Thermodynamic characterization of substrate and inhibitor binding to Trypanosoma brucei 6-phosphogluconate dehydrogenase

6-Phosphogluconate dehydrogenase is a potential target for new drugs against African trypanosomiasis. Phosphorylated aldonic acids are strong inhibitors of 6-phosphogluconate dehydrogenase, and 4-phospho-d-erythronate (4PE) and 4-phospho-d-erythronohydroxamate are two of the strongest inhibitors of the Trypanosoma brucei enzyme. Binding of the substrate 6-phospho-d-gluconate (6PG), the inhibitors 5-phospho-d-ribonate (5PR) and 4PE, and the coenzymes NADP, NADPH and NADP analogue 3-amino-pyridine adenine dinucleotide phosphate to 6-phospho-d-gluconate dehydrogenase from T. brucei was studied using isothermal titration calorimetry. Binding of the substrate (K(d) = 5 microm) and its analogues (K(d) =1.3 microm and K(d) = 2.8 microm for 5PR and 4PE, respectively) is entropy driven, whereas binding of the coenzymes is enthalpy driven. Oxidized coenzyme and its analogue, but not reduced coenzyme, display a half-site reactivity in the ternary complex with the substrate or inhibitors. Binding of 6PG and 5PR poorly affects the dissociation constant of the coenzymes, whereas binding of 4PE decreases the dissociation constant of the coenzymes by two orders of magnitude. In a similar manner, the K(d) value of 4PE decreases by two orders of magnitude in the presence of the coenzymes. The results suggest that 5PR acts as a substrate analogue, whereas 4PE mimics the transition state of dehydrogenation. The stronger affinity of 4PE is interpreted on the basis of the mechanism of the enzyme, suggesting that the inhibitor forces the catalytic lysine 185 into the protonated state

Changes in protein expression in two cholangiocarcinoma cell lines undergoing formation of multicellular tumor spheroids In vitro

Epithelial-to-Mesenchymal Transition (EMT) is relevant in malignant growth and frequently correlates with worsening disease progression due to its implications in metastases and re- sistance to therapeutic interventions. Although EMT is known to occur in several types of solid tumors, the information concerning tumors arising from the epithelia of the bile tract is still limited. In order to approach the problem of EMT in cholangiocarcinoma, we decided to investigate the changes in protein expression occurring in two cell lines under conditions leading to growth as adherent monolayers or to formation of multicellular tumor spheroids (MCTS), which are considered culture models that better mimic the growth characteristics of in-vivo solid tumors. In our system, changes in phenotypes occur with only a decrease in transmembrane E-cadherin and vimentin expression, minor changes in the transglutami- nase protein/activity but with significant differences in the proteome profiles, with declining and increasing expression in 6 and in 16 proteins identified by mass spectrometry. The aris- ing protein patterns were analyzed based on canonical pathways and network analysis. These results suggest that significant metabolic rearrangements occur during the conver- sion of cholangiocarcinomas cells to the MCTS phenotype, which most likely affect the car- bohydrate metabolism, protein folding, cytoskeletal activity, and tissue sensitivity to oxygen

A Calcium- and GTP-Dependent Transglutaminase in Leishmania infantum

While human and animal leishmaniasis affect several millions of people worldwide, L. infantum is the species responsible for visceral leishmaniasis in Europe, Middle East, and America. Antileishmanial drugs present issues associated with drug toxicity and increasing parasite resistance. Therefore, the study of this parasite with a focus on new potential drug targets is extremely useful. Accordingly, we purified and characterized a transglutaminase (TGase) from L. infantum promastigotes. While Tgases are known to be involved in cell death and autophagy, it appears that these functions are very important for parasites' virulence. For the first time, we showed a Ca2+- and GTP-dependent TGase in Leishmania corresponding to a 54 kDa protein, which was purified by two chromatographic steps: DEAE-Sepharose and Heparin-Sepharose. Using polyclonal antibodies against a 50-amino-acid conserved region of the catalytic core of human TGase 2, we revealed two other bands of 66 and 75 kDa. The 54 kDa band appears to be different from the previously reported TGase, which was shown to be Ca2+- independent. Future research should address the identification of the purified enzyme sequence and, subsequently, its cloning to more comprehensively investigate its pathophysiological function and possible differences from mammal enzymes

Predictive Factors of Surgical Site Infection in Prosthetic Joint Surgery: A Prospective Study on 760 Arthroplasties

Purpose. The success of total joint arthroplasty (TJA) has led to consistent growth in the use of arthroplasty in progressivelyyounger patients. However, more than 10 percent of patients require revision surgery due to implant failure caused by asepticor septic inflammation. Among the latter, surgical site infection (SSI) represents one of the worst complications of TJA,potentially resulting in the removal of the prosthesis. The aim of our study was to identify potential risk factors for SSIs in apopulation of patients undergoing TJA. Methods. TJA were prospectively recruited at Casa di Cura Santa Maria Maddalenafrom February 2019 to April 2020. Age, sex, major comorbidities, American Society of Anesthesiologists (ASA) class, length ofsurgery, type of surgical suture, total hospital length of stay, and clinical laboratory data were collected. The study populationwas then divided into two groups: Group A, normal postoperative course, and Group B, patients who developed SSI at follow-up (17-25 days). Results. 25/760 (3.3%) patients developed SSIs at follow-up. Clinical and demographic parameters were notdifferent between the two groups. Total leucocyte and neutrophil values at discharge resulted to be significatively higher inGroup B compared to Group A (p = 0:025 and p = 0:016, respectively). Values of 7860/μL for total leucocyte and 5185/μL forneutrophil count at discharge significantly predicted the future development of SSI (AUC 0.623 and AUC 0.641, respectively; p< 0:05) independently from confounding factors (total leukocytes: O:R: = 3, 69 [95% C.I. 1,63-8,32]; neutrophils: O:R: = 3, 98[95% C.I. 1,76-8,97]). Deep SSIs has been diagnosed significantly before superficial SSIs (p = 0,008), with a median advance of9 days. Conclusion. Total leukocytes and neutrophils at discharge seem useful to identify a population at risk for thedevelopment of septic inflammation at the surgical site following TJA. Further studies with larger populations are needed to develop a predictive SSIs risk score that should include those variables

Vaginal Lactoferrin Modulates PGE 2

Inflammation plays an important role in pregnancy, and cytokine and matrix metalloproteases (MMPs) imbalance has been associated with premature rupture of membranes and increased risk of preterm delivery. Previous studies have demonstrated that lactoferrin (LF), an iron-binding protein with anti-inflammatory properties, is able to decrease amniotic fluid (AF) levels of IL-6. Therefore, we aimed to evaluate the effect of vaginal LF administration on amniotic fluid PGE2 level and MMP-TIMP system in women undergoing genetic amniocentesis. One hundred and eleven women were randomly divided into controls (n = 57) or treated with LF 4 hours before amniocentesis (n = 54). Amniotic fluid PGE2, active MMP-9 and MMP-2, and TIMP-1 and TIMP-2 concentrations were determined by commercially available assays and the values were normalized by AF creatinine concentration. PGE2, active MMP-9, and its inhibitor TIMP-1 were lower in LF-treated group than in controls (p < 0.01, p < 0.005, and p < 0.001, resp.). Conversely, active MMP-2 (p < 0.0001) and MMP-2/TIMP-2 molar ratio (p < 0.001) were increased, whilst TIMP-2 was unchanged. Our data suggest that LF administration is able to modulate the inflammatory response following amniocentesis, which may counteract cytokine and prostanoid imbalance that leads to abortion. This trial is registered with Clinical Trial number NCT02695563

Oxidative stress and menopause-related hot flashes may be independent events.

Abstract Objective At present, there is growing demand for alternative, or additional, treatments to hormone replacement therapy for menopause-related hot flashes (HF). Antioxidant supplements have been recently proposed as possible candidates for this purpose, regardless of the absence of clear evidence in support of a link between these vasomotor symptoms and oxidative stress (OxS). The aim of our study was to evaluate the association between HF and OxS serum markers in a large sample of middle-aged women. Materials and methods We conducted a cross-sectional study on 245 perimenopausal and early postmenopausal women (age 45–60 years). The variables examined were presence of self-reported HF and levels of 8-iso-prostaglandin F2α, 8-OH-deoxy-2′-guanosine, advanced oxidation protein products, total antioxidant power, uric acid, thiols, and paroxonase-1. Results Seventy-six women (31%) reported to suffer from HF (either medium or high intensity). None of the peripheral markers of OxS examined was found to be significantly associated with the presence of HF. Conclusion Taken together, our data suggest that systemic OxS might not be implicated with the onset of the climacteric vasomotor symptoms that most commonly affect women experiencing perimenopause and early postmenopause

Targeted lipidomics distinguishes patient subgroups in mild cognitive impairment (MCI) and late onset Alzheimer's disease (LOAD)

AbstractBackgroundDiverse research approaches support the concept that a clinical diagnosis of Late-Onset Alzheimer's Disease (LOAD) does not distinguish between subpopulations with differing neuropathologies, including dementia patients with amyloid deposition and dementia patients without amyloid deposition but with cortical thinning. Mild cognitive impairment (MCI) is generally considered the prodromal phase for LOAD, however, while a number of studies have attempted to define plasma biomarkers for the conversion of MCI to LOAD, these studies have not taken into account the heterogeneity of patient cohorts within a clinical phenotype.MethodsStudies of MCI and LOAD in several laboratories have demonstrated decrements in ethanolamine plasmalogen levels in plasma and brain and increased levels of diacylglycerols in plasma and brain. To further extend these studies and to address the issue of heterogeneity in MCI and LOAD patient groups we investigated the levels of diacylglycerols and ethanolamine plasmalogens in larger cohorts of patients utilizing, high-resolution (0.2 to 2ppm mass error) mass spectrometry.ResultsFor the first time, our lipidomics data clearly stratify both MCI and LOAD subjects into 3 different patient cohorts within each clinical diagnosis. These include i) patients with lower circulating ethanolamine plasmalogen levels; ii) patients with augmented plasma diacylglycerol levels; and iii) patients with neither of these lipid alterations.ConclusionsThese represent the first serum biochemical data to stratify MCI and LOAD patients, advancing efforts to biochemically define patient heterogeneity in cognitive disorders.General significanceLipidomics offers a new approach for identifying biomarkers and biological targets in cognitive disorders

Increased Serum Beta-Secretase 1 Activity is an Early Marker of Alzheimer's Disease

Background: Beta-site APP cleaving enzyme 1 (BACE1) is the rate-limiting enzyme in amyloid-beta (A beta) plaques formation. BACE1 activity is increased in brains of patients with Alzheimer's disease (AD) and mild cognitive impairment (MCI) and plasma levels of BACE1 appears to reflect those in the brains.Objective: In this work, we investigated the role of serum BACE1 activity as biomarker for AD, estimating the diagnostic accuracy of the assay and assessing the correlation of BACE1 activity with levels of A beta(1-40), A beta(1-42), and A beta(40/42) ratio in serum, known biomarkers of brain amyloidosis.Methods: Serum BACE1 activity and levels of A beta(1-40), A beta(1-42), were assessed in 31 AD, 28 MCI, diagnosed as AD at follow-up (MCI-AD), and 30 controls. The BACE1 analysis was performed with a luciferase assay, where interpolation of relative fluorescence units with a standard curve of concentration reveals BACE1 activity. Serum levels of A beta(1-40), A beta(1-42) were measured with the ultrasensitive Single Molecule Array technology.Results: BACE1 was increased (higher than 60%) in AD and MCI-AD: a cut-off of 11.04 kU/L discriminated patients with high sensitivity (98.31%) and specificity (100%). Diagnostic accuracy was higher for BACE1 than A beta(40/42) ratio. High BACE1 levels were associated with worse cognitive performance and earlier disease onset, which was anticipated by 8 years in patients with BACE1 values above the median value (&gt; 16.67 kU/L).Conclusion: Our results provide new evidence supporting serum/plasma BACE1 activity as an early biomarker of AD

Fermentation of Vaccinium floribundum Berries with Lactiplantibacillus plantarum Reduces Oxidative Stress in Endothelial Cells and Modulates Macrophages Function

Accumulating evidence suggests that high consumption of natural antioxidants promotes health by reducing oxidative stress and, thus, the risk of developing cardiovascular diseases. Similarly, fermentation of natural compounds with lactic acid bacteria (LAB), such as Lactiplantibacillus plantarum, enhances their beneficial properties as regulators of the immune, digestive, and cardiovascular system. We investigated the effects of fermentation with Lactiplantibacillus plantarum on the antioxidant and immunomodulatory effects of Pushgay berries (Vaccinium floribundum, Ericaceae family) in human umbilical vein endothelial cells (HUVECs) and macrophage cell line RAW264.7. Polyphenol content was assayed by Folin-Ciocalteu and HPLC-MS/MS analysis. The effects of berries solutions on cell viability or proliferation were assessed by WST8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt and Lactate dehydrogenase (LDH) release, Trypan blue exclusion test, and Alamar blue assay. Antioxidant activity was evaluated by a cell-based chemiluminescent probe for the detection of intracellular H2O2 production in HUVECs. Heme oxygenase-1 (HO-1) expression levels were investigated by RT-qPCR. Glutathione reductase (GR), glutathione peroxidase (Gpx), superoxide dismutase (SOD), and catalase (CAT) activities, as markers of intracellular antioxidant defense, were evaluated by spectrophotometric analysis. The immunomodulatory activity was examined in RAW 264.7 by quantification of inducible nitric oxide synthase (iNOS) and Tumor Necrosis Factor-alpha (TNF alpha) by RT-qPCR. Data showed that fermentation of Pushgay berries (i) enhances the content of quercetin aglycone, and (ii) increases their intracellular antioxidant activity, as indicated by the reduction in H2O2-induced cell death and the decrease in H2O2-induced HO-1 gene expression in HUVECs treated for 24 h with fermented berries solution (10 mu g/mL). Moreover, treatment with Pushgay berries for 72 h (10 mu g/mL) promotes cells growth in RAW 264.7, and only fermented Pushgay berries increase the expression of iNOS in the same cell line. Taken together, our results show that LAB fermentation of Pushgay berries enhances their antioxidant and immunomodulatory properties