Pan-cancer Genomic Analysis of AXL Mutations Reveals a Novel, Recurrent, Functionally Activating AXL W451C Alteration Specific to Myxofibrosarcoma.

Myxofibrosarcoma (MFS) is a common soft tissue sarcoma of the elderly that typically shows low tumor mutational burden, with mutations in TP53 and in genes associated with cell cycle checkpoints ( RB1 , CDKN2A ). Unfortunately, no alterations or markers specific to MFS have been identified and, as a consequence, there are no effective targeted therapies. The receptor tyrosine kinase AXL, which drives cellular proliferation, is targetable by new antibody-based therapeutics. Expression of AXL messenger RNA is elevated in a variety of sarcoma types, with the highest levels reported in MFS, but the pathogenic significance of this finding remains unknown. To assess a role for AXL abnormalities in MFS, we undertook a search for AXL genomic alterations in a comprehensive genomic profiling database of 463,546 unique tumors (including 19,879 sarcomas, of which 315 were MFS) interrogated by targeted next-generation DNA and/or RNA sequencing. Notably, the only genomic alterations recurrent in a specific sarcoma subtype were AXL W451C (n = 8) and AXL W450C (n = 2) mutations. The tumors involved predominantly older adults (age: 44 to 81 [median: 72] y) and histologically showed epithelioid and spindle-shaped cells in a variably myxoid stroma, with 6 cases diagnosed as MFS, 3 as undifferentiated pleomorphic sarcoma (UPS), and 1 as low-grade sarcoma. The AXL W451C mutation was not identified in any non-sarcoma malignancy. A review of publicly available data sets revealed a single AXL W451C-mutant case of UPS that clustered with MFS/UPS by methylation profiling. Functional studies revealed a novel activation mechanism: the W451C mutation causes abnormal unregulated dimerization of the AXL receptor tyrosine kinase through disulfide bond formation between pairs of mutant proteins expressing ectopic cysteine residues. This dimerization triggers AXL autophosphorylation and activation of downstream ERK signaling. We further report sarcomas of diverse histologic subtypes with AXL gene amplifications, with the highest frequency of amplification identified in MFS cases without the W451C mutation. In summary, the activating AXL W451C mutation appears highly specific to MFS, with a novel mechanism to drive unregulated signaling. Moreover, AXL gene amplifications and messenger RNA overexpression are far more frequent in MFS than in other sarcoma subtypes. We conclude that these aberrations in AXL are distinct features of MFS and may aid diagnosis, as well as the selection of available targeted therapies.

SIRT5 variants from patients with mitochondrial disease are associated with reduced SIRT5 stability and activity, but not with neuropathology.

SIRT5 is a sirtuin deacylase that represents the major activity responsible for removal of negatively-charged lysine modifications, in the mitochondrial matrix and elsewhere in the cell. In benign cells and mouse models, under basal non-stressed conditions, the phenotypes of SIRT5 deficiency are generally quite subtle. Here, we identify two homozygous SIRT5 variants in human patients suffering from severe mitochondrial disease. Both variants, P114T and L128V, are associated with reduced SIRT5 protein stability and impaired biochemical activity, with no evidence of neomorphic or dominant negative properties. The crystal structure of the P114T enzyme was solved and shows only subtle deviations from wild-type. Via CRISPR-Cas9, we generate a mouse model that recapitulates the human P114T mutation; homozygotes show reduced SIRT5 levels and activity, but no obvious metabolic abnormalities, neuropathology or other gross evidence of severe disease. We conclude that these human SIRT5 variants most likely represent severe hypomorphs, and are likely not the primary pathogenic cause of the neuropathology observed in the patients.

Mycoplasma pneumoniae: Among the smallest bacterial pathogens with great clinical significance in children.

BACKGROUND: Mycoplasmas are the smallest prokaryotic microorganisms found in nature. Mycoplasma pneumoniae (M. pneumoniae) is the most commonly studied among human mycoplasmas. OBJECTIVES: In this review, we briefly focus on the recent developments that have enhanced our understanding of M. pneumoniae, one of the smallest pathogenic bacteria of great clinical importance in children. CONTENT: M. pneumoniae infections may involve either upper or lower respiratory tract or both of them. Extrapulmonary manifestations have been reported in almost every organ, including the skin and the hematologic, cardiovascular, musculoskeletal, and nervous system due to direct local effects, after dissemination of bacteria or indirect effects. The correct identification of M. pneumoniae infections is vital for prescription of the appropriate therapy.There are scarce specific findings of clinical laboratory results for the diagnosis of M. pneumoniae infection. Detection of M. pneumoniae infections can be achieved using culture, serology, or molecular-based methods. Culture is time-consuming, laborious, and expensive. The major types of serological tests for M. pneumoniae include the microtiter plate enzyme immunoassay (EIA), the membrane EIA, indirect immunofluorescence, and particle agglutination. Nucleic acid amplification tests (NAATs) include traditional PCR, nested PCR, real-time quantitative PCR, nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP) technology, and RNA simultaneous amplification and testing (SAT). Macrolides have been the drug of choice for treating M. pneumoniae infection in past years. Clinically significant acquired macrolide-resistant M. pneumoniae (MRMP)has emerged worldwide which may be associated with more extrapulmonary complications, and severe clinical and radiological features. Since molecular-based assays can detect M. pnueumoniae in clinical specimens, there is a need for real point of care testing for fast detection of M. pneumoniae or its DNA and mutations in macrolide resistance gene. It is necessary to develop safe vaccines that provide protective immunity against M.pneumoniae infection.

Cdc73 protects Notch-induced T-cell leukemia cells from DNA damage and mitochondrial stress.

ABSTRACT: Activated Notch signaling is highly prevalent in T-cell acute lymphoblastic leukemia (T-ALL), but pan-Notch inhibitors showed excessive toxicity in clinical trials. To find alternative ways to target Notch signals, we investigated cell division cycle 73 (Cdc73), which is a Notch cofactor and key component of the RNA polymerase-associated transcriptional machinery, an emerging target in T-ALL. Although we confirmed previous work that CDC73 interacts with NOTCH1, we also found that the interaction in T-ALL was context-dependent and facilitated by the transcription factor ETS1. Using mouse models, we showed that Cdc73 is important for Notch-induced T-cell development and T-ALL maintenance. Mechanistically, chromatin and nascent gene expression profiling showed that Cdc73 intersects with Ets1 and Notch at chromatin within enhancers to activate expression of known T-ALL oncogenes through its enhancer functions. Cdc73 also intersects with these factors within promoters to activate transcription of genes that are important for DNA repair and oxidative phosphorylation through its gene body functions. Consistently, Cdc73 deletion induced DNA damage and apoptosis and impaired mitochondrial function. The CDC73-induced DNA repair expression program co-opted by NOTCH1 is more highly expressed in T-ALL than in any other cancer. These data suggest that Cdc73 might induce a gene expression program that was eventually intersected and hijacked by oncogenic Notch to augment proliferation and mitigate the genotoxic and metabolic stresses of elevated Notch signaling. Our report supports studying factors such as CDC73 that intersect with Notch to derive a basic scientific understanding on how to combat Notch-dependent cancers without directly targeting the Notch complex.

Biallelic variants in COQ7 cause distal hereditary motor neuropathy with upper motor neuron signs.

COQ7 encodes a hydroxylase responsible for the penultimate step of coenzyme Q10 (CoQ10) biosynthesis in mitochondria. CoQ10 is essential for multiple cellular functions, including mitochondrial oxidative phosphorylation, lipid metabolism, and reactive oxygen species homeostasis. Mutations in COQ7 have been previously associated with primary CoQ10 deficiency, a clinically heterogeneous multisystemic mitochondrial disorder. We identified COQ7 biallelic variants in nine families diagnosed with distal hereditary motor neuropathy with upper neuron involvement, expending the clinical phenotype associated with defects in this gene. A recurrent p.Met1? change was identified in five families from Brazil with evidence of a founder effect. Fibroblasts isolated from patients revealed a substantial depletion of COQ7 protein levels, indicating protein instability leading to loss of enzyme function. High-performance liquid chromatography assay showed that fibroblasts from patients had reduced levels of CoQ10, and abnormal accumulation of the biosynthetic precursor DMQ10. Accordingly, fibroblasts from patients displayed significantly decreased oxygen consumption rates in patients, suggesting mitochondrial respiration deficiency. Induced pluripotent stem cell-derived motor neurons from patient fibroblasts showed significantly increased levels of extracellular neurofilament light protein, indicating axonal degeneration. Our findings indicate a molecular pathway involving CoQ10 biosynthesis deficiency and mitochondrial dysfunction in patients with distal hereditary motor neuropathy. Further studies will be important to evaluate the potential benefits of CoQ10 supplementation in the clinical outcome of the disease.

Cdc73 protects Notch-induced T-cell leukemia cells from DNA damage and mitochondrial stress.

Activated Notch signaling is highly prevalent in T-cell acute lymphoblastic leukemia (T-ALL) but pan-Notch inhibitors were toxic in clinical trials. To find alternative ways to target Notch signals, we investigated Cell division cycle 73 (Cdc73), which is a Notch cofactor and component of transcriptional machinery, a potential target in T-ALL. While we confirmed previous work that CDC73 interacts with NOTCH1, we also found that the interaction in T-ALL was context-dependent and facilitated by the lymphoid transcription factor ETS1. Using mouse models, we showed that Cdc73 is important for Notch-induced T-cell development and T-ALL maintenance. Mechanistically, Cdc73, Ets1, and Notch intersect chromatin at promoters and enhancers to activate oncogenes and genes that are important for DNA repair and oxidative phosphorylation. Consistently, Cdc73 deletion in T-ALL cells induced DNA damage and impaired mitochondrial function. Our data suggests that Cdc73 might promote a gene expression program that was eventually intersected by Notch to mitigate the genotoxic and metabolic stresses of elevated Notch signaling. We also provide mechanistic support for testing inhibitors of DNA repair, oxidative phosphorylation, and transcriptional machinery. Inhibiting pathways like Cdc73 that intersect with Notch at chromatin might constitute a strategy to weaken Notch signals without directly targeting the Notch complex.

Potent and Specific Activators for Mitochondrial Sirtuins Sirt3 and Sirt5.

Sirtuins are NAD+-dependent protein deacylases involved in metabolic regulation and aging-related diseases. Specific activators for seven human Sirtuin isoforms would be important chemical tools and potential therapeutic drugs. Activators have been described for Sirt1 and act via a unique N-terminal domain of this isoform. For most other Sirtuin isoforms, including mitochondrial Sirt3-5, no potent and specific activators have yet been identified. We here describe the identification and characterization of 1,4-dihydropyridine-based compounds that either act as pan Sirtuin activators or specifically stimulate Sirt3 or Sirt5. The activators bind to the Sirtuin catalytic cores independent of NAD+ and acylated peptides and stimulate turnover of peptide and protein substrates. The compounds also activate Sirt3 or Sirt5 in cellular systems regulating, e.g., apoptosis and electron transport chain. Our results provide a scaffold for potent Sirtuin activation and derivatives specific for Sirt3 and Sirt5 as an excellent basis for further drug development.

Identification of 2-hydroxybenzoic acid derivatives as selective SIRT5 inhibitors.

The sirtuin deacetylase SIRT5 plays important roles in regulating multiple metabolic pathways, and potentially represents an attractive target for the treatment of several human diseases, especially cancer. In this study, we report the identification of the hit compound 11 bearing a 2-hydroxybenzoic acid functional group as a novel SIRT5-selective inhibitor via our medium-throughput thermal shift screening assay. Hit 11 stabilizes SIRT5 in a dose-dependent manner and shows moderate inhibitory activity against SIRT5 and high subtype selectivity over SIRT1, 2, and 3 in a trypsin coupled enzyme-based assay. The carboxylic acid and the adjacent hydroxyl group of 11 are essential for maintaining activity. To further improve the potency of compound 11, a lead optimization was carried out, resulting in compound 43 with a 10-fold improved potency. Overall, compound 11 represents a promising new chemical scaffold for further investigation to develop SIRT5-selective inhibitors.

Sirtuin 5 levels are limiting in preserving cardiac function and suppressing fibrosis in response to pressure overload.

Heart failure (HF) is the inability of the heart to pump blood sufficiently to meet the metabolic demands of the body. HF with reduced systolic function is characterized by cardiac hypertrophy, ventricular fibrosis and remodeling, and decreased cardiac contractility, leading to cardiac functional impairment and death. Transverse aortic constriction (TAC) is a well-established model for inducing hypertrophy and HF in rodents. Mice globally deficient in sirtuin 5 (SIRT5), a NAD+-dependent deacylase, are hypersensitive to cardiac stress and display increased mortality after TAC. Prior studies assessing SIRT5 functions in the heart have all employed loss-of-function approaches. In this study, we generated SIRT5 overexpressing (SIRT5OE) mice, and evaluated their response to chronic pressure overload using TAC. Compared to littermate controls, SIRT5OE mice were protected against adverse functional consequences of TAC, left ventricular dilation and impaired ejection fraction. Transcriptomic analysis revealed that SIRT5 suppresses key HF sequelae, including the metabolic switch from fatty acid oxidation to glycolysis, immune activation, and fibrotic signaling pathways. We conclude that SIRT5 is a limiting factor in the preservation of cardiac function in response to experimental pressure overload.

Preventing erosion of X-chromosome inactivation in human embryonic stem cells.

X-chromosome inactivation is a paradigm of epigenetic transcriptional regulation. Female human embryonic stem cells (hESCs) often undergo erosion of X-inactivation upon prolonged culture. Here, we investigate the sources of X-inactivation instability by deriving new primed pluripotent hESC lines. We find that culture media composition dramatically influenced the expression of XIST lncRNA, a key regulator of X-inactivation. hESCs cultured in a defined xenofree medium stably maintained XIST RNA expression and coating, whereas hESCs cultured in the widely used mTeSR1 medium lost XIST RNA expression. We pinpointed lithium chloride in mTeSR1 as a cause of XIST RNA loss. The addition of lithium chloride or inhibitors of GSK-3 proteins that are targeted by lithium to the defined hESC culture medium impeded XIST RNA expression. GSK-3 inhibition in differentiating female mouse embryonic stem cells and epiblast stem cells also resulted in a loss of XIST RNA expression. Together, these data may reconcile observed variations in X-inactivation in hESCs and inform the faithful culture of pluripotent stem cells.

Human Epidermal Growth Factor Receptor 2 (HER2) Expression in Colorectal Carcinoma: A Potential Area of Focus for Future Diagnostics.

Objective In this study, we aimed to explore the potential diagnostic utility of human epidermal growth factor receptor 2 (HER2) expression in colorectal carcinoma. We investigated the association of HER2 expression with the type and grade of the tumor along with the pattern, staining intensity, and the percentage of cells stained. Methods This was an observational study involving 50 cases of colorectal carcinoma that underwent immunohistochemistry to analyze the HER2 expression. Results The positive expression of HER2 was seen in 16 (32%) cases. The majority of the study population was between the fifth-seventh decades of life. The most commonly diagnosed tumor was conventional adenocarcinoma with grade II, cytoplasmic pattern, +2 positivity, and moderate intensity. The maximum positivity for HER2 was seen in tumors of the rectum in eight (16%) cases. Conclusion A substantial rate of HER2 overexpression paves the way for it to become a potential future target in cancer therapeutics.

Detection of Mycoplasma pneumoniae by two polymerase chain reactions and role of Mycoplasma pneumoniae in pediatric community-acquired lower respiratory tract infections.

PURPOSE: The study was conducted to evaluate the role of Mycoplasma pneumoniae (M. pneumoniae) in children with community-acquired lower respiratory tract infections (LRTIs). METHODS: Seventy five children aged 2 months -12 years with community-acquired LRTIs were investigated for M. pneumoniae etiology employing paired serum samples to assay M. pneumoniae antibodies. Nasopharyngeal aspirates were obtained for the detection of M. pneumoniae by using polymerase chain reaction(PCR) and nested PCR. RESULTS: M. pneumoniae infection was positive in 24(85.71%) children aged <5 years and 4 (14.29%) â€‹≥ â€‹5-12 years and the difference was statistically insignificant (P â€‹= â€‹0.18). Difference in prevalence of M. pneumoniae infection across male and female groups was statistically insignificant (P â€‹= â€‹0.69). Clinical and radiological profiles across M. pneumoniae positive and negative cases were comparable except bronchopneumonia which was statistically significant (P â€‹= â€‹0.04). Serological evidence of M. pneumoniae infection was observed in 26(33%); PCR was positive in 9 (12%) and nested PCR in 10 (13.33%) children. Together, serology, PCR and nested PCR diagnosed M. pneumoniae infection in 28(37.33%) patients. Sensitivity of serology was 77.78%: specificity 68.18%; positive predictive value 25.00% and negative predictive value at 95.74%. CONCLUSIONS: Serological and molecular methods in combination is useful for detection of M. pneumoniae. Our data underline the role of M. pneumoniae in community-acquired LRTIs in children of all ages.

ER stress protein PERK promotes inappropriate innate immune responses and pathogenesis during RSV infection.

The activation of dendritic cells (DC) during respiratory viral infections is central to directing the immune response and the pathologic outcome. In these studies, the effect of RSV infection on development of ER stress responses and the impact on innate immunity was examined. The upregulation of ER stress was closely associated with the PERK pathway through the upregulation of CHOP in RSV infected DC. The inhibition of PERK corresponded with decreased EIF2a phosphorylation but had no significant effect on Nrf2 in DC, two primary pathways regulated by PERK. Subsequent studies identified that by blocking PERK activity in infected DC an altered ER stress response and innate cytokine profile was observed with the upregulation of IFNß and IL-12, coincident to the down regulation of IL-1ß. When mitochondria respiration was assessed in PERK deficient DC there were increased dysfunctional mitochondria after RSV infection that resulted in reduced oxygen consumption rates (OCR) and ATP production indicating altered cellular metabolism. Use of a CD11c targeted genetic deleted murine model, RSV infection was characterized by reduced inflammation and diminished mucus staining as well as reduced mucus-associated gene gob5 expression. The assessment of the cytokine responses showed decreased IL-13 and IL-17 along with diminished IL-1ß in the lungs of PERK deficient infected mice. When PERK-deficient animals were assessed in parallel for lung leukocyte numbers, animals displayed significantly reduced myeloid and activated CD4 and CD8 T cell numbers. Thus, the PERK activation pathway may provide a rational target for altering the severe outcome of an RSV infection through modifying immune responses.

Epigenetic mechanisms of cadmium-induced nephrotoxicity.

Cadmium (Cd) is a widespread toxic pollutant that affects millions of individuals worldwide. Cd exposure in humans occurs primarily through consumption of contaminated food and water, cigarette smoking, and industrial applications. The kidney proximal tubular (PT) epithelial cells are the primary target of Cd toxicity. Cd-induced injury to PT cells impedes tubular reabsorption. Despite the many long-term sequelae of Cd exposure, molecular mechanisms of Cd toxicity are poorly understood, and no specific therapies exist to mitigate the effects of Cd exposure. In this review, we summarize recent work linking Cd-mediated damage to epigenetic perturbations - DNA methylation, and levels of histone modifications, including methylation and acetylation. New insights into the links between Cd intoxication and epigenetic damage will contribute to an improved understanding of Cd's pleiotropic impacts on cells, and perhaps lead to new, mechanism-based treatments for this condition.

Assessment of Cellular Bioenergetics in Mouse Hematopoietic Stem and Primitive Progenitor Cells using the Extracellular Flux Analyzer.

Under steady state, hematopoietic stem cells (HSCs) remain largely quiescent and are believed to be predominantly reliant on glycolysis to meet their energetic needs. However, under stress conditions such as infection or blood loss, HSCs become proliferative and rapidly produce downstream progenitor cells, which in turn further differentiate, ultimately producing mature blood cells. During this transition and differentiation process, HSCs exit from quiescence and rapidly undergo a metabolic switch from glycolysis to oxidative phosphorylation (OxPHOS). Various stress conditions, such as aging, cancer, diabetes, and obesity, can negatively impact mitochondrial function and thus can alter the metabolic reprogramming and differentiation of HSCs and progenitors during hematopoiesis. Valuable insights into glycolytic and mitochondrial functions of HSCs and progenitors under normal and stress conditions can be gained through the assessment of their extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), which are indicators of cellular glycolysis and mitochondrial respiration, respectively. Here, a detailed protocol is provided to measure ECAR and OCR in mouse bone marrow-derived lineage-negative cell populations, which include both hematopoietic stem and primitive progenitor cells (HSPCs), using the extracellular flux analyzer. This protocol describes approaches to isolate lineage-negative cells from mouse bone marrow, explains optimization of cell seeding density and concentrations of 2-deoxy-D-glucose (2-DG, a glucose analog that inhibits glycolysis) and various OxPHOS-targeted drugs (oligomycin, FCCP, rotenone, and antimycin A) used in these assays, and describes drug treatment strategies. Key parameters of glycolytic flux, such as glycolysis, glycolytic capacity, and glycolytic reserve, and OxPHOS parameters, such as basal respiration, maximal respiration, proton leak, ATP production, spare respiratory capacity, and coupling efficiency, can be measured in these assays. This protocol allows ECAR and OCR measurements on non-adherent HSPCs and can be generalized to optimize analysis conditions for any type of suspension cells.

Demographic Dividend in the Age of Neoliberal Capitalism: An Analysis of Employment and Employability in India.

After examining the concept of demographic dividend and in-depth analysis of the changing demographic profile of Indian population in a comparative framework, in this study it has been argued that the transformation of demographic potential into demographic dividend is predicated on the premise that India adopts state sponsored social-economic policy regime for public health and education for its youth to acquire skills which will be required in the twenty-first century and adopts macroeconomic policies which ensures optimal use of human resources. Otherwise demographic dividend will be a myth and a mirage.

Demystifying R Naught: Understanding What Does it Hide?

Since the onset of the pandemic in Wuhan city, China, forecasting and projections of the pandemic are the areas of interest for the investigators, and the basic reproduction rate R0 always stayed the favorite tool. The basic reproduction number (R0) is either ratio or rate or the basic reproductive rate. This dimensionless number was calculated in the past to describe the contagiousness or transmissibility of infectious agents for many communicable diseases. Its importance in the context of COVID-19 is not less, it tells us about the public health measures to be undertaken for disease prevention, and how the transmission of COVID-19 will be affected or eliminated. R0 is affected by several biological, sociobehavioral, and environmental factors which decide agent transmission. R0 is estimated by using complex mathematical models, the results of which are easily distorted, misjudged, and misused. R0 is not a biological constant for an agent or pathogen, it is a rate over time. It can measure the disease severity and also gives an estimate about the herd immunity required for the reversal of epidemic. R0 cannot be altered through vaccination campaigns though it can tell us about the relationship between the population's immune status and epidemic curve. Modeled R0 values are dependent on the model structures and assumptions made. Some R0 values reported in the scientific literature are likely outdated as assumptions are frequently changing in the current pandemic. R0 must be predicted and applied with great caution as this basic metric is far from simple.

The deacylase SIRT5 supports melanoma viability by influencing chromatin dynamics.

Cutaneous melanoma remains the most lethal skin cancer, and ranks third among all malignancies in terms of years of life lost. Despite the advent of immune checkpoint and targeted therapies, only roughly half of patients with advanced melanoma achieve a durable remission. Sirtuin 5 (SIRT5) is a member of the sirtuin family of protein deacylases that regulates metabolism and other biological processes. Germline Sirt5 deficiency is associated with mild phenotypes in mice. Here we showed that SIRT5 was required for proliferation and survival across all cutaneous melanoma genotypes tested, as well as uveal melanoma, a genetically distinct melanoma subtype that arises in the eye and is incurable once metastatic. Likewise, SIRT5 was required for efficient tumor formation by melanoma xenografts and in an autochthonous mouse Braf Pten-driven melanoma model. Via metabolite and transcriptomic analyses, we found that SIRT5 was required to maintain histone acetylation and methylation levels in melanoma cells, thereby promoting proper gene expression. SIRT5-dependent genes notably included MITF, a key lineage-specific survival oncogene in melanoma, and the c-MYC proto-oncogene. SIRT5 may represent a druggable genotype-independent addiction in melanoma.

Food Safety in Eating Establishments: Assessing Conformance of Eating Establishments to Food Safety and Standards Regulations.

INTRODUCTION: In view of the rising burden of the foodborne illnesses and the rise of eating out culture in India, food safety has assumed greater significance. The Food Safety and Standards Authority of India has prescribed food safety and standards regulations (FSSRs) for commercial eating establishments (EEs). The present study was carried out to ascertain conformance of the EEs to these regulations. METHODOLOGY: It was a cross-sectional study conducted on 74 EEs in a metro city in western Maharashtra from May to October 2019 using an interviewer-administered study tool based on FSSR 2011. The study tool covered critical domains such as food hygiene, equipments, health and personal cleanliness, training of food handlers, and product information. RESULTS: Seventy-four EEs included 29 restaurants, 21 bakeries, and 24 snack bars. The score ranged between 42.3% and 73.3%. Of 74, 20 (27%) EEs were placed in poor category (score <50%), Only 3 EE scored >70% and were rated as very good. Only 13 (17.6%) EEs were cleaning the food contact surfaces adequately, i.e. before and after each use, whereas 38 (51.4%) were not cleaning food contact surfaces at least daily. The knowledge regarding food handlers as potential carriers of disease was poor with 60.81% of the respondents having no knowledge about it. CONCLUSION: The study found significant gaps in EEs with respect to studied food safety domains of FSSR 2011.