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1.  Natural populations of lactic acid bacteria in douchi from Yunnan Province, China*  
This research was aimed at isolating and identifying the predominant lactic acid bacteria (LAB) in the traditional Chinese salt-fermented soybean food, douchi, from Yunnan, China. The predominant LAB present were isolated and identified by conventional culture-dependent methods combined with molecular biological methods. Two hundred and sixty isolates were obtained from thirty kinds of traditional fermented douchi from six cities and counties in Yunnan, and those strains were divided into twelve groups by their morphological and biochemical characteristics. Based on 16S ribosomal DNA (rDNA) sequencing and analysis, 56 representative strains were identified as belonging to 6 genera and 14 species: Lactobacillus (4 spp.), Weissella (3 spp.), Pediococcus (2 spp.), Staphylococcus (2 spp.), Enterococcus (1 sp.), and Bacillus (2 spp.). The results show that douchi contains a large natural population of LAB of diverse composition from which some strains could be selected as starters for functional fermented foods. This is the first study on the original douchi from Yunnan, and the results suggest that it may be a useful source for the isolation of LAB. This study has also laid a foundation for further research on developing functional douchi products.
PMCID: PMC3323945  PMID: 22467371
Fermented food; Douchi; Lactic acid bacteria; Culture-dependent methods; 16S rDNA
2.  Thrombolysis with Human Extrinsic (Tissue-Type) Plasminogen Activator in Rabbits with Experimental Jugular Vein Thrombosis. EFFECT OF MOLECULAR FORM AND DOSE OF ACTIVATOR, AGE OF THE THROMBUS, AND ROUTE OF ADMINISTRATION 
Journal of Clinical Investigation  1983;71(2):368-376.
A simple venous thrombosis model in rabbits was used for the quantitative evaluation of the thrombolytic effect of human extrinsic (tissue-type) plasminogen activator as compared with urokinase.
A thrombus was formed in an isolated segment of the jugular vein from a mixture of 125I-labeled fibrinogen, whole rabbit blood, and thrombin. In order to immobilize the thrombus during lysis, it was formed around a woolen thread introduced longitudinally in the lumen of the vein. Thrombotic extension of the clot was prevented by subcutaneous injection of heparin. The extent of thrombolysis was measured as the difference between the radioactivity introduced in the clot and that recovered in the vein segment at the end of the experiment. In control animals the extent of thrombolysis was 5.6±1.4% (n = 5) after 6 h, 14.5±1.7% (n = 10) after 30 h, 16.0±1.5% (n = 11) after 78 h, and 48.1±2.7% (n = 10) after 174 h (mean±SEM).
Extrinsic (tissue-type) plasminogen activator, highly purified from the culture fluid of a human melanoma cell line, was administered systemically or locally over a time period of 4 h and the percent thrombolysis measured 2 h after the end of the infusion. One- and two-chain extrinsic plasminogen activator had very similar thrombolytic potency. Systemic infusion resulted in a dose-dependent degree of thrombolysis. The activator-induced thrombolysis, after infusion of 100,000 IU (≅1 mg protein), was ∼75% for fresh clots, 35% for 1-d-old clots, 30% for 3-d-old clots, and 50% for 7-d-old clots. The thrombolytic activity of urokinase was more than five times lower than that of extrinsic plasminogen activator: Infusion of 500,000 IU resulted in ∼40% lysis of fresh clots and 25% of 1-3-d-old clots, while 7-d-old clots appeared to have become resistent to urokinase. Local infusion resulted in a 5-10 times higher thrombolytic effect of both extrinsic plasminogen activator and urokinase.
Thrombolysis with extrinsic plasminogen activator was not associated with systemic activation of the fibrinolytic system as evidenced by unaltered plasma levels of fibrinogen, plasminogen, and α2-antiplasmin. Systemic infusion of urokinase resulted in significant thrombolysis only at doses that were associated with disseminated plasminogen activation. Local infusion of urokinase required a 5-10-fold higher dose than extrinsic plasminogen activator to obtain a similar degree of thrombolysis, which also occurred in the absence of systemic activation of the fibrinolytic system.
It is concluded that the extent of thrombolysis by extrinsic plasminogen activator is mainly determined by the dose of activator and its delivery in the vicinity of the thrombus and much less by the age of the thrombus or the molecular form of the activator. Extrinsic plasminogen activator appears to be superior to urokinase because of its higher (5-10-fold) specific thrombolytic activity and the absence of systemic activation of the fibrinolytic system, which results in defibrinogenation and a bleeding tendency.
PMCID: PMC436876  PMID: 6681615
3.  Multifunctional nanoagent for thrombus-targeted fibrinolytic therapy 
Nanomedicine (London, England)  2012;7(7):1017-1028.
Current thrombolytic therapies rely upon exogenous plasminogen activators (PA) to effectively lyse clots, thereby restoring blood flow and preventing tissue and organ death. Yet, these PAs may also impair normal hemostasis which may lead to life-threatening bleeding, including intracerebral hemorrhage. Thus, the aim of this current study is to develop new thrombus-targeted fibrinolytic agents that harness the multifunctional theranostic capabilities of nanomaterials, potentially allowing for the generation of efficacious thrombolytics while minimizing deleterious side effects.
Materials and Methods
A thrombus-targeted nano-fibrinolytic agent (CLIO-FXIII-PEG-tPA) was synthesized using a magnetofluorescent crosslinked dextran-coated iron oxide (CLIO) nanoparticle platform that was conjugated to recombinant tissue plasminogen activator (tPA). Thrombus-targeting was achieved by derivatizing the nanoparticle with an activated factor XIII (FXIIIa)-sensitive peptide based on the amino terminus of α2-antiplasmin. Human plasma clot binding ability of the targeted and control agents was assessed by fluorescence reflectance imaging. Next, the in vitro enzymatic activity of the agents was assessed by S2288-based amidolytic activity, and an ELISA D-dimer assay for fibrinolysis. In vivo targeting of the nanoagent was next examined by intravital fluorescence microscopy of murine arterial and venous thrombosis. The fibrinolytic activity of the targeted nanoagent compared to free tPA was then evaluated in vivo in murine pulmonary embolism.
In vitro, the targeted thrombolytic nanoagent demonstrated binding to fresh frozen plasma (FFP) clots superior to control nanoagents (ANOVA p < 0.05). On a weight (mg) basis, the S2288 amidolytic efficiency of the targeted nanoagent was approximately 15% reduced compared to free tPA. When normalized by S2288-based activity, targeted, control, and free tPA samples demonstrated equivalent in vitro fibrinolytic activity against human plasma clots, as determined by ELISA D-dimer assays. The FXIIIa targeted fibrinolytic nanoagent efficiently bound the margin of intravascular thrombi as detected by IVFM. In in vivo fibrinolysis studies normalized for activity, the FXIIIa-targeted agent lysed pulmonary emboli with similar efficacy as free tPA (p>0.05).
The applicability of a FXIIIa-targeted thrombolytic nanoagent in the treatment of thromboembolism was demonstrated in vitro and in vivo. Future studies are planned to investigate the safety profile and overall efficacy of this class of nanoagents, and to further optimize their thrombus-targeting profile and lytic action.
PMCID: PMC3360120  PMID: 22348271
Fibrinolytic; iron oxide; therapy; thrombosis; multimodal; theranostic; imaging
4.  Cost effectiveness of a telephone intervention to promote dilated fundus examination in adults with diabetes mellitus 
In a clinical trial, we have previously shown that a telephone intervention can significantly increase participation in dilated fundus examination (DFE) screening among low-income adults with diabetes. Here the costs and cost-effectiveness ratio of this intervention are calculated. Intervention effectiveness was estimated as the difference in DFE utilization between the telephone intervention and print groups from the clinical trial multiplied by the size of the telephone intervention group. A micro-costing approach was used. Personnel time was aggregated from logs kept during the clinical trial of the intervention. Wage rates were taken from a commercial compensation database. Telephone charges were estimated based on prevailing fees. The cost-effectiveness ratio was calculated as the ratio of total costs of the intervention to the number of DFEs gained by the intervention. A sensitivity analysis estimated the cost-effectiveness of a more limited telephone intervention. A probabilistic sensitivity analysis using bootstrap samples from the clinical trial results quantified the uncertainties in resource utilization and intervention effectiveness. Net intervention costs were US$18,676.06, with an associated gain of 43.7 DFEs and 16.4 new diagnoses of diabetic retinopathy. The cost-effectiveness ratio is US$427.37 per DFE gained. A restricted intervention limiting the number of calls to 5, as opposed to 7, would achieve the same results, but would cost approximately 17% less. In the probabilistic sensitivity analysis, the 5th and 95th percentiles of the cost-effectiveness ratio were US$304.05 and US$692.52 per DFE gained, respectively. Our telephone intervention is more expensive than simple mail or telephone reminders used in other settings to promote preventive care; it is, however, also considerably more effective, and is effective in a low-income minority population at greater risk for diabetes complications. The costs are dominated by labor costs, and may be substantially defrayed, without loss of effectiveness, by restricting the number of telephone calls to 5 per patient.
PMCID: PMC2699788  PMID: 19668428
cost-effectiveness; diabetes mellitus; dilated fundus examination
5.  Knowledge, Attitudes, and Beliefs about Dilated Eye Examinations among African-Americans 
To understand factors that influence African-Americans’ attitude toward eye examinations.
Ten focus groups were conducted with 86 African-Americans. Four focus groups were conducted with people 65 years of age and older who had not received a dilated fundus examination (DFE) in the past 2 years, two groups were held with people 65 years of age and older who had had a recent DFE, and two groups each were held with people 40 to 64 years of age, with and without recent DFEs. Focus group interviews were conducted by using a moderator guide to address perceived benefits of and barriers to getting an eye examination; motivators for getting DFEs; and knowledge of eye examinations, glaucoma, and diabetic retinopathy. Participants also completed a questionnaire that provided demographic information. Quantitative and qualitative analyses were conducted.
Cost or lack of sufficient insurance was identified as the most important barrier to getting a DFE. Also frequently mentioned was not having any symptoms and being too busy. The most frequently cited benefit of getting a DFE was to help prevent eye disease, whereas the most frequently reported motivating factor was experiencing a vision problem. Regarding knowledge, many people did not know the risk factors for glaucoma, but seemed to have a better understanding of how to reduce the effects of diabetes on their eyes.
Study findings identified important links between financial resources and experiencing a vision problem and the adoption of preventive eye care in an urban African-American population.
PMCID: PMC1978096  PMID: 17460251
6.  How frequently should asymptomatic patients be dilated? 
Journal of Optometry  2013;7(1):57-61.
To determine if routine dilated fundus examination (DFE) should be performed sooner than at 10-year intervals in asymptomatic patients.
Records for all patients consecutively evaluated in a one-year time frame were systematically reviewed. Of those patients who received initial DFE and were living 10 years later, records for sequential DFE were again evaluated to determine presence of clinically-significant, peripheral retinal findings. Databases were also searched in order to determine the number of patients during the same 10-year time period who developed vision or life-threatening peripheral retinal findings. The two groups were cross-matched to determine effectiveness of routine DFE.
Only 10 of 592 patients were deemed to have “clinically-significant” peripheral retinal findings—none of whom developed untoward outcomes. Of the 29 new retinal detachments and four intraocular tumors discovered during ten years of clinical follow-up, nearly 90% were symptomatic at the time of discovery. Three detachments and one tumor were detected as incidental findings in asymptomatic patients. No further treatment was recommended for the three detachments and the patient with the tumor survives, although with profound loss of vision in the involved eye.
In the absence of symptoms, routine DFE seems to have a very low yield for discovery of serious ocular events and appears to be ineffective in altering the course of incidental findings. Routine DFE is not indicated for older, asymptomatic patients—even at decade intervals. The findings of this study should be prospectively confirmed in population-based studies.
PMCID: PMC3938739  PMID: 24646902
Pupillary dilation; Frequency; Diagnostic yield; Dilatación pupilar; Frecuencia; Rendimiento diagnóstico
7.  Fibrin-Targeted Magnetic Resonance Imaging Allows In Vivo Quantification of Thrombus Fibrin Content and Identifies Thrombi Amenable for Thrombolysis 
Deep venous thrombosis is a major health problem. Thrombolytic therapies are effective in recanalizing the veins and preventing post-thrombotic complications, but there is no consensus on selection criteria. The aim of this study was to investigate a fibrin-specific MRI contrast agent (EP-2104R) for the accurate quantification of thrombus’ fibrin content in vivo and for the identification of thrombus suitable for thrombolysis.
Approach and Results
Venous thrombosis was induced in the inferior vena cava of 8- to 10-week-old male BALB/C mice and MRI performed 2, 4, 7, 10, 14, and 21 days later. Eighteen mice were scanned at each time point pre and 2 hours post injection of EP-2104R (8.0 μmol/kg) with 12 mice at each time point used to correlate fibrin contrast uptake with thrombus’ histological stage and fibrin content. Six mice at each time point were immediately subjected to intravascular thrombolytic therapy (10 mg/kg of tissue-type plasminogen activator). Mice were imaged to assess response to lytic therapy 24 hours after thrombolytic treatment. Two mice at each time point were scanned post injection of 0.2 mmol/kg of Gd-DTPA (gadolinium with diethylenetriaminepentacetate, Magnevist, Schering AG, Berlin, Germany) for control purpose. Contrast uptake was correlated positively with the fibrin content of the thrombus measured by Western blotting (R2=0.889; P<0.001). Thrombus relaxation rate (R1) post contrast and the change in visualized thrombus size on late gadolinium enhancement inversion recovery MRI pre–EP-2104R and post–EP-2104R injection were the best predictors for successful thrombolysis (area under the curve, 0.989 [95% confidence interval, 0.97–1.00] and 0.994 [95% confidence interval, 0.98–1.00] respectively).
MRI with a fibrin-specific contrast agent accurately estimates thrombus fibrin content in vivo and identifies thrombi that are amenable for thrombolysis.
PMCID: PMC4195984  PMID: 24723557
fibrin; magnetic resonance imaging; molecular imaging; thrombolytic therapy; venous thrombosis
8.  Telephone Intervention to Promote Diabetic Retinopathy Screening Among the Urban Poor 
Participation in diabetic retinopathy screening is suboptimal. The Vision is Precious study (2001–2005) tested the hypothesis that a tailored telephone intervention in urban minority diabetes populations, offered in English or Spanish, would result in greater screening for retinopathy than a standard print intervention.
Randomized controlled trial
Subjects (N=598) were adults with diabetes without a dilated fundus examination (DFE) in >1 year from three healthcare centers in Bronx NY.
A tailored telephone intervention to promote retinopathy screening compared to a standard print intervention over a 6-month period.
Main outcome measures
Documentation of a DFE within 6 months was the main outcome. Data on risk perceptions using the Risk Perception Survey for Diabetes were collected pre- and post-intervention. Electronic databases were used to obtain hemoglobin A1c information.
Subjects were 40% men, mean age 57 years; 39% reported household incomes as <$15K; 45% reported their race as black, and 42% reported ethnicity as Hispanic/Latino; 23% chose Spanish as their preferred language. Data were analyzed in 2006. There was a 74% increase in retinopathy screening in the telephone versus print group (p<0.0005), with no differences by intervention language or by gender. Predictors of undergoing a DFE included: telephone intervention, baseline risk-perception scores indicating less worry and more realism about diabetes complications, and the interaction of self-reported worry and being in the telephone intervention. Subjects who had poor diabetes control responded with greater success to telephone interventions.
A limited telephone intervention can improve significantly participation in retinopathy screening in a minority, low-income population. This intervention influenced risk perceptions about diabetes complications. Further research is needed to develop effective risk communications to prevent the complications of diabetes.
PMCID: PMC2272534  PMID: 18312805
9.  Tailored and targeted interventions to encourage dilated fundus examinations in older African Americans 
Archives of ophthalmology  2011;129(12):1592-1598.
To compare the effects of a tailored and targeted print intervention in promoting dilated fundus examinations (DFEs) in older African Americans, and to determine if other factors are associated with getting a DFE.
African Americans, 65 years of age or older, who had not had a DFE in at least two years were recruited from community settings. Participants were randomized to receive either a tailored or targeted newsletter. Telephone follow-up was conducted at one, three, and six months to ascertain eye examination status. All self-reported DFEs were confirmed by contacting their eye doctor by telephone.
Main outcome measure
Doctor-confirmed DFE at six months.
Of the 329 participants enrolled, 128 (38.9%) had a doctor-confirmed DFE. There was no difference in doctor-confirmed DFEs by intervention group (RR=1.07, 0.82–1.40 CI), with 66 participants in the tailored group (40.2%) and 62 (37.6%) participants in the targeted group having a doctor-confirmed DFE. Based on logistic regression analysis, reading the newsletter (OR=1.76, 1.08–2.87 CI) and planning on making an appointment for a DFE (OR=2.46, 1.42–4.26 CI) were significant predictors for getting a DFE.
The tailored and targeted interventions were equally effective in promoting doctor-confirmed DFEs at six months. Given the increases cost and effort associated with tailoring, our results suggest that well-designed targeted print messages can motivate older African Americans to get DFEs.
PMCID: PMC3534730  PMID: 22159679
10.  Thrombolytic Effects of the Snake Venom Disintegrin Saxatilin Determined by Novel Assessment Methods: A FeCl3-Induced Thrombosis Model in Mice 
PLoS ONE  2013;8(11):e81165.
Saxatilin, a novel disintegrin purified and cloned from the venom of the Korean snake Gloydius saxatilis, strongly inhibits activation and aggregation of platelets. Glycoprotein (GP) IIb/IIIa receptor antagonists can resolve thrombus, so saxatilin might also have thrombolytic effects. We investigated the thrombolytic effects of saxatilin in mice using a ferric chloride-induced carotid arterial thrombosis model. Thrombotic occlusion and thrombus resolution were evaluated quantitatively by measuring blood flow in the carotid artery with an ultrasonic flow meter and calculating the degree of flow restoration on a minute-by-minute basis; results were confirmed by histological examination. Saxatilin dissolved thrombi in a dose-dependent manner. Saxatilin at 5 mg/kg restored blood flow to baseline levels. As saxatilin dose increased, time to recanalization decreased. A bolus injection of 10% of a complete dose with continuous infusion of the remaining dose for 60 minutes resulted in effective recanalization without reocclusion. The thrombolytic effect of saxatilin was also demonstrated in vitro using platelet aggregometry by administering saxatilin in preformed thrombi. Bleeding complications were observed in 2 of 71 mice that received saxatilin. Fibrin/fibrinogen zymography and platelet aggregometry studies indicated that saxatilin does not have fibrinolytic activity, but exerted its action on platelets. Integrin-binding assays showed that saxatilin inhibited multiple integrins, specifically α2bβ3 (GP IIb/IIIa), α5β1, αvβ3, αvβ1, and αvβ5, which act on platelet adhesion/aggregation. Saxatilin inhibited multiple integrins by acting on platelets, and was safe and effective in resolving thrombi in mice.
PMCID: PMC3832438  PMID: 24260554
11.  Date Fruit Extract Is a Neuroprotective Agent in Diabetic Peripheral Neuropathy in Streptozotocin-Induced Diabetic Rats: A Multimodal Analysis 
Background. To study the effects of an aqueous extract of date fruit (Phoenix dactylifera L. Arecaceae) diet on diabetic polyneuropathy (DPN) in streptozotocin- (STZ-) induced diabetic rats. Methods. The effects of a date fruit extract (DFE) diet on diabetic neuropathy in STZ-induced diabetic rats were evaluated and compared with a nondiabetic control group, diabetic control group (sham), and vehicle group with respect to the following parameters: open field behavioral test, motor nerve conduction velocity (MNCV), and morphological observations. Results. In the model of STZ-induced of diabetic neuropathy, chronic treatment for 6 weeks with DFE counteracted the impairment of the explorative activity of the rats in an open field behavioral test and of the conduction velocity of the sciatic nerve (MNCV). In addition, pretreatment with DFE significantly reversed each nerve diameter reduction in diabetic rats. Conclusion. DFE treatment shows efficacy for preventing diabetic deterioration and for improving pathological parameters of diabetic neuropathy in rats, as compared with control groups.
PMCID: PMC3236446  PMID: 22191015
12.  Biochemical toxicology of unsaturated halogenated monomers. 
Previous inhalation toxicity studies from our laboratory have shown that 1,1-dichloroethylene (1,1-DCE), 1,1-dibromoethylene (1,1-DBE), and 2-chloro-1,3,-butadiene (2-CBD) are more toxic to fasted rats than to fed rats. Vinyl chloride monomer (VCM) and 1,1-difluoroethylene (1,1-DFE) were not acutely hepatotoxic at 46,500 and 82,000 ppm, respectively, in normal male rats, whether fed or fasted. On a molar basis, 1,1-DBE and 1,1-DCE have similar toxicities while 2-CBD is less toxic. All three compounds produce similar elevation of serum transaminase and bloody ascites, although at differing times following differing exposure concentrations. 1,1-DCE produces massive midzonal hepatic necrosis with hepatic thrombosis and chromatolysis within 2 hr after a 4 hr exposure of fasted rats to 200 ppm. Subsequent to formation of this midzonal lesion, the central portion of the lobule collapses, accompanied by congestion, ascites, and in increased hematocrit in the rat. Serum transaminase and sorbital dehydrogenase are greatly elevated at 6 hr. This effect in fasted rats is associated with glutathione (GSH) depletion. Diethyl maleate (DEM) which depletes GSH in fed rats potentiates the injury associated with 1,1-DCE exposure as well as that produced by 2-CBD. Rats fed ad libitum and exposed to 1,1-DCE or 2-CBD at night, a time of low hepatic GSH concentration, exhibit enhancement of hepatotoxic response when compared to animals exposed during the day when GSH is high.
PMCID: PMC1475211  PMID: 1175549
13.  The Year in Evolutionary Biology 
The rates and properties of new mutations affecting fitness have implications for a number of outstanding questions in evolutionary biology. Obtaining estimates of mutation rates and effects has historically been challenging, and little theory has been available for predicting the distribution of fitness effects (DFE); however, there have been recent advances on both fronts. Extreme-value theory predicts the DFE of beneficial mutations in well-adapted populations, while phenotypic fitness landscape models make predictions for the DFE of all mutations as a function of the initial level of adaptation and the strength of stabilizing selection on traits underlying fitness. Direct experimental evidence confirms predictions on the DFE of beneficial mutations and favors distributions that are roughly exponential but bounded on the right. A growing number of studies infer the DFE using genomic patterns of polymorphism and divergence, recovering a wide range of DFE. Future work should be aimed at identifying factors driving the observed variation in the DFE. We emphasize the need for further theory explicitly incorporating the effects of partial pleiotropy and heterogeneity in the environment on the expected DFE.
PMCID: PMC4282485  PMID: 24891070
mutation; distribution of fitness effects; experimental evolution; population genomics; mutational landscape models
14.  Effect of dragon fruit extract on oxidative stress and aortic stiffness in streptozotocin-induced diabetes in rats 
Pharmacognosy Research  2010;2(1):31-35.
Cardiovascular complications are consistently observed in diabetic patients across all age groups. The objective of the present study was to investigate the effect of aqueous extract of the fruit pulp of Hylocereus undatus (DFE) on aortic stiffness and oxidative stress in streptozotocin (STZ)-induced diabetes in rats. Twenty-four male, Sprague-Dawley rats were randomized into four groups: I (control), II (diabetic), III (DFE, 250 mg/kg) and IV (DFE 500 mg/kg). Diabetes was induced in groups II, III and IV by intraperitoneal (i.p.) injection of STZ (40 mg/kg). After confirmation of diabetes, group III and IV received DFE for 5 weeks. Pulse wave velocity (PWV) was used as a marker of aortic stiffness and was determined at the end of 5 weeks. DFE significantly decreased (P < 0.05) the fasting blood glucose levels in diabetic rats, but not to normal levels. Systolic blood pressure, pulse pressure and PWV were significantly increased (P < 0.05) in diabetic rats at the end of 5 weeks in comparison with control group. DFE treatment significantly decreased (P < 0.05) these elevations. Oxidative damage was observed in group II after 5 weeks. Plasma malondialdehyde levels significantly decreased (P < 0.05), while superoxide dismutase and total antioxidant capacity significantly increased (P < 0.05) with DFE treatment in comparison with group II. These data demonstrate that DFE treatment was effective in controlling oxidative damage and decreasing the aortic stiffness measured by PWV in STZ-induced diabetes in rats.
PMCID: PMC3140125  PMID: 21808536
Arterial stiffness; diabetes; Hylocereus undatus; pulse wave velocity; streptozotocin
15.  Thrombolysis with human extrinsic (tissue-type) plasminogen activator in dogs with femoral vein thrombosis. 
Journal of Clinical Investigation  1982;69(3):573-580.
Extrinsic (tissue-type) plasminogen activator (plasminogen activator) was isolated either as a single-chain or as a two-chain molecule from the culture medium of a human melanoma cell line. The thrombolytic activity of both molecular forms of activator was investigated in beagle dogs with an experimental femoral vein thrombosis and compared with that of urokinase. The 125I-fibrinogen-labeled thrombus was formed in an isolated 4-cm segment of the vein, aged for 30 min, and the thrombolytic substances were infused over a 4-h period. The degree of thrombolysis was measured 2 h later as the difference between the injected and recovered 125I. In six control animals with a saline infusion the extent of thrombolysis was 16.3 +/- 3.8% (mean +/- SEM), in five dogs receiving 100,000 IU urokinase, 17.4 +/- 3.7% (P less than 0.4) and in four dogs with 1,000,000 IU urokinase 40.6 +/- 4.8% (P less than 0.001). Infusion of 100.000 IU single-chain plasminogen activator in five dogs resulted in 3.5 +/- 7.8% lysis (P less than 0.05) and of 100,000 IU two-chain plasminogen activator in five dogs in 60.1 +/- 10.8% (P less than 0.001). Infusion of 300,000 IU one-chain plasminogen activator yielded 57.5% lysis and of the same amount of two-chain plasminogen activator 72.9%. Significant activation of plasminogen, consumption of alpha 2-antiplasmin, and fibrinogen breakdown in plasma was only observed in animals receiving the high doses of urokinase but not in the saline, plasminogen activator, or the low-dose urokinase groups. It is thus concluded that in this thrombosis model human extrinsic plasminogen activator has a higher specific thrombolytic effect that urokinase. Plasminogen activator also appears to induce thrombolysis without systemic fibrinolytic activation and fibrinogen breakdown.
PMCID: PMC371013  PMID: 7199539
16.  Monocyte urokinase-type plasminogen activator up-regulation reduces thrombus size in a model of venous thrombosis 
Journal of Vascular Surgery  2009;50(5):1127-1134.
Our previous studies showed that the direct injection of an adenovirus construct expressing urokinase-type plasminogen activator (uPA) into experimental venous thrombi significantly reduces thrombus weight. The systemic use of adenovirus vectors is limited by inherent hepatic tropism and inflammatory response. As macrophages are recruited into venous thrombi, it is reasonable to speculate that these cells could be used to target the adenovirus uPA (ad-uPA) gene construct to the thrombus. The aims of this study were to determine whether macrophages transduced with ad-uPA have increased fibrinolytic activity and whether systemic injection of transduced cells could be used to target uPA expression to the thrombus and reduce its size.
The effect of up-regulating uPA was examined in an immortalized macrophage cell line (MM6) and macrophages differentiated from human blood monocyte-derived macrophages (HBMMs). Cells were infected with ad-uPA or blank control virus (ad-blank). Fibrinolytic mediator expression, cell viability, and cytokine expression were measured by activity assays and enzyme-linked immunosorbent assays. Monocyte migration was measured using a modified Boyden chamber assay. A model of venous thrombosis was developed and characterized in mice with severe combined immunodeficiency (SCID). This model was used to study whether systemically administered macrophages over-expressing uPA reduced thrombus size. Uptake of HBMMs into the thrombus induced in these mice was confirmed by a combination of PKH2-labeled cell tracking and colocalization with human leukocyte antigen (HLA) by immunohistology.
Compared with ad-blank, treated HBMMs transduction with ad-uPA increased uPA production by >1000-fold (P = .003), uPA activity by 150-fold (P = .0001), and soluble uPA receptor (uPAR) by almost twofold (P = .043). Expression of plasminogen activator inhibitor (PAI-1) and PAI-2 was decreased by about twofold (P = .011) and threefold (P = .005), respectively. Up-regulation of uPA had no effect on cell viability or inflammatory cytokine production compared with ad-blank or untreated cells. Ad-uPA transduction increased the migration rate of HBMMs (about 20%, P = .03) and MM6 cells (>twofold, P = .005) compared with ad-blank treated controls. Human macrophage recruitment into the mouse thrombus was confirmed by the colocalization of HLA with the PKH2-marked cells. Systemic injection of uPA-up-regulated HBMMs reduced thrombus weight by approximately 20% compared with ad-blank (P = .038) or sham-treated controls (P = .0028).
Transduction of HBBM with ad-uPA increases their fibrinolytic activity. Systemic administration of uPA up-regulated HBBMs reduced thrombus size in an experimental model of venous thrombosis. Alternative methods of delivering fibrinolytic agents are worth exploring.
Clinical Relevance
The use of thrombolysis in the treatment of acute iliofemoral deep vein thrombosis is not suitable for all patients. Our previous studies have shown that direct injection of an adenovirus construct expressing urokinase plasminogen activator (uPA) into experimental venous thrombi significantly reduced thrombus weight. The systemic use of adenovirus vectors is, however, limited by both their inherent hepatic tropism, which precludes targeted delivery to disease sites, and by the associated host inflammatory response. As macrophages are recruited into venous thrombi, these cells could be used to target uPA gene constructs to the thrombus after systemic administration.
PMCID: PMC2778796  PMID: 19703758
17.  The distribution of fitness effects of new beneficial mutations in Pseudomonas fluorescens 
Biology Letters  2010;7(1):98-100.
Theoretical studies of adaptation emphasize the importance of understanding the distribution of fitness effects (DFE) of new mutations. We report the isolation of 100 adaptive mutants—without the biasing influence of natural selection—from an ancestral genotype whose fitness in the niche occupied by the derived type is extremely low. The fitness of each derived genotype was determined relative to a single reference type and the fitness effects found to conform to a normal distribution. When fitness was measured in a different environment, the rank order changed, but not the shape of the distribution. We argue that, even with detailed knowledge of the genetic architecture underpinning the adaptive types (as is the case here), the DFEs remain unpredictable, and we discuss the possibility that general explanations for the shape of the DFE might not be possible in the absence of organism-specific biological details.
PMCID: PMC3030884  PMID: 20659918
mutation; adaptation; evolution
18.  Statistical Optimization of Fibrinolytic Enzyme Production Using Agroresidues by Bacillus cereus IND1 and Its Thrombolytic Activity In Vitro 
BioMed Research International  2014;2014:725064.
A potent fibrinolytic enzyme-producing Bacillus cereus IND1 was isolated from the Indian food, rice. Solid-state fermentation was carried out using agroresidues for the production of fibrinolytic enzyme. Among the substrates, wheat bran supported more enzyme production and has been used for the optimized enzyme production by statistical approach. Two-level full-factorial design demonstrated that moisture, supplementation of beef extract, and sodium dihydrogen phosphate have significantly influenced enzyme production (P < 0.05). A central composite design resulted in the production of 3699 U/mL of enzyme in the presence of 0.3% (w/w) beef extract and 0.05% (w/w) sodium dihydrogen phosphate, at 100% (v/w) moisture after 72 h of fermentation. The enzyme production increased fourfold compared to the original medium. This enzyme was purified to homogeneity by ammonium sulfate precipitation, diethylaminoethyl-cellulose ion-exchange chromatography, Sephadex G-75 gel filtration chromatography, and casein-agarose affinity chromatography and had an apparent molecular mass of 29.5 kDa. The optimum pH and temperature for the activity of fibrinolytic enzyme were found to be 8.0 and 60°C, respectively. This enzyme was highly stable at wide pH range (7.0–9.0) and showed 27% ± 6% enzyme activity after initial denaturation at 60°C for 1 h. In vitro assays revealed that the enzyme could activate plasminogen and significantly degraded the fibrin net of blood clot, which suggests its potential as an effective thrombolytic agent.
PMCID: PMC4070475  PMID: 25003130
19.  Effects of organic extracts of six Bangladeshi plants on in vitro thrombolysis and cytotoxicity 
Thrombus formed in blood vessels lead to atherothrombotic diseases such as myocardial or cerebral infarction. Thrombolytic agents are used to dissolve the already formed clots in the blood vessels; however, these drugs sometimes cause serious and fatal consequences. Herbal preparations have been used since ancient times for the treatment of several diseases although they show little toxicity in some cases. Aqueous extracts of herbs used in thrombolysis have been reported before with cytotoxic data, however, the organic extracts of herbs have not been documented. This study aims to investigate whether organic extracts possess thrombolytic properties with minimal or no toxicity.
An in vitro thrombolytic model was used to check the clot lysis effect of six Bangladeshi herbal extracts viz., Ageratum conyzoides L., Clausena suffruticosa, Leea indica (Burm.f.) Merr., Leucas aspera Willd., Senna sophera L. Roxb., and Solanum torvum Swartz. using streptokinase as a positive control and water as a negative control. Briefly, venous blood drawn from twenty healthy volunteers was allowed to form clots which were weighed and treated with the test plant materials to disrupt the clots. Weight of clot after and before treatment provided a percentage of clot lysis. Cytotoxicity was screened by brine shrimp lethality bioassay using vincristine sulfate as positive control.
Using an in vitro thrombolytic model, Ageratum conyzoides, Clausena suffruticosa, Leea indica, Leucas aspera, Senna sophera and Solanum torvum showed 18.12 ± 2.34%, 48.9 ± 2.44%, 39.30 ± 0.96%, 37.32 ± 2.00%, 31.61 ± 2.97% and 31.51 ± 0.57% and clot lysis respectively. Among the herbs studied Clausena suffruticosa, Leea indica and Leucas aspera showed very significant (p < 0.0001) percentage (%) of clot lysis compared to reference drug streptokinase (75.00 ± 3.04%). In brine shrimp cytotoxic assay, the extracts Ageratum conyzoides, Clausena suffruticosa, Leea indica, Leucas aspera, Senna sophera and Solanum torvum showed LC50 values 508.86 ± 6.62,41.16 ± 1.26, 2.65 ± 0.16, 181.67 ± 1.65, 233.37 ± 7.74 and 478.40 ± 3.23 μg/ml, respectively, with reference to vincristine sulfate (LC50 0.76 ± 0.04).
Through our study it was found that Clausena suffruticosa, Leea indica and Leucas aspera possessed effective thrombolytic properties whereas Senna sophera and Solanum torvum showed moderate to mild thrombolytic effects while Ageratum conyzoides showed no significant effect. No extract was found cytoxic compared to positive control. Clausena suffruticosa, Leea indica and Leucas aspera could be incorporated as a thrombolytic agent with in vivo effects to improve the atherothrombotic patients. However, Clausena suffruticosa could be the best one to use in this purpose.
PMCID: PMC3567938  PMID: 23363538
Thrombolysis; Clausena suffruticosa; Leea indica; Leucas aspera; Streptokinase
20.  Coronary thrombosis: In vivo, ex vivo and in vitro 
BMJ Case Reports  2009;2009:bcr09.2008.0983.
Acute stent thrombosis remains one of the most important concerns in clinical cardiology. The mechanism is not fully understood but a prothrombotic state is a key component. We describe a case of acute stent thrombosis, within an hour of rescue angioplasty, despite use of full dose fibrinolytic (reteplase) and antiplatelet therapy (aspirin and clopidogrel). Risk of acute stent thrombosis was predicted an hour earlier, when the patient was clinically well, by a novel near-patient test of thrombotic and thrombolytic status (in vitro). Subsequent stent thrombosis was visualised angiographically (in vivo) and confirmed by extraction of the thrombus (ex vivo). The near-patient test sensitively detected reversal of the prothrombotic state after abciximab treatment. We believe this is the first description of the clinical use of a near-patient test within the cardiac catheterisation laboratory to predict risk of imminent stent thrombosis.
PMCID: PMC3027665  PMID: 21686525
21.  Approximation to the Distribution of Fitness Effects across Functional Categories in Human Segregating Polymorphisms 
PLoS Genetics  2014;10(11):e1004697.
Quantifying the proportion of polymorphic mutations that are deleterious or neutral is of fundamental importance to our understanding of evolution, disease genetics and the maintenance of variation genome-wide. Here, we develop an approximation to the distribution of fitness effects (DFE) of segregating single-nucleotide mutations in humans. Unlike previous methods, we do not assume that synonymous mutations are neutral or not strongly selected, and we do not rely on fitting the DFE of all new nonsynonymous mutations to a single probability distribution, which is poorly motivated on a biological level. We rely on a previously developed method that utilizes a variety of published annotations (including conservation scores, protein deleteriousness estimates and regulatory data) to score all mutations in the human genome based on how likely they are to be affected by negative selection, controlling for mutation rate. We map this and other conservation scores to a scale of fitness coefficients via maximum likelihood using diffusion theory and a Poisson random field model on SNP data. Our method serves to approximate the deleterious DFE of mutations that are segregating, regardless of their genomic consequence. We can then compare the proportion of mutations that are negatively selected or neutral across various categories, including different types of regulatory sites. We observe that the distribution of intergenic polymorphisms is highly peaked at neutrality, while the distribution of nonsynonymous polymorphisms has a second peak at . Other types of polymorphisms have shapes that fall roughly in between these two. We find that transcriptional start sites, strong CTCF-enriched elements and enhancers are the regulatory categories with the largest proportion of deleterious polymorphisms.
Author Summary
The relative frequencies of polymorphic mutations that are deleterious, nearly neutral and neutral is traditionally called the distribution of fitness effects (DFE). Obtaining an accurate approximation to this distribution in humans can help us understand the nature of disease and the mechanisms by which variation is maintained in the genome. Previous methods to approximate this distribution have relied on fitting the DFE of new mutations to a single probability distribution, like a normal or an exponential distribution. Generally, these methods also assume that a particular category of mutations, like synonymous changes, can be assumed to be neutral or nearly neutral. Here, we provide a novel method designed to reflect the strength of negative selection operating on any segregating site in the human genome. We use a maximum likelihood mapping approach to fit these scores to a scale of neutral and negative fitness coefficients. Finally, we compare the shape of the DFEs we obtain from this mapping for different types of functional categories. We observe the distribution of polymorphisms has a strong peak at neutrality, as well as a second peak of deleterious effects when restricting to nonsynonymous polymorphisms.
PMCID: PMC4222666  PMID: 25375159
22.  Fibrolase: Trials and Tribulations 
Toxins  2010;2(4):793-808.
Fibrolase is the fibrinolytic enzyme isolated from Agkistrodon contortrix contortrix (southern copperhead snake) venom. The enzyme was purified by a three-step HPLC procedure and was shown to be homogeneous by standard criteria including reverse phase HPLC, molecular sieve chromatography and SDS-PAGE. The purified enzyme is a zinc metalloproteinase containing one mole of zinc. It is composed of 203 amino acids with a blocked amino-terminus due to cyclization of the terminal Gln residue. Fibrolase shares a significant degree of homology with enzymes of the reprolysin sub-family of metalloproteinases including an active site homology of close to 100%; it is rapidly inhibited by chelating agents such as EDTA, and by alpha2-macroglobulin (α2Μ). The enzyme is a direct-acting thrombolytic agent and does not rely on plasminogen for clot dissolution. Fibrolase rapidly cleaves the A(α)-chain of fibrinogen and the B(β)-chain at a slower rate; it has no activity on the γ-chain. The enzyme exhibits the same specificity with fibrin, cleaving the α-chain more rapidly than the β-chain. Fibrolase was shown to have very effective thrombolytic activity in a reoccluding carotid arterial thrombosis model in the canine. A recombinant version of the enzyme was made in yeast by Amgen, Inc. (Thousand Oaks, CA, USA) and called alfimeprase. Alfimeprase is identical to fibrolase except for a two amino acid truncation at the amino-terminus and the insertion of a new amino-terminal amino acid in the truncated protein; these changes lead to a more stable enzyme for prolonged storage. Alfimeprase was taken into clinical trials by Nuvelo, Inc. (San Carlos, CA), which licensed the enzyme from Amgen. Alfimeprase was successful in Phase I and II clinical trials for peripheral arterial occlusion (PAO) and central venous access device (CVAD) occlusion. However, in Phase III trials alfimeprase did not meet the expected end points in either PAO or CVAD occlusion and in a Phaase II stroke trial, and Nuvelo dropped further development in 2008.
PMCID: PMC3153196  PMID: 22069611
fibrolase; alfimeprase; thrombolysis; peripheral arterial occlusion; animal models; central venous access device occlusion; stroke; alpha2 macroglobulin; metalloproteinase
23.  Mutant Prourokinase with Adjunctive C1-Inhibitor Is an Effective and Safer Alternative to tPA in Rat Stroke 
PLoS ONE  2011;6(7):e21999.
A single-site mutant (M5) of native urokinase plasminogen activator (prouPA) induces effective thrombolysis in dogs with venous or arterial thrombosis with a reduction in bleeding complications compared to tPA. This effect, related to inhibition of two-chain M5 (tcM5) by plasma C1-inhibitor (C1I), thereby preventing non-specific plasmin generation, was augmented by the addition of exogenous C1I to plasma in vitro. In the present study, tPA, M5 or placebo +/− C1I were administered in two rat stroke models. In Part-I, permanent MCA occlusion was used to evaluate intracranial hemorrhage (ICH) by the thrombolytic regimens. In Part II, thromboembolic occlusion was used with thrombolysis administered 2 h later. Infarct and edema volumes, and ICH were determined at 24 h, and neuroscore pre (2 h) and post (24 h) treatment. In Part I, fatal ICH occurred in 57% of tPA and 75% of M5 rats. Adjunctive C1I reduced this to 25% and 17% respectively. Similarly, semiquantitation of ICH by neuropathological examination showed significantly less ICH in rats given adjunctive C1I compared with tPA or M5 alone. In Part-II, tPA, M5, and M5+C1I induced comparable ischemic volume reductions (>55%) compared with the saline or C1I controls, indicating the three treatments had a similar fibrinolytic effect. ICH was seen in 40% of tPA and 50% of M5 rats, with 1 death in the latter. Only 17% of the M5+C1I rats showed ICH, and the bleeding score in this group was significantly less than that in either the tPA or M5 group. The M5+C1I group had the best Benefit Index, calculated by dividing percent brain salvaged by the ICH visual score in each group. In conclusion, adjunctive C1I inhibited bleeding by M5, induced significant neuroscore improvement and had the best Benefit Index. The C1I did not compromise fibrinolysis by M5 in contrast with tPA, consistent with previous in vitro findings.
PMCID: PMC3136496  PMID: 21779364
24.  A 2.8 Å Fe-Fe Separation in the Fe2III/IV Intermediate (X) from Escherichia coli Ribonucleotide Reductase 
Journal of the American Chemical Society  2013;135(45):16758-16761.
A class Ia ribonucleotide reductase (RNR) employs a µ-oxo-Fe2III/III/tyrosyl radical cofactor in its β subunit to oxidize a cysteine residue ~ 35 Å away in its α subunit; the resultant cysteine radical initiates substrate reduction. During self-assembly of the Escherichia coli RNR-β cofactor, reaction of the protein’s Fe2II/II complex with O2 results in accumulation of an Fe2III/IV cluster, termed X, which oxidizes the adjacent tyrosine (Y) 122 to the radical (Y122•) as the cluster is converted to the µ-oxo-Fe2III/III product. As the first high-valent non-heme-iron enzyme complex to be identified and the key activating intermediate of class Ia RNRs, X has been the focus of intensive efforts to determine its structure. Initial characterization by extended X-ray absorption fine structure (EXAFS) spectroscopy yielded a 2.5 Å Fe-Fe separation (dFe-Fe), which was interpreted to imply the presence of three single-atom bridges (O2−, HO−, and/or µ-1,1-carboxylates). This short dFe-Fe has been irreconcilable with computational and synthetic models, which all have dFe-Fe ≥ 2.7 Å. To resolve this conundrum, we revisited the EXAFS characterization of X. Assuming that samples containing increased concentrations of the intermediate would yield EXAFS data of improved quality, we applied our recently developed method of generating O2 in situ from chlorite using the enzyme chlorite dismutase to prepare X at ~ 2.0 mM, > 2.5 times the concentration realized in the previous EXAFS study. The measured dFe-Fe of 2.78 Å is fully consistent with computational models containing a (µ-oxo)2-Fe2III/IV core. The correction of dFe–Fe brings the experimental data and computational models into full conformity and thus informs analysis of the mechanism by which X generates Y122•.
PMCID: PMC4209742  PMID: 24094084
25.  A Bayesian MCMC Approach to Assess the Complete Distribution of Fitness Effects of New Mutations: Uncovering the Potential for Adaptive Walks in Challenging Environments 
Genetics  2014;196(3):841-852.
The role of adaptation in the evolutionary process has been contentious for decades. At the heart of the century-old debate between neutralists and selectionists lies the distribution of fitness effects (DFE)—that is, the selective effect of all mutations. Attempts to describe the DFE have been varied, occupying theoreticians and experimentalists alike. New high-throughput techniques stand to make important contributions to empirical efforts to characterize the DFE, but the usefulness of such approaches depends on the availability of robust statistical methods for their interpretation. We here present and discuss a Bayesian MCMC approach to estimate fitness from deep sequencing data and use it to assess the DFE for the same 560 point mutations in a coding region of Hsp90 in Saccharomyces cerevisiae across six different environmental conditions. Using these estimates, we compare the differences in the DFEs resulting from mutations covering one-, two-, and three-nucleotide steps from the wild type—showing that multiple-step mutations harbor more potential for adaptation in challenging environments, but also tend to be more deleterious in the standard environment. All observations are discussed in the light of expectations arising from Fisher’s geometric model.
PMCID: PMC3948810  PMID: 24398421
adaptation; experimental evolution; Fisher’s geometric model (FGM); adaptive walk; distribution of fitness effects

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