Placing the keystone: the LIMBIC Military and Tactical Athlete Research Study.

OBJECTIVE: The LIMBIC Military and Tactical Athletic Research Study (MATARS) framework was established to confirm and extend understanding of concussion with initial studies driven by clinical data collected between 2015 and 2020 in a collegiate sports setting. The LIMBIC MATARS framework will be leveraged to apply gold-standard and innovative research designs to advance the science of concussion. This manuscript provides the background, methodology, and initial demographic data associated with the LIMBIC MATARS. METHODS: Consensus-based common data elements were used to conduct a retrospective chart review, specific to collegiate athletes diagnosed with concussions between 2015 and 2020 at 11 universities. RESULTS: A final sample of 1,311 (47.8% female) concussions were diagnosed during the five-year study period from athletes participating in a variety of National Collegiate Athlete Association (NCAA) sports. The LIMBIC MATARS demographic data, align with the NCAA and other pioneering multi-site concussion-related studies in terms of biological sex, race and ethnicity, and sport participation. CONCLUSION: This pragmatic, methodological approach was used to address several a priori hypotheses related to concussion, align with other multi-site studies of concussion, and establish a consortium for future investigations.

Lost in translation: the association of international status and native language on concussion in collegiate athletes in the United States.

OBJECTIVE: The purpose of this study was to characterize the associations of international student status and native language on time (in days) with the date of injury to (i) diagnosis, (ii) symptom resolution, and (iii) return to sport. METHODS: Utilizing data from a cross-sectional cohort of 1,044 concussion cases from LIMBIC MATARS member institutions (n = 11) in the US, we conducted two, matched case-control designs. Cases were divided into two groups: (i) international (n = 32) or domestic students (n = 32) and (ii) English as an Additional Language (EAL) speakers (n = 18) or Native English language speakers (n = 18). Both groups were individually matched to their respective controls based on gender, age, sport, and preexisting health conditions. RESULTS: There were no significant differences in days from injury to diagnosis (p = 0.94), symptom resolution (p = 0.64), or return to sport (p = 0.15) between international and domestic athletes. EAL speakers experienced symptom resolution approximately 7.5 days sooner (Md = 4.50; IQR = 4.00, 8.00) than Native English language speakers (Md = 12.00; IQR = 7.00, 21.00, p = 0.01). CONCLUSIONS: Our findings suggest that native language is associated with symptom resolution in collegiate athletes. Healthcare professionals should consider barriers related to native language that may impact symptom reporting and the overall injury experience of diverse collegiate athletes.

Racial identity and concussion diagnosis and recovery trajectories in collegiate athletes: a LIMBIC MATARS investigation.

OBJECTIVE: To determine if there were concussion diagnosis and recovery disparities between collegiate athletes with Black and White racial identities. DESIGN: Retrospective cohort study. METHODS: Concussion information was extracted from NCAA athlete medical files at LIMBIC MATARS member institutions from the 2015-16' to 2019-20' academic years. A total of 410 concussions from 9 institutions were included that provided all independent (i.e. racial identity of Black or White) and dependent variable information (i.e. dates of injury, diagnosis, symptom resolution, and return to sport) that were analyzed using Mann-Whitney U tests. The sample consisted of 114 (27.8%) concussions sustained by Black athletes and 296 (72.1%) sustained by White athletes. RESULTS: The overall sample had a median of 0 days between injury occurrence to diagnosis, 7 days to symptom resolution, and 12 days to return to sport. No significant timing differences were observed for concussion diagnosis (p = .14), symptom resolution (p = .39), or return to sport (p = 0.58) between collegiate athletes with Black versus White racial identities. CONCLUSIONS: These findings may reflect equitable access to onsite sports medicine healthcare resources that facilitate concussion management in the collegiate sport setting. Future work should explore these associations with a larger and more diverse sample of collegiate athletes.

The 2020 International Alliance for the Control of Scabies Consensus Criteria for the Diagnosis of Scabies.

BACKGROUND: Scabies is a common parasitic skin condition that causes considerable morbidity globally. Clinical and epidemiological research for scabies has been limited by a lack of standardization of diagnostic methods. OBJECTIVES: To develop consensus criteria for the diagnosis of common scabies that could be implemented in a variety of settings. METHODS: Consensus diagnostic criteria were developed through a Delphi study with international experts. Detailed recommendations were collected from the expert panel to define the criteria features and guide their implementation. These comments were then combined with a comprehensive review of the available literature and the opinion of an expanded group of international experts to develop detailed, evidence-based definitions and diagnostic methods. RESULTS: The 2020 International Alliance for the Control of Scabies (IACS) Consensus Criteria for the Diagnosis of Scabies include three levels of diagnostic certainty and eight subcategories. Confirmed scabies (level A) requires direct visualization of the mite or its products. Clinical scabies (level B) and suspected scabies (level C) rely on clinical assessment of signs and symptoms. Evidence-based, consensus methods for microscopy, visualization and clinical symptoms and signs were developed, along with a media library. CONCLUSIONS: The 2020 IACS Criteria represent a pragmatic yet robust set of diagnostic features and methods. The criteria may be implemented in a range of research, public health and clinical settings by selecting the appropriate diagnostic levels and subcategories. These criteria may provide greater consistency and standardization for scabies diagnosis. Validation studies, development of training materials and development of survey methods are now required. What is already known about this topic? The diagnosis of scabies is limited by the lack of accurate, objective tests. Microscopy of skin scrapings can confirm the diagnosis, but it is insensitive, invasive and often impractical. Diagnosis usually relies on clinical assessment, although visualization using dermoscopy is becoming increasingly common. These diagnostic methods have not been standardized, hampering the interpretation of findings from clinical research and epidemiological surveys, and the development of scabies control strategies. What does this study add? International consensus diagnostic criteria for common scabies were developed through a Delphi study with global experts. The 2020 International Alliance for the Control of Scabies (IACS) Criteria categorize diagnosis at three levels of diagnostic certainty (confirmed, clinical and suspected scabies) and eight subcategories, and can be adapted to a range of research and public health settings. Detailed definitions and figures are included to aid training and implementation. The 2020 IACS Criteria may facilitate the standardization of scabies diagnosis.

Infliximab is associated with an increased risk of serious infection in patients with psoriasis in the U.K. and Republic of Ireland: results from the British Association of Dermatologists Biologic Interventions Register (BADBIR).

BACKGROUND: Patients with psoriasis and clinicians are concerned that infliximab may be associated with a risk of serious infections. OBJECTIVES: To compare the risk of serious infections associated with infliximab in patients with chronic plaque psoriasis against a cohort on nonbiologic systemic therapies. METHODS: A prospective cohort study was performed using data from the British Association of Dermatologists Biologic Interventions Register (BADBIR). Infliximab was compared with nonbiologic systemic therapies, inclusive of any exposure to methotrexate, ciclosporin, acitretin, fumaric acid esters, psoralen-ultraviolet A or hydroxycarbamide. Serious infections were those associated with hospitalization, the use of intravenous antimicrobial therapy and/or those that led to death. Propensity score inverse probability treatment weights were used to adjust for potential confounding from a priori identified covariates. Cox proportional hazards models were calculated to obtain hazard ratios (HRs). RESULTS: In total, 3843 participants were included for analysis up to October 2016. The incidence rates were significantly higher in the infliximab cohort (47·8 per 1000 person-years) [95% confidence interval (CI) 35·7-64·0], compared with 14·2 per 1000 person-years (95% CI 11·5-17·4) in the nonbiologic systemic cohort. Infliximab was associated with an overall increase in the risk of serious infection compared with nonbiologics [adjusted HR (adjHR) 1·95, 95% CI 1·01-3·75] and methotrexate only (adjHR 2·96, 95% CI 1·58-5·57) and a higher risk of serious infection in the first 6 months of therapy (adjHR 3·49, 95% CI 1·14-10·70). CONCLUSIONS: Infliximab is associated with an increased risk of serious infections compared with nonbiologic systemic therapies in patients with psoriasis in the U.K. and the Republic of Ireland.

Vitrification of in vitro-derived bovine embryos: targeting enhancement of quality by refining technology and standardising procedures.

Bovine invitro fertilisation technology has been widely exploited in commercial settings. The majority of invitro-derived cattle embryos are transferred into recipient cows as recently collected (i.e. 'fresh') embryos due to the lack of a reliable cryopreservation method that results in favourable pregnancy rates following transfer of thawed embryos. This is a primary reason for the poor industry uptake of this extreme temperature freezing process. Numerous investigations into vitrification have revealed the importance of rapid cooling and warming rates, enhancing embryo viability after cryopreservation compared with conventional slow freezing. Those studies spawned a considerable assortment of cryovessels and diversity of procedures, delivering variable rates of success, which makes performing vitrification consistently a practical challenge. Hence, further research is required in order to both optimise and standardise vitrification methodology and to design a cryovessel that enables direct transfer of vitrified embryos to recipients after warming. In parallel with improvements in vitrification, it is important to continue to raise the quality of invitro-derived cattle embryos through modifications in laboratory culture techniques. The twin goals of methodology refinement and standardisation, leading to embryo quality enhancement, are each imperative if invitro fertilisation technology is to be adopted in the field.

Doxycycline compared with prednisolone therapy for patients with bullous pemphigoid: cost-effectiveness analysis of the BLISTER trial.

BACKGROUND: Bullous pemphigoid (BP) is an autoimmune blistering skin disorder associated with significant morbidity and mortality. Doxycycline and prednisolone to treat bullous pemphigoid were compared within a randomized controlled trial (RCT). OBJECTIVES: To compare the cost-effectiveness of doxycycline-initiated and prednisolone-initiated treatment for patients with BP. METHODS: Quality-of-life (EuroQoL-5D-3L) and resource data were collected as part of the BLISTER trial: a multicentre, parallel-group, investigator-blinded RCT. Within-trial analysis was performed using bivariate regression of costs and quality-adjusted life-years (QALYs), with multiple imputation of missing data, informing a probabilistic assessment of incremental treatment cost-effectiveness from a health service perspective. RESULTS: In the base case, there was no robust difference in costs or QALYs per patient at 1 year comparing doxycycline- with prednisolone-initiated therapy [net cost £959, 95% confidence interval (CI) -£24 to £1941; net QALYs -0·024, 95% CI -0·088 to 0·041]. However, the findings varied by baseline blister severity. For patients with mild or moderate blistering (≤ 30 blisters) net costs and outcomes were similar. For patients with severe blistering (> 30 blisters) net costs were higher (£2558, 95% CI -£82 to £5198) and quality of life poorer (-0·090 QALYs, 95% CI -0·22 to 0·042) for patients starting on doxycycline. The probability that doxycycline would be cost-effective for those with severe pemphigoid was 1·5% at a willingness to pay of £20 000 per QALY. CONCLUSIONS: Consistently with the clinical findings of the BLISTER trial, patients with mild or moderate blistering should receive treatment guided by the safety and effectiveness of the drugs and patient preference - neither strategy is clearly a preferred use of National Health Service resources. However, prednisolone-initiated treatment may be more cost-effective for patients with severe blistering.

Prospective study in bullous pemphigoid: association of high serum anti-BP180 IgG levels with increased mortality and reduced Karnofsky score.

BACKGROUND: Bullous pemphigoid (BP) is a subepidermal blistering disease characterized by autoantibodies against the two hemidesmosomal proteins, BP180 (type XVII collagen) and BP230. The multicentre prospective BLISTER (Bullous Pemphigoid Steroids and Tetracyclines) trial randomized 253 patients with BP to compare the benefits and harms between initial treatment with doxycycline or prednisolone. OBJECTIVES: To analyse distinct autoantibody profiles for the prediction of the disease course in a well-characterized cohort of BP sera. METHODS: One hundred and forty-three patients of the BLISTER trial consented to participate in this serological study. Sera taken at baseline were analysed by (i) indirect immunofluorescence, (ii) anti-BP180 NC16A (16th noncollagenous domain) and anti-BP230 enzyme-linked immunosorbent assay and (iii) immunoblotting with various substrates. Results were then linked with clinical parameters including age, Karnofsky score, number of blisters, related adverse events and mortality. RESULTS: Disease activity correlated with immunoglobulin (Ig)G anti-BP180 levels but not with levels of anti-BP230 IgG and anti-BP180 IgE. High levels of both anti-BP180 IgG and anti-BP230 IgG were associated with a low Karnofsky score. The presence of anti-BP230 IgG was more frequent in older patients. Those with higher total IgE serum levels suffered from fewer adverse events. Higher IgG anti-BP180 levels were associated with an increased 1-year mortality rate. CONCLUSIONS: Analysis of the autoantibody profile is not only of diagnostic relevance but may also be helpful in predicting the course of the disease.

Does the Addition of Salubrinal to in vitro Maturation Medium Enhance Bovine Blastocyst Yields and Embryo Cryotolerance?

BACKGROUND: Modifications to in vitro maturation (IVM) media are made to improve rates of blastocyst formation and quality of mammalian embryos. Embryo quality is an important factor in the viability of embryos following cryopreservation. Salubrinal is a specific inhibitor of endoplasmic reticulum stress-induced apoptosis in eukaryotic cells. Here, it was added to IVM medium in an attempt to increase blastocyst formation and to enhance embryo quality in cattle. OBJECTIVE: To assess the effect on blastocyst formation and cryotolerance of the supplementation of salubrinal to bovine IVM medium. MATERIALS AND METHODS: Cumulus-oocyte complexes (COCs) collected from slaughterhouse ovaries were assigned randomly to two groups, either cultured in IVM medium that was supplemented with 400 mM salubrinal (treated, 262 COCs) or that was not supplemented (control, 263 COCs). All oocytes of the matured COCs were fertilized in vitro with sperm from the same proven bull and cultured for 6-7 d. At the time of blastocyst collection, expanded blastocysts were chosen for cryopreservation, while early, hatching and hatched blastocysts and those of poor quality were not used. There were 83 expanded blastocysts classified to be of good quality in both the control and salubrinal-treated groups that were subjected to vitrification. After 5 to 10 months of cold storage, the embryos were warmed and cultured in vitro for 24 h to assess the survival rate and for 48 h to assess the hatching rate. RESULTS: The blastocyst developmental rate in the salubrinal-treated group was similar to that in the control group, 61.5% compared with 62.7% (P > 0.05). The survival rate of blastocysts after vitrification was also similar, at or very close to 100%. In addition, there was no statistically significant difference in the hatching rate of expanded blastocysts derived from the COCs cultured with (treated) and without (control) addition of salubrinal to the IVM medium (91.6% compared with 85.5%; P > 0.05). CONCLUSION: Supplementation of salubrinal to the IVM medium neither improved nor reduced rates of bovine blastocyst formation and of embryo cryotolerance as determined by post-warming viability.

Generating new evidence, improving clinical practice and developing research capacity: the benefits of recruiting to the U.K. Dermatology Clinical Trials Network's STOP GAP and BLISTER trials.

Clinical trials may benefit clinical practice in three ways: firstly, clinicians may change their practice according to the new trial evidence; secondly, clinical processes can improve when working on a trial; and thirdly, research capacity is increased. We held a meeting to present and discuss the results of two large multicentre randomized controlled trials delivered through the U.K. Dermatology Clinical Trials Network. Investigators gave reflections on how the trials had changed their clinical practice. The STOP GAP trial showed that prednisolone and ciclosporin are equally effective as first-line systemic treatment for pyoderma gangrenosum. The final decision of which treatment to use should be based on the different adverse event profiles of the two drugs in relation to comorbidities, along with age, disease severity and patient preference. The BLISTER trial showed that starting people with pemphigoid on doxycycline produces acceptable short-term effectiveness and a superior safety profile to oral corticosteroids. Recruiting to these trials has led to the development of new specialist clinics with improved documentation. It has increased the profile of participating departments and embedded research in the department's activities. Helping to design and run these trials has also allowed trial staff to develop new skills in research design, which has been beneficial for career development. These and other benefits of recruiting to the trials are summarized here. We hope that these reflections will inspire wider involvement in clinical research.

Use of Brevibacillus choshinensis for the production of biologically active brain-derived neurotrophic factor (BDNF).

Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family critical for neuronal cell survival and differentiation, with therapeutic potential for the treatment of neurological disorders and spinal cord injuries. The production of recombinant, bioactive BDNF is not practical in most traditional microbial expression systems because of the inability of the host to correctly form the characteristic cystine-knot fold of BDNF. Here, we investigated Brevibacillus choshinensis as a suitable expression host for bioactive BDNF expression, evaluating the effects of medium type (2SY and TM), temperature (25 and 30 °C), and culture time (48-120 h). Maximal BDNF bioactivity (per unit mass) was observed in cultures grown in 2SY medium at extended times (96 h at 30 °C or >72 h at 25 °C), with resulting bioactivity comparable to that of a commercially available BDNF. For cultures grown in 2SY medium at 25 °C for 72 h, the condition that led to the greatest quantity of biologically active protein in the shortest culture time, we recovered 264 µg/L of BDNF. As with other microbial expression systems, BDNF aggregates did form in all culture conditions, indicating that while we were able to recover biologically active BDNF, further optimization of the expression system could yield still greater quantities of bioactive protein. This study provides confirmation that B. choshinensis is capable of producing biologically active BDNF and that further optimization of culture conditions could prove valuable in increasing BDNF yields.

Immunization of looked-after children and young people: a review of the literature.

BACKGROUND: Numbers of looked-after children and young people (LACYP) in the UK have risen over the last seven years. Looked-after children and young people should receive regular health assessments, including establishing immunization status and, if needed, developing a health plan to achieve full immunization. The Department for Education publish data on immunizations among LACYP to monitor both how well they are immunized and service performance. METHODS: A literature review was conducted using four databases (PubMed, Embase, Scopus and Web of Science) on immunization status of LACYP, factors affecting uptake and challenges to immunization, and interventions to improve immunization rates. RESULTS: Thirty-two papers were identified, 16 of which were UK based. Looked-after children and young people are less likely to be 'up-to-date' with their immunizations than children in the general population. Looked-after children and young people are less likely to receive timely immunizations, and older LACYP are less likely to be 'up-to-date' than younger LACYP. Barriers to immunization include failure to attend health checks, absence from school and frequent placement moves. Unknown and discrepant immunization histories, name changes, sharing of information between organizations and obtaining consent for immunizations are also challenges. CONCLUSIONS: In recent years, immunization of LACYP has been given a higher priority. However, the immunization figures produced by the Department for Education are problematic because of challenges in determining whether the child is 'up-to-date', and data are not comparable with the general population; ideally, this should be changed to correspond to routine immunization data. In the interim, for reporting purposes, the use of a tool to assist with determining a child's immunization status would be beneficial. When a child's immunization status is incomplete or unknown, Public Health England's algorithm for vaccination of individuals with uncertain or incomplete status should be used. Practice to improve immunization uptake amongst LACYP needs to be evaluated to develop evidence-based recommendations.

Characterization of transcription factor response kinetics in parallel.

BACKGROUND: Transcription factors (TFs) are effectors of cell signaling pathways that regulate gene expression. TF networks are highly interconnected; one signal can lead to changes in many TF levels, and one TF level can be changed by many different signals. TF regulation is central to normal cell function, with altered TF function being implicated in many disease conditions. Thus, measuring TF levels in parallel, and over time, is crucial for understanding the impact of stimuli on regulatory networks and on diseases. RESULTS: Here, we report the parallel analysis of temporal TF level changes due to multiple stimuli in distinct cell types. We have analyzed short-term dynamic changes in the levels of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), signal transducer and activator of transcription 3 (Stat3), cAMP response element-binding protein (CREB), glucocorticoid receptor (GR), and TATA binding protein (TBP), in breast and liver cancer cells after tumor necrosis factor-alpha (TNF-α) and palmitic acid (PA) exposure. In response to both stimuli, NF-kB and CREB levels were increased, Stat3 decreased, and TBP was constant. GR levels were unchanged in response to TNF-α stimulation and increased in response to PA treatment. CONCLUSIONS: Our results show significant overlap in signaling initiated by TNF-α and by PA, with the exception that the events leading to PA-mediated cytotoxicity likely also include induction of GR signaling. These results further illuminate the dynamics of TF responses to cytokine and fatty acid exposure, while concomitantly demonstrating the utility of parallel TF measurement approaches in the analysis of biological phenomena.

Certifications for sight impairment due to age related macular degeneration in England.

OBJECTIVES: To examine variability across England in certification rates for age related macular degeneration (AMD) between 1st April 2011 and 31st March 2012. STUDY DESIGN: Cross-sectional survey. METHODS: An electronic version of the CVI, the ECVI, was used at the Certifications Office, London, to transfer information from paper based certificates into a database. The electronic certifications data set was queried for all certificates completed in England between April 1st 2011 and March 31st 2012 with the main cause of certifiable visual loss being AMD or with the main cause of certifiable visual loss being multiple pathology but a contributory cause being AMD. Data were explored by type of AMD, visual status, age and sex and then directly standardized rates were computed by English region. RESULTS: The Certifications Office received 23,616 CVIs for England between April 2011 and March 2012, of which 10,481 (44%) were people certified severely sight-impaired (blind) (SSI) and 12,689 (54%) were certified as sight-impaired (partial sight) (SI). The remainder did not have visual status classified. AMD contributed to 11546 causes of certification on the CVI forms during this period, 53% of forms being for geographic atrophy (GA)/dry AMD which is currently mostly untreatable. The median (interquartile) age at certification for AMD was 86 (81, 90) years and women were more commonly certified than men (66%). Considerable variability was seen across English regions, although there was consistency in that GA was the more common form in all areas. CONCLUSIONS: There is considerable regional variability in CVI rates in England, which are not attributable to differences in age or sex. Reasons for such variability need examination yet this should not undermine the value of these data in terms of describing those newly registered with sight impairment due to AMD who are predominantly female and over 85 years of age.

Mid- to late term hypoxia in the mouse alters placental morphology, glucocorticoid regulatory pathways and nutrient transporters in a sex-specific manner.

Maternal hypoxia is a common perturbation that can disrupt placental and thus fetal development, contributing to neonatal impairments. Recently, evidence has suggested that physiological outcomes are dependent upon the sex of the fetus, with males more susceptible to hypoxic insults than females. This study investigated the effects of maternal hypoxia during mid- to late gestation on fetal growth and placental development and determined if responses were sex specific. CD1 mice were housed under 21% or 12% oxygen from embryonic day (E) 14.5 until tissue collection at E18.5. Fetuses and placentas were weighed before collection for gene and protein expression and morphological analysis. Hypoxia reduced fetal weight in both sexes at E18.5 by 7% but did not affect placental weight. Hypoxia reduced placental mRNA levels of the mineralocorticoid and glucocorticoid receptors and reduced the gene and protein expression of the glucocorticoid metabolizing enzyme HSD11B2. However, placentas of female fetuses responded differently to maternal hypoxia than did placentas of male fetuses. Notably, morphology was significantly altered in placentas from hypoxic female fetuses, with a reduction in placental labyrinth blood spaces. In addition mRNA expression of Glut1, Igf2 and Igf1r were reduced in placentas of female fetuses only. In summary, maternal hypoxia altered placental formation in a sex specific manner through mechanisms involving placental vascular development, growth factor and nutrient transporter expression and placental glucocorticoid signalling. This study provides insight into how sex differences in offspring disease development may be due to sex specific placental adaptations to maternal insults.

Crusted scabies is associated with increased IL-17 secretion by skin T cells.

Scabies is an ectoparasitic infestation by the mite Sarcoptes scabiei. Although commonly self-limiting, a fraction of patients develop severely debilitating crusted scabies. The immune mechanisms underlying the development of crusted scabies are unclear, and undertaking longitudinal infection studies in humans is difficult. We utilized a porcine model to compare cellular immune responses in peripheral blood and skin of pigs with different clinical manifestations of scabies (n = 12), and in uninfected controls (n = 6). Although clinical symptoms were not evident until at least 4 weeks post-infestation, the numbers of peripheral IFNγ-secreting CD4(+) T cells and γδ T cells increased in infected pigs from week 1 post-infestation. γδ T cells remained increased in the blood at week 15 post-infestation. At week 15, skin cell infiltrates from pigs with crusted scabies had significantly higher CD8(+) T cell, γδ T cell and IL-17(+) cell numbers than those with ordinary scabies. Peripheral IL-17 levels were not increased, suggesting that localized skin IL-17-secreting T cells may play a critical role in the pathogenesis of crusted scabies development. Given the potential of anti-IL-17 immunotherapy demonstrated for other inflammatory skin diseases, this study may provide a novel therapeutic avenue for patients with recurrent crusted scabies.

Quantitative, solution-phase profiling of multiple transcription factors in parallel.

Transcription factors are regulatory proteins that bind to specific sites of chromosomal DNA to enact responses to intracellular and extracellular stimuli. Transcription factor signalling networks are branched and interconnected so that any single transcription factor can activate many different genes and one gene can be activated by a combination of different transcription factors. Thus, trying to characterize a cellular response to a stimulus by measuring the level of only one transcription factor potentially ignores important simultaneous events that contribute to the response. Hence, parallel measurements of transcription factors are necessary to capture the breadth of valuable information about cellular responses that would not be obtained by measuring only a single transcription factor. We have sought to develop a new, scalable, flexible, and sensitive approach to analysis of transcription factor levels that complements existing parallel approaches. Here, we describe proof-of-principle analyses of purified human transcription factors and breast cancer nuclear extracts. Our assay can successfully quantify transcription factors in parallel with ~10-fold better sensitivity than current techniques. Sensitivity of the assay can be further increased by 200-fold through the use of PCR for signal amplification.

Latest developments in experimental and computational approaches to characterize protein-lipid interactions.

Understanding the functional roles of all the molecules in cells is an ultimate goal of modern biology. An important facet is to understand the functional contributions from intermolecular interactions, both within a class of molecules (e.g. protein-protein) or between classes (e.g. protein-DNA). While the technologies for analyzing protein-protein and protein-DNA interactions are well established, the field of protein-lipid interactions is still relatively nascent. Here, we review the current status of the experimental and computational approaches for detecting and analyzing protein-lipid interactions. Experimental technologies fall into two principal categories, namely solution-based and array-based methods. Computational methods include large-scale data-driven analyses and predictions/dynamic simulations based on prior knowledge of experimentally identified interactions. Advances in the experimental technologies have led to improved computational analyses and vice versa, thereby furthering our understanding of protein-lipid interactions and their importance in biological systems.

Molecular mechanism by which palmitate inhibits PKR autophosphorylation.

PKR (double-stranded RNA-activated protein kinase) is an important component of the innate immunity, antiviral, and apoptotic pathways. Recently, our group found that palmitate, a saturated fatty acid, is involved in apoptosis by reducing the autophosphorylation of PKR at the Thr451 residue; however, the molecular mechanism by which palmitate reduces PKR autophosphorylation is not known. Thus, we investigated how palmitate affects the phosphorylation of the PKR protein at the molecular and biophysical levels. Biochemical and computational studies show that palmitate binds to PKR, near the ATP-binding site, thereby inhibiting its autophosphorylation at Thr451 and Thr446. Mutation studies suggest that Lys296 and Asp432 in the ATP-binding site on the PKR protein are important for palmitate binding. We further confirmed that palmitate also interacts with other kinases, due to the conserved ATP-binding site. A better understanding of how palmitate interacts with the PKR protein, as well as other kinases, could shed light onto possible mechanisms by which palmitate mediates kinase signaling pathways that could have implications on the efficacy of current drug therapies that target kinases.