Cryptochromes (CRYs) are flavoproteins important for the molecular clocks of animals. The Drosophila cryptochrome (dCRY) is a circadian photoreceptor, whereas mouse cryptochromes (mCRY1 and mCRY2) are essential negative elements of circadian clock transcriptional feedback loops. It has been proposed that reduction/oxidation (redox) reactions are important for dCRY light responsiveness and mCRY1 transcriptional inhibition. We therefore evaluated the role of redox in light-dependent activation of dCRY and in mCRY1 transcriptional inhibition in Drosophila Schneider 2 cells. Using site-directed mutagenesis, three of the four conserved flavin binding residues in dCRY were found to be essential for light responses, whereas three of the four corresponding residues in mCRY1 did not abolish transcriptional responses. Two tryptophan residues in dCRY are critical for its function and are likely involved in an intramolecular redox reaction. The corresponding tryptophan residues do not play a redox-mediated role in mCRY1 function. The data provide a multistep redox model for the light-dependent activities of dCRY and suggest that such a model does not apply to mCRY1 transcriptional responses.
The molecular dissection of the mammalian circadian clock continues to yield exciting and important discoveries. Here we review the recent progress in the field, highlighting work on the central clock mechanism itself, the photopigments involved in the entrainment of the central clock by light, and the signals that entrain peripheral oscillators.
Research Participation Decision-Making Among Youth and Parents of Youth With Chronic Health Conditions
The aims of this qualitative descriptive study were to describe how past experiences with research (including communication, information, values, and support) may contribute to research fatigue among youth and parents of youth with HIV, cystic fibrosis, and Type 1 diabetes. Eighteen parents and youth were purposively recruited from outpatient subspecialty clinics at a major academic medical center. They took part in qualitative interviews and completed a demographics form and the Decisional Conflict Scale. Youth participants also completed the Erikson Psychosocial Stage Inventory. Two major themes emerged: Blurred Lines and Hope for the Future. Research fatigue was not found in this sample. Results point to challenges with informed consent in settings where research and clinical care are integrated and suggest that protective factors allow for continued participation without excess burden on youth and parents. Strategies to minimize research fatigue and support engagement in research are offered.
Single-Cell Transcriptional Analysis Reveals Novel Neuronal Phenotypes and Interaction Networks Involved in the Central Circadian Clock
Single-cell heterogeneity confounds efforts to understand how a population of cells organizes into cellular networks that underlie tissue-level function. This complexity is prominent in the mammalian suprachiasmatic nucleus (SCN). Here, individual neurons exhibit a remarkable amount of asynchronous behavior and transcriptional heterogeneity. However, SCN neurons are able to generate precisely coordinated synaptic and molecular outputs that synchronize the body to a common circadian cycle by organizing into cellular networks. To understand this emergent cellular network property, it is important to reconcile single-neuron heterogeneity with network organization. In light of recent studies suggesting that transcriptionally heterogeneous cells organize into distinct cellular phenotypes, we characterized the transcriptional, spatial, and functional organization of 352 SCN neurons from mice experiencing phase-shifts in their circadian cycle. Using the community structure detection method and multivariate analytical techniques, we identified previously undescribed neuronal phenotypes that are likely to participate in regulatory networks with known SCN cell types. Based on the newly discovered neuronal phenotypes, we developed a data-driven neuronal network structure in which multiple cell types interact through known synaptic and paracrine signaling mechanisms. These results provide a basis from which to interpret the functional variability of SCN neurons and describe methodologies toward understanding how a population of heterogeneous single cells organizes into cellular networks that underlie tissue-level function.
Assessing the Effectiveness of an Evidence-based Practice Pharmacology Course Using the Fresno Test.
Objective. To assess the effectiveness of an evidence-based practice (EBP) pharmacology elective course to teach EBP skills using the Fresno Test (FT).
Methods. Pharmacy faculty members and medical librarians developed the elective course and offered it to two cohorts of doctor of pharmacy (PharmD) students. A pre/post intervention study design was used. Seven of 12 FT items were chosen to measure specific EBP skills: Ask, Access, Appraise and Apply. Pre/postcomposite and FT item mean scores were compared using Student's t test with p < 0.05 set as significant a priori.
Results. Composite FT mean scores increased significantly for both cohorts. Mean scores for both cohorts increased significantly in four of the seven FT items but on different FT items.
Conclusion. As a profession that commonly uses evidence-based guidelines, developing and integrating an EBP course in the PharmD curriculum is worth considering.
The circadian clock generates daily cycles of gene expression that regulate physiological processes. The liver plays an important role in xenobiotic metabolism and also has been shown to possess its own cell-based clock. The liver clock is synchronized by the master clock in the brain, and a portion of rhythmic gene expression can be driven by behavior of the organism as a whole even when the hepatic clock is suppressed. So far, however, there is relatively little evidence indicating whether the liver clock is functionally important in modulating xenobiotic metabolism. Thus, mice lacking circadian clock function in the whole body or specifically in liver were challenged with pentobarbital and acetaminophen, and pentobarbital sleep time (PBST) and acetaminophen toxicity, respectively, was assessed at different times of day in mutant and control mice. The results suggest that the liver clock is essential for rhythmic changes in xenobiotic detoxification. Surprisingly, it seems that the way in which the clock is disrupted determines the rate of xenobiotic metabolism in the liver. CLOCK-deficient mice are remarkably resistant to acetaminophen and exhibit a longer PBST, while PERIOD-deficient mice have a short PBST. These results indicate an essential role of the tissue-intrinsic peripheral circadian oscillator in the liver in regulating xenobiotic metabolism.
Brain aging is associated with diminished circadian clock output and decreased expression of the core clock proteins, which regulate many aspects of cellular biochemistry and metabolism. The genes encoding clock proteins are expressed throughout the brain, though it is unknown whether these proteins modulate brain homeostasis. We observed that deletion of circadian clock transcriptional activators aryl hydrocarbon receptor nuclear translocator-like (Bmal1) alone, or circadian locomotor output cycles kaput (Clock) in combination with neuronal PAS domain protein 2 (Npas2), induced severe age-dependent astrogliosis in the cortex and hippocampus. Mice lacking the clock gene repressors period circadian clock 1 (Per1) and period circadian clock 2 (Per2) had no observed astrogliosis. Bmal1 deletion caused the degeneration of synaptic terminals and impaired cortical functional connectivity, as well as neuronal oxidative damage and impaired expression of several redox defense genes. Targeted deletion of Bmal1 in neurons and glia caused similar neuropathology, despite the retention of intact circadian behavioral and sleep-wake rhythms. Reduction of Bmal1 expression promoted neuronal death in primary cultures and in mice treated with a chemical inducer of oxidative injury and striatal neurodegeneration. Our findings indicate that BMAL1 in a complex with CLOCK or NPAS2 regulates cerebral redox homeostasis and connects impaired clock gene function to neurodegeneration.
Objective: To understand the experience of the informationist recipients of NLM-funded Administrative Supplements for Informationist Services and gather evidence for their impact on NIH-funded biomedical research
Methods: A mixed methods approach consisting of a survey of principal investigators and a focus group of informationists.
Results: Informationists appeared to have a positive impact on their team’s research, especially in the areas of data storage, data management planning, data organization, and literature searching. In addition, many informationists felt that their involvement increased their research skills and made them true research partners. Assessing their own impact was a challenge for the award recipients, and questions remain about the best evaluation methods. The overall experience of the informationists and researchers was mixed but largely positive.
Conclusion: The NLM-funded informationist supplement award appears to be a successful mechanism for immersing informationists into research teams and improving data management in the supported projects.
As librarians move into new roles, such as research data management, they sometimes find themselves moving, both organizationally and physically, out of the library. While this dislocation can be professionally unsettling, the shift in perspective that it brings can heighten their and their colleagues’ appreciation for the particular skills and talents that librarians can bring to the research enterprise.
Metagenomic Sequencing with Strain-Level Resolution Implicates Uropathogenic E. coli in Necrotizing Enterocolitis and Mortality in Preterm Infants
Necrotizing enterocolitis (NEC) afflicts approximately 10% of extremely preterm infants with high fatality. Inappropriate bacterial colonization with Enterobacteriaceae is implicated, but no specific pathogen has been identified. We identify uropathogenic E. coli (UPEC) colonization as a significant risk factor for the development of NEC and subsequent mortality. We describe a large-scale deep shotgun metagenomic sequence analysis of the early intestinal microbiome of 144 preterm and 22 term infants. Using a pan-genomic approach to functionally subtype the E. coli, we identify genes associated with NEC and mortality that indicate colonization by UPEC. Metagenomic multilocus sequence typing analysis further defined NEC-associated strains as sequence types often associated with urinary tract infections, including ST69, ST73, ST95, ST127, ST131, and ST144. Although other factors associated with prematurity may also contribute, this report suggests a link between UPEC and NEC and indicates that further attention to these sequence types as potential causal agents is needed.
Expansion of the Knockdown Resistance Frequency Map for Human Head Lice (Phthiraptera: Pediculidae) in the United States Using Quantitative Sequencing
Pediculosis is a prevalent parasitic infestation of humans, which is increasing due, in part, to the selection of lice resistant to either the pyrethrins or pyrethroid insecticides by the knockdown resistance (kdr) mechanism. To determine the extent and magnitude of thekdr-type mutations responsible for this resistance, lice were collected from 138 collection sites in 48 U.S. states from 22 July 2013 to 11 May 2015 and analyzed by quantitative sequencing. Previously published data were used for comparisons of the changes in the frequency of thekdr-type mutations over time. Mean percent resistance allele frequency (mean % RAF) values across the three mutation loci were determined from each collection site. The overall mean % RAF (+/-SD) for all analyzed lice was 98.3 +/- 10%. 132/138 sites (95.6%) had a mean % RAF of 100%, five sites (3.7%) had intermediate values, and only a single site had no mutations (0.0%). Forty-two states (88%) had a mean % RAF of 100%. The frequencies ofkdr-type mutations did not differ regardless of the human population size that the lice were collected from, indicating a uniformly high level of resistant alleles. The loss of efficacy of the Nix formulation (Prestige Brand, Tarrytown, NY) from 1998 to 2013 was correlated to the increase inkdr-type mutations. These data provide a plausible reason for the decrease in the effectiveness of permethrin in the Nix formulation, which is the parallel increase ofkdr-type mutations in lice over time.
The Combined Deficiency of Immunoproteasome Subunits Affects Both the Magnitude and Quality of Pathogen- and Genetic Vaccination-Induced CD8+ T Cell Responses to the Human Protozoan Parasite Trypanosoma cruzi
The beta1i, beta2i and beta5i immunoproteasome subunits have an important role in defining the repertoire of MHC class I-restricted epitopes. However, the impact of combined deficiency of the three immunoproteasome subunits in the development of protective immunity to intracellular pathogens has not been investigated. Here, we demonstrate that immunoproteasomes play a key role in host resistance and genetic vaccination-induced protection against the human pathogen Trypanosoma cruzi (the causative agent of Chagas disease), immunity to which is dependent on CD8+ T cells and IFN-gamma (the classical immunoproteasome inducer). We observed that infection with T. cruzi triggers the transcription of immunoproteasome genes, both in mice and humans. Importantly, genetically vaccinated or T. cruzi-infected beta1i, beta2i and beta5i triple knockout (TKO) mice presented significantly lower frequencies and numbers of splenic CD8+ effector T cells (CD8+CD44highCD62Llow) specific for the previously characterized immunodominant (VNHRFTLV) H-2Kb-restricted T. cruzi epitope. Not only the quantity, but also the quality of parasite-specific CD8+ T cell responses was altered in TKO mice. Hence, the frequency of double-positive (IFN-gamma+/TNF+) or single-positive (IFN-gamma+) cells specific for the H-2Kb-restricted immunodominant as well as subdominant T. cruzi epitopes were higher in WT mice, whereas TNF single-positive cells prevailed among CD8+ T cells from TKO mice. Contrasting with their WT counterparts, TKO animals were also lethally susceptible to T. cruzi challenge, even after an otherwise protective vaccination with DNA and adenoviral vectors. We conclude that the immunoproteasome subunits are key determinants in host resistance to T. cruzi infection by influencing both the magnitude and quality of CD8+ T cell responses.
BACKGROUND/AIM: Unilateral choroidal infiltration as the initial manifestation of leukemic relapse in adults is rare, particularly after an extended period of remission. This report describes this unique ophthalmic presentation, highlights the associated diagnostic challenges, and reviews the literature.
METHODS: Two cases are described and an extensive literature review was conducted.
RESULTS: A 59-year-old male with acute lymphoid leukemia, in remission for 18 months, presented with unilateral scleritis, exudative retinal detachment, and choroidal thickening. A 57-year-old male with a history of acute myeloid leukemia, in remission for 4 years, presented with unilateral choroidal thickening leading to secondary angle closure. In both cases, there was a significant lag from the onset of eye symptoms to establishing a systemic diagnosis of acute leukemia, leading to a delay in definitive systemic treatment, despite a high suspicion of disease based on ophthalmic findings.
CONCLUSIONS: These two cases illustrate the fundus findings consistent with leukemic choroidal infiltration that can represent the first sign of relapsed leukemia. The successful treatment of these patients hinges on collaboration between ophthalmologists and oncologists to optimize patient outcomes, highlighting the need for both groups to be aware of this rare ophthalmic presentation.
Microbial sphingomyelinase induces RhoA-mediated reorganization of the apical brush border membrane and is protective against invasion
The apical brush border membrane (BBM) of intestinal epithelial cells forms a highly structured and dynamic environmental interface that serves to regulate cellular physiology and block invasion by intestinal microbes and their products. How the BBM dynamically responds to pathogenic and commensal bacterial signals can define intestinal homeostasis and immune function. We previously found that in model intestinal epithelium, the conversion of apical membrane sphingomyelin to ceramide by exogenous bacterial sphingomyelinase (SMase) protected against the endocytosis and toxicity of cholera toxin. Here we elucidate a mechanism of action by showing that SMase induces a dramatic, reversible, RhoA-dependent alteration of the apical cortical F-actin network. Accumulation of apical membrane ceramide is necessary and sufficient to induce the actin phenotype, and this coincides with altered membrane structure and augmented innate immune function as evidenced by resistance to invasion by Salmonella.
We report a case of a 75-year-old female with a history of acute deep vein thrombosis (DVT) 6 years ago who presented with unilateral calf swelling and pain. D-dimer was normal, and compression ultrasound revealed findings typical of DVT, including an incompressible dilated and hypoechoic peroneal vein. Despite 4 months of anticoagulation for supposed recurrent DVT, pain symptoms persisted and repeat D-dimer and compression ultrasound were unchanged. A magnetic resonance imaging scan to investigate the leg demonstrated a 6-cm dissecting Baker's cyst extending posterolaterally resulting in venous compression and distal dilation, which appeared to have been confused with a DVT. Ultrasound-guided aspiration of the cyst provided immediate and sustained relief. Herein we provide a review of the literature for the management of this rare scenario.
Performance of rapid SOFIA Influenza A+B test compared to Luminex x-TAG respiratory viral panel assay in the diagnosis of influenza A, B, and subtype H3
Influenza is an acute respiratory illness caused by influenza A or B viruses that occur in outbreaks, mainly during the winter season. Rapid laboratory diagnosis of influenza can help guide the clinical management of suspected patients effectively. Clinical sensitivities and specificities of the rapid influenza diagnostic tests have varied considerably in the literature. Most of these studies are evaluated using previously frozen or stored specimens that had previously tested positive. This study compares the performance of the rapid SOFIA Influenza A+B test to nucleic acid multiplex test x-TAG respiratory viral panel (RVP) assay in freshly collected nasal aspirates and measured simultaneously by both assays. Retrospective data from 1649 nasal aspirates (September 2014 to May 2015) collected from adults as well as from children tested simultaneously by both rapid SOFIA Influenza A+B FIA immunofluorescence (Quidel, San Diego, CA) and qualitative nucleic acid multiplex RVP assay X-TAG Luminex technology (Luminex, Austin, Texas, USA) were analyzed. Concordance, and analytical sensitivity and specificity were evaluated for influenza A, subtypes H1 and H3, and influenza B. Prevalence for influenza A by RVP was 15%, for subtype H3 it was 11.2%, and for influenza B, 2.9%. None of the aspirates were positive for influenza A subtype H1. SOFIA Influenza rapid test demonstrated good specificity and low sensitivity compared with a nucleic acid test for influenza A, subtype H3, and for influenza B. SOFIA Influenza A + B test performed well in providing a rapid diagnosis, however, confirmatory molecular testing is recommended for negative test results. Re-evaluation of test performance should be periodically carried out during outbreaks with the emergence and circulation of new influenza strains.
BACKGROUND: Long-term incidence of endocrine and exocrine insufficiency after pancreatectomy is poorly described. We analyze the long-term risks of pancreatic insufficiency after pancreatectomy.
METHODS: Subjects who underwent pancreatectomy from 2002 to 2012 were identified from a prospective database (n = 227). Subjects who underwent total pancreatectomy or pancreatitis surgery were excluded. New post-operative endocrine and exocrine insufficiency was defined as the need for new pharmacologic intervention within 1000 days from resection.
RESULTS: 28 (16%) of 178 subjects without pre-existing endocrine insufficiency developed post-operative endocrine insufficiency: 7 (25%) did so within 30 days, 8 (29%) between 30 and 90 days, and 13 (46%) after 90 days. 94 (43%) of 214 subjects without pre-operative exocrine insufficiency developed exocrine insufficiency: 20 (21%) did so within 30 days, 29 (31%) between 30 and 90 days, and 45 (48%) after 90 days. Adjuvant radiation was associated with new endocrine insufficiency. On multivariate regression, pancreaticoduodenectomy and chemotherapy were associated with a greater risk of exocrine insufficiency.
CONCLUSION: Reporting 30-day functional outcomes for pancreatic resection is insufficient, as nearly 45% of subjects who develop disease do so after 90 days. Reporting of at least 90-day outcomes may more reliably assess risk for post-operative endocrine and exocrine insufficiency.
CA19-9 decrease at 8 weeks as a predictor of overall survival in a randomized phase III trial (MPACT) of weekly nab-paclitaxel plus gemcitabine versus gemcitabine alone in patients with metastatic pancreatic cancer
BACKGROUND: A phase I/II study and subsequent phase III study (MPACT) reported significant correlations between CA19-9 decreases and prolonged overall survival (OS) with nab-paclitaxel plus gemcitabine (nab-P + Gem) treatment for metastatic pancreatic cancer (MPC). CA19-9 changes at week 8 and potential associations with efficacy were investigated as part of an exploratory analysis in the MPACT trial.
PATIENTS AND METHODS: Untreated patients with MPC (N = 861) received nab-P + Gem or Gem alone. CA19-9 was evaluated at baseline and every 8 weeks.
RESULTS: Patients with baseline and week-8 CA19-9 measurements were analyzed (nab-P + Gem: 252; Gem: 202). In an analysis pooling the treatments, patients with any CA19-9 decline (80%) versus those without (20%) had improved OS (median 11.1 versus 8.0 months; P = 0.005). In the nab-P + Gem arm, patients with (n = 206) versus without (n = 46) any CA19-9 decrease at week 8 had a confirmed overall response rate (ORR) of 40% versus 13%, and a median OS of 13.2 versus 8.3 months (P = 0.001), respectively. In the Gem-alone arm, patients with (n = 159) versus without (n = 43) CA19-9 decrease at week 8 had a confirmed ORR of 15% versus 5%, and a median OS of 9.4 versus 7.1 months (P = 0.404), respectively. In the nab-P + Gem and Gem-alone arms, by week 8, 16% (40/252) and 6% (13/202) of patients, respectively, had an unconfirmed radiologic response (median OS 13.7 and 14.7 months, respectively), and 79% and 84% of patients, respectively, had stable disease (SD) (median OS 11.1 and 9 months, respectively). Patients with SD and any CA19-9 decrease (158/199 and 133/170) had a median OS of 13.2 and 9.4 months, respectively.
CONCLUSION: This analysis demonstrated that, in patients with MPC, any CA19-9 decrease at week 8 can be an early marker for chemotherapy efficacy, including in those patients with SD. CA19-9 decrease identified more patients with survival benefit than radiologic response by week 8.
Combining statistics from two national complex surveys to estimate injury rates per hour exposed and variance by activity in the USA
BACKGROUND: A common issue in descriptive injury epidemiology is that in order to calculate injury rates that account for the time spent in an activity, both injury cases and exposure time of specific activities need to be collected. In reality, few national surveys have this capacity. To address this issue, we combined statistics from two different national complex surveys as inputs for the numerator and denominator to estimate injury rate, accounting for the time spent in specific activities and included a procedure to estimate variance using the combined surveys.
METHODS: The 2010 National Health Interview Survey (NHIS) was used to quantify injuries, and the 2010 American Time Use Survey (ATUS) was used to quantify time of exposure to specific activities. The injury rate was estimated by dividing the average number of injuries (from NHIS) by average exposure hours (from ATUS), both measured for specific activities. The variance was calculated using the 'delta method', a general method for variance estimation with complex surveys.
RESULTS: Among the five types of injuries examined, 'sport and exercise' had the highest rate (12.64 injuries per 100 000 h), followed by 'working around house/yard' (6.14), driving/riding a motor vehicle (2.98), working (1.45) and sleeping/resting/eating/drinking (0.23). The results show a ranking of injury rate by activity quite different from estimates using population as the denominator.
CONCLUSIONS: Our approach produces an estimate of injury risk which includes activity exposure time and may more reliably reflect the underlying injury risks, offering an alternative method for injury surveillance and research.
Chimeric 2C10R4 anti-CD40 antibody therapy is critical for long-term survival of GTKO.hCD46.hTBM pig-to-primate cardiac xenograft
Preventing xenograft rejection is one of the greatest challenges of transplantation medicine. Here, we describe a reproducible, long-term survival of cardiac xenografts from alpha 1-3 galactosyltransferase gene knockout pigs, which express human complement regulatory protein CD46 and human thrombomodulin (GTKO.hCD46.hTBM), that were transplanted into baboons. Our immunomodulatory drug regimen includes induction with anti-thymocyte globulin and alphaCD20 antibody, followed by maintenance with mycophenolate mofetil and an intensively dosed alphaCD40 (2C10R4) antibody. Median (298 days) and longest (945 days) graft survival in five consecutive recipients using this regimen is significantly prolonged over our recently established survival benchmarks (180 and 500 days, respectively). Remarkably, the reduction of alphaCD40 antibody dose on day 100 or after 1 year resulted in recrudescence of anti-pig antibody and graft failure. In conclusion, genetic modifications (GTKO.hCD46.hTBM) combined with the treatment regimen tested here consistently prevent humoral rejection and systemic coagulation pathway dysregulation, sustaining long-term cardiac xenograft survival beyond 900 days.