Identification of extracellular miRNA in archived serum samples by next-generation sequencing from RNA extracted using multiple methods

miRNAs act as important regulators of gene expression by promoting mRNA degradation or by attenuating protein translation. Since miRNAs are stably expressed in bodily fluids, there is growing interest in profiling these miRNAs, as it is minimally invasive and cost-effective as a diagnostic matrix. A technical hurdle in studying miRNA dynamics is the ability to reliably extract miRNA as small sample volumes and low RNA abundance create challenges for extraction and downstream applications. The purpose of this study was to develop a pipeline for the recovery of miRNA using small volumes of archived serum samples. The RNA was extracted employing several widely utilized RNA isolation kits/methods with and without addition of a carrier. The small RNA library preparation was carried out using Illumina TruSeq small RNA kit and sequencing was carried out using Illumina platform. A fraction of five microliters of total RNA was used for library preparation as quantification is below the detection limit. We were able to profile miRNA levels in serum from all the methods tested. We found out that addition of nucleic acid based carrier molecules had higher numbers of processed reads but it did not enhance the mapping of any miRBase annotated sequences. However, some of the extraction procedures offer certain advantages: RNA extracted by TRIzol seemed to align to the miRBase best; extractions using TRIzol with carrier yielded higher miRNA-to-small RNA ratios. Nuclease free glycogen can be carrier of choice for miRNA sequencing. Our findings illustrate that miRNA extraction and quantification is influenced by the choice of methodologies. Addition of nucleic acid- based carrier molecules during extraction procedure is not a good choice when assaying miRNA using sequencing. The careful selection of an extraction method permits the archived serum samples to become valuable resources for high-throughput applications.     

Design,synthesis and molecular modeling of new 4-phenylcoumarin derivatives as tubulin polymerization inhibitors targeting MCF-7 breast cancer cells

A new set of 4-phenylcoumarin derivatives was designed and synthesized aiming to introduce new tubulin polymerization inhibitors as anti-breast cancer candidates. All the target compounds were evaluated for their cytotoxic effects against MCF-7 cell line, where compounds 2f, 3a, 3b, 3f, 7a and 7b, showed higher cytotoxic effect (IC₅₀?=?4.3–21.2?μg/mL) than the reference drug doxorubicin (IC₅₀?=?26.1?μg/mL), additionally, compounds 1 and 6b exhibited the same potency as doxorubicin (IC₅₀?=?25.2 and 28.0?μg/mL, respectively). The thiazolidinone derivatives 3a, 3b and 3f with potent and selective anticancer effects towards MCF-7 cells (IC₅₀?=?11.1, 16.7 and 21.2?μg/mL) were further assessed for tubulin polymerization inhibition effects which showed that the three compounds were potent tubulin polymerization suppressors with IC₅₀ values of 9.37, 2.89 and 6.13?μM, respectively, compared to the reference drug colchicine (IC₅₀?=?6.93?μM). The mechanistic effects on cell cycle progression and induction of apoptosis in MCF-7 cells were determined for compound 3a due to its potent and selective cytotoxic effects in addition to its promising tubulin polymerization inhibition potency. The results revealed that compound 3a induced cell cycle cessation at G2/M phase and accumulation of cells in pre-G1 phase and prevented its mitotic cycle, in addition to its activation of caspase-7 mediating apoptosis of MCF-7 cells. Molecular modeling studies for compounds 3a, 3b and 3f were carried out on tubulin crystallography, the results indicated that the compounds showed binding mode similar to the co-crystalized ligand; colchicine. Moreover, pharmacophore constructed models and docking studies revealed that thiazolidinone, acetamide and coumarin moieties are crucial for the activity. Molecular dynamics (MD) studies were carried out for the three compounds over 100?ps. MD results of compound 3a showed that it reached the stable state after 30?ps which was in agreement with the calculated potential and kinetic energy of compound 3a.     

Validated spectrofluorimetric method for the determination of clonazepam in pharmaceutical preparations

A simple, highly sensitive and validated spectrofluorimetric method was applied in the determination of clonazepam (CLZ). The method is based on reduction of the nitro group of clonazepam with zinc/CaCl₂, and the product is then reacted with 2‐cyanoacetamide (2‐CNA) in the presence of ammonia (25%) yielding a highly fluorescent product. The produced fluorophore exhibits strong fluorescence intensity at ?_em = 383 nm after excitation at ?_ex = 333 nm. The method was rectilinear over a concentration range of 0.1–0.5 ng/mL with a limit of detection (LOD) of 0.0057 ng/mL and a limit of quantification (LOQ) of 0.017 ng/mL. The method was fully validated and successfully applied to the determination of CLZ in its tablets with a mean percentage recovery of 100.10 ± 0.75%. Method validation according to ICH Guidelines was evaluated. Statistical analysis of the results obtained using the proposed method was successfully compared with those obtained using a reference method, and there was no significance difference between the two methods in terms of accuracy and precision. Copyright © 2015 John Wiley & Sons, Ltd.     

A comparative gametogenesis and biochemical differentiation of goatfish (Family: Mullidae) from the Red Sea

Comparative study between few species of goatfishes (Mullidae), locally called Sultan Ibrahim, has been investigated from Jeddah coast. The present study compares gonad structure, oogenesis and spermatogenesis in three species of goatfish. The various stages are similar in the different species, but differ in time of spawning. The reproductive biology of three goatfishes, Mulloidichthys flavolineatus (1 black blotch), Mulloidichthys flavolineatus (3 black blotches), Mulloidichthys vanicolensis, were examined. A macroscopic and histological examination of the gonads demonstrated that all the three species are typically gonochoristic. Species-specific reproductive parameters including male stages, changes in gonado-somatic index (GSI), classification of oocyte stages were analyzed for each of the three targeted species. All three species have asynchronous ovary. The spawning season for the three studied species differ from one species to another i.e., the spawning season for M. vanicolensis females and males from April to July; M. flavolineatus, (1 black blotches) for both sexes from May until December; for M. flavolineatus (3 black blotches) showed a peak in May to early October for both sexes. Ultrastructurally the mature spermatozoon’s head shape appears like a computermouse, the tiny mid-piece consist two mitochondria and a flagellum. The chromatin, within the nucleus is electron-dense and homogenous in M. flavolineatus (with one black blotch), while the chromatin is granular in (M. vanicolensis and M. flavolineatus (3 black blotches)). The posterior region of the nucleus is deeply penetrated by the nuclear fossa which contains, the basal part of flagellum and centriolar complex. The ultrastructure of the eggs of the three Mullidae species is circular in shape and contains a simple, circular micropyle. The ovarian follicular epithelium of the three Mullidae species consists of two layers, an outer theca layer and an inner granulosa layer with zona radiata under them. Zona radiata consists of 2 layer-interna and externa. Zona radiata interna is thicker than zona radiata externa. Differences in protein patterns of muscles and gonads were observed in the present study.     

Phenotype-genotype updates from familial Mediterranean fever database registry of Mansoura University Children’ Hospital,Mansoura, Egypt

BACKGROUND:

Familial Mediterranean fever (FMF) is autosomal recessive disease that affects people from Mediterranean region, Europe and Japan. Its gene (Mediterranean fever [MEFV]) has more than 100 mostly non-sense mutations.

OBJECTIVES:

The objective of the following study is to provide some phenotype-genotype correlates in FMF by categorizing the Egyptian FMF cases from Delta governorates after analysis of the four most common mutations of MEFV gene (M680I, M694I, M694V, V726A).

SUBJECTS AND METHODS:

Clinically, suspected FMF cases using Tel-Hashomer criteria were enrolled in the study. Cases were referred to Mansoura University Children''s Hospital that serves most of the most middle Delta governorates, in the period from 2006 to 2011. Subjects included 282 males and 144 females, mean age of onset 9.3 ± 2.2 years. All cases were analyzed for these mutations using amplification refractory mutation system based on the polymerase chain reaction technique. Five FMF patients agreed to undergo renal biopsy to check for development of amyloidosis. Analysis of data was carried out using SPSS (SPSS, Inc., Chicago, IL, USA).

RESULTS:

Mutation was found in 521 out of 852 studies alleles, the most frequent is M694V (35.4%) followed by M694I, V726A and M680I. 11 cases were homozygous; 7 M694V, 3 M680I and only one M694I case. Severe abdominal pain occurred in 31 (7.28%) but severe arthritis in 103 cases (24.2%). Strong association was found between arthritis and homozygous mutant compared with single and double heterozygous (72.7% vs. 33.3% and 20.24%, P < 0.001). Four amyloid cases were M694V positive.

CONCLUSION:

M694V allele is the most common among Egyptian FMF especially those with amyloidosis. We recommend routine check for amyloidosis in FMF cases to statistically validate this link.     

Autophagy in osteoblasts is involved in mineralization and bone homeostasis

Bone remodeling is a tightly controlled mechanism in which osteoblasts (OB), the cells responsible for bone formation, osteoclasts (OC), the cells specialized for bone resorption, and osteocytes, the multifunctional mechanosensing cells embedded in the bone matrix, are the main actors. Increased oxidative stress in OB, the cells producing and mineralizing bone matrix, has been associated with osteoporosis development but the role of autophagy in OB has not yet been addressed. This is the goal of the present study. We first show that the autophagic process is induced in OB during mineralization. Then, using knockdown of autophagy-essential genes and OB-specific autophagy-deficient mice, we demonstrate that autophagy deficiency reduces mineralization capacity. Moreover, our data suggest that autophagic vacuoles could be used as vehicles in OB to secrete apatite crystals. In addition, autophagy-deficient OB exhibit increased oxidative stress and secretion of the receptor activator of NFKB1 (TNFSF11/RANKL), favoring generation of OC, the cells specialized in bone resorption. In vivo, we observed a 50% reduction in trabecular bone mass in OB-specific autophagy-deficient mice. Taken together, our results show for the first time that autophagy in OB is involved both in the mineralization process and in bone homeostasis. These findings are of importance for mineralized tissues which extend from corals to vertebrates and uncover new therapeutic targets for calcified tissue-related metabolic pathologies.     

An integrative review of systematic reviews related to the management of breathlessness in respiratory illnesses

Background

Breathlessness is a debilitating and distressing symptom in a wide variety of diseases and still a difficult symptom to manage. An integrative review of systematic reviews of non-pharmacological and pharmacological interventions for breathlessness in non-malignant disease was undertaken to identify the current state of clinical understanding of the management of breathlessness and highlight promising interventions that merit further investigation.

Methods

Systematic reviews were identified via electronic databases between July 2007 and September 2009. Reviews were included within the study if they reported research on adult participants using either a measure of breathlessness or some other measure of respiratory symptoms.

Results

In total 219 systematic reviews were identified and 153 included within the final review, of these 59 addressed non-pharmacological interventions and 94 addressed pharmacological interventions. The reviews covered in excess of 2000 trials. The majority of systematic reviews were conducted on interventions for asthma and COPD, and mainly focussed upon a small number of pharmacological interventions such as corticosteroids and bronchodilators, including beta-agonists. In contrast, other conditions involving breathlessness have received little or no attention and studies continue to focus upon pharmacological approaches. Moreover, although there are a number of non-pharmacological studies that have shown some promise, particularly for COPD, their conclusions are limited by a lack of good quality evidence from RCTs, small sample sizes and limited replication.

Conclusions

More research should focus in the future on the management of breathlessness in respiratory diseases other than asthma and COPD. In addition, pharmacological treatments do not completely manage breathlessness and have an added burden of side effects. It is therefore important to focus more research on promising non-pharmacological interventions.     

Staphylococcal Enterotoxin B Initiates Protein Kinase C Translocation and Eicosanoid Metabolism While Inhibiting Thrombin-Induced Aggregation in Human Platelets

Staphylococcal enterotoxin (SE) B, a heat-stable toxin secreted by Staphylococcus aureus, has been implicated in the pathogenesis and exacerbation of several critical illnesses. It has been hypothesized that enterotoxins may interact with blood products such as platelets, in addition to T-lymphocytes and renal proximal tubule cells. The aim of this present study was to elucidate whether SEB directly alters human platelet function. Human platelet rich plasma (PRP) was pre-incubated with SEA, SEB, SEC or TSST-1, (at various concentrations and incubation times). After incubation, PRP was exposed to thrombin and aggregation was assessed. Incubation with all toxins tested resulted in decreased aggregation, specifically; exposure to 10μ g/ml of SEB for 30 min caused a 20% decrease and a 49% decrease at 90 min. A similar reduction in aggregation was seen in samples incubated with phorbol myristate acetate, a known stimulator of protein kinase C (PKC). Further, platelets exposed to SEB exhibited an increased plasma membrane PKC activity. Sphingosine, an inhibitor of PKC proved to block the SEB-induced reduction in aggregation. SEB effects on platelet metabolism were investigated using high performance liquid chromatography showing up to a 2-fold increase of active metabolites lipoxin A4 and 12-HETE, as compared to control. These data indicate that SEB is able to induce platelet dysfunction, and these effects may be mediated through activation of PKC.The views of the authors do not purport to reflect the position of the Department of the Army or the Department of Defense (Para, 4–3) AR360-5.