Noodles, the all-in-one system for on-target efficiency analysis of CRISPR guide RNAs.

The efficiency of clustered regularly interspaced short palindromic repeats (CRISPR) guide RNA (gRNA) targeting is critical for CRISPR associated protein 9 (Cas9)-dependent genomic modifications. Here, we developed Noodles, an all-in-one system to test the on-target activity of gRNAs easily and efficiently. Single-strand annealing repair mechanism of the split luciferase gene allows a positive selection of gRNAs efficiently driving nuclease activity of Cas9 from Streptococcus pyogenes (SpCas9). Our system can reliably validate in silico-predicted gRNAs before implementing them for in vitro and in vivo applications. Altogether, Noodles might be an asset for researchers and bioengineers, saving their time and efforts, while keeping the screening efficient and sensitive. •All-in-one dual-luciferase system to easily probe on-target activity of gRNAs based on homology-directed repair mechanism.•Easy-to-subclone spCas9-based 2-plasmid system comprising Renilla luciferase for transfection efficiency control.

In Vivo Reductionist Approach Identifies miR-15a Protecting Mice From Obesity.

Obesity is a growing medical and social problem worldwide. The control of energy homeostasis in the brain is achieved by various regions including the arcuate hypothalamic nucleus (ARH). The latter comprises a number of neuronal populations including the first order metabolic neurons, appetite-stimulating agouti-related peptide (AgRP) neurons and appetite-suppressing proopiomelanocortin (POMC) neurons. Using an in vivo reductionist approach and POMCCre-dependent CRISPR-Cas9, we demonstrate that miR-15a-5p protects from obesity. Moreover, we have identified Bace1, a gene previously linked to energy metabolism imbalance, as a direct target of miR-15a-5p. This work warrants further investigations of non-coding RNA-mediated regulation of energy homeostasis and might contribute to the development of novel therapeutic approaches to treat metabolic diseases.

Neuronal miR-29a protects from obesity in adult mice.

OBJECTIVE: Obesity, a growing threat to the modern society, represents an imbalance of metabolic queues that normally signal to the arcuate hypothalamic nucleus, a critical brain region sensing and regulating energy homeostasis. This is achieved by various neurons many of which developmentally originate from the proopiomelanocortin (POMC)-expressing lineage. Within the mature neurons originating from this lineage, we aimed to identify non-coding genes in control of metabolic function in the adulthood. METHODS: In this work, we used microRNA mimic delivery and POMCCre-dependent CRISPR-Cas9 knock-out strategies in young or aged mice. Importantly, we also used CRISPR guides directing suicide cleavage of Cas9 to limit the off-target effects. RESULTS: Here we found that mature neurons originating from the POMC lineage employ miR-29a to protect against insulin resistance obesity, hyperphagia, decreased energy expenditure and obesity. Moreover, we validated the miR-29 family as a prominent regulator of the PI3K-Akt-mTOR pathway. Within the latter, we identified a direct target of miR-29a-3p, Nras, which was up-regulated in those and only those mature POMCCreCas9 neurons that were effectively transduced by anti-miR-29 CRISPR-equipped construct. Moreover, POMCCre-dependent co-deletion of Nras in mature neurons attenuated miR-29 depletion-induced obesity. CONCLUSIONS: Thus, the first to our knowledge case of in situ Cre-dependent CRISPR-Cas9-mediated knock-out of microRNAs in a specific hypothalamic neuronal population helped us to decipher a critical metabolic circuit in adult mice. This work significantly extends our understanding about the involvement of neuronal microRNAs in homeostatic regulation.

Dicer and microRNAs protect adult dopamine neurons.

MicroRNAs (miRs) are important post-transcriptional regulators of gene expression implicated in neuronal development, differentiation, aging and neurodegenerative diseases, including Parkinson's disease (PD). Several miRs have been linked to PD-associated genes, apoptosis and stress response pathways, suggesting that deregulation of miRs may contribute to the development of the neurodegenerative phenotype. Here, we investigate the cell-autonomous role of miR processing RNAse Dicer in the functional maintenance of adult dopamine (DA) neurons. We demonstrate a reduction of Dicer in the ventral midbrain and altered miR expression profiles in laser-microdissected DA neurons of aged mice. Using a mouse line expressing tamoxifen-inducible CreERT2 recombinase under control of the DA transporter promoter, we show that a tissue-specific conditional ablation of Dicer in DA neurons of adult mice led to decreased levels of striatal DA and its metabolites without a reduction in neuronal body numbers in hemizygous mice (DicerHET) and to progressive loss of DA neurons with severe locomotor deficits in nullizygous mice (DicerCKO). Moreover, we show that pharmacological stimulation of miR biosynthesis promoted survival of cultured DA neurons and reduced their vulnerability to thapsigargin-induced endoplasmic reticulum stress. Our data demonstrate that Dicer is crucial for maintenance of adult DA neurons, whereas a stimulation of miR production can promote neuronal survival, which may have direct implications for PD treatment.

Role of Leptin Deficiency, Inefficiency, and Leptin Receptors in Obesity.

Leptin protein consists of 167 amino acids, which is mainly secreted from the white adipose tissue. This protein acts on the hypothalamic regions of the brain which control eating behavior, thus playing a significant role in maintaining body's metabolism. Leptin receptors belong to glycoprotein 130 (gp130) family of cytokine receptors and exist in six isoforms (LEPR a-f), and all the isoforms are encoded by LEPR gene; out of these isoforms, the LEPR-b receptor is the 'longest form,' and in most of the cases, mutations in this isoform cause severe obesity. Also, mutations in the leptin gene (LEP) or its receptors gene can lead to obesity. Some biochemical pathways affect the bioactivity of leptin and/or its receptors. To date, eleven pathogenic mutations have been reported in the LEP which are p.L72S, p.N103K, p.R105W, p.H118L, p.S141C, p.W121X c.104_106delTCA, c.135del3bp, c.398delG, c.481_482delCT, and c.163C>T. Different mutations in the LEPR have also been reported as c.2396-1 G>T, c.1675 G>A, p.P316T, etc. In some studies, where leptin was deficient, leptin replacement therapy has shown positive impact by preventing weight gain and obesity.

Elevated serum level of human alkaline phosphatase in obesity.

OBJECTIVE: To investigate a correlation between serum alkaline phosphatase level and body mass index in human subjects. METHODS: The comparative cross-sectional study was carried out at the National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan, from April 2012 to June 2013. Blood serum alkaline phosphatase levels were estimated and the subjects were divided into three sub-groups on the basis of their body mass. INDEX: normal weight (<25kg/m2), overweight (25-27kg/m2) and obese (>27kg/m2) subjects. The serum samples were used for the estimation of clinically important biochemical parameters, using commercial kits on clinical chemistry analyser. RESULTS: Of the 197 subjects, 97(49%) were obese and 100(51%) were non-obese. The serum alkaline phosphatase level increased in obese (214±6.4 IU/L) compared to the non-obese subjects (184.5±5 IU/L). Furthermore, a significant linear relationship (r=0.3;p-0.0001) was found between serum alkaline phosphatase and body mass index. Other biochemical variables were not correlated to the body mass index. CONCLUSIONS: Over activity and higher amounts of alkaline phosphatase were linked to the development of obesity.

Serum adiponectin levels in diabetes, obesity and gender in Punjabi subjects from Faisalabad, Pakistan.

Adiponectin has been associated with common metabolic disorders. The current study was conducted to measure and compare levels of adiponectin with obesity, type 2 diabetes mellitus (T2DM) and gender in Punjabi subjects from Faisalabad, Pakistan. Serum adiponectin was measured by enzyme-linked immunosorbent assay (ELISA) along with measurements of some clinically important analytes (fasting blood glucose, cholesterol, triglycerides) as well as body mass index (BMI) in 80 subjects. The main results were significantly (p < 0.003) decreased serum adiponectin level in T2DM patients (n = 40) compared to non-diabetic controls (n = 40). In obese subjects, (n = 40) also, there was a decrease, but it was not significant. Adiponectin levels in the subgroups of diabetic and obese patients were also observed, but no significant gender-based differences were found.

Serum total bilirubin levels and prevalence of diabetic retinopathy in a Chinese population.

BACKGROUND: Patients with type 2 diabetes (T2D) are at a high risk of developing microvascular complications, such as diabetic retinopathy (DR). Previous studies have shown that low serum bilirubin concentrations in T2D patients may increase the risk of diabetic complications. Thus, the aim of the present was to investigate the association between the prevalence of DR and serum concentrations of total bilirubin in a Chinese population. METHODS: The present study was a population-based cross-sectional study on 1761 T2D patients aged ≥40 years from the Jiading district of Shanghai, China. Fundus photographs were taken to confirm the presence and severity of DR. Subjects were assigned to quartiles based on serum total bilirubin concentrations (Quartile (Q) 1 <0.60 mg/dL; Q2 0.60-0.76 mg/dL; Q3 0.77-0.99 mg/dL; Q4 >0.99 mg/dL). Logistic regression models were used to explore the association between bilirubin concentrations and the prevalence of DR. RESULTS: The prevalence of DR in the entire study population was 9.6%. The prevalence of DR was significantly lower in Q4 compared with the other three quartiles (Ptrend = 0.004). After adjustment for multiple confounding factors, T2D patients in Q4 (i.e. serum bilirubin >0.99 mg/dL) were less likely (odds ratio 0.55; 95% confidence interval 0.33-0.91) to suffer from DR than patients in Q1 (i.e. serum bilirubin <0.60 mg/dL). CONCLUSION: Serum bilirubin concentrations were inversely associated with DR in an elderly Chinese population, independent of traditional risk factors for microvascular complications.