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Multistep synthesis performed on solid support is a powerful means to generate small-molecule libraries for the discovery of chemical probes to dissect biological mechanisms as well as for drug discovery. Therefore, expansion of the collection of robust chemical transformations amenable to solid-phase synthesis is desirable for achieving chemically diverse libraries for biological testing. Here, we show that sulfur(VI) fluoride exchange (SuFEx) chemistry, exemplified by pairing phenols with aryl fluorosulfates, can be used for the solid-phase synthesis of biologically active compounds. As a case study, we designed and synthesized a library of 84 hydroxamic acid-containing small molecules, providing a rich source of inhibitors with diverse selectivity profiles across the human histone deacetylase enzyme family. Among other discoveries, we identified a scaffold that furnished inhibitors of HDAC11 with exquisite selectivity in vitro and a selective inhibitor of HDAC6 that was shown to affect the acetylation of α-tubulin over histone sites H3K18, H3K27, as well as SMC3 in cultured cells. Our results encourage the further use of SuFEx chemistry for the synthesis of diverse small-molecule libraries and provide insight for future design of selective HDAC inhibitors., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Thioamides are naturally occurring isosteres of amide bonds in which the chalcogen atom of the carbonyl is changed from oxygen to sulfur. This substitution gives rise to altered nucleophilicity and hydrogen bonding properties with importance for both chemical reactivity and non-covalent interactions. As such, thioamides have been introduced into biologically active compounds to achieve improved target affinity and/or stability towards hydrolytic enzymes but have also been applied as probes of protein and peptide folding and dynamics. Recently, a series of new methods have been developed for the synthesis of thioamides as well as their utilization in peptide chemistry. Further, novel strategies for the incorporation of thioamides into proteins have been developed, enabling both structural and functional studies to be performed. In this Review, we highlight the recent developments in the preparation of thioamides and their applications for peptide modification and study of protein function., (© 2023 Wiley-VCH GmbH.)

The sirtuins are NAD + -dependent lysine deacylases, comprising seven isoforms (SIRT1-7) in humans, which are involved in the regulation of a plethora of biological processes, including gene expression and metabolism. The sirtuins share a common hydrolytic mechanism but display preferences for different ϵ-N-acyllysine substrates. SIRT7 deacetylates targets in nuclei and nucleoli but remains one of the lesser studied of the seven isoforms, in part due to a lack of chemical tools to specifically probe SIRT7 activity. Here we expressed SIRT7 and, using small-angle X-ray scattering, reveal SIRT7 to be a monomeric enzyme with a low degree of globular flexibility in solution. We developed a fluorogenic assay for investigation of the substrate preferences of SIRT7 and to evaluate compounds that modulate its activity. We report several mechanism-based SIRT7 inhibitors as well as de novo cyclic peptide inhibitors selected from mRNA-display library screening that exhibit selectivity for SIRT7 over other sirtuin isoforms, stabilize SIRT7 in cells, and cause an increase in the acetylation of H3 K18., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

The sirtuin enzymes are a family of lysine deacylases that regulate gene transcription and metabolism. Sirtuin 5 (SIRT5) hydrolyzes malonyl, succinyl, and glutaryl ϵ-N-carboxyacyllysine posttranslational modifications and has recently emerged as a vulnerability in certain cancers. However, chemical probes to illuminate its potential as a pharmacological target have been lacking. Here we report the harnessing of aryl fluorosulfate-based electrophiles as an avenue to furnish covalent inhibitors that target SIRT5. Alkyne-tagged affinity-labeling agents recognize and capture overexpressed SIRT5 in cultured HEK293T cells and can label SIRT5 in the hearts of mice upon intravenous injection of the compound. This work demonstrates the utility of aryl fluorosulfate electrophiles for targeting of SIRT5 and suggests this as a means for the development of potential covalent drug candidates. It is our hope that these results will serve as inspiration for future studies investigating SIRT5 and general sirtuin biology in the mitochondria., (© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Sirtuin 5 (SIRT5) is a protein lysine deacylase enzyme that regulates diverse biology by hydrolyzing ϵ-N-carboxyacyllysine posttranslational modifications in the cell. Inhibition of SIRT5 has been linked to potential treatment of several cancers but potent compounds with activity in cells have been lacking. Here we developed mechanism-based inhibitors that incorporate isosteres of a carboxylic acid residue that is important for high-affinity binding to the enzyme active site. By masking of the tetrazole moiety of the most potent candidate from our initial SAR study, we achieved potent and cytoselective growth inhibition for the treatment of SIRT5-dependent leukemic cancer cell lines in culture. Thus, we provide an efficient, cellularly active small molecule that targets SIRT5, which can help elucidate its function and potential as a future drug target. This work shows that masked isosteres of carboxylic acids are viable chemical motifs for the development of inhibitors that target mitochondrial enzymes, which may have applications beyond the sirtuin field., (© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

The glucose-dependent insulinotropic polypeptide (GIP) is a 42-residue metabolic hormone that is actively being targeted for its regulatory role of glycemia and energy balance. Limited structural data of its receptor has made ligand design tedious. This study investigates the structure and function of the GIP receptor (GIPR), using a homology model based on the GLP-1 receptor. Molecular dynamics combined with in vitro mutational data were used to pinpoint residues involved in ligand binding and/or receptor activation. Significant differences in binding mode were identified for the naturally occurring agonists GIP(1-30)NH 2 and GIP(1-42) compared with high potency antagonists GIP(3-30)NH 2 and GIP(5-30)NH 2 . Residues R183 2.60 , R190 2.67 , and R300 5.40 are shown to be key for activation of the GIPR, and evidence suggests that a disruption of the K293 ECL2 -E362 ECL3 salt bridge by GIPR antagonists strongly reduces GIPR activation. Combinatorial use of these findings can benefit rational design of ligands targeting the GIPR., Competing Interests: Declaration of interests This study was financially co-supported by Antag Therapeutics ApS. A.H.S.-U. is the CEO and co-founder of Antag Therapeutics ApS. M.M.R. is co-founder of Antag Therapeutics ApS and of Bainan Biotech ApS. M.M.R. is board member at Bainan Biotech. Patents related to this work: The rights to the two PCT applications above and any patents derived therefrom are licensed to Antag Therapeutics ApS, co-founded by A.H.S.-U. and M.M.R. The rights to the two PCT applications above and any patents derived therefrom are licensed to Bainan Biotech ApS, co-founded by M.M.R., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Objective: To test the hypothesis that infants born <30 weeks' gestation supported by Seattle-PAP will have lower rates of continuous positive airway pressure (CPAP) failure than infants supported with conventional, Fisher&Paykel-CPAP (FP-CPAP)., Study Design: Randomized trial (3/2017-01/2019) at 5 NICUs. The primary outcome was CPAP failure; subgroup analyses (gestational age, receipt antenatal corticosteroids) were performed., Results: A total of 232 infants were randomized. Infants in the Seattle-PAP and FP-CPAP groups had mean gestational ages of 27.0 and 27.2 weeks, respectively. We observed no differences in rates of treatment failure between Seattle-PAP (40/112, 35.7%) and FP-CPAP (38/120, 31.7%; risk difference, 4.1%; 95% CI, -8.1-16.2; P = 0.51). Subgroup analysis indicated no differences in rates of CPAP failure. We observed no differences between the two groups in frequencies of adverse events or duration of respiratory support., Conclusions: Among infants born <30 weeks' gestation, rates of CPAP failure did not differ between Seattle-PAP and FP-CPAP.

Ethnopharmacological Relevance: Species of the genus Cinchona (Rubiaceae) have been used in traditional medicine, and as a source for quinine since its discovery as an effective medicine against malaria in the 17th century. Despite being the sole cure of malaria for almost 350 years, little is known about the chemical diversity between and within species of the antimalarial alkaloids found in the bark. Extensive historical Cinchona bark collections housed at the Royal Botanic Gardens, Kew, UK, and in other museums may shed new light on the alkaloid chemistry of the Cinchona genus and the history of the quest for the most effective Cinchona barks., Aim of the Study: We used High-Pressure Liquid Chromatography (HPLC) coupled with fluorescence detection (FLD) to reanalyze a set of Cinchona barks originally annotated for the four major quinine alkaloids by John Eliot Howard and others more than 150 years ago., Materials and Methods: We performed an archival search on the Cinchona bark collections in the Economic Botany Collection housed in Kew, focusing on those with historical alkaloid content information. Then, we performed HPLC analysis of the bark samples to separate and quantify the four major quinine alkaloids and the total alkaloid content using fluorescence detection. Correlations between historic and current annotations were calculated using Spearman's rank correlation coefficient, before paired comparisons were performed using Wilcox rank sum tests. The effects of source were explored using generalized linear modelling (GLM), before the significance of each parameter in predicting alkaloid concentrations were assessed using chi-square tests as likelihood ratio testing (LRT) models., Results: The total alkaloid content estimation obtained by our HPLC analysis was comparatively similar to the historical chemical annotations made by Howard. Additionally, the quantity of two of the major alkaloids, quinine and cinchonine, and the total content of the four alkaloids obtained were significantly similar between the historical and current day analysis using linear regression., Conclusions: This study demonstrates that the historical chemical analysis by Howard and current day HPLC alkaloid content estimations are comparable. Current day HPLC analysis thus provide a realistic estimate of the alkaloid contents in the historical bark samples at the time of sampling more than 150 years ago. Museum collections provide a powerful but underused source of material for understanding early use and collecting history as well as for comparative analyses with current day samples., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: At birth, the majority of neonates born at <30 weeks of gestation require respiratory support to facilitate transition and ensure adequate gas exchange. Although the optimal approach to the initial respiratory management is uncertain, the American Academy of Pediatrics endorses noninvasive respiratory support with nasal continuous positive airway pressure (nCPAP) for premature neonates with respiratory insufficiency. Despite evidence for its use, nCPAP failure, requiring intubation and mechanical ventilation, is common. Recently, investigators have described a novel method to deliver bubble nCPAP, termed Seattle-PAP. While preclinical and pilot studies are encouraging regarding the potential value of Seattle-PAP, a large trial is needed to compare Seattle-PAP directly with the current standard of care for bubble nCPAP (Fisher & Paykel CPAP or FP-CPAP)., Methods/design: We designed a multicenter, non-blinded, randomized controlled trial that will enroll 230 premature infants (22 0/7 to 29 6/7 weeks of gestation). Infants will be randomized to receive Seattle-PAP or FP-CPAP. The primary outcome is respiratory failure requiring intubation and mechanical ventilation. Secondary outcomes include measures of short- and long-term respiratory morbidity and cost-effectiveness., Discussion: This trial will assess whether Seattle-PAP is more efficacious and cost-effective than FP-CPAP in real-world practice among premature neonates., Trial Registration: ClinicalTrials.gov, NCT03085329 . Registered on 21 March 2017.

Background: Almost one million prematurely born infants die annually from respiratory insufficiency, predominantly in countries with limited access to respiratory support for neonates. The primary hypothesis tested in the present study was that a modified device for bubble nasal continuous positive airway pressure (Bn-CPAP) would provide lower work of spontaneous breathing, estimated by esophageal pressure-rate products., Methods: Infants born <32 weeks gestation and stable on Bn-CPAP with FiO2 <0.30 were studied within 72 h following delivery. Esophageal pressures during spontaneous breathing were measured during 2 h on standard Bn-CPAP, then 2 h with Bn-CPAP using a modified bubble device presently termed Seattle-PAP, which produces a different pattern of pressure fluctuations and which provided greater respiratory support in preclinical studies, then 2 h on standard Bn-CPAP., Results: All 40 infants enrolled completed the study and follow-up through 36 wks post menstrual age or hospital discharge, whichever came first. No infants were on supplemental oxygen at completion of follow-up. No infants developed pneumothoraces or nasal trauma, and no adverse events attributed to the study were observed. Pressure-rate products on the two devices were not different, but effort of breathing, assessed by areas under esophageal pressure-time curves, was lower with Seattle-PAP than with standard Bn-CPAP., Conclusion: Use of Seattle-PAP to implement Bn-CPAP lowers the effort of breathing exerted even by relatively healthy spontaneously breathing premature neonates. Whether the lower effort of breathing observed with Seattle-PAP translates to improvements in neonatal mortality or morbidity will need to be determined by studies in appropriate patient populations.

Developmental topographical disorientation (DTD) is a newly discovered cognitive disorder in which individuals experience a lifelong history of getting lost in both novel and familiar surroundings. Recent studies have shown that such a selective orientation defect relies primarily on the inability of the individuals to form cognitive maps, i.e., mental representations of the surrounding that allow individuals to get anywhere from any location in the environment, although other orientation skills are additionally affected. To date, the neural correlates of this developmental condition are unknown. Here, we tested the hypothesis that DTD may be related to ineffective functional connectivity between the hippocampus (HC; known to be critical for cognitive maps) and other brain regions critical for spatial orientation. A group of individuals with DTD and a group of control subjects underwent a resting-state functional magnetic resonance imaging (rsfMRI) scan. In addition, we performed voxel-based morphometry to investigate potential structural differences between individuals with DTD and controls. The results of the rsfMRI study revealed a decreased functional connectivity between the right HC and the prefrontal cortex (PFC) in individuals with DTD. No structural differences were detected between groups. These findings provide evidence that ineffective functional connectivity between HC and PFC may affect the monitoring and processing of spatial information while moving within an environment, resulting in the lifelong selective inability of individuals with DTD to form cognitive maps that are critical for orienting in both familiar and unfamiliar surroundings., (Copyright © 2014 Wiley Periodicals, Inc.)

The presence of PCR inhibitors in extracted DNA may interfere with the subsequent quantification and short tandem repeat (STR) reactions used in forensic genetic DNA typing. DNA extraction from fabric for forensic genetic purposes may be challenging due to the occasional presence of PCR inhibitors that may be co-extracted with the DNA. Using 120 forensic trace evidence samples consisting of various types of fabric, we compared three automated DNA extraction methods based on magnetic beads (PrepFiler Express Forensic DNA Extraction Kit on an AutoMate Express, QIAsyphony DNA Investigator kit either with the sample pre-treatment recommended by Qiagen or an in-house optimized sample pre-treatment on a QIAsymphony SP) and one manual method (Chelex) with the aim of reducing the amount of PCR inhibitors in the DNA extracts and increasing the proportion of reportable STR-profiles. A total of 480 samples were processed. The highest DNA recovery was obtained with the PrepFiler Express kit on an AutoMate Express while the lowest DNA recovery was obtained using a QIAsymphony SP with the sample pre-treatment recommended by Qiagen. Extraction using a QIAsymphony SP with the sample pre-treatment recommended by Qiagen resulted in the lowest percentage of PCR inhibition (0%) while extraction using manual Chelex resulted in the highest percentage of PCR inhibition (51%). The largest number of reportable STR-profiles was obtained with DNA from samples extracted with the PrepFiler Express kit (75%) while the lowest number was obtained with DNA from samples extracted using a QIAsymphony SP with the sample pre-treatment recommended by Qiagen (41%)., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Clozapine remains the drug of choice for patients with treatment-resistant schizophrenia, who show a response rate of about 50% despite their unresponsiveness to other antipsychotics. Although treatment with clozapine can lead to considerable savings on bed days, the drug is underutilized for several reasons, perhaps most importantly because of the mandatory hematological monitoring. The Chempaq Express Blood Counter (Chempaq XBC) is a point-of-care device providing counts of white blood cells (WBC) and granulocytes based on a capillary blood sampling. A randomized cross-over trial design was used comparing capillary blood sampling using a point-of-care device with traditional venous blood sampling. Patients were randomized to two sequences starting with either capillary or venous blood sampling followed by a repeated sequence. Primary outcome was measured on a 10-cm visual analog scale. Eighty-five patients were included in the test. Eight (9.4%) dropped out before completion. Patients indicated that they found capillary blood monitoring less painful than venous sampling (VAS ratings: 0.55 cm 25-75 percentiles: 0.1-1.4 cm vs. 1.75 cm 25-75 percentiles: 0.7-2.6, p<0.001). They also felt less inconvenienced by the point-of-care method than the traditional blood sampling, which involved traveling to the laboratory clinical (0.3 cm 25-75 percentiles: 0.05-0.7 vs. 2.3 cm 25-75 percentiles: 0.75-4.5, p<0.001). For hematological monitoring of clozapine patients a point-of-care device based on capillary blood sampling is better tolerated than traditional venous blood sampling., (Copyright © 2011 Elsevier B.V. All rights reserved.)

We have developed two devices: a high-amplitude bubble continuous positive airway pressure (HAB-CPAP) and an inexpensive bubble intermittent mandatory ventilator (B-IMV) to test the hypotheses that simple, inexpensive devices can provide gas exchange similar to that of bubble CPAP (B-CPAP) and conventional mechanical ventilation (CMV). Twelve paralyzed juvenile rabbits were intubated, stabilized on CMV, and then switched to CPAP. On identical mean airway pressures (MAPs), animals were unable to maintain pulse oximeter oxygen saturation (SpO2) >80% on conventional B-CPAP, but all animals oxygenated well (97.3 ± 2.1%) on HAB-CPAP. In fact, arterial partial pressures of O2 (Pao2) were higher during HAB-CPAP than during CMV (p = 0.01). After repeated lung lavages, arterial partial pressures of CO2 (Paco2) were lower with B-IMV than with CMV (p < 0.0001), despite identical ventilator settings. In lavaged animals, when HAB-CPAP was compared with CMV at the same MAP and 100% O2, no differences were observed in Pao2, but Paco2 levels were higher with HAB-CPAP (70 ± 7 versus 50 ± 5 mm Hg; p < 0.05). Arterial blood pressures were not impaired by HAB-CPAP or B-IMV. The results confirm that simple inexpensive devices can provide respiratory support in the face of severe lung disease and could extend the use of respiratory support for preterm infants into severely resource-limited settings.

Bubble continuous positive airway pressure (B-CPAP) applies small-amplitude, high-frequency oscillations in airway pressure (DeltaPaw) that may improve gas exchange in infants with respiratory disease. We developed a device, high-amplitude B-CPAP (HAB-CPAP), which provides greater DeltaPaw than B-CPAP provides. We studied the effects of different operational parameters on DeltaPaw and volumes of gas delivered to a mechanical infant lung model. In vivo studies tested the hypothesis that HAB-CPAP provides noninvasive respiratory support greater than that provided by B-CPAP. Lavaged juvenile rabbits were stabilized on ventilator nasal CPAP. The animals were then supported at the same mean airway pressure, bias flow, and fraction of inspired oxygen (FiO2) required for stabilization, whereas the bubbler angle was varied in a randomized crossover design at exit angles, relative to vertical, of 0 (HAB-CPAP0; equivalent to conventional B-CPAP), 90 (HAB-CPAP90), and 135 degrees (HAB-CPAP135). Arterial blood gases and pressure-rate product (PRP) were measured after 15 min at each bubbler angle. Pao2 levels were higher (p<0.007) with HAB-CPAP135 than with conventional B-CPAP. PaCO2 levels did not differ (p=0.073) among the three bubbler configurations. PRP with HAB-CPAP135 were half of the PRP with HAB-CPAP0 or HAB-CPAP90 (p=0.001). These results indicate that HAB-CPAP135 provides greater respiratory support than conventional B-CPAP does.

Introduction: Interventions directed toward mothers before and during pregnancy and childbirth may help reduce preterm births and stillbirths. Survival of preterm newborns may also be improved with interventions given during these times or soon after birth. This comprehensive review assesses existing interventions for low- and middle-income countries (LMICs)., Methods: Approximately 2,000 intervention studies were systematically evaluated through December 31, 2008. They addressed preterm birth or low birth weight; stillbirth or perinatal mortality; and management of preterm newborns. Out of 82 identified interventions, 49 were relevant to LMICs and had reasonable amounts of evidence, and therefore selected for in-depth reviews. Each was classified and assessed by the quality of available evidence and its potential to treat or prevent preterm birth and stillbirth. Impacts on other maternal, fetal, newborn or child health outcomes were also considered. Assessments were based on an adaptation of the Grades of Recommendation Assessment, Development and Evaluation criteria., Results: Most interventions require additional research to improve the quality of evidence. Others had little evidence of benefit and should be discontinued. The following are supported by moderate- to high-quality evidence and strongly recommended for LMICs: Two interventions prevent preterm births--smoking cessation and progesterone. Eight interventions prevent stillbirths--balanced protein energy supplementation, screening and treatment of syphilis, intermittant presumptive treatment for malaria during pregnancy, insecticide-treated mosquito nets, birth preparedness, emergency obstetric care, cesarean section for breech presentation, and elective induction for post-term delivery. Eleven interventions improve survival of preterm newborns--prophylactic steroids in preterm labor, antibiotics for PROM, vitamin K supplementation at delivery, case management of neonatal sepsis and pneumonia, delayed cord clamping, room air (vs. 100% oxygen) for resuscitation, hospital-based kangaroo mother care, early breastfeeding, thermal care, and surfactant therapy and application of continued distending pressure to the lungs for respiratory distress syndrome, Conclusion: The research paradigm for discovery science and intervention development must be balanced to address prevention as well as improve morbidity and mortality in all settings. This review also reveals significant gaps in current knowledge of interventions spanning the continuum of maternal and fetal outcomes, and the critical need to generate further high-quality evidence for promising interventions.

An unknown number of asylum seekers arriving in Denmark have been exposed to torture. Amnesty International's Danish Medical Group examined 142 asylum seekers, of whom 45% had been exposed to torture. Physical and psychological symptoms were 2-3 times as frequent among torture survivors as among non-tortured asylum seekers. Among the torture survivors, 63% fulfilled the criteria of post-traumatic stress disorder, 58% had objective psychological findings, and 42% had torture-related scars. Identification of torture survivors is important in order to initiate the necessary medical treatment.

Recently, next-generation sequencing has been introduced as a promising, new platform for assessing the copy number of transcripts, while the existing microarray technology is considered less reliable for absolute, quantitative expression measurements. Nonetheless, so far, results from the two technologies have only been compared based on biological data, leading to the conclusion that, although they are somewhat correlated, expression values differ significantly. Here, we use synthetic RNA samples, resembling human microRNA samples, to find that microarray expression measures actually correlate better with sample RNA content than expression measures obtained from sequencing data. In addition, microarrays appear highly sensitive and perform equivalently to next-generation sequencing in terms of reproducibility and relative ratio quantification.

To recruit and train the next generations of pediatric clinician-scientists, the American Pediatric Society and Society for Pediatric Research initiated a program in 1991 to support medical students with interests in research and pediatrics to conduct research at institutions other than their respective medical schools. Since 1991, the American Pediatric Society-Society for Pediatric Research Medical Student Research Program (MSRP) has funded 732 of 2209 applicants from 132 US or Canadian medical schools for 8-12 wk of research under the direction of experienced investigators. PubMed-attributable publications tabulated in 2001 for MSRP applicants through 2000 indicated that participants had published more actively than had nonparticipant applicants. Male nonparticipants exhibited greater publication activities than did female nonparticipants, but female and male participants published equally. Of all MSRP participants between 1991 and 1996, as of 2008, 36% were in pediatrics, and a remarkable 29% were in academic pediatrics.

Background: An unknown number of asylum seekers arriving in Denmark have been exposed to torture or have experienced other traumatising events in their country of origin. The health of traumatised asylum seekers, both physically and mentally, is affected upon arrival to Denmark, and time in asylum centres leads to further deterioration in health., Methods: One hundred forty-two (N=142) newly arrived asylum seekers were examined at Center Sandholm by Amnesty International Danish Medical Group from the 1st of September until the 31st of December 2007., Findings: The asylum seekers came from 33 different countries, primarily representing Afghanistan, Iraq, Iran, Syria, and Chechnya. Of the asylum seekers, 45 percent had been exposed to torture--approximately one-third within the year of arrival to Denmark. Unsystematic blows, personal threats or threats to family, degrading treatment, isolation, and witnessing torture of others were the main torture methods reported. The majority of the asylum seekers had witnessed armed conflict, persecution, and imprisonment. The study showed that physical symptoms were approximately twice as frequent and psychological symptoms were approximately two to three times as frequent among torture survivors as among non-tortured asylum seekers. However, even the health of non-tortured asylum seekers was affected. Among the torture survivors, 63 percent fulfilled the criteria for post-traumatic stress disorder, and 30-40 percent of the torture survivors were depressed, in anguish, anxious, and tearful in comparison to 5-10 percent of the non-tortured asylum seekers. Further, 42 percent of torture survivors had torture-related scars., Interpretation: Torture survivors amid newly arrived asylum seekers are an extremely vulnerable group, hence examination and inquiry about the torture history is extremely important in order to identify this population to initiate the necessary medical treatment and social assistance. Amnesty International Danish Medical group is currently planning a follow-up study of the present population which will focus on changes in health status during their time in Denmark.

In the present study, we tested the hypothesis that exposure of newborn mice to sublethal hyperoxia would alter lung development and expressions of fibroblast growth factor receptors (FGFRs)-3 and FGFR-4. Newborn FVB mice were exposed to 85% O2 or maintained in room air for up to 14 d. No animal mortality was observed, and body weight gains were not affected by hyperoxia. At postnatal d 7 and 14 (P7, P14), lungs of mice exposed to 85% O2 showed fewer alveolar secondary crests and larger alveoli or terminal air spaces than did mice in room air. In pups kept in room air, lung levels of FGFR-3 and FGFR-4 mRNA were greater at P3 than at P1, but similar increases were not observed in hyperoxic mice. Immunoreactivity of FGFR-3 and FGFR-4 was lower in lungs of hyperoxic mice than in controls at P14. In pups kept in room air, lung fibroblast growth factor (FGF)-7 mRNA levels were greater at P14 than at P1, but similar changes were not observed in hyperoxic mice. The temporally and spatially specific alterations in the expressions of FGFR-3, FGFR-4, and FGF-7 in the mice exposed to hyperoxia may contribute to aberrant lung development.

Reduction of glutathione disulfide (GSSG) to glutathione (GSH) by glutathione reductase (GR) enhances the efficiency of GSH-dependent antioxidant activities. However, GR-deficient (a1Neu) mice are less susceptible to acute lung injury from continuous exposure to > 95% O(2) (96 h: 6.9 +/- 0.1 g right lung/kg body versus room air 3.6 +/- 0.3) than are C3H/HeN control mice (10.6 +/- 1.3 versus 4.2 +/- 0.3, P < 0.001). a1Neu mice have greater hepatic thioredoxin (Trx)1 and Trx2 levels than do C3H/HeN mice, suggesting compensation for the absence of GR. a1Neu mice exposed to hyperoxia for 96 hours showed lower levels of inflammatory infiltrates in lungs than did similarly exposed C3H/HeN mice. Pretreatment with aurothioglucose (ATG), a thioredoxin reductase (TrxR) inhibitor, exacerbated the effects of hyperoxia on lung injury in a1Neu mice (11.6 +/- 0.8, P < 0.001), but attenuated hyperoxic lung edema and inflammation in C3H/HeN mice (6.3 +/- 0.4, P < 0.001). No consistent alterations were observed in lung GSH contents or liver GSH or GSSG levels after ATG pretreatment. The data suggest that modulation of Trx/TrxR systems might provide therapeutically useful alterations of cellular resistance to oxidant stresses. The protective effects of ATG against hyperoxic lung injury could prove to be particularly useful therapeutically.

We have observed large-amplitude strain waves following a rapid change in density of InSb due to nonthermal melting. The strain has been measured in real time via time-resolved x-ray diffraction, with a temporal resolution better than 2 ps. The change from the solid to liquid density of the surface layer launches a high-amplitude strain wave into the crystalline material below. This induces an effective plane rotation in the asymmetrically cut crystal leading to deflection of the diffracted beam. The uniform strain in the layer below the molten layer is 2.0(+/-0.2)%. A strain of this magnitude develops within 5 ps of the incident pulse showing that the liquid has reached the equilibrium density within this time frame. Both the strain amplitude and the depth of the strained material in the solid can be explained by assuming a reduction in the speed of sound in the nonequilibrium liquid compared to measured equilibrium values.

The melting dynamics of laser excited InSb have been studied with femtosecond x-ray diffraction. These measurements observe the delayed onset of diffusive atomic motion, signaling the appearance of liquidlike dynamics. They also demonstrate that the root-mean-squared displacement in the [111] direction increases faster than in the [110] direction after the first 500 fs. This structural anisotropy indicates that the initially generated fluid differs significantly from the equilibrium liquid.

The motion of atoms on interatomic potential energy surfaces is fundamental to the dynamics of liquids and solids. An accelerator-based source of femtosecond x-ray pulses allowed us to follow directly atomic displacements on an optically modified energy landscape, leading eventually to the transition from crystalline solid to disordered liquid. We show that, to first order in time, the dynamics are inertial, and we place constraints on the shape and curvature of the transition-state potential energy surface. Our measurements point toward analogies between this nonequilibrium phase transition and the short-time dynamics intrinsic to equilibrium liquids.

Linear-accelerator-based sources will revolutionize ultrafast x-ray science due to their unprecedented brightness and short pulse duration. However, time-resolved studies at the resolution of the x-ray pulse duration are hampered by the inability to precisely synchronize an external laser to the accelerator. At the Sub-Picosecond Pulse Source at the Stanford Linear-Accelerator Center we solved this problem by measuring the arrival time of each high energy electron bunch with electro-optic sampling. This measurement indirectly determined the arrival time of each x-ray pulse relative to an external pump laser pulse with a time resolution of better than 60 fs rms.

The potentially detrimental influence of parenchymal cells on endothelial function during preservation in UW solution was examined by co-storage of rat abdominal aortic rings with isolated liver cells. Cold storage of rings in UW solution alone for up to 96 h had no effect on the response to acetylcholine, though constriction was progressively lost. Co-storage of rings with liver cells resulted in no loss of sodium nitroprusside response, but the relaxation response to acetylcholine was reduced. The loss of acetylcholine response could not be attributed to Kupffer cells, the lowering of pH, oxygen depletion, or the loss of constriction. A similar loss of endothelial function was observed in rings stored in pieces of liver, kidney or heart. We conclude that parenchymal cells exude factors during preservation by cold storage which reversibly inhibit vascular NO production. These factors could significantly impair whole organ function on reperfusion.

Mitogen-activated protein (MAP) kinases play a pivotal role in the macrophages in the production of proinflammatory cytokines triggered by lipopolysaccharides. However, their function in the responses of macrophages to Gram-positive bacteria is poorly understood. Even less is known about the attenuation of MAP kinase signaling in macrophages exposed to Gram-positive bacteria. In the present study, we have investigated the regulation of MAP kinases and the role of MAP kinase phosphatase (MKP)-1 in the production of pro-inflammatory cytokines using murine RAW264.7 and primary peritoneal macrophages after peptidoglycan stimulation. Treatment of macrophages with peptidoglycan resulted in a transient activation of JNK, p38, and extracellular signal-regulated kinase. Most interestingly, MKP-1 expression was potently induced by peptidoglycan, and this induction was concurrent with MAP kinase dephosphorylation. Triptolide, a diterpenoid triepoxide, potently blocked the induction of MKP-1 by peptidoglycan and prolonged the activation of JNK and p38. Overexpression of MKP-1 substantially attenuated the production of tumor necrosis factor (TNF)-alpha induced by peptidoglycan, whereas knockdown of MKP-1 by small interfering RNA substantially increased the production of both TNF-alpha and interleukin-1 beta. Finally, we found that in primary murine peritoneal macrophages, MKP-1 induction following peptidoglycan stimulation also coincided with inactivation of JNK and p38. Blockade of MKP-1 induction resulted in a sustained activation of both JNK and p38 in primary macrophages. Our results reveal that MKP-1 critically regulates the expression of TNF-alpha and interleukin-1 beta in RAW264.7 cells and further suggest a central role for this phosphatase in controlling the inflammatory responses of primary macrophages to Gram-positive bacterial infection.

A strain of mice (Gr1a1Neu) that exhibited tissue glutathione reductase (GR) activities that were substantially lower (less than 10% in liver) than the corresponding activities in control mice has been reported. The present report describes characterization of the mutation(s) in the GR gene of these mice. RT-PCR of mRNA from the Neu mice indicated a substantial deletion in the normal GR coding sequence. Southern blots revealed that the deletion involved a region spanning from intron 1 through intron 5. The exact breakpoints of the deletion were characterized by PCR and sequencing through the region encompassing the deletion. The deletion involves nucleotides 10840 through 23627 of the genomic GR gene and functionally deletes exons 2 through 5. In addition, the deletion produces a frame shift in exon 6 and introduces a stop codon in exon 7 that would prevent translation of the remainder of the protein. Consequently, the Neu mice are incapable of producing a functional GR protein and appear to be genetic knockouts for GR. The Neu mice offer live animal models with which to test hypotheses regarding oxidant mechanisms of tissue injury in vivo.

Glutathione (GSH) plays vital roles in antioxidant defense mechanisms. To determine whether gene transfection strategies could be used to enhance GSH synthetic capacities and protect mammalian cells against oxidant stresses, we used liposome-mediated transfer of the cDNA for rat glutamate-cysteine ligase (GLCL) catalytic subunit (GLCLC) to transfect Chinese hamster ovary (CHO) cells. CHO cell lines (CHOhi) with stably enhanced GLCL activities (14.61+/-0.82 mU/mg protein) and greater GSH contents (45.7+/-1.37 nmol/mg protein) than observed in wild-type CHO K1 cells (0.26+/-0.01 mU/mg protein and 20.7+/-1.15 nmol/mg protein, respectively) were developed and were confirmed to have integrated the GLCLC cDNA into their genomic DNA and to exhibit increased GLCLC mRNA levels, by Southern and northern analyses, respectively. Similarly treated and selected CHO cell lines that showed no increases in GLCL activities (CHOun) were studied as controls for the effects of GLCLC transgene expression. CHOhi cells showed significantly greater resistance to oxidant stress caused by exposure to tert-butyl hydroperoxide (tBuOOH) than did CHO or CHOun cells. Twenty-four hours after exposure to 400 or 800 microM tBuOOH, wild-type CHO cells had released more cellular lactate dehydrogenase (67.3+/-14.5% and 94.4+/-2%) than had CHOhi cells (5.11+/-0.5% and 46.0+/-5.4%, n=4, P<0.05). The present data demonstrate improved resistance to oxidant injury of CHO cells stably transfected with the GLCLC cDNA. Although additional enhancements in GLCL activities are possible by transfection with cDNAs for both catalytic and regulatory GLCL subunits, our results demonstrate that the increases in GLCL activities that can be attained by transfection of the GLCLC cDNA alone can enhance cellular antioxidant defense function.

The present studies were to test the hypotheses that glutathione reductase (GR), glutathione peroxidase (GPX), and glutathione S-transferase (GST) activities are expressed in nuclei and nucleoli of rat liver cells, and that differences in activities of these enzymes would correlate with the greater resistance of female than of male Fischer-344 rats to hepatic necrosis in vivo, mediated by reactive oxygen species generated by redox-cycling metabolism of diquat. Adult male and female Fischer-344 rats were treated with comparably hepatotoxic doses of diquat (0.1 or 0.2 mmol/kg, respectively), or equal volumes of saline, ip. Six hours later, the livers were harvested, and purified nuclei and nucleoli were isolated by differential centrifugation. Nuclear GR activities in male and female rats were 12 and 15 mU/mg protein, and nucleolar activities were 30 and 51 mU/mg protein, respectively, p < 0.05. Some differences between male and female rats in nuclear and nucleolar activities of GPXs and GSTs were observed, as were some differences in the respective diquat-treated animals, but implications of these differences for susceptibility to diquat-induced oxidant stress effects are not apparent. Nuclear GR, GPX, and GST probably contribute to antioxidant defense mechanisms, but the functions served by localization of GR and GPX in nucleoli are less evident.

The angular distribution of electron temperature and density in a laser-ablation plume has been studied for the first time. The electron temperature ranges from 0.1 to 0.5 eV and is only weakly dependent on the angle in the low-intensity range studied here. In contrast, the typical ion energy is about 2 orders of magnitude larger, and its angular distribution is more peaked about the target normal. The derived values of the electron density are in agreement with the measured values of ion density.

Background: Neonatal sepsis is a low incidence, high-risk disease with many sepsis work-ups performed to detect a single case. Seventy-two hours of antibiotic therapy have been traditionally recommended pending negative culture results. Improved culture media and new technology integrated into blood culture systems could shorten incubation time required to detect positive culture results. This would then change the length of antibiotic therapy in the management of the newborn infant with suspected sepsis. In addition, previous data supporting the 72-hour recommendation were retrospectively acquired, utilized nonautomated systems, and reported in an era with a different population of microorganisms cultured in special care nurseries., Objective: Evaluate the time of incubation to detect positive blood cultures from newborn infants with suspected sepsis using a computer-assisted, automated blood culture system, ESP (Trek Diagnostic Systems, Inc, Westlake, OH)., Design: Prospective, observational study., Patients and Setting: All positive blood culture results that were obtained from term and preterm newborn infants born from November 1993 through June 1997 at a publicly funded hospital with over 6000 live births per year., Methods: As positive blood culture results were identified, data were prospectively obtained from the patient's medical record. The computer algorithm in the automated blood culture system determined the time to positivity. Time to positivity was determined for blood cultures obtained before the initiation antimicrobial therapy and compared with those cultures obtained after beginning therapy. Time to positivity was also evaluated for clinically important Gram-positive and Gram-negative bacteria and yeast., Results: Four hundred fifty-five positive blood culture results were obtained from 222 patients. Gram-positive organisms accounted for 80% (366/455) of the positive culture results, Gram-negative organisms accounted for 11% (48/455), and yeast for 9% (41/455). Virtually all cultures growing clinically significant Gram-positive and Gram-negative organisms were positive by 24 to 36 hours of incubation. Cultures growing Staphylococcus epidermidis were virtually all positive after 36 to 48 hours of incubation. Of cultures growing yeast, 88% (36/41) were positive by 48 hours of incubation. There was no difference in time to positivity in pretherapy or posttherapy obtained positive blood cultures. Prenatally administered antibiotics did not affect time to positivity in positive cultures drawn on the first day of life. In a selected group of microorganisms that are the frequent cause of bacteremia in term infants, 97% and 99% of cultures were positive by 24 to 36 hours of incubation when only pretherapy cultures are evaluated., Conclusions: The ESP blood culture system identified 77%, 89% and 94% of all microorganisms at 24, 36, and 48 hours of incubation in aerobic cultures obtained from both term and preterm infants. Introduction of antimicrobial therapy did not affect time to positivity. Reducing duration of antibiotic therapy to 24 to 36 hours should be considered in term, asymptomatic newborn infants undergoing evaluation for suspected sepsis for maternal indications. Confirmation of similar rapidity of detection using other blood culture systems should be undertaken.

Rat fetal lung cells (RFL-6) were transiently transfected with a full-length rat heme oxygenase (HO)-1 cDNA construct and then exposed to hyperoxia (95% O2-5% CO2) for 48 h. Total HO activity and HO-1 protein were measured as well as cell viability, lactate dehydrogenase (LDH) release, protein oxidation, lipid peroxidation, and total glutathione to measure oxidative injury. HO-1 overexpression resulted in increased total HO activity (2-fold), increased HO-1 protein (1.5-fold), and increased cell proliferation. Immunohistochemistry revealed perinuclear HO-1 localization, followed by migration to the nucleus by day 3. Decreased cell death, protein oxidation, and lipid peroxidation but increased LDH release and glutathione depletion were seen with HO-1 overexpression. Reactive iron content could not explain the apparent loss of cell membrane integrity. With the addition of tin mesoporphyrin, total HO activity was decreased and all changes in injury parameters were normalized to control values. We conclude that moderate overexpression of HO-1 is protective against oxidative injury, but we speculate that there is a beneficial threshold of HO-1 expression.

Objective: To determine if premature infants greater than 31 weeks of gestation with established hyaline membrane disease (HMD) can be treated with endotracheal continuous positive airway pressure (ETCPAP) after rescue surfactant replacement therapy., Study Design: Retrospective study of 46 premature infants (>31 weeks of gestation) admitted to Texas Children's Hospital with HMD. Tolerance to ETCPAP after surfactant replacement was evaluated. Prenatal and postnatal characteristics and outcome were compared in the success and failure groups. Multiple logistic regression was used to determine predictive factors associated with failure., Results: Thirty infants (65.2%) were successfully treated with rescue surfactant and ETCPAP. Cesarean section, higher 1-minute Apgar score, and higher FiO2 level at entry were independent predictors of failure to remain on CPAP due to hypoxemia (56.3%), hypercapnia (31.2%), and apnea (12.5%). In the success group duration of intubation (p < 0.001), oxygen administration (p < 0.01), >40% oxygen requirement (p < 0.001), hospital stay (p < 0.05), and respiratory support on day 7 (p < 0.001) were significantly favorable., Conclusion: Two thirds of infants greater than 31 weeks of gestation, with HMD needing rescue surfactant treatment, can be successfully managed with ETCPAP.

Glutathione reductase (GR) is an important component of cellular antioxidant defense functions. Although GR activities are found in mitochondria and cytoplasm, the sorting mechanisms of mammalian GR into mitochondria have not been elucidated. To identify the mouse GR gene structure, including a sequence for a potential mitochondrial targeting signal (MTS), we screened a mouse genomic library and isolated four contiguous clones that covered the entire coding region of the gene. The coding region is composed of 13 exons. Exon 1 has two in-frame start codons separated by a sequence for an arginine-rich peptide segment. Expression studies, in which Chinese hamster ovary cells were transiently transfected with a plasmid containing the first 78 bp of the mouse exon 1 attached 5' to the human GR cDNA, showed marked and selective increases in mitochondrial GR activities. The data indicate that this 78 bp sequence encodes a potential MTS for GR in mice.