Isolation and characterization of novel 1,3-propanediol-producing Lactobacillus panis PM1 from bioethanol thin stillage.

Conversion of glycerol to 1,3-propanediol (1,3-PDO) is an attractive option to increase the economic efficiency of the biofuel industry. A bacterial strain that produced 1,3-PDO in the presence of glycerol was isolated from thin stillage, the fermentation residue of bioethanol production. This 1,3-PDO-producing organism was identified as Lactobacillus panis through biochemical characteristics and by 16S rRNA sequencing. Characterization of the L. panis strain hereafter designated as PM1 revealed it was an aerotolerant acidophilic anaerobe able to grow over a wide range of temperatures; tolerant to high concentrations of sodium chloride, ethanol, acetic acid, and lactic acid; and resistant to many common antibiotics. L. panis PM1 could utilize glucose, lactose, galactose, maltose, xylose, and arabinose, but could not grow on sucrose or fructose. Production of 1,3-PDO by L. panis PM1 occurred only when glucose was available as the carbon source in the absence of oxygen. These metabolic characteristics strongly suggested NADH recycling for glucose metabolism is achieved through 1,3-PDO production by this strain. These characteristics classified L. panis PM1 within the group III heterofermentative lactic acid bacteria, which includes the well-characterized 1,3-PDO-producing strain, Lactobacillus reuteri. Metabolite production profiles showed that L. panis PM1 produced considerable amounts of succinic acid (~11-12 mM) from normal MRS medium, which distinguishes this strain from L. reuteri strains.

Alkaline conditions stimulate the production of 1,3-propanediol in Lactobacillus panis PM1 through shifting metabolic pathways.

A novel Lactobacillus panis PM1 isolate was found to be capable of converting glycerol to 1,3-propanediol (1,3-PDO), an increasingly valuable commodity chemical. In this study the effects of various process parameters, including glucose and glycerol concentrations, inoculum size, temperature, aeration, pH, and carbon source were examined to determine the optimal conditions for the production of 1,3-PDO using a culture method simulating late log to early stationary phases. Inoculum size did not influence the production of 1,3-PDO, and temperature variance showed similar 1,3-PDO production between 25 and 37 °C under the examined conditions. Glycerol concentration and pH played a primary role in the final concentration of 1,3-PDO. The highest production occurred at 150-250 mM glycerol when 50 mM glucose was available. Alkaline initial conditions (pH 9-10) stimulated the production of 1,3-PDO which concurrently occurred with increased acetic acid production. Under these conditions, 213.6 mM of 1,3-PDO were produced from 300 mM glycerol (conversion efficiency was 71 %). These observations indicated that the production of 1,3-PDO was associated with the shift of the metabolic end-product ethanol to acetic acid, and that this shift resulted in an excess concentration of NADH available for the processing of glycerol to 1,3-PDO.

Influence of positional isomers on the macroscale and nanoscale architectures of aggregates of racemic hydroxyoctadecanoic acids in their molecular gel, dispersion, and solid states.

Inter/intramolecular hydrogen bonding of a series of hydroxystearic acids (HSAs) are investigated. Self-assembly of molecular gels obtained from these fatty acids with isomeric hydroxyl groups is influenced by the position of the secondary hydroxyl group. 2-Hydroxystearic acid (2HSA) does not form a molecular dimer, as indicated by FT-IR, and growth along the secondary axis is inhibited because the secondary hydroxyl group is unable to form intermolecular H-bonds. As well, the XRD long spacing is shorter than the dimer length of hydroxystearic acid. 3-Hydroxystearic acid (3HSA) forms an acyclic dimer, and the hydroxyl groups are unable to hydrogen bond, preventing the crystal structure from growing along the secondary axis. Finally, isomers 6HSA, 8HSA, 10HSA, 12HSA, and 14HSA have similar XRD and FT-IR patterns, suggesting that these molecules all self-assemble in a similar fashion. The monomers form a carboxylic cyclic dimer, and the secondary hydroxyl group promotes growth along the secondary axis.

The Relationship Between Cerebral Reperfusion And Regional Expression Of Matrix Metalloproteinase-9 In Rat Brain Following Focal Cerebral Ischemia.

We investigated the effect of full and partial mechanical reperfusion on MMP-9 expression in rat brain following middle cerebral artery occlusion, mimicking mechanical thrombectomy. Using percentage hemispheric lesion volume and oedema as measures, partial reperfusion reduced extent of brain damage caused by MCA occlusion, but the protective effect was less pronounced than with complete reperfusion. Using ELISA quantification in fresh frozen tissue, confirmed by immunofluorescence in perfusion fixed tissue, increased MMP-9 expression was observed in infarcted tissue. MMP-9 was increased in lesioned tissue of the anterior and posterior temporal cortex and underlying striatal tissue, but also the normal appearing frontal cortex. No significant increase in MMP-9 in the hippocampus was observed, nor in the unlesioned contralateral hemisphere. Both partial reperfusion and full reperfusion reduced the regional MMP expression significantly. The highest levels of MMP-9 were observed in lesioned brain regions in the non-reperfused group. MMP-9 expression was evident in microvessels and in neuronal cell bodies of affected tissue. This study shows that MMP-9 brain levels are reduced relative to the extent of reperfusion. These observations suggest targeting early increases in MMP-9 expression as a possible neuroprotective therapeutic strategy and highlight the rat MCA occlusion model as an ideal model in which to study candidate therapeutics.

Improving the alkaline stability of pepsin through rational protein design using renin, an alkaline-stable aspartic protease, as a structural and functional reference.

The present study sought to identify the structural determinants of aspartic protease structural stability and activity at elevated pH. Various hypotheses have been published regarding the features responsible for the unusual alkaline structural stability of renin, however, few structure-function studies have verified these claims. Using pepsin as a model system, and renin as a template for functional and structural alkaline stability, a rational re-design of pepsin was undertaken to identify residues contributing to the alkaline instability of pepsin-like aspartic proteases in regards to both structure and function. We constructed 13 mutants based on this strategy. Among them, mutants D159 L and D60A led to an increase in activity at elevated pH levels (p ≤ 0.05) and E4V and H53F were shown to retain native-like structure at elevated pH (p ≤ 0.05). Previously suggested carboxyl groups Asp11, Asp118, and Glu13 were individually shown not to be responsible for the structural instability or lack of activity at neutral pH in pepsin. The importance of the ß-barrel to structural stability was highlighted as the majority of the stabilizing residues identified, and 39% of the weakly conserved residues in the N-terminal lobe, were located in ß-sheet strands of the barrel. The results of the present study indicate that alkaline stabilization of pepsin will require reduction of electrostatic repulsions and an improved understanding of the role of the hydrogen bonding network of the characteristic ß-barrel.

Novel Human Acute Ischemic Stroke Blood Clot Analogs for In Vitro Thrombectomy Testing.

BACKGROUND AND PURPOSE: Previous studies have successfully created blood clot analogs for in vitro endovascular device testing using animal blood of various species. Blood components vary greatly among species; therefore, creating clot analogs from human blood is likely a more accurate representation of thrombi formed in the human vasculature. MATERIALS AND METHODS: Following approval from the Mayo Clinic institutional review board, human whole-blood and platelet donations were obtained from the blood transfusion service. Twelve clot analogs were created by combining different ratios of red blood cells + buffy coat, plasma, and platelets. Thrombin and calcium chloride were added to stimulate coagulation. Clot composition was assessed using histologic and immunohistochemical staining. To assess the similarities of mechanical properties to patient clots, 3 types of clot analogs (soft, elastic, and stiff) were selected for in vitro thrombectomy testing. RESULTS: The range of histopathologic compositions produced is representative of clots removed during thrombectomy procedures. The red blood cell composition ranged from 8.9% to 91.4%, and fibrin composition ranged from 3.1% to 53.4%. Platelets (CD42b) and von Willebrand Factor ranged from 0.5% to 47.1% and 1.0% to 63.4%, respectively. The soft clots had the highest first-pass effect and successful revascularization rates followed by the elastic and stiff clots. Distal embolization events were observed when clot ingestion could not be achieved, requiring device pullback. The incidence rate of distal embolization was the highest for the stiff clots due to the weak clot/device integration. CONCLUSIONS: Red blood cell-rich, fibrin-rich, and platelet-rich clot analogs that mimic clots retrieved from patients with acute ischemic stroke were created in vitro. Differing retrieval outcomes were confirmed using in vitro thrombectomy testing in a subset of clots.

Animal Models of Focal Cerebral Ischaemia and Haemorrhagic Transformation: Considerations in Experimental Stroke Study Design.

BACKGROUND: Ischaemic stroke is often complicated with haemorrhage within the infarct zone or in a remote location especially when treated with intravenous thrombolysis and/or thrombectomy. While these early recanalisation treatments are highly effective, some of the benefit is lost because of haemorrhagic complications and consequential neurological deterioration of the patients. A number of mechanisms have been described that mediate the haemorrhagic changes and several agents have been tested in experimental models for inhibiting post-stroke haemorrhage. METHODS: Here, we review and discuss the small animal models of focal cerebral ischaemia and postischaemic stroke haemorrhagic transformation and how these models can best be utilised for developing further insights as well as potential treatment approaches for this serious clinical complication. RESULTS: The need to use appropriate animal models with relevant stroke risk factors to improve the clinical relevance and applicability of findings is becoming ever more apparent. Current focal ischaemia models can be adapted for the study of haemorrhagic transformation post-stroke. CONCLUSION: A number of factors can be added to the animal model design to increase the incidence and/or severity of haemorrhagic transformation post-ischaemic stroke, which can improve clinical relevance, aid the study of the pathophysiology and the future development of novel interventions.

Production of eosinophil chemoattractant activity by ovine gastrointestinal nematodes.

Eosinophilia is a well documented feature of helminth infections but the precise nature of the interaction between parasite and eosinophil remains an enigma. This paper describes experiments demonstrating that ruminant gastrointestinal trichostrongyles produce potent chemoattractant activity for ovine bone marrow-derived eosinophils in vitro. This activity was initially identified as a constituent of whole worm extracts of third and fourth larval (L3, L4), and adult stages of Teladorsagia circumcincta, and adult Haemonchus contortus. Similar activity was detected in excretory/secretory (E/S) material derived from live T. circumcincta L3. Subsequently, by adapting the assay technique to incorporate live worms directly into the system, it was shown that L3 of both T. circumcincta and H. contortus produced eosinophil chemoattractant activity. In contrast, neither whole worm extracts, or E/S preparations from mixed stages of the free-living nematode Caenorhabditis elegans contained eosinophil chemoattractant activity, and there was no evidence of chemoattractant production by live C. elegans. The results described are challenging to the traditional dogma that eosinophils are host-protective effector cells, and raise the intriguing possibility that ovine nematodes actively encourage recruitment of eosinophils. Local eosinophil-mediated mucosal damage, comparable to that seen in the asthmatic lung, may then provide a permissive local microenvironment for the parasite. Moreover, if they prove important for pathogenicity, nematode chemoattractants could offer future potential as novel therapeutic targets.

Aspirin and other antiplatelet drugs in the prophylaxis of thrombosis.

Aspirin is of proven value as an antithrombotic drug. In unstable angina it reduces the risk of death and myocardial infarction by half. After a myocardial infarction it reduces the risk of death by about 10% and of coronary incidence (coronary death or definite myocardial infarction) by about 25%. These effects appear to be additive with those of beta-blocking drugs. Aspirin also reduces the risk of occlusion of aortocoronary saphenous vein grafts by about half. In transient cerebral ischaemia, aspirin may reduce the risk of stroke and death by 50%. In most clinical trials to date the daily dose of aspirin ranges from 325 mg to 1400 mg. Interest in very low doses of aspirin (less than 60 mg daily) is considerable but has yet to be translated into proven clinical benefit. Dipyridamole has not been shown to be effective as an antithrombotic when used alone. Its antiplatelet action ex vivo may be enhanced by combination with aspirin but clinical trials have shown relatively little advantage of the combination over aspirin alone. Sulphinpyrazone has not become established as a first line antithrombotic drug. Epoprostenol is useful in extracorporeal circulations to prevent platelet consumption and possibly in severe inoperable peripheral vascular disease.

Carboxylation of osteocalcin in post-menopausal osteoporotic women following vitamin K and D supplementation.

The effect of vitamin supplements on bone metabolism indices in patients with osteoporosis has received scant attention in the literature. Over a 2-week period, vitamin supplements of K and K+D were given to 20 post-menopausal osteoporotic women with previous Colles fractures. Osteoporosis was confirmed by bone mass measurements that demonstrated that broadband ultrasound attenuation (os calcis) was almost as discriminatory as dual energy X-ray absorptiometry (spine and hip) in Colles fracture patients compared with matched controls. Vitamin K corrected the carboxylation defect in osteocalcin and while less marked 4 weeks later, the improvement was still detectable. The result after K+D was similar. The level of carboxylation became the same as in premenopausal women. Total osteocalcin level (bound) osteocalcin. While there was vitamin K correctable undercarboxylation of osteocalcin, simultaneously there was no evidence of undercarboxylation of prothrombin.

The effeCt of vitamin c supplements on serum cholesterol, coagulation, fibrinolysis and platelet adhesiveness.

The administration of ascorbic acid (1g/day) to healthy adults did not significantly influence the levels of serum cholesterol, plasminogen activator activity, plasminogen, fibrinogen, FR-antigen, partial thromboplastin time, platelet adhesiveness, a-1-antitrypsin or a-2-macroglobulin over the 3-month period of study.

Lipoprotein fractions and antithrombin III consumption during clotting.

Plasma and serum antithrombin levels were measured in functional (initial rate measurement) and immunological assays together with serum lipid levels in normal subjects and patients with coronary artery disease. Specific antithrombin activity in plasma showed a negative correlation with triglyceride levels. The consumption of antithrombin activity during blood clotting was negatively correlated with both serum total triglyceride and heparin precipitable lipoprotein and positively correlated with serum high density lipoprotein cholesterol. Different blood lipoprotein fractions may influence the activity of the antithrombin III molecule.

Platelet adhesiveness and fibrinolysis after recent cerebro-vascular accidents and their relationship with subsequent deep venous thrombosis of the legs.

In fifteen patients with a cerebro-vascular accident resulting in an acute hemiplegia there was a subsequent rise in the platelet count and plasma fibrinogen level. There were no significant alterations in platelet adhesiveness, plasminogen activator, plasminogen, FR-antigen and haematocrit. Patients diagnosed as developing deep venous thrombosis with the 125I-fibrinogen technique had a significantly lower platelet adhesiveness and plasminogen level than those who were not.

Individualisation of oral anticoagulant therapy.

The hepatic synthesis of vitamin K dependent coagulation factors is modified by oral anticoagulant drugs, resulting in the release of functionally deficient coagulation factors into the circulation and consequently anticoagulation. Since their introduction into clinical medicine over 30 years ago, both clinical and scientific evidence has demonstrated the value of oral anticoagulants in the treatment and prophylaxis of venous thrombosis. In the treatment of arterial disease, however, both the indications for and usefulness of oral anticoagulants remain very much in doubt despite their widespread use in the 1950s and 1960s and in numerous clinical trials. The initiation and continuation of oral anticoagulant therapy is a co-operative venture involving the patient, the clinician and the laboratory. The clinician must have a thorough knowledge of the indications for and contraindications to the use of these drugs, and regular, accurate laboratory control is essential if haemorrhage, the major side effect, is to be avoided or reduced to a minimum. The patient must bear the responsibility for regular clinic attendance, abstinence from proprietary medications, and must immediately seek medical advice if any sign of haemorrhage occurs.

An international and interregional comparison of haemostatic variables in the study of ischaemic heart disease. Report of a working group.

Levels of haemostatic variables that may be involved in thrombogenesis have been compared in groups of men of similar mean age in communities at very low (Gambia), high (England and Czechoslovakia) or very high (Scotland and Finland) risk of ischaemic heart disease (IHD). There was a consistent gradient of higher factor VII levels with higher IHD risk and also suggestive gradients in the case of two other vitamin K dependent factors, factors II and X. Mean platelet counts were lower and mean fibrinolytic activity was greater in Gambian men than in European men. There was a suggestive though not entirely consistent association between mean fibrinogen levels and IHD risk in the groups from IHD-endemic countries. The results as a whole, and particularly those on factor VII, strengthen the case for the increasingly detailed epidemiological as well as laboratory investigation of the role of the haemostatic system in thrombogenesis and IHD.

Preoperative antithrombin III activities and lipoprotein concentrations as predictors of venous thrombosis in patients with fracture of neck of femur.

The presence or absence of venous thrombosis was determined by the technique of autologous 111indium-labelled platelets in 64 patients following reduction and fixation of fractures of the neck of femur. Venous thrombosis was found in 41 (64%) of these patients. Compared to the thrombosis-negative group, the thrombosis-positive group had a significantly lower mean serum antithrombin, and higher mean antithrombin consumption during clotting in the immediate preoperative period. No significant differences were seen between the two groups in plasma ATIII activities, fibrinogen, alpha-2-macroglobulin, serum lipoproteins or age. Significant correlations were seen between ATIII activities and lipoprotein fractions; these are discussed.

The effect of age on the fibrinolytic enzyme system.

Components of the fibrinolytic enzyme system, FR-antigen and fibrinogen, were measured in 20 healthy volunteers aged 20-40 years and in 61 elderly subjects aged 66-96 years. Plasminogen activator levels did not significantly differ between the 20-40 and 66-75 age groups, but were higher in those over 75. Plasminogen showed no change with age except for a fall in those over 75. Fibrinogen, FR-antigen, alpha(1)-antitrypsin, and alpha(2)-macroglobulin all rose with age, but the mean fibrinogen concentration fell in the very elderly.

Relationships between platelet function tests in normal and uraemic subjects.

IN TESTS OF PLATELET FUNCTION IN NORMAL SUBJECTS, THE FOLLOWING RELATIONSHIPS WERE FOUND: the greater the platelet adhesiveness the less the ability to disaggregate after challenge with adenosine diphosphate (ADP), and the greater the disaggregation after ADP, the longer the clotting time in the test for platelet factor 3 availability. Such correlations were disturbed in uraemic patients.

Platelet adhesiveness, coagulation, and fibrinolytic activity in obesity.

In a study of 41 fasting subjects it was confirmed that fibrinolytic activity was reduced in obese persons: an increase in fibrinogen was also associated with obesity. There was no correlation between obesity and the platelet count, platelet adhesiveness to glass, the level of serum fibrin degradation products, or the whole blood clotting time in plastic tubes.