A conserved π-helix plays a key role in thermoadaptation of catalysis in the glycoside hydrolase family 4

Here, we characterize the role of a π-helix in the molecular mechanisms underlying thermoadaptation in the glycoside hydrolase family 4 (GH4). The interspersed π-helix present in a subgroup is evolutionarily related to a conserved α-helix in other orthologs by a single residue insertion/deletion event. The insertional residue, Phe407, in a hyperthermophilic α-glucuronidase, makes specific interactions across the inter-subunit interface. In order to establish the sequence-structure-stability implications of the π-helix, the wild-type and the deletion variant (Δ407) were characterized. The variant showed a significant lowering of melting temperature and optimum temperature for the highest activity. Crystal structures of the proteins show a transformation of the π-helix to a continuous α-helix in the variant, identical to that in orthologs lacking this insertion. Thermodynamic parameters were determined from stability curves representing the temperature dependence of unfolding free energy. Though the proteins display maximum stabilities at similar temperatures, a higher melting temperature in the wild-type is achieved by a combination of higher enthalpy and lower heat capacity of unfolding. Comparisons of the structural changes, and the activity and thermodynamic profiles allow us to infer that specific non-covalent interactions, and the existence of residual structure in the unfolded state, are crucial determinants of its thermostability. These features permit the enzyme to balance the preservation of structure at a higher temperature with the thermodynamic stability required for optimum catalysis.     

Study of p16 promoter methylation in Egyptian colorectal cancer patients

Many tumor-suppressor genes contain CpG islands in their promoter regions which raised the necessity of investigating the role of methylation in silencing these genes. We examined p16 methylation as a potential biomarker in the peripheral blood of colorectal cancer (CRC) patients. Using methylation-specific polymerase chain reaction method, the methylation status of p16 was investigated in the tumor tissue and blood of 65 CRC patients and blood samples from 70 healthy control individuals. Also, the relationship between p16 methylation level and the clinical-pathological findings in CRC was evaluated. The frequency of blood p16 methylation in CRC cases was significantly higher than in control (P = 0.0001). The sensitivity and specificity of p16 methylation in diagnosing CRC was 55.38% and 98.5%, respectively, with 77.7% diagnostic accuracy. There was significant association between p16 methylation and age, sex, Dukes staging, lymph node involvement, and carcinoembryonic antigen levels. Our study revealed that p16 promoter methylation could be considered as both potential diagnostic and prognostic biomarker of CRC.     

Exploring the Support Needs of Family Caregivers of Patients with Brain Cancer Using the CSNAT: A Comparative Study with Other Cancer Groups

A substantial burden is placed on family caregivers of patients diagnosed with brain cancers. Despite this, the support needs of the caregivers are often under-recognised and not addressed adequately in current routine and patient centred clinical care. The Care Support Needs Assessment Tool (CSNAT) is a validated instrument designed to systematically identify and address caregiver needs. It has been trialled in an Australian palliative care community setting using a stepped wedge cluster design involving 322 family carers of terminally ill patients. The current article reports on a subset from this trial, 29 caregivers of patients with primary brain cancer, and compares their profile and outcomes to those of other cancer groups. Caregiver strain was assessed using the Family Appraisal of Caregiving Questionnaire, caregiver physical and mental wellbeing using SF12 and caregiver workload using a questionnaire on support with activities of daily living (ADL). In comparison to caregivers of patients with all other cancers, the primary brain cancer group had significantly higher levels of caregiver strain, lower levels of mental wellbeing and a higher level of ADL workload. Their physical wellness also deteriorated significantly over time. An action plan approach led to practical solutions for addressing highlighted concerns. Four themes evolved from the family caregivers’ feedback interviews: The extremely challenging caregiver experience with brain cancer; the systematic and practical approach of the CSNAT during rapid changes; connection with health professionals, feeling acknowledged and empowered; and timely advice and assurance of support during the caregiving journey. This preliminary study has demonstrated that the CSNAT provides a practical and useful tool for assessing the support needs of family caregivers of patients with brain cancer and has provided the basis for a larger scale, longitudinal study that allows a more detailed characterisation of the evolving caregiver needs at different stages of the disease.     

Morphological characterization of testicular cells,spermatogenesis and formation of spermatophores in a fish ectoparasite Argulus bengalensis Ramakrishna, 1951 (Crustacea: Branchiura)

The present study has been carried out to describe the cell morphology of the developing male gametes in a fish ectoparasite, Argulus bengalensis Ramakrishna, 1951. With respect to cell volume and nucleoplasmic index, spermatogonia are the smallest and primary spermatocytes are the largest in this lineage. The spermatogonia and the differentiating spermatogenic cells are in separate niches and confined to different enclaves within each testicular lobe. Spermiogenesis occurs within the inner enclave of each testicular lobe. During this process the nucleus becomes streamlined; an acrosome is formed, axoneme is originated, and residual cytoplasm is discarded through the flagellum. The sperm cell morphology displays a general pattern comprising head, mid-piece, and a full length flagellum. In the axoneme 9 + 2 arrangement of the microtubule is conserved. In addition to the axoneme, some more singlet microtubules are found surrounding a fiber sheath and around one of the mitochondria adjacent to the axoneme. This arrangement indicates a close phylogenetic relationship with pentastomida. In the present study, structure and formation of spermatophore are described in this branchiuran parasite.     

Enhanced prostaglandin F2α formation in human pregnancy and the effect of increased oily fish intake: results from the Salmon in Pregnancy Study

Oily fish intake during pregnancy may reduce the risk of allergic diseases in infancy possibly by shifts in the fatty acid balance and subsequent altered prostaglandin (PG) formation. This intervention is the first study to evaluate if increased oily fish intake affects in vivo PGF(2α) formation during pregnancy. British pregnant women were randomised to two portions of farmed salmon weekly (n=47), or maintenance of their normal diet low in fish (n=41), from pregnancy week 20 until parturition. The concentrations of eicosapentaenoic and docosahexaenoic acids in plasma phosphatidylcholine (PC) were higher and the concentration of arachidonic acid in plasma PC was lower in the salmon group than the control group at weeks 34 and 38 of pregnancy. PGF(2α) formation was evaluated by urinary measurement of 15-keto-dihydro-PGF(2α), a major PGF(2α) metabolite, at 20, 34 and 38 weeks. In both the salmon and control groups urinary 15-keto-dihydro-PGF(2α) concentrations increased significantly during pregnancy, which may be of physiological importance. Oily fish intervention altered fatty acid concentrations but did not affect urinary 15-keto-dihydro-PGF(2α) concentrations in pregnant women.     

A novel injectable in situ forming poly-DL-lactide and DL-lactide/glycolide implant containing lipospheres for controlled drug delivery

One of the greatest challenges in in situ forming implant (ISFI) systems by polymer precipitation is the large burst release during the first 1-24 hours after implant injection. The aim of this study was to decrease the burst-release effect of a water-soluble model drug, donepezil HCl, with a molecular weight of 415.96?Da, from in situ forming implants using a novel in situ implant containing lipospheres (ISILs). In situ implant suspensions were prepared by dispersing cetyl alcohol and glyceryl stearate lipospheres in a solution of poly-DL-lactide (PDL) or DL-lactide/glycolide copolymer (PDLG). Also, in situ implant solutions were prepared using different concentrations of PDL or PDLG solutions in N-methyl-2-pyrrolidone (NMP). Triacetin and Pluronic L121 were used to modify the release pattern of donepezil from the in situ implant solutions. In vitro release, rheological measurement, and injectability measurement were used to evaluate the prepared in situ implant formulae. It was found that ISIL decreased the burst effect as well as the rate and extent of drug release, compared to lipospheres, PDL, and PDLG in situ implant. The amount of drug released in the first day was 37.75, 34.99, 48.57, 76.3, and 84.82% for ISIL in 20% PDL (IL-1), ISIL in 20% PDLG (IL-2), lipospheres (L), 20% PDL ISFI (I5), and 20% PDLG ISFI (I8), respectively. The prepared systems showed Newtonian flow behavior. ISIL (IL-1 and IL-2) had a flow rate of 1.94 and 1.40?mL/min, respectively. This study shows the potential of using in situ implants containing lipospheres in controlling the burst effect of ISFI.     

Associations between oxidative parameters in pregnancy and birth anthropometry in a cohort of women and children in rural Bangladesh: the MINIMat-cohort

Oxidative stress is suggested as a potential mechanism in impaired foetal growth, smaller birth size and thus subsequently adult chronic diseases. We have investigated associations between oxidative stress in pregnancy and birth anthropometry (weight, height, head and chest circumferences). In the MINIMat-trial (Maternal and Infant Nutrition Interventions, Matlab) in rural Bangladesh, free 8-iso-prostaglandin F(2α) (lipid peroxidation) was analysed in pregnancy week 14 and 30 and 8-Hydroxy-2 -Deoxyguanosine (DNA oxidation) in week 19. We found that higher levels of lipid peroxidation in early pregnancy were associated with larger infant size (birth length and chest circumference). In late pregnancy, no clear pattern of associations was found. Increasing level of DNA oxidation was associated with lower birth length in girls but no other associations were found. In conclusion, a higher level of lipid peroxidation in early (but not late) pregnancy was associated with a favourable larger birth size suggesting that timing of lipid peroxidation is of importance.     

Bioactive eicosanoids: Role of prostaglandin F2α and F2-isoprostanes in inflammation and oxidative stress related pathology

Oxidative stress and inflammation are supposed to be the key players of several acute and chronic diseases, and also for progressive aging process. Eicosanoids, especially prostaglandin F_2α (PGF_2α) and F₂-isoprostanes are endogenous compounds that are involved both in physiology and the above mentioned pathologies. These compounds are biosynthesized mainly from esterified arachidonic acid through both enzymatic and non-enzymatic free radical-catalysed reactions in vivo, respectively. They have shown to possess potent biological activities in addition to their application as biomarkers of oxidative stress and inflammation. Recent advancement of methodologies has made it possible to quantify these compounds more reliably and apply them in various in vivo studies successfully. Today, experimental and clinical studies have revealed that both PGF_2α and F₂-isoprostanes are involved in severe acute or chronic inflammatory conditions such as rheumatic diseases, asthma, risk factors of atherosclerosis, diabetes, ischemia-reperfusion, septic shock and many others. These evidences promote that assessment of bioactive PGF_2α and F₂-isoprostanes simultaneously in body fluids offers unique non-invasive analytical opportunity to study the function of these eicosanoids in physiology, oxidative stress-related and inflammatory diseases, and also in the determination of potency of various radical scavengers, anti-inflammatory compounds, drugs, antioxidants and diet.     

Fibrinolysis and anticoagulant potential of a metallo protease produced by <Emphasis Type="Italic">Bacillus subtilis</Emphasis> K42

In this study, a potent fibrinolytic enzyme-producing bacterium was isolated from soybean flour and identified as Bacillus subtilis K42 and assayed in vitro for its thrombolytic potential. The molecular weight of the purified enzyme was 20.5 kDa and purification increased its specific activity 390-fold with a recovery of 14%. Maximal activity was attained at a temperature of 40°C (stable up to 65°C) and pH of 9.4 (range: 6.5–10.5). The enzyme retained up to 80% of its original activity after pre-incubation for a month at 4°C with organic solvents such as diethyl ether (DE), toluene (TO), acetonitrile (AN), butanol (BU), ethyl acetate (EA), ethanol (ET), acetone (AC), methanol (ME), isopropanol (IP), diisopropyl fluorophosphate (DFP), tosyl-lysyl-chloromethylketose (TLCK), tosyl-phenylalanyl chloromethylketose (TPCK), phenylmethylsulfonylfluoride (PMSF) and soybean trypsin inhibitor (SBTI). Aprotinin had little effect on this activity. The presence of ethylene diaminetetraacetic acid (EDTA), a metal-chelating agent and two metallo protease inhibitors, 2,2′-bipyridine and o-phenanthroline, repressed the enzymatic activity significantly. This, however, could be restored by adding Co²⁺ to the medium. The clotting time of human blood serum in the presence of this enzyme reached a relative PTT of 241.7% with a 3.4-fold increase, suggesting that this enzyme could be an effective antithrombotic agent.     

The structural plasticity of the human copper chaperone for SOD1: insights from combined size-exclusion chromatographic and solution X-ray scattering studies

The incorporation of copper into biological macromolecules such as SOD1 (Cu,Zn superoxide dismutase) is essential for the viability of most organisms. However, copper is toxic and therefore the intracellular free copper concentration is kept to an absolute minimum. Several proteins, termed metallochaperones, are charged with the responsibility of delivering copper from membrane transporters to its intracellular destination. The CCS (copper chaperone for SOD1) is the major pathway for SOD1 copper loading. We have determined the first solution structure of hCCS (human CCS) by SAXS (small-angle X-ray scattering) in conjunction with SEC (size-exclusion chromatography). The findings of the present study highlight the importance of this combined on-line chromatographic technology with SAXS, which has allowed us to unambiguously separate the hCCS dimer from other oligomeric and non-physiological aggregated states that would otherwise adversely effect measurements performed on bulk solutions. The present study exposes the dynamic molecular conformation of this multi-domain chaperone in solution. The metal-binding domains known to be responsible for the conveyance of copper to SOD1 can be found in positions that would expedite this movement. Domains I and III of a single hCCS monomer are able to interact and can also move into positions that would facilitate initial copper binding and ultimately transfer to SOD1. Conversely, the interpretation of our solution studies is not compatible with an interaction between these domains and their counterparts in an hCCS dimer. Overall, the results of the present study reveal the plasticity of this multi-domain chaperone in solution and are consistent with an indispensable flexibility necessary for executing its dual functions of metal binding and transfer.