Aspergillus awamori MK788209 cellulase: production, statistical optimization, pea peels saccharification and textile applications.

BACKGROUND: The demand for low-cost cellulolytic enzyme synthesis is rising in the enzyme market. This work aims to produce cellulase by utilizing various agricultural wastes and investigating the use of enzyme in saccharification and textile industries. RESULTS: Solid state fermentation (SSF) was applied to produce industrial enzymes, particularly cellulase, through utilizing Molokhia (Corchorus olitorius) stems by Aspergillus awamori MK788209 isolate. Two stages of statistical factorial designs Plackett-Burman (PB) and Central Composite Design (CCD) were applied to enhance the A. awamori MK788209 cellulase production from Molokhia stems (MS). The fold increase of enzyme production by PB followed by CCD was 2.51 and 4.86, respectively. Additionally, the A. awamori MK788209 culture filtrate was highly effective in saccharifying various agricultural wastes, particularly pea peels (PP) (yielding 98.33 mg reducing sugar/ml), due to its richness in cellulase, laccase, xylanase, pectinase, and amylase. By optimizing the three main variables; pea peel weight, culture filtrate volume added, and saccharification time by CCD, the sugar recovery from PP was enhanced, leading to a 3.44-fold increase in reducing sugar recovery (338 mg reducing sugar /ml). Furthermore, the A. awamori MK788209 culture filtrate showed high efficacy in textile applications, enhancing the roughness, weight loss, white index, and printing capability of treated cotton fabrics. CONCLUSIONS: A. Awamori MK788209 produced cellulase which was effective in PP saccharification. The enzyme was also capable of enhancing cotton fabric properties.

Macrophage Migration Inhibitory Factor on Apoptotic Extracellular Vesicles Regulates Compensatory Proliferation.

Apoptotic cells can signal to neighboring cells to stimulate proliferation and compensate for cell loss to maintain tissue homeostasis. While apoptotic cell-derived extracellular vesicles (AEVs) can transmit instructional cues to mediate communication with neighboring cells, the molecular mechanisms that induce cell division are not well understood. Here we show that macrophage migration inhibitory factor (MIF)-containing AEVs regulate compensatory proliferation via ERK signaling in epithelial stem cells of larval zebrafish. Time-lapse imaging showed efferocytosis of AEVs from dying epithelial stem cells by healthy neighboring stem cells. Proteomic and ultrastructure analysis of purified AEVs identified MIF localization on the AEV surface. Pharmacological inhibition or genetic mutation of MIF, or its cognate receptor CD74, decreased levels of phosphorylated ERK and compensatory proliferation in the neighboring epithelial stem cells. Disruption of MIF activity also caused decreased numbers of macrophages patrolling near AEVs, while depletion of the macrophage lineage resulted in a reduced proliferative response by the epithelial stem cells. We propose that AEVs carrying MIF directly stimulate epithelial stem cell repopulation and guide macrophages to cell non-autonomously induce localized proliferation to sustain overall cell numbers during tissue maintenance.

Analysis of MTNR1A Genetic Polymorphisms and Their Association with the Reproductive Performance Parameters in Two Mediterranean Sheep Breeds.

Sheep farming plays an important economic role, and it contributes to the livelihoods of many rural poor in several regions worldwide and particularly in Tunisia. Therefore, the steady improvement of ewes' reproductive performance is a pressing need. The MTNR1A gene has been identified as an important candidate gene that plays a key role in sheep reproduction and its sexual inactivity. It is involved in the control of photoperiod-induced seasonality mediated by melatonin secretion. The aim of this study was to identify SNPs in the MTNR1A gene in two Tunisian breeds, Barbarine (B) and Queue Fine de l'Ouest (QFO). DNA extracted from the blood of 77 adult ewes was sequenced. Selected ewes were exposed to adult fertile rams. A total of 26 SNPs were detected; 15 SNPs in the promoter region and 11 SNPs in the exon II were observed in both (B) and (QFO) breeds. The SNP rs602330706 in exon II is a novel SNP detected for the first time only in the (B) breed. The SNPs rs430181568 and rs40738822721 (SNP18 and SNP20 in our study, respectively) were totally linked in this study and can be considered a single marker. DTL was associated with SNP18 and SNP20 in (B) ewes (p < 0.05); however, no significant difference was detected between the three genotypes (G/G, G/A, and A/A) at these two SNPs. Fertility rate and litter size parameters were not affected by SNP18 and SNP20. There was an association between these two polymorphisms and (B) lambs' birth weights (p < 0.05). Furthermore, the ewes with the A/A genotype gave birth to lambs with a higher weight compared to the other two genotypes for this breed (p < 0.05). There was not an association between SNP 18 and SNP20 and (QFO) ewes' reproductive parameters. These results might be considered in future sheep selection programs for reproductive genetic improvement.

Utility of Circulating Cell-Free DNA in Assessing Microsatellite Instability and Loss of Heterozygosity in Breast Cancer Using Human Identification Approach.

The diagnostic and prognostic utility of circulating cell-free DNA (cfDNA) in breast cancer (BC) patients was recently reported. Here, we investigated the use of cfDNA to examine microsatellite instability (MSI) and loss of heterozygosity (LOH) for early BC diagnosis. cfDNA and genomic DNA from 41 female BC patients and 40 healthy controls were quantified using NanoDrop spectrophotometry and real-time PCR. The stability of genomic and cfDNA was assessed using a high-resolution AmpFlSTR MiniFiler human identification kit. Significant increases in cfDNA plasma concentrations were observed in BC patients compared to controls. The genotype distribution of the eight autosomal short tandem repeat (STR) loci D7S820, D13S317, D21S11, D2S1338, D18S51, D16S539, FGA, and CSF1PO were in Hardy-Weinberg equilibrium. Significant differences in the allele frequencies of D7S820 allele-8, D21S11 allele-29, allele-30.2, allele-32.2, and CSF1PO allele-11 were seen between BC patients and controls. LOH and MSI were detected in 36.6% of the cfDNA of patients compared to genomic DNA. This study highlights the utility of plasma-derived cfDNA for earlier, less invasive, and cost-effective cancer diagnosis and molecular stratification. It also highlights the potential value of cfDNA in molecular profiling and biomarkers discovery in precision and forensic medicine.

Detection of genetic mutations in patients with breast cancer from Saudi Arabia using Ion AmpliSeq™ Cancer Hotspot Panel v.2.0.

Next-Generation Sequencing allows for quick and precise sequencing of multiple genes concurrently. Recently, this technology has been employed for the identification of novel gene mutations responsible for disease manifestation among breast cancer (BC) patients, the most common type of cancer amongst Arabian women, and the major cause of disease-associated death in women worldwide. Genomic DNA was extracted from the peripheral blood of 32 Saudi Arabian BC patients with histologically confirmed invasive BC stages I-III and IV, as well from 32 healthy Saudi Arabian women using a QIAamp® DNA Mini Kit. The isolated DNA was quantified using a Qubit™ dsDNA BR Assay Kit with a Qubit 2.0 Fluorometer. Ion semiconductor sequencing technology with an Ion S5 System and AmpliSeq™ Cancer Hotspot Panel v2 were utilized to analyze ~2,800 mutations described in the Catalogue of Somatic Mutations in Cancer from 50 oncogenes and tumor suppressor genes. Ion Reporter Software v.5.6 was used to evaluate the genomic alterations in all the samples after alignment to the hg19 human reference genome. The results showed that out of the 50 genes, 26 mutations, including 17 (65%) missense point mutations (single nucleotide variants), and 9 (35%) frameshift (insertion/deletion) mutations, were identified in 11 genes across the cohort in 61 samples (95%). Mutations were predominantly focused on two genes, PIK3CA and TP53, in the BC genomes of the sample set. PIK3CA mutation, c.1173A>G located in exon 9, was identified in 15 patients (46.9%). The TP53 mutations detected were a missense mutation (c.215C>G) in 26 patients (86.70%) and 1 frameshift mutation (c.215_216insG) in 1 patient (3.33%), located within exon 3 and 5, respectively. This study revealed specific mutation profiles for every BC patient, Thus, the results showed that Ion Torrent DNA Sequencing technology may be a possible diagnostic and prognostic method for developing personalized therapy based on the patient's individual BC genome.

Population genetic data for 12 X-STR loci in the Central Saudi region using investigator Argus X-12 amplification kit.

BACKGROUND: X-chromosome short tandem repeat (X-STR) markers are important in forensic identity investigations and kinship analysis. SUBJECT AND METHODS: In the current study, the distribution of 12 X-STR loci located in four linkage groups was evaluated using Investigator® Argus X-12 Amplification Kit in 200 unrelated healthy individuals (105 males and 95 females) from the central region of Saudi Arabia in order to develop an allelic frequency database for the markers included in the kit. RESULTS: DXS10146 locus was the most informative with 21 alleles, while DXS8378 locus was the least with five alleles. Forensic parameters showed that all X-STRs loci, either as individual markers or as linkage groups, provide genetic information with high discrimination that is appropriate for forensic purposes with polymorphism information content (PIC), power of exclusion (PE), and paternity index (PI) varying from 0.61211 to 0.917979, 0.38722 to 0.842949, and 0.038416 to 0.16367, respectively. The pairwise genetic distance fixation index (Fst) results showed that the Saudi population is genetically closer to the Egyptian and Emirati populations and distant to the Turkish population. CONCLUSION: The current study revealed that Investigator® Argus 12 X-STR kit would support the forensic application, kinship testing involving female offspring, and human identification in the Saudi population.

Failure to engage the temporoparietal junction/posterior superior temporal sulcus predicts impaired naturalistic social cognition in schizophrenia.

Schizophrenia is associated with marked impairments in social cognition. However, the neural correlates of these deficits remain unclear. Here we use naturalistic stimuli to examine the role of the right temporoparietal junction/posterior superior temporal sulcus (TPJ-pSTS)-an integrative hub for the cortical networks pertinent to the understanding complex social situations-in social inference, a key component of social cognition, in schizophrenia. Twenty-seven schizophrenia participants and 21 healthy control subjects watched a clip of the film The Good, the Bad and the Ugly while high resolution multiband functional MRI images were collected. We used inter-subject correlation to measure the evoked activity, which we then compared to social cognition as measured by The Awareness of Social Inference Test (TASIT). We also compared between groups the TPJ-pSTS blood oxygen level-dependent activity (i) relationship with the motion content in the film; (ii) synchronization with other cortical areas involved in the viewing of the movie; and (iii) relationship with the frequency of saccades made during the movie. Activation deficits were greatest in middle TPJ (TPJm) and correlated significantly with impaired TASIT performance across groups. Follow-up analyses of the TPJ-pSTS revealed decreased synchronization with other cortical areas, decreased correlation with the motion content of the movie, and decreased correlation with the saccades made during the movie. The functional impairment of the TPJm, a hub area in the middle of the TPJ-pSTS, predicts deficits in social inference in schizophrenia participants by disrupting the integration of visual motion processing into the TPJ. This disrupted integration then affects the use of the TPJ to guide saccades during the visual scanning of the movie clip. These findings suggest that the TPJ may be a treatment target for improving deficits in a key component of social cognition in schizophrenia participants.

Population genetics of 30 insertion/deletion polymorphisms in the Bahraini population.

This paper evaluates the forensic utility of 30 insertion-deletion polymorphism (indel) markers in a sample from the Bahraini population using the Qiagen Investigator DIPplex Kit. Allele frequencies and forensic stats of the 30 indels were investigated in 293 unrelated individuals from different governorates of the Kingdom of Bahrain. None of the markers showed significant deviation from Hardy Weinberg equilibrium except for HLD88 locus and no linkage disequilibrium were detected between all possible pair of the indel loci, assuming that these markers are independent and their allele frequencies can be used to calculate the match probabilities in the Bahraini population. The high power of discrimination (CPD = 0.9999999999998110) and the low combined match probability (CPM = 1.89 × 10-13) indicate that these markers are informative and can be successfully used for human identification in terms of forensics and paternity. Genetic distances and relatedness were displayed through multidimensional plotting and phylogenetic tree using various populations in the region. Our study showed that the Bahraini population was clustered with neighboring countries such as Kuwait and Emirates which indicates that these closely geographical regions share similar allele frequencies and are more genetically related than other reference population studied.

The impact of chimerism on DNA-based human identification from skin surface cells of post-allogenic hematopoietic stem cell transplantation (HCST) patients.

The use of biological traces recovered from touched or handled items increased with the advance of the forensic analysis system. Thus, DNA profiles obtained from touch DNA became a useful tool in forensic investigation. However, a chimeric person with more than one chromosomal population can be challenging for a forensic analyst. We investigated the genetic profile in blood, buccal swab, and skin swabs from twenty-four recipients aged 21-63 years who underwent a matched sibling allogeneic hematopoietic stem cell transplantation with no sign of skin graft versus host disease. Autosomal short tandem repeats genotyping was performed to evaluate chimerism status at 15 loci along with gender marker Amelogenin. According to our results, donor chimerism was detected in all recipient's blood samples, while in buccal swabs, five recipients showed no presence of donor-derived cells in their genotype. Epithelial cells swabbed from hand fingertips were not devoid of donor-derived cells since all recipients showed high chimerism (39.69%-96.66%) in their genotypes. A significant change in chimerism was seen among various types of biological samples (p<0.05). No correlations were observed between chimerism and recipient age, gender, or time after transplant (p> 0.05). The loci D21S11, D8S1179, and FGA were the most informative, whereas D13S317, Vwa, and TOPX were the least informative STR markers. We concluded that touch DNA from a person who has undergone a successful allogeneic HSCTs should not be considered as reliable evidence for human identifications. Therefore, necessary precautions must be taken to avoid false identification and miscarriage of justice.

Anastatica hierochuntica (L.) methanolic and aqueous extracts exert antiproliferative effects through the induction of apoptosis in MCF-7 breast cancer cells.

Breast cancer therapy using anticancer bioactive compounds derived from natural products as adjuvant treatment has gained recognition due to expensive and toxic conventional chemotherapeutic drugs. The whole plant of Anastatica hierochuntica (L.) (A. hierochuntica) has been investigated for its pharmacologically important anticancer properties but without categorizing the biological activities of the plant parts. We assessed the anticancer potential of different parts of A. hierochuntica (seeds, stems and leaves) and explored their mechanisms of action using the human breast cancer cell line, MCF-7. Currently, we investigated the antiproliferative effects of methanolic (MSD, MST, ML) and aqueous (ASD, AST, AL) extracts of A. hierochuntica plant parts on the MCF-7 cells using cell viability assays. Flow cytometry, Western Blot, DNA fragmentation, and gene expression assays were performed to evaluate apoptosis and cell cycle regulatory proteins. The results indicate that the methanolic and aqueous extracts decreased MCF-7 cell viability in a dose-dependent manner. The induction of apoptosis was observed in all the methanolic and aqueous-treated MCF-7 cells. The cell death process was confirmed by the visualization of DNA fragmentation and cleavage of the intrinsic apoptotic pathways, caspase-9 and caspase-3, the key enzyme causing apoptosis hallmarks. In addition, the most pro-apoptotic extracts, ASD and ML, up-regulated the expression of pro-apoptotic Bax, tumor suppressor TP53 genes and the cyclin inhibitor CDKN1A gene. In conclusion, of the aqueous and methanolic extracts of A. hierochuntica plant parts exerting antiproliferative effects through the induction of apoptosis in breast cancer MCF-7 cells, ASD and ML extracts were the most promising natural-based drugs for the treatment of breast cancer.

Identification of Potential Genes for Benign Prostatic Hyperplasia and Prostate Cancer Susceptibility in Four X-chromosome Regions with High Frequency of Microvariant Alleles.

BACKGROUND: The X-chromosome has been suggested to play a role in prostate cancer (PrCa) since epidemiological studies have provided evidence for an X-linked mode of inheritance for PrCa based on the higher relative risk among men who report an affected brother(s) as compared to those reporting an affected father. The aim of this study was to examine the potential association between the forensic STR markers located at four regions Xp22.31, Xq11.2-12, Xq26.2, and Xq28 and the risk of BPH and PrCa to confirm the impact of ChrX in the PrCa incidence. This may be helpful in the incorporation of STRs genetic variation in the early detection of men population at risk of developing PrCa. METHODS: DNA samples from 92 patients and 156 healthy controls collected from two medical centers in Riyadh, Saudi Arabia were analyzed for four regions located at X-chromosome using the Investigator® Argus X-12 QS Kit. RESULTS: The results demonstrated that microvariant alleles of (DXS7132, DXS10146, HPRTB, DXS10134, and DXS10135) are overrepresented in the BPH group (p < 0.00001). Allele 28 of DXS10135 and allele 15 of DXS7423 could have a protective effect, OR 0.229 (95%CI, 0.066-0.79); and OR 0.439 (95%CI, 0.208-0.925). On the other hand, patients carrying allele 23 of DXS10079 and allele 26 of DXS10148 presented an increased risk to PrCa OR 4.714 (95%CI, 3.604-6.166). CONCLUSION: The results are in concordance with the involvement of the X chromosome in PrCa and BPH development. STR allele studies may add further information from the definition of a genetic profile of PrCa resistance or susceptibility. As TBL1, AR, LDOC1, and RPL10 genes are located at regions Xp22.31, Xq11.2-12, Xq26.2, and Xq28, respectively, these genes could play an essential role in PrCa or BPH.

Geographical structuring and low diversity of paternal lineages in Bahrain shown by analysis of 27 Y-STRs.

We have determined the distribution of Y-chromosomal haplotypes and predicted haplogroups in the ethnically diverse Kingdom of Bahrain, a small archipelago in the Arabian Gulf. Paternal population structure within Bahrain was investigated using the 27 Y-STRs (short tandem repeats) in the Yfiler Plus kit to generate haplotypes from 562 unrelated Bahraini males, sub-divided into four geographical regions-Northern, Capital, Southern and Muharraq. Yfiler Plus provided a significant improvement over the 17-locus Yfiler kit in discrimination capacity (from 77% to 87.5% overall), but discrimination capacity differed widely between regions from 98.4% in Muharraq to 75.2% in the Northern region, an unusually low value possibly resulting from recent rapid population expansion. Clusters of closely related male lineages were seen, with only 79.4% of donors displaying unique haplotypes and 59% of instances of shared haplotypes occurring within, rather than between, regions. Haplogroup prediction indicated diverse origins of the population with a predominance of haplogroups J2 and J1, both typical of the Arabian Peninsula, but also haplogroups such as B2 and E1b1a likely originating in Africa, and H, L and R2 likely indicative of migration from South Asia. Haplogroup frequencies differed significantly between regions, with J2 significantly more common in the Northern region compared with the Southern, possibly due to differential settlement by Baharna and Arabs. Our study shows that paternal lineage population structure can exist even over small geographical scales, and that highly discriminating genetic tools are required where rapid expansions have occurred within tightly bounded populations.

Tube shunt surgery in pig eyes: a wet lab teaching model.

OBJECTIVE: To describe a teaching model for aqueous tube shunt surgery using freshly enucleated pig eyes. DESIGN: A descriptive methods report, containing a small prospective noncomparative interventional case series. PARTICIPANTS: Six ophthalmology residents from the University of Alberta program. METHODS: A descriptive report outlines the materials and methods required for creating a surgical wet lab that teaches aqueous draining device implantation. Residents practiced each step associated with the insertion of plated aqueous tube shunt devices in porcine eyes. RESULTS: The porcine model worked well to demonstrate and perform steps associated with tube implant surgery. Trainee comfort improved in all surgical steps practiced during the session when reassessed at 3-month follow-up: priming and anchoring the drainage device (p = 0.042), inserting the tube into the eye (p = 0.025), creating and securing a scleral patch graft (p = 0.034), and closure of the conjunctiva (p = 0.034). Overall confidence in performing tube shunt surgery also remained above baseline at follow-up (p = 0.042). CONCLUSIONS: Implantation of tube shunt devices in the porcine model closely resembles surgery in human eyes. Practicing each step associated with tube shunt surgery on porcine eyes in a supervised wet-lab environment improves trainee confidence in the procedure.

Ab interno trabeculectomy with Trabectome: outcomes in African American versus Caucasian patients.

OBJECTIVE: To compare the intraocular pressure-lowering efficacy and complication rate of ab interno trabeculectomy with the Trabectome between African American (AA) and Caucasian patients with open-angle glaucoma. METHODS: A total of 164 patients (82 AA and 82 Caucasian) who underwent ab interno trabeculectomy over an 8-year period were included in this prospective, case-control study. The Neomedix database was used to look for AA or Caucasian patients with open-angle glaucoma who underwent Trabectome with or without phacoemulsification. RESULTS: The average IOP of AA patients was reduced from 21.2 ± 6.8 mm Hg to 16.1 ± 4.1 mm Hg at 12 M (p < 0.01), and the mean number of glaucoma medications was reduced from 2.4 ± 1.3 to 2.0 ± 1.4 (p = 0.13). Among Caucasians, the mean IOP was reduced from 21.2 ± 6.8 mm Hg to 15.7 ± 4.2 mm Hg at 12 M (p < 0.01), and the number of medications dropped from 2.4 ± 1.2 to 1.7 ± 1.3 (p < 0.01). No statistically significant difference was found between these 2 race groups in IOP, number of medications, and complications. For complications with Trabectome alone, 13% of AA and 9% of Caucasian patients needed secondary surgery. There was only 1 case of hypotony which was in the AA group. For complications with Trabectome combined with phacoemulsification, 4% of AA as well as 4% of Caucasian patients required secondary surgery. There was only 1 case of hypotony which was in the Caucasian group. CONCLUSION: Ab interno trabeculectomy with Trabectome is associated with a reduction in IOP in both race groups with a similar complication and survival profiles.

Cell type-dependent effects of ellagic acid on cellular metabolism.

Ellagic acid is a botanical polyphenol which has been shown to have numerous effects on cellular function. Ellagic acid can induce apoptosis and inhibit the proliferation of various cancer cell types in vitro and in vivo. As such, ellagic acid has attracted significant interest as a potential chemotherapeutic compound. One mechanism by which ellagic acid has been proposed to affect cellular physiology is by regulating metabolic pathways. Here we show the dose-dependent effects of ellagic acid on cellular energy production and downstream induction of the apoptotic program in HEK293, HeLa, MCF7, and HepG2 cells. At physiologically relevant doses, ellagic acid has pleiotropic and cell-type specific effects on mitochondrial function. At high doses ellagic acid can also influence glycolytic pathways and induce cell death. Our results demonstrate that ellagic acid can influence mitochondrial function at therapeutically relevant concentrations. The observed effects of ellagic acid on cellular respiration are complex and cell type-specific, which may limit the chemotherapeutic utility of this compound.

Interaction between nitric oxide and renal α1-adrenoreceptors mediated vasoconstriction in rats with left ventricular hypertrophyin Wistar Kyoto rats.

Left ventricular hypertrophy (LVH) is associated with decreased responsiveness of renal α1-adrenoreceptors subtypes to adrenergic agonists. Nitric oxide donors are known to have antihypertrophic effects however their impact on responsiveness of renal α1-adrenoreceptors subtypes is unknown. This study investigated the impact of nitric oxide (NO) and its potential interaction with the responsiveness of renal α1-adrenoreceptors subtypes to adrenergic stimulation in rats with left ventricular hypertrophy (LVH). This study also explored the impact of NO donor on CSE expression in normal and LVH kidney. LVH was induced using isoprenaline and caffeine in drinking water for 2 weeks while NO donor (L-arginine, 1.25g/Lin drinking water) was given for 5 weeks. Intrarenal noradrenaline, phenylephrine and methoxamine responses were determined in the absence and presence of selective α1-adrenoceptor antagonists, 5- methylurapidil (5-MeU), chloroethylclonidine (CeC) and BMY 7378. Renal cortical endothelial nitric oxide synthase mRNA was upregulated 7 fold while that of cystathione γ lyase was unaltered in the NO treated LVH rats (LVH-NO) group compared to LVH group. The responsiveness of renal α1A, α1B and α1D-adrenoceptors in the low dose and high dose phases of 5-MeU, CEC and BMY7378 to adrenergic agonists was increased along with cGMP in the kidney of LVH-NO group. These findings suggest that exogenous NO precursor up-regulated the renal eNOS/NO/cGMP pathway in LVH rats and resulted in augmented α1A, α1B and α1D adrenoreceptors responsiveness to the adrenergic agonists. There is a positive interaction between H2S and NO production in normal animals but this interaction appears absent in LVH animals.

p53 loss-of-heterozygosity is a necessary prerequisite for mutant p53 stabilization and gain-of-function in vivo.

Missense mutations in TP53 comprise >75% of all p53 alterations in cancer, resulting in highly stabilized mutant p53 proteins that not only lose their tumor-suppressor activity, but often acquire oncogenic gain-of-functions (GOFs). GOF manifests itself in accelerated tumor onset, increased metastasis, increased drug resistance and shortened survival in patients and mice. A known prerequisite for GOF is mutant p53 protein stabilization, which itself is linked to aberrant protein conformation. However, additional determinants for mutant p53 stabilization likely exist. Here we show that in initially heterozygous mouse tumors carrying the hotspot GOF allele R248Q (p53Q/+), another necessary prerequisite for mutant p53 stabilization and GOF in vivo is loss of the remaining wild-type p53 allele, termed loss-of-heterozygosity (LOH). Thus, in mouse tumors with high frequency of p53 LOH (osteosarcomas and fibrosarcomas), we find that mutant p53 protein is stabilized (16/17 cases, 94%) and tumor onset is significantly accelerated compared with p53+/- tumors (GOF). In contrast, in mouse tumors with low frequency of p53 LOH (MMTV-Neu breast carcinomas), mutant p53 protein is not stabilized (16/20 cases, 80%) and GOF is not observed. Of note, human genomic databases (TCGA, METABRIC etc.) show a high degree of p53 LOH in all examined tumor types that carry missense p53 mutations, including sarcomas and breast carcinomas (with and without HER2 amplification). These data - while cautioning that not all genetic mouse models faithfully represent the human situation - demonstrate for the first time that p53 LOH is a critical prerequisite for missense mutant p53 stabilization and GOF in vivo.

Cystathione gamma lyase/Hydrogen Sulphide Pathway Up Regulation Enhances the Responsiveness of α1A and α1B-Adrenoreceptors in the Kidney of Rats with Left Ventricular Hypertrophy.

The purpose of the present study was to investigate the interaction between H2S and NO (nitric oxide) in the kidney and to evaluate its impact on the functional contribution of α1A and α1B-adrenoreceptors subtypes mediating the renal vasoconstriction in the kidney of rats with left ventricular hypertrophy (LVH). In rats the LVH induction was by isoprenaline administration and caffeine in the drinking water together with intraperitoneal administration of H2S. The responsiveness of α1A and α1B to exogenous noradrenaline, phenylephrine and methoxaminein the absence and presence of 5-methylurapidil (5-MeU) and chloroethylclonidine (CEC) was studied. Cystathione gamma lyase (CSE), cystathione ß synthase (CBS), 3-mercaptopyruvate sulphar transferase (3-MST) and endothelial nitric oxide synthase (eNOS) were quantified. There was significant up regulation of CSE and eNOS in the LVH-H2S compared to the LVH group (P<0.05). Baseline renal cortical blood perfusion (RCBP) was increased (P<0.05) in the LVH-H2S compared to the LVH group. The responsiveness of α1A-adrenergic receptors to adrenergic agonists was increased (P<0.05) after administration of low dose 5-Methylurapidil in the LVH-H2S group while α1B-adrenergic receptors responsiveness to adrenergic agonists were increased (P<0.05) by both low and high dose chloroethylclonidine in the LVH-H2S group. Treatment of LVH with H2S resulted in up-regulation of CSE/H2S, CBS, and 3-MST and eNOS/NO/cGMP pathways in the kidney. These up regulation of CSE/H2S, CBS, and 3-MST and eNOS/NO/cGMP pathways enhanced the responsiveness of α1A and α1B-adrenoreceptors subtypes to adrenergic agonists in LVH-H2S. These findings indicate an important role for H2S in modulating deranged signalling in the renal vasculature resulting from LVH development.