[The assessment value of urinary NGAL combined with blood NLR in the early prognosis of patients with acute paraquat poisoning].

Objective: To explore the evaluate of neutrophil gelatinase-associated lipocalin (NGAL) , combined with neutrophil/lymphocyte ratio (NLR) in the early prognosis of patients with acute paraquat poisoning (APP) . Methods: In March 2019, 108 APP patients admitted to Emergency Medicine Department of Hebei Medical University from January 2017 to December 2018 were selected as the observation group, 60 healthy people in the same period were see as the control group according to the results of diagnosis and 28-day survival, the observation group was divided into 51 death group and 57 survival group. The correlation between NGAL, NLR and the death of APP patients was analyzed, to explore the value of NGAL and NLR in predicting the death of APP patients. Results: Compared with the Control group, the NGAL and NLR in the observation group were significantly higher (P<0.01) , and the NGAL and NLR in the death group were significantly higher (P<0.05) . The results showed that NGAL and NLR were positively correlated with the death of APP patients on 28th day, and the Correlation Coefficient was 0.456 and 0.638 at 2nd Day (P<0.01) The area under the ROC curve of NGAL, NLR and their combination were 0.764, 0.869 and 0.905, respectively. Conclusion: The combined detection of NGAL and NLR has important clinical significance in the early prediction of 28-day mortality in APP patients.

[The role of noninvasive hemodynamic monitoring in the evaluation of acute and severe pesticide poisoning].

Objective: To explore the method of noninvasive hemodynamic monitoring system (NICaS) in monitoring the hemodynamics of patients with acute pesticide poisoning, and to analyze the clinical guiding value of NICaS in hemodynamics of patients with severe pesticide poisoning. Methods: In August 2019, 200 patients with severe acute organophosphorus pesticide poisoning (AOPP) or moderate severe acute paraquat pesticide poisoning (APP) admitted to Harrison international peace hospital from January 2017 to August 2019 were randomly divided into NICaS group (n=68) , transpulmonary thermodilution method (n=67) and empirical treatment group (n=65) . The relationship between acute physiology and chronic health score (APACHE Ⅱ) , heart rate, hemodynamic indexes, survival rate and complications were analyzed. Results: There were no significant differences in age, sex ratio, body mass index, heart rate, systolic blood pressure, no treatment period and admission APACHE II score between NICaS group, Picco group and experience group (P>0.05) ; Compared with the experience group, the mortality of AOPP and app in NICaS group and Picco group were lower, and the differences were statistically significant (P<0.05) .The cardiac output (CO) had a significant correlation in the interval of 2.8-6.7 L·min(-1) (r=0.738, r(2)=0.545, P<0.01) , and peripheral vascular resistance index (SVRI) had a significant correlation in the interval of 410-1 950 d·s·cm(-5)·m(2) (r=0.792, r(2)=0.627, P<0.01) . Bland Altman analysis showed that CO and SVRI measured by Picco and NICaS had 97.01% and 95.52% consistency, respectively. Compared with the experience group, the average daily infusion volume and daily colloid infusion volume of NICaS group and Picco group were lower, the differences were statistically significant (P<0.05) . Conclusion: NICaS can effectively monitor the hemodynamic indexes of patients with acute pesticide poisoning.

[Effects of transcranial direct current stimulation on cognitive function in delayed encephalopathy after carbon monoxide poisoning].

Objective: To investigate the effect of transcranial direct current stimulation (tDCS) on cognitive function of delayed encephalopathy after carbon monoxide poisoning (DEACMP) . Methods: A total of 58 patients with DEACMP admitted to the Emergency Medicine Department of Hudson International Peace Hospital from January 2018 to January 2020 were included. According to the random number table, the patients were randomly divided into study group and control group, with 29 patients in each group. Patients in both groups were given nutritional nerve therapy, improved microcirculation, adrenal cortical hormone and other drugs, as well as hyperbaric oxygen and rehabilitation training, once per day. The study group was treated with tDCS (electrode pads were placed and current stimulation was given 30 min/time) 1/d on the basis of conventional treatment, while the control group was treated with tDCS pseudo stimulation (electrode pads were placed and current stimulation was given for 10 s) 1/d for 30 days consecutively. The auditory event-related potential P300 (ERP-P300) was tested before and 30 days after treatment, and the Mini-Mental State Examination (MMSE) scale, Barthel index (BI) and the National Institutes of Health Neurological Impairment score (NIHSS) were used to evaluate and analyze the patients. Results: There was no significant difference in scores between the two groups before treatment (all P>0.05) . After 30 days of treatment, the MMSE score, orientation, memory, attention and computing power, and language ability of the study group were all higher than those of the control group, and the differences were statistically significant (P<0.05) . 90 days after treatment, the BI of the study group was higher than that of the control group (P<0.05) . After 30 days of treatment, the NIHSS score of the study group was significantly lower than that of the control group (P<0.05) . The latency of ERP-P300 in the study group was shorter than that in the control group, and the amplitude was higher than that in the control group at 30 days after treatment, and the difference was statistically significant (P<0.05) . The latency of ERP-P300 was negatively correlated with the total MMSE score before and after DEACMP treatment (r=-0.837, -0.819, P<0.05) . The latency of ERP-P300 was negatively correlated with orientation, attention and computing power, and language ability before treatment (r=-0.698, -0.675, -0.742, P<0.05) . Conclusion: TDCS treatment can help improve the cognitive function of DEACMP patients, and ERP-P300 test can help determine the cognitive function severity of patients.

[Value of APACHE.II score and DIC score in predicting the death of patients with heat stroke].

Objective: To investigate the predictive values of the acute physiology and chronic health evaluation II (APACHE.II) score and disseminated intravascular coagulation (DIC) score on death in patients with heat stroke. Methods: A total of 76 patients with heat stroke who were treated in Emergency Department of Harrison International Peace Hospital from June 2013 to September 2017 were studied. According to the outcome of patients, we distributed the patients to death group and survival group. APACHE.II score and DIC score were calculated according to the clinical data and the test results at admission. Evaluate the correlation between the two indicators associated with death. Results: There were 76 patients, with 23 deaths (30.3%) and 53 survivors (69.7%) . The APACHE-II score and DIC score were 26.26±6.48 and 4.00±1.38 in the death group.significantly higher than 20.74±4.17 and 2.28±1.21 in the survival group, and there were significant difference (P< 0.01) . The APACHE. II score was positively correlated with the DIC score, and the higher the score, the higher the mortality rate.Both indicators are significant for the Logitic regression analysis of death (P<0.01) .The sensitivity and specificity of the APACHE.II score were 65.2% and 81.1% in prediction of mortality, The sensitivity and specificity of DIC score were 65.2% and 84.9% in prediction of mortality. The specificity of the APACHE II score plus DIC score were higher than that of single APACHE. II score or DIC score in prediction of mortality (P<0.05) . Conclusion: The APACHE.II score and DIC score are significantly increased in the early stage of the patients with heat stroke, and the APACHE. II score combined with DIC score may improve the value in prediction of mortality with heat stroke.

Exposure Duration Is a Determinant of the Effect of Sinusoidal Electromagnetic Fields on Peak Bone Mass of Young Rats.

We proposed a three-step strategy to obtain the optimal therapeutic parameters, which is composed of large-scale screening at cellular level, verification in animal experiments, and confirmation by a clinical trial. The objective of the current study was to test the feasibility of our strategy. Newborn rat calvarial osteoblasts were treated by 50 Hz 1.8 mT sinusoidal electromagnetic fields (SEMFs) with 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 h/days, respectively. The osteogenic differentiation and maturation of the osteoblast were assayed and compared to obtain the optimal duration. One-month-old growing rats were then treated by the same SEMFs with 0.5, 1.5, and 2.5 h/days, respectively, and the peak bone mass was analyzed after 2 months. It was found that the optimal exposure duration to promote the osteogenic differentiation and maturation of osteoblasts was 1.5 h/days, judging by the increasing degrees of ALP activity, calcified nodules formed, the gene and protein expression levels of Runx-2, BMP-2, and Col-I, as well as the expression levels of signaling proteins of the BMP-2/Smad1/5/8 pathway. The highest increase of peak bone mass after 2 months was also obtained by 1.5 h/days, judging by the results of X-ray dual-energy absorptiometry, mechanical property analysis, micro-CT scanning, and serum bone turnover marker examinations. The above results indicated that exposure duration is a determinant for the therapeutic effect of EMFs, and the optimal therapeutic effects only can be obtained by the optimal exposure duration.

[Effects of Ginaton on nitric oxide and nitric oxide synthase in patients with delayed encephalopathy after carbon monoxide poisoning].

Objective: To observe the effects of Ginaton on blood nitric oxide (NO) and nitric oxide synthase (NOS) in patients with delayed encephalopathy after acute carbon monoxide poisoning (DEACMP). Methods: A total of 116 patients with DEACMP who were treated in Emergency Department of Harrison International Peace Hospital Affiliated to Hebei Medical University from January 2012 to April 2016 were enrolled and ran-domly divided into control group and treatment group using a random number table, with 58 patients in each group. The patients in the control group were given conventional treatment including hyperbaric oxygen, preven-tion and treatment of cerebral edema, and promotion of brain cell metabolism, and those in the treatment group were given Ginaton in addition to the conventional treatment. The course of treatment was 2 weeks for both groups. The levels of neuron-specific enolase (NSE) , NO, NOS, and inducible nitric oxide synthase (iNOS) were measured before treatment and at 2 weeks after treatment, and the change in Mini-Mental State Examina-tion (MMSE) score and clinical outcome were observed in both groups. The correlation between the blood NO level on admission and the MMSE score was analyzed. Results: There was a significant difference in the overall response rate between the treatment group and the control group (81.03% vs 62.07%, χ(2) = 5.124, P=0.024). Be-fore treatment, there were no significant differences in the levels of NO and NSE, the activity of NOS and iN-OS, and MMSE score between the two groups (P>0.05). After treatment, both groups showed reductions in the levels of NO and NSE and the activity of NOS and iNOS, but the treatment group had significantly greater reduc-tions compared with the control group (P<0.05). Both groups showed a significant increase in the MMSE score after treatment, while the treatment group had a significantly greater increase compared with the control group (P<0.05). In the patients with DEACMP, the blood NO level on admission was negatively correlated with the MMSE score (r=-0.268, P=0.004). Conclusion: In the treatment of patients with DEACMP, Ginaton can effectively reduce the levels of NO and NSE and the activity of NOS and iNOS, increase the MMSE score, and promote the recovery of neurological function.

[Effects of extract of Ginkgo biloba on magnetic resonance imaging and electroencephalography in patients with delayed encephalopathy after acute carbon monoxide poisoning].

Objective: To observe the effects of extract of Ginkgo biloba (Ginaton) on magnetic resonance imaging (MRI) and electroencephalography (EEG) in patients with delayed encephalopathy after acute carbon monoxide poisoning. Methods: The 84 patients with delayed encephalopathy after acute carbon monoxide poisoning treated in our hospital from Jan. 2011 to Apr. 2016 were randomly divied into therapy group and observation group. The therapy group received routine treatments of hyperbaric oxygen, cure cerebral edema and promote brain cell metabolism, and observation group was given intravenous injection (intravenous drip) Ginaton 70 mg (adding 0.9% sodium chloride injection 250 ml) , once a day, 2 weeks for one therapeutic course. The changes of MRI and EEG before and after treatment between therapy group and observation group were observed. Results: In the observation group, the white matter and globus pallidus lesions of 14 d after treatment were smaller than those in the treatment group, and the abnormal signal intensity was decreased. At 14 days after treatment the improvement of EEG in observation group were better than therapy group (P<0.05) . Conclusion: Early treatment of extract of Ginkgo biloba (Ginaton) in delayed encephalopathy after acute carbon monoxide poisoning can effectively improve lesion and signal on MRI and abnormal rate on EEG. It has a certain therapeutic effect in clinical.

[Clinical value of serum presepsin (SCD14-ST) concentration measurement in patients with pesticide poisoning].

OBJECTIVE: To investigate the value of serum presepsin concentration measurement in the clinical diagnosis and treatment of patients with pesticide poisoning patients. METHODS: A total of 160 patients with pesticide poisoning were enrolled as study subjects and divided into moderate organophosphate pesticide poisoning group (40 patients) , severe organophosphate pesticide poisoning group (40 patients) , abamectin pesticide poisoning group (40 patients) , and paraquat poisoning group (40 patients). A total of 20 healthy volunteers were enrolled as the control group. All the patients with poisoning received conventional treatment of pesticide poisoning immediately after admission, and serum presepsin concentration was measured on days 1 (within 24 hours after poisoning) , 3, and 7 of admission, and biochemical and radiological parameters related to the patient's condition were also examined. The patients with a Presepsin concentration of >800 pg/ml on day 1 of admission were randomly divided into conventional treatment group and ulinastatin treatment group, and the treatment outcome was compared between the two groups. RESULTS: Compared with the healthy control group, the groups with pesticide poisoning showed significant increases in serum Presepsin concentrations, with the highest degree of increase on day 1 (P <0.05). The serum Presepsin concentration was positively correlated with alanine aminotransferase, aspartate aminotransferase, creatine kinase, creatine kinase MB, lactate dehydrogenase, serum creatinine, blood urea nitrogen, interleukin-18, and white blood cell count, but negatively correlated with cholinesterase. In the conventional treatment group and ulinastatin treatment group, the overall response rate was 68% and 78.8%, respectively, with a significant difference between the two groups (P<0.05). In 40 patients with paraquat poisoning, 32 experienced an increase in serum presepsin concentration, and among these 32 patients, 27 (83%) experienced exudation on lung CT. CONCLUSION: Serum Presepsin concentration measurement can assist early diagnosis, evaluation of disease severity, and guidance for clinical medication in patients with pesticide poisoning, especially in those with severe pesticide poisoning and a tendency to multiple organ failure.

[Prognostic value of the age-adjusted Charlson comorbidity index in patients over 60 years old with laryngeal squamous cell carcinoma].

Objective: To evaluate the value of the age-adjusted Charlson comorbidity Index (ACCI) in predicting the prognosis and guiding the clinical treatment of laryngeal squamous cell carcinoma (LSCC) in patients over 60 years old. Methods: Retrospective analysis of 249 cases of LSCC in Shanxi Provincial Cancer Hospital and First Hospital of Shanxi Medical University from 2008 to 2015 was performed. There were 234 males and 15 females, aged from 60 to 88 years. The clinical characteristics, treatment information and follow-up data were collected. ACCI was used to score the comorbidities of the patients. Receiver operating characteristic (ROC) curve was drawn and the patients were divided into high ACCI group and low ACCI group according to the cut-off value of ACCI. Prognostic factors were analyzed. Kaplan-Meier method was used for survival analysis, rank sum test was used for comparison between groups, χ2 test was used for enumeration data. Results: Overall survival (OS) was 54.6%, progression-free survival (PFS) was 59.4%, and cancer-specific survival (CSS) was 58.6%. Both the median survival time and PFS time were 60 months. The best cutoff point of the ACCI group was 5. Cox multivariate analysis showed that ACCI was an independent risk factor for OS, PFS and CSS (OR=1.553, 1.499 and 1.534,respectively, all P<0.05). In the high ACCI group, OS (χ2=4.120 and 4.115,P<0.05) and CSS (χ2=4.510 and 5.009,P<0.05) of patients treated with surgery plus radiotherapy and patients with radiotherapy alone were better than those of patients with surgery alone (P<0.05). But in the low ACCI group, there was no significant difference in prognosis among the three treatment regimens (P>0.05). Conclusion: High ACCI offors important prognostic information for LSCC in patients over 60 years old, and can guide clinical treatment options.

MiR-137 affects bone mineral density in osteoporosis rats through regulating RUNX2.

OBJECTIVE: To study the influence of micro ribonucleic acid (miR)-137 on osteoporosis rats by regulating runt-related transcription factor 2 (RUNX2). MATERIALS AND METHODS: A total of 36 Sprague-Dawley rats were randomly assigned to the normal group (n=12), model group (n=12), and inhibitor group (n=12). No treatment was performed in the normal group. The osteoporosis model in rats was prepared in the model group, and miR-137 inhibitor was administered in osteoporosis rats of inhibitor group. Following 12 weeks of intervention, sampling was conducted. The expression of RUNX2 was detected via immunohistochemistry, and its protein expression level was determined via Western blotting. Quantitative Polymerase Chain Reaction (qPCR) was carried out to detect the mRNA level of miR-137. The contents of serum bone Gla protein (BGP) and total alkaline phosphatase (TALP) were measured using enzyme-linked immunosorbent assay (ELISA). Finally, bone mineral density was determined with a dual-energy X-ray absorptiometry instrument. RESULTS: According to the immunohistochemistry detection, the rats in model group and inhibitor group had a notably lower positive expression level of RUNX2 than normal group (p<0.05), and its expression level in the inhibitor group was substantially higher than that in the model group (p<0.05). Western blotting results showed that compared with that in the normal group, the protein expression level of RUNX2 was notably lowered in the model and inhibitor group (p<0.05), which was markedly higher in the inhibitor group than that in the model group (p<0.05). It was found through the qPCR that the expression level of miR-137 was remarkably raised in both model group and inhibitor group compared with that in the normal group, showing statistically significant differences (p<0.05). The rats in the inhibitor group had a remarkably lower expression level of miR-137 than the model group (p<0.05). ELISA results revealed that the model group and inhibitor group had substantially lower contents of serum BGP and TALP than the normal group (p<0.05), and that their contents rose dramatically in the inhibitor group compared with that in the model group (p<0.05). Additionally, based on the measurement of bone mineral density, compared with that in the normal group, bone mineral density declined considerably in the model group and inhibitor group (p<0.05). It was markedly elevated in inhibitor group in comparison with that in the model group (p<0.05). CONCLUSIONS: MiR-137 regulates RUNX2 to affect the bone mineral density of osteoporosis model rats.

LncRNA LINC00707 promotes osteogenic differentiation of hBMSCs through the Wnt/ß-catenin pathway activated by LINC00707/miR-145/LRP5 axis.

OBJECTIVE: Human bone marrow mesenchymal stem cells (hBMSCs) have a strong self-renewal potential and osteogenic differentiation ability, thus providing a new method for bone defect repair research. LncRNA LINC00707 participates in the regulation of osteogenic differentiation of hBMSCs and our aim was to explore the potential regulatory mechanism. MATERIALS AND METHODS: Firstly, quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression levels of LINC00707, miR-145, the low-density lipoprotein receptor-related protein 5 (LRP5) and osteogenesis-related genes. Next, alkaline phosphatase (ALP) activity assay was used to measure the relative activity of ALP in hBMSCs. The protein levels of LRP5 and osteogenesis-related genes were detected by Western blot. Finally, the relationship among LINC00707, miR-145 and LRP5 were predicted by online software and verified by Dual-Luciferase reporter assay, RNA pull-down and RNA immunoprecipitation (RIP). RESULTS: LINC00707 and osteogenesis-related genes were gradually upregulated during osteogenesis of hBMSCs. Meanwhile, overexpression of LINC00707 promoted osteogenic differentiation of hBMSCs. Interestingly, we found that LINC00707 negatively regulated the miR-145 expression and osteogenic differentiation functions by directly interacting with miR-145, and LINC00707 affected the functions of LRP5 by sponging miR-145 in hBMSCs. Moreover, LINC00707 promoted the Wnt/ß-catenin pathway through the LINC00707/miR-145/LRP5 axis. CONCLUSIONS: LncRNA LINC00707 promoted osteogenic differentiation of hBMSCs by targeting LRP5 mediated by miR-145 through the activation of the Wnt/ß-catenin pathway.

Correlation between neutrophil gelatinase-associated lipocalin and soluble CD14 subtype on the prognosis evaluation of acute paraquat poisoning patients.

OBJECTIVE: The objective of this article is to study the correlation between neutrophil gelatinase-associated lipocalin (NGAL) and soluble CD14 subtype (presepsin) on the severity and prognosis evaluation of acute paraquat poisoning (APP) patients. MATERIALS AND METHODS: We studied 120 APP patients who were divided into three groups: light (28 cases), moderate (52 cases), and heavy poisoning (40 cases) groups. Twenty healthy volunteers were enrolled as controls. RESULTS: Acute kidney injury (AKI) occurred in 86 APP patients (71.7%, 86 of 120). In AKI group, urine NGAL was elevated 3 h after treatment, serum NGAL was elevated 24 h after treatment, and serum creatine (SCr) was elevated 2 days after treatment, which were all significantly higher than non-AKI group. Compared with control group, there were significant differences in presepsin and acute physiology and chronic health status (APACHE) II score of different poisoning groups. There were significant differences in detection indices 24 h, 3 days, and 7 days after treatment among different poisoning groups. There was a positive correlation between urine NGAL and serum paraquat concentration, urine NGAL, and AKI morbidity (r 1 = 0.974, r 2 = 0.766, p < 0.001), suggesting higher urine NGAL level indicated higher AKI morbidity. Receiver operating characteristic curves analysis suggested serum presepsin level and urine NGAL level had higher sensitivity and specificity than APACHE II score when predicting 28-day mortality of APP patients. CONCLUSION: Serum and urine NGAL level is elevated earlier than SCr, which is important for the early diagnosis of APP. Serum presepsin and urine NGAL levels can be used as markers to diagnose the severity of AKI and predict the mortality of APP patients.

Detection of acute HIV infections among sexually transmitted disease clinic patients: a practice in Guangxi Zhuang Autonomous Region, China.

Detection of people with acute HIV infection (AHI) affords an important opportunity for early HIV treatment and prevention. HIV RNA reverse transcriptase-polymerase chain reaction (RT-PCR) testing with two-stage pooling scheme was used to detect the AHI in specimens collected from sexually transmitted disease (STD) clinic patients in Guangxi, China. A total of 246 HIV RNA tests were required to screen 11 395 samples negative for conventional enzyme immunoassay (EIA) and Western blot assays, and five AHI cases (0.04%, 95%CI 0.02% to 0.10%) with a high viral load (median of 265,677 copies per ml) were detected. The total expenditure for RT-PCR testing reflected an added cost of $2.9 per specimen screened and $6575 per additional case of AHI identified among the study population. This study supports the feasibility of pooled RNA testing in addition to detection of HIV infections among patients at STD clinics in China, but the cost effectiveness should be carefully considered.

Tracheobronchial brush cytology and bronchoalveolar lavage in dogs and cats with chronic cough: 45 cases (2012-2014).

BACKGROUND: Animals with chronic cough can have normal bronchoalveolar lavage fluid cytology when small airway disease is absent. Cytology of a tracheobronchial brushing can detect inflammation in larger airways; however, evaluation of this technique has been limited in veterinary medicine. OBJECTIVE: To compare airway brush cytology to bronchoalveolar lavage fluid analysis in dogs and cats with chronic cough. ANIMALS: Forty dogs and five cats undergoing bronchoscopic investigation of chronic cough. METHODS: Prospective study. Bronchoscopy and bronchoalveolar lavage were performed followed by tracheobronchial brushing of central airways. Results of cytologic assessment of BAL fluid and brush cytology were compared for the presence or absence of inflammation and concordance of inflammatory cell type. RESULTS: Brush cytology detected central airway inflammation in 34 of 40 (85%) dogs with inflammatory BAL fluid. However, the type of inflammation reported differed in 23 of 34 dogs. In five cats with inflammation in BAL fluid, brush cytology detected inflammation in four; the type of inflammation was discordant in all cats. CONCLUSIONS AND CLINICAL RELEVANCE: Brush cytology has good agreement with BAL regarding the presence of inflammation, although the type of inflammation detected with the different sampling techniques commonly varies. Brush cytology can provide supplementary information to BAL, and additional studies will provide further information on the role of tracheobronchial brush cytology in the diagnosis and management of respiratory conditions.

Packing and hydrophobicity effects on protein folding and stability: effects of beta-branched amino acids, valine and isoleucine, on the formation and stability of two-stranded alpha-helical coiled coils/leucine zippers.

The aim of this study was to examine the differences between hydrophobicity and packing effects in specifying the three-dimensional structure and stability of proteins when mutating hydrophobes in the hydrophobic core. In DNA-binding proteins (leucine zippers), Leu residues are conserved at positions "d," and beta-branched amino acids, Ile and Val, often occur at positions "a" in the hydrophobic core. In order to discern what effect this selective distribution of hydrophobes has on the formation and stability of two-stranded alpha-helical coiled coils/leucine zippers, three Val or three Ile residues were simultaneously substituted for Leu at either positions "a" (9, 16, and 23) or "d" (12, 19, and 26) in both chains of a model coiled coil. The stability of the resulting coiled coils was monitored by CD in the presence of Gdn.HCl. The results of the mutations of Ile to Val at either positions "a" or "d" in the reduced or oxidized coiled coils showed a significant hydrophobic effect with the additional methylene group in Ile stabilizing the coiled coil (delta delta G values range from 0.45 to 0.88 kcal/mol/mutation). The results of mutations of Leu to Ile or Val at positions "a" in the reduced or oxidized coiled coils showed a significant packing effect in stabilizing the coiled coil (delta delta G values range from 0.59 to 1.03 kcal/mol/mutation). Our results also indicate the subtle control hydrophobic packing can have not only on protein stability but on the conformation adopted by the amphipathic alpha-helices. These structural findings correlate with the observation that in DNA-binding proteins, the conserved Leu residues at positions "d" are generally less tolerant of amino acid substitutions than the hydrophobic residues at positions "a."

Role of interchain alpha-helical hydrophobic interactions in Ca2+ affinity, formation, and stability of a two-site domain in troponin C.

We have previously shown that a 34-residue synthetic peptide representing the calcium-binding site III of troponin C formed a symmetric two-site dimer consisting of two helix-loop-helix motifs arranged in a head-to-tail fashion (Shaw, G.S., Hodges, R.S., & Sykes, B.D., 1990, Science 249, 280-283). In this study the hydrophobicities of the alpha-helices were altered by replacing L-98 and F-102 in the N-terminal region and/or I-121 and L-122 in the C-terminal region with alanine residues. Our results showed that substitution of hydrophobic residues either in the N- or C-terminal region have little effect on alpha-helix formation but resulted in a 100- and 300-fold decrease in Ca2+ affinity, respectively. Simultaneous substitution of both hydrophobes in the N- and C-terminal region resulted in a 1,000-fold decrease in Ca2+ affinity. Data from guanidine hydrochloride denaturation studies suggested that intermolecular interactions occur and that the less hydrophobic analogs had a lower overall conformational stability. These data support the contention that the hydrophobic residues are important in the formation of the two-site domain in troponin C, and this hydrophobic association stabilizes Ca2+ affinity.

Discovery of transition state factor Xa inhibitors as potential anticoagulant agents.

Factor Xa is an attractive biological target in the discovery and development of either parenteral or orally active anticoagulant agents. Several strategies have been utilized at COR Therapeutics in the pursuit of tri-peptide based transition state mimetic factor Xa inhibitors with high aqueous solubility. Some of these inhibitors have displayed excellent in vitro potency in inhibiting factor Xa in the prothrombinase complex. More importantly, these compounds showed strong in vivo antithrombotic efficacy without significant bleeding complications in several animal thrombosis models. These results demonstrated that small molecule factor Xa inhibitors could be advantageous over Warfarin and LMWH. For the discovery and development of orally active anticoagulant agents, small organic molecules as reversible factor Xa inhibitors were explored. From a medicinal chemistry perspective, significant insight has been gained regarding the in vivo antithrombotic efficacy and pharmacokinetic behaviors of each class of factor Xa inhibitors. This review will focus on the design and discovery of transition state factor Xa inhibitors as potential parenteral anticoagulant agents. Several excellent comprehensive review articles on factor Xa inhibitors have appeared recently [1-4].

Potent bivalent thrombin inhibitors: replacement of the scissile peptide bond at P(1)-P(1)' with arginyl ketomethylene isosteres.

We have designed highly potent synthetic bivalent thrombin inhibitors, which consist of an active site blocking segment, a fibrinogen recognition exosite blocking segment, and a linker connecting these segments. The bivalent inhibitors bind to the active site and the fibrinogen recognition exosite simultaneously. As a result, the inhibitors showed much higher affinity for thrombin than the individual blocking segments. Various arginyl ketomethylene isosteres ArgPsi[CO-CH(2)-X]P(1)' were incorporated into the bivalent inhibitors as P(1)-P(1)' segment to eliminate the scissile bond. The P(1)' residue is a natural or unnatural amino acid; specifically, the incorporation of mercaptoacetic acid exhibited superiority in synthesis and affinity for thrombin. Inhibitor 16, (D-cyclohexylalanine)-Pro-ArgPsi[CO-CH(2)-S]Gly-(Gly)(4)-Asp-Tyr-G lu- Pro-Ile-Pro-Glu-Glu-Tyr-cyclohexylalanine-(D-Glu)-OH, showed the lowest K(i) value of 3.5 +/- 0.5 x 10(-13) M, which is comparable to that (K(i) = 2.3 x 10(-13) M) of recombinant hirudin. Consequently we successfully reduced the size of the inhibitor from approximately 7 kDa of recombinant hirudin to approximately 2 kDa without losing the affinity.