Recovery of the Decorin-Enriched Fraction, Extract (D), From Human Skin: An Accelerated Protocol

The original extraction procedure of Engel and Catchpole [1] has often been used to recover decorin-enriched material from the skin. This material has a strong inhibitory effect on fibroblast proliferation, and clearly suppresses it in skin except after the first 5–6 days of wounding when new scaffold material is required. The aim of our present study has been to find and evaluate the product of a faster recovery method, and to check its consistency as a more reliable means of regularly obtaining sufficient material for topical application in wounds that might become hypertrophic. Modifications of the original Toole and Lowther [2] extraction procedure have been carefully evaluated in an attempt to cut preparation time without compromising biological activity of the inhibitory extract. We have devised a faster recovery procedure without compromising biological activity, even if initial recovery has been somewhat reduced. The latter problem could be offset by repeated cycles of the final extraction step. The main inhibitory activity is shown to be within the decorin-enriched “extract D,” as the core protein and DSPG II. Adjustment of the extract towards neutrality after dialysis against water keeps most of the extracted protein in solution and yielded a decorin-enriched preparation that had a specific activity equivalent to that of the old method. It also yielded a fraction that was readily lyophilised to give a small amount of material that could be stored indefinitely without loss of activity and readily redissolved in aqueous solution. A reliable and relatively quick method is presented for the production, from human skin, of a decorin-enriched preparation that has strong fibroblast inhibitory action. The value of the procedure is that it is inexpensive and can produce the quantities that might be used topically in reducing hypertrophic scarring of wounds

Tissue-specific mRNA expression profiling in grape berry tissues

<p>Abstract</p> <p>Background</p> <p>Berries of grape (<it>Vitis vinifera</it>) contain three major tissue types (skin, pulp and seed) all of which contribute to the aroma, color, and flavor characters of wine. The pericarp, which is composed of the exocarp (skin) and mesocarp (pulp), not only functions to protect and feed the developing seed, but also to assist in the dispersal of the mature seed by avian and mammalian vectors. The skin provides volatile and nonvolatile aroma and color compounds, the pulp contributes organic acids and sugars, and the seeds provide condensed tannins, all of which are important to the formation of organoleptic characteristics of wine. In order to understand the transcriptional network responsible for controlling tissue-specific mRNA expression patterns, mRNA expression profiling was conducted on each tissue of mature berries of <it>V. vinifera </it>Cabernet Sauvignon using the Affymetrix GeneChip^®<it>Vitis </it>oligonucleotide microarray ver. 1.0. In order to monitor the influence of water-deficit stress on tissue-specific expression patterns, mRNA expression profiles were also compared from mature berries harvested from vines subjected to well-watered or water-deficit conditions.</p> <p>Results</p> <p>Overall, berry tissues were found to express approximately 76% of genes represented on the <it>Vitis </it>microarray. Approximately 60% of these genes exhibited significant differential expression in one or more of the three major tissue types with more than 28% of genes showing pronounced (2-fold or greater) differences in mRNA expression. The largest difference in tissue-specific expression was observed between the seed and pulp/skin. Exocarp tissue, which is involved in pathogen defense and pigment production, showed higher mRNA abundance relative to other berry tissues for genes involved with flavonoid biosynthesis, pathogen resistance, and cell wall modification. Mesocarp tissue, which is considered a nutritive tissue, exhibited a higher mRNA abundance of genes involved in cell wall function and transport processes. Seeds, which supply essential resources for embryo development, showed higher mRNA abundance of genes encoding phenylpropanoid biosynthetic enzymes, seed storage proteins, and late embryogenesis abundant proteins. Water-deficit stress affected the mRNA abundance of 13% of the genes with differential expression patterns occurring mainly in the pulp and skin. In pulp and seed tissues transcript abundance in most functional categories declined in water-deficit stressed vines relative to well-watered vines with transcripts for storage proteins and novel (no-hit) functional assignments being over represented. In the skin of berries from water-deficit stressed vines, however, transcripts from several functional categories including general phenypropanoid and ethylene metabolism, pathogenesis-related responses, energy, and interaction with the environment were significantly over-represented.</p> <p>Conclusion</p> <p>These results revealed novel insights into the tissue-specific expression mRNA expression patterns of an extensive repertoire of genes expressed in berry tissues. This work also establishes an extensive catalogue of gene expression patterns for future investigations aimed at the dissection of the transcriptional regulatory hierarchies that govern tissue-specific expression patterns associated with tissue differentiation within berries. These results also confirmed that water-deficit stress has a profound effect on mRNA expression patterns particularly associated with the biosynthesis of aroma and color metabolites within skin and pulp tissues that ultimately impact wine quality.</p

Randomized multicentre pilot study of sacubitril/valsartan versus irbesartan in patients with chronic kidney disease: United Kingdom Heart and Renal Protection (HARP)- III—rationale, trial design and baseline data

BACKGROUND: Patients with chronic kidney disease (CKD) are at risk of progression to end-stage renal disease and cardiovascular disease. Data from other populations and animal experiments suggest that neprilysin inhibition (which augments the natriuretic peptide system) may reduce these risks, but clinical trials among patients with CKD are required to test this hypothesis. METHODS: UK Heart and Renal Protection III (HARP-III) is a multicentre, double-blind, randomized controlled trial comparing sacubitril/valsartan 97/103 mg two times daily (an angiotensin receptor-neprilysin inhibitor) with irbesartan 300 mg one time daily among 414 patients with CKD. Patients ≥18 years of age with an estimated glomerular filtration rate (eGFR) of ≥45 but &lt;60 mL/min/1.73 m2 and urine albumin:creatinine ratio (uACR) &gt;20 mg/mmol or eGFR ≥20 but &lt;45 mL/min/1.73 m2 (regardless of uACR) were invited to be screened. Following a 4- to 7-week pre-randomization single-blind placebo run-in phase (during which any current renin-angiotensin system inhibitors were stopped), willing and eligible participants were randomly assigned either sacubitril/valsartan or irbesartan and followed-up for 12 months. The primary aim was to compare the effects of sacubitril/valsartan and irbesartan on measured GFR after 12 months of therapy. Important secondary outcomes include effects on albuminuria, change in eGFR over time and the safety and tolerability of sacubitril/valsartan in CKD. RESULTS: Between November 2014 and January 2016, 620 patients attended a screening visit and 566 (91%) entered the pre-randomization run-in phase. Of these, 414 (73%) participants were randomized (mean age 63 years; 72% male). The mean eGFR was 34.0 mL/min/1.73 m2 and the median uACR was 58.5 mg/mmol. CONCLUSIONS: UK HARP-III will provide important information on the short-term effects of sacubitril/valsartan on renal function, tolerability and safety among patients with CKD

Water deficit alters differentially metabolic pathways affecting important flavor and quality traits in grape berries of Cabernet Sauvignon and Chardonnay

<p>Abstract</p> <p>Background</p> <p>Water deficit has significant effects on grape berry composition resulting in improved wine quality by the enhancement of color, flavors, or aromas. While some pathways or enzymes affected by water deficit have been identified, little is known about the global effects of water deficit on grape berry metabolism.</p> <p>Results</p> <p>The effects of long-term, seasonal water deficit on berries of Cabernet Sauvignon, a red-wine grape, and Chardonnay, a white-wine grape were analyzed by integrated transcript and metabolite profiling. Over the course of berry development, the steady-state transcript abundance of approximately 6,000 Unigenes differed significantly between the cultivars and the irrigation treatments. Water deficit most affected the phenylpropanoid, ABA, isoprenoid, carotenoid, amino acid and fatty acid metabolic pathways. Targeted metabolites were profiled to confirm putative changes in specific metabolic pathways. Water deficit activated the expression of numerous transcripts associated with glutamate and proline biosynthesis and some committed steps of the phenylpropanoid pathway that increased anthocyanin concentrations in Cabernet Sauvignon. In Chardonnay, water deficit activated parts of the phenylpropanoid, energy, carotenoid and isoprenoid metabolic pathways that contribute to increased concentrations of antheraxanthin, flavonols and aroma volatiles. Water deficit affected the ABA metabolic pathway in both cultivars. Berry ABA concentrations were highly correlated with 9-cis-epoxycarotenoid dioxygenase (<it>NCED1</it>) transcript abundance, whereas the mRNA expression of other <it>NCED </it>genes and ABA catabolic and glycosylation processes were largely unaffected. Water deficit nearly doubled ABA concentrations within berries of Cabernet Sauvignon, whereas it decreased ABA in Chardonnay at véraison and shortly thereafter.</p> <p>Conclusion</p> <p>The metabolic responses of grapes to water deficit varied with the cultivar and fruit pigmentation. Chardonnay berries, which lack any significant anthocyanin content, exhibited increased photoprotection mechanisms under water deficit conditions. Water deficit increased ABA, proline, sugar and anthocyanin concentrations in Cabernet Sauvignon, but not Chardonnay berries, consistent with the hypothesis that ABA enhanced accumulation of these compounds. Water deficit increased the transcript abundance of lipoxygenase and hydroperoxide lyase in fatty metabolism, a pathway known to affect berry and wine aromas. These changes in metabolism have important impacts on berry flavor and quality characteristics. Several of these metabolites are known to contribute to increased human-health benefits.</p

Long-term follow-up of brain-damaged children requiring feeding gastrostomy: Should an antireflux procedure always be performed?

Due to the frequent presence of latent gastroesophageal reflux (GER) in mentally impaired children, it is now standard to perform upper gastrointestinal contrast (UGI) and pH probe studies in all children referred for feeding gastrostomy, even if they are without clinical evidence for GER. For patients with documented GER, an antireflux operation performed in conjunction with gastrostomy is usually considered mandatory. Some authors have suggested that a "protective" antireflux operation be performed at the time of gastrostomy placement in all brain-damaged children, citing a high incidence of postoperative GER in this group of patients following gastrostomy, even with a negative preoperative evaluation for GER. To evaluate this theory, we prospectively studied, over the past 6 years, all mentally retarded children referred for feeding gastrostomy with UGI contrast and esophageal pH probe studies. In total, 148 children were studied; 105 had a positive evaluation for GER and underwent gastrostomy and antireflux surgery. Of the 43 children with a negative preoperative evaluation for GER, 37 are doing well following gastrostomy alone without clinical reflux at an average follow-up of 21 months. Six of the 43 (14%) developed symptomatic GER occurring at an average of 10 months following gastrostomy placement. Five of these children have been successfully treated with an antireflux operation and the sixth patient has been successfully man-aged nonoperatively. Given the significant morbidity and mortality that has been documented with antireflux operations in mentally retarded children, we conclude that a protective antireflux operation at the time of gastrostomy placement is not indicated because the data suggest that 86% of children with a negative preoperative evaluation for GER would undergo the operation needlessly.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29436/1/0000517.pd

Hirschsprung's disease in adolescents and adults

Hirschsprung's disease in the adolescent and adult is a rare and often misdiagnosed cause of lifelong refractory constipation. Two adolescent and three adult patients with Hirschsprung's disease treated between 1973 and 1987 at the University of Michigan Medical Center are reported. Each patient presented with chronic constipation requiring enemas, cathartics, and multiple hospital admissions for management. Diagnosis in each case was made with barium enema and full-thickness rectal biopsy. Four patients underwent endorectal pull-through procedures, all with good long-term results. The fifth patient, initially treated with a Duhamel retrorectal pull-through procedure, required reoperation for constipation secondary to a retained rectal septum. Review of 199 cases of adult Hirschsprung's disease enables comparison of the various operative procedures for this disorder with respect to postoperative complications and functional outcomes. Anorectal myectomy with low anterior resection, the Duhamel-Martin procedure, and the Soave endorectal pull-through procedure are the most acceptable methods for surgical management .Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41392/1/10350_2005_Article_BF02052222.pd

LC-MS based stability-indicating method for studying the degradation of lonidamine under physical and chemical stress conditions

Background and purpose: Lonidamine is a hexokinase II inhibitor, works as an anticancer molecule, and is extensively explored in clinical trials. Limited information prevails about the stability-indicating methods which could determine the forced degradation of lonidamine under stressed conditions. Hence, we report the use of a rapid, sensitive, reproducible, and highly accurate liquid chromatography and mass spectrometry method to analyze lonidamine degradation. Experimental approach: The Xbridge BEH shield reverse phase C18 column (2.5 μm, 4.6 × 75 mm) using isocratic 50:50 water: acetonitrile with 0.1% formic acid can detect lonidamine with help of mass spectrometer in tandem with an ultraviolet (UV) detector at 260 nm wavelength. Findings/ Results: A linear curve with r2\u3e 0.99 was obtained for tandem liquid chromatography-mass spectrometry (LC-MS)-UV based detections. This study demonstrated (in the present set up of isocratic elution) that LC-MS based detection has a relatively high sensitivity (S/N (10 ng/mL): 220 and S/N (20 ng/mL): 945) and accuracy at lower detection and quantitation levels, respectively. In addition to developing the LC-MS method, we also report that the current method is stability-indicating and shows that lonidamine gets degraded over time under all three stress conditions; acidic, basic, and oxidative. Conclusion and implications: LC-MS based quantitation of lonidamine proved to be a better method compared to high-performance liquid chromatography (HPLC)-UV detections for mapping lonidamine degradation. This is the first report on the stability-indicating method for studying the forced degradation of lonidamine using LC-MS method

Transcriptomic and metabolite analyses of Cabernet Sauvignon grape berry development

BACKGROUND: Grape berry development is a dynamic process that involves a complex series of molecular genetic and biochemical changes divided into three major phases. During initial berry growth (Phase I), berry size increases along a sigmoidal growth curve due to cell division and subsequent cell expansion, and organic acids (mainly malate and tartrate), tannins, and hydroxycinnamates accumulate to peak levels. The second major phase (Phase II) is defined as a lag phase in which cell expansion ceases and sugars begin to accumulate. Véraison (the onset of ripening) marks the beginning of the third major phase (Phase III) in which berries undergo a second period of sigmoidal growth due to additional mesocarp cell expansion, accumulation of anthocyanin pigments for berry color, accumulation of volatile compounds for aroma, softening, peak accumulation of sugars (mainly glucose and fructose), and a decline in organic acid accumulation. In order to understand the transcriptional network responsible for controlling berry development, mRNA expression profiling was conducted on berries of V. vinifera Cabernet Sauvignon using the Affymetrix GeneChip® Vitis oligonucleotide microarray ver. 1.0 spanning seven stages of berry development from small pea size berries (E-L stages 31 to 33 as defined by the modified E-L system), through véraison (E-L stages 34 and 35), to mature berries (E-L stages 36 and 38). Selected metabolites were profiled in parallel with mRNA expression profiling to understand the effect of transcriptional regulatory processes on specific metabolite production that ultimately influence the organoleptic properties of wine. RESULTS: Over the course of berry development whole fruit tissues were found to express an average of 74.5% of probes represented on the Vitis microarray, which has 14,470 Unigenes. Approximately 60% of the expressed transcripts were differentially expressed between at least two out of the seven stages of berry development (28% of transcripts, 4,151 Unigenes, had pronounced (≥2 fold) differences in mRNA expression) illustrating the dynamic nature of the developmental process. The subset of 4,151 Unigenes was split into twenty well-correlated expression profiles. Expression profile patterns included those with declining or increasing mRNA expression over the course of berry development as well as transient peak or trough patterns across various developmental stages as defined by the modified E-L system. These detailed surveys revealed the expression patterns for genes that play key functional roles in phytohormone biosynthesis and response, calcium sequestration, transport and signaling, cell wall metabolism mediating expansion, ripening, and softening, flavonoid metabolism and transport, organic and amino acid metabolism, hexose sugar and triose phosphate metabolism and transport, starch metabolism, photosynthesis, circadian cycles and pathogen resistance. In particular, mRNA expression patterns of transcription factors, abscisic acid (ABA) biosynthesis, and calcium signaling genes identified candidate factors likely to participate in the progression of key developmental events such as véraison and potential candidate genes associated with such processes as auxin partitioning within berry cells, aroma compound production, and pathway regulation and sequestration of flavonoid compounds. Finally, analysis of sugar metabolism gene expression patterns indicated the existence of an alternative pathway for glucose and triose phosphate production that is invoked from véraison to mature berries. CONCLUSION: These results reveal the first high-resolution picture of the transcriptome dynamics that occur during seven stages of grape berry development. This work also establishes an extensive catalog of gene expression patterns for future investigations aimed at the dissection of the transcriptional regulatory hierarchies that govern berry development in a widely grown cultivar of wine grape. More importantly, this analysis identified a set of previously unknown genes potentially involved in critical steps associated with fruit development that can now be subjected to functional testing.National Science Foundation Plant Genome Project (DBI-0217653); Bioinformatics program (DBI-0136561); National Institute of Health Biomedical Research Infrastructure Network (NIH-NCRR P20 RR16464; National Institute of Health IDeA Network of Biomedical Research Excellence (INBRE, RR-03-008); Nevada Agricultural Experimental Statio

Ligand-induced conformational changes in a SMALP-encapsulated GPCR.

The adenosine 2A receptor (A2AR), a G-protein-coupled receptor (GPCR), was solubilised and purified encapsulated in styrene maleic acid lipid particles (SMALPs). The purified A2AR-SMALP was associated with phospholipids characteristic of the plasma membrane of Pichia pastoris, the host used for its expression, confirming that the A2AR-SMALP encapsulated native lipids. The fluorescence spectrum of the A2AR-SMALP showed a characteristic broad emission peak at 330 nm, produced by endogenous Trp residues. The inverse agonist ZM241385 caused 30% increase in fluorescence emission, unusually accompanied by a red-shift in the emission wavelength. The emission spectrum also showed sub-peaks at 321 nm, 335 nm and 350 nm, indicating that individual Trp inhabited different environments following ZM241385 addition. There was no effect of the agonist NECA on the A2AR-SMALP fluorescence spectrum. Substitution of two Trp residues by Tyr suggested that ZM241385 affected the environment and mobility of Trp2466.48 in TM6 and Trp2687.33 at the extracellular face of TM7, causing transition to a more hydrophobic environment. The fluorescent moiety IAEDANS was site-specifically introduced at the intracellular end of TM6 (residue 2316.33) to report on the dynamic cytoplasmic face of the A2AR. The inverse agonist ZM241385 caused a concentration-dependent increase in fluorescence emission as the IAEDANS moved to a more hydrophobic environment, consistent with closing the G-protein binding crevice. NECA generated only 30% of the effect of ZM241385. This study provides insight into the SMALP environment; encapsulation supported constitutive activity of the A2AR and ZM241385-induced conformational transitions but the agonist NECA generated only small effects