CRISPR Knockout of the HuR Gene Causes a Xenograft Lethal Phenotype.

Pancreatic ductal adenocarcinoma (PDA) is the third leading cause of cancer-related deaths in the United States, whereas colorectal cancer is the third most common cancer. The RNA-binding protein HuR (ELAVL1) supports a pro-oncogenic network in gastrointestinal (GI) cancer cells through enhanced HuR expression. Using a publically available database, HuR expression levels were determined to be increased in primary PDA and colorectal cancer tumor cohorts as compared with normal pancreas and colon tissues, respectively. CRISPR/Cas9 technology was successfully used to delete the HuR gene in both PDA (MIA PaCa-2 and Hs 766T) and colorectal cancer (HCT116) cell lines. HuR deficiency has a mild phenotype

Posttranscriptional regulation of PARG mRNA by HuR facilitates DNA repair and resistance to PARP inhibitors

The majority of pancreatic ductal adenocarcinomas (PDAC) rely on the mRNA stability factor HuR (ELAV-L1) to drive cancer growth and progression. Here, we show that CRISPR-Cas9–mediated silencing of the HuR locus increases the relative sensitivity of PDAC cells to PARP inhibitors (PARPi). PDAC cells treated with PARPi stimulated translocation of HuR from the nucleus to the cytoplasm, specifically promoting stabilization of a new target, poly (ADP-ribose) glycohydrolase (PARG) mRNA, by binding a unique sequence embedded in its 30 untranslated region. HuR-dependent upregulation of PARG expression facilitated DNA repair via hydrolysis of polyADP-ribose on related repair proteins. Accordingly, strategies to inhibit HuR directly promoted DNA damage accumulation, inefficient PAR removal, and persistent PARP-1 residency on chromatin (PARP-1 trapping). Immunoprecipitation assays demonstrated that the PARP-1 protein binds and posttranslationally modifies HuR in PARPi-treated PDAC cells. In a mouse xenograft model of human PDAC, PARPi monotherapy combined with targeted silencing of HuR significantly reduced tumor growth compared with PARPi therapy alone. Our results highlight the HuR–PARG axis as an opportunity to enhance PARPi-based therapies. ©2017 AACR

Posttranscriptional Upregulation of IDH1 by HuR Establishes a Powerful Survival Phenotype in Pancreatic Cancer Cells.

Cancer aggressiveness may result from the selective pressure of a harsh nutrient-deprived microenvironment. Here we illustrate how such conditions promote chemotherapy resistance in pancreatic ductal adenocarcinoma (PDAC). Glucose or glutamine withdrawal resulted in a 5- to 10-fold protective effect with chemotherapy treatment. PDAC xenografts were less sensitive to gemcitabine in hypoglycemic mice compared with hyperglycemic mice. Consistent with this observation, patients receiving adjuvant gemcitabine (n = 107) with elevated serum glucose levels (HgbA1C \u3e 6.5%) exhibited improved survival. We identified enhanced antioxidant defense as a driver of chemoresistance in this setting. ROS levels were doubled in vitro by either nutrient withdrawal or gemcitabine treatment, but depriving PDAC cells of nutrients before gemcitabine treatment attenuated this effect. Mechanistic investigations based on RNAi or CRISPR approaches implicated the RNA binding protein HuR in preserving survival under nutrient withdrawal, with or without gemcitabine. Notably, RNA deep sequencing and functional analyses in HuR-deficient PDAC cell lines identified isocitrate dehydrogenase 1 (IDH1) as the sole antioxidant enzyme under HuR regulation. HuR-deficient PDAC cells lacked the ability to engraft successfully in immunocompromised mice, but IDH1 overexpression in these cells was sufficient to fully restore chemoresistance under low nutrient conditions. Overall, our findings highlight the HuR–IDH1 regulatory axis as a critical, actionable therapeutic target in pancreatic cancer

Intelligent Assistant Language Understanding On Device

It has recently become feasible to run personal digital assistants on phones and other personal devices. In this paper we describe a design for a natural language understanding system that runs on device. In comparison to a server-based assistant, this system is more private, more reliable, faster, more expressive, and more accurate. We describe what led to key choices about architecture and technologies. For example, some approaches in the dialog systems literature are difficult to maintain over time in a deployment setting. We hope that sharing learnings from our practical experiences may help inform future work in the research community

Global investments in pandemic preparedness and COVID-19: development assistance and domestic spending on health between 1990 and 2026

Background The COVID-19 pandemic highlighted gaps in health surveillance systems, disease prevention, and treatment globally. Among the many factors that might have led to these gaps is the issue of the financing of national health systems, especially in low-income and middle-income countries (LMICs), as well as a robust global system for pandemic preparedness. We aimed to provide a comparative assessment of global health spending at the onset of the pandemic; characterise the amount of development assistance for pandemic preparedness and response disbursed in the first 2 years of the COVID-19 pandemic; and examine expectations for future health spending and put into context the expected need for investment in pandemic preparedness. Methods In this analysis of global health spending between 1990 and 2021, and prediction from 2021 to 2026, we estimated four sources of health spending: development assistance for health (DAH), government spending, out-of-pocket spending, and prepaid private spending across 204 countries and territories. We used the Organisation for Economic Co-operation and Development (OECD)'s Creditor Reporting System (CRS) and the WHO Global Health Expenditure Database (GHED) to estimate spending. We estimated development assistance for general health, COVID-19 response, and pandemic preparedness and response using a keyword search. Health spending estimates were combined with estimates of resources needed for pandemic prevention and preparedness to analyse future health spending patterns, relative to need. Findings In 2019, at the onset of the COVID-19 pandemic, US$9·2 trillion (95$7·3 trillion (95% UI 7·2–7·4) in 2019; 293·7 times the $24·8 billion (95$43·1 billion in development assistance was provided to maintain or improve health. The pandemic led to an unprecedented increase in development assistance targeted towards health; in 2020 and 2021, $1·8 billion in DAH contributions was provided towards pandemic preparedness in LMICs, and$37·8 billion was provided for the health-related COVID-19 response. Although the support for pandemic preparedness is 12·2% of the recommended target by the High-Level Independent Panel (HLIP), the support provided for the health-related COVID-19 response is 252·2% of the recommended target. Additionally, projected spending estimates suggest that between 2022 and 2026, governments in 17 (95% UI 11–21) of the 137 LMICs will observe an increase in national government health spending equivalent to an addition of 1% of GDP, as recommended by the HLIP. Interpretation There was an unprecedented scale-up in DAH in 2020 and 2021. We have a unique opportunity at this time to sustain funding for crucial global health functions, including pandemic preparedness. However, historical patterns of underfunding of pandemic preparedness suggest that deliberate effort must be made to ensure funding is maintained

Integration of Molecular Networks in the Shoot Apical Meristem that Controls Floral Specification in Arabidopsis thaliana

Post-embryonic development in plants derives from the Shoot Apical Meristem (SAM). The floral transition is a major developmental phase change that transforms the identity of SAM from vegetative to inflorescence. The transition from juvenile to adult vegetative phase is regulated by miRNA156 (miR156) and miRNA172 (miR172). In Arabidopsis, the endogenous flowering time pathway is mediated by miR156 and a subset of SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL) genes including SPL3, SPL4 and SPL5. During shoot development, a subset of SPLs is post-transcriptionally regulated by miR156. The miR156/SPLs module functions to specify floral meristem identity by activating floral integrators and floral meristem identity genes including APETELLA1 (AP1). The universal florigen FLOWERING LOCUS T (FT) promotes floral induction by activating, in part, SPL3, SPL4 and SPL5. In turn, SPLs act in parallel with FT to promote floral meristem identity. Two related BELL1-like homeobox genes, PENNYWISE (PNY) and POUND-FOOLISH (PNF), which are expressed in the SAM, are required for floral specification. Previous genetic studies indicate that the floral specification function of FT depends upon PNY and PNF. However, the relationship between these homeodomain proteins and miR156/SPLs is not known. Results from this study indicate that the photoperiodic floral induction of SPL3, SPL4 and SPL5 is dependent upon PNY and PNF. Moreover, the levels of miR156 fail to decline in pny pnf apices under floral inductive conditions. Therefore, PNY and PNF appear to regulate levels of miR156 during reproductive development. In addition, PNY and PNF control SPL3, SPL4, and SPL5 expression by negatively regulating miR156. Furthermore, results demonstrate that ectopic expression of miR156 resistant SPL4 partially restores reproductive development in pny pnf plants whereas SPL3 and SPL5 failed to promote floral specification. This suggests that the function of SPL3, SPL4, and SPL5 is dependent upon PNY and PNF as well as expression of multiple SPLs is required for completing floral specification in pny pnf plants. Lastly, GA application activates SOC1 and AGL24 in pny pnf plants but failed to promote flower formation. Therefore, PNY and PNF acts downstream of GA signaling pathway and SOC1 and AGL24 are dependent upon PNY and PNF for flower specification

Integration of Molecular Networks in the Shoot Apical Meristem that Controls Floral Specification in Arabidopsis thaliana

Post-embryonic development in plants derives from the Shoot Apical Meristem (SAM). The floral transition is a major developmental phase change that transforms the identity of SAM from vegetative to inflorescence. The transition from juvenile to adult vegetative phase is regulated by miRNA156 (miR156) and miRNA172 (miR172). In Arabidopsis, the endogenous flowering time pathway is mediated by miR156 and a subset of SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL) genes including SPL3, SPL4 and SPL5. During shoot development, a subset of SPLs is post-transcriptionally regulated by miR156. The miR156/SPLs module functions to specify floral meristem identity by activating floral integrators and floral meristem identity genes including APETELLA1 (AP1). The universal florigen FLOWERING LOCUS T (FT) promotes floral induction by activating, in part, SPL3, SPL4 and SPL5. In turn, SPLs act in parallel with FT to promote floral meristem identity. Two related BELL1-like homeobox genes, PENNYWISE (PNY) and POUND-FOOLISH (PNF), which are expressed in the SAM, are required for floral specification. Previous genetic studies indicate that the floral specification function of FT depends upon PNY and PNF. However, the relationship between these homeodomain proteins and miR156/SPLs is not known. Results from this study indicate that the photoperiodic floral induction of SPL3, SPL4 and SPL5 is dependent upon PNY and PNF. Moreover, the levels of miR156 fail to decline in pny pnf apices under floral inductive conditions. Therefore, PNY and PNF appear to regulate levels of miR156 during reproductive development. In addition, PNY and PNF control SPL3, SPL4, and SPL5 expression by negatively regulating miR156. Furthermore, results demonstrate that ectopic expression of miR156 resistant SPL4 partially restores reproductive development in pny pnf plants whereas SPL3 and SPL5 failed to promote floral specification. This suggests that the function of SPL3, SPL4, and SPL5 is dependent upon PNY and PNF as well as expression of multiple SPLs is required for completing floral specification in pny pnf plants. Lastly, GA application activates SOC1 and AGL24 in pny pnf plants but failed to promote flower formation. Therefore, PNY and PNF acts downstream of GA signaling pathway and SOC1 and AGL24 are dependent upon PNY and PNF for flower specification

Inter-Relationship Between Cooking Time and Some Physico Chemical Characteristics in Pigeon Pea (Cajanus cajan) Genotypes

Thirteen genotypes of pigeon pea were evaluated for their physical and physico-chemical characteristics, namely, seed size, seed volume, seed density, hydration capacity, hydration index, swelling capacity, swelling index and cooking time. Cooking time of whole pigeon pea pulse ranged between 51 and 63 min indicating a large variation. However, the cooking time of pigeon pea dhal ranged between 19 and 31 min. Correlation coefficients between seed weight and hydration capacity (0.77), seed volume and seed weight (-0.86) and that of swelling capacity and swelling index (0.90) were found to be significant at I% level. Alinear regression model was also fitted between the cooking time of whole pulse and other parameters under study. The coefficient of determination (R2 ) was observed as 81%. These inter-relationships amongst variables enable to predict the cooking time of whole pulse for a large number of pigeon pea genotypes

Safety assessment of Withania somnifera extract standardized for Withaferin A: Acute and sub-acute toxicity study

Background: The use of Withania somnifera is increasing due to a number of its chemical constituents found useful for health. Objective: The present study was carried out to investigate the potential adverse effects (if any) of a standardized Withania somnifera extract (WSE) in rats following acute and sub chronic administration. Materials and methods: The toxicity study was performed in Wistar rats by oral administration. An acute toxicity study was done at the dose of 2000 mg/kg. In the sub-acute study, Wistar rats (10/sex/group) were administered via gavage 0 (control), 500, 1000, 2000 mg/kg body weight/day of WSE for 28 days. Among two additional satellite groups, one group did not receive any drug while the second group received 2000 mg/kg/day for 28 days. At the end of study, the animals sacrificed and their body weight, hematology, serum chemistry, and histopathology evaluation was done. Results: In acute toxicity studies, oral LD50 of WSE in Wistar rats was greater than 2000 mg/kg body weight. Compared to the control group in sub-acute toxicity study, administration of extract did not show any toxicologically significant treatment related changes in clinical observations, ophthalmic examination, body weight gain, feed consumption, clinical pathology evaluation, and organ weight. Hematological and serum chemistry parameters were within the normal limits. Terminal necropsy did not reveal any treatment related gross or histopathological findings. Conclusion: Based on this study, the no-observed-adverse-effect-level of WSE is 2000 mg/kg body weight, the highest level tested