High-throughput sequencing has become a standard and powerful tool for analyzing nucleic acids primarily due to its sensitivity and convenience. Small RNAs play important roles in regulating cellular and viral genes. The conventional methods for small RNA analyses are tedious and often lack accuracy, specificity and sensitivity for many small RNA species. Therefore, high-throughput sequencing becomes an indispensable tool for analyzing small RNAs. However, it is challenging to generate a reliable and representative small RNA library for high-throughput sequencing since small RNAs are usually expressed at extremely low levels and often contain modifications which affect library construction, usually causing biased readouts. This review compares various strategies for generating small RNA libraries of high quality and reliability, and provides recommendations on best practice in preparing high-throughput sequencing RNA libraries.

Bone marrow transplantation (BMT) or hematopoietic stem cell transplantation (HSCT) is an archetype of cellular therapy. However, to date BMT still suffers from several complications. Recent technological advances have encouraged us to think about an alternative to traditional BMT. Specifically, we propose in utero HSCT (IUHSCT). For this purpose, we suggest that induced fetal-like hematopoietic stem cells (ifHSCs) might be suitable for IUHSCT, and should be seriously evaluated.

The aim was to study the polymorphisms in GH, GHR, IGF-I and IGFBPII genes and their association with productive and reproductive traits. DNA was extracted from blood samples of 380 breeder hens of Mazandaran native fowls breeding station by modified salting out method. For genotyping, PCR products of GH, GHR, IGF-I and IGFBPII loci were digested with specific restriction enzymes. We found six genotypes of AA, BB, CC, AB, AC and BC with the frequency of 0.10, 0.01, 0.36, 0.07, 0.34 and 0.12 in GH, two haploid alleles and genotypes of B+ and B- with the frequency of 0.72 and 0.28 in GHR, three genotypes of BB, Bb, bb with the frequency of 0.49, 0.44 and 0.07 in IGF-I and three genotypes of CC, CT and TT with the frequency of 0.14, 0.62 and 0.24 in IGFBPII loci, respectively. Significant associations were found between SNPs in IGF-I locus and egg number at 120-270 and 345-375 days of age, egg weight at puberty and percentage of hatchability traits (P<0.05). We also found significant effects of GHR and IGFBPII loci on egg number at 345-375 days of age and average egg weight at 345-375 days of age traits, respectively (P<0.05). Mean comparisons analysis between different genotypes of GH gene for body weight at day one and puberty, age at sexual maturity, egg weight at puberty and 30 weeks of age, average egg weight and egg number at 345-375 days of age and also percentage of fertility traits and IGFBPII genotypes for laying intensity trait were observed to be significant (P<0.05). Regarding the significant effects of GHR, IGF-I and IGFBPII genes on egg production traits, it could be resulted that these candidate genes may be used as the candidate markers in poultry breeding programs to improve productive and reproductive traits in this population.

Six psychotropic strains of Carnobacterium divergens were isolated from Tunisian minced raw beef meat packed and stored at 6°C. They were first identified by biochemical methods. Using biochemical reactions and carbohydrate fermentation before their characterisation by molecular techniques. The strain of Carnobacterium divergens is a nonmotile, Gram-positive psychotropic rod that lacks catalase, oxidase and mannitol. It grows at pH 9.1 (D-MRS agar), but not on acetate agar (pH ≤ 5.4). For all these isolates, the phenotypic identification, using the API 50 CHL system, revealed a variability in the fermentation abilities of some sugars (glycerol, amygdaline, arbutine, D-trehalose and potassium gluconate). Species-specific polymerase chain reaction (PCR) primers were used to ensure identification of these isolated strains at the species level. Moreover, the sequencing of 16S rRNA gene confirmed that all the six isolates are identified as C. divergens. The Rep-PCR technique was performed to investigate intra-specific diversity within these six strains of C. divergens, commonly identified in meat. The Rep-PCR method distinguished the C. divergens strains, demonstrating their biodiversity and their evident similarities. BOX and REP primers allowed amplification with 85% and 80% similarities between the strains, respectively. The BOXA1R and REP primers were proven to be useful for the differentiation of C. divergens strains.

Background and objectives: Mutation analysis in Egyptian children with clinical diagnosis of short limb dwarfism (Achondroplasia). In addition, to adopt a molecular method for both prenatal and postnatal diagnosis. Materials and methods: Eight sporadic cases of short limb dwarfism were studied. A mutation analysis was done by PCR/RFLP (Polymerase chain reaction/Restriction fragment length polymorphism). Results of RFLP were confirmed using sequencing of PCR products. Results: The G380R mutation was positive in all eight probands (100%) and negative in all parents (0%). Results of RFLP were confirmed using sequencing that revealed substitution of guanine by adenine at the nucleotide (nt. 1138). Interpretation and conclusions: All cases had the G-A transition at nt. 1138 of the FGFR3 gene. Our conclusion is that vast majority of Egyptian achondroplasia patients have the same mutation. Mutation analysis is useful for both prenatal and postnatal diagnosis.

5-carboxylcytosine (5caC) is an important derivative of cytosine modification in DNA demethylation pathway. However, the accurate quantification of 5caC is a challenge, especially in mammalian tissue. This plight probably results from the trace amount of 5caC in mammalian and the inadequate sensitivity of the existing technologies. Herein, we report a novel LC-MS/MS method to precisely quantify 5caC in mammalian cells. The 5caC standard reagent was synthesized, and the genomic DNA was hydrolyzed with formic acid and the target compound 5caC was detected by HILIC LC-MS/MS. The results showed 5caC generally exist in mice organs and cancer tissues. The content of 5caC in mice brain was higher than in lungs and liver, and was obviously decreased in colorectal cancer tissues compared with the adjacent tissue. This result suggests that 5caC probably associates with tumorigenesis and plays a dominated role in epigenetic control of neuronal function.

Development of novel monoclonal antibodies, vaccines and oncolytic virus therapies have relied on analysis of biomarkers as potential predictors of success. One well studied biomarker is the CD16/ FcγRIIIa receptor residue 158 F/V. Identifying variants through genotyping of the FcγRIIIa locus is widely practiced and highly varied with commonly used methods including: Sanger sequencing, flow-cytometry, PCR/RFLP, Goldengate (replaced by Infinium) and TaqMAN analysis. While each of these methods have considerable backing in publications related to CD16 FcγRIIIa 158 F/V, the majority present significant short comings in identifying both homozygotes (wild-type and mutant) and heterozygotes in a time and cost-efficient manner. Utilization of droplet-digital PCR with FcγRIIIa-F158V specific probes results in the accurate genotyping using direct recognition of sequence in genomic samples at a lower average cost and faster turnaround. Here we demonstrate the use of ddPCR to accurately identify FcγRIIIa-F158V genotypes with confirmation by Illumina sequencing in 128 patient samples.

Virus-like particles or what is called VLPs for short are considered a useful tool in the development of vaccines. These VLPs-based vaccines have attracted the attention of many biotech and pharmaceutical companies as effective treatment for many types of non-infectious chronic diseases. VLPs simply resemble viruses; however they are noninfectious due to the lack of viral genome. The expression of envelop or capsid proteins result in the self-assembly of the particle, this assembly can easily be carried out in bacteria, yeast, plant cells or insect cell lines. The presence of repetitive viral surface protein epitopes is successful in eliciting strong B cell responses. VLPs have also high safety profile due to the lack of viral genome and lack of the ability to replicate. This area of research is not really new, the discovery of hepatitis B virus derived VLPs goes back to 1976.

Barley is one of the world's most important crops with uses ranging from food and feed production representing the fourth most abundant cereal in both area and harvested tonnage. Barley is the best suitable crop for genetic studies, because of its diploid nature with a relatively small number of large chromosomes and has been widely used as a genetic model. With the advance of new genomics platforms, transcriptome, metabolome analyses and bioinformatics, the dissection of various quantitative traits and the determination of their chromosomal locations which in turn led to the identification of a number of marker–trait associations in various crop species including barley become feasible.

The R-spondin 2 (Rspo2) belongs to the Rspo family, which is a cluster of secreted molecules possessing pleiotropic functions in development and stem cell growth by strongly enhancing Wnt signaling activation. In this study, porcine Rspo2 (pRspo2) was amplified from cDNA derived from porcine brain tissue. DNA sequencing result indicates that the open reading frame (ORF) of pRspo2 consists of 732 base pairs (bp), encodes 243 amino acids protein with a putative molecular weight of 28.19 kDa and is high identity to those of horse (98%), cattle (98%), human (96%) and rat (92%). pRspo2 is highly expressed in the brain tissue, whereas with comparatively lower level of expression in duodenum, thymus, liver, lung, lymph node, kidney and ileum tissues, which is characterized by reverse transcriptionpolymerase chain reaction (RT-PCR) analysis. All the results above will provide the fundamental research on the pleiotropic functions of pRspo2.