The influence of instructions on generalised valence - conditional stimulus instructions after evaluative conditioning update the explicit and implicit evaluations of generalisation stimuli.

Generalisation in evaluative conditioning occurs when the valence acquired by a conditional stimulus (CS), after repeated pairing with an unconditional stimulus (US), spreads to stimuli that are similar to the CS (generalisation stimuli, GS). CS evaluations can be updated via CS instructions that conflict with prior conditioning (negative conditioning + positive instruction). We examined whether CS instructions can update GS evaluations after conditioning. We used alien stimuli where one alien (CSp) from a fictional group was paired with pleasant US images and another alien (CSu) from a different group was paired with unpleasant US images. The other members from the two groups were used as GSs. After conditioning, participants received negative CSp instructions and positive CSu instructions. In Experiment 1, explicit and implicit GS evaluations were measured before and after the instructions. In Experiment 2, we used a between-participants design where one group received positive/negative CS instructions while a control group received neutral instructions. In both experiments, the positive/negative CS instructions caused a reversal of explicit GS evaluations and an elimination of implicit GS evaluations. The findings suggest that generalised evaluations can change after CS instructions which may have implications for interventions aimed at reducing negative group attitudes.

Conceptual generalisation in fear conditioning using single and multiple category exemplars as conditional stimuli - electrodermal responses and valence evaluations generalise to the broader category.

Conceptual generalisation occurs when conditional responses generalise to novel stimuli from the same category. Past research demonstrates that physiological fear responses generalise across categories, however, conceptual generalisation of stimulus valence evaluations during fear conditioning has not been examined. We investigated whether conceptual generalisation, as indexed by electrodermal responses and stimulus evaluations, would occur, and differ after training with single or multiple conditional stimuli (CSs). Stimuli from two of four categories (vegetables, farm animals, clothing, and office supplies) were used as the CS+ (followed by an electric stimulus) or CS- (presented alone). Generalisation was assessed by presenting novel stimuli from the CS categories after acquisition, extinction, and reinstatement. One category exemplar was used as the CS+ and CS- in the single group, whereas three exemplars were used as the CS+ and CS- in the multiple group. Electrodermal responses generalised in acquisition and extinction but did not differ between groups. In the multiple group, CS evaluations generalised in acquisition and extinction, whereas generalisation was not evident in the single group. Training with multiple CSs also resulted in the extinction of stimulus valence. The current findings have implications for future research examining the generalisation of valence and for exposure-based treatments of anxiety.

National Trends and Outcomes of Nonautoimmune Hemolytic Anemia in Alcoholic Liver Disease: Analysis of the Nationwide Inpatient Sample.

GOAL: The aim of this study was to determine the burden of nonautoimmune hemolytic anemia (NAHA) in hospitalized patients with coexisting alcoholic liver disease (ALD), identify risk factors for NAHA in ALD and describe the hospitalization outcomes. BACKGROUND: ALD can result in structural and metabolic alterations in the red-blood cell membrane leading to premature destruction of erythrocytes and hemolytic anemia of varying severity. STUDY: Hospitalized ALD patients with concomitant NAHA were identified in the Nationwide Inpatient Sample database using International Classification of Diseases-9 codes from 2009 to 2014. The primary outcome was to determine the nationwide prevalence and risk factors of NAHA in patients hospitalized with ALD. RESULTS: The prevalence of NAHA was 0.17% (n=3585) among all ALD patients (n=2,125,311) that were hospitalized. Multivariate analysis indicated higher odds of NAHA in ALD patients in the following groups: female gender [adjusted odds ratio (AOR) AOR 1.80, P<0.0001]; highest quartile of median household income (AOR 1.88, P<0.0001); increasing Charlson-Deyo Comorbidity Index (3 to 4 vs. 0, AOR 2.16, P=0.0042) and cirrhosis (AOR 2.74, P<0.0001). Discharges of ALD with anemia had a significantly longer average length of stay (8.8 vs. 6.0 d, P<0.0001), increased hospital charges ($38,961 vs. $25,244, P<0.0001) and higher mortality (9.0% vs. 5.6%, P<0.0001) when compared with ALD with no anemia. CONCLUSION: NAHA in patients with ALD is an important prognostic marker, predicting a longer, costlier hospitalization and increased inpatient mortality in ALD.

Be careful what you say! - Evaluative change based on instructional learning generalizes to other similar stimuli and to the wider category.

In evaluative conditioning, a conditional stimulus (CS; e.g. a neutral picture) acquires positive/negative valence if it is paired with a pleasant/unpleasant unconditional stimulus (US; e.g. a positive/negative picture). This valence generalises to other stimuli similar to the CS and to the wider CS category. Being informed that the CS will be paired with the US induces a similar change in valence (instructional learning), but it is not clear whether instructional learning would also generalise. In Experiment 1, participants were informed that one shape would be paired with pleasant and another with unpleasant images. These instructions instilled conditional valence to the CSs which generalised to different shapes from the same category (generalisation stimuli). In Experiment 2, we replicated this finding in an implicit measure using stimuli varying in perceptual features. Participants were informed that three CSs from one category (e.g. vegetables) would be paired with pleasant images and three CSs from a second category (e.g. office supplies) would be paired with unpleasant images. This instruction instilled conditional valence to the CSs which generalised to novel exemplars from the same categories. This suggests that conditional valence instilled via instructions generalises to other stimuli - a finding with implications for prejudice and racism.

Toll pathway is required for wound-induced expression of barrier repair genes in the Drosophila epidermis.

The epidermis serves as a protective barrier in animals. After epidermal injury, barrier repair requires activation of many wound response genes in epidermal cells surrounding wound sites. Two such genes in Drosophila encode the enzymes dopa decarboxylase (Ddc) and tyrosine hydroxylase (ple). In this paper we explore the involvement of the Toll/NF-κB pathway in the localized activation of wound repair genes around epidermal breaks. Robust activation of wound-induced transcription from ple and Ddc requires Toll pathway components ranging from the extracellular ligand Spätzle to the Dif transcription factor. Epistasis experiments indicate a requirement for Spätzle ligand downstream of hydrogen peroxide and protease function, both of which are known activators of wound-induced transcription. The localized activation of Toll a few cell diameters from wound edges is reminiscent of local activation of Toll in early embryonic ventral hypoderm, consistent with the hypothesis that the dorsal-ventral patterning function of Toll arose from the evolutionary cooption of a morphogen-responsive function in wound repair. Furthermore, the combinatorial activity of Toll and other signaling pathways in activating epidermal barrier repair genes can help explain why developmental activation of the Toll, ERK, or JNK pathways alone fail to activate wound repair loci.

Duox, Flotillin-2, and Src42A are required to activate or delimit the spread of the transcriptional response to epidermal wounds in Drosophila.

The epidermis is the largest organ of the body for most animals, and the first line of defense against invading pathogens. A breach in the epidermal cell layer triggers a variety of localized responses that in favorable circumstances result in the repair of the wound. Many cellular and genetic responses must be limited to epidermal cells that are close to wounds, but how this is regulated is still poorly understood. The order and hierarchy of epidermal wound signaling factors are also still obscure. The Drosophila embryonic epidermis provides an excellent system to study genes that regulate wound healing processes. We have developed a variety of fluorescent reporters that provide a visible readout of wound-dependent transcriptional activation near epidermal wound sites. A large screen for mutants that alter the activity of these wound reporters has identified seven new genes required to activate or delimit wound-induced transcriptional responses to a narrow zone of cells surrounding wound sites. Among the genes required to delimit the spread of wound responses are Drosophila Flotillin-2 and Src42A, both of which are transcriptionally activated around wound sites. Flotillin-2 and constitutively active Src42A are also sufficient, when overexpressed at high levels, to inhibit wound-induced transcription in epidermal cells. One gene required to activate epidermal wound reporters encodes Dual oxidase, an enzyme that produces hydrogen peroxide. We also find that four biochemical treatments (a serine protease, a Src kinase inhibitor, methyl-ß-cyclodextrin, and hydrogen peroxide) are sufficient to globally activate epidermal wound response genes in Drosophila embryos. We explore the epistatic relationships among the factors that induce or delimit the spread of epidermal wound signals. Our results define new genetic functions that interact to instruct only a limited number of cells around puncture wounds to mount a transcriptional response, mediating local repair and regeneration.

Not missing at random: Missing data are associated with clinical status and trajectories in an electronic monitoring longitudinal study of bipolar disorder.

There is limited information on the association between participants' clinical status or trajectories and missing data in electronic monitoring studies of bipolar disorder (BD). We collected self-ratings scales and sensor data in 145 adults with BD. Using a new metric, Missing Data Ratio (MDR), we assessed missing self-rating data and sensor data monitoring activity and sleep. Missing data were lowest for participants in the midst of a depressive episode, intermediate for participants with subsyndromal symptoms, and highest for participants who were euthymic. Over a mean ± SD follow-up of 246 ± 181 days, missing data remained unchanged for participants whose clinical status did not change throughout the study (i.e., those who entered the study in a depressive episode and did not improve, or those who entered the study euthymic and remained euthymic). Conversely, when participants' clinical status changed during the study (e.g., those who entered the study euthymic and experienced the occurrence of a depressive episode), missing data for self-rating scales increased, but not for sensor data. Overall missing data were associated with participants' clinical status and its changes, suggesting that these are not missing at random.

Neurophysiological and other features of working memory in older adults at risk for dementia.

Theta-gamma coupling (TGC) is a neurophysiological process that supports working memory. Working memory is associated with other clinical and biological features. The extent to which TGC is associated with these other features and whether it contributes to working memory beyond these features is unknown. Two-hundred-and-three older participants at risk for Alzheimer's dementia-98 with mild cognitive impairment (MCI), 39 with major depressive disorder (MDD) in remission, and 66 with MCI and MDD (MCI + MDD)-completed a clinical assessment, N-back-EEG, and brain MRI. Among them, 190 completed genetic testing, and 121 completed [11C] Pittsburgh Compound B ([11C] PIB) PET imaging. Hierarchical linear regressions were used to assess whether TGC is associated with demographic and clinical variables; Alzheimer's disease-related features (APOE ε4 carrier status and ß-amyloid load); and structural features related to working memory. Then, linear regressions were used to assess whether TGC is associated with 2-back performance after accounting for these features. Other than age, TGC was not associated with any non-neurophysiological features. In contrast, TGC (ß = 0.27; p = 0.006), age (ß = - 0.29; p = 0.012), and parietal cortical thickness (ß = 0.24; p = 0.020) were associated with 2-back performance. We also examined two other EEG features that are linked to working memory-theta event-related synchronization and alpha event-related desynchronization-and found them not to be associated with any feature or performance after accounting for TGC. Our findings suggest that TGC is a process that is independent of other clinical, genetic, neurochemical, and structural variables, and supports working memory in older adults at risk for dementia. Supplementary Information: The online version contains supplementary material available at 10.1007/s11571-023-09938-y.

Use and efficacy of nebulized naloxone in patients with suspected opioid intoxication.

OBJECTIVE: To describe the use and efficacy of nebulized naloxone in patients with suspected opioid intoxication. METHODS: This was an observational study conducted at an inner city emergency department. Patients were eligible if they had self-reported or suspected opioid intoxication and a spontaneous respiratory rate ≥6 breaths/minute. Nebulized naloxone (2 mg in 3 mL normal saline) was administered through a standard face mask at the discretion of the treating physician. Structured data collection included demographics, vital signs pre and post naloxone administration and adverse events. The primary outcome was level of consciousness, which was recorded pre and 15 minutes postnaloxone administration using the Glasgow Coma Scale (GCS) and the Richmond Agitation Sedation Scale (RASS). RESULTS: Of the 73 patients who presented with suspected opioid intoxication and were given naloxone over the study period, 26 were initially treated with nebulized naloxone. After nebulized naloxone administration, median GCS improved from 11 [interquartile range (IQR) 3.5] to 13 (IQR, 2.5), P = .001. Median RASS improved from -3.0 (IQR, -1.0) to -2.0 (IQR, -1.5), P < .0001. Need for supplemental oxygen decreased from 81% to 50%, P = .03. Vital signs did not differ pre/post therapy. There were few adverse effects from nebulized naloxone administration: 12% experienced moderate-severe agitation, 8% were diaphoretic and none vomited. Eleven required subsequent administrations of naloxone, nine of whom self-reported using either heroin, methadone or both. Of these, 5 underwent urine drug screening and all 5 tested positive for either opiates or methadone. CONCLUSIONS: Nebulized naloxone was well-tolerated and led to a reduction in the need for supplemental oxygen as well as improved median GCS and RASS scores in patients with suspected opioid intoxication.

The influence of cross unconditional stimulus reinstatement on electrodermal responding and conditional stimulus valence in differential fear conditioning.

We examined whether the inhibitory Conditional Stimulus (CS)-no Unconditional Stimulus (US) association formed during extinction can be triggered by a novel US during the reinstatement of conditional electrodermal responding and self-reported CS valence in human differential fear conditioning. Participants were trained with either a shock or an aversive scream US before undergoing extinction. Participants then received either the same (i.e., shock_shock or scream_scream) or a different US during reinstatement (i.e., shock_scream, scream_shock). Differential conditioning across all indices was stronger when a shock US was used during acquisition. After reinstatement, electrodermal responding to both the CS+ and the CS- increased regardless of the type of US used during reinstatement (non-differential reinstatement). Differential CS valence evaluations were larger after reinstatement in the groups that received the same US during acquisition and reinstatement (differential reinstatement), but differential evaluations did not increase in the groups receiving a different US at reinstatement. This dissociation suggests that the reinstatement of negative stimulus valence and the reinstatement of expectancy learning may differ.

Quantitative Assessment of Cortical Excitability in Alzheimer's Dementia and Its Association with Clinical Symptoms: A Systematic Review and Meta-Analyses.

BACKGROUND: Alzheimer's disease (AD) is characterized by cognitive and neuropsychiatric symptoms (NPS) due to underlying neurodegenerative pathology. Some studies using electroencephalography (EEG) have shown increased epileptiform and epileptic activity in AD. OBJECTIVE: This review and meta-analyses aims to synthesize the existing evidence for quantitative abnormalities of cortical excitability in AD and their relationship with clinical symptoms. METHODS: We systematically searched and reviewed publications that quantitatively assessed cortical excitability, using transcranial magnetic stimulation (TMS) resting motor threshold (rMT), active motor threshold (aMT), motor evoked potential (MEP) or directly from the cortex using TMS-EEG via TMS-evoked potential (TEP). We meta-analyzed studies that assessed rMT and aMT using random effects model. RESULTS: We identified 895 publications out of which 37 were included in the qualitative review and 30 studies using rMT or aMT were included in the meta-analyses. The AD group had reduced rMT (Hedges' g = -0.99, 95% CI [-1.29, -0.68], p < 0.00001) and aMT (Hedges' g = -0.87, 95% CI [-1.50, -0.24], p < 0.00001) as compared with control groups, indicative of higher cortical excitability. Qualitative review found some evidence of increased MEP amplitude, whereas findings related to TEP were inconsistent. There was some evidence supporting an inverse association between cortical excitability and global cognition. No publications reported on the relationship between cortical excitability and NPS. CONCLUSION: There is strong evidence of increased motor cortex excitability in AD and some evidence of an inverse association between excitability and cognition. Future studies should assess cortical excitability from non-motor areas using TMS-EEG and examine its relationship with cognition and NPS.

A comparison of normal versus low dietary carbohydrate intake on substrate oxidation during and after moderate intensity exercise in women.

We compared the effects of consuming a 2-day low-carbohydrate (CHO) diet (low-CHO; 20% CHO, 40% protein, 40% fat) versus an isocaloric 2-day moderate-CHO diet (mod-CHO; 55% CHO, 15% protein, 30% fat) on substrate oxidation during and after exercise in ten active, young women. Subjects were 24.9 ± 6.2% body fat with a VO(2max) of 68.8 ± 13.8 ml/kg FFM/min. For 2 days prior to exercise, subjects consumed either the mod-CHO or the low-CHO diet and then completed treadmill exercise at 55% of VO(2max) until 350 kcal of energy was expended. During exercise and for 2 h post-exercise, expired gases were analyzed to determine oxidation rates for CHO (CHO-OX) and fat (FAT-OX). Significant differences (p < 0.05) were found between diets for CHO-OX and FAT-OX (mg/kg FFM/min) during exercise, 1 h post-ex, and 2 h post-ex. During exercise, FAT-OX was higher (low-CHO 8.7 ± 2.2 vs. mod-CHO 6.2 ± 2.2) and CHO-OX was lower (low-CHO 25.1 ± 5.6 vs. mod-CHO 31.1 ± 6.2) following the low-CHO diet. A similar trend was observed during 1 h post-ex for FAT-OX (low-CHO 2.2 ± 0.5 vs. mod-CHO 1.6 ± 0.5) and CHO-OX (low-CHO 2.5 ± 1.2 vs. mod-CHO 4.1 ± 1.9), as well as 2 h post-ex for FAT-OX (low-CHO vs. 1.9 ± 0.5 mod-CHO 1.7 ± 0.4) and CHO-OX (low-CHO 2.5 ± 0.9 vs. mod-CHO 3.1 ± 1.1). Significant positive correlations were observed between VO(2max) and CHO-OX during exercise and post-exercise, as well as significant negative correlations between VO(2max) and FAT-OX post-exercise in the low-CHO condition. Waist circumference and FAT-OX exhibited a significant negative correlation during exercise in the low-CHO condition. Dietary macronutrient intake influenced substrate oxidation in active young women during and after moderate intensity exercise.

Physician Wellness During a Pandemic.

INTRODUCTION: We are currently in the midst of the coronavirus disease 2019 (COVID-19) pandemic. Research into previous infectious disease outbreaks has shown that healthcare workers are at increased risk for burnout during these dire times, with those on the front lines at greatest risk. The purpose of this prospective study was to determine the effect that the COVID-19 pandemic has had on the wellness of emergency physicians (EP). METHODS: A survey was sent to 137 EPs in a multi-hospital network in eastern Pennsylvania. We compared 10 primary and two supplemental questions based on how the physicians had been feeling in the prior 2-3 weeks (COVID-19 period) to the same questions based on how they were feeling in the prior 4-6 months (pre-COVID-19 period). RESULTS: We received 55 responses to the survey (40.1% response rate). The study found that during the pandemic, EPs felt less in control (p-value = 0.001); felt decreased happiness while at work (p-value 0.001); had more trouble falling asleep (p-value = 0.001); had an increased sense of dread when thinking of work needing to be done (p-value = 0.04); felt more stress on days not at work (p-value <0.0001); and were more concerned about their own health (p-value <0.0001) and the health of their families and loved ones (p-value <0.0001). CONCLUSION: This study showed a statistically significant decrease in EP wellness during the COVID-19 pandemic when compared to the pre-pandemic period. We need to be aware of evidence-based recommendations to help mitigate the risks and prevent physician burnout.

"Prepared" fear or socio-cultural learning? Fear conditioned to guns, snakes, and spiders is eliminated by instructed extinction in a within-participant differential fear conditioning paradigm.

Across three experiments, we investigated whether electrodermal responses conditioned to ontogenetic fear-relevant (pointed guns) and phylogenetic fear-relevant stimuli (snakes and spiders) would resist instructed extinction in a within-participant differential fear conditioning paradigm. Instructed extinction involves informing participants before extinction that the unconditional stimulus (US) will no longer be presented. This manipulation has been shown to abolish fear conditioned to fear-irrelevant conditional stimuli, but is said to leave fear conditioned to images of snakes and spiders intact. The latter finding, however, has only been demonstrated when fear-relevance is manipulated between-groups. It is also not known whether instructed extinction affects fear conditioned to ontogenetic fear-relevant stimuli, such as pointed guns. In Experiment 1, we demonstrated that fear conditioned to images of pointed guns does not resist instructed extinction. In Experiment 2, we detected some evidence to suggest that fear conditioned to images of snakes and spiders survives instructed extinction but this evidence was not conclusive. In Experiment 3, we directly compared the effects of instructed extinction on fear conditioned to snakes and spiders and to guns and provide strong evidence that fear conditioned to both classes of stimuli is reduced after instructed extinction with no differences between ontogenetic and phylogenetic stimuli. The current results suggest that when fear relevance is manipulated within-participants fear conditioned to both phylogenetic and ontogenetic, fear-relevant stimuli responds to instructed extinction providing evidence in favor of a socio-cultural explanation for "preparedness" effects.

Lack of evidence for contact sensitization by Pfiesteria extract.

BACKGROUND: Members of the estuarine dinoflagellate genus Pfiesteria are reported to have been responsible for massive fish kills in the southeastern United States. Some reports suggest that exposure to waters having Pfiesteria blooms or occupation-related exposure might result in Pfiesteria-induced dermal irritation and inflammation. Although the toxin has not been isolated and purified, the original data suggested both hydrophilic and hydrophobic toxic components. Some investigators propose that dermonecrotic properties are associated with a hydrophobic fraction. OBJECTIVES: A bioactive C18-bound putative toxin (CPE) extracted from Pfiesteria-laden aquarium water during active fish-killing conditions was examined in the present study to evaluate its potential to produce inflammation and dermal sensitization and to determine whether the inflammation and dermatitis reported in early human exposure studies were allergic or irritant in nature. RESULTS: This fraction was cytotoxic to mouse Neuro-2A cells and primary human epidermal keratinocytes (NHEK) at a concentration of 1 mg/mL. Balb/C mice exposed to 50-200% CPE by skin painting exhibited a 6-10% increase in ear swelling relative to vehicle-treated mice in a primary irritancy assay. There was no increase in lymph node cell proliferation as measured using the local lymph node assay. Exposure to CPE in culture up-regulated interleukin-8 in NHEK, whereas granulocyte macrophage-colony-stimulating factor and tumor necrosis factor alpha were only minimally altered. CONCLUSIONS: This study suggests that CPE is cytotoxic to keratinocytes in culture at high concentrations and that it induces mild, localized irritation but not dermal sensitization.

Responses to Treatment With Teriparatide in Patients With Atypical Femur Fractures Previously Treated With Bisphosphonates.

If oversuppression of bone turnover explained the association between bisphosphonate use and atypical subtrochanteric femur fractures (AFF), this could be reversed with anabolic treatment such as teriparatide. We conducted a prospective, open-label study in patients previously treated with bisphosphonates who sustained AFF, examining the response to 24-month treatment with teriparatide on bone mineral density (BMD), trabecular bone score (TBS), bone turnover markers (BTM), and fracture healing as well as quantitative histomorphometry. We studied 14 patients. Baseline BMD, BTM, and TBS varied widely. On initial bone biopsies, 12 of 14 patients showed tetracycline labels, but mineralizing surface/bone surface was below published normal values in all but 2. Lumbar spine BMD increased significantly at month 24 (6.1% ± 4.3%, p < 0.05 versus baseline), whereas total hip BMD and TBS did not change significantly. Changes in BTM occurred as reported previously for patients without AFF treated with teriparatide after prior bisphosphonate treatment. At month 24, fractures were healed in 6 patients, showed partial healing in 3, were unchanged in 2, and showed nonunion in 1. In a patient with two fractures, the fracture that occurred before teriparatide treatment was reported as healed, but the fracture that occurred while on treatment showed only partial healing. Bisphosphonate-treated patients who sustain AFF show heterogeneity of bone turnover. Treatment with teriparatide resulted in increases in BTM and lumbar spine BMD, as has been reported for patients without AFF. There was no significant effect of teriparatide on hip BMD, mineralizing surface to bone surface (MS/BS), or TBS and no consistent effect on fracture healing. In the context of a patient who has experienced an AFF after receiving bisphosphonate treatment, therapy with teriparatide for 24 months would be expected to increase BMD and BTM (and probably reduce the risk of fractures resulting from osteoporosis) but should not be relied on to aid in healing of the AFF. © 2017 American Society for Bone and Mineral Research.

Gene expression alterations in immune system pathways following exposure to immunosuppressive chemicals.

Exposure to environmental agents can affect a number of adverse immunological outcomes, including changes in the incidence of infectious disease. Diethylstilbestrol (DES), dexamethasone (DEX), cyclophosphamide, and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are immunosuppressive chemicals that can induce similar pathophysiological end points in the thymus; however, the mechanism of toxicity is different for each compound. We examined differential gene expression in the spleen and thymus following chemical exposure and correlated these changes with alterations in functional immune end points and our knowledge of the known mechanisms of action. RNA from the spleen and thymus has been analyzed using Illumina Sentrix arrays and BeadStudio software. Preliminary data suggest that DES induced the greatest number of gene changes in the spleen, while DEX induced the most changes in the thymus. In both spleen and thymus, genomic analysis revealed gene expression changes that were common to multiple chemicals and that may be associated with xenobiotic-induced immune system perturbations, including alterations in genes associated with apoptosis, antigen processing and presentation, and response to biotic stimulus. This was particularly evident in the thymus, where there were many similarities in the expression profiles, as well as gene alterations unique to a single compound. In contrast, expression profiles in spleen were more distinct. The category of genes most profoundly affected by all four chemicals was response to biotic stimulus: there were both clusters of genes modulated by multiple chemicals and genes altered by a single chemical. The distinct gene profiles may specifically relate to cellular targets and mechanism of action.

Microinjection wound assay and in vivo localization of epidermal wound response reporters in Drosophila embryos.

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to maintain cellular homeostasis. Puncture injury with glass needles provides a direct method to trigger a rapid epidermal wound response that activates wound transcriptional reporters, which can be visualized by a localized reporter signal in living embryos or larvae. Puncture or laser injury also provides signals that promote the recruitment of hemocytes to the wound site. Surprisingly, severe (through and through) puncture injury in late stage embryos only rarely disrupts normal embryonic development, as greater than 90% of such wounded embryos survive to adulthood when embryos are injected in an oil medium that minimizes immediate leakage of hemolymph from puncture sites. The wound procedure does require micromanipulation of the Drosophila embryos, including manual alignment of the embryos on agar plates and transfer of the aligned embryos to microscope slides. The Drosophila epidermal wound response assay provides a quick system to test the genetic requirements of a variety of biological functions that promote wound healing, as well as a way to screen for potential chemical compounds that promote wound healing. The short life cycle and easy culturing routine make Drosophila a powerful model organism. Drosophila clean wound healing appears to coordinate the epidermal regenerative response, with the innate immune response, in ways that are still under investigation, which provides an excellent system to find conserved regulatory mechanisms common to Drosophila and mammalian epidermal wounding.

MT2-MMP expression during early avian morphogenesis.

Membrane-type 2 matrix metalloproteinase (MT2-MMP; also called MMP15) is a membrane-bound protease that degrades extracellular matrix and activates proMMPs such as proMMP-2. MMP-2 expression in avian embryos is well documented, but it is not clear how proMMP-2 is activated during avian embryogenesis. Herein, we report that MT2-MMP mRNA is expressed in several tissues including the neural folds and epidermal ectoderm, intermediate mesoderm, pharyngeal arches, limb buds, and dermis. Several, but not all, of these tissues are known to express MMP-2. These observations suggest MT2-MMP may play a role during embryonic development not only through its own proteolytic activity but also by activating proMMP-2.

Serine proteolytic pathway activation reveals an expanded ensemble of wound response genes in Drosophila.

After injury to the animal epidermis, a variety of genes are transcriptionally activated in nearby cells to regenerate the missing cells and facilitate barrier repair. The range and types of diffusible wound signals that are produced by damaged epidermis and function to activate repair genes during epidermal regeneration remains a subject of very active study in many animals. In Drosophila embryos, we have discovered that serine protease function is locally activated around wound sites, and is also required for localized activation of epidermal repair genes. The serine protease trypsin is sufficient to induce a striking global epidermal wound response without inflicting cell death or compromising the integrity of the epithelial barrier. We developed a trypsin wounding treatment as an amplification tool to more fully understand the changes in the Drosophila transcriptome that occur after epidermal injury. By comparing our array results with similar results on mammalian skin wounding we can see which evolutionarily conserved pathways are activated after epidermal wounding in very diverse animals. Our innovative serine protease-mediated wounding protocol allowed us to identify 8 additional genes that are activated in epidermal cells in the immediate vicinity of puncture wounds, and the functions of many of these genes suggest novel genetic pathways that may control epidermal wound repair. Additionally, our data augments the evidence that clean puncture wounding can mount a powerful innate immune transcriptional response, with different innate immune genes being activated in an interesting variety of ways. These include puncture-induced activation only in epidermal cells in the immediate vicinity of wounds, or in all epidermal cells, or specifically in the fat body, or in multiple tissues.