Ethanolic extract of sharah, Plectranthus aegyptiacus, enhances healing of skin wound in rats

Sharah, Plectranthus aegyptiacus (Forssk.) C. Chr. is a common native plant in the Taif region of Saudi Arabia. An ethanolic extract of freeze dried sharah leaves was added as 10% (w/w) to an ointment base of beeswax and sesame oil. The resultant ointment was examined as a potential enhancer of wound healing. Excision wounds in the nape region of the skin were induced in sixty albino Wistar rats. Animals were allocated in 4 groups (n = 15) and kept individually in clean cages. The first group served as negative untreated controls without medication; the second group was treated with ointment base (vehicle); the third group represented the positive control and was treated with a reference ointment and the fourth one served as the experimental group and received the test plant extract (as ointment). Animal groups received the respective medications for 14 successive days. Wounds were measured and photographed every 3 days till the end of the experiment (day 21) in order to determine the wound closure rate (WCR). Specimens from wounds and surrounding skin were collected from sacrificed animals for histological and molecular studies. Both morphometric (based on WCR) and histological findings showed that the healing in animals treated with the sharah plant extract was better than those in control group or vehicle-treated group and was similar to that in the group that received the reference ointment. Moreover, the molecular findings concerning the expression levels of hepatocyte growth factor (HGF) and its receptor (c-Met) displayed a reasonable healing enhancing effect of the plant extract with the expression levels of both being higher in the extract-treated group than in the control group.     

Hypotensive but not normotensive haemorrhage increases tryptophan hydroxylase-2 mRNA in caudal midline medulla

Severe blood loss triggers shock, a precipitous hypotension and bradycardia. The integrity of (i) neurons in the vasodepressor region of the caudal midline medulla and (ii) central 5-HT neurotransmission are critical for the expression of haemorrhagic shock. This study investigated whether progressive blood loss triggers altered synthesis of 5-HT in the vasodepressor region of the caudal midline medulla by measuring changes in relative expression levels of tryptophan hydroxylase 2 (TpH 2) mRNA, the rate-limiting enzyme in the synthesis of neuronal 5-HT. Hypotensive but not normotensive haemorrhage triggered a significant increase in TpH 2 mRNA in the vasodepressor region of the caudal midline medulla, identifying an important role for 5-HT-containing caudal midline medullary neurons in haemorrhagic shock.     

Analysis of biological and technical variability in gene expression assays from formalin-fixed paraffin-embedded classical Hodgkin lymphomas

Formalin-fixed paraffin-embedded (FFPE) tissues are invaluable sources of biological material for research and diagnostic purposes. In this study, we aimed to identify biological and technical variability in RT-qPCR TaqMan® assays performed with FFPE-RNA from lymph nodes of classical Hodgkin lymphoma samples. An ANOVA-nested 6-level design was employed to evaluate BCL2, CASP3, IRF4, LYZ and STAT1 gene expression. The most variable genes were CASP3 (low expression) and LYZ (high expression). Total variability decreased after normalization for all genes, except by LYZ. Genes with moderate and low expression were identified and suffered more the effects of the technical manipulation than high-expression genes. Pre-amplification was shown to introduce significant technical variability, which was partially alleviated by lowering to a half the amount of input RNA. Ct and Cy₀ quantification methods, based on cycle-threshold and the kinetic of amplification curves, respectively, were compared. Cy₀ method resulted in higher quantification values, leading to the decrease of total variability in CASP3 and LYZ genes. The mean individual noise was 0.45 (0.31 to 0.61 SD), indicating a variation of gene expression over ~ 1.5 folds from one case to another. We showed that total variability in RT-qPCR from FFPE-RNA is not higher than that reported for fresh complex tissues, and identified gene-, and expression level-sources of biological and technical variability, which can allow better strategies for designing RT-qPCR assays from highly degraded and inhibited samples.     

Novel mutations in the mitochondrial complex I assembly gene NDUFAF5 reveal heterogeneous phenotypes

Primary mitochondrial complex I deficiency is the most common defect of the mitochondrial respiratory chain. It is caused by defects in structural components and assembly factors of this large protein complex. Mutations in the assembly factor NDUFAF5 are rare, with only five families reported to date. This study provides clinical, biochemical, molecular and functional data for four unrelated additional families, and three novel pathogenic variants. Three cases presented in infancy with lactic acidosis and classic Leigh syndrome. One patient, however, has a milder phenotype, with symptoms starting at 27?months and a protracted clinical course with improvement and relapsing episodes. She is homozygous for a previously reported mutation, p.Met279Arg and alive at 19?years with mild neurological involvement, normal lactate but abnormal urine organic acids. We found the same mutation in one of our severely affected patients in compound heterozygosity with a novel p.Lys52Thr mutation. Both patients with p.Met279Arg are of Taiwanese descent and had severe hyponatremia. Our third and fourth patients, both Caucasian, shared a common, newly described, missense mutation p.Lys109Asn which we show induces skipping of exon 3. Both Caucasian patients were compound heterozygotes, one with a previously reported Ashkenazi founder mutation while the other was negative for additional exonic variants. Whole genome sequencing followed by RNA studies revealed a novel deep intronic variant at position c.223-907A>C inducing an exonic splice enhancer. Our report adds significant new information to the mutational spectrum of NDUFAF5, further delineating the phenotypic heterogeneity of this mitochondrial defect.