Structure activity relationship towards design of cryptosporidium specific thymidylate synthase inhibitors.

Cryptosporidiosis is a human gastrointestinal disease caused by protozoans of the genus Cryptosporidium, which can be fatal in immunocompromised individuals. The essential enzyme, thymidylate synthase (TS), is responsible for de novo synthesis of deoxythymidine monophosphate. The TS active site is relatively conserved between Cryptosporidium and human enzymes. In previous work, we identified compound 1, (2-amino-4-oxo-4,7-dihydro-pyrrolo[2,3-d]pyrimidin-methyl-phenyl-l-glutamic acid), as a promising selective Cryptosporidium hominis TS (ChTS) inhibitor. In the present study, we explore the structure-activity relationship around 1 glutamate moiety by synthesizing and biochemically evaluating the inhibitory activity of analogues against ChTS and human TS (hTS). X-Ray crystal structures were obtained for compounds bound to both ChTS and hTS. We establish the importance of the 2-phenylacetic acid moiety methylene linker in optimally positioning compounds 23, 24, and 25 within the active site. Moreover, through the comparison of structural data for 5, 14, 15, and 23 bound in both ChTS and hTS identified that active site rigidity is a driving force in determining inhibitor selectivity.

[Feeding of infants based on age. Practice guidelines]. / Alimentation du nourrisson et de l'enfant en bas âge. Réalisation pratique.

This paper presents practical guidelines for nutrition and feeding of infants and toddlers including vitamin D, vitamin K and fluoride supplementations and preventive measures at risk for food allergy based on family history.

[Efficacy and tolerance of Algosteril (calcium alginate) versus Jelonet (paraffin gauze) in the treatment of scalp graft donor sites in children. Results of a randomized study]. / Evaluation de l'efficacité et de la tolérance d'Algostéril (compresse d'alginate de calcium) versus Jelonet (gaze paraffinée) dans le traitement du site donneur de greffe dermo-épidermique du cuir chevelu. Résultats d'un essai pédiatrique.

Split skin graft is frequently needed in the treatment of burned patients. Scalp is often free of burns. Due to its good skin quality and important surface, scalp is a very interesting skin donor site, specially in case of children. A controlled, randomised clinical trial was carried out in 10 French Plastic Surgery or Burns Units. It assessed the efficacy and the acceptability of calcium alginate dressing (Algosteril) versus paraffin gauze dressing (Jelonet) in the treatment of scalp donor sites in children. 67 children (mean age 54 months) entered the study, 34 in the alginate group and 33 in the control group. Follow-up visits were on day 2/d3, d5/d6, Day complete healing, d30 and d60 after surgery. The two groups were comparable on inclusion (demographic characteristics, burn nature and surface, donor site surface and thickness of split skin graft). The mean healing time was 10 and 11 days for Algosteril and Jelonet group respectively (ns). The quality of the newly formed tissue was estimated to permit a sooner skin reharvesting in the Algosteril group than in the control group (p = 0.003). Bleeding through dressing was significantly less important in the Algosteril group (p = 0.02). Changes were considered by investigators less painful with Algosteril on day complete healing (p = 0.0096). Hair growth is homogenous in both groups on day 30 and day 60 (ns). These results showed that scalp is a very interesting skin donor site and that Algosteril is of a real interest in donor site treatment.

Antiviral drug design: computational analyses of the effects of the L100I mutation for HIV-RT on the binding of NNRTIs.

Monte Carlo/free energy perturbation (MC/FEP) calculations were used to evaluate the binding free energy change for HIV-RT/inhibitor complexes upon L100I mutation. Inhibitor size and flexibility adjacent to hydrogen-bonding sites are evident as important considerations for antiviral drug design.

In vitro regulated expression of tyrosine hydroxylase in ventral midbrain neurons from Nurr1-null mouse pups.

The transcription factor Nurr1, an orphan member of the steroid-thyroid hormone nuclear receptor superfamily, is essential for the proper terminal differentiation of ventral midbrain dopaminergic neurons. Disruption of the Nurr1 gene in mice by homologous recombination abolishes synthesis of dopamine (DA) and expression of DA biosynthetic enzymes, including tyrosine hydroxylase (TH), in the ventral midbrain without affecting the synthesis of DA in other areas of the brain. At birth, however, dopaminergic neuron precursors in Nurr1 null (-/-) pups remain as shown by continued expression of residual, untranslated Nurr1 mRNA not altered by homologous recombination. Since Nurr1 disruption is lethal shortly after birth, to further investigate the developmental properties of these neurons, dissociated ventral midbrain neurons from newborn pups were grown for 5 days on an astrocyte feeder layer, subjected to various treatments and then evaluated for expression of TH by fluorescent immunocytochemistry. Initially, a small percentage of neurons (0.26% +/- 0.07%) from the ventral midbrain of Nurr1 -/- pups were TH-immunoreactive (TH-IR). No change in TH expression was observed in the presence of glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), or DA alone or in combination. Treatment with forskolin (Fsk), however, significantly increased the percentage of TH-IR neurons (1.36% +/- 0.15%). Combination of Fsk, BNDF, and DA further increased the percentage of TH-IR neurons (2.58% +/- 0.50%). Therefore, these data suggest that dopaminergic neuron precursors, which develop in vivo without Nurr1, remain in an undifferentiated condition that is permissive to the induction of TH in vitro. J. Neurosci. Res. 64:322-330, 2001. Published 2001 Wiley-Liss, Inc.

Involvement of cholinergic mechanisms in the central control of respiration in the cane toad, Bufo marinus.

Chemical substrates, central sites and central mechanisms underlying the regulation of breathing in lower vertebrates have not been well characterized. The present study was undertaken to determine the effect of pH changes and cholinergic agents on the central control of respiration in the cane toad, Bufo marinus. Adult toads were anesthetized, catheterized and unidirectionally ventilated before exposing the brainstem. An airtight buccal cannula was also inserted through the tympanum to record buccal pressure. The animal was decerebrated, anesthetic removed and the responses to pH changes of solutions bathing the ventral surface of the medulla (VSM) were tested by superfusing the VSM with mock cerebrospinal fluid (mCSF) of pH 7.8-normal, 7.2-acidic and 8.4-basic. The acidic solution increased respiratory activity, the basic solution decreased activity and the normal solution had no effect. In addition, cholinergeric agents (acetylcholine-ACh, physostigmine-Phy, nicotine-Nic, and atropine-Atr) dissolved in mCSF were applied bilaterally onto the VSM using filter paper pledgets. ACh, Phy and Nic increased episodic breathing frequency by 14.3+/-9.7, 9.4+/-5.4 and 29.1+/-11.8 %, respectively, whereas, Atr caused a decrease (-26.6+/-16.6%). These agents had no effect on blood pressure. It is therefore, concluded that the VSM is pH sensitive and a cholinergic mechanism is involved in the central modulation of respiration in Bufo.

Alpha2 adrenergic receptors and the central control of breathing in the cane toad, Bufo marinus.

The effect of adrenergic agents on the central control of breathing in the cane toad, Bufo marinus, was tested by applying adrenergic agents to the ventral medullary surface of decerebrate adult toads. Toads were unidirectionally ventilated while recording lung, buccal, and artery blood pressures (BPI), as well as heart rate (HR). Following a control period, filter paper pledgets soaked in the appropriate solution (epinephrine - 0.023, 0.05, 0.10, and 0.23 mM; norepinephrine - 0.002, 0.016, 0.032, and 0.16 mM; clonidine - 0.00375, 0.0375, and 0.375 mM; or yohimbine - 0.43) mM) were placed bilaterally on the ventral medullary surface. Epinephrine significantly increased the number of breaths (26%), lung amplitude (9%), and episode duration (21%), but had no effect on BP or HR. The alpha2-agonist. clonidine, significantly increased respiratory activity at moderate doses (0.0375 mM) and decreased activity at high doses (0.375 mM), however, it failed to elicit significant changes in BP or HR. Pretreatment with the alpha2-adrenoceptor antagonist, yohimbine (0.43 mM), blocked the clonidine induced changes in respiratory activity. Yohimbine had no effect on cardiorespiratory parameters. Norepinephrine had no effect on either cardiovascular or respiratory variables. Thus, it appears that an alpha2 adrenergic mechanism is involved in the central control of respiration in this lower vertebrate.

Estimation of binding affinities for HEPT and nevirapine analogues with HIV-1 reverse transcriptase via Monte Carlo simulations.

The interactions and energetics associated with the binding of 20 HEPT and 20 nevirapine nonnucleoside inhibitors of HIV-1 reverse transcriptase (RT) have been explored in an effort to establish simulation protocols and methods that can be used in the development of more effective anti-HIV drugs. Using crystallographic structures as starting points, all 40 inhibitors were modeled in the bound and unbound states via Monte Carlo (MC) statistical mechanics methods. Potentially useful descriptors of binding affinity were configurationally averaged for each inhibitor during the MC simulations, and correlations were sought with reported experimental activities. A viable regression equation was obtained using only four descriptors to correlate the 40 experimental activities with an r(2)() of 0.75 and cross-validated q(2)() of 0.69. The computed activities show a rmsd of 0.94 kcal/mol in comparison with experiment and an average unsigned error of 0.69 kcal/mol. The MC results reveal three physically reasonable parameters that control the binding affinities: (1) loss of hydrogen bonds with the inhibitor is unfavorable, (2) burial of hydrophobic surface area is favorable, and (3) a good geometrical fit without steric clashes is needed for the protein-inhibitor complex. It is gratifying that the corresponding descriptors are statistically the most important quantities for determining the anti-HIVRT activity for the 40 compounds. Representative examples are also given in which structural and thermodynamic information from the MC simulations is used to help understand binding differences for related compounds. A key pi-type hydrogen bond has been identified between secondary-amide nevirapine analogues and Tyr188A of HIVRT that explains their otherwise surprising activity and the ineffectiveness of nevirapine against the Y188C mutant.

Monte Carlo calculations on HIV-1 reverse transcriptase complexed with the non-nucleoside inhibitor 8-Cl TIBO: contribution of the L100I and Y181C variants to protein stability and biological activity.

A computational model of the non-nucleoside inhibitor 8-Cl TIBO complexed with HIV-1 reverse transcriptase (RT) was constructed in order to determine the binding free energies. Using Monte Carlo simulations, both free energy perturbation and linear response calculations were carried out for the transformation of wild-type RT to two key mutants, Y181C and L100I. The newer linear response method estimates binding free energies based on changes in electrostatic and van der Waals energies and solvent-accessible surface areas. In addition, the change in stability of the protein between the folded and unfolded states was estimated for each of these mutations, which are known to emerge upon treatment with the inhibitor. Results from the calculations revealed that there is a large hydrophobic contribution to protein stability in the native, folded state. The calculated absolute free energies of binding from both the linear response, and also the more rigorous free energy perturbation method, gave excellent agreement with the experimental differences in activity. The success of the relatively rapid linear response method in predicting experimental activities holds promise for estimating the activity of the inhibitors not only against the wild-type RT, but also against key protein variants whose emergence undermines the efficacy of the drugs.

Cultured epithelial autografts in extensive burn coverage of severely traumatized patients: a five year single-center experience with 30 patients.

OBJECTIVE: We report recent five-year experience in a large, single center series of severely burned and otherwise traumatized patients given cultured epithelial autografts (CEA) from a single commercial laboratory. SUMMARY BACKGROUND DATA: Initial optimism over CEA application has been tempered by subsequent reports asserting that this modality is unreliable and expensive. Discussion continues over its clinical role. METHODS: From 1991 to 1996, CEA were applied to a mean 37+/-17% of total body surface area (TBSA) of 30 patients. These patients had 78+/-10% average burn size, 65+/-16% average third-degree burn size, 90% prevalence of endoscopically confirmed inhalation injury and 37% prevalence of other serious conditions. RESULTS: CEA achieved permanent coverage of a mean 26+/-15% of TBSA, an area greater than that covered by conventional autografts (a mean 25+/-10% of TBSA). Survival was 90% in these severely burned and otherwise traumatized patients. Final CEA take was a mean 69+/-23%. In subset analyses, only younger age was significantly associated with better CEA take (p = 0.0001 in univariate analysis, p<0.04 in multivariate analysis, Student's t-test). CONCLUSIONS: Epicel CEA successfully provided extensive, permanent burn coverage in severely traumatized patients, proving an important adjunct to achievement of a high survival rate in a patient population whose prognosis previously had been poor. In our experience CEA appear to have a very high beneficial value in the management of bur ns >60% TBSA. In some cases studied it is very likely that CEA was a life-saving treatment.

Long-term regeneration of human epidermis on third degree burns transplanted with autologous cultured epithelium grown on a fibrin matrix.

BACKGROUND: Extensive third degree burn wounds can be permanently covered by the transplantation of autologous cultured keratinocytes. Many modifications to Green and colleagues' original technique have been suggested, including the use of a fibrin matrix. However, the properties of the cultured cells must be assessed using suitable criteria before a modified method of culture for therapeutic purposes is transferred to clinical use, because changes in culture conditions may reduce keratinocyte lifespan and result in the loss of the transplanted epithelium. METHODS: To evaluate the performances of human keratinocytes grown on a fibrin matrix, we assay for their colony-forming ability, their growth potential and their ability to generate an epidermis when grafted onto athymic mice. The results of these experiments allowed us to compare side by side the performance for third degree burn treatment of autologous cultured epithelium grafts grown according to Rheinwald and Green on fibrin matrices with that of grafts grown directly on plastic surfaces. RESULTS: We found that human keratinocytes cultured on a fibrin matrix had the same growth capacity and transplantability as those cultured on plastic surfaces and that the presence of a fibrin matrix greatly facilitated the preparation, handling, and surgical transplantation of the grafts, which did not need to be detached enzymatically. The rate of take of grafts grown on fibrin matrices was high, and was similar to that of conventionally cultured grafts. The grafted autologous cells are capable of generating a normal epidermis for many years and favor the regeneration of a superficial dermis. CONCLUSION: We have demonstrated that: 1) fibrin matrices have considerable advantages over plastic for the culture of skin cells for grafting and that it is now possible to generate and transplant enough cultured epithelium from a small skin biopsy to restore completely the epidermis of an adult human in 16 days; and 2) the generated epidermis self-renews itself for years. The use of fibrin matrices thus significantly improves the transplantation of cultured epithelium grafts for extensive burns as recently demonstrated in a follow-up work.

[Nursing report for patient discharge. A practical service experience]. / Informe de enfermería al alta. Una experiencia en la práctica asistencial.

The report for Medical Release Certificates which nurses fill out for patients after a period of hospitalization is one of the indispensable requirements that guarantee the continuity of treatment. Nursing professionals have the obligation to develop the mechanisms which allow for their services to be provided to the general public in an integrated, continuous manner. On the other hand, health service clients demand rapid, individualized, high quality treatment. Health service administrators, echoing these demands, pursue interdepartmental coordination and efficiency from health service system. This purpose of this article is to supply regulated information to the patient and his/her family as well as to health professionals which will serve as a guide while filling out the hospital medical release certificate. To this end, by means of a consensus among groups of experts, a document for medical release certificate reports, emission circuits, transfer of forms and their filing was elaborated. Furthermore, an attempt was made to introduce these reports as a habitual part in the procedures nurses follow while treating patients. The document designed and the circuits correspond to, in reality, the needs for regulated information. Other organizations have not stated any inconveniences to accepting this document and nurses are filling out these reports as the final part of the treatment process they lend to their patients. This document and its procedures guarantee its availability to all interested parties while increasing and accelerating interprofessional communication; in addition, this information is differentiated according to the needs of the receiver.

Estimation of the binding affinities of FKBP12 inhibitors using a linear response method.

A series of non-immunosuppressive inhibitors of FK506 binding protein (FKBP12) are investigated using Monte Carlo statistical mechanics simulations. These small molecules may serve as scaffolds for chemical inducers of protein dimerization, and have recently been found to have FKBP12-dependent neurotrophic activity. A linear response model was developed for estimation of absolute binding free energies based on changes in electrostatic and van der Waals energies and solvent-accessible surface areas, which are accumulated during simulations of bound and unbound ligands. With average errors of 0.5 kcal/mol, this method provides a relatively rapid way to screen the binding of ligands while retaining the structural information content of more rigorous free energy calculations.

[Latissimus dorsi free flap with "Y" anastomoses. Technical variant and use in leg reconstruction]. / Lambeau libre de grand dorsal avec anastomoses en "Y". Variante technique et intérêt en reconstruction de jambe.

The authors report a new technique for harvesting and anastomosis of the latissimus dorsi free flap. The latissimus dorsi free flap is elevated with the subscapular and circumflex scapular vessels forming a "Y" pedicle. The recipient artery is sectioned and the arterial tree of the flap is anastomosed to the recipient vessel by two end-to-end anastomoses. This technique is particularly useful in leg reconstructive surgery when only one vessel remains: it simplifies transfer (end-to-end anastomosis), anastomoses are easier because more superficial.

Prediction of binding affinities for TIBO inhibitors of HIV-1 reverse transcriptase using Monte Carlo simulations in a linear response method.

Monte Carlo (MC) simulations in combination with a linear response approach were used to estimate the free energies of binding for a series of 12 TIBO nonnucleoside inhibitors of HIV-1 reverse transcriptase. Separate correlations were made for the R6 and S6 absolute conformations of the inhibitors, as well as for the analogous N6-monoprotonated species. Models based upon the neutral unbound inhibitors produced overall better fits to experimental values than did those using the protonated unbound inhibitors, with only slight differences between the neutral R6 and S6 cases. The best results were obtained with a three-parameter linear response equation containing van der Waals (alpha), electrostatic (beta), and solvent accessible surface area (SASA, gamma) terms. The averaged (R6 and S6) rms error was approximately 0.88 kcal/mol for the observed range of 4.06 kcal/mol in inhibitor activities. The averaged values of alpha, beta, and gamma were -0.150, 0.114, and 0. 0286, respectively. Omission of the alpha term gave beta 0.152 and gamma 0.022 with a rms of 0.92. The unweighted van der Waals components were found to be highly attractive but failed to correlate well across the series of inhibitors. Contrastingly, while the electrostatic components are all repulsive, they show a direct correlation with inhibitor activity as measured by DeltaGbinding. The role of gamma is primarily to produce an overall negative binding energy, and it can effectively be replaced with a negative constant. During the MC simulations of the unbound solvated inhibitors, the R6 and S6 absolute conformations do not interconvert due to the formation of a favorable hydrogen bond to solvent. In the complex, however, interconversion of these conformations of the inhibitor is observed during the course of the simulations, a phenomenon which is apparently not observed in the crystalline state of the complex. Hydrogen bonding of the inhibitor to the backbone NH of K101 and the lack of such an interaction with the C=O of K101 or with solvent correlate with enhanced activity, as does the ability to assume a number of different orientations of the inhibitor dimethylallyl moiety with respect to residues Y181 and Y188 while retaining contact with W229. Overall, the use of a combination of MC simulation with a linear response method shows promise as a relatively rapid means of estimating inhibitor activities. This approach should be useful in the preliminary evaluation of potential modifications to known inhibitors to enhance activity.

Towards the unification of inference structures in medical diagnostic tasks.

The central purpose of artificial intelligence applied to medicine is to develop models for diagnosis and therapy planning at the knowledge level, in the Newell sense, and software environments to facilitate the reduction of these models to the symbol level. The usual methodology (KADS, Common-KADS, GAMES, HELIOS, Protégé, etc) has been to develop libraries of generic tasks and reusable problem-solving methods with explicit ontologies. The principal problem which clinicians have with these methodological developments concerns the diversity and complexity of new terms whose meaning is not sufficiently clear, precise, unambiguous and consensual for them to be accessible in the daily clinical environment. As a contribution to the solution of this problem, we develop in this article the conjecture that one inference structure is enough to describe the set of analysis tasks associated with medical diagnoses. To this end, we first propose a modification of the systematic diagnostic inference scheme to obtain an analysis generic task and then compare it with the monitoring and the heuristic classification task inference schemes using as comparison criteria the compatibility of domain roles (data structures), the similarity in the inferences, and the commonality in the set of assumptions which underlie the functionally equivalent models. The equivalences proposed are illustrated with several examples. Note that though our ongoing work aims to simplify the methodology and to increase the precision of the terms used, the proposal presented here should be viewed more in the nature of a conjecture.

Molecular dynamics simulations of the unfolding of barnase in water and 8 M aqueous urea.

Molecular dynamics simulations of barnase have been conducted both in water and in 8 M urea solution for 500 ps at 25 degrees C and for 2000 ps at 85 degrees C. The final structure of the aqueous simulation at room temperature matches closely the structure obtained by NMR and the experimentally observed protections from isotopic exchange. The comparison of the structures generated by the aqueous simulation at 85 degrees C reveals a trajectory composed of groups of geometrically related structures separated by narrow regions of rapid change in structure. The first of these regions displays changes in backbone rmsd to the crystal structure and solvent-accessible area suggestive of a transition state, while the properties observed during the final 300 ps of the simulation are consistent with a stable intermediate. These assignments were confirmed by calculation of the "progress along the reaction coordinate" phi-values using an empirical equation based on a linear response method. The pathway of unfolding defined in this fashion agrees well with the experimental results of site-directed mutagenesis in terms of secondary structure content of the transition state and the intermediate and reproduces the relative stability of the different elements of secondary structure. The results of the simulations in urea suggest a mechanism at the molecular level for its well-known enhancement of the denaturation of proteins. The analysis of radial distribution functions shows that the first solvation shell of the protein is enriched in urea relative to the bulk solvent. The displacement of water molecules allows greater exposure of hydrophobic side chains, as witnessed particularly in the analysis of solvent-accessible surface areas at the higher temperature. Almost all urea molecules in the first shell form at least one hydrogen bond with the protein. They provide a more favorable environment for accommodation of the remaining water molecules, and they facilitate the separation of secondary structure elements by acting as a bridge between groups previously forming intraprotein hydrogen bonds.

[Objectives, results and future prospects of burn treatment in 1997]. / Objectifs, résultats et perspectives du traitement des brûlés en 1997.

When burn injuries to the skin are extensive, delays in wound closure contribute to multiple organ failure because the availability of donor sites does not allow early and permanent coverage of excised wounds. From 1991 to 1996, 30 patients with a mean burn size of 78% total body surface area (65% full-thickness) underwent skin grafting with autologous cultured epidermis (AEC) performed in the labs of Genzyme Tissue Repair Company. Twenty three were adults and seven children under 15 (mean age 29, range 2.5 to 70); 27 suffered inhalation injury; 3 presented with multiple trauma and 2 with blast injury. As soon as possible wound beds were excised and temporarily covered with allografts or with sandwich or meshed autografts; the mean surface covered with autografts was 28 +/- 12%. Keratinocytes grafts were applied to a mean of 37 +/- 16.5%, an average of 210 grafts of 25 to 30 cm2. Three patients died respectively at day 67, 81 and 90. At time of gaze backing removal, the mean percentage of culture engraftment was 69% (range 25 to 95); this engraftment was higher for children (74%) and very bad above 60 (25%). The mean length of hospitalisation was 114 +/- 30 days. The definitive coverage by AEC was evaluated through the percentage of secondary autografted area: 10 +/- 9.5% (range 0 to 46). The average cost by patient was 98,500$ or 16$ by cm2 of culture. The weakness of epithelialisation makes essential a dermal support to the keratinocytes cultures, allodermis is now currently used, perhaps the new skin substitutes will give the ideal missing piece.

Clinical interest of cutaneous models reproduced in vitro for severe burn treatment: histopathological and ultrastructural study.

The healing of minimal skin lesions is usually obtained by epidermal migration and proliferation from peripheral wound margins. However, cutaneous grafts or reconstituted skin are necessary for severe injuries. Various models have recently been reproduced for this purpose. The aim of this work is to report the histopathologic evolution of burn lesions treated two years ago by autologous epidermis (Genzyme Tissue Repair, Boston, USA). Fifteen patients with severe burns (more than 80% of surface) have been treated. These observations have been based exclusively on biopsies of grafted wounds. Cultured epidermis is rapidly fully differentiated after grafting with temporary hyperplasia and normal strata. At 18 months, rete ridges formation is present only in young patients. Melanocytes and Langerhans' cells repopulated grafts rapidly. The use of cultured epidermis nowadays represents an important improvement in burn treatment.