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γδ T cells are potent anticancer effectors with the potential to target tumours broadly, independent of patient-specific neoantigens or human leukocyte antigen background1–5. γδ T cells can sense conserved cell stress signals prevalent in transformed cells2,3, although the mechanisms behind the targeting of stressed target cells remain poorly characterized. Vγ9Vδ2 T cells—the most abundant subset of human γδ T cells4—recognize a protein complex containing butyrophilin 2A1 (BTN2A1) and BTN3A1 (refs. 6–8), a widely expressed cell surface protein that is activated by phosphoantigens abundantly produced by tumour cells. Here we combined genome-wide CRISPR screens in target cancer cells to identify pathways that regulate γδ T cell killing and BTN3A cell surface expression. The screens showed previously unappreciated multilayered regulation of BTN3A abundance on the cell surface and triggering of γδ T cells through transcription, post-translational modifications and membrane trafficking. In addition, diverse genetic perturbations and inhibitors disrupting metabolic pathways in the cancer cells, particularly ATP-producing processes, were found to alter BTN3A levels. This induction of both BTN3A and BTN2A1 during metabolic crises is dependent on AMP-activated protein kinase (AMPK). Finally, small-molecule activation of AMPK in a cell line model and in patient-derived tumour organoids led to increased expression of the BTN2A1–BTN3A complex and increased Vγ9Vδ2 T cell receptor-mediated killing. This AMPK-dependent mechanism of metabolic stress-induced ligand upregulation deepens our understanding of γδ T cell stress surveillance and suggests new avenues available to enhance γδ T cell anticancer activity.

Cold atmospheric plasmas (CAPs) have shown great promise for medical applications through their synergistic chemical, electrical, and thermal effects, which can induce therapeutic outcomes. However, safe and reproducible plasma treatment of complex biological surfaces poses a major hurdle to the widespread adoption of CAPs for medical applications. Predictive modeling of the mutual interactions between the plasma and biological surfaces and, thus, systematic approaches to quantify and predict plasma treatment outcomes remain largely elusive due to the lack of mechanistic understanding of plasma–surface interactions that can span across vastly different length scales and timescales. In addition, real-time sensing capabilities in biomedical CAP devices are often limited, which can be detrimental to plasma treatment due to the intrinsic plasma and surface variability during the treatment, as well as sensitivity to external perturbations. All of these challenges can make reproducible and effective plasma treatment of biological surfaces difficult to realize, which is further compounded by errors due to the human operation of hand-held CAP devices. Machine learning and data-driven approaches can be particularly useful in addressing these challenges in three major ways: 1) data-driven modeling of hard-to-model plasma–surface interactions and plasma treatment outcomes; 2) learning data analytics for plasma and surface diagnostics in real time; and 3) developing predictive controllers that enable reliable and effective CAP treatments. This article discusses the promise of machine learning to accelerate plasma medicine research in these areas, toward machine learning-assisted and automated CAP treatment of complex biological surfaces.

Atmospheric pressure plasma jets (APPJs) are increasingly used for biomedical applications. Reproducible and effective operation of APPJs hinges on controlling the nonlinear effects of plasma on a target substrate in the face of intrinsic variabilities of the plasma as well as exogenous disturbances. This paper presents a low-memory fast approximate nonlinear model predictive control (NMPC) strategy for an APPJ with prototypical applications in plasma medicine. The NMPC objective is to regulate the delivery of the cumulative thermal effects of plasma to a substrate, while adhering to constraints pertaining to a patient’s safety and comfort. Deep neural networks are used to approximate the implicit NMPC law with a cheap-to-evaluate explicit control law that has low memory requirements. Robust constraint satisfaction is guaranteed by projecting the output of the neural network onto a set that ensures the state stays within an appropriately defined invariant set. Closed-loop simulations and real-time control experiments indicate that the proposed approximate NMPC strategy is effective in handling nonlinear control costs at fast sampling times, while guaranteeing satisfaction of safety-critical system constraints. This work takes a crucial step toward fast NMPC of safety-critical plasma applications using resource-limited embedded control hardware.

The electronic structures and spectroscopic properties of four bisanthracene bis(dicarboxylic imide)s (M1–M4) have been investigated theoretically by using density functional theory (DFT) and its time-dependent extension (TDDFT) in view of their potential use as photosensitizers in photodynamic therapy (PDT). The optimized geometries, electronic absorption transitions, singlet–triplet energy gaps, spin–orbit matrix elements, ionization potentials, and electron affinities have been determined in gas phase and in solvent. Both type I and II PDT mechanisms have been considered. In addition, the variation of a series of relevant properties upon heavy atom substitution (Br and I) have been determined and discussed. Results show that only M4 is able to support the type I reaction, and one of its brominated and iodinated derivatives can produce cytotoxic singlet oxygen (type II reaction).

Real-time diagnostics of cold atmospheric plasma (CAP) sources can be challenging due to the requirement for expensive equipment and complicated analysis. Data analytics that rely on machine learning (ML) methods can help address this challenge. In this paper, we demonstrate the application of several ML methods for real-time diagnosis of CAPs using information-rich optical emission spectra and electro-acoustic emission. We show that data analytics based on ML can provide a simple and effective means for estimation of operation-relevant parameters such as rotational and vibrational temperature and substrate characteristic in real-time. Our findings indicate a great potential promise for ML for real-time diagnostics of CAPs.

This paper presents a comparative study between two constraint-tightening approaches for tube-based stochastic nonlinear model predictive control (SNMPC) with and without terminal constraints. A simple constraint-tightening method based on the exponential decay rate of a δ-Lyapunov function is extended to the stochastic setting. This method uses the notion of incremental stabilizability to alleviate the need for offline, but involved computation of terminal constraints. The proposed method is compared to a SNMPC formulation that employs terminal constraints and Lipschitz constant-based constraint tightening. A comparative analysis is presented on a benchmark continuous stirred-tank reactor problem. Practical approximations for computing terminal sets are discussed in the context of this comparison.

Introduction:Isatuximab (Isa), a humanized monoclonal antibody directed against the surface antigen CD38, has been available in Italy since October 2021 for the treatment of relapsed or refractory Multiple Myeloma (RRMM) in combination with Pomalidomide (P) and Dexamethasone (d) (IsaPd) after two lines of therapy with lenalidomide and a proteosome inhibitor, and from April 2022 in combination with carfilzomib (K) after one line of treatment. Patients eligible for these therapies must not be refractory to Daratumumab. Here, we report preliminary efficacy and safety data for Isatuximab in a Sardinian study of RRMM patients treated with IsaPd and IsaKda as salvage therapy outside of controlled clinical trials. The primary objective of this study was to evaluate the toxicity profile and quality of response to Isatuximab-based regimens administered as salvage therapy in a real-world setting.

The local atomic configuration of graphene oxide (GO) was investigated by identifying the different oxygen functionalities and following their evolution induced by thermal treatments in various environments (vacuum, nitrogen or argon flow). X-ray photoelectron spectroscopy and scanning transmission electron microscopy analyses were performed, and electron energy-loss (EEL) spectra were acquired in different regions of GO and thermally reduced GO flakes. Experimental results show a series of characteristic peaks related to C and O K-edge shells and different features of GO thermally annealed at the same temperature but in different environments. In order to understand the experimental results, density functional theory calculations of core-loss EEL spectra of GO (C and O K-edges) in the presence of oxygen functional groups have been performed for different combinations and/or concentrations. Such calculations have allowed for the association of the observed experimental peaks to the presence of specific oxygen functional groups, giving the opportunity to establish the atomic configurations that prevail in different ranges of annealing temperatures and environments.

•Amp(1q), HRCA ≥2, high CTC levels, and a longer time to reach first MRD negativity are significant predictors of unsustained MRD negativity.•Maintenance treatment with carfilzomib-lenalidomide reduces the risk of unsustained MRD negativity vs lenalidomide alone.

The Borexino detector has convincingly shown its outstanding performance in the in the sub-MeV regime through its unprecedented accomplishments in the solar and geo-neutrinos detection, which make it the ideal tool to unambiguously test the long-standing issue of the existence of a sterile neutrino, as suggested by several anomalies: the outputs of the LSND and Miniboone experiments, the results of the source calibration of the two Gallium solar νexperiments, and the recently hinted reactor anomaly. The SOX project will exploit two sources, based on chromium and cerium, which deployed under the experiment will emit two intense beams of νe(Cr) and νe‾(Ce). Interacting in the active volume of the liquid scintillator, each beam would create a spatial wave pattern in case of oscillation of the νe(or νe‾) into the sterile state, which would be the smoking gun proving the existence of the new sterile member of the neutrino family. Otherwise, its absence will allow setting very stringent limit on its existence.

The Borexino detector was built starting from 1996 in the underground hall C of Gran Sasso National Laboratory (LNGS) in Italy under about 1400 m of rock (3800 m.w.e) and it is mostly aimed to the study in real-time of the low-energy solar neutrinos.

DarkSide-50 (DS-50) at Gran Sasso underground laboratory (LNGS), Italy, is a direct dark matter search experiment based on a TPC with liquid argon. DS-50 has completed its first dark matter run using atmospheric argon as target. The DS-50 detector performances and the results of the first physics run are reviewed in this proceeding.

National Committee for Quality Assurance recommends patient-centered medical homes incorporate input from patient populations; however, many health care organizations do not. This qualitative study used two open-ended questions from 148 active duty Army Soldiers and their family members to illicit recommendations for primary care providers and clinic leadership that would improve their health care experiences. Content analysis and descriptive statistics were used to analyze responses. Participant responses were related to four major themes: Access to Care, Interpersonal Interaction, Satisfaction of Care, and Quality of Care. Participants were overall satisfied with their care; however, spending less time waiting for appointments and to see the provider or specialist were the most frequently requested improvements related to Access to Care. For Interpersonal Interaction, 82% of the responses recommended that providers be more attentive listeners, courteous, patient, caring, and respectful. Decreasing wait times and improving interpersonal skills would improve health care experiences and patient satisfaction.

The Sun is fueled by a series of nuclear reactions that produce the energy that makes it shine. The primary reaction is the fusion of two protons into a deuteron, a positron and a neutrino. These neutrinos constitute the vast majority of neutrinos reaching Earth, providing us with key information about what goes on at the core of our star. Several experiments have now confirmed the observation of neutrino oscillations by detecting neutrinos from secondary nuclear processes in the Sun; this is the first direct spectral measurement of the neutrinos from the keystone proton-proton fusion. This observation is a crucial step towards the completion of the spectroscopy of pp-chain neutrinos, as well as further validation of the LMA-MSW model of neutrino oscillations.

Plasma spray techniques have been used to increase the Al content on existing NiCoCrAlY films in order to obtain high temperature resistant coatings on Ni-base superalloys for gas turbine applications. Surface alloying has been produced by thermal diffusion under vacuum of the Al and MCrAlY films. This treatment reduces the surface roughness and increases the coating density by sealing its porosity. The aluminides formed have been analyzed in terms of their structural composition and oxidation resistance. Al additions are effective in reducing the oxidation rates, in particular for the MCrAlY coatings which are APS deposited. By increasing the temperature over 1100 °C, the coating efficiency rapidly decreases.

The coherent signal of Small Angle Scattering superimposes to a flat background, which is given by incoherent scattering, by electronic noise and, sometimes, by radiation background around the instrument. In order to analyze the experimental data, the background level has to be taken into account and quite often theoretical estimations do not give values comparable to the experimental evidences. In this paper it is shown a new representation which allows in a simple way the background level determination ; in particular, in a convenient plot, the experimental data are fitted by a straight line passing through the origin, the slope of which is equal to the fourth root of the background.

Density functional theory and its time-dependent extension (DFT, TDDFT) has been herein employed to elucidate the structural and electronic properties for a series of isoindole-boron dipyrromethene (isoindole-BODIPY) derivatives. The role played by both the nature and the positions of the substituents on intersystem spin-crossing has been investigated computing the spin–orbit matrix elements between singlet and triplet excited state wave functions weighted by the TDDFT transition coefficients. Their potential therapeutic use as photosensitizers in photodynamic therapy (PDT) is proposed on the basis of their strong absorbance in the red part of the visible spectrum, vertical triplet energies resulting higher than 0.98 eV, and the spin–orbit matrix elements that result to be comparable with different drugs already used in PDT.

To determine which individual characteristics (religious participation, mistrust, racism/discrimination, spirituality, perceived access to care, and continuity of care) were predictors of patient outcomes (patient satisfaction, physical health, and mental health status) for an Army health care clinic transitioning to the Patient-Centered Medical Home (PCMH).

Background.In multiple myeloma (MM), the role of minimal residual disease (MRD) by multiparameter flow cytometry (MFC) and next-generation sequencing (NGS) is well established (H. Avet-Loiseau et al. IMW 2019, S. Oliva et al. EHA 2020).

The use of large animals (e.g., pig and sheep) in human medicine, and the need to develop new therapeutic strategies for domestic animal diseases related to platelet disorders, require better characterization of the physiology of animal platelets. In this study, the ability of platelets from buffaloes, horses, pigs and sheep to adhere to immobilized autologous fibrinogen was compared with that of human platelets. Blood samples were collected from the jugular vein of six healthy subjects of each species and platelet-rich plasma (PRP) was obtained by centrifugation. Platelets, isolated by further centrifugation of PRP, were washed by gel-filtration on Sepharose-2B, counted and added to the wells of 96-well plates pre-coated with autologous fibrinogen. After different times of incubation, non-adherent platelets were removed, and the number of adherent platelets was assessed by measuring endogenous acid phosphatase activity. Horse platelets showed the strongest ability to adhere to autologous immobilized fibrinogen, being 1·7-, 3·1- and 2·3-fold more active than human, buffalo and porcine platelets, respectively. Sheep platelets were unable to adhere to autologous immobilized fibrinogen. Platelet activation by adenosine 5-diphosphate (ADP) increased both human and animal platelet adhesive response. ADP-stimulated sheep platelets were able to adhere to autologous immobilized fibrinogen, albeit to a lesser extent than platelets from the other animal species. The observed interspecies variability in adhesive properties of platelets may reflect structural differences, or differences in the availability of the fibrinogen receptor (glycoprotein IIb/IIIa) on the platelet surface.

The prototype of the Borexino detector Counting Test Facility, located in the Gran-Sasso laboratory, has been used to obtain a bound on the stability of the electron. The new lower limit on the mean lifetime defined on 32.1 days of data set is τ(e−→νe+γ)⩾4.6×1026yr (90% c.l.).

Failure of chemotherapy with anthracyclines as a result of drug resistance and toxicity is a major problem in the clinical management of neoplasia. The aim of the present study was to evaluate the activity of medroxyprogesterone acetate (MPA) as a chemosensitiser on anthracycline cytotoxicity. The study investigated whether such an effect could be related to an increase in lipid peroxidation, nitric oxide production, membrane fluidity and intracellular anthracycline concentration. The results showed that anthracyclines decreased nitric oxide production but increased membrane viscosity (polarisation constant) and lipid hydroperoxide formation in canine mammary tumour cells. Moreover, it was found that both drug-induced cytotoxicity and membrane viscosity increased in the presence of MPA. Conversely, lipid hydroperoxides decreased in MPA-supplemented cells. Medroxyprogesterone acetate did not show any effect on nitric oxide production. The two anthracyclines used (doxorubicin and idarubicin) showed differential intranuclear accumulation in canine mammary tumour cells, and MPA significantly modified intracellular concentration of anthracyclines.

Monofilament synthetic absorbable suture materials offer excellent glide characteristics and cause minimal tissue trauma as a result of their smooth monofilament structure and gradual bio-absorption. An investigation was conducted on 72 rats to compare three types of monofilament absorbable suture material (Polydioxanone, Poliglecaprone 25, Glycomer 631), with respect to their clinical characteristics, tissue inflammatory reaction and suture absorption times. The results identified different qualities for each suture: Poliglecaprone 25 and Glycomer 631 suture materials were found to be less reactive than Polydioxanone in rat skin. However, because of their extremely low tissue reaction values, all three materials were deemed particularly suitable for use as intracuticular sutures. Absorption times in rat skin were less than 3 months for Poliglecaprone 25, between 3 and 6 months for Glycomer 631 and 6 months for Polydioxanone. The differences in suture characteristics which were detected in our study can help in the surgical selection of the most appropriate suture material. Copyright 2000 The British Association of Plastic Surgeons

Echistatin, flavoridin and kistrin belong to a family of low molecular weight snake-venom proteins, termed “disintegrins” because of their ability to bind integrin receptors on the cell surfaces. Most disintegrins contain the tripeptide arginine-glycine-aspartic acid (RGD) sequence, which represents a common cell adhesion recognition site. Here we report the differing activity of echistatin, flavoridin and kistrin on adenosine 5′-diphosphate (ADP)-induced aggregation of platelets from the buffalo, dog and horse. The three disintegrins inhibited the aggregation of platelets from all three animal species at nanomolar concentrations. Echistatin was the most active of the disintegrins towards equine platelets, but flavoridin and kistrin showed a higher potency than echistatin in inhibiting aggregation of platelets from the buffalo and dog. Kistrin was 1·6-fold more effective than flavoridin in inhibiting ADP-induced aggregation of platelets from either the buffalo or dog, whereas flavoridin was 2·1-fold more active than kistrin in inhibiting aggregation of equine platelets. The species-dependent platelet sensitivity to these snake-venom proteins may reflect structural differences of the integrin receptor GP IIb/IIIa on the platelet surface in different mammalian species.

Prediction of gene structure in newly sequenced DNA becomes very important in large genome sequencing projects. This problem is complicated due to the exon-intron structure of eukaryotic genes and because gene expression is regulated by many different short nucleotide domains. In order to be able to analyse the full gene structure in different organisms, it is necessary to combine information about potential functional signals (promoter region, splice sites, start and stop codons, 3' untranslated region) together with the statistical properties of coding sequences (coding potential), information about homologous proteins, ESTs and repeated elements.

Motivation: Prediction of gene structure in newly sequenced DNA becomes very important in large genome sequencing projects. This problem is complicated due to the exon-intron structure of eukaryotic genes and because gene expression is regulated by many different short nucleotide domains. In order to be able to analyse the full gene structure in different organisms, it is necessary to combine information about potential functional signals (promoter region, splice sites, start and stop codons, 3′ untranslated region) together with the statistical properties of coding sequences (coding potential), information about homologous proteins, ESTs and repeated elements.
Results: We have developed the GeneBuilder system which is based on prediction of functional signals and coding regions by different approaches in combination with similarity searches in proteins and EST databases. The potential gene structure models are obtained by using a dynamic programming method. The program permits the use of several parameters for gene structure prediction and refinement. During gene model construction, selecting different exon homology levels with a protein sequence selected from a list of homologous proteins can improve the accuracy of the gene structure prediction. In the case of low homology, GeneBuilder is still able to predict the gene structure. The GeneBuilder system has been tested by using the standard set (Burset and Guigo, Genomics, 34, 353-367, 1996) and the performances are: 0.89 sensitivity and 0.91 specificity at the nucleotide level. The total correlation coefficient is 0.88.
Availability: The GeneBuilder system is implemented as a part of the WebGene a the URL: http://www.itba.mi.cnr.it/webgene and TRADAT (TRAncription Database and Analysis Tools) launcher URL: http://www.itba.mi.cnr.it/tradat.
Contact: milanesi@itba.mi.cnr.it

Pharmacologic and molecular evidence conflicts in regard to the existence of tissue-specific subtypes of thromboxane A2 receptors (TXR). The full length TXR complementary DNA (cDNA) was cloned from a platelet-like cell line. It was expressed and its pharmacology was characterized. Northern analysis of TXR transcripts in multiple tissues showed strong hybridization to K562 chronic myelogenous leukemia messenger RNA. Therefore, a K562 cDNA library was screened and a full-length TXR cDNA (K562TXR) was isolated. K562TXR encodes a protein identical to the previously characterized placenta TXR cDNA, except for a single amino acid substitution (Glu21-->Lys). Similar to thromboxane receptors on K562 cells, K562TXR transiently expressed in HEK 293 cells (K562TXR/293) bound the thromboxane agonist 125I-labeled [1S-(1 alpha,2 beta(5Z),3 alpha-(1E,3S),4 alpha]-7-[3-(3-hydroxy-4-(p- iodophenoxy)-1-butenyl)-7-oxabicyclo-[2.2.1]heptane-2-yl]-5- heptenoic acid ([125I]BOP) with a Kd of 5.5 +/- 1.1 nM, a Bmax of 289,056 +/- 60,220 sites/cell and a Hill coefficient of -0.94 +/- 0.01 (n = 6). K562TXR/293 cells also demonstrated concentration-dependent increases in intracellular calcium in response to the thromboxane agonist (15S-hydroxy-11 alpha,9 alpha(epoxymethano)-prosta-5Z,13E-dienoic acid. In contrast to the single [125I]BOP binding site observed in K562TXR/293, [125I]BOP binding to placental membranes resulted in a Hill coefficient significantly less than unity with a statistically superior two-site model for binding [KdH 0.63 +/- 0.18 nM and KdL of 12.5 +/- 5.0 nM, with Bmaxs of 29 +/- 9 and 212 +/- 41 fmol/mg of protein, respectively (n = 7)].(ABSTRACT TRUNCATED AT 250 WORDS)

The first commercially available practical gradient field-portable HPLC system is described. Solvalub Technologies, a Russian company engaged in the design of equipment for the analysis of environmental pollutants, specializing in the analysis of pollutants resulting from accidental leakages, developed the MINICHROM system. The instrument was developed for in situ qualitative and quantitative analysis of a broad spectrum of organic and some inorganic substances. Currently the instruments have been further enhanced in concert with Savannah Scientific Instruments of Northern Ireland. The new design has included both column heating and increased spectrum analysis, and has become more cost effective to produce. Furthermore, it was modified to meet EC (European Community) requirements for commercial products. The particular areas of interest for field chemistry would be the new EC environmental, agricultural, and field screening controls as well as forensic in situ investigation. MINICHROM is a portable (9.5 kg) gradient computer-controlled system. With immediate field screening and analysis, the user downgrades the risk of contamination introduced in handling, storage, and transport. With time-critical testing (i.e., perishable produce) this allows for measurements at the source of the contaminated or test areas. © 1998 John Wiley & Sons, Inc. Field Analyt Chem Technol 2:281–285, 1998

Platelet protein makeup is determined during transformation of megakaryoblasts to mature megakaryocytes, the immediate precursor of circulating platelets. To better understand the molecular mechanisms of megakaryocyte formation, gene expression was characterized by Northern analysis and RNA fingerprinting of cultured human CHRF-288 megakaryoblastic cells undergoing phorbol ester-stimulated megakaryocytic differentiation or serum-stimulated megakaryoblast proliferation. Protooncogenes c-fos and c-jun were coordinately upregulated in both proliferating and differentiating cells, whereas c-myc transcripts were upregulated during proliferation only. In contrast, mRNAs for transforming growth factor-beta 1 (TGF-beta 1) and thromboxane receptors were coordinately upregulated during differentiation but differentially regulated during proliferation. RNA fingerprinting revealed multiple transcripts specific to either proliferating or differentiated cells. Three of these were identified by homology to known DNA sequence: CDw44 adhesion molecule (upregulated during differentiation), glutathione sulfhydryl peroxidase (downregulated during differentiation), and plectin cytoskeletal protein (upregulated during differentiation). Thus, although megakaryoblast proliferation and megakaryocyte differentiation both involve DNA and protein synthesis, each growth response is characterized by a distinct pattern of gene expression.

Alpha 2-adrenergic receptors comprise a heterogeneous population based on pharmacologic and molecular evidence. We have isolated a cDNA clone (pRNG alpha 2) encoding a rat alpha 2-adrenergic receptor. A rat kidney cDNA library was screened with an oligonucleotide complementary to a highly conserved region found in all biogenic amine receptors described to date. The deduced amino acid sequence displays many features of guanyl nucleotide-binding protein-coupled receptors except it does not have a consensus N-linked glycosylation site near the amino terminus. Membranes prepared from COS cells transfected with pRNG alpha 2 DNA display high affinity and saturable binding to [3H]rauwolscine (Kd = 2 nM). Competition curve data analysis shows that RNG alpha 2 protein binds to a variety of adrenergic drugs with the following rank order of potency: yohimbine greater than or equal to chlorpromazine greater than or equal to prazosin greater than or equal to clonidine greater than norepinephrine greater than or equal to oxymetazoline. RNG alpha 2 RNA accumulates in both rat kidney and neonatal rat lung (predominant species is 4000 nucleotides). When a cysteine residue (Cys-169) that is conserved among all members of the seven-transmembrane-region superfamily is changed to phenylalanine, the RNG alpha 2 protein fails to bind [3H]rauwolscine after expression in COS cells. We conclude that pRNG alpha 2 likely represents a cDNA for a rat alpha 2B-adrenergic receptor.

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The two most extensively characterized thromboxane/prostaglandin endoperoxide (TP) receptors, from human platelets and rat vascular smooth muscle, exhibit thromboxane agonist [15-(1alpha,2beta(5Z), 3alpha-(1E,3S), 4alpha)]-7-[3-hydroxy-4-(p-iodophenoxy)-1-butenyl-7-oxabi cyclohepteno ic acid (I-BOP) binding affinities that differ by an order of magnitude, rat TP having the higher affinity. We utilized this difference in I-BOP affinity to identify structural determinants of TP receptor heterogeneity. No significant difference was found in the rank order of affinities for a series of thromboxane receptor ligands to bind to cloned human TPalpha versus rat TP, indicating that these represent species homologs, not distinct TP subtypes. Structural determinants for observed differences in I-BOP binding Kd were localized by creating chimeric human/rat TP followed by mutational substitution of specific critical amino acids. Initially, seven chimeric receptors with splice sites in transmembranes 1, 2, 4, or 7 were constructed and expressed in HEK293 cells for analysis of ligand binding properties. Substitution of any part except the carboxyl tail of the human TP into the rat TP resulted in a receptor with I-BOP binding affinity intermediate between the two. Analysis of chimeras in which only the extracellular amino terminus and a portion of transmembrane 1 were switched localized the determinant of high affinity binding to the region between amino acids 3 and 40. Using this chimera, amino acids in the human portion (extracellular amino terminus and part of transmembrane 1) were replaced with analogous amino acids from rat TP to regain high affinity I-BOP binding. Only when amino acid Val37 and either Val36 or Ala40 were reverted to their respective rat TP counterparts (Ala36, Leu37, and Gly40, respectively) was high affinity I-BOP binding recovered. The mechanism for the increased I-BOP affinity may be the lengthening of the amino acid side chain at position 37, thus extending this group further into the putative I-BOP binding pocket, with compensatory shortening of side chains in spatially adjacent amino acids.

We have defined conditions permitting the in vitro growth of human laryngeal papilloma cells at the air-liquid interface. Using this model system, retinoic acid has been found to modulate the differentiation of human laryngeal papilloma cells along two different pathways. At low concentrations of retinoic acid [10×9mol/L and 10 8mol/L], the cells formed a stratified squamous epithelium with a differentiation-specific protein staining pattern identical to that found in vivo. At higher concentrations of retinoic acid [10×7mol/L and 10×6mol/L], the cells differentiated into a columnar epithelium with occasional ciliated cells, lacking the markers of squamous differentiation. Analysis of the human papillomavirus DNA content revealed that as the concentration of retinoic acid increased, the viral DNA content decreased. This system is proposed as a model to further investigate the differentiation defects of human laryngeal papilloma cells and the regulatory role of retinoic acid in the clinical expression of human laryngeal papillomatosis.

Organic matter dynamics were studied in five streams at Coweeta Hydrologic Laboratory. Three of these streams drained logged watersheds, and two drained reference deciduous forest watersheds. Litter inputs to the streams draining disturbed watersheds were significantly lower than to reference streams. Additionally, while undisturbed litterfall consisted primarily of relatively refractory leaf species, litterfall in the disturbed watersheds was composed of more labile leaf material. Non-woody benthic organic material was generally lower in disturbed streams than in reference streams, and woody benthic material was substantially lower in disturbed streams. Particulate organic matter transport was measured intensively during storms. These data were used in a computer model to estimate annual particulate organic matter transport. The model was driven by empirical equations relating particulate concentration to the rate of increase in flow during storms, time since peak storm discharge, and average baseflow concentation. Results showed that disturbed streams exported significantly more particulate organic matter and that most of this transport occurred during storms. In order to place our results in perspective, the model was also used to estimate transport over a 47-year period. Transport during our study was not significantly different from the long-term average. Organic matter budgets were calculated from input, standing crop, and export data. This synthesis showed that forest disturbance has increased export, has accelerated turnover of benthic particulate organic matter, and is depleting benthic material. These changes are related primarily to the decline of woody debris dams in the disturbed streams.

Using the spiralling concept, this study assessed the relative importance of temperature, current velocity, leaf biomass, fine particulate organic matter, and leaf type in determining phosphorus retention in woodland streams. To accomplish this objective, streamside artificial streams and two contrasting leaf types were used. A labile leaf type (dogwood), typical of successional forest vegetation, and a more refractory leaf type (oak) characteristic of mature forest species were chosen. Leaves were picked just before abscission and air dried. Dogwood leaves were added to three streams and oak leaves to three other streams. Phosphorus uptake experiments were conducted in the streams during November 1987-June 1988 and November 1988-June 1989. All streams were least retentive of phosphorus in December, the coldest month of the year, and more retentive in the warmer spring and summer months. Phosphorus uptake was not correlated with microbial biomass or activity on leaves or with leaf biomass. However, streams with oak leaves were more retentive than streams with dogwood leaves. Measurements of penetrance (i.e., weight required to push a metal rod through the leaf) revealed that dogwood leaves were softer than oak leaves. The soft dogwood leaves were less effective at retarding water flow and therefore velocity was typically faster in dogwood streams (p < 0.05; Pearson's correlation). This higher velocity apparently contributed to lower retention. These results suggest that phosphorus retention under conditions of this experiment was governed primarily by temperature and water velocity. Coarse and fine particulate organic matter biomass and composition did influence retention, but their impact was secondary to the effects of temperature and velocity.

Soluble barium salts, when ingested in a toxic quantity, produce a characteristic muscular paralysis that can lead to respiratory paralysis and death. Detailed modern accounts of barium intoxication of such severity report survival in four1-4 of ten hospitalized individuals.1-7 The following report of barium intoxication demonstrates how correction of profound hypokalemia, saline diuresis, and respiratory assistance contributed greatly to the survival of the patient. A review of the literature on the subject of barium ion toxicity is included. PATIENT SUMMARY A 26-year-old black man, with suicidal intent, consumed one can of facial depilatory (Magic Shave) containing 15.8 gm of barium sulfide (12.8 gm of barium ion and 3 gm of sulfide). Ipecac, immediately given, induced an unknown amount of vomiting. He complained of weakness in his right arm, then paralysis of that limb, three hours after the ingestion. He then was taken to the District of Columbia General

We described previously the molecular characterization of a rat alpha 2B-adrenergic receptor and have shown also that the rat genome contains three closely related alpha 2-adrenergic receptor genes. To characterize the ligand-binding properties of these receptor gene products, we expressed the DNAs encoding these receptors individually in COS-1 cells and studied their binding to a wide variety of typical and atypical adrenergic ligands. The receptors displayed high affinity binding to the radioligand [3H] rauwolscine, with equilibrium dissociation constants ranging from 1.4 to 2.8 nM. Kinetic analysis of the binding of [3H]rauwolscine to membranes from transfected cells was in very good agreement with data obtained from saturation analysis. We examined the ability of a number of agents to compete for the binding of [3H]rauwolscine to the alpha 2-adrenergic receptor-transfected membranes. Whereas one of these receptors displayed a pharmacological profile typical of an alpha 2A-adrenergic receptor, the other two receptors showed similar pharmacological properties characteristic of an alpha 2B-adrenergic receptor. The two alpha 2B-like adrenergic receptors differed, however, in the ratios of Ki values for oxymetazoline and prazosin, as well as the Ki ratio of prazosin and yohimbine. In addition, the two alpha 2B-like adrenergic receptors had a 9-fold difference in affinity for chlorpromazine. The pharmacological characterization of the three rat alpha 2-adrenergic receptor gene products is consistent with the known pharmacology of alpha 2-adrenergic receptors, as documented using tissues and cell lines.

The human platelet thromboxane A2 receptor is a member of the G-protein-coupled superfamily of receptors. Previous pharmacologic studies examining the effects of biochemical reduction, oxidation, or sulfhydryl alkylation on thromboxane receptors have suggested a role for cysteines in determining receptor binding characteristics. To characterize the roles of individual cysteines, we employed site-directed mutagenesis to substitute serines for cysteines at seven positions throughout the human K562 thromboxane receptor and analyzed mutant receptor radioligand ([1S-(1alpha,2beta(5Z),3alpha- (1E,3S),4alpha]-7-[3-(3-hydroxy-4-(p-iodophenoxy)-l-butenyl)-7-oxabicyclo-[2. 2.1]heptane-2-yl]-5-heptenoic acid) binding and calcium signaling. Replacing cysteines in the amino terminus (amino acid position 11), and transmembrane domains two and six (positions 68 and 257) had little effect on thromboxane receptor binding or signaling. Introduction of serines for cysteines in the first (position 105) or the second (position 183) extracellular loop eliminated thromboxane receptor binding, consistent with the existence of a critical disulfide bond between these positions. Mutation of a second cysteine in extracellular loop one (position 102) resulted in a receptor with decreased binding affinity and low binding capacity that transduced only a low amplitude calcium signal, suggesting the involvement of a free sulfhydryl group at this location in receptor-ligand interactions. Finally, mutation of the cysteine at position 223, located in intracellular loop three, resulted in a receptor with normal ligand binding characteristics, but which did not transduce a calcium signal. Some additional amino acid substitutions in this region of the receptor (Cys-223 --> Ala, Thr-221 --> Met) resulted in receptors that had normal binding but transduced low amplitude calcium signals, while other mutations in the same region (His-224 --> Arg and His-227 --> Arg) exhibited normal binding and calcium signaling characteristics. These findings demonstrate that cysteines in extracellular loops one and two contribute to proper ligand binding to thromboxane receptors and show the importance of discrete amino acid sequences in the third intracellular loop, especially cysteine 223, in thromboxane receptor-effector coupling.

Expression of platelet thromboxane receptors is transcriptionally increased during megakaryocytic differentiation stimulated by phorbol 12-myristate 13-acetate (PMA). We previously cloned and characterized the promoter region of the human thromboxane receptor gene and localized PMA-responsive elements to a region between 1.84 and 1.95 kilobase pairs (kb) 5' of the transcription initiation site (D'Angelo, D. D., Davis, M. G., Houser, W. A., Eubank, J. J., Ritchie, M. E., and Dorn, G. W., II (1995) Circ. Res. 77, 466-474). Herein we report the localization of the PMA response element to a 14-nucleotide C-rich sequence, flanked by an octanucleotide inverted repeat, located -1.938 to -1.925 kb 5' of the transcription start site of this gene. We further identify the PMA-responsive enhancer factor that binds to this C-rich sequence as Sp1. Heterologous thromboxane receptor gene promoter/thymidilate kinase reporter constructs transfected into K562 cells exhibited PMA responsiveness when the C-rich element was included with additional 3' sequence from -1.924 to -1.84 kb. However, mutations of the C-rich element that disrupted a GC box located on the inverse strand eliminated PMA responsiveness and, in gel mobility shift assays, eliminated binding of Sp1. PMA treatment of K562 cells significantly increased, by 5-fold, Sp1 binding to the C-rich element and increased both phosphorylated and nonphosphorylated Sp1 protein levels by 2-fold. Furthermore, PMA treatment transiently increased Sp1 mRNA levels prior to increasing thromboxane receptor mRNA, suggesting that up-regulation of Sp1 contributes to up-regulation of thromboxane receptors. Finally, we have detected an unidentified K562 nuclear protein that binds specifically to the sense strand of the C-rich sequence overlapping the Sp1 binding site and that, by stabilizing a double stem-loop conformation of this DNA segment, may also play a role in Sp1 regulation of this gene. These studies are the first to describe regulatory and regulated roles for Sp1 in PMA-responsive gene expression and suggest that modulation of Sp1 levels controls thromboxane receptor expression during megakaryocytic differentiation.