Kinetic role of helix caps in protein folding is context-dependent.
Secondary structure punctuation through specific backbone and side chain interactions at the beginning and end of alpha-helices has been proposed to play a key role in hierarchical protein folding mechanisms [Baldwin, R. L., and Rose, G. D. (1999) Trends Biochem. Sci. 24, 26-33; Presta, L. G., and Rose, G. D. (1988) Science 240, 1632-1641]. We have made site-specific substitutions in the N- and C-cap motifs of the 5-helix protein monomeric lambda repressor (lambda(6-85)) and have measured the rate constants for folding and unfolding of each variant. The consequences of C-cap changes are strongly context-dependent. When the C-cap was located at the chain terminus, changes had little energetic and no kinetic effect. However, substitutions in a C-cap at the boundary between helix 4 and the subsequent interhelical loop resulted in large changes to the stability and rate constants of the variant, showing a substantial kinetic role for this interior C-cap and suggesting a general kinetic role for interior helix C-caps. Statistical preferences tabulated separately for internal and terminal C-caps also show only weak residue preferences in terminal C-caps. This kinetic distinction between interior and terminal C-caps can explain the discrepancy between the near-absence of stability and kinetic effects seen for C-caps of isolated peptides versus the very strong C-cap effects seen for proteins in statistical sequence preferences and mutational energetics. Introduction of consensus, in-register N-capping motifs resulted in increased stability, accelerated folding, and slower unfolding. The kinetic measurements indicate that some of the new native-state capping interactions remain unformed in the transition state. The accelerated folding rates could result from helix stabilization without invoking a specific role for N-caps in the folding reaction.


Plasma waveguide formation in predissociated clustering gases.
We report on the use of a novel technique to create a plasma waveguide suitable for guiding high-intensity laser pulses in underdense plasmas. A narrow channel of a clustering gas is dissociated with a low-intensity prepulse. This prepulse is followed by a high-intensity, focused laser pulse. The high absorption of the clusters surrounding the dissociated atomic channel causes the remaining annulus of clusters to become highly ionized, leaving low-density plasma in the center. We have interferometrically probed the formation of this channel with picosecond laser pulses.


Chicken albumen-based whispering gallery mode microlasers.
Microlasers based on biomaterials have attracted enormous interest because of their promising potential for future applications in medical treatments, bio-tracking, and biosensing. In this work, we demonstrate chicken albumen as a novel and excellent low-cost biomaterial for a laser cavity. By using a simple but effective emulsion process, rhodamine B-doped chicken albumen microspheres with various diameters ranging from 20 μm to 100 μm can be fabricated. Under optical pulse excitation, these microspheres emit lasing emission. The lasing mechanism is investigated and ascribed to the whispering gallery mode (WGM). A threshold of 23.2 μJ mm-2 and a high Q-factor of approximately 2400 are obtained from an 82 μm-diameter microsphere. Size-dependent lasing characteristics are also examined, and the result shows good agreement with the WGM theory. Interestingly, these microsphere biolasers can operate in aqueous and biological environments such as water and human blood serum, which makes them a promising candidate for laser-based biosensing and biological applications.


T cell activation and proliferation following acute exercise in human subjects is altered by storage conditions and mitogen selection.
Recent work investigating exercise induced changes in immunocompetence suggests that some of the ambiguity in the literature is resultant from different cell isolation protocols and mitogen selection. To understand this effect, we compared post-exercise measures of T cell activation and proliferation using two different stimulation methods (costimulation through CD28 or stimulation with phytohaemagglutinin [PHA]). Further, we investigated whether exercise induced changes are maintained when T cell isolation from whole blood is delayed overnight in either a room temperature or chilled (4°C) environment. As expected, an increased proliferation response was observed post-exercise in T cells isolated from whole blood of previously trained individuals immediately after blood collection. Also, cells stimulated with PHA after resting overnight in whole blood were not adversely impacted by the storage conditions. In contrast, allowing cells to rest overnight in whole blood prior to stimulation through CD28, lessened the proliferation observed by cells following exercise rendering both the room temperature and chilled samples closer to the results seen in the control condition. Changes in early markers of activation (CD25), followed a similar pattern, with activation in PHA stimulated cells remaining fairly robust after overnight storage; whereas cell activation following stimulation through CD3+CD28 was disproportionately decreased by the influence of overnight storage. These findings indicate that decisions regarding cell stimulation methods need to be paired with the timeline for T cell isolation from whole blood. These considerations will be especially important for field based studies of immunocompetence where there is a delay in getting whole blood samples to a lab for processing as well as clinical applications where a failure to isolate T cells in a timely manner may result in loss of the response of interest.


Inhibitory effects of curcumin and capsaicin on phorbol ester-induced activation of eukaryotic transcription factors, NF-kappaB and AP-1.
Recently, considerable attention has been focused on identifying dietary and medicinal phytochemicals that can inhibit, retard or reverse the multi-stage carcinogenesis. Spices and herbs contain phenolic substances with potent antioxidative and chemopreventive properties. Curcumin, a yellow colouring agent from turmeric and capsaicin, a pungent principle of red pepper exhibit profound anticarcinogenic and antimutagenic activities. Two well-defined eukaryotic transcription factors, nuclear factor-kappa B (NF-kappaB) and activator protein 1 (AP-1) have been implicated in pathogenesis of many human diseases including cancer. These transcription factors are known to be activated by a wide array of external stimuli, such as tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), tumor necrosis factor, reactive oxygen species, bacterial lipopolysaccharide, and ultraviolet. In the present study, we found that topical application of TPA onto dorsal skin of female ICR mice resulted in marked activation of epidermal NF-kappaB and AP-1. Curcumin and capsaicin, when topically applied prior to TPA, significantly attenuated TPA-induced activation of each transcription factor in mouse skin. Likewise, both compounds inhibited NF-kappaB and AP-1 activation in cultured human promyelocytic leukemia (HL-60) cells stimulated with TPA. Based on these findings, it is likely that curcumin and capsaicin exert anti-tumor promotional effects through suppression of the tumor promoter-induced activation of transcription factors, NF-kappaB and AP-1.


A genome-wide screen in 1119 relative pairs with autoimmune thyroid disease.
Autoimmune thyroid diseases (AITD), comprising Graves' disease and autoimmune hypothyroidism, are characterized by loss of immunological self-tolerance to thyroid antigens. These are complex diseases arising from a combination of genetic and environmental factors. An understanding of the genetic susceptibility factors for AITD could help to target treatments more effectively and identify people at risk for these conditions. The objective of this study was to identify regions of genetic linkage to AITD that could potentially harbor genetic susceptibility factors for these conditions. The study design was a genome-wide screen performed on affected relative pairs with AITD. Patients were recruited through hospital endocrinology clinics. Some 1119 Caucasian relative pairs affected with AITD (Graves' disease or autoimmune hypothyroidism) were recruited into the study. Blood samples were obtained from each participant for DNA analysis, and clinical questionnaires were completed. The study aimed to identify regions of genetic linkage to AITD. Three regions of suggestive linkage were obtained on chromosomes 18p11 (maximum LOD score, 2.5), 2q36 (maximum LOD score, 2.2), and 11p15 (maximum LOD score, 2.0). No linkage to human leukocyte antigen was found. The absence of significant evidence of linkage at any one locus in such a large dataset argues that genetic susceptibility to AITD reflects a number of loci, each with a modest effect. Linkage analysis may be limited in defining such loci, and large-scale association studies may prove to be more useful in identifying genetic susceptibility factors for AITD.


Antimicrobial and antiproliferative activities of stingless bee Melipona scutellaris geopropolis.
Geopropolis is a type of propolis containing resin, wax, and soil, collected by threatened stingless bee species native to tropical countries and used in folk medicine. However, studies concerning the biological activity and chemical composition of geopropolis are scarce. In this study, we evaluated the antimicrobial and antiproliferative activity of the ethanolic extract of geopropolis (EEGP) collected by Melipona scutellaris and its bioactive fraction against important clinical microorganisms as well as their in vitro cytotoxicity and chemical profile. The antimicrobial activity of EEGP and fractions was examined by determining their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against six bacteria strains as well as their ability to inhibit Streptococcus mutans biofilm adherence. Total growth inhibition (TGI) was chosen to assay the antiproliferative activity of EEGP and its bioactive fraction against normal and cancer cell lines. The chemical composition of M. scutellaris geopropolis was identified by reversed-phase high-performance liquid chromatography and gas chromatography-mass spectrometry. EEGP significantly inhibited the growth of Staphylococcus aureus strains and S. mutans at low concentrations, and its hexane fraction (HF) presented the highest antibacterial activity. Also, both EEGP and HF inhibited S. mutans biofilm adherence (p < 0.05) and showed selectivity against human cancer cell lines, although only HF demonstrated selectivity at low concentrations. The chemical analyses performed suggest the absence of flavonoids and the presence of benzophenones as geopropolis major compounds. The empirical use of this unique type of geopropolis by folk medicine practitioners was confirmed in the present study, since it showed antimicrobial and antiproliferative potential against the cancer cell lines studied. It is possible that the major compounds found in this type of geopropolis are responsible for its properties.


Therapeutic Inhibition of the MDM2-p53 Interaction Prevents Recurrence of Adenoid Cystic Carcinomas.
Purpose: Conventional chemotherapy has modest efficacy in advanced adenoid cystic carcinomas (ACC). Tumor recurrence is a major challenge in the management of ACC patients. Here, we evaluated the antitumor effect of a novel small-molecule inhibitor of the MDM2-p53 interaction (MI-773) combined with cisplatin in patient-derived xenograft (PDX) ACC tumors.Experimental Design: Therapeutic strategies with MI-773 and/or cisplatin were evaluated in SCID mice harboring PDX ACC tumors (UM-PDX-HACC-5) and in low passage primary human ACC cells (UM-HACC-2A, -2B, -5, -6) in vitro The effect of therapy on the fraction of cancer stem cells (CSC) was determined by flow cytometry for ALDH activity and CD44 expression.Results: Combined therapy with MI-773 with cisplatin caused p53 activation, induction of apoptosis, and regression of ACC PDX tumors. Western blots revealed induction of MDM2, p53 and downstream p21 expression, and regulation of apoptosis-related proteins PUMA, BAX, Bcl-2, Bcl-xL, and active caspase-9 upon MI-773 treatment. Both single-agent MI-773 and MI-773 combined with cisplatin decreased the fraction of CSCs in PDX ACC tumors. Notably, neoadjuvant MI-773 and surgery eliminated tumor recurrences during a postsurgical follow-up of more than 300 days. In contrast, 62.5% of mice that received vehicle control presented with palpable tumor recurrences within this time period (P = 0.0097).Conclusions: Collectively, these data demonstrate that therapeutic inhibition of MDM2-p53 interaction by MI-773 decreased the CSC fraction, sensitized ACC xenograft tumors to cisplatin, and eliminated tumor recurrence. These results suggest that patients with ACC might benefit from the therapeutic inhibition of the MDM2-p53 interaction. Clin Cancer Res; 23(4); 1036-48. ©2016 AACR.


New Permian Insects Discovered in Kansas and Oklahoma.
The Midco insect bed of Oklahoma and a newly discovered insect bed above this were traced across Kay County, Okla., into Sumner County, Kan. As a result, a greater time span is available for study of insect evolution during the mid-continent Permian, and the exact stratigraphic correlation of the Wellington of Oklahoma and Kansas can now be demonstrated. Four insect orders have thus far been identified from the new insect bed: Protodonata, Odonata, Protoperlaria, and Ephemeroptera. Numerous new species and higher categories are included in the collections from the two insect beds.


Risk of epithelial ovarian cancer in relation to benign ovarian conditions and ovarian surgery.
Some forms of ovarian neoplasms may be preventable through the removal of precursor lesions. We assessed the risk associated with a prior diagnosis of, and ovarian surgery following, ovarian cysts and endometriosis, with a focus on characterizing risk among tumor subgroups. Information was collected during in-person interviews with 812 women with ovarian cancer diagnosed in western Washington State from 2002 to 2005 and 1,313 population-based controls. Logistic regression was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs). The risk of a borderline mucinous ovarian tumor associated with a history of an ovarian cyst was increased (OR=1.7, 95% CI: 1.0-2.8), but did not vary notably according to receipt of subsequent ovarian surgery. While risk of invasive epithelial ovarian cancer was slightly increased among women with a cyst who had no subsequent ovarian surgery, it was reduced when a cyst diagnosis was followed by surgery (OR = 0.6, 95% CI: 0.4-0.9). This reduction in risk was most evident for serous invasive tumors. Women with a history of endometriosis had a threefold increased risk of endometrioid and clear cell invasive tumors, with a lesser risk increase among women who underwent subsequent ovarian surgery. Our results suggest differences in the relation of ovarian cysts and endometriosis with risk of specific subtypes of ovarian cancer as well as the possibility that ovarian surgery in women with these conditions may lower the risk of invasive disease.


Delayed school entry and academic performance: a natural experiment.
Recent reports suggest that delayed school entry (DSE) may be beneficial for children with developmental delays. However, studies of the effects of DSE are inconclusive. This study investigated the effects of DSE versus age-appropriate school entry (ASE) on children's academic achievement and attention in middle childhood. In total, 999 children (492 females, 507 males; 472 born preterm) were studied as part of a prospective population-based longitudinal study in Germany. Using a natural experimental design, propensity score matching was applied to create two matched groups who differed only in terms of DSE versus ASE. Teacher ratings of achievement in mathematics, reading, writing, and attention were obtained in Year 1, and standardized tests were administered at 8 years of age. There was no evidence of a difference in the odds of DSE versus ASE children being rated as above average by teachers in Year 1. In contrast, the standardized mean test scores for DSE children were lower than ASE children's mean scores in all domains (mathematics: B=-0.28 [-0.51 to -0.06)], reading: B=-0.39 [-0.65 to -0.14], writing: B=-0.90 [-1.07 to -0.74], and attention: B=-0.58 [-0.79 to -0.36]). DSE did not affect teacher-rated academic performance. However, missing 1 year of learning opportunities was associated with poorer average performance in standardized tests at 8 years of age. Future research is needed to determine the long-term effect of DSE on academic achievement.


From exercise physiology to preventive medicine.
The study of the normal human individual provides an important baseline for the study of disease. Exercise physiology is particularly important because an exercise situation provides a unique opportunity to study how different functions are coordinated and integrated. In fact, most functions and structures are in one way or another affected by acute and chronic (i.e. training) exercise. There is unanimous agreement that regular exercise is essential for optimal function of the human body. It is evident that extrinsic factors, such as diet and exercise habits, are reflected in the morbidity and mortality statistics, especially in aging. Both healthy individuals and those with chronic illnesses and functional handicaps can improve their performances and, therefore, their quality of life by increased physical activity. In my opinion, epidemiological research has established that physical inactivity from a medical viewpoint is a risk factor threatening health and an optimal life style. Adverse effects of non-competitive exercises are very small in comparison with health benefits.


Cartilage repair: past and future--lessons for regenerative medicine.
Since the first cell therapeutic study to repair articular cartilage defects in the knee in 1994, several clinical studies have been reported. An overview of the results of clinical studies did not conclusively show improvement over conventional methods, mainly because few studies reach level I of evidence for effects on middle or long term. However, these explorative trials have provided valuable information about study design, mechanisms of repair and clinical outcome and have revealed that much is still unknown and further improvements are required. Furthermore, cellular and molecular studies using new technologies such as cell tracking, gene arrays and proteomics have provided more insight in the cell biology and mechanisms of joint surface regeneration. Besides articular cartilage, cartilage of other anatomical locations as well as progenitor cells are now considered as alternative cell sources. Growth Factor research has revealed some information on optimal conditions to support cartilage repair. Thus, there is hope for improvement. In order to obtain more robust and reproducible results, more detailed information is needed on many aspects including the fate of the cells, choice of cell type and culture parameters. As for the clinical aspects, it becomes clear that careful selection of patient groups is an important input parameter that should be optimized for each application. In addition, the study outcome parameters should be improved. Although reduced pain and improved function are, from the patient's perspective, the most important outcomes, there is a need for more structure/tissue-related outcome measures. Ideally, criteria and/or markers to identify patients at risk and responders to treatment are the ultimate goal for these more sophisticated regenerative approaches in joint surface repair in particular, and regenerative medicine in general.


A comparison of impact of fatigue on cognitive, physical, and psychosocial status in patients with fibromyalgia and rheumatoid arthritis.
This study was performed to compare the impact of fatigue on different aspects of quality of life in patients with rheumatoid arthritis (RA) and fibromyalgia syndrome (FM). This study involved subjects with FM (n = 45) and RA (n = 44). Impact of fatigue on physical, cognitive, and psychosocial status was measured with Fatigue Impact Scale (FIS) and health-related quality of life (HRQoL) with the Medical Outcome Study Short Form 36 (SF-36). Multiple regression analyses were used to evaluate impact of fatigue on quality of life by taking into account clinical symptoms and disease activity scores in these two patient groups. Although the severity of fatigue assessed by FSS was the same in FM and RA; according to Fatigue Impact Scale, fatigue has higher impact on cognitive function in FM (mean ± SD; 28.8 ± 19.9), and on the other hand, it has higher impact on mainly physical component (mean ± SD; 26.3 ± 4.9) in RA. Regarding all the clinical symptoms and disease activity scores, multiple regression models showed that fatigue together with pain affected the HRQoL (SF-36) in both patient groups. Fatigue has different impacts on QoL in FM and RA, respectively. Together with pain, fatigue lead FM patients to see disease as having worse health in terms of mental function, whereas it leads to poor health in terms of physical function in RA.


Dynamics of the putative RNA helicase Spb4 during ribosome assembly in Saccharomyces cerevisiae.
Spb4 is a putative ATP-dependent RNA helicase that is required for proper processing of 27SB pre-rRNAs and therefore for 60S ribosomal subunit biogenesis. To define the timing of association of this protein with preribosomal particles, we have studied the composition of complexes that copurify with Spb4 tagged by tandem affinity purification (TAP-tagged Spb4). These complexes contain mainly the 27SB pre-rRNAs and about 50 ribosome biogenesis proteins, primarily components of early pre-60S ribosomal particles. To a lesser extent, some protein factors of 90S preribosomal particles and the 35S and 27SA pre-rRNAs also copurify with TAP-tagged Spb4. Moreover, we have obtained by site-directed mutagenesis an allele that results in the R360A substitution in the conserved motif VI of the Spb4 helicase domain. This allele causes a dominant-negative phenotype when overexpressed in the wild-type strain. Cells expressing Spb4(R360A) display an accumulation of 35S and 27SB pre-rRNAs and a net 40S ribosomal subunit defect. TAP-tagged Spb4(R360A) displays a greater steady-state association with 90S preribosomal particles than TAP-tagged wild-type Spb4. Together, our data indicate that Spb4 is a component of early nucle(ol)ar pre-60S ribosomal particles containing 27SB pre-rRNA. Apparently, Spb4 binds 90S preribosomal particles and dissociates from pre-60S ribosomal particles after processing of 27SB pre-rRNA.


Hydrothermal ceramic for porcelain-fused-to-metal crowns: an initial experience report from clinical practice.
The development of hydrothermal ceramics was an important requirement for fusing hydrolysis-resistant ceramics to precious metal alloys with low melting interval. Clinical findings since 1992 confirm the wide range of application of these metal-ceramic restorations in dentistry. Initial problems concerning temperature regulation of the ceramic ovens were rectified by checking and adjusting them for lower sintering temperatures. The golden yellow color is only one advantage of the ceramic-veneered alloy. Other advantages include its nonporous, smooth surface, translucence, and suitability for use in all types of dental restorations and reconstructions. Hydrothermal ceramic veneers thus can help to substantially reduce the number of different alloys used in a patient's mouth.


Thoughts and emotions during traumatic birth: a qualitative study.
Previous research shows that 1 to 6 percent of women will develop symptoms of posttraumatic stress disorder after childbirth. The objective of this study was to examine thoughts and emotions during birth, cognitive processing after birth, and memories of birth that might be important in the development of postnatal posttraumatic stress symptoms. In a qualitative study, women with posttraumatic stress symptoms (n= 25) and without (n = 25) were matched for obstetric events to examine the nonmedical aspects of birth that made it traumatic. Women were interviewed 3 months after birth. The following themes emerged for all women: thoughts during birth included mental coping strategies, wanting labor to end, poor understanding of what was going on, and mental defeat. More negative than positive emotions were described during birth, primarily feeling scared, frightened, and upset. Postnatal cognitive processing included retrospective appraisal of birth, such as taking a fatalistic view and focusing on the present, for example, concentrating on the baby. Memories of birth included not remembering parts of the birth and forgetting how bad it was. Women with posttraumatic stress symptoms reported more panic, anger, thoughts of death, mental defeat, and dissociation during birth; after birth, they reported fewer strategies that focused on the present, more painful memories, intrusive memories, and rumination, than women without symptoms. The results provide a useful first step toward identifying aspects of birth and postnatal processing that might determine whether women develop postnatal posttraumatic stress symptoms. Further research is needed to broaden knowledge of posttraumatic stress disorder before drawing definite conclusions.


Marine ω-3 polyunsaturated fatty acids and risk of colorectal cancer according to microsatellite instability.
Chronic inflammation is involved in the development of colorectal cancer (CRC) and microsatellite instability (MSI), a distinct phenotype of CRC. Experimental evidence indicates an anti-inflammatory and antineoplastic effect of marine ω-3 polyunsaturated fatty acids (PUFAs). However, epidemiologic data remain inconclusive. We investigated whether the association between marine ω-3 PUFAs and CRC varies by MSI-defined subtypes of tumors in the Nurses' Health Study and Health Professionals Follow-up Study. We documented and classified 1125 CRC cases into either MSI-high tumors, in which 30% or more of the 10 microsatellite markers demonstrated instability, or microsatellite-stable (MSS) tumors. Cox proportional hazards model was used to estimate the hazard ratios (HRs) and 95% confidence intervals (CIs) of MSS tumors and MSI-high tumors in relation to marine ω-3 PUFA intake. All statistical tests were two-sided. Marine ω-3 PUFA intake was not associated with overall incidence of CRC. However, a statistically significant difference was detected by MSI status (P heterogeneity = .02): High marine ω-3 PUFA intake was associated with a lower risk of MSI-high tumors (comparing ≥0.30g/d with <0.10g/d: multivariable HR = 0.54, 95% CI = 0.35 to 0.83, P linearity = .03) but not MSS tumors (HR = 0.97, 95% CI = 0.78 to 1.20, P linearity = .28). This differential association appeared to be independent of CpG island methylator phenotype and BRAF mutation status. High marine ω-3 PUFA intake is associated with lower risk of MSI-high CRC but not MSS tumors, suggesting a potential role of ω-3 PUFAs in protection against CRC through DNA mismatch repair. Further research is needed to confirm our findings and elucidate potential underlying mechanisms.


Failure of chemoprophylaxis with standard antituberculosis agents in child contacts of multidrug-resistant tuberculosis cases.
There is little published information on optimal chemoprophylaxis for children with multidrug-resistant tuberculosis (MDR-TB) contacts. Current guidelines of World Health Organization suggest that isoniazid (INH), the standard first-line chemoprophylaxis, be used for those exposed to MDR-TB. This is a retrospective review of medical records of 5 children residing in the Western Cape Province, South Africa, who developed MDR-TB while receiving conventional chemoprophylaxis with either INH or a combination of INH, rifampin, and pyrazinamide. Adult MDR-TB source cases were identified for all children and resistance patterns of patient and source case isolates matched in all cases. The median age of the patients was 0.4 years. One patient participated in a trial of INH chemoprophylaxis for HIV-infected children. Four HIV-uninfected infants presented with TB-related symptoms several months after being given chemoprophylaxis because of a known source case. Stigmata of TB were cough >3 weeks in 4, weight loss or a history of failing to thrive in 3, fever in 2 infants, and reported night sweats in 1. Chest radiographs at diagnosis revealed lymphadenopathy, lobar opacification, and airway narrowing. All patients were treated for varying time periods at a TB referral institution in the Western Cape. Standard, first-line anti-TB agents were inadequate to prevent MDR-TB in children exposed to MDR-TB contacts. Second-line chemoprophylaxis, reflecting the susceptibility profile of the source case's isolate, with at least 2 drugs with activity against the drug-resistant isolate for 6-12 months should be considered.


Picomolar-Level Sensing of Cannabidiol by Metal Nanoparticles Functionalized with Chemically Induced Dimerization Binders.
Simple and fast detection of small molecules is critical to health and environmental monitoring. Methods for chemical detection often use mass spectrometers or enzymes; the former relies on expensive equipment and the latter is limited to those that can act as enzyme substrates. Affinity reagents like antibodies can target a variety of small-molecule analytes, but the detection requires successful design of chemically conjugated targets or analogs for competitive binding assays. Here, we developed a generalizable method for highly sensitive and specific in-solution detection of small molecules, using cannabidiol (CBD) as an example. Our sensing platform uses gold nanoparticles (AuNPs) functionalized with a pair of chemically induced dimerization (CID) nanobody binders (nano-binders), where CID triggers AuNPs aggregation and sedimentation in the presence of CBD. Despite moderate binding affinities of the two nano-binders to CBD (KDs of ~6 and ~56 μM), a scheme consisting of CBD-AuNP pre-analytical incubation, centrifugation, and electronic detection (ICED) was devised to demonstrate a high sensitivity (limit of detection of ~100 picomolar) in urine and saliva, a relatively short assay time (~2 hours), a large dynamic range (5 logs), and a sufficiently high specificity to differentiate CBD from its analog, tetrahydrocannabinol. The high sensing performance was achieved with the multivalency of AuNP sensing, the ICED scheme that increases analyte concentrations in a small assay volume, and a portable electronic detector. This sensing system is readily coupled to other binders for wide molecular diagnostic applications.


Plasma cannabinoid pharmacokinetics following controlled oral delta9-tetrahydrocannabinol and oromucosal cannabis extract administration.
Sativex(®), a cannabis extract oromucosal spray containing Δ(9)-tetrahydrocannabinol (THC) and cannabidiol (CBD), is currently in phase III trials as an adjunct to opioids for cancer pain treatment, and recently received United Kingdom approval for treatment of spasticity. There are indications that CBD modulates THC's effects, but it is unclear if this is due to a pharmacokinetic and/or pharmacodynamic interaction. Cannabis smokers provided written informed consent to participate in this randomized, controlled, double-blind, double-dummy institutional review board-approved study. Participants received 5 and 15 mg synthetic oral THC, low-dose (5.4 mg THC and 5.0 mg CBD) and high-dose (16.2 mg THC and 15.0 mg CBD) Sativex, and placebo over 5 sessions. CBD, THC, 11-hydroxy-THC, and 11-nor- 9-carboxy-THC were quantified in plasma by 2-dimensional GC-MS. Lower limits of quantification were ≤0.25 μg/L. Nine cannabis smokers completed all 5 dosing sessions. Significant differences (P < 0.05) in maximum plasma concentrations (C(max)) and areas under the curve from 0-10.5 h postdose (AUC(0→10.5)) for all analytes were found between low and high doses of synthetic THC and Sativex. There were no statistically significant differences in C(max), time to maximum concentration or in the AUC(0→10.5) between similar oral THC and Sativex doses. Relative bioavailability was calculated to determine the relative rate and extent of THC absorption; 5 and 15 mg oral THC bioavailability was 92.6% (13.1%) and 98.8% (11.0%) of low- and high-dose Sativex, respectively. These data suggest that CBD modulation of THC's effects is not due to a pharmacokinetic interaction at these therapeutic doses.


Antipsychotic adherence patterns and health care utilization and costs among patients discharged after a schizophrenia-related hospitalization.
This study aimed to assess antipsychotic adherence patterns and all-cause and schizophrenia-related health care utilization and costs sequentially during critical clinical periods (i.e., before and after schizophrenia-related hospitalization) among Medicaid-enrolled patients experiencing a schizophrenia-related hospitalization. All patients aged ≥ 18 years with a schizophrenia-related inpatient admission were identified from the MarketScan Medicaid database (2004-2008). Adherence (proportion of days covered [PDC]) to antipsychotics and schizophrenia-related and all-cause health care utilization and costs were assessed during preadmission (182- to 121-day, 120- to 61-day, and 60- to 0-day periods; overall, 6 months) and postdischarge periods (0- to 60-day, 61- to 120-day, 121- to 180-day, 181- to 240-day, 241- to 300-day, and 301- to 365-day periods; overall, 12 months). Health care utilization and costs (2010 US dollars) were compared between each adjacent 60-day follow-up period after discharge using univariate and multivariable regression analyses. No adjustment was made for multiplicity. Of the 2,541 patients with schizophrenia (mean age: 41.2 years; 57% male; 59% black) who were identified, approximately 89% were "discharged to home self-care." Compared with the 60- to 0-day period before the index inpatient admission, greater mean adherence as measured by PDC was observed during the 0- to 60-day period immediately following discharge (0.46 vs. 0.78, respectively). The mean PDC during the overall 6-month preadmission period was lower than during the 6-month postdischarge period (0.53 vs. 0.69; P < 0.001). Compared with the 0- to 60-day postdischarge period, schizophrenia-related health care costs were significantly lower during the 61- to 120-day postdischarge period (mean: $2,708 vs. $2,102; P < 0.001); the primary cost drivers were rehospitalization (mean: $978 vs. $660; P < 0.001) and pharmacy (mean: $959 vs. $743; P < 0.001). Following the initial 60-day period, both all-cause and schizophrenia-related costs declined and remained stable for the remaining postdischarge periods (days 121-365). Although long-term (e.g., 365-day) adherence measures are important, estimating adherence over shorter intervals may clarify the course of vulnerability to risk and enable clinicians to better design adherence/risk-related interventions. The greatest risk of rehospitalization and thus greater resource utilization were observed during the initial 60-day postdischarge period. Physicians should consider tailoring management and treatment strategies to help mitigate the economic and humanistic burden for patients with schizophrenia during this period.


Role of Cytokine Combinations on CD4+ T Cell Differentiation, Partial Polarization, and Plasticity: Continuous Network Modeling Approach.
Purpose: We put forward a theoretical and dynamical approach for the semi-quantitative analysis of CD4+ T cell differentiation, the process by which cells with different functions are derived from activated CD4+ T naïve lymphocytes in the presence of particular cytokine microenvironments. We explore the system-level mechanisms that underlie CD4+ T plasticity-the conversion of polarized cells to phenotypes different from those originally induced. Methods: In this paper, we extend a previous study based on a Boolean network to a continuous framework. The network includes transcription factors, signaling pathways, as well as autocrine and exogenous cytokines, with interaction rules derived using fuzzy logic. Results: This approach allows us to assess the effect of relative differences in the concentrations and combinations of exogenous and endogenous cytokines, as well as of the expression levels of diverse transcription factors. We found either abrupt or gradual differentiation patterns between observed phenotypes depending on critical concentrations of single or multiple environmental cytokines. Plastic changes induced by environmental cytokines were observed in conditions of partial phenotype polarization in the T helper 1 to T helper 2 transition. On the other hand, the T helper 17 to induced regulatory T-cells transition was highly dependent on cytokine concentrations, with TGFβ playing a prime role. Conclusion: The present approach is useful to further understand the system-level mechanisms underlying observed patterns of CD4+ T differentiation and response to changing immunological challenges.


A monoclonal antibody to the interleukin-2 receptor enhances the survival of neural allografts: a time-course study.
A time-course study of the survival and immunological characteristics of rat neural allografts was undertaken in animals treated with a murine monoclonal antibody to the alpha-chain (p55) of the rat interleukin-2 receptor. This antibody, NDS 63, was administered for ten days following grafting beginning on the day of operation. Inbred rat strains differing at both major and minor histocompatibility loci were selected as donor and host. Furthermore, the recipient strain displayed a high responder major histocompatibility complex haplotype. All grafts were placed in the lateral ventricle. Comparison was drawn between NDS 63-treated recipients and two groups of controls; an untreated group and a second group treated with the monoclonal antibody NDS 66, directed at a second epitope on the alpha-chain of the interleukin-2 receptor, which has been shown to be ineffective in competing with interleukin-2 for binding. Immunocytochemical analysis of the transplants was performed at several time-points up to 150 days following grafting. Grafts of NDS 63-treated recipients exhibited 100% survival with minimal induction of major histocompatibility complex antigens (both class I and class II) and negligible leukocyte infiltration at all time-points studied. In contrast grafts from both groups of controls showed evidence of a chronic immune response with most grafts undergoing rejection as shown by markedly elevated major histocompatibility complex antigen expression accompanied by specific immune cell infiltration. This was a protracted process with several grafts undergoing complete rejection by 60 days and a majority, but not all, by 150 days after transplantation. It is concluded that NDS 63, a monoclonal antibody to the interleukin-2 receptor, may diminish the immune response to transplanted allogeneic neural tissue and thereby enhance its prospects for long-term survival.


Quality of life, urogynecological morbidity, and lymphedema after radical vaginal trachelectomy for early-stage cervical cancer.
Radical vaginal trachelectomy (RVT) offers a possibility for future childbearing for young women with early-stage cervical cancer. However, the literature on quality of life and self-reported morbidity in patients undergoing RVT is scarce. The aim of this study was to prospectively assess quality of life after RVT with focus on urogynecological morbidity and lymphedema. Furthermore, the aim of this study was to compare results with those in women treated with radical abdominal hysterectomy (RAH) and with age-matched control women from the general population. Eighteen patients with early-stage cervical cancer operated with RVT were prospectively included and assessed preoperatively, 3, 6, and 12 months postoperatively using validated questionnaires. Thirty-two patients treated with RAH were included consecutively and assessed once at 12 months postsurgery, whereas an age-matched control group of 30 healthy women was assessed once. Fifty percent of the RVT group and 41% of the RAH reported any grade of incomplete bladder emptying problems at 1 year postsurgery assessment. Eleven percent of the RVT patients and 12.5% of the RAH patients reported severe lymphedema of the legs as assessed by the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire Cervical Cancer Module. The Global Health Status scores of the RVT patients improved over time but were significantly lower than in the healthy controls during the entire observation time (P = 0.029). Patients treated with RVT for early-stage cervical cancer had persistent bladder emptying problems and lymphedema comparable to those experienced by patients treated with RAH and significantly higher than those reported by healthy control women.


Current Status and Needs of Long-Term Follow-Up Clinics for Hematopoietic Cell Transplantation Survivors: Results of a Nationwide Survey in Japan.
With increasing focus on the importance of long-term survivorship care after allogeneic hematopoietic cell transplantation (allo-HCT), more institutions have been establishing long-term follow-up (LTFU) clinics. Currently, however, with varying volumes of HCT procedures and resources, there is no standardized operation of these clinics in HCT centers. We conducted a nationwide questionnaire survey to characterize the current operation of LTFU clinics in Japan. We targeted 271 HCT centers (189 adult and 82 pediatric) that registered allo-HCT cases to the national transplant registry database. The response rate was 69%, and 117 of the 188 participating centers (62%) had an established LTFU clinic. The most frequent reason cited for not operating an LTFU clinic was a "lack of human resources," especially nurses. Most centers with an LTFU clinic targeted allo-HCT recipients, although autologous HCT survivors were followed up at 18% of adult centers and 48% of pediatric centers. Ninety-two percent of centers did not terminate LTFU at a specific time point, and 56% recommended that patients visit the LTFU clinic beyond 5 years after HCT. Fifteen of 20 pediatric centers indicated that they did not routinely refer survivors who underwent HCT at a young age to an adult HCT center for their adulthood LTFU. We found that staffing and standard practices varied widely among centers, and that most centers continued to see long-term HCT survivors at their own outpatient clinics. The development of common LTFU tools may help standardize LTFU practices and facilitate efficient transitions.


On the coordination of highly dynamic human movements: an extension of the Uncontrolled Manifold approach applied to precision jump in parkour.
The human body generally has more degrees of freedom than necessary for generating a given movement. According to the motor abundance principle, this redundancy is beneficial as it provides the central nervous system with flexibility and robustness for the generation of movements. Under the hypothesis of the Uncontrolled Manifold, the additional degrees of freedom are used to produce motor solutions by reducing the variability that affects the motion performance across repetitions. In this paper, we present a general mathematical framework derived from robotics to formulate kinematic and dynamic tasks in human movement. On this basis, an extension of the Uncontrolled Manifold approach is introduced to deal with dynamic movements. This extension allows us to present a complex experimental application of the proposed framework to highly dynamic task variables in parkour movements. This experiment involves dynamic tasks expressed in terms of linear and angular momenta. The results show that the central nervous system is able to coordinate such skilled tasks which appear to be preferentially controlled and hierarchically organized. The proposed extension is promising for the study of motion generation in anthropomorphic systems and provides a formal description to investigate kinematics and dynamics tasks in human motions.


Invariant NKT cells metabolically adapt to the acute myeloid leukaemia environment.
Acute myeloid leukaemia (AML) creates an immunosuppressive environment to conventional T cells through Arginase 2 (ARG2)-induced arginine depletion. We identify that AML blasts release the acute phase protein serum amyloid A (SAA), which acts in an autocrine manner to upregulate ARG2 expression and activity, and promote AML blast viability. Following in vitro cross-talk invariant natural killer T (iNKT) cells become activated, upregulate mitochondrial capacity, and release IFN-γ. iNKT retain their ability to proliferate and be activated despite the low arginine AML environment, due to the upregulation of Large Neutral Amino Acid Transporter-1 (LAT-1) and Argininosuccinate Synthetase 1 (ASS)-dependent amino acid pathways, resulting in AML cell death. T cell proliferation is restored in vitro and in vivo. The capacity of iNKT cells to restore antigen-specific T cell immunity was similarly demonstrated against myeloid-derived suppressor cells (MDSCs) in wild-type and Jα18-/- syngeneic lymphoma-bearing models in vivo. Thus, stimulation of iNKT cell activity has the potential as an immunotherapy against AML or as an adjunct to boost antigen-specific T cell immunotherapies in haematological or solid cancers.


Association between -276 C/T polymorphism of the ERBB3 gene and lung cancer risk in a Korean population.
The associations between the polymorphisms in the promoter region of the V-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (ERBB3) gene and lung cancer risk were investigated. By direct sequencing, we first found two known polymorphisms (-536 A/G and -276 C/T). Further TaqMan assays and logistic regression analyses were performed in order to characterize the association between the -276 C/T polymorphism of ERBB3 and lung cancer risk in 425 patients with lung cancer and 411 healthy controls. To examine the potential effects of the -276 C/T polymorphism on ERBB3 transcription, luciferase reporter assays were performed in non-small cell lung cancer (NSCLC) cell lines. The -276 C/T polymorphism was associated with the risk of lung cancer in a recessive model of never-smokers. Interestingly, the T allele conferred significantly lower promoter activity by 32% and 33% than the C allele in H2009 and H358 cell lines. ERBB3 promoter polymorphisms affect ERBB3 gene expression, and contribute to genetic susceptibility to lung cancer in never-smokers.


Maneuvering Together, Apart, and at Odds: Residents' Care Convoys in Assisted Living.
Frail and disabled individuals such as assisted living residents are embedded in "care convoys" comprised of paid and unpaid caregivers. We sought to learn how care convoys are configured and function in assisted living and understand how and why they vary and with what resident and caregiver outcomes. We analyzed data from a qualitative study involving formal in-depth interviews, participant observation and informal interviewing, and record review. We prospectively studied 28 residents and 114 care convoy members drawn from four diverse assisted living communities over 2 years. Care convoys involved family and friends who operated individually or shared responsibility, assisted living staff, and multiple external care workers. Residents and convoy members engaged in processes of "maneuvering together, apart, and at odds" as they negotiated the care landscape routinely and during health crises. Based on consensus levels, and the quality of collaboration and communication, we identified three main convoy types: cohesive, fragmented, and discordant. Care convoys clearly shape care experiences and outcomes. Identifying strategies for establishing effective communication and collaboration practices and promoting convoy member consensus, particularly over time, is essential to the creation and maintenance of successful and supportive care partnerships.


Early second-trimester plasma levels of NT-proBNP in women who subsequently develop early-onset preeclampsia.
Plasma levels of NT-proBNP are elevated in women with preeclampsia at the time of diagnosis. The objective of this case-control study was to evaluate N-terminal proBNP (NT-proBNP) in maternal plasma as an early second-trimester biomarker for prediction of early-onset preeclampsia. In early second-trimester samples, women who later developed preeclampsia at gestational age 34 wk + 0 or earlier (n = 16) had similar plasma levels of NT-proBNP (median 51.8, range 26.1-131.9 pg/ml) as women with uncomplicated pregnancy outcomes (n = 43) (53.0, 14.9-184.2 pg/ml). The early second-trimester level of NT-proBNP cannot therefore be used as a predictive biomarker of early-onset preeclampsia.


Predicting self-care behaviours of patients with type 2 diabetes: the importance of beliefs about behaviour, not just beliefs about illness.
There is evidence that perceptions of treatment may be more predictive than illness perceptions, e.g. medication adherence is often better predicted by beliefs about medication than by beliefs about illness. The present study aims to assess the generality of this finding, by comparing the extent to which self-care behaviours of patients with type 2 diabetes are predicted by patients' beliefs about those behaviours, compared with their illness perceptions. This study is a one year prospective cohort analysis of 453 patients recruited to a randomised trial of blood glucose self-monitoring. Behaviour was assessed by the medication adherence report scale (MARS) and diabetes self-care activities (DSCA) scales; illness perceptions by IPQ-R; study-specific scales of beliefs about diet and physical activity were constructed by factor analysing items based on beliefs elicited in an earlier interview study involving patients with type 2 diabetes. Past behaviour, trial group allocation, and clinical and demographic factors predicted between 16% and 35% variance in medication adherence, exercise, and diet scales. Illness perceptions added between 0.9% and 4.5% additional variance; beliefs about behaviour added a further 1.1% to 6.4% additional variance. Beliefs regarding, respectively, the importance of exercise in controlling diabetes, the need to east less, and enjoyment from eating sweet or fatty food, added unique variance. Beliefs about behaviour are at least as important as beliefs about illness in predicting several health-related behaviours. This suggests the possibility that behaviour change interventions with patient groups would be more effective by targeting beliefs about behaviour, rather than beliefs about illness.


Human infection with a reassortment avian influenza A H3N8 virus: an epidemiological investigation study.
A four-year-old boy developed recurrent fever and severe pneumonia in April, 2022. High-throughput sequencing revealed a reassortant avian influenza A-H3N8 virus (A/Henan/ZMD-22-2/2022(H3N8) with avian-origin HA and NA genes. The six internal genes were acquired from Eurasian lineage H9N2 viruses. Molecular substitutions analysis revealed the haemagglutin retained avian-like receptor binding specificity but that PB2 genes possessed sequence changes (E627K) associated with increased virulence and transmissibility in mammalian animal models. The patient developed respiratory failure, liver, renal, coagulation dysfunction and sepsis. Endotracheal intubation and extracorporeal membrane oxygenation were administered. H3N8 RNA was detected from nasopharyngeal swab of a dog, anal swab of a cat, and environmental samples collected in the patient's house. The full-length HA sequences from the dog and cat were identical to the sequence from the patient. No influenza-like illness was developed and no H3N8 RNA was identified in family members. Serological testing revealed neutralizing antibody response against ZMD-22-2 virus in the patient and three family members. Our results suggest that a triple reassortant H3N8 caused severe human disease. There is some evidence of mammalian adaptation, possible via an intermediary mammalian species, but no evidence of person-to-person transmission. The potential threat from avian influenza viruses warrants continuous evaluation and mitigation.


The fate of absorbed and exogenous ammonia as influenced by forage or forage-concentrate diets in growing sheep.
Changes in splanchnic energy and N metabolism were studied in sheep, prepared with vascular catheters across the portal-drained viscera (PDV) and the liver, and maintained on supramaintenance intakes of either gross or grass + barley pellets. The animals were challenged, on both diets, with 4 d intramesenteric vein infusions of NH4Cl (25 mumol/min) plus NH4HCO3 (at either 0 or 125 mumol/min). On the final day of each treatment the natural abundance NH4Cl was replaced with 15NH4Cl over a 10 h infusion while over the same period [1-13C]leucine was infused via a jugular vein. Measurements were made of blood flow plus mass transfers of NH3, urea, free amino acids and O2 across the PDV and liver. Enrichments of [14N15N]urea and [15N15N]urea plus [15N]glutamine, aspartate and glutamate were also monitored. Whole-body urea flux was determined by infusion of [14C]urea. At the end of the study the animals were infused for 3 h with 15nH4Cl, killed and liver samples assayed for intracellular free amino acid enrichments and concentrations. Blood flows across the splanchnic region were unaffected by either diet or level of ammonium salt infusion. At the lower ammonium salt infusion there was a trend for greater absorption of NH3 across the PDV (P < 0.10) with grass + barley than with the grass diet, while removal of urea was unaltered. At the higher ammonium salt infusions there was a significantly greater appearance of NH3 across the PDV and this exceeded the extra infused. Urea-N removal, however, was also elevated and by more than that required to account for the additional NH3. The PDV contributed 19-28% to whole-body O2 consumption and the liver 23-32%. Hepatic extraction of absorbed NH3 was complete on all treatments and systemic pH remained constant. The fractions of urea-N apparently derived from NH3 were similar on the grass (0.50-0.64) and grass + barley (0.64-0.67) diets. Hepatic production of urea agreed well with urea flux measurements. Between the two levels of ammonium salt infusion and within diets the additional NH3 removed across the PDV was accounted for by the increased urea-N production. The [14N15N]:[15N15N] ratio of the urea produced was 97:3, while the enrichment of hepatic intracellular free aspartate was lower than that of [14N15N]urea. Glutamine enrichments were 0.23-0.37 those of [14N15N]urea, indicating a minor role for those hepatocytes (probably perivenous) which contain glutamine synthetase (EC 220.127.116.11). Leucine kinetics, either for the whole body or splanchnic tissues, were not different between diets or level of ammonium salt infusion, except for oxidation which was less on the grass + barley ration. Amino acid concentrations were lower on the grass + barley diet but net PDV absorptions were similar. The pattern of essential amino acids absorbed into the PDV showed good agreement with the published composition of mixed rumen microbial protein. Fractional disappearances of absorbed free essential amino acids across the liver varied from 0.4 (branched chains) to near unity (histidine, phenylalanine).


Fast and highly chemoselective alkynylation of thiols with hypervalent iodine reagents enabled through a low energy barrier concerted mechanism.
Among all functional groups, alkynes occupy a privileged position in synthetic and medicinal chemistry, chemical biology, and materials science. Thioalkynes, in particular, are highly useful, as they combine the enhanced reactivity of the triple bond with a sulfur atom frequently encountered in bioactive compounds and materials. Nevertheless, general methods to access these compounds are lacking. In this article, we describe the mechanism and full scope of the alkynylation of thiols using ethynyl benziodoxolone (EBX) hypervalent iodine reagents. Computations led to the discovery of a new, three-atom concerted transition state with a very low energy barrier, which rationalizes the high reaction rate. On the basis of this result, the scope of the reaction was extended to the synthesis of aryl- and alkyl-substituted alkynes containing a broad range of functional groups. New sulfur nucleophiles such as thioglycosides, thioacids, and sodium hydrogen sulfide were also alkynylated successfully to lead to the most general and practical method yet reported for the synthesis of thioalkynes.


A c-type cytochrome and a transcriptional regulator responsible for enhanced extracellular electron transfer in Geobacter sulfurreducens revealed by adaptive evolution.
The stimulation of subsurface microbial metabolism often associated with engineered bioremediation of groundwater contaminants presents subsurface microorganisms, which are adapted for slow growth and metabolism in the subsurface, with new selective pressures. In order to better understand how Geobacter species might adapt to selective pressure for faster metal reduction in the subsurface, Geobacter sulfurreducens was put under selective pressure for rapid Fe(III) oxide reduction. The genomes of two resultant strains with rates of Fe(III) oxide reduction that were 10-fold higher than those of the parent strain were resequenced. Both strains contain either a single base-pair change or a 1 nucleotide insertion in a GEMM riboswitch upstream of GSU1761, a gene coding for the periplasmic c-type cytochrome designated PgcA. GSU1771, a gene coding for a SARP regulator, was also mutated in both strains. Introduction of either of the GEMM riboswitch mutations upstream of pgcA in the wild-type increased the abundance of pgcA transcripts, consistent with increased expression of pgcA in the adapted strains. One of the mutations doubled the rate of Fe(III) oxide reduction. Interruption of GSU1771 doubled the Fe(III) oxide reduction rate. This was associated with an increased in expression of pilA, the gene encoding the structural protein for the pili thought to function as microbial nanowires. The combination of the GSU1771 interruption with either of the pgcA mutations resulted in a strain that reduced Fe(III) as fast as the comparable adapted strain. These results suggest that the accumulation of a small number of beneficial mutations under selective pressure, similar to that potentially present during bioremediation, can greatly enhance the capacity for Fe(III) oxide reduction in G. sulfurreducens. Furthermore, the results emphasize the importance of the c-type cytochrome PgcA and pili in Fe(III) oxide reduction and demonstrate how adaptive evolution studies can aid in the elucidation of complex mechanisms, such as extracellular electron transfer.


Effects of human growth hormone in men over 60 years old.
The declining activity of the growth hormone--insulin-like growth factor I (IGF-I) axis with advancing age may contribute to the decrease in lean body mass and the increase in mass of adipose tissue that occur with aging. To test this hypothesis, we studied 21 healthy men from 61 to 81 years old who had plasma IGF-I concentrations of less than 350 U per liter during a six-month base-line period and a six-month treatment period that followed. During the treatment period, 12 men (group 1) received approximately 0.03 mg of biosynthetic human growth hormone per kilogram of body weight subcutaneously three times a week, and 9 men (group 2) received no treatment. Plasma IGF-I levels were measured monthly. At the end of each period we measured lean body mass, the mass of adipose tissue, skin thickness (epidermis plus dermis), and bone density at nine skeletal sites. In group 1, the mean plasma IGF-I level rose into the youthful range of 500 to 1500 U per liter during treatment, whereas in group 2 it remained below 350 U per liter. The administration of human growth hormone for six months in group 1 was accompanied by an 8.8 percent increase in lean body mass, a 14.4 percent decrease in adipose-tissue mass, and a 1.6 percent increase in average lumbar vertebral bone density (P less than 0.05 in each instance). Skin thickness increased 7.1 percent (P = 0.07). There was no significant change in the bone density of the radius or proximal femur. In group 2 there was no significant change in lean body mass, the mass of adipose tissue, skin thickness, or bone density during treatment. Diminished secretion of growth hormone is responsible in part for the decrease of lean body mass, the expansion of adipose-tissue mass, and the thinning of the skin that occur in old age.


Paracoccidioides brasiliensis-gp43 used as paracoccidioidin.
A purified glycoprotein of 43,000 daltons from Paracoccidioides brasiliensis (gp43) was tested as paracoccidioidin in delayed-type hypersensitivity (DTH) tests in both experimental animals (guinea pig and mice) and patients with paracoccidioidomycosis (PCM). The gp43 paracoccidioidin was compared with the traditional Fava Netto antigen (AgFN). In guinea pigs, the intradermal injection of 2 micrograms of gp43 showed a similar response to those obtained with AgFN, showing in histological sections a population of lymphoid cells that participate in DTH. In mice, gp43 at a dose of 3.75 micrograms showed positive DTH response. The use of gp43 as paracoccidioidin in humans showed that this molecule can be used to evaluate the DTH response in patients with PCM. Of 25 PCM patients studied, 48% were positive to gp43 while only 28% were positive to AgFN; 12 PCM patients were completely anergic to both antigens. Considering only those 13 PCM patients who were responsive to gp43 and/or to AgFN, 92.3% reacted against gp43 and 53.8% reacted against AgFN (P < 0.05). Gp43 skin test responses (13.67 +/- 9.56 mm) were significantly larger than those obtained with AgFN (8.43 +/- 3.69 mm). Immunohistochemical study of the human skin showed a perivascular inflammatory response constituted predominantly by T lymphocytes, macrophages and polymorphonuclear leukocytes.


[Disseminated histoplasmosis and haemophagocytic syndrome in HIV patients: A case series in a Peruvian hospital].
Disseminated histoplasmosis (DH) is an opportunistic fungal infection in severely immunocompromised patients with HIV infection. Haemophagocytic syndrome (HFS), which can occur in these co-infected patients when the immune response is significantly altered, is often associated with high mortality. To describe the epidemiological, clinical, analytical and microbiological characteristics, along with studying the presence of HFS, in patients with DH-HIV. A retrospective study was conducted on a case series using data from the clinical records of patients diagnosed with DH and HIV infection during the years 2014 and 2015. DH was diagnosed in 8 (1.3%) of 597 HIV patients. All patients were in stage C3, and 75% (6/8) were not receiving combined antiretroviral therapy (CART). The remaining two patients had recently begun CART (possible immune reconstitution syndrome). Five (62.5%) of the 8 patients met criteria for HFS. The most frequent clinical symptoms were lymphoproliferative and consumptive syndrome, respiratory compromise, and cytopenia. Histoplasma was isolated in lymph nodes of 75% (6/8) of the patients, in blood samples in 25% (2/8), and also in intestinal tissue in one patient. The antifungal therapy was amphotericin B deoxycholate, without adjuvants. The overall mortality was 50%. In this case series, DH-HIV co-infection frequently progressed to HFS with high mortality. The clinical picture may resemble that of other systemic opportunistic infections, such as tuberculosis, or can take place simultaneously with other infections. Clinical suspicion is important in patients with severe cytopenia and lymphoproliferative and consumptive syndrome in order to establish an early diagnosis and prescribing a timely specific therapy.


Intracranial pressure at high altitude and acute mountain sickness.
1. Raised intracranial pressure has been noted in severe forms of acute mountain sickness and high-altitude cerebral oedema, but the role of intracranial pressure in the pathogenesis of mild to moderate acute mountain sickness is unknown. 2. Serial measurements of intracranial pressure were made indirectly by assessing changes in tympanic membrane displacement in 24 healthy subjects on rapid ascent to 5200 m. 3. Acute hypoxia at 3440 m was associated with a rise in intracranial pressure, but no difference was found in pressure changes at 4120 or 5200 m in subjects with or without symptoms of acute mountain sickness. 4. Raised intracranial pressure, though temporarily associated with acute hypoxia, is not a feature of acute mountain sickness with mild or moderate symptoms.


N-terminal variant Asp14Asn of the human p70 S6 Kinase 1 enhances translational signaling causing different effects in developing and mature neuronal cells.
The ribosomal p70 S6 Kinase 1 (S6K1) has been implicated in the etiology of complex neurological diseases including autism, depression and dementia. Though no major gene disruption has been reported in humans in RPS6KB1, single nucleotide variants (SNVs) causing missense mutations have been identified, which have not been assessed for their impact on protein function. These S6K1 mutations have the potential to influence disease progression and treatment response. We mined the Simon Simplex Collection (SSC) and SPARK autism database to find inherited SNVs in S6K1 and characterized the effect of two missense SNVs, Asp14Asn (allele frequency = 0.03282%) and Glu44Gln (allele frequency = 0.0008244%), on S6K1 function in HEK293, human ES cells and primary neurons. Expressing Asp14Asn in HEK293 cells resulted in increased basal phosphorylation of downstream targets of S6K1 and increased de novo translation. This variant also showed blunted response to the specific S6K1 inhibitor, FS-115. In human embryonic cell line Shef4, Asp14Asn enhanced spontaneous neural fate specification in the absence of differentiating growth factors. In addition to enhanced translation, neurons expressing Asp14Asn exhibited impaired dendritic arborization and increased levels of phosphorylated ERK 1/2. Finally, in the SSC families tracked, Asp14Asn segregated with lower IQ scores when found in the autistic individual rather than the unaffected sibling. The Glu44Gln mutation showed a milder, but opposite phenotype in HEK cells as compared to Asp14Asn. Although the Glu44Gln mutation displayed increased neuronal translation, it had no impact on neuronal morphology. Our results provide the first characterization of naturally occurring human S6K1 variants on cognitive phenotype, neuronal morphology and maturation, underscoring again the importance of translation control in neural development and plasticity.


Individual differences in cardiovascular response to social challenge.
An important determinant of cardiovascular stress reactivity and morbidity is the individual behavioral strategy of coping with social challenge. This review summarizes the results of a number of studies that we performed in rats, aimed at investigating the relationship between aggression and cardiovascular responsivity under social stress conditions. We show that rats belonging to the 'aggressive tail' of a population are characterized by a higher sympathetic-adrenomedullary activation during social and non-social stress episodes. Wild-type rats are characterized by a larger sympathetic dominance and a higher susceptibility to cardiac arrhythmias during defeat as compared to Wistars. Cardiovascular habituation takes place when social challenge is an intermittent victory experience, whereas no habituation is observed across repeated defeat episodes. Dominant rats whose social dominance is challenged by the aggression of another subject display long-term alterations of heart rate circadian rhythmicity. Such changes are linked to individual proness to defend social dominance: the more the animal counterattacks the aggressor, the smaller the subsequent rhythm disturbance. These data underline how important it is to carefully consider individual differences in aggression and the context in which aggression is expressed, when studying cardiovascular effects of social interactions.


Genome-wide associations between genetic and epigenetic variation influence mRNA expression and insulin secretion in human pancreatic islets.
Genetic and epigenetic mechanisms may interact and together affect biological processes and disease development. However, most previous studies have investigated genetic and epigenetic mechanisms independently, and studies examining their interactions throughout the human genome are lacking. To identify genetic loci that interact with the epigenome, we performed the first genome-wide DNA methylation quantitative trait locus (mQTL) analysis in human pancreatic islets. We related 574,553 single nucleotide polymorphisms (SNPs) with genome-wide DNA methylation data of 468,787 CpG sites targeting 99% of RefSeq genes in islets from 89 donors. We identified 67,438 SNP-CpG pairs in cis, corresponding to 36,783 SNPs (6.4% of tested SNPs) and 11,735 CpG sites (2.5% of tested CpGs), and 2,562 significant SNP-CpG pairs in trans, corresponding to 1,465 SNPs (0.3% of tested SNPs) and 383 CpG sites (0.08% of tested CpGs), showing significant associations after correction for multiple testing. These include reported diabetes loci, e.g. ADCY5, KCNJ11, HLA-DQA1, INS, PDX1 and GRB10. CpGs of significant cis-mQTLs were overrepresented in the gene body and outside of CpG islands. Follow-up analyses further identified mQTLs associated with gene expression and insulin secretion in human islets. Causal inference test (CIT) identified SNP-CpG pairs where DNA methylation in human islets is the potential mediator of the genetic association with gene expression or insulin secretion. Functional analyses further demonstrated that identified candidate genes (GPX7, GSTT1 and SNX19) directly affect key biological processes such as proliferation and apoptosis in pancreatic β-cells. Finally, we found direct correlations between DNA methylation of 22,773 (4.9%) CpGs with mRNA expression of 4,876 genes, where 90% of the correlations were negative when CpGs were located in the region surrounding transcription start site. Our study demonstrates for the first time how genome-wide genetic and epigenetic variation interacts to influence gene expression, islet function and potential diabetes risk in humans.


Incomplete transfer of accessory loci influencing SbMATE expression underlies genetic background effects for aluminum tolerance in sorghum.
Impaired root development caused by aluminum (Al) toxicity is a major cause of grain yield reduction in crops cultivated on acid soils, which are widespread worldwide. In sorghum, the major Al-tolerance locus, AltSB , is due to the function of SbMATE, which is an Al-activated root citrate transporter. Here we performed a molecular and physiological characterization of various AltSB donors and near-isogenic lines harboring various AltSB alleles. We observed a partial transfer of Al tolerance from the parents to the near-isogenic lines that was consistent across donor alleles, emphasizing the occurrence of strong genetic background effects related to AltSB . This reduction in tolerance was variable, with a 20% reduction being observed when highly Al-tolerant lines were the AltSB donors, and a reduction as great as 70% when other AltSB alleles were introgressed. This reduction in Al tolerance was closely correlated with a reduction in SbMATE expression in near-isogenic lines, suggesting incomplete transfer of loci acting in trans on SbMATE. Nevertheless, AltSB alleles from the highly Al-tolerant sources SC283 and SC566 were found to retain high SbMATE expression, presumably via elements present within or near the AltSB locus, resulting in significant transfer of the Al-tolerance phenotype to the derived near-isogenic lines. Allelic effects could not be explained by coding region polymorphisms, although occasional mutations may affect Al tolerance. Finally, we report on the extensive occurrence of alternative splicing for SbMATE, which may be an important component regulating SbMATE expression in sorghum by means of the nonsense-mediated RNA decay pathway.


Vitamin D, race, and cardiovascular mortality: findings from a national US sample.
Findings are conflicting about the relationship between vitamin D levels and cardiovascular mortality. We wanted to determine the contribution of vitamin D levels to black-white disparities in cardiovascular mortality. We examined the association of serum 25(OH)D levels with cardiovascular mortality and its contribution to elevated risk among blacks through a retrospective cohort using baseline data from the third National Health and Nutrition Examination Survey 1988-1994 and cause-specific mortality through 2001 using the National Death Index. Using piecewise Poisson regression models, we examined the risk of cardiovascular death (coronary heart disease, heart failure, and stroke) by sample 25(OH)D quartile, adjusting for cardiovascular risk factors, and compared models of adjusted race-related cardiovascular mortality with and without further adjustment for 25(OH)D levels. Participants with 25(OH)D levels in the lowest quartile (mean = 13.9 ng/mL) compared with those in the 3 higher quartiles (mean = 21.6, 28.4, and 41.6 ng/mL) had higher adjusted risk of cardiovascular death (incident rate ratio [IRR] = 1.40; 95% confidence interval [CI], 1.16-1.70). The higher age- and sex-adjusted cardiovascular mortality observed in blacks vs whites (IRR = 1.38; 95% CI, 1.13-1.70) was attenuated (IRR = 1.14; 95% CI, 0.91-1.44) by adjustment for 25(OH)D levels and fully eliminated with further adjustment for income (IRR=1.01; 95% CI, 0.82-1.24). Low serum levels of 25(OH)D are associated with increased cardiovascular mortality in a nationally representative US sample. Black-white differences in 25(OH)D levels may contribute to excess cardiovascular mortality in blacks. Interventional trials among persons with low vitamin D levels are needed to determine whether oral supplementation improves cardiovascular outcomes.


Advanced X-ray absorption and emission spectroscopy: in situ catalytic studies.
Knowledge of the structure of catalysts is essential to understand their behavior, which further facilitates development of an active, selective, and stable catalyst. Determining the structure of a functioning catalyst is essential in this regard. The structure of a catalyst is prone to change during the catalytic process and needs to be determined in its working conditions. In this tutorial review, we have summarized studies done at synchrotron radiation facilities that illustrate the capability to determine catalyst structure using X-ray absorption spectroscopy (XAS) and X-ray emission spectroscopy (XES). These studies aim at facilitating the determination of the dynamic structure-performance relationships during a catalytic process.


Targeting mutant RAS in patient-derived colorectal cancer organoids by combinatorial drug screening.
Colorectal cancer (CRC) organoids can be derived from almost all CRC patients and therefore capture the genetic diversity of this disease. We assembled a panel of CRC organoids carrying either wild-type or mutant RAS, as well as normal organoids and tumor organoids with a CRISPR-introduced oncogenic KRAS mutation. Using this panel, we evaluated RAS pathway inhibitors and drug combinations that are currently in clinical trial for RAS mutant cancers. Presence of mutant RAS correlated strongly with resistance to these targeted therapies. This was observed in tumorigenic as well as in normal organoids. Moreover, dual inhibition of the EGFR-MEK-ERK pathway in RAS mutant organoids induced a transient cell-cycle arrest rather than cell death. In vivo drug response of xenotransplanted RAS mutant organoids confirmed this growth arrest upon pan-HER/MEK combination therapy. Altogether, our studies demonstrate the potential of patient-derived CRC organoid libraries in evaluating inhibitors and drug combinations in a preclinical setting.


Effects of Droplet Composition on Nanodroplet-Mediated Histotripsy.
Nanodroplet-mediated histotripsy (NMH) is a targeted ablation technique combining histotripsy with nanodroplets that can be selectively delivered to tumor cells. In two previous studies, polymer-encapsulated perfluoropentane nanodroplets were used to generate well-defined ablation similar to that obtained with histotripsy, but at significantly lower pressure, when NMH therapy was applied at a pulse repetition frequency (PRF) of 10 Hz. However, cavitation was not maintained over multiple pulses when ultrasound was applied at a lower PRF (i.e., 1-5 Hz). We hypothesized that nanodroplets with a higher-boiling-point perfluorocarbon core would provide sustainable cavitation nuclei, allowing cavitation to be maintained over multiple pulses, even at low PRF, which is needed for efficient and complete tissue fractionation via histotripsy. To test this hypothesis, we investigated the effects of droplet composition on NMH therapy by applying histotripsy at various frequencies (345 kHz, 500 kHz, 1.5 MHz, 3 MHz) to tissue phantoms containing perfluoropentane (PFP, boiling point ∼29°C, surface tension ∼9.5 mN/m) and perfluorohexane (PFH, boiling point ∼56°C, surface tension ∼11.9 mN/m) nanodroplets. First, the effects of droplet composition on the NMH cavitation threshold were investigated, with results revealing a significant decrease (>10 MPa) in the peak negative pressure (p-) cavitation threshold for both types of nanodroplets compared with controls. A slight decrease (∼1-3 MPa) in threshold was observed for PFP phantoms compared with PFH phantoms. Next, the ability of nanodroplets to function as sustainable cavitation nuclei over multiple pulses was investigated, with results revealing that PFH nanodroplets were sustainable cavitation nuclei over 1,000 pulses, whereas PFP nanodroplets were destroyed during the first few pulses (<50 pulses), likely because of the lower boiling point. Finally, tissue phantoms containing a layer of embedded red blood cells were used to compare the damage generated for NMH treatments using PFP and PFH droplets, with results indicating that PFH nanodroplets significantly improved NMH ablation, allowing for well-defined lesions to be generated at all frequencies and PRFs tested. Overall, the results of this study provide significant insight into the role of droplet composition in NMH therapy and provide a rational basis to tailor droplet parameters to improve NMH tissue fractionation.


An online randomized controlled trial evaluating HIV prevention digital media interventions for men who have sex with men.
As HIV infection continues unabated, there is a need for effective interventions targeting at-risk men who have sex with men (MSM). Engaging MSM online where they meet sexual partners is critical for HIV prevention efforts. A randomized controlled trial (RCT) conducted online among U.S. MSM recruited from several gay sexual networking websites assessed the impact of 2 HIV prevention videos and an HIV prevention webpage compared to a control condition for the study outcomes HIV testing, serostatus disclosure, and unprotected anal intercourse (UAI) at 60-day follow-up. Video conditions were pooled due to reduced power from low retention (53%, n = 1,631). No participant incentives were provided. Follow-up was completed by 1,631 (53%) of 3,092 eligible men. In the 60 days after the intervention, men in the pooled video condition were significantly more likely than men in the control to report full serostatus disclosure ('asked and told') with their last sexual partner (OR 1.32, 95% CI 1.01-1.74). Comparing baseline to follow-up, HIV-negative men in the pooled video (OR 0.70, 95% CI 0.54-0.91) and webpage condition (OR 0.43, 95% CI 0.25-0.72) significantly reduced UAI at follow-up. HIV-positive men in the pooled video condition significantly reduced UAI (OR 0.38, 95% CI 0.20-0.67) and serodiscordant UAI (OR 0.53, 95% CI 0.28-0.96) at follow-up. Findings from this online RCT of MSM recruited from sexual networking websites suggest that a low cost, brief digital media intervention designed to engage critical thinking can increase HIV disclosure to sexual partners and decrease sexual risk. Effective, brief HIV prevention interventions featuring digital media that are made widely available may serve as a complementary part of an overall behavioral and biomedical strategy for reducing sexual risk by addressing the specific needs and circumstances of the target population, and by changing individual knowledge, motivations, and community norms. ClinicalTrials.gov NCT00649701.


Trachoma Mapping in Gombe State, Nigeria: Results of 11 Local Government Area Surveys.
To determine the need or otherwise for establishment of a trachoma elimination program in Gombe State, Nigeria, by estimating the population-based prevalence of trachoma in each Local Government Area (LGA) of Gombe. Using a multi-stage, systematic, random, and quasi-random sampling approach and Global Trachoma Mapping Project support, we selected 25 clusters in each LGA. In each cluster, we selected 25 households and all consenting residents aged 1 year and older were examined for trachomatous inflammation-follicular (TF) and trichiasis, using the World Health Organization simplified grading scheme. No LGA in Gombe State had a TF prevalence ≥5% in 1-9-year-olds. All LGAs had trichiasis prevalences above the elimination threshold and should be targeted for community-based delivery of trichiasis surgery. Only three LGAs had household-level improved wash water access of greater than 80%. Access to improved sanitation facilities was also poor as household access to improved sanitation facilities was above 80% in only one LGA. A trachoma program focused on delivery of trichiasis surgery is required in Gombe. Improvements in water and sanitation, through engagement with other sectors, are necessary.


Chinese translation norms for 1,429 English words.
We present Chinese translation norms for 1,429 English words. Chinese-English bilinguals (N = 28) were asked to provide the first Chinese translation that came to mind for 1,429 English words. The results revealed that 71 % of the English words received more than one correct translation indicating the large amount of translation ambiguity when translating from English to Chinese. The relationship between translation ambiguity and word frequency, concreteness and language proficiency was investigated. Although the significant correlations were not strong, results revealed that English word frequency was positively correlated with the number of alternative translations, whereas English word concreteness was negatively correlated with the number of translations. Importantly, regression analyses showed that the number of Chinese translations was predicted by word frequency and concreteness. Furthermore, an interaction between these predictors revealed that the number of translations was more affected by word frequency for more concrete words than for less concrete words. In addition, mixed-effects modelling showed that word frequency, concreteness and English language proficiency were all significant predictors of whether or not a dominant translation was provided. Finally, correlations between the word frequencies of English words and their Chinese dominant translations were higher for translation-unambiguous pairs than for translation-ambiguous pairs. The translation norms are made available in a database together with lexical information about the words, which will be a useful resource for researchers investigating Chinese-English bilingual language processing.


Inhibition of miR-302 Suppresses Hypoxia-Reoxygenation-Induced H9c2 Cardiomyocyte Death by Regulating Mcl-1 Expression.
MicroRNAs play important roles in cell proliferation, differentiation, and apoptosis, and their expression influences cardiomyocyte apoptosis resulting from ischemia-induced myocardial infarction. Here, we determined the role of miR expression in cardiomyocyte apoptosis during hypoxia and reoxygenation. The rat cardiomyocyte cell line H9c2 was incubated for 3 h in normal or hypoxia medium, followed by reoxygenation for 24 h and transfection with a miR-302 mimic or antagomir. The effect of miR-302 on myeloid leukemia cell-differentiation protein-1 (Mcl-1) expression was determined by western blot, real-time polymerase chain reaction, and luciferase reporter assays, with cell viability assays. We observed that miR-302 expression was elevated by hypoxia/reoxygenation injury and increased further or decreased by transfection of the miR-302 mimic or miR-302 antagomir, respectively. Additionally, elevated miR-302 levels increased apoptosis-related protein levels and cardiomyocyte apoptosis, and luciferase reporter assays revealed miR-302 binding to the Mcl-1 mRNA 3' untranslated region. Our findings suggested that miR-302 overexpression aggravated hypoxia/reoxygenation-mediated cardiomyocyte apoptosis by inhibiting antiapoptotic Mcl-1 expression, thereby activating proapoptotic molecules. Furthermore, results indicating cardiomyocyte rescue from hypoxia/reoxygenation injury following treatment with miR-302 antagomir suggested that miR-302 inhibition might constitute a therapeutic strategy for protection against cardiomyocyte apoptosis during hypoxia/reoxygenation injury.


Cell population kinetics of collagen scaffolds in ex vivo oral wound repair.
Biodegradable collagen scaffolds are used clinically for oral soft tissue augmentation to support wound healing. This study sought to provide a novel ex vivo model for analyzing healing kinetics and gene expression of primary human gingival fibroblasts (hGF) within collagen scaffolds. Sponge type and gel type scaffolds with and without platelet-derived growth factor-BB (PDGF) were assessed in an hGF containing matrix. Morphology was evaluated with scanning electron microscopy, and hGF metabolic activity using MTT. We quantitated the population kinetics within the scaffolds based on cell density and distance from the scaffold border of DiI-labled hGFs over a two-week observation period. Gene expression was evaluated with gene array and qPCR. The sponge type scaffolds showed a porous morphology. Absolute cell number and distance was higher in sponge type scaffolds when compared to gel type scaffolds, in particular during the first week of observation. PDGF incorporated scaffolds increased cell numbers, distance, and formazan formation in the MTT assay. Gene expression dynamics revealed the induction of key genes associated with the generation of oral tissue. DKK1, CYR61, CTGF, TGFBR1 levels were increased and integrin ITGA2 levels were decreased in the sponge type scaffolds compared to the gel type scaffold. The results suggest that this novel model of oral wound healing provides insights into population kinetics and gene expression dynamics of biodegradable scaffolds.


Resident work hours: what they are really doing.
We attempted to better quantitate resident work within our system of care. Survey. Academic training program. Surgical residents. A work-hour survey was developed defining 5 areas of activity: patient care related to educational objectives, required educational activities, patient care activities unrelated to educational objectives, off-duty educational activity, and off-duty hours. Total work hours and noneducational work hours were analyzed by resident level, rotation, and category. The survey response rate was 52%, covering 110 workweeks. Residents worked 80 hours or less for 57 weeks and more than 80 hours for 53 weeks. The mean number of hours worked was 77. Fewer than one quarter (21.9%) of work hours were unrelated to educational activities. The amount of time spent in noneducational activities was lowest at community hospitals (17%) and similar at the Veterans Affairs (23%) and academic (22%) medical centers. It did not vary by total hours worked, averaging 21% for rotations of more than 80 h/wk and 23% for rotations of 80 h/wk or less. Residents spend a large amount of time in noneducational activities. Eliminating these activities would bring our rotations into compliance with the 80-hour workweek. It would also generate a large amount of time for educational activities within our training program.


Phosphate alkylation in different DNA substrates: the role of local DNA sequence and electrophile character in determining the nonrandom nature of phosphotriester adduct formation.
DNA phosphate oxygens are sites for alkylation leading to DNA phosphotriester adduct (PTE) formation. Previously, we have reported that the manifestation of PTEs was nonrandom in mouse liver DNA treated in vivo [Guichard et al. (2000) Cancer Res. 60, 1276-1282], and while further studies revealed possible PTE repair, this was determined not to play a role in the observed nonrandom manifestation in vivo [Le Pla et al. (2004) Chem. Res. Toxicol. 17, 1491-1500]. In the present study, to determine whether the nonrandom manifestation of PTEs in vivo was specifically due to their nonrandom formation, we have compared the in vitro formation of diethylsulfate (DES)-induced PTEs in h2E1/OR human B-lymphoblastoid cells, their isolated nuclei, and their isolated DNA, using the 5' nearest neighbor analysis postlabeling procedure developed by Le Pla et al.. Furthermore, to determine the role of electrophile character in PTE manifestation, prepared oligonucleotides ([dT](20)[dG](20):[dC](20)[dA](20)) were treated with three alkylating agents of differing electrophilic character (DES, methylnitrosourea, and ethylnitrosourea), and PTE manifestation was assessed by postlabeling. The formation of PTEs was determined to be nonrandom in the whole cells, nuclei, and DNA, with PTEs being formed to a greater extent 3' to pyrimidine moieties than 3' to purine moieties. The studies with the oligonucleotides confirm these observations and demonstrate that the nonrandom formation of PTEs is primarily determined by DNA sequence, and not by DNA packaging/chromatin factors, and that the extent of the nonrandom formation of PTEs is also governed by electrophile reactivity, with the more reactive electrophiles yielding a more random formation of PTEs. From our observations, we propose a model for the nonrandom formation of PTEs, which is governed by (i) the phosphate oxygens having to compete with adjacent nucleophilic sites for the alkylating electrophile and (ii) the electrophile's inherent reactivity.


Autoantibodies against triosephosphate isomerase. A possible clue to pathogenesis of hemolytic anemia in infectious mononucleosis.
In sera from patients with acute EBV, infection and the clinical symptoms of infectious mononucleosis antibodies of the Ig class M were found that are directed against two cellular proteins. The molecular mass of these proteins was determined to be 29 (p29) and 26 kD (p26), respectively, in SDS-PAGE. P29 was identified as part of the glycolytic enzyme triosephosphate isomerase (TPI) by comparison of the NH2-terminal amino acid sequences. A purified antibody against TPI induces a 51Cr release from human erythrocytes. Possibly, anti-TPI causes hemolysis, which is an infrequent but serious symptom of infectious mononucleosis.


Transcriptome analysis of IL-10-stimulated (M2c) macrophages by next-generation sequencing.
Alternatively activated "M2" macrophages are believed to function during late stages of wound healing, behaving in an anti-inflammatory manner to mediate the resolution of the pro-inflammatory response caused by "M1" macrophages. However, the differences between two main subtypes of M2 macrophages, namely interleukin-4 (IL-4)-stimulated "M2a" macrophages and IL-10-stimulated "M2c" macrophages, are not well understood. M2a macrophages are characterized by their ability to inhibit inflammation and contribute to the stabilization of angiogenesis. However, the role and temporal profile of M2c macrophages in wound healing are not known. Therefore, we performed next generation sequencing (RNA-seq) to identify biological functions and gene expression signatures of macrophages polarized in vitro with IL-10 to the M2c phenotype in comparison to M1 and M2a macrophages and an unactivated control (M0). We then explored the expression of these gene signatures in a publicly available data set of human wound healing. RNA-seq analysis showed that hundreds of genes were upregulated in M2c macrophages compared to the M0 control, with thousands of alternative splicing events. Following validation by Nanostring, 39 genes were found to be upregulated by M2c macrophages compared to the M0 control, and 17 genes were significantly upregulated relative to the M0, M1, and M2a phenotypes (using an adjusted p-value cutoff of 0.05 and fold change cutoff of 1.5). Many of the identified M2c-specific genes are associated with angiogenesis, matrix remodeling, and phagocytosis, including CD163, MMP8, TIMP1, VCAN, SERPINA1, MARCO, PLOD2, PCOCLE2 and F5. Analysis of the macrophage-conditioned media for secretion of matrix-remodeling proteins showed that M2c macrophages secreted higher levels of MMP7, MMP8, and TIMP1 compared to the other phenotypes. Interestingly, temporal gene expression analysis of a publicly available microarray data set of human wound healing showed that M2c-related genes were upregulated at early times after injury, similar to M1-related genes, while M2a-related genes appeared at later stages or were downregulated after injury. While further studies are required to confirm the timing and role of M2c macrophages in vivo, these results suggest that M2c macrophages may function at early stages of wound healing. Identification of markers of the M2c phenotype will allow more detailed investigations into the role of M2c macrophages in vivo.


Disorders of B cells and helper T cells in the pathogenesis of the immunoglobulin deficiency of patients with ataxia telangiectasia.
The pathogenesis of the immunoglobulin deficiency of 20 patients with ataxia telangiectasia was studied using an in vitro immunoglobulin biosynthesis system. 10 patients had no detectable IgA in their serum as assessed by radial diffusion in agar and 3 had a reduced serum IgA concentration. The peripheral blood mononuclear cells of 17 of the patients and 17 normal controls were cultured with pokeweed mitogen for 12 d and the immunoglobulin in the supernatants measured. The immunoglobulin synthesis was below the lower limit of the normal 95% confidence interval for IgM in 5 patients, for IgG in 8, and for IgA in 14. The mononuclear cells from 9 of the 10 patients with a serum IgA concentration less than 0.1 mg/ml failed to synthesize IgA in vitro. None of the patients manifested excessive suppressor cell activity. All patients had reduced but measurable helper T cell activity for immunoglobulin synthesis by co-cultured normal pokeweed mitogen-stimulated B cells (geometric mean 22% of normal). Furthermore, the addition of normal irradiated T cells to patient peripheral blood mononuclear cells led to an augmentation of IgM synthesis in 15 of 17 and to increased IgG synthesis in 9 of the 17 patients studied, including 9 of the 12 patients who had synthesized IgG before the addition of the irradiated T cells. In addition, IgA synthesis was increased in all eight patients examined that had serum IgA concentrations greater than 0.1 mg/ml. These studies suggest that a helper T cell defect contributes to the diminished immunoglobulin synthesis. However, a helper T cell defect does not appear to be the sole cause since there was no IgA synthesis by the peripheral blood mononuclear cells of 9 of the 10 patients with a profoundly reduced serum IgA even when co-cultured with normal T cells. Furthermore, the cells of the nine patients with profoundly reduced IgA levels examined also failed to produce IgA when stimulated with the relatively helper T cell-independent polyclonal activators, Nocardia water soluble mitogen or Epstein-Barr virus. Taken together these data support the view that the reduced immunoglobulin synthesis of these patients is due to defects of both B cells and helper T cells. Such a broad defect in lymphocyte maturation taken in conjunction with our demonstration of persistent alpha fetoprotein production by ataxia telangiectasia patients provides support for the proposal that these patients exhibit a generalized defect in tissue differentiation.


Physical Activity and Anger or Emotional Upset as Triggers of Acute Myocardial Infarction: The INTERHEART Study.
Physical exertion, anger, and emotional upset are reported to trigger acute myocardial infarction (AMI). In the INTERHEART study, we explored the triggering association of acute physical activity and anger or emotional upset with AMI to quantify the importance of these potential triggers in a large, international population. INTERHEART was a case-control study of first AMI in 52 countries. In this analysis, we included only cases of AMI and used a case-crossover approach to estimate odds ratios for AMI occurring within 1 hour of triggers. Of 12 461 cases of AMI 13.6% (n=1650) engaged in physical activity and 14.4% (n=1752) were angry or emotionally upset in the case period (1 hour before symptom onset). Physical activity in the case period was associated with increased odds of AMI (odds ratio, 2.31; 99% confidence interval [CI], 1.96-2.72) with a population-attributable risk of 7.7% (99% CI, 6.3-8.8). Anger or emotional upset in the case period was associated with an increased odds of AMI (odds ratio, 2.44; 99% CI, 2.06-2.89) with a population-attributable risk of 8.5% (99% CI, 7.0-9.6). There was no effect modification by geographical region, prior cardiovascular disease, cardiovascular risk factor burden, cardiovascular prevention medications, or time of day or day of onset of AMI. Both physical activity and anger or emotional upset in the case period were associated with a further increase in the odds of AMI (odds ratio, 3.05; 99% CI, 2.29-4.07; P for interaction <0.001). Physical exertion and anger or emotional upset are triggers associated with first AMI in all regions of the world, in men and women, and in all age groups, with no significant effect modifiers.