Global transcriptional response of Saccharomyces cerevisiae to the deletion of SDH3

<p>Abstract</p> <p>Background</p> <p>Mitochondrial respiration is an important and widely conserved cellular function in eukaryotic cells. The succinate dehydrogenase complex (Sdhp) plays an important role in respiration as it connects the mitochondrial respiratory chain to the tricarboxylic acid (TCA) cycle where it catalyzes the oxidation of succinate to fumarate. Cellular response to the Sdhp dysfunction (i.e. impaired respiration) thus has important implications not only for biotechnological applications but also for understanding cellular physiology underlying metabolic diseases such as diabetes. We therefore explored the physiological and transcriptional response of <it>Saccharomyces cerevisiae </it>to the deletion of <it>SDH3</it>, that codes for an essential subunit of the Sdhp.</p> <p>Results</p> <p>Although the Sdhp has no direct role in transcriptional regulation and the flux through the corresponding reaction under the studied conditions is very low, deletion of <it>SDH3 </it>resulted in significant changes in the expression of several genes involved in various cellular processes ranging from metabolism to the cell-cycle. By using various bioinformatics tools we explored the organization of these transcriptional changes in the metabolic and other cellular functional interaction networks.</p> <p>Conclusion</p> <p>Our results show that the transcriptional regulatory response resulting from the impaired respiratory function is linked to several different parts of the metabolism, including fatty acid and sterol metabolism.</p

High cell density cultivation of Escherichia coli K4 in a microfiltration bioreactor: a step towards improvement of chondroitin precursor production

<p>Abstract</p> <p>Background</p> <p>The bacteria <it>Escherichia coli </it>K4 produces a capsular polysaccharide (K4 CPS) whose backbone is similar to the non sulphated chondroitin chain. The chondroitin sulphate is one of the major components of the extra-cellular matrix of the vertebrate connective tissues and a high value molecule, widely employed as active principle in the treatment of osteoarthritis. It is usually obtained by extraction from animal tissues, but the risk of virus contaminations, as well as the scarceness of raw material, makes this productive process unsafe and unable to satisfy the growing market demand. In previous studies a new biotechnological process to produce chondroitin from <it>Escherichia coli </it>K4 capsular polysaccharide was investigated and a 1.4 g·L^-1K4 CPS concentration was reached using fed-batch fermentation techniques. In this work, on the trail of these results, we exploited new fermentation strategies to further improve the capsular polysaccharide production.</p> <p>Results</p> <p>The inhibitory effect of acetate on the bacterial cells growth and K4 CPS production was studied in shake flask conditions, while a new approach, that combined the optimization of the feeding profiles, the improvement of aeration conditions and the use of a microfiltration bioreactor, was investigated in three different types of fermentation processes. High polysaccharide concentrations (4.73 ± 0.2 g·L^-1), with corresponding average yields (0.13 ± 0.006 g_{K4 CPS}·g_cdw^-1), were obtained; the increase of K4 CPS titre, compared to batch and fed-batch results, was of 16-fold and 3.3-fold respectively, while average yield was almost 3.5 and 1.4 fold higher.</p> <p>Conclusion</p> <p>The increase of capsular polysaccharide titre confirmed the validity of the proposed fermentation strategy and opened the way to the use of the microfiltration bioreactor for the biotechnological production of chondroitin.</p

Biophysical and biological characterization of a new line of hyaluronan-based dermal fillers: A scientific rationale to specific clinical indications

Chemico-physical and biological characterization of hyaluronan-based dermal fillers is of key importance to differentiate between numerous available products and to optimize their use. These studies on fillers are nowadays perceived as a reliable approach to predict their performance in vivo. The object of this paper is a recent line of hyaluronic acid (HA)-based dermal fillers, Aliaxin®, available in different formulations that claim a complete facial restoration. The aim of the study is to provide biophysical and biological data that may support the clinical indications and allow to predict performance possibly with respect to similar available products. Aliaxin® formulations were tested for their content in soluble HA, water uptake capacity, rheological behavior, stability to enzymatic degradation, and for in vitro capacity to stimulate extracellular matrix components production. The formulations were found to contain a low amount of soluble HA and were equivalent to each other regarding insoluble hydrogel concentration. The different crosslinking degree declared by the producer was consistent with the trend in water uptake capacity, rigidity, viscosity. No significant differences in stability to enzymatic hydrolysis were found. In vitro experiments, using a full thickness skin model, showed an increase in collagen production in the dermoepidermal junction. Results support the claims of different clinical indications, the classification of products regarding hydro-, lift-action and the specifically suggested needle gauge for the delivery. The biological outcomes also support products effectiveness in skin structure restoration. These data predicted a better performance regarding hydro-action, tissue integration, clinical management during delivery, and a high durability of the aesthetic effect when compared to data on marketed similar products

Hyaluronan and Derivatives: An In Vitro Multilevel Assessment of Their Potential in Viscosupplementation

In this research work, viscosupplements based on linear, derivatized, crosslinked and complexed HA forms were extensively examined, providing data on the hydrodynamic parameters for the water-soluble-HA-fraction, rheology, sensitivity to enzymatic hydrolysis and capacity to modulate specific biomarkers’ expression in human pathological chondrocytes and synoviocytes. Soluble HA ranged from 0 to 32 mg/mL and from 150 to 1330 kDa MW. The rheological behavior spanned from purely elastic to viscoelastic, suggesting the diversity of the categories that are suitable for restoring specific/different features of the healthy synovial fluid. The rheological parameters were reduced in a diverse manner upon dilution and hyaluronidases action, indicating different durations of the viscosupplementation effect. Bioactivity was found for all the samples, increasing the expression of different matrix markers (e.g., hyaluronan-synthase); however, the hybrid cooperative complexes performed better in most of the experiments. Hybrid cooperative complexes improved COLII mRNA expression (~12-fold increase vs. CTR), proved the most effective at preserving cell phenotype. In addition, in these models, the HA samples reduced inflammation. IL-6 was down-regulated vs. CTR by linear and chemically modified HA, and especially by hybrid complexes. The results represent the first comprehensive panel of data directly comparing the diverse HA forms for intra-articular injections and provide valuable information for tailoring products’ clinical use as well as for designing new, highly performing HA-formulations that can address specific needs

Potential of Biofermentative Unsulfated Chondroitin and Hyaluronic Acid in Dermal Repair

Chondroitin obtained through biotechnological processes (BC) shares similarities with both chondroitin sulfate (CS), due to the dimeric repetitive unit, and hyaluronic acid (HA), as it is unsulfated. In the framework of this experimental research, formulations containing BC with an average molecular size of about 35 KDa and high molecular weight HA (HHA) were characterized with respect to their rheological behavior, stability to enzymatic hydrolysis and they were evaluated in different skin damage models. The rheological characterization of the HHA/BC formulation revealed a G' of 92 ± 3 Pa and a G″ of 116 ± 5 Pa and supported an easy injectability even at a concentration of 40 mg/mL. HA/BC preserved the HHA fraction better than HHA alone. BTH was active on BC alone only at high concentration. Assays on scratched keratinocytes (HaCaT) monolayers showed that all the glycosaminoglycan formulations accelerated cell migration, with HA/BC fastening healing 2-fold compared to the control. In addition, in 2D HaCaT cultures, as well as in a 3D skin tissue model HHA/BC efficiently modulated mRNA and protein levels of different types of collagens and elastin remarking a functional tissue physiology. Finally, immortalized human fibroblasts were challenged with TNF-α to obtain an in vitro model of inflammation. Upon HHA/BC addition, secreted IL-6 level was lower and efficient ECM biosynthesis was re-established. Finally, co-cultures of HaCaT and melanocytes were established, showing the ability of HHA/BC to modulate melanin release, suggesting a possible effect of this specific formulation on the reduction of stretch marks. Overall, besides demonstrating the safety of BC, the present study highlights the potential beneficial effect of HHA/BC formulation in different damage dermal models

Production of succinic acid from Basfia succiniciproducens up to the pilot scale from Arundo donax hydrolysate.

Abstract In the present work the recently isolated strain Basfia succiniciproducens BPP7 was evaluated for the production of succinic acid up to the pilot fermentation scale in separate hydrolysis and fermentation experiments on Arundo donax, a non-food dedicated energy crop. An average concentration of about 17 g/L of succinic acid and a yield on consumed sugars of 0.75 mol/mol were obtained demonstrating strain potential for further process improvement. Small scale experiments indicated that the concentration of acetic acid in the medium is crucial to improve productivity; on the other hand, interestingly, short-term (24 h) adaptation to higher acetic acid concentrations, and strain recovery, were also observed

Antioxidant and Hypolipidemic Activity of Açai Fruit Makes It a Valuable Functional Food

Several plant extracts are acquiring increasing value because of their antioxidant activity and hypolipidemic properties. Among them, great interest has been recently paid to acai fruit as a functional food. The aim of this study was to test the ability of acai extract in reducing oxidative stress and modulating lipid metabolism in vitro using different cell models and different types of stress. In fact, lipid peroxidation as evaluated in a HepG2 model was reduced five-fold when using 0.25 mu g/mL of extract, and it was further reduced (20-fold) with the concentration increase up to 2.5 mu g/mL. With the nonalcoholic fatty liver disease (NAFLD)in vitro model, all concentrations tested showed at least a two-fold reduced fat deposit. In addition, primary adipocytes challenged with TNF-alpha under hypoxic conditions to mimic the persistent subcutaneous fat, treated with acai extract showed an approximately 40% reduction of fat deposit. Overall, our results show that acai is able to counteract oxidative states in all the cell models analysed and to prevent the accumulation of lipid droplets. No toxic effects and high stability overtime were highlighted at the concentrations tested. Therefore, acai can be considered a suitable support in the prevention of different alterations of lipid and oxidative metabolism responsible for fat deposition and metabolic pathological conditions

Epigenetic modulator UVI5008 inhibits MRSA by interfering with bacterial gyrase

The impact of multi-drug resistant bacterial strains on human health is reaching worrisome levels. Over 2 million people are infected by resistant bacteria, and more than 700,000 people die each year because of the continuous spread of resistant strains. The development of new antibiotics and the prudent use of existing ones to prolong their lifespan require a constant effort by drug industries and healthcare workers. The re-purposing of existing drugs for use as antimicrobial agents would streamline the development of new antibacterial strategies. As part of this effort, we screened a panel of drugs previously characterized to be epigenetic modulators/pro-apoptotic/differentiative drugs. We selected a few compounds that alter Gram-positive growth. Among these, UVI5008, a derivative of the natural compound psammaplin A (Psa_A), was identified. The interaction of Psa_A with the DNA gyrase enzyme has been shown, and here, we hypothesized and confirmed the gyrase-specific activity by biochemical assays. UVI5008 exhibited growth inhibition activity against Staphylococcus aureus via structural modification of the cell wall, which was observed by SEM electron microscopy. Based on our findings, we propose UVI5008 as an alternative antibacterial compound against methicillin-resistant (Met.R) S. aureus strainsMinisterio de Economía | Ref. SAF2016-77620-R-FEDERXunta de Galicia | Ref. ED431C 29017/61Xunta de Galicia | Ref. ED-431G/02-FEDERProgramma di Ricerca Scientifica di Rilevante Interesse Nazionale | Ref. PRIN-20152TE5PK_00

Beta-Defensin-2 and Beta-Defensin-3 Reduce Intestinal Damage Caused by Salmonella typhimurium

The intestinal microbiota is a major factor in human health and disease. This microbial community includes autochthonous (permanent inhabitants) and allochthonous (transient inhabitants) microorganisms that contribute to maintaining the integrity of the intestinal wall, modulating responses to pathogenic noxae and representing a key factor in the maturation of the immune system. If this healthy microbiota is disrupted by antibiotics, chemotherapy, or a change in diet, intestinal colonization by pathogenic bacteria or viruses may occur, leading to disease. To manage substantial microbial exposure, epithelial surfaces of the intestinal tract produce a diverse arsenal of antimicrobial peptides (AMPs), including, of considerable importance, the β-defensins, which directly kill or inhibit the growth of microorganisms. Based on the literature data, the purpose of this work was to create a line of intestinal epithelial cells able to stably express gene encoding human β-defensin-2 (hBD-2) and human β-defensin-3 (hBD-3), in order to test their role in S. typhimurium infections and their interaction with the bacteria of the gut microbiota