Characterization of energetically functional inverted membrane vesicles from Corynebacterium glutamicum

AbstractWe show that inverted membrane vesicles from Corynebacteriwn glutamicum, a Gram-positive bacterium, are able to generate and maintain an electrochemical gradient of protons in response to the addition of NADH. This result indicates that the respiratory chain is intact and that the vesicles are reasonably impermeable to protons. These membrane vesicles may be the starting point for in vitro translocation studies of proteins in Gram-positive bacteria

HEPATOCYTE APOPTOSIS INDUCTION BY ACETAMINOPHEN THROUGH MODULATION OF CASPASE/BAX PATHWAY IN MICE

Objective: Acetaminophen (APAP) overdose contributes to liver damage through modulation of pro-apoptotic processing. This study evaluated the involvement of caspase/Bax factors in APAP hepatotoxicity in vivo and in vitro. Methods: The involvement of caspase/Bax factors in APAP hepatotoxicity was evaluated in BALB/c mice and on isolated primary mouse hepatocytes. In vitro MTT assay was carried out on primary cultured mouse hepatocytes treated with APAP (2.5, 5, 10 mmol) and Annexin V/PI staining was employed to cell suspension for imaging under fluorescence microscopy. In addition, caspase-3 concentrations were determined in cell lysates. In vivo, mice were treated with a toxic dose of APAP (700 mg/kg) and immunodetection of Bax was made by Western Blot. Vitamin C (Vit C) was used as a hepato-protectant due to its known antioxidant activities. Results: In vitro dose-dependent increase in mitochondrial electron transport capacity was evident in isolated mouse primary hepatocytes incubated with the high dose of APAP (10 mmol) compared to both nontreated cells and cells pre-treated with Vitamin C (Vit C) (0.5 mmol) (p<0.05). Apoptosis was confirmed in hepatocytes through Annexin V staining after APAP treatment and the signal was reduced when hepatocytes were pre-treated with Vit C. In addition, caspase-3 concentration was decreased in cells pretreated with Vit C prior to APAP exposure. In vivo, Bax immunodetection utilizing western blotting was increased in mice treated with the toxic dose of APAP (700 mg/kg) and attenuated through pre-treatment with Vit C. Conclusion: Modulation of apoptotic caspase/Bax pathway is present in hepatocytes undergoing APAP-induced toxicity

ISO-1 Binding to the Tautomerase Active Site of MIF Inhibits Its Pro-inflammatory Activity and Increases Survival in Severe Sepsis

MIF is a proinflammatory cytokine that has been implicated in the pathogenesis of sepsis, arthritis, and other inflammatory diseases. Antibodies against MIF are effective in experimental models of inflammation, and there is interest in strategies to inhibit its deleterious cytokine activities. Here we identify a mechanism of inhibiting MIF pro-inflammatory activities by targeting MIF tautomerase activity. We designed small molecules to inhibit this tautomerase activity; a lead molecule, "ISO-1 ((S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester)," significantly inhibits the cytokine activity in vitro. Moreover, ISO-1 inhibits tumor necrosis factor release from macrophages isolated from LPStreated wild type mice but has no effect on cytokine release from MIFdeficient macrophages. The therapeutic importance of the MIF inhibition by ISO-1 is demonstrated by the significant protection from sepsis, induced by cecal ligation and puncture in a clinically relevant time frame. These results identify ISO-1 as the first small molecule inhibitor of MIF proinflammatory activities with therapeutic implications and indicate the potential of the MIF active site as a novel target for therapeutic interventions in human sepsis

Comparative studies on haemato-biochemical changes following pre-emptive analgesia with tramadol, pentazocine lactate and meloxicam inpain management of canine ovariohysterectomy

The present study was conducted to evaluate the effects of tramadol, pentazocine lactate and meloxicam as pre- emptive analgesics in dogs premedicated with glycopyrrolate, inducted with propofol and maintained with propofol continuous rate infusion (CRI) for certain haematological and biochemical parameters. The animals were randomly divided into three equal groups,viz. Group-T, Group-P and Group-M comprising six animals in each group and all the animals were premedicated with glycopyrrolate. After 10 min of pre-anaesthetic administration, pre-emptive analgesia was given. Blood was collected from cephalic or saphenous vein at intervals 0 (baseline) min before premedication, thereafter at 10 min, 30 min, 1 h, 2 h and 3 h after pre-emptive analgesic administration and haemato- biochemical parameters were recorded. Hb, PCV and TEC were significantly decreased at 30 min and 1 h interval in all the three groups. TLC and glucose concentration were significantly higher in group-M as compared to group-T and group-P at different time intervals. GGT level increased significantly at 30 min in all the three groups. CRP concentration was significantly higher in group-M as compared to group-T. Total protein was significantly decreased at 1 h interval in group-T and group-P, but in group-M such finding was noticed at 2 h interval. Cortisol was significantly lower in group-T in entire study period. The alterations in physiological and haematological parameters caused by tramadol, pentazocine lactate and meloxicam were found to be minimal and within the physiological limits. Tramadol produced less significant rise in CRP and cortisol concentrations which indicated better pain management. Based on the findings of the present study, it is concluded that tramadol is more effective as compared to pentazocine lactate and meloxicam in the management of post-operative pain due to canine ovariohysterectomy

Amyloid fibril formation by macrophage migration inhibitory factor

We demonstrate herein that human macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine expressed in the brain and not previously considered to be amyloidogenic, forms amyloid fibrils similar to those derived from the disease associated amyloidogenic proteins beta-amyloid and alpha-synuclein. Acid denaturing conditions were found to readily induce MIF to undergo amyloid fibril formation. MIF aggregates to form amyloid-like structures with a morphology that is highly dependent on pH. The mechanism of MIF amyloid formation was probed by electron microscopy, turbidity, Thioflavin T binding, circular dichroism spectroscopy, and analytical ultracentrifugation. The fibrillar structures formed by MIF bind Congo red and exhibit the characteristic green birefringence under polarized light. These results are consistent with the notion that amyloid fibril formation is not an exclusive property of a select group of amyloidogenic proteins, and contribute to a better understanding of the factors which govern protein conformational changes and amyloid fibril formation in vivo

Pedagogical Agents for Fostering Question-Asking Skills in Children

Question asking is an important tool for constructing academic knowledge, and a self-reinforcing driver of curiosity. However, research has found that question asking is infrequent in the classroom and children's questions are often superficial, lacking deep reasoning. In this work, we developed a pedagogical agent that encourages children to ask divergent-thinking questions, a more complex form of questions that is associated with curiosity. We conducted a study with 95 fifth grade students, who interacted with an agent that encourages either convergent-thinking or divergent-thinking questions. Results showed that both interventions increased the number of divergent-thinking questions and the fluency of question asking, while they did not significantly alter children's perception of curiosity despite their high intrinsic motivation scores. In addition, children's curiosity trait has a mediating effect on question asking under the divergent-thinking agent, suggesting that question-asking interventions must be personalized to each student based on their tendency to be curious.Comment: Accepted at CHI 202

Structural and Functional Insights into the Pilotin-Secretin Complex of the Type II Secretion System

Gram-negative bacteria secrete virulence factors and assemble fibre structures on their cell surface using specialized secretion systems. Three of these, T2SS, T3SS and T4PS, are characterized by large outer membrane channels formed by proteins called secretins. Usually, a cognate lipoprotein pilot is essential for the assembly of the secretin in the outer membrane. The structures of the pilotins of the T3SS and T4PS have been described. However in the T2SS, the molecular mechanism of this process is poorly understood and its structural basis is unknown. Here we report the crystal structure of the pilotin of the T2SS that comprises an arrangement of four α-helices profoundly different from previously solved pilotins from the T3SS and T4P and known four α-helix bundles. The architecture can be described as the insertion of one α-helical hairpin into a second open α-helical hairpin with bent final helix. NMR, CD and fluorescence spectroscopy show that the pilotin binds tightly to 18 residues close to the C-terminus of the secretin. These residues, unstructured before binding to the pilotin, become helical on binding. Data collected from crystals of the complex suggests how the secretin peptide binds to the pilotin and further experiments confirm the importance of these C-terminal residues in vivo

Two-Photon Microscopy for Non-Invasive, Quantitative Monitoring of Stem Cell Differentiation

BACKGROUND: The engineering of functional tissues is a complex multi-stage process, the success of which depends on the careful control of culture conditions and ultimately tissue maturation. To enable the efficient optimization of tissue development protocols, techniques suitable for monitoring the effects of added stimuli and induced tissue changes are needed. METHODOLOGY/PRINCIPAL FINDINGS: Here, we present the quantitative use of two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) as a noninvasive means to monitor the differentiation of human mesenchymal stem cells (hMSCs) using entirely endogenous sources of contrast. We demonstrate that the individual fluorescence contribution from the intrinsic cellular fluorophores NAD(P)H, flavoproteins and lipofuscin can be extracted from TPEF images and monitored dynamically from the same cell population over time. Using the redox ratio, calculated from the contributions of NAD(P)H and flavoproteins, we identify distinct patterns in the evolution of the metabolic activity of hMSCs maintained in either propagation, osteogenic or adipogenic differentiation media. The differentiation of these cells is mirrored by changes in cell morphology apparent in high resolution TPEF images and by the detection of collagen production via SHG imaging. Finally, we find dramatic increases in lipofuscin levels in hMSCs maintained at 20% oxygen vs. those in 5% oxygen, establishing the use of this chromophore as a potential biomarker for oxidative stress. CONCLUSIONS/SIGNIFICANCE: In this study we demonstrate that it is possible to monitor the metabolic activity, morphology, ECM production and oxidative stress of hMSCs in a non-invasive manner. This is accomplished using generally available multiphoton microscopy equipment and simple data analysis techniques, such that the method can widely adopted by laboratories with a diversity of comparable equipment. This method therefore represents a powerful tool, which enables researchers to monitor engineered tissues and optimize culture conditions in a near real time manner

Coronary Artery Bypass Grafting (CABG) versus Percutaneous Coronary Intervention (PCI) in treatment of left main coronary artery disease

BackgroundCoronary artery bypass graft surgery (CABG) has been widely used for left main coronary artery disease (LMCAD). Percutaneous coronary intervention (PCI) has become an option for this condition.AimsTo summarize the current evidence that compare between CABG vs. PCI in regards to ‎cardiac death, stroke, and myocardial infarction.‎Methods We searched randomized trials of treatment of LMCAD with PubMed, Google Scholar, and EBSCO.Results Five randomized studies were retrieved, which compared the efficacy between CABG vs. PCI in treatment of LMCAD.ConclusionPCI may be reasonable management of patients with LM stenosis involving distal bifurcation or with coexisting multivessel disease

An Anomalous Type IV Secretion System in Rickettsia Is Evolutionarily Conserved

Bacterial type IV secretion systems (T4SSs) comprise a diverse transporter family functioning in conjugation, competence, and effector molecule (DNA and/or protein) translocation. Thirteen genome sequences from Rickettsia, obligate intracellular symbionts/pathogens of a wide range of eukaryotes, have revealed a reduced T4SS relative to the Agrobacterium tumefaciens archetype (vir). However, the Rickettsia T4SS has not been functionally characterized for its role in symbiosis/virulence, and none of its substrates are known.Superimposition of T4SS structural/functional information over previously identified Rickettsia components implicate a functional Rickettsia T4SS. virB4, virB8 and virB9 are duplicated, yet only one copy of each has the conserved features of similar genes in other T4SSs. An extraordinarily duplicated VirB6 gene encodes five hydrophobic proteins conserved only in a short region known to be involved in DNA transfer in A. tumefaciens. virB1, virB2 and virB7 are newly identified, revealing a Rickettsia T4SS lacking only virB5 relative to the vir archetype. Phylogeny estimation suggests vertical inheritance of all components, despite gene rearrangements into an archipelago of five islets. Similarities of Rickettsia VirB7/VirB9 to ComB7/ComB9 proteins of epsilon-proteobacteria, as well as phylogenetic affinities to the Legionella lvh T4SS, imply the Rickettsiales ancestor acquired a vir-like locus from distantly related bacteria, perhaps while residing in a protozoan host. Modern modifications of these systems likely reflect diversification with various eukaryotic host cells.We present the rvh (Rickettsiales vir homolog) T4SS, an evolutionary conserved transporter with an unknown role in rickettsial biology. This work lays the foundation for future laboratory characterization of this system, and also identifies the Legionella lvh T4SS as a suitable genetic model