A new forensic tool to date human blood pools.

Courtrooms are asking for reliable scientific evidence in order to prevent wrongful convictions. Thus, a more rigorous approach to forensic science approved by scientific methods is promoted. The study of human blood dynamics in the context of forensic science is becoming a widespread research topic, although the physics behind wetting and drying of blood is not completely understood. Based on the morphological changes of drying blood pools, the following work presents a patentable method to quantitatively date these blood pools for forensic purposes. As for drying drops of blood, cracking patterns are observed but they are more disordered. Similar disordered crack patterns are observed in the case of gels, their evaporation process is, therefore, presented since this topic has been thoroughly investigated. We aim to find reliable patterns that could give information concerning the evolution of a blood pool over time to lead to practical application of this knowledge. An empirical model is established between final dried blood patterns and the generating mechanism, yielding application in bloodstain pattern analysis for forensic investigations.

Transdermal drug delivery using electroporation. I. Factors influencing in vitro delivery of terazosin hydrochloride in hairless rats.

The use of electroporation pulses as a physical means of enhancing the permeability of skin to deliver drugs is in the early stages of development. In this article, a systematic study examining the parameters influencing electroporative transdermal delivery of terazosin hydrochloride to hairless rat skin are reported. It was found that voltage, pulse length (tau), and number of pulses were the three most important parameters, in that order. For creating a significant enhancement in drug delivery to the skin, without causing any apparent change in its external appearance, it was necessary to deliver five or more exponentially decaying electroporation pulses, at 88 +/- 2.5 V (voltage across the skin), with a decay time constant of 20 ms. Electrodes with larger area could attain the same voltages across the skin with a much lower applied voltage and possessed other advantages with regard to performance of the drug delivery system.

Transdermal drug delivery using electroporation. II. Factors influencing skin reversibility in electroporative delivery of terazosin hydrochloride in hairless rats.

A previous study indicated that the parameters governing the performance of electroporative delivery to the skin, are voltage, pulse length, number of pulses and electrode area.1 This article describes a study in which the reversibility of the electroporation technique is evaluated with in vitro methods. The skin's reversal from an enhanced permeation mode as a result of electroporation to the base level was used as an index to understand the mechanism of drug delivery and also as a preliminary indicator of safety. Maximum delivery of the model drug, terazosin hydrochloride, occurred during the pulsing. Electroporative delivery with a wire electrode (small-area electrode, 0.56 cm(2)) using 20 pulses at U(skin,0) 88 V, and pulse length 20 ms, did not cause any damage to the skin. Increasing the pulse length to 60 ms, while keeping the rest of the parameters fixed, caused a visible change in the external appearance of the skin. However, with the use of a spiral electrode (large-area electrode, 2.74 cm(2)) at 60-ms pulse length, there was minimal damage to the skin. This may be attributed to the more uniform flow of current over the whole skin area. The large-area electrode required a smaller electrode voltage, U(electrode,0) for any given U(skin,0) and also delivered nearly double the instantaneous power density compared with the small-area electrode. These findings indicate that using shorter pulses and large-area electrodes is a safer technique than large pulses and small-area electrodes when electroporation is used to enhance skin's permeability for drug delivery.

Ligand linked assembly of Scapharca dimeric hemoglobin.

The assembly of Scapharca dimeric hemoglobin as a function of ligation has been explored by analytical gel chromatography, sedimentation equilibrium, and oxygen binding experiments to test the proposal that its cooperativity is based on quaternary enhancement. This hypothesis predicts that the liganded form would be assembled more tightly into a dimer than the unliganded form and that dissociation would lead to lower oxygen affinity. Our experiments demonstrate that although the dimeric interface is quite tight in this hemoglobin, dissociation can be clearly detected in the liganded states with monomer to dimer association constants in the range of 10(8) M-1 for the CO-liganded state and lower association constants measured in the oxygenated state. In contrast, the deoxy dimer shows no detectable dissociation by analytical ultracentrifugation. Thus, the more highly hydrated deoxy interface of this dimer is also the more tightly assembled. Equilibrium oxygen binding experiments reveal an increase in oxygen affinity and decrease in cooperativity as the concentration is lowered (in the muM range). These experiments unambiguously refute the hypothesis of quaternary enhancement and indicate that, as in the case of human hemoglobin and other allosteric proteins, quaternary constraint underlies cooperativity in Scapharca dimeric hemoglobin.

Education in primary care psychiatry: research-based audit.

This paper describes some of the difficulties faced by the KCGP Mental Health Education Fellows in designing an educational initiative to enhance the recognition and management of psychiatric illness in general practice. The initiative was evaluated using a consensus technique over six meetings which took place between 1993 and 1995. The results of the exercise illustrated the value of utilizing a validated instrument in identifying mental health problems, but when employed without support created disillusionment and a lack of confidence in the learner. However, when the instrument was applied as a part of a wide educational process examining current practice, learners were stimulated to examine and change their behaviour by synthesizing newly acquired knowledge with their own previous clinical experience.

Cyanomet human hemoglobin crystallized under physiological conditions exhibits the Y quaternary structure.

Cyanomet human hemoglobin has been crystallized at a chloride ion concentration and pH similar to physiological conditions. Molecular replacement calculations definitively show that the hemoglobin subunits are arranged in the Y quaternary form recently discovered in carbon monoxy hemoglobin Yp-silanti (beta 99 Asp-Tyr), and subsequently observed in carbon monoxy normal human hemoglobin crystallized at low ionic strength and low pH. The structure has been refined at 2.09 A resolution to an R-value of 0.232, and further refinement is currently underway. Although the refinement is not yet complete, our results are the first indication that the Y structure may represent an important quaternary form of liganded hemoglobin under physiological buffer conditions. These results suggest the need for a reexamination of structure-function correlations in the hemoglobin system.

Expected duration of hospital stay of low birth weight infants: graphic depiction in relation to birth weight and gestational age.

Neonatal duration of hospital stay correlated with both birth weight and gestational age in positively skewed, nonlinear relationships. Within increments of birth weight, gestational age had a semi-independent influence on length of stay. Log length of stay correlated with both birth weight and gestational age in linear, normally distributed relationships. A nomogram is provided for predicting individual lengths of stay.

Perceptual processing of pattern goodness by left and right hemispheres.

Two experiments tested perceptual processing of Garner and Clement's (1963) good and poor five-dot patterns by the left and right hemispheres. Two (good) patterns were from four-member equivalence sets, and the other (poor) pattern was from an eight-member equivalence set. One of the good patterns formed a T shape that could be processed as a linguistic unit. In Experiment 1, 80 right-handed subjects made same-different judgments for lateralized 200-msec paired presentations of these patterns. When both presentations were to the RVF/LH, response latency was faster for the T pattern than for the other two. When both presentations were to the LVF/RH, response latency was faster for the two good patterns than for the poor pattern. When the first pattern was presented to the RVF/LH and the second was presented to the LVF/RH, response latencies were lower for the two good patterns than for the poor pattern. Also, when the first pattern was presented to the LVF/RH and the second was presented to the RVF/LH, response latency was faster for the T pattern than for the other good pattern which was, in turn, faster than for the poor pattern. Experiment 2 used a duration judgment task (Avant & Lyman, 1975) to test effects of pattern goodness on apparent durations of pre- and postmasked 10-msec pattern presentations. With left hemisphere inputs, presentations of good patterns were judged to be longer than presentations of the poor pattern. When each hemisphere compared the T and the other good pattern, presentations of the T pattern were judged to be longer, and the right hemisphere further discriminated among pattern orientations. Presentations of the T pattern to each hemisphere were judged to be longer than presentations of the poor pattern, and both hemispheres discriminated among orientations of both patterns. These results indicate that the two hemispheres can, during perceptual processing, function cooperatively, and that both prerecognition and conscious perceptual operations are guided by task demands.

Mutagenic dissection of hemoglobin cooperativity: effects of amino acid alteration on subunit assembly of oxy and deoxy tetramers.

Free energies of oxygen-linked subunit assembly and cooperative interaction have been determined for 34 molecular species of human hemoglobin, which differ by amino acid alterations as a result of mutation or chemical modification at specific sites. These studies required the development of extensions to our earlier methodology. In combination with previous results they comprise a data base of 60 hemoglobin species, characterized under the same conditions. The data base was analyzed in terms of the five following issues. (1) Range and sensitivity to site modifications. Deoxy tetramers showed greater average energetic response to structural modifications than the oxy species, but the ranges are similar for the two ligation forms. (2) Structural localization of cooperative free energy. Difference free energies of dimer-tetramer assembly (oxy minus deoxy) yielded delta Gc for each hemoglobin, i.e., the free energy used for modulation of oxygen affinity over all four binding steps. A structure-energy map constructed from these results shows that the alpha 1 beta 2 interface is a unique structural location of the noncovalent bonding interactions that are energetically coupled to cooperativity. (3) Relationship of cooperativity to intrinsic binding. Oxygen binding energetics for dissociated dimers of mutants strongly indicates that cooperativity and intrinsic binding are completely decoupled by tetramer to dimer dissociation. (4) Additivity, site-site coupling and adventitious perturbations. All these are exhibited by individual-site modifications of this study. Large nonadditivity may be correlated with global (quaternary) structure change. (5) Residue position vs. chemical nature. Functional response is solely dictated by structural location for a subset of the sites, but varies with side-chain type at other sites. The current data base provides a unique framework for further analyses and modeling of fundamental issues in the structural chemistry of proteins and allosteric mechanisms.

The mutation beta 99 Asp-Tyr stabilizes Y--a new, composite quaternary state of human hemoglobin.

Carbonmonoxy hemoglobin Ypsilanti (beta 99 Asp-Tyr) exhibits a quaternary form distinctly different from any structures previously observed for human hemoglobins. The relative orientation of alpha beta dimers in the new quaternary form lies well outside the range of values observed for normal unliganded and liganded tetramers (Baldwin, J., Chothia, C., J. Mol. Biol. 129:175-220, 1979). Despite this large quaternary structural difference between carbonmonoxy hemoglobin Ypsilanti and the two canonical structures, the new quaternary structure's hydrogen bonding interactions in the "switch" region, and packing interactions in the "flexible joint" region, show noncovalent interactions characteristic of the alpha 1 beta 2 contacts of both unliganded and liganded normal hemoglobins. In contrast to both canonical structures, the beta 97 histidine residue in carbonmonoxy hemoglobin Ypsilanti is disengaged from quaternary packing interactions that are generally believed to enforce two-state behavior in ligand binding. These features of the new quaternary structure, denoted Y, may therefore be representative of quaternary states that occur transiently along pathways between the normal unliganded, T, and liganded, R, hemoglobin structures.

The use of hyaluronidase in the treatment of intravenous extravasation injuries.

Intravenous extravasation injuries are a major cause of morbidity in the pediatric population. A variety of commonly used drugs and intravenous fluids have been shown to cause injury, particularly when infants are infused subcutaneously. Optimal management of intravenous extravasations remains controversial. The enzyme hyaluronidase degrades hyaluronic acid, a constituent of the normal interstitial barrier; by degrading interstitial bonds, it can increase the distribution and absorption of locally injected substances. To test the hypothesis that hyaluronidase might prevent skin injury associated with extravasations, a reliable skin injury model in immature pigs was created using subcutaneous CaCl2 injections. Hyaluronidase, in a concentration of 150 U/mL injected subcutaneously in a circumferential fashion immediately following the injection of CaCl2, significantly reduced the area of necrosis (P less than .01). As a control for the diluent volume administered with the hyaluronidase injection, the effect of a circumferential injection of 1.0 mL normal saline was compared with a similar injection of 1.0 mL normal saline with 150 units of hyaluronidase. Again, the area of skin necrosis following injection with hyaluronidase was statistically smaller (P less than .01). We have created a reliable skin injury model using immature porcine skin, which resembles human skin. Our data using this model suggest that the use of hyaluronidase may decrease the morbidity associated with intravenous extravasation injuries.

Superior vena cava syndrome in infants.

Superior vena cava syndrome is uncommon in infants. With the increased use of central venous catheters, however, there has been an increased incidence of central venous occlusion. We report a case of superior vena cava syndrome occurring on two separate occasions in a premature infant with an indwelling central line. We discuss current concepts of prevention and management.

Congenital absence of the left coronary artery.

Congenital absence of the left coronary artery system and the prevalence of a single right coronary artery to supply the heart is a rare anomaly. There is not yet enough data to determine its importance because only a few have been found in living patients. This is just such a case, diagnosed premortem in an otherwise healthy coronary system with no other physical abnormalities.

Three-state combinatorial switching in hemoglobin tetramers: comparison between functional energetics and molecular structures.

In a previous study on cyanomethemoglobin the 10 tetrameric species (each with a unique combination of ligated and unligated subunits) were found to exhibit three distinct free energies of cooperative interaction. The distribution of these free energies among the partially ligated species is incompatible with a two-state mechanism of molecular switching and requires a minimum of three molecular structures with distinctly different free energies of heme-heme interaction. Ligand-linked transitions between the three cooperativity states were found to be "combinatorial"--i.e., dependent upon changes in both the number and specific configuration of bound ligands. Here we present results from two other chemical systems that mimic intermediate oxygenation states. In these systems the heme iron is replaced by manganese in certain of the subunits. We find the same distribution of cooperative free energies as reported for the cyanomethemoglobin system. These results demonstrate that the three-state combinatorial nature of cooperative switching is neither a special feature of the cyanomet reactions nor of the substitution of manganese for iron, but reflects a fundamental property of hemoglobin. These findings are compared with crystallographic structural results on partially ligated hemoglobins.

Selective immunomodulation by the antineoplastic agent mitoxantrone. II. Nonspecific adherent suppressor cells derived from mitoxantrone-treated mice.

Mitoxantrone exerts a potent suppressive influence upon humoral immune responses. The B cell is a likely target for this inhibitory effect, and we have reported evidence supporting this possibility. The impact of mitoxantrone upon T lymphocyte reactivity was assessed as a second mode of action of this novel antineoplastic drug. TH and TS lymphocyte induction were tested in the in vitro anti-sheep erythrocyte response, and a surprising differential effect of mitoxantrone was observed. Helper activity was abrogated and suppressor function was enhanced. In apparent disagreement with this result, mitoxantrone inhibited the in vivo induction of TS cells using trinitrophenylated spleen cells. Macrophages were investigated as potential mediators of these effects upon immunoregulatory function. Replacement of macrophages in mitoxantrone-treated spleen cell preparations by normal adherent cells allowed the induction and complete expression of TH lymphocyte function. Conversely, replacement of mitoxantrone-treated macrophages with normal adherent cells before induction of TS cells failed to generate TS cell function. Thus, TH cells were resistant and TS cells were completely susceptible to mitoxantrone. Furthermore, supplementation of normal TH cell cultures with splenic macrophages from mitoxantrone-treated mice inhibited the induction of helper function. Production of the lymphokines IL 2 and TRF in mitoxantrone-treated mice was normal. This is consistent with the retention of functional TH cells in drug-treated spleens. Macrophages in the spleens of mitoxantrone-treated mice were responsible for the abrogated helper function and the enhanced suppressor activity. Although TS cell induction was directly inhibited by the drug, the effect upon TH cell function was secondary to the action of mitoxantrone-induced suppressor macrophages. Mitogen-stimulated lymphokine production was normal. Thus, mitoxantrone is a selective immunomodulator. The macrophage-mediated suppression of TH cell induction and humoral immunity investigated in spleens from mitoxantrone-treated mice is an intriguing finding that may have significant implications for immunotherapy.