPANTS: Help-seekers (N = 1,005) with subjective cognitive complaints or complaints from an informant. MEASUREMENTS: Neurocognitive testing, including the Cantonese Mini-Mental State Examination (MMSE), Clock Drawing Test, Digit Span, and Fuld Object Memory Evaluation and other clinical and functioning assessments, including the Clinical Dementia Rating (CDR), activities of daily living (ADLs), instrumental ADLs (IADLs), and depressive symptoms. Time since the person or an informant reported that they first noticed symptoms. RESULTS: Eighty-six percent of help-seekers had at least very mild dementia (CDR score ≥0.5). Cognitive performance was moderately impaired (mean MMSE score 18.4 ± 6.1). They required some assistance with IADLs, had very mild ADL impairments, and had few depressive symptoms. Median time to seek assessment was 12 months (interquartile range 7-30 months) according to the person or the informant (an adult child in 75% of the sample). Using the median-split method, time to seek assessment was classified as early (0-12 months) and late (>12 months). Worse cognitive and IADL performance but not ADL performance or depressive symptoms were observed in late than in early help-seekers. Longer time intervals between symptom recognition and early assessment showed a trend of further impairments on all measures except ADLs. CONCLUSION: A time interval of more than 12 months between symptom recognition and early assessment appears to be associated with worse cognitive function upon presentation.

Development of Six Arts, a Culturally Appropriate Multimodal Nonpharmacological Intervention in Dementia.

PURPOSE: There is accumulating evidence for the efficacy of nonpharmacological multimodal stimulation interventions in maintaining cognition and improving quality of life in people with mild-to-moderate dementia. However, the complex nature of these interventions limits their application in practice and research. We report here the design and development of a culturally appropriate framework, the Six Arts, to guide delivery of multimodal interventions in a Chinese community. DESIGN AND DEVELOPMENT: The Six Arts are a core set of Confucian philosophy comprising 6 disciplines of rites, music, archery, charioteering, literacy, and numeracy. They correspond to major mind-body functional domains of social functioning; music and rhythm; visuospatial and fine motor skills; kinesthetic and gross motor skills; language and verbal skills; and executive function. Using Six Arts as a framework, we mapped theoretical principles and evidence-based nonpharmacological interventions of cognitive stimulation, physical exercise, and social activities against the 6 functional domains. From 2011, we field-tested the use of Six Arts in structuring intervention programs in 263 people in a dementia day center in Hong Kong. RESULTS: The Six Arts was operationalized through the development of an intervention activity database, a scoring system for intensity level, and a service delivery model for application in dementia day centers. IMPLICATIONS: Six Arts can be used as framework for structuring nonpharmacological group intervention programs in dementia day center in a metropolitan Chinese city. Its cultural appropriateness may facilitate communication and shared decision making with families with dementia in communities influenced by Confucian philosophy.

A case of young-onset dementia.

Dementia is a major worldwide public health concern in view of the global ageing phenomenon. Dementia usually occurs in old age. However, if the symptoms occur in young patients, the diagnosis can be challenging. Posterior cortical atrophy is a variant of the Alzheimer's disease, which is described as a presenile disease affecting relatively late-middle-aged patients. A combination of clinical, neuropsychological, and neuroimaging techniques may facilitate making a diagnosis of this particular patient group, as demonstrated in this report. Although there is no effective disease-modifying agent for treating these patients to date, there may be considerable pressure to arrive at a quick and accurate diagnosis from the perspective of employment and insurance.

Effect of early pulmonary rehabilitation on health care utilization and health status in patients hospitalized with acute exacerbations of COPD.

BACKGROUND AND OBJECTIVE: Acute exacerbations of chronic obstructive pulmonary disease (AECOPD) incur heavy utilization of health-care resources for patients who require hospitalization. We evaluated whether an early outpatient pulmonary rehabilitation programme (PRP) after hospitalization for AECOPD could reduce acute health-care utilization over the succeeding year. METHODS: Sixty patients admitted with AECOPD were randomized to either PRP or usual care (UC). The PRP group received 8weeks of outpatient rehabilitation programme 2-3weeks after discharge from hospital. Lung function, 6min walk test and dyspnoea score were assessed at baseline, 3, 6, 9 and 12months, while St George's respiratory questionnaire and cardiopulmonary exercise test were assessed at baseline, 3, 6 and 12months. RESULTS: The PRP and UC groups demonstrated a 53.3% and 43.3% risk of readmissions at 12months (incident risk ratio 0.97 (95% CI: 0.57-1.60), P=0.90). The mean readmission rates were 1.00±1.20 and 1.03±1.87 (P=0.47) for the PRP versus UC groups respectively. The rates of AECOPD and emergency department visits were similar between the two groups. The St George's respiratory questionnaire total score was lower in the PRP group (40.15±19.10 vs 46.91±18.21, P=0.01 and 42.3±20.06 vs 51.44±18.98 P=0.01 at 3 and 6months respectively). There were no statistically significant differences in the FEV(1) % predicted, dyspnoea score, 6min walk test and maximal oxygen consumption during exercise test between PRP and UC at different time points. CONCLUSIONS: An early rehabilitation programme following AECOPD led to improvement in quality of life up to 6months, but did not reduce health-care utilization at 1year.

Tuberculin skin test reaction and body mass index in old age home residents in Hong Kong.

OBJECTIVES: To study the relationship between body mass index (BMI) and tuberculin skin test (TST) reaction in predicting the development of active tuberculosis (TB). DESIGN: A follow-up study. SETTING: Old age homes. PARTICIPANTS: Three thousand six hundred five residents who took part in a screening program for TB and had two-step TST using two units of the tuberculin PPD-RT23. MEASUREMENTS: Rate of development of active TB in these residents over an average follow-up period of 2.5+/-1.25 years. RESULTS: After one-step and two-step testing, 46.3% and 69.6% of residents, respectively, had positive TST reactions (> or =10 mm). Thirty-four residents developed active TB (323 per 100,000 person-years) during follow-up. The only significant risk factors associated with development of active TB were positive TST according to one-step testing (adjusted odds ratio (OR)=2.91, 95% confidence interval (CI)=1.26-6.74) and a BMI less than 18.5 (adjusted OR=3.15, 95% CI=1.45-6.86). Residents with a BMI less than 18.5 and a negative TST also had greater risk of active TB than residents with a BMI greater than 18.5 and negative TST (adjusted OR=4.36, 95% CI=1.04-18.3), whereas those with a positive TST had the highest risk (adjusted OR=10.2, 95% CI=2.63-39.4). Two-step testing increased the sensitivity but reduced the specificity of TST in identifying active TB on follow-up. CONCLUSION: In the elderly population, interpretation of TST should take into consideration the BMI of the individual. A positive TST according to one-step but not two-step testing was useful in predicting the development of active TB on follow-up.

Prevalence of tuberculous infection and active tuberculosis in old age homes in Hong Kong.

OBJECTIVES: To assess the prevalence of tuberculous infection and active tuberculosis (TB) in old age homes in Hong Kong and to determine whether there is institutional transmission in these homes. DESIGN: Cross-sectional. SETTING: Old age homes. PARTICIPANTS: Total of 2,243 residents, representing 84.6% of all residents in 15 old age homes; 1,698 were women, and 545 were men, with an average age of 82. MEASUREMENTS: All residents had a questionnaire-based interview, medical record review, two-stage tuberculin testing using two units purified protein derivative-RT23, and a chest x-ray. Those with radiological abnormalities had sputum examined for acid-fast bacilli. RESULTS: The estimated prevalence rate of active TB in this population was 669 per 100,000, significantly higher in men than in women (1,101 per 100,000 vs 530 per 100,000). The proportion with positive tuberculin reactivity (> or =10 mm induration) after two-stage testing was 68.6%, significantly higher in men than in women. There was no evidence of active transmission of disease in these old age homes, with restriction fragment length polymorphism (RFLP) analysis performed on five cases of active pulmonary TB in the home with the highest rate of TB showing unique RFLP patterns. CONCLUSION: The rate of active TB and TB infection in old age homes in Hong Kong is still high. Because treatment for latent TB carries a high risk for liver dysfunction in this population, clinicians and other healthcare workers need a high index of suspicion and to diagnose and treat this disease as early as possible to prevent transmission.