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.

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.

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.

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.

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.

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.

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.

The actuation of a biomimetic poly(vinyl alcohol)poly(acrylic acid) gel.

Active polymer gels expand and contract in response to certain environmental stimuli, such as the application of an electric field or a change in the pH level of the surroundings. This ability to achieve large, reversible deformations with no external mechanical loading has generated much interest in the use of these gels as biomimetic actuators and "artificial muscles". In previous work, a thermodynamically consistent finite-elastic constitutive model has been developed to describe the mechanical and actuation behaviours of active polymer gels. The mechanical properties were characterized by a free-energy function, and the model uses an evolving internal variable to describe the actuation state. In this work, an evolution law for the internal variable is determined from free actuation experiments on a poly(vinyl alcohol)poly(acrylic acid) (PVAPAA) gel. The complete finite-elastic/evolution law constitutive model is then used to predict the response of the PVA-PAA gel to isotonic and isometric loading and actuation. The model is shown to give relatively good agreement with experimental results.

Modeling tongue surface contours from Cine-MRI images.

This study demonstrated that a simple mechanical model of global tongue movement in parallel sagittal planes could be used to quantify tongue motion during speech. The goal was to represent simply the differences in 2D tongue surface shapes and positions during speech movements and in subphonemic speech events such as coarticulation and left-to-right asymmetries. The study used tagged Magnetic Resonance Images to capture motion of the tongue during speech. Measurements were made in three sagittal planes (left, midline, right) during movement from consonants (/k/, /s/) to vowels (/i/, /a/, /u/). MR image-sequences were collected during the C-to-V movement. The image-sequence had seven time-phases (frames), each 56 ms in duration. A global model was used to represent the surface motion. The motions were decomposed into translation, rotation, homogeneous stretch, and in-plane shear. The largest C-to-V shape deformation was from /k/ to /a/. It was composed primarily of vertical compression, horizontal expansion, and downward translation. Coarticulatory effects included a trade-off in which tongue shape accommodation was used to reduce the distance traveled between the C and V. Left-to-right motion asymmetries may have increased rate of motion by reducing the amount of mass to be moved.

Modeling the motion of the internal tongue from tagged cine-MRI images.

A new technique, tagged Cine-Magnetic Resonance Imaging (tMRI), was used to develop a mechanical model that represented local, homogeneous, internal tongue deformation during speech. The goal was to infer muscle activity within the tongue from tissue deformations seen on tMRI. Measurements were made in three sagittal slices (left, middle, right) during production of the syllable /ka/. Each slice was superimposed with a grid of tag lines, and the approximately 40 tag line intersections were tracked at 7 time-phases during the syllable. A local model, similar to a finite element analysis, represented planar stretch and shear between the consonant and vowel at 110 probed locations within the tongue. Principal strains were calculated at these locations and revealed internal compression and extension patterns from which inferences could be drawn about the activities of the Verticalis, Hyoglossus, and Superior Longitudinal muscles, among others.

Changing incidence of Achilles tendon rupture in Scotland: a 15-year study.

OBJECTIVE: To determine the incidence of Achilles tendon rupture in Scotland from 1980 to 1995. DESIGN: Retrospective analysis of prospectively collected data. SETTING: Data were obtained from the National Health Service Information and Statistics Division and analyzed in terms of age- and gender-specific incidence rates and time trends by age group. PARTICIPANTS: A total of 4,201 patients with Achilles tendon ruptures occurring during the study period. MAIN OUTCOME MEASURES: Calculation of incidence and of seasonality. RESULTS: The overall incidence of Achilles tendon rupture increased from 4.7/100,000 in 1981 to 6/100,000 in 1994, with a peak in 1986. In men, the incidence rose from 6.3/100,000 to 7.3/100,000. In women, the increase in incidence was more pronounced, from 3/100,000 to 4.7/100,000. In men, peak incidence rate occurred in the 30- to 39-year age group, whereas in women, the peak age-specific incidence occurred in those aged 80 years and older, with a steady increase after age 60. There was no evidence of a seasonality effect in the rate of occurrence of Achilles tendon rupture. A bimodal distribution of age at time of Achilles tendon rupture was noted. CONCLUSION: There was a significant increase in the incidence of Achilles tendon rupture during the period from 1980 to 1995. This reflects the increased incidence of the injury noted in other Northern European countries.

Seasonality, but not prevalence of sudden infant death syndrome varies by region in mainland Britain.

This study aimed to investigate whether seasonal variation in day length contributed to winter/summer variation in sudden infant death syndrome (SIDS) at different latitudes in mainland Britain. Over 11 yrs 13,973 deaths were studied. Using appropriate analytic techniques a sine curve was fitted to monthly rates with the amplitude indicating magnitude of seasonal change. The rate of SIDS per 1,000 live births was the same (1.73) in the north as in the south. The amplitude was a quarter less in the north (41.3%) than in the south (54.2%) (p<0.001). While annual rates did not differ, the within year distribution did. The findings for seasonality of SIDS births were similar (amplitudes: north 213%, south 32.3%). Correlations were made between SIDS amplitude and individual environmental factors, particularly temperature and day length. These complex issues, while reported briefly, do not allow firm conclusions. In the north the winter day length is shorter, sunshine hours are less and temperature is lower, but the winter increment in SIDS is less. The extent of seasonal variation of sudden infant death syndrome is greater in the south as compared with the colder, darker north but this has no effect on sudden infant death syndrome rates. Changing photoperiod by latitude, amongst other environmental influences, may hold clues to the aetiology of sudden infant death syndrome.

Seasonality of sudden infant death syndrome (SIDS) by age at death.

Seasonality of sudden infant death syndrome (SIDS) is a well-established epidemiological finding. The purpose of the study was to determine whether this feature varied significantly with age at death. In total, 13990 cases of SIDS in Scotland, England and Wales during 1982-1992 were studied by age group at death. Seasonality was established by fitting a sinusoidal curve and for each set of monthly data the peak position in the year and its magnitude were determined. Weighted regression revealed significant differences in peak position and amplitude of seasonal variation between those dying at < or = 4 months and those aged > or = 5 months at death. Those infants in the younger age group were more likely to die earlier in the winter months and had a smaller variation in seasonality. The peak (acrophase) months were January for < or = 4 months and February for > or = 5 months at death. Weighted regressions of peak position and amplitude on age at death had p-values of <0.001 and <0.01, respectively. A log linear model relating SIDS incidence to month of birth, month of death and age was able to explain some of these findings. The findings support the hypothesis that in SIDS there may be more than one infant cohort, each of which passes through a vulnerable developmental window at different ages.

Seasonality of sudden infant death syndrome in mainland Britain and Ireland 1985-95.

By the end of 1995 four years had passed since the dramatic fall in the incidence of sudden infant death syndrome (SIDS), following the "back to sleep" campaign. This time lag permitted a more definitive epidemiological reassessment than had been possible before. The extent of seasonal variation from 1992-5 fell by about half, occurring mainly as a single step down in 1992. The fall was relatively greater in winter than in summer. Before 1992 the extent of seasonal variation (amplitude) was greater in those age > or = 5 months compared with those aged < or = 4 months. Since 1992 the falls in incidence and amplitude have been greater in the younger group, suggesting that SIDS deaths in younger and older babies may have different causes.

Does vitamin D deficiency account for ethnic differences in tuberculosis seasonality in the UK?

OBJECTIVES: Notifications of tuberculosis in England and Wales are reported to peak in the summer season. The purpose of this study was to confirm that finding and to determine to what extent patients of Indian Subcontinent (ISC) ethnic origin contributed to the seasonality. The clinical presentation of the disease is presumed to occur some months following reactivation of the endogenous latent focus of tuberculosis infection. There arises the possibility of vitamin D deficiency producing immunological inadequacy at the end of winter and beginning of spring. PATIENTS AND METHODS: Monthly (or 4-weekly) aggregated data over 7 years were collected from the three countries of mainland Britain, England, Wales, Scotland and from the city of Birmingham in England. The notifications from Birmingham were divided into those of ISC ethnic origin and 'whites'. The presence or absence of seasonality was determined by fitting a sinusoidal curve by the technique called 'cosinor analysis'. In this method amplitude gives a measure of the extent of the seasonal variation. RESULTS: The summer peak of clinical diagnosis was confirmed in the UK series from England, Wales and Scotland. In England and Wales without Scotland a larger seasonal variation was present. Scotland, with a lower proportion of population of ISC ethnic origin, was examined separately and the results in Scotland alone failed to confirm seasonality. In the data from Birmingham, seasonality was confirmed with a greater amplitude, particularly in those over 60 years of age. The finding was influenced by those of ISC ethnic origin, seasonality not being present in the 'white' population. CONCLUSION: The results from Birmingham are very striking, but there were almost three times as many patients in the ISC ethnic group as in indigenous 'white' patients. A series with larger numbers of 'white' patients would be necessary to confirm the absence of seasonality in the 'white' population. The discussion reviews the evidence that vitamin D may have an important hormonal role in immunological defence in the prevention of tuberculosis.

Seasonal changes in psychological well-being in an elderly population.

BACKGROUND: Little is known about seasonal fluctuations in psychological well-being among elderly people. METHOD: Over a period of 21 months, 1466 elderly people completed the General Health Questionnaire and the Leeds Scales for Depression and Anxiety. Scores during the winter months (December to February) were compared with those during other months of the year. RESULTS: Scores on all scales were significantly higher during the winter months, but there was no difference in rates of caseness. Unlike younger populations, elderly women did not exhibit greater seasonality in well-being than did elderly men. CONCLUSIONS: Elderly people exhibit a small seasonal fluctuation in psychological well-being, which is probably of little clinical importance, and there is no gender difference. The findings support the contention that seasonal mood changes are most pronounced among females of reproductive age.

Circannual rhythm of measles subsequent to immunisation.

This is an epidemiological study of biennial and annual rhythms of measles in England and Wales between 1959-1994 highlighting changes following immunisation. The study describes annual rhythms starting in autumn (Week 41) and the statistical method tests the fit of a sine curve to annual data. Before immunisation the previously established biennial rhythm was seen: the two years differed in magnitude of seasonal variation, position of the peak, "visibility of school holidays" and peak breadth. After immunisation (1968) these biennial features tended to disappear, one annual rhythm becoming the predominant feature. By the late eighties adjacent years had very similar sine curve fits. About 1990 (89-91) the fit of the sine curve was no longer significant, was of low amplitude and numbers were at an all time low. In 1992 significant annual variation returned, numbers rose and magnitude of seasonal variation increased. At this time a new epidemic was being predicted on other evidence and this renewed sine curve fit may be an additional warning signal. A possible influence of normal birth rhythm on numbers of susceptibles is described.

Month of birth as an independent variable in the sudden infant death syndrome.

Well-known epidemiological features of sudden infant death syndrome (SIDS) are age at death and the increased numbers in winter. There are more SIDS deaths in late autumn/early winter and there is a seasonal rhythm of births with a peak in late summer and early autumn. The data set was 14033 SIDS deaths from Scotland, England and Wales over the 11 years 1982-92. Using log-linear models, which accounted for age at death and month of death, birth month was found to be a statistically significant risk factor for SIDS independent of age at death and winter environment (P < 0.001). Although winter season had the largest effect (relative risk 2.7 in January compared with August), the independent effect of birth month was of clinical as well as statistical significance with a relative risk for August births of 1.37 compared with those born in April. The analysis of each birth month cohort revealed a change in age distribution with infants born in early winter (December) dying at a younger age (mean 108 days) than those born in midsummer (June) (mean 146 days). Although winter season and age are the most influential factors, the substantial effect of month of birth requires explanation and points to as yet unidentified environmental influences during pregnancy.