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El trasplante renal es la terapia de elección para el manejo de la Enfermedad Renal Crónica Terminal (ERCT) en pediatría. La evaluación previa debe ser cuidadosa, para minimizar los riesgos del trasplante y de las condiciones que afecten la sobrevida del paciente y del injerto. El estudio comprende una adecuada selección del donante, y una evaluación completa del paciente, que debe considerar la etiología de la ERCT, las infecciones e inmunizaciones que ha presentado el paciente a lo largo de su vida, así como el estado serológico para ciertas infecciones relevantes como el CMV y EBV. El estudio inmunológico comprende antecedentes de eventos sensibilizantes, tipificación de grupo sanguíneo y HLA, estudio de anticuerpos y pruebas cruzadas (XM), que permita predecir los riesgos de rechazo y planificar estrategias de inmunosupresión individualizadas, de acuerdo a cada situación particular. Se describen los nuevos métodos de estudio, en fase sólida, de la respuesta aloinmune.

Summary: Tissue boundaries and interfaces are engines of morphogenesis in vivo. However, despite a wealth of micropatterning approaches available to control tissue size, shape, and mechanical environment in vitro, fine-scale spatial control of cell positioning within tissue constructs remains an engineering challenge. To address this, we augment DNA “velcro” technology for selective patterning of ssDNA-labeled cells on mechanically defined photoactive polyacrylamide hydrogels. Hydrogels bearing photopatterned single-stranded DNA (ssDNA) features for cell capture are then co-functionalized with extracellular matrix (ECM) proteins to support subsequent adhesion of patterned tissues. ECM protein co-functionalization does not alter ssDNA pattern fidelity, cell capture, or hydrogel elastic stiffness. This approach enables mechanobiology studies and measurements of signaling activity at dynamic cell interfaces with precise initial patterning. Combining DNA velcro patterning and ECM functionalization provides independent control of initial cell placement, adhesion, and mechanics, constituting a new tool for studying biological interfaces and for programming multicellular interactions in engineered tissues.

Summary: The regulation of mitochondrial RNA life cycles and their roles in ribosome biogenesis and energy metabolism are not fully understood. We used CRISPR/Cas9 to generate heart- and skeletal-muscle-specific knockout mice of the pentatricopeptide repeat domain protein 1, PTCD1, and show that its loss leads to severe cardiomyopathy and premature death. Our detailed transcriptome-wide and functional analyses of these mice enabled us to identify the molecular role of PTCD1 as a 16S rRNA-binding protein essential for its stability, pseudouridylation, and correct biogenesis of the mitochondrial large ribosomal subunit. We show that impaired mitoribosome biogenesis can have retrograde signaling effects on nuclear gene expression through the transcriptional activation of the mTOR pathway and upregulation of cytoplasmic protein synthesis and pro-survival factors in the absence of mitochondrial translation. Taken together, our data show that impaired assembly of the mitoribosome exerts its consequences via differential regulation of mitochondrial and cytoplasmic protein synthesis. : Perks et al. engineered intron-exon boundaries using CRISPR/Cas9 to conditionally knock out Ptcd1 in mice. The RNA-binding protein PTCD1 is essential for heart function and regulates the stability and maturation of the 16S rRNA. PTCD1 is required for mitoribosome biogenesis, mitochondrial function, and coordinated nuclear transcription. Keywords: RNA, cardiomyopathy, regulatory RNAs, RNA-seq, mitochondrial ribosome, RNA-binding proteins, mitochondria, mitochondrial gene expression, ribosome biogenesis

Summary: Sugars and refined carbohydrates are major components of the modern diet. ATP-citrate lyase (ACLY) is upregulated in adipocytes in response to carbohydrate consumption and generates acetyl-coenzyme A (CoA) for both lipid synthesis and acetylation reactions. Here, we investigate the role of ACLY in the metabolic and transcriptional responses to carbohydrates in adipocytes and unexpectedly uncover a sexually dimorphic function in maintaining systemic metabolic homeostasis. When fed a high-sucrose diet, AclyFAT−/− females exhibit a lipodystrophy-like phenotype, with minimal fat accumulation, insulin resistance, and hepatic lipid accumulation, whereas AclyFAT−/− males have only mild metabolic phenotypes. We find that ACLY is crucial for nutrient-dependent carbohydrate response element-binding protein (ChREBP) activation in adipocytes and plays a key role, particularly in females, in the storage of newly synthesized fatty acids in adipose tissue. The data indicate that adipocyte ACLY is important in females for the systemic handling of dietary carbohydrates and for the preservation of metabolic homeostasis. : Sugar is a major component of the modern diet. Fernandez et al. identify ATP-citrate lyase (ACLY) as a key facilitator of transcriptional and metabolic responses to dietary carbohydrates in adipocytes. Lack of adipocyte ACLY disrupts proper storage of lipids in adipose versus liver and promotes insulin resistance, particularly in females. Keywords: acetyl-CoA, ATP-citrate lyase, adipocyte, adipose tissue, liver, sexual dimorphism, carbohydrate, glucose, fatty acid synthesis, ChREBP

Mechanisms of metabolic flexibility enable cells to survive under stressful conditions and can thwart therapeutic responses. Acetyl-coenzyme A (CoA) plays central roles in energy production, lipid metabolism, and epigenomic modifications. Here, we show that, upon genetic deletion of Acly, the gene coding for ATP-citrate lyase (ACLY), cells remain viable and proliferate, although at an impaired rate. In the absence of ACLY, cells upregulate ACSS2 and utilize exogenous acetate to provide acetyl-CoA for de novo lipogenesis (DNL) and histone acetylation. A physiological level of acetate is sufficient for cell viability and abundant acetyl-CoA production, although histone acetylation levels remain low in ACLY-deficient cells unless supplemented with high levels of acetate. ACLY-deficient adipocytes accumulate lipid in vivo, exhibit increased acetyl-CoA and malonyl-CoA production from acetate, and display some differences in fatty acid content and synthesis. Together, these data indicate that engagement of acetate metabolism is a crucial, although partial, mechanism of compensation for ACLY deficiency.

Heterozygous mice (αMHC403/+) expressing the human disease-causing mutation Arg403Gln exhibit cardinal features of hypertrophic cardiomyopathy (HCM) including hypertrophy, myocyte disarray, and increased myocardial fibrosis. Treatment of αMHC403/+mice with the L-type calcium channel (ICa-L) antagonist diltiazem has been shown to decrease left ventricular anterior wall thickness, cardiac myocyte hypertrophy, disarray, and fibrosis. However, the role of the ICa-L in the development of HCM is not known. In addition to maintaining cardiac excitation and contraction in myocytes, the ICa-L also regulates mitochondrial function through transmission of movement of ICa-L via cytoskeletal proteins to mitochondrial voltage-dependent anion channel. Here, the authors investigated the role of ICa-L in regulating mitochondrial function in αMHC403/+mice. Whole-cell patch clamp studies showed that ICa-L current inactivation kinetics were significantly increased in αMHC403/+cardiac myocytes, but that current density and channel expression were similar to wild-type cardiac myocytes. Activation of ICa-L caused a significantly greater increase in mitochondrial membrane potential and metabolic activity in αMHC403/+. These increases were attenuated with ICa-L antagonists and following F-actin or β-tubulin depolymerization. The authors observed increased levels of fibroblast growth factor-21 in αMHC403/+mice, and altered mitochondrial DNA copy number consistent with altered mitochondrial activity and the development of cardiomyopathy. These studies suggest that the Arg403Gln mutation leads to altered functional communication between ICa-L and mitochondria that is associated with increased metabolic activity, which may contribute to the development of cardiomyopathy. ICa-L antagonists may be effective in reducing the cardiomyopathy in HCM by altering metabolic activity.

Context: Efforts to reduce the psychological distress of surrogate decision-makers of critically ill patients have had limited success, and some have even exacerbated distress., Objectives: The aim of this study was to determine the feasibility, acceptability, and preliminary efficacy of EMPOWER (Enhancing and Mobilizing the POtential for Wellness and Resilience), an ultra-brief (∼2-hour), 6-module manualized psychological intervention for surrogates., Methods: Surrogates who reported significant anxiety and/or an emotionally close relationship with the patient (n=60) were randomized to receive EMPOWER or enhanced usual care (EUC) at one of three metropolitan hospitals. Participants completed evaluations of EMPOWER's acceptability and measures of psychological distress pre-intervention, immediately post-intervention, and at 1- and 3-month follow-up assessments., Results: Delivery of EMPOWER appeared feasible, with 89% of participants completing all 6 modules, and acceptable, with high ratings of satisfaction (mean=4.5/5, SD = .90). Compared to EUC, intent-to-treat analyses showed EMPOWER was superior at reducing peritraumatic distress (Cohen's d = -0.21, small effect) immediately post-intervention and grief intensity (d = -0.70, medium-large effect), posttraumatic stress (d = -0.74, medium-large effect), experiential avoidance (d = -0.46, medium effect), and depression (d = -0.34, small effect) 3 months post-intervention. Surrogate satisfaction with overall critical care (d = 0.27, small effect) was higher among surrogates randomized to EMPOWER., Conclusions: EMPOWER appeared feasible and acceptable, increased surrogates' satisfaction with critical care, and prevented escalation of posttraumatic stress, grief, and depression 3 months later., (Copyright © 2024 American Academy of Hospice and Palliative Medicine. Published by Elsevier Inc. All rights reserved.)

Selection bias can arise through many aspects of a study, including recruitment, inclusion/exclusion criteria, input-level exclusion and outcome-level exclusion, and often reflects the underrepresentation of populations historically disadvantaged in medical research. The effects of selection bias can be further amplified when non-representative samples are used in artificial intelligence (AI) and machine learning (ML) applications to construct clinical algorithms. Building on the "Data Cards" initiative for transparency in AI research, we advocate for the addition of a participant flow diagram for AI studies detailing relevant sociodemographic and/or clinical characteristics of excluded participants across study phases, with the goal of identifying potential algorithmic biases before their clinical implementation. We include both a model for this flow diagram as well as a brief case study explaining how it could be implemented in practice. Through standardized reporting of participant flow diagrams, we aim to better identify potential inequities embedded in AI applications, facilitating more reliable and equitable clinical algorithms., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Hyaluronan (HA) is a ubiquitous extracellular matrix component playing a crucial role in the regulation of cell behaviors, including cancer. Aggressive breast cancer cells tend to proliferate, migrate and metastatize. Notably, triple-negative breast cancer cells lacking the expression of estrogen receptor (ER) as well as progesterone receptor and HER2 are more aggressive than ER-positive ones. As currently no targeted therapy is available for triple-negative breast cancer, the identification of novel therapeutic targets has a high clinical priority. In ER-negative cells, tumoral behavior can be reduced by inhibiting HA synthesis or silencing the enzymes involved in its metabolism, such as HA synthase 2 (HAS2). HAS2-AS1 is a long non-coding RNA belonging to the natural antisense transcript family which is known to favor HAS2 gene expression and HA synthesis, thus bolstering malignant progression in brain, ovary, and lung tumors. As the role of HAS2-AS1 has not yet been investigated in breast cancer, in this work we report that ER-positive breast cancers had lower HAS2-AS1 expression compared to ER-negative tumors. Moreover, the survival of patients with ER-negative tumors was higher when the expression of HAS2-AS1 was elevated. Experiments with ER-negative cell lines as MDA-MB-231 and Hs 578T revealed that the overexpression of either the full-length HAS2-AS1 or its exon 2 long or short isoforms alone, strongly reduced cell viability, migration, and invasion, whereas HAS2-AS1 silencing increased cell aggressiveness. Unexpectedly, in these ER-negative cell lines, HAS2-AS1 is involved neither in the regulation of HAS2 nor in HA deposition. Finally, transcriptome analysis revealed that HAS2-AS1 modulation affected several pathways, including apoptosis, proliferation, motility, adhesion, epithelial to mesenchymal transition, and signaling, describing this long non-coding RNA as an important regulator of breast cancer cells aggressiveness., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Associations between early traffic-related air pollution (TRAP) exposure and respiratory and allergic morbidity in adolescents are inconsistent. However, sub-groups might be more vulnerable to the health effects of this exposure., Objectives: We investigated associations between early exposure to TRAP and respiratory and allergic morbidity at age 13 years in the PARIS birth cohort, and potential modifying effects of sex, parental allergy, stressful family event and lower respiratory tract infections (LRTI)., Methods: This study deals with data from 732 children of the PARIS birth cohort followed up using repeated questionnaires until 13 years of age. Prenatal TRAP exposure was assessed by measuring daily concentrations of nitrogen dioxide at the nearest station to mother's home. Early postnatal TRAP exposure was calculated for each child during the first year of life by a nitrogen oxides (NO x ) air dispersion model taking into account both residence and daycare. Associations between TRAP exposures and asthma, rhinitis and related symptoms were assessed using multivariable logistic regression models adjusted for potential confounding factors. Effect modification was explored by testing multiplicative interactions., Results: An increase in interquartile range (17.0 μg/m 3 ) of early postnatal NO x exposure was positively related to current asthma (adjusted odds ratio aOR = 1.21; 95% confidence interval CI: 1.02, 1.43), severe wheeze (aOR = 1.23; 95% CI: 1.02, 1.47) and persistent asthma at 13 years old (aOR = 1.26; 95% CI: 1.03, 1.55) and tended to be associated with asthma ever. Parental history of allergy, asthma, early stressful family event and LRTI modified these associations with TRAP exposure. No relationship with rhinitis was found. Prenatal TRAP exposure did not show any association with respiratory and allergic morbidity., Discussion: This study is one of the first to show several modifiers of the association between early postnatal TRAP exposure and asthma at adolescence. Not all adolescents seem equally affected by early postnatal TRAP exposure: those presenting parental history of allergy, especially asthma, those with early stressful family event or LRTI appear to be more vulnerable., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Hyaluronan (HA) is a glycosaminoglycan composed by repeating units of D-glucuronic acid (GlcUA) and N-acetylglucosamine (GlcNAc) that is ubiquitously present in the extracellular matrix (ECM) where it has a critical role in the physiology and pathology of several mammalian tissues. HA represents a perfect environment in which cells can migrate and proliferate. Moreover, several receptors can interact with HA at cellular level triggering multiple signal transduction responses. The control of the HA synthesis is therefore critical in ECM assembly and cell biology; in this review we address the metabolic regulation of HA synthesis. In contrast with other glycosaminoglycans, which are synthesized in the Golgi apparatus, HA is produced at the plasma membrane by HA synthases (HAS1-3), which use cytoplasmic UDP-glucuronic acid and UDP-N-acetylglucosamine as substrates. UDP-GlcUA and UDP-hexosamine availability is critical for the synthesis of GAGs, which is an energy consuming process. AMP activated protein kinase (AMPK), which is considered a sensor of the energy status of the cell and is activated by low ATP:AMP ratio, leads to the inhibition of HA secretion by HAS2 phosphorylation at threonine 110. However, the most general sensor of cellular nutritional status is the hexosamine biosynthetic pathway that brings to the formation of UDP-GlcNAc and intracellular protein glycosylation by O-linked attachment of the monosaccharide β-N-acetylglucosamine (O-GlcNAcylation) to specific aminoacid residues. Such highly dynamic and ubiquitous protein modification affects serine 221 residue of HAS2 that lead to a dramatic stabilization of the enzyme in the membranes., (© 2013.)

The small leucine-rich proteoglycan, decorin, has incrementally been shown to be a powerful inhibitor of growth in a wide variety of tumour cells, an effect specifically mediated by the interaction of decorin core protein with the epidermal growth factor receptor (EGFR) and other ErbB family proteins. Nowadays, this matrikine has become the main focus of various cancer studies. Decorin is an important component of the cellular microenvironment or extracellular matrix (ECM). Its interactions with matrix and cell membrane components have been implicated in many physiological and pathophysiological processes including matrix organisation, signal transduction, wound healing, cell migration, inhibition of metastasis, and angiogenesis. This review summarises recent findings on decorin's interactions and behaviour related to cancer. Highlighted are key functions of decorin such as interaction with cell surface receptors, as well as with ECM components, and the therapeutic potential of this multifunctional molecule., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Mutations in the sulfate transporter gene, SCL26A2, lead to cartilage proteoglycan undersulfation resulting in chondrodysplasia in humans; the phenotype is mirrored in the diastrophic dysplasia (dtd) mouse. It remains unclear whether bone shortening and deformities are caused solely by changes in the cartilage matrix, or whether chondroitin sulfate proteoglycan undersulfation affects also signalling pathways involved in cell proliferation and differentiation. Therefore we studied macromolecular sulfation in the different zones of the dtd mouse growth plate and these data were related to growth plate histomorphometry and proliferation analysis. A 2-fold increase of non-sulfated disaccharide in dtd animals compared to wild-type littermates in the resting, proliferative and hypertrophic zones was detected indicating proteoglycan undersulfation; among the three zones the highest level of undersulfation was in the resting zone. The relative height of the hypertrophic zone and the average number of cells per column in the proliferative and hypertrophic zones were significantly reduced compared to wild-types; however the total height of the growth plate was within normal values. The chondrocyte proliferation rate, measured by bromodeoxyuridine labelling, was also significantly reduced in mutant mice. Immunohistochemistry combined with expression data of the dtd growth plate demonstrated that the sulfation defect alters the distribution pattern, but not expression, of Indian hedgehog, a long range morphogen required for chondrocyte proliferation and differentiation. These data suggest that in dtd mice proteoglycan undersulfation causes reduced chondrocyte proliferation in the proliferative zone via the Indian hedgehog pathway, therefore contributing to reduced long bone growth., (Copyright © 2010 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.)

Polycyclic aromatic compounds (PAHs) are of concern in environmental chemistry and toxicology. In the present work, a QSRR study was performed for 209 previously reported PAHs using quantum mechanics and other sources descriptors estimated by different approaches. The B3LYP/6-31G* level of theory was used for geometrical optimization and quantum mechanics related variables. A good linear relationship between gas-chromatographic retention index and electronic or topologic descriptors was found by stepwise linear regression analysis. The molecular polarizability (alpha) and the second order molecular connectivity Kier and Hall index ((2)chi) showed evidence of significant correlation with retention index by means of important squared coefficient of determination, (R(2)), values (R(2)=0.950 and 0.962, respectively). A one variable QSRR model is presented for each descriptor and both models demonstrates a significant predictive capacity established using the leave-many-out LMO (excluding 25% of rows) cross validation method's q(2) cross-validation coefficients q(2)(CV-LMO25%), (obtained q(2)(CV-LMO25%) 0.947 and 0.960, respectively). Furthermore, the physicochemical interpretation of selected descriptors allowed detailed explanation of the source of the observed statistical correlation. The model analysis suggests that only one descriptor is sufficient to establish a consistent retention index-structure relationship. Moderate or non-significant improve was observed for quantitative results or statistical validation parameters when introducing more terms in predictive equation. The one parameter QSRR proposed model offers a consistent scheme to predict chromatographic properties of PAHs compounds., (Copyright 2010 Elsevier B.V. All rights reserved.)

Fats are indispensable to life not only as an energy source but also for their structural role in the skin, retina, nervous system, lipoproteins, and biologic membranes. They are also precursors of important hormones and constitute the vehicle for the absorption of liposoluble vitamins. Nutritionists recommend a balanced lipid intake corresponding to a total amount of fats equal to 25% to 30% of total calories with a ratio in monounsaturated and polyunsaturated fatty acids. Thus, olive oil, with its balanced fatty acid composition, is of high nutritional value. Moreover, extra virgin olive oil, extracted from a fruit, has an important value related to the antioxidant power of minor components. Extra virgin olive oil contains 98% to 99% triglycerides and 1% to 2% minor components. In the triglycerides, the main fatty acids are represented by monounsaturates (oleic), with a slight amount of saturates and an adequate amount of polyunsaturates. The minor components are alpha-tocopherol, phenol compounds, carotenoids, squalene, phytosterols, and chlorophyll. Factors that can influence olive oil's composition, especially in regard to its minor components, are the cultivar, area of production, time of harvesting, and degree of technology used in its production. Therefore, an evaluation of the biologic value of extra virgin olive oil and its use as a topical raw material in cosmetic dermatology is reported.

Indolyl aryl sulfone (IAS) non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) have been previously shown to effectively inhibit wild-type (wt) and drug-resistant human immunodeficiency virus type 1 (HIV-1) replication. IASs proved to act through different mechanisms of action, depending on the nature and position of their chemical substituents. Here we describe selected novel IAS derivatives (di-halo-IASs). Our results show that these compounds are selective for the enzyme-substrate complex. The molecular basis for this selectivity was a different dissociation rate of the drug to a particular enzymatic form along the reaction pathway. By comparing the activities of the different compounds against wild-type RT and the resistant enzymes carrying the single mutations Lys103Asn, Leu100Ile, and Tyr181Ile (K103N, L100I, and Y181I), we found that one compound (RS1914) dissociated from the mutated enzymes almost 10-fold slower than from the wild type RT. These results demonstrate that IASs are very flexible molecules, interacting dynamically with the viral RT, and that this property can be successfully exploited to design inhibitors endowed with an enhanced binding to common NNRTI-resistant mutants.

The remodelling of the endometrial architecture is fundamental to create a suitable environment for the establishment of pregnancy. During this process, substantial alterations in the composition of maternal extracellular matrix play an important role by providing a prosperous medium for implantation as well as modulating trophoblast invasion leading to the formation of a functional placental unit. Hyaluronan is a conspicuous component of the extracellular matrix, particularly in remodelling tissues undergoing regeneration and repair. During gestation, changes in HA deposition and distribution indicate that this molecule may participate in preparation of the endometrial stroma for reception and implantation of the embryo. However, little is known about the role of hyaluronan at the fetomaternal interface, specially regarding its influence in pregnancy outcome. In the present study we show increased decidual hyaluronan levels in spontaneous abortion compared with normal pregnancy mice on gestation day 7.5. Both in normal and pathologic pregnancies, high molecular size hyaluronan was found at the fetomaternal unit. However, hyaluronan metabolism (which results from the activity of hyaluronan synthases and hyaluronidases) seems to be altered in spontaneous abortion as shown by a decrease in Hyal-3 expression as well as by differences in hyaluronan molecular size spectrum. This alteration in hyaluronan metabolism in spontaneous abortion could explain its increased concentration observed in decidua and the abnormal distribution of hyaluronan around the embryo implantation crypt. Thus, increased decidual hyaluronan levels resulting from abnormal deposition and turn over may contribute to the pathogenesis of pregnancy failure.

Intranuclear lipid metabolism modifications in relation to cell proliferation and/or apoptosis were demonstrated in hepatocytes. The aim of this study was to establish whether nuclear lipid metabolites influence cell function in different experimental models using a rat thyroid cell line (FRTL-5) treated with UV-C radiation. After UV-C irradiation cells proliferate and undergo apoptosis in the presence of thyrotropin, are quiescent and resistant to radiation-induced apoptosis in its absence and finally are proapoptotic for nutrition withdrawal. In nuclei purified from proliferating cells, irradiation stimulates neutral-sphingomyelinase activity and inhibits sphingomyelin-synthase, phosphatidylcholine-specific phospholipase C and phosphatidylinositol-specific phospholipase C activity with a consequent increase in the ceramide/diacylglycerol ratio. This effect is marked in proapoptotic cell nuclei and low in quiescent cell nuclei. In conclusion, UV-C radiation induces apoptosis, modifying nuclear lipid metabolism in relation to the physiological state of cells.

To quantify the amount of the 3460G-->A/ND1 point mutation responsible for Leber's hereditary optic neuropathy, we developed a quantitative real-time polymerase chain reaction method based on the SYBR Green assay and a new approach using the TaqMan assay. Both methods were based on the amplification refractory mutation system, comparing the heteroplasmic load quantified by restriction fragment length polymorphism in 15 Leber's hereditary optic neuropathy family members, with the results obtained using quantitative real-time polymerase chain reaction methods. The comparative evaluation of mitochondrial DNA (mtDNA) heteroplasmy from blood samples showed significant correlation between restriction fragment length polymorphism analysis, real-time SYBR Green assay, and TaqMan assay. We validated the last method by measuring experimental samples composed by a known proportion of cloned plasmids containing either the wild-type or mutant sequence, giving a correlation coefficient of 0.999 (P < 0.0001). The real-time amplification refractory mutation system polymerase chain reaction by TaqMan assay provides a rapid, reliable, sensitive, reproducible, and one-step quantitative method to detect heteroplasmic mutant mtDNA. This method allows the quantitation of a broad range of mutational load (up to 100%, down to 0.01%) on the basis of in vitro calibration, thus rendering the TaqMan assay suitable for the diagnostic analysis of heteroplasmic load in mtDNA-related disorders.

An "intranuclear complex" characterized by the presence of a new synthesised double-strand RNA stabilized by sphingomyelin and cholesterol has been recently isolated from hepatocyte nuclei. In the present research the existence of STAT3 in the intranuclear complex and its behaviour in relation to cyclin D and sphingomyelin metabolism during liver regeneration were studied with the aim to see if the sphingomyelinase could have a role in cell proliferation. Our data demonstrate that the transcription factor is present in the intranuclear complex either as unphosphorylated or phosphorylated monomeric form. After partial hepatectomy, unphosphorylated STAT3 is very low during G1/S transition of the cell cycle and it increases in correspondence of the of S-phase of the cell cycle when cyclin D1 is reduced. The phosphorylated form increases at the beginning of the S-phase when the neutral sphingomyelinase activity is stimulated with consequent enrichment of ceramide pool. In order to see if the two phenomena could be correlated, the intranuclear complex extracted from normal liver was treated with exogenous sphingomyelinase or ceramide and STAT3 was evaluated. The results show an increase of the phosphorylated transcription factor as happens in liver regeneration, suggesting that sphingomyelinase present in the intranuclear complex is responsible for the STAT3 activation during cell proliferation.

Objective: Blunt carotid artery injury resulting in either dissection or pseudoaneurysm is a rare entity that can result in significant morbidity and mortality if undiagnosed. Although unavoidable in certain traumatic situations such as motor vehicle collisions, direct neck trauma in sports may be preventable with the use of proper protective equipment., Clinical Presentation: The following report documents a case of extracranial carotid artery dissection resulting from a foul ball striking the upper, lateral neck of an umpire. The patient had transient right upper extremity monoparesis. He later presented with right hemiparesis and aphasia that resolved over several hours. Imaging studies demonstrated a left carotid pseudoaneurysm with evidence of left hemispheric embolic infarcts., Intervention: Systemic intravenous anticoagulation with heparin followed by oral anticoagulation with warfarin for 6 months, subsequently followed by aspirin monotherapy., Conclusion: The use of standard protective equipment did not adequately cover the patient's neck in the region of the cervical carotid artery. This case illustrates that modification in current catcher and umpire equipment could potentially avoid this rare but significant vascular injury.

Cross-reactivity among drugs is either mediated by immunologic mechanisms or not. The former kind is usually explained by the presence of common antigenic determinants in the cross-reacting drugs. In the case of compounds provoking non-allergic hypersensitivity reactions, cross-reactivity is explained by a common pharmacological characteristic, such as the inhibitory effect of non-steroidal anti-inflammatory drugs on cyclooxygenase-1 and the capability of muscle relaxants or contrast media to release histamine through a non-immunologic mechanism. The main clinical problem deriving from cross-reactivity among drugs is the compelling need to choose a potentially cross-reactive compound and, therefore, to assess cross-reactivity by diagnostic tests. In choosing alternative compounds, skin testing has been used in evaluating IgE-mediated cross-reactivity between penicillins and cephalosporins, as well as among muscle relaxants. In assessing T cell-mediated cross-reactivity among contrast media, corticosteroids, anticonvulsants and heparins, delayed-reading intradermal tests and patch tests, together with lymphocyte transformation tests, can be performed. Because of the limited sensitivity of in vivo and in vitro testing, the most prudent way of establishing the tolerability of a compound of the same group in patients who especially require one is a graded challenge when other allergologic tests are negative.

The mechanisms by which the excess genetic material of chromosome 21 results in the dysmorphologic features of Down syndrome (DS) are largely unknown. It has been found that the extracellular matrix of nuchal skin of DS fetuses exhibits an higher content of hyaluronan (HA) compared to that of euploid fetuses. Since HA plays a central role in many morphogenetic processes during embryogenesis, an alteration in its metabolism could be involved in the pathogenesis of several structural defects of DS. The extracellular matrix of umbilical cord (UC) is the mammalian tissue with one of the highest content of HA. Therefore we sought to explore the quantitative HA modifications during gestation, tissue distribution and HA metabolism in euploid and DS UCs. Euploid UCs (n=28) and UCs from DS fetuses (n=13) were obtained after termination of pregnancy, spontaneous abortion, or at delivery. Quantitative and molecular size analysis were performed using HPLC and FPLC. Tissue distribution was visualized by immunohistochemistry. Gene expression for HA synthases (HAS) and hyaluronidases (HYAL) were quantified by real-time PCR techniques and HYAL activity was detected by zymography. In euploid UC only HA of a molecular weight of 1700 kDA was present while in DS UC an additional lower weight HA molecule of 1100 kDA was found. Immunohistochemistry showed a larger amount of Wharton's jelly HA in DS UCs than in euploid UC. Real-time PCR analysis showed that HAS 2 and HYAL 2 were expressed at significant levels in all specimens. A higher expression of HAS 2 and a lower expression of HYAL 2 was found in the Wharton's jelly of DS fetuses compared to that of euploid fetuses at 14 weeks of gestation. On the contrary, at term HYAL 2 expression was higher in DS specimens than in those from euploid fetuses. Zymographic studies showed a similar behavior with a lower HYAL activity at early gestation and a higher HYAL activity at term gestation in DS UCs compared to euploid specimens. Therefore we can conclude that HA is more represented in DS UCs than in euploid UCs. A complex alteration of the HA metabolism characterized by an increased synthesis of lower weight HA molecules is a peculiarity of DS UCs.

The content of hyaluronan (HA), a polymer of the extracellular matrix involved in a variety of physiological and pathological processes, depends on the activity of synthetic (HAS) and degrading enzymes. Since HA is also involved in embryogenesis, we have used Xenopus as a model organism because information is available for HAS1 and HAS2, but not for HAS3. We report the sequence of xlHAS3 mRNA, its genomic organization and its expression in adult tissues as well as during embryonic development. Interestingly, evidence from in situ hybridization indicates that xlHAS3 expression is restricted to the developing inner ear and cement gland. In addition, we have correlated the expression pattern of the enzymes involved in HA metabolism with the HA content during development.

Decorin is a small leucine-rich proteoglycan able to interact with several molecules of the subendothelial matrix, such as collagen and fibronectin. In this work, we investigated the ability of purified decorin to support adhesion of human platelets. We found that gel-filtered platelets were actually able to interact with immobilized decorin. Platelet adhesion to decorin was time dependent, required the presence of Mg(2+) ions, and was totally mediated by the protein core of the proteoglycan. Platelet stimulation with either adenosine diphosphate (ADP) or a thrombin receptor-activating peptide significantly increased interaction of these cells with the proteoglycan. Upon adhesion to immobilized decorin a number of platelet proteins were found to become tyrosine-phosphorylated. By immunoprecipitation experiments with specific antibodies, the tyrosine phosphorylation of the tyrosine kinase Syk and the phospholipase Cgamma2 (PLCgamma2) isozyme was demonstrated in decorin-adherent platelets. Interaction of platelets with decorin was selectively prevented by 2 different antibodies against membrane integrin alpha(2)beta(1), but not by a number of antibodies against other membrane receptors. In addition, integrin alpha(2)beta(1), purified from platelet membranes, was able to specifically interact with immobilized decorin. Finally, purified decorin bound to Sepharose beads could precipitate integrin alpha(2)beta(1) from a platelet membrane glycoprotein preparation. Therefore, these results demonstrate that human platelets can bind to immobilized decorin through integrin alpha(2)beta(1), and that this interaction results in the tyrosine phosphorylation of intracellular proteins.

Background: A prospective multi-institutional phase II trial was undertaken to define the activity and toxicity of a unique decrescendo infusion of interleukin-2 (IL-2) in combination with interferon (IFN) in patients with metastatic renal cell carcinoma. The identical regimen has shown promise in advanced melanoma., Patients and Methods: Between February 1997 and March 1999, 47 patients with metastatic renal cell carcinoma, from five institutions, were treated with outpatient s.c. IFN (10 mU/m2/day) on days 1-5, followed by inpatient IL-2 via continuous i.v. decrescendo infusion [18 million International Units (MIU) (I mg)/m2/6 h, followed by 18 MIU/m2/12 h, then 18 MIU/m2/24 h and 4.5 MIU/m2/24 h for the following 3 days] on days 8-12, in a hospital ward without intensive care unit (ICU)-type monitoring. Treatment was repeated every 4 weeks. In contrast to high dose IL-2 protocols, patient eligibility did not require pulmonary function tests and allowed serum creatinine up to 2 mg/dl., Results: Among 44 eligible patients, 57% (25) had their primary in place, 57% (25) had bone or visceral involvement, and only 4% (2) had lung as their only site of disease. The overall response rate in 43 response-evaluable patients was 16.3% [95% confidence interval (CI) 5.3 to 27.3], with three complete responses and four partial responses observed. The median survival was 13 months; nine patients remain alive at >23 months. The median duration of response is 36 months (range 11.5 to 48+ months). Toxicity was modest, consisting of typical cytokine-induced systemic symptoms and rare organ dysfunction. Severe grade 4 toxicity occurred in only 13% of the 130 cycles., Conclusions: This unique, reasonably well tolerated IL-2/IFN combination induced a modest response rate with a number of durable remissions. While the optimal IL-2-based regimen for the treatment of advanced renal cell carcinoma remains elusive, the present regimen should attract considerable interest. This is based on tumor activity very similar to high dose IL-2 in a patient population not as carefully selected for optimal organ function.

In the last few years Cupressus sempervirens has been identified as the cause of an increasing number of cases of late winter-early spring pollinosis in Mediterranean countries. We conducted a 4-year retrospective study of a large group of subjects with documented allergic respiratory disease in order to determine the prevalence, clinical significance and annual rate of sensitization to C. sempervirens pollen. Anamnestic data and skin prick tests (SPT) with common aeroallergens and C. sempervirens extract were collected from 1397 subjects (712 male and 685 female) resident in Latium, a region in central Italy, with complaints related to upper- or lower-respiratory-tract disorders or conjunctival disease. Two hundred and forty-three subjects (17.4%) showed positive results to C. sempervirens extract: 47 (19.3%) of them were monosensitized. The annual sensitization rate of SPT positivity to C. sempervirens varied from 7.2% in 1995 to 22% in 1998. All the subjects monosensitized to cypress pollen had symptoms from January through April. Our study suggests that sensitivity to C. sempervirens is responsible for respiratory symptoms in an increasing percentage of subjects. Further studies are needed to determine its frequency at the national level.

Phosphatidylcholine-dependent phospholipase C is an enzyme which hydrolyses phosphatidylcholine giving origin to diacylglicerol and phosphorylcholine. Diacylglicerol has many effect and activates also protein kinase C. Since the presence of protein kinase C in the hepatocyte nuclei and the existence of a phospholipidic fraction in the chromatin have been demonstrated, we investigated if phosphatidylcholine-dependent phospholipase C could be present in the nuclei. The results obtained have shown the presence of this enzyme in the chromatin fraction which differs with respect to that of nuclear membrane in pH and Km. The activity has been also evaluated during liver regeneration. In the chromatin an increase of activity has been shown 12 h and 30 h after hepatectomy, i.e. at the beginning of hepatocyte S-phase. No similar behaviour has been observed in the nuclear membrane. It has been suggested that diacylglicerol, produced by the hydrolysis of chromatin phosphatidylcholine, may have a role in initiating DNA synthesis through the prolonged activation of the nuclear form of protein kinase C., (Copyright 1999 Academic Press.)

Tyrosine aminotransferase activity in rat liver increases during the first 24 hrs after partial hepatectomy with two peaks, one at 10 hrs and another at 18 hrs. This behaviour is due to an increase in TATmRNA synthesis. Expression of serine deydratase is also enhanced during the first 5 hrs after hepatectomy. It is suggested that the enhanced expression of the two genes is due to an increase in hormone incretion particularly glucagon and glucocorticoids.

The activity of the enzyme tyrosine aminotransferase and the synthesis of its specific mRNA were evaluated at different hours of the day in the liver of 3-, 12- and 24-month old BN rats. The enzyme activity has a circadian rhythm with a peak at midnight in 3- and 12-month old, which shifts to 03.00 hrs in 24-month old animals, in agreement with previous results. The expression of TATmRNA also changes during the day indicating circadian fluctuations which change with age. In 3-month old rats the TATmRNA peak is at 19.00 hrs, preceding that of the enzyme activity. In 12-month old rats the TATmRNA synthesis reaches a maximum at midnight and in 24-month old rats at 03.00 hrs. The results show that the circadian rhythm of tyrosine aminotransferase activity is due to a different gene expression throughout the day, which is influenced by age.