Structure-based substitutions for increased solubility of a designed protein

Manipulation of protein solubility is important for many aspects of protein design and engineering. Previously, we designed a series of consensus ankyrin repeat proteins containing one, two, three and four identical repeats (1ANK, 2ANK, 3ANK and 4ANK). These proteins, particularly 4ANK, are intended for use as a universal scaffold on which specific binding sites can be constructed. Despite being well folded and extremely stable, 4ANK is soluble only under acidic conditions. Designing interactions with naturally occurring proteins requires the designed protein to be soluble at physiological pH. Substitution of six leucines with arginine on exposed hydrophobic patches on the surface of 4ANK resulted in increased solubility over a large pH range. Study of the pH dependence of stability demonstrated that 4ANK is one of the most stable ankyrin repeat proteins known. In addition, analogous leucine to arginine substitutions on the surface of 2ANK allowed the partially folded protein to assume a fully folded conformation. Our studies indicate that replacement of surface-exposed hydrophobic residues with positively charged residues can significantly improve protein solubility at physiological pH.     

Chemoradiotherapy or chemotherapy as adjuvant treatment for resected gastric cancer: should we use selection criteria?

BackgroundThe management of gastric adenocarcinoma is essentially based on surgery followed by adjuvant treatment. Adjuvant chemotherapy (CT) as well as chemoradiotherapy (CTRT) have proven their effectiveness in survival outcomes compared to surgery alone. However, there is little data comparing the two adjuvant approaches. This study aimed to compare the prognosis and survival outcomes of patients with gastric adenocarcinoma operated and treated by adjuvant radio-chemotherapy or chemotherapyMaterials and methodsWe retrospectively evaluated 80 patients with locally advanced gastric cancer (LGC) who received adjuvant treatment. We compared survival outcomes and patterns of recurrence of 53 patients treated by CTRT and those of 27 patients treated by CT.ResultsAfter a median follow-up of 38.48 months, CTRT resulted in a significant improvement of the 5-year PFS (60.9% vs. 36%, p = 0.03) and the 5-year OS (55.9% vs. 33%, p = 0.015) compared to adjuvant CT. The 5-year OS was significantly increased by adjuvant CTRT (p = 0.046) in patients with lymph node metastasis, and particularly those with advanced pN stage (p = 0.0078) and high lymph node ratio (LNR) exceeding 25% (p = 0.012). Also, there was a significant improvement of the PFS of patients classified pN2–N3 (p = 0.022) with a high LNR (p = 0.018). CTRT was also associated with improved OS and PFS in patients with lymphovascular and perineural invasion (LVI and PNI) compared to chemotherapy.ConclusionThere is a particular survival benefit of adding radiotherapy to chemotherapy in patients with selected criteria such as lymph node involvement, high LNR LVI, and PNI.     

Dicer Is Required for Normal Cerebellar Development and to Restrain Medulloblastoma Formation

Dicer, a ribonuclease III enzyme, is required for the maturation of microRNAs. To assess its role in cerebellar and medulloblastoma development, we genetically deleted Dicer in Nestin-positive neural progenitors and in mice lacking one copy for the Sonic Hedgehog receptor, Patched 1. We found that conditional loss of Dicer in mouse neural progenitors induced massive Trp53-independent apoptosis in all proliferative zones of the brain and decreased proliferation of cerebellar granule progenitors at embryonic day 15.5 leading to abnormal cerebellar development and perinatal lethality. Loss of one copy of Dicer significantly accelerated the formation of mouse medulloblastoma of the Sonic Hedgehog subgroup in Patched1-heterozygous mice. We conclude that Dicer is required for proper cerebellar development, and to restrain medulloblastoma formation.     

Trend of Cardio-Metabolic Risk Factors in Polycystic Ovary Syndrome: A Population-Based Prospective Cohort Study

Objective

To see the changes of cardio-metabolic risk factors overtime in polycystic ovary syndrome vs. control women.

Methods

This study was conducted on 637 participants (85 PCOS and 552 control reproductive aged, 18–45 years) of Tehran Lipid and Glucose Study (TLGS), an ongoing population-based cohort study with 12 years of follow-up. The cardiovascular risk factors of these groups were assessed in three-year intervals using standard questionnaires, history taking, anthropometric measures, and metabolic/endocrine evaluation. Generalized estimating equation was used to analyze the data.

Results

Overall mean of insulin (3.55, CI: 0.66–6.45), HOMA-IR (0.63, CI: 0.08–1.18), and HOMA-β (45.90, CI: 0.86–90.93) were significantly higher in PCOS than in healthy women after adjustment for age, BMI, and baseline levels. However, the negative interaction (follow-up years × PCOS status) of PCOS and normal women converged overtime. Comparing third follow-up with first, insulin and HOMA-IR decreased 10.6% and 5%, respectively in PCOS women; and increased 6.7% and 14.6%, respectively in controls (P<0.05). The results did not show any significant result for other cardio-metabolic variables including WC, lipid profile, FPG, 2-h PG, SBP, and DBP.

Conclusion

While the insulin level and insulin resistance rate were higher in reproductive aged PCOS than in healthy women, the difference of these risk factors decreased overtime. Thus, the metabolic consequences of PCOS women in later life may be lower than those initially anticipated.     

Amphibious Shelter-Builder Oniscidea Species from the New World with Description of a New Subfamily,a New Genus and a New Species from Brazilian Cave (Isopoda,Synocheta, Styloniscidae)

The new subfamily Iuiuniscinae, Styloniscidae, is erected for the new genus Iuiuniscus and the new species I. iuiuensis, which is described from cave of the State of Bahia, Northeastern Brazil. A special ecological character is shown here for the first time for a New World Oniscidea: the construction of mud shelters. An introduction addressing the systematics of Synocheta with emphasis on Styloniscidae Vandel, 1952 is provided, as well as general comments about the dependence of water in some Oniscidea and ecological traits of amphibious Synocheta. The problems referring to nomenclature, taxonomy and the interrelationships in Styloniscidae are discussed.     

Camptosemin, a tetrameric lectin of Camptosema ellipticum: structural and functional analysis

Lectins have been classified into a structurally diverse group of proteins that bind carbohydrates and glycoconjugates with high specificity. They are extremely useful molecules in the characterization of saccharides, as drug delivery mediators, and even as cellular surface makers. In this study, we present camptosemin, a new lectin from Camptosema ellipticum. It was characterized as an N-acetyl-d-galactosamine-binding homo-tetrameric lectin, with a molecular weight around 26 kDa/monomers. The monomers were stable over a wide range of pH values and exhibited pH-dependent oligomerization. Camptosemin promoted adhesion of breast cancer cells and hemagglutination, and both activities were inhibited by its binding of sugar. The stability and unfolding/folding behavior of this lectin was characterized using fluorescence and far-UV circular dichroism spectroscopies. The results indicate that chemical unfolding of camptosemin proceeds as a two-state monomer-tetramer process. In addition, small-angle X-ray scattering shows that camptosemin behaves as a soluble and stable homo-tetramer molecule in solution.     

Partitioning of acidic, basic and neutral amino acids into imidazolium-based ionic liquids

In this paper, partitioning behaviors of typical neutral (Alanine), acidic (Glutamic acid) and basic (Lysine) amino acids into imidazolium-based ionic liquids [C₄mim][PF₆], [C₆mim][PF₆], [C₈mim][PF₆], [C₆mim][BF₄] and [C₈mim][BF₄] as extracting solvents were examined. [C₆mim][BF₄] showed the best efficiency for partitioning of amino acids. The partition coefficients of amino acids in ionic liquids were found to depend strongly on pH of the aqueous solution, amino acid and ionic liquid chemical structures. Different chemical forms of amino acids in aqueous solutions were pH dependent, so the pH value of the aqueous phase was a determining factor for extraction of amino acids into ionic liquid phase. Both water content of ionic liquids and charge densities of their anionic and cationic parts were important factors for partitioning of cationic and anionic forms of amino acids into ionic liquid phase. Extracted amino acids were back extracted into phosphate buffer solutions adjusted on appropriate pH values. The results showed that ionic liquids could be used as suitable modifiers on the stationary phase of an HPLC column for efficient separation of acidic, basic, and neutral amino acids.     

Kinetic study of Lactococcus lactis strains (SLT6 and SLT10) growth on papain-hydrolysed whey

The effect of controlled whey hydrolysis by papain on growth of two lactic acid bacteria isolated from artisanal Leben: Lactococcus lactis var. diacetylactis (SLT6 and SLT10) was investigated. The higher biomass and maximum specific growth rate (μ _max) were obtained after 30 min of hydrolysis. HPLC analysis of peptides showed that whey hydrolysis reduced the amount of peptides of MW > 400 Da and increased those peptides of MW < 400 Da. The two studied strains exhibited different peptide requirements. The pH-controlled batch cultures in 30 min hydrolysed whey followed the Monod kinetic for growth and for lactate production. The values of the key kinetic constants were: maximum specific growth rate (μ _max), 1.08 and 0.56 h^?1; yield biomass on lactose (Y _x/s), 0.20 and 0.18 g g^?1 and saturation constant K _s, 4.2 and 2.8 g L^?1 for SLT6 and SLT10, respectively. When compared with batch experimental data, the model provided good predictions for growth, lactose utilisation and lactate production profiles.     

Antimutagenic and Antioxidant Potentials of Teucrium ramosissimum Essential Oil

The mutagenic and antimutagenic effects of the essential oil extracted from the aerial parts of Teucrium ramosissimum were evaluated by the bacterial reverse mutation assay in Salmonella typhimurium TA98, TA100, and TA1535, with and without exogenous metabolic activation (S9 fraction). The T. ramosissimum essential oil showed no mutagenic effect. In contrast, our results established that it possessed antimutagenic effects against sodium azide (SA), aflatoxin B1 (AFB1), benzo[a]pyrene (B[a]P), and 4‐nitro‐o‐phenylenediamine (NOPD). The antioxidant capacity of the tested essential oil was evaluated using enzymatic, i.e., the xanthine/xanthine oxidase (X/XOD) assay, and nonenzymatic systems, i.e., the nitro‐blue tetrazolium (NBT)/riboflavin and the DPPH assays. A moderate free radical‐scavenging activity was observed towards DPPH^. and O$\rm{{_{2}^{{^\cdot} -}}}$ . In contrast, T. ramosissimum essential oil showed no effect for all the tested concentrations in the X/XOD assay.