A method for characterizing Cas9 variants via a one-million target sequence library of self-targeting sgRNAs.

Detailed target-selectivity information and experiment-based efficacy prediction tools are primarily available for Streptococcus pyogenes Cas9 (SpCas9). One obstacle to develop such tools is the rarity of accurate data. Here, we report a method termed 'Self-targeting sgRNA Library Screen' (SLS) for assaying the activity of Cas9 nucleases in bacteria using random target/sgRNA libraries of self-targeting sgRNAs. Exploiting more than a million different sequences, we demonstrate the use of the method with the SpCas9-HF1 variant to analyse its activity and reveal motifs that influence its target-selectivity. We have also developed an algorithm for predicting the activity of SpCas9-HF1 with an accuracy matching those of existing tools. SLS is a facile alternative to the much more expensive and laborious approaches used currently and has the capability of delivering sufficient amount of data for most of the orthologs and variants of SpCas9.

Caps and progression-free sets in Z m n.

We study progression-free sets in the abelian groups G = ( Z m n , + ) . Let r k ( Z m n ) denote the maximal size of a set S ⊂ Z m n that does not contain a proper arithmetic progression of length k. We give lower bound constructions, which e.g. include that r 3 ( Z m n ) ≥ C m ( ( m + 2 ) / 2 ) n n , when m is even. When m = 4 this is of order at least 3 n / n ≫ | G | 0.7924 . Moreover, if the progression-free set S ⊂ Z 4 n satisfies a technical condition, which dominates the problem at least in low dimension, then | S | ≤ 3 n holds. We present a number of new methods which cover lower bounds for several infinite families of parameters m, k, n, which includes for example: r 6 ( Z 125 n ) ≥ ( 85 - o ( 1 ) ) n . For r 3 ( Z 4 n ) we determine the exact values, when n ≤ 5 , e.g.  r 3 ( Z 4 5 ) = 124 , and for r 4 ( Z 4 n ) we determine the exact values, when n ≤ 4 , e.g.  r 4 ( Z 4 4 ) = 128 . With regard to affine caps, i.e. sets without 3 points on a line, the new methods asymptotically improve the known lower bounds, when m = 4 and m = 5 : in Z 4 n from 2 . 519 n to ( 3 - o ( 1 ) ) n , and when m = 5 from 2 . 942 n to ( 3 - o ( 1 ) ) n . This last improvement modulo 5 appears to be the first asymptotic improvement of any cap in AG(n, m), when m ≥ 5 over a tensor lifting from dimension 6 (see Edel, in Des Codes Crytogr 31:5-14, 2004).

Sex influenced association of directly measured insulin sensitivity and serum transaminase levels: Why alanine aminotransferase only predicts cardiovascular risk in men?

BACKGROUND: Non alcoholic fatty liver disease (NAFLD) is an independent cardiovascular (CV) risk factor which is closely associated with insulin resistance measured by both direct or indirect methods. Gender specific findings in the relationship between alanine aminotransferase (ALT) and CV disease, the prevalence of NAFLD and type 2 diabetes (T2DM) have been published recently. The aim of the present study was to explore the gender aspects of the association between insulin sensitivity, liver markers and other metabolic biomarkers in order to elucidate the background behind the sex influenced difference in both NAFLD, T2DM and their association with CV risk. PATIENTS AND METHODS: 158 female (47 normal and 111 impaired glucose intolerant) and 148 male (74 normal and 74 impaired glucose tolerant) subjects were included (mean age: 46.5 ± 8.31 vs. 41.6 ± 11.3, average Hba1c < 6.1 %, i.e. prediabetic population, drug naive at the time of the study). Subjects underwent a hyperinsulinemic normoglycemic clamp to determine muscle glucose uptake (M3), besides liver function tests and other fasting metabolic and anthropometric parameters were determined. RESULTS: Significant bivariate correlations were found between clamp measured M3 and all three liver enzymes (ALT, aspartate aminotransferase and gamma-glutamyl transferase) in both sexes. When data were adjusted for possible metabolic confounding factors correlations ceased in the male population but stayed significant in the female group. Feature selection analysis showed that ALT is an important attribute for M3 in the female but not in male group (mean Z: 3.85 vs. 0.107). Multiple regression analysis confirmed that BMI (p < 0.0001) and ALT (p = 0.00991) significantly and independently predicted clamp measured muscle glucose uptake in women (R(2) = 0.5259), while in men serum fasting insulin (p = 0.0210) and leptin levels (p = 0.0294) but none of the liver enzymes were confirmed as significant independent predictors of M3 (R(2) = 0.4989). CONCLUSION: There is a gender specific association between insulin sensitivity, metabolic risk factors and liver transaminase levels. This might explain the sex difference in the predictive role of ALT elevation for CV disease. Moreover, ALT may be used as a simple diagnostic tool to identify insulin resistant subjects only in the female population according to our results.

[Metabolic alteration in healthy men with first degree type 2 diabetic relatives]. / Anyagcsere-eltérések a 2-es típusú cukorbetegek egészséges, elso fokú férfi rokonaiban.

INTRODUCTION: The recognition of prediabetic patients with the genetic risk of type 2 diabetes is very important as prediabetes is the last stage when manifestation of diabetes could be prevented by life style modification or drug intervention. This suggests the need for diagnostic processes to trace the risk of patients in time. AIMS: The authors looked for metabolic differences between age and BMI in adjusted healthy men with or without first degree type 2 diabetic relatives. METHODS: The study included 73 healthy men (21 with and 52 without) first-degree relatives with type 2 diabetes. RESULTS: Total body and muscle tissue glucose utilization, glucose tolerance did not differ between the two groups, but free fatty acid levels were not suppressed by glucose load in subjects with diabetic relatives. In addition the body fat content, leptin and IL-6 levels were higher, while adiponectin and the free fatty acid/adiponectin ratio were significantly lover in healthy men with diabetic relatives. In this group HDL cholesterol, and the large buoyant LDL fraction were lower whereas the high density LDL - small molecular lipid fraction was higher than those measured in subjects without diabetic relatives. CONCLUSIONS: These data suggest that deteriorations of insulin sensitivity and glucose tolerance is preceded by disturbances of fatty acid metabolism. The observed alteration in free fatty acid/adiponectin ratio, and/or the absence of free fatty acid suppression during glucose tolerance tests could be a screening tool for diabetes risk among men.

[Metabolic differences in healthy first-degree female relatives of type 2 diabetic patients]. / Anyagcsere-eltérések a 2-es típusú cukorbetegek egészséges, elsofokú norokonaiban.

INTRODUCTION: Today the prevalence of type 2 diabetes reached an epidemic level. It is known that type 2 diabetes could only be prevented before the manifestation, during the "prediabetic" state, urging the development of diagnostic tests to recognize the group at risk in time. AIM: The authors explored metabolic differences between healthy, normal glucose tolerant, normal insulin resistant females having first degree relatives with and without type 2 diabetes. METHOD: Healthy, normal insulin sensitive females without (n = 26) and with (n = 18) type 2 diabetic relatives were investigated. RESULTS: Healthy females with first degree diabetic relatives had lower low density lipoproteins and higher high density lipoproteins as well as higher glucose and insulin levels at the 120 min of oral glucose test as compared to those without first degree diabetic relatives. CONCLUSIONS: These results suggest that the appearance of insulin resistance is preceded by hepatic insulin resistance and impaired lipid metabolism in the symptom-free prediabetic period of genetically susceptible females.

Draft genome sequence of Methylibium sp. strain T29, a novel fuel oxygenate-degrading bacterial isolate from Hungary.

Methylibium sp. strain T29 was isolated from a gasoline-contaminated aquifer and proved to have excellent capabilities in degrading some common fuel oxygenates like methyl tert-butyl ether, tert-amyl methyl ether and tert-butyl alcohol along with other organic compounds. Here, we report the draft genome sequence of M. sp. strain T29 together with the description of the genome properties and its annotation. The draft genome consists of 608 contigs with a total size of 4,449,424 bp and an average coverage of 150×. The genome exhibits an average G + C content of 68.7 %, and contains 4754 protein coding and 52 RNA genes, including 48 tRNA genes. 71 % of the protein coding genes could be assigned to COG (Clusters of Orthologous Groups) categories. A formerly unknown circular plasmid designated as pT29A was isolated and sequenced separately and found to be 86,856 bp long.