Novel expression of coat proteins from thermophilic bacteriophage ΦIN93 and evaluation for assembly into virus-like particles.

Virus-like particles (VLPs) have the potential to be used as display platforms to develop vaccines against infectious and non-infectious agents. However, most VLPs used as vaccine display platforms are derived from viruses that infect humans; unfortunately, most humans already have pre-existing antibodies against these platforms and thus, the immunogenicity of these vaccines may be compromised. VLP platforms derived from viruses that infect bacteria (bacteriophages), especially bacteriophages that infect bacteria, which do not colonize humans are less likely to have pre-existing antibodies against the platforms in the human population. In this study, we assessed whether two putative coat proteins (ORF13 and ORF14) derived from a thermophilic bacteriophage (ΦIN93) can be expressed and purified from a mesophilic bacterium such as E. coli. We also assessed whether expressed coat proteins can assemble to form VLPs. Truncated versions of ORF13 and ORF14 were successfully co-expressed in bacteria; the co-expressed truncated proteins formed oval structures that look like VLPs, but their sizes were less than those of an authentic ΦIN93 virus.

Self-adaptive amplified spontaneous emission suppression with a photorefractive two-beam coupling filter.

Optical bandpass filters can be utilized to suppress parasitic broadband spectral power prior to laser amplification but are typically designed around specific frequencies or require manual adjustment, thus limiting their compatibility with highly tunable or integrated laser systems. In this Letter, we introduce a self-adaptive volume holographic filter using the dynamic two-beam coupling interaction in photorefractive BaTiO3, demonstrating -10dB suppression of amplified spontaneous emission noise surrounding a tunable 780 nm diode laser peak, with <2nm filter bandwidth and 50% power throughput. The spectral filtering is automatically centered on the lasing mode, with an estimated auto-tuning rate of 100 GHz/s under typical conditions. Furthermore, the filter suppression and bandwidth can be optimized via the two-beam coupling intensity ratio and angle, respectively, for versatile control over the self-adaptive filter characteristics.

Impairment of wound healing by reactive skin decontamination lotion (RSDL®) in a Göttingen minipig® model.

Reactive Skin Decontamination Lotion (RSDL®) is an FDA-approved skin decontamination kit carried by service members for removal and neutralisation of vesicants and nerve agents. The RSDL kit, comprised of a lotion-impregnated sponge, was shown to be the superior medical decontamination device for chemical warfare agent (CWA) exposure on intact skin. In the event of a chemical exposure situation (i.e. terrorism, battlefield) physical injuries are probable, and preservation of life will outweigh the risk associated with application of RSDL to compromised skin. The purpose of this study was to quantify the rate and quality of wound healing in epidermal skin wounds treated with RSDL in a porcine model. Degree of wound healing was assessed using bioengineering methods to include ballistometry, colorimetry, evaporimetry, and high-frequency ultrasonography. Clinical observation, histopathology and immunohistochemistry were also utilised. All pigs received four bilateral superficial abdominal wounds via a pneumatic dermatome on their ventral abdomen, then were treated with the following dressings over a seven-day period: RSDL sponge, petroleum based Xeroform® gauze, 3 M™ Tegaderm™ Film, and 3 M™ Tegaderm™ Foam. Two additional non-wounded sites on the flank were used as controls. Two groups of pigs were then evaluated for a 21- or 56-day time period, representing short- and long-term wound-healing progression. Our findings indicated RSDL had a negative impact on wound-healing progression at both 21 and 56 days post-injury. Wounds receiving RSDL demonstrated a decreased skin elasticity, significant transepidermal water loss, and altered skin colouration and thickness. In addition, the rate of wound healing was delayed, and return to a functional skin barrier was altered when compared to non-RSDL-treated wounds. In conclusion, wound management care and clinical therapeutic intervention plans should be established to account for a prolonged duration of healing in patients with RSDL-contaminated wounds.

Bronchiolitis Obliterans and Pulmonary Fibrosis after Sulfur Mustard Inhalation in Rats.

Inhalation of powerful chemical agents, such as sulfur mustard (SM), can have debilitating pulmonary consequences, such as bronchiolitis obliterans (BO) and parenchymal fibrosis (PF). The underlying pathogenesis of disorders after SM inhalation is not clearly understood, resulting in a paucity of effective therapies. In this study, we evaluated the role of profibrotic pathways involving transforming growth factor-ß (TGF-ß) and platelet-derived growth factor (PDGF) in the development of BO and PF after SM inhalation injury using a rat model. Adult Sprague-Dawley rats were intubated and exposed to SM (1.0 mg/kg), then monitored daily for respiratory distress, oxygen saturation changes, and weight loss. Rats were killed at 7, 14, 21, or 28 days, and markers of injury were determined by histopathology; pulmonary function testing; and assessment of TGF-ß, PDGF, and PAI-1 concentrations. Respiratory distress developed over time after SM inhalation, with progressive hypoxemia, respiratory distress, and weight loss. Histopathology confirmed the presence of both BO and PF, and both gradually worsened with time. Pulmonary function testing demonstrated a time-dependent increase in lung resistance, as well as a decrease in lung compliance. Concentrations of TGF-ß, PDGF, and PAI-1 were elevated at 28 days in lung, BAL fluid, and/or plasma. Time-dependent development of BO and PF occurs in lungs of rats exposed to SM inhalation, and the elevated concentrations of TGF-ß, PDGF, and PAI-1 suggest involvement of these profibrotic pathways in the aberrant remodeling after injury.

Experimental Demonstration of Shaken-Lattice Interferometry.

We experimentally demonstrate a shaken-lattice interferometer. Atoms are trapped in the ground Bloch state of a red-detuned optical lattice. Using a closed-loop optimization protocol based on the dcrab algorithm, we phase-modulate (shake) the lattice to transform the atom momentum state. In this way, we implement an atom beam splitter and build five interferometers of varying interrogation times T_{I}. The sensitivity of shaken-lattice interferometry is shown to scale as T_{I}^{2}, consistent with simulation (2C. A. Weidner, H. Yu, R. Kosloff, and D. Z. Anderson, Phys. Rev. A 95, 043624 (2017).PLRAAN2469-992610.1103/PhysRevA.95.043624). Finally, we show that we can measure the sign of an applied signal and optimize the interferometer in the presence of a bias signal.

Socioeconomic status and hippocampal volume in children and young adults.

An individual's socioeconomic status (SES) is often viewed as a proxy for a host of environmental influences. SES disparities have been linked to variance in brain structures particularly the hippocampus, a neural substrate of learning and memory. However, it is unclear whether the association between SES and hippocampal volume is similar in children and adults. We investigated the relationship between hippocampal volume and SES in a group of children (n = 31, age 8-12 years) and a group of young adults (n = 32, age 18-25 years). SES was assessed with four indicators that loaded on a single factor, therefore a composite SES scores was used in the main analyses. Hippocampal volume was measured using manual demarcation on high resolution structural images. SES was associated with hippocampal volume in the children, but not in adults, suggesting that in childhood, but not adulthood, SES-related environmental factors influence hippocampal volume. In addition, hippocampal volume, but not SES, was associated with scores on a memory task, suggesting that net effects of postnatal environmental factors, captured by SES, are more distal determinants of memory performance than hippocampal volume. Longitudinal investigation of the association between SES, hippocampal volume and cognitive functioning may further our understanding of the putative neural mechanisms underlying SES-related environmental effects on cognitive development.

Statistical Modeling of Fire Occurrence Using Data from the Tohoku, Japan Earthquake and Tsunami.

In this article, we develop statistical models to predict the number and geographic distribution of fires caused by earthquake ground motion and tsunami inundation in Japan. Using new, uniquely large, and consistent data sets from the 2011 Tohoku earthquake and tsunami, we fitted three types of models-generalized linear models (GLMs), generalized additive models (GAMs), and boosted regression trees (BRTs). This is the first time the latter two have been used in this application. A simple conceptual framework guided identification of candidate covariates. Models were then compared based on their out-of-sample predictive power, goodness of fit to the data, ease of implementation, and relative importance of the framework concepts. For the ground motion data set, we recommend a Poisson GAM; for the tsunami data set, a negative binomial (NB) GLM or NB GAM. The best models generate out-of-sample predictions of the total number of ignitions in the region within one or two. Prefecture-level prediction errors average approximately three. All models demonstrate predictive power far superior to four from the literature that were also tested. A nonlinear relationship is apparent between ignitions and ground motion, so for GLMs, which assume a linear response-covariate relationship, instrumental intensity was the preferred ground motion covariate because it captures part of that nonlinearity. Measures of commercial exposure were preferred over measures of residential exposure for both ground motion and tsunami ignition models. This may vary in other regions, but nevertheless highlights the value of testing alternative measures for each concept. Models with the best predictive power included two or three covariates.

On-chip optical lattice for cold atom experiments.

An atom-chip-based integrated optical lattice system for cold and ultracold atom applications is presented. The retroreflection optics necessary for forming the lattice are bonded directly to the atom chip, enabling a compact and robust on-chip optical lattice system. After achieving Bose-Einstein condensation in a magnetic chip trap, we load atoms directly into a vertically oriented 1D optical lattice and demonstrate Landau-Zener tunneling. The atom chip technology presented here can be readily extended to higher dimensional optical lattices.

Mustard Gas Inhalation Injury: Therapeutic Strategy.

Mustard gas (sulfur mustard [SM], bis-[2-chloroethyl] sulfide) is a vesicating chemical warfare agent and a potential chemical terrorism agent. Exposure of SM causes debilitating skin blisters (vesication) and injury to the eyes and the respiratory tract; of these, the respiratory injury, if severe, may even be fatal. Therefore, developing an effective therapeutic strategy to protect against SM-induced respiratory injury is an urgent priority of not only the US military but also the civilian antiterrorism agencies, for example, the Homeland Security. Toward developing a respiratory medical countermeasure for SM, four different classes of therapeutic compounds have been evaluated in the past: anti-inflammatory compounds, antioxidants, protease inhibitors and antiapoptotic compounds. This review examines all of these different options; however, it suggests that preventing cell death by inhibiting apoptosis seems to be a compelling strategy but possibly dependent on adjunct therapies using the other drugs, that is, anti-inflammatory, antioxidant, and protease inhibitor compounds.

Postexposure application of Fas receptor small-interfering RNA to suppress sulfur mustard-induced apoptosis in human airway epithelial cells: implication for a therapeutic approach.

Sulfur mustard (SM) is a vesicant chemical warfare and terrorism agent. Besides skin and eye injury, respiratory damage has been mainly responsible for morbidity and mortality after SM exposure. Previously, it was shown that suppressing the death receptor (DR) response by the dominant-negative Fas-associated death domain protein prior to SM exposure blocked apoptosis and microvesication in skin. Here, we studied whether antagonizing the Fas receptor (FasR) pathway by small-interfering RNA (siRNA) applied after SM exposure would prevent apoptosis and, thus, airway injury. Normal human bronchial/tracheal epithelial (NHBE) cells were used as an in vitro model with FasR siRNA, FasR agonistic antibody CH11, and FasR antagonistic antibody ZB4 as investigative tools. In NHBE cells, both SM (300 µM) and CH11 (100 ng/ml) induced caspase-3 activation, which was inhibited by FasR siRNA and ZB4, indicating that SM-induced apoptosis was via the Fas response. FasR siRNA inhibited SM-induced caspase-3 activation when added to NHBE cultures up to 8 hours after SM. Results using annexin V/propidium iodide-stained cells showed that both apoptosis and necrosis were involved in cell death due to SM; FasR siRNA decreased both apoptotic and necrotic cell populations. Bronchoalveolar lavage fluid (BALF) of rats exposed to SM (1 mg/kg, 50 minutes) revealed a significant (P < 0.05) increase in soluble Fas ligand and active caspase-3 in BALF cells. These findings suggest an intervention of Fas-mediated apoptosis as a postexposure therapeutic strategy with a therapeutic window for SM inhalation injury and possibly other respiratory diseases involving the Fas response.

Overlapping and distinct gray and white matter abnormalities in schizophrenia and bipolar I disorder.

OBJECTIVES: Schizophrenia and bipolar disorder may share common neurobiological mechanisms, but few studies have directly compared gray and white matter structure in these disorders. We used diffusion-weighted magnetic resonance imaging and a region of interest based analysis to identify overlapping and distinct gray and white matter abnormalities in 35 patients with schizophrenia and 20 patients with bipolar I disorder in comparison to 56 healthy volunteers. METHODS: We examined fractional anisotropy within the white matter and mean diffusivity within the gray matter in 42 regions of interest defined on a probabilistic atlas following non-linear registration of the images to atlas space. RESULTS: Patients with schizophrenia had significantly lower fractional anisotropy in temporal (superior temporal and parahippocampal) and occipital (superior and middle occipital) white matter compared to patients with bipolar disorder and healthy volunteers. By contrast, both patient groups demonstrated significantly higher mean diffusivity in frontal (inferior frontal and lateral orbitofrontal) and temporal (superior temporal and parahippocampal) gray matter compared to healthy volunteers, but did not differ from each other. CONCLUSIONS: Our study implicates overlapping gray matter frontal and temporal lobe structural alterations in the neurobiology of schizophrenia and bipolar I disorder, but suggests that temporal and occipital lobe white matter deficits may be an additional risk factor for schizophrenia. Our findings may have relevance for future diagnostic classification systems and the identification of susceptibility genes for these disorders.

The quality of survey research in burn care: A systematic review.

INTRODUCTION: Burn care is a relatively small, mutidisciplinary field with variability in practices between centers. Given these factors, survey studies are frequently used to better understand practice variations, establish guidelines, and direct future research. If survey research is poorly designed or reported, it limits the ability to form meaningful conclusions. This study evaluates the quality of survey studies published in burn care and determines areas of improvement to increase generalizability. METHODS: A systematic review was performed by two independent reviewers. Three databases (PubMed, Scopus, Web of Science) were queried between January 1, 2000 and March 19, 2020. Studies were included if they surveyed any member of the multidisciplinary burn team on a topic related to burn care, and surveys of non-clinicians were excluded. Data related to survey content, methodology, and quality was extracted for analysis. RESULTS: Of 247 citations, 144 met inclusion criteria. The number of published surveys increased by an average of 23% annually over the study period (p < 0.001). Studies represented a breadth of countries, scopes, themes, and disciplines. Few studies reported using reminders or incentives. The majority did not report survey development steps or validity/reliability, and half did not include the questionnaire in the publication. The median (IQR) response rate of all studies was 54% (32-83). A subgroup analysis of surveys to North American burn directors (N = 28) had a response rate of 40% (26-50). CONCLUSION: Survey reporting in the burn care literature is generally inconsistent, limiting the ability to apply this research into practice.

Pathological studies on the protective effect of a macrolide antibiotic, roxithromycin, against sulfur mustard inhalation toxicity in a rat model.

Macrolide antibiotics have been shown to protect airway epithelial cells and macrophages from sulfur mustard (SM)-induced cytotoxicity. In the current study, the efficacy of roxithromycin in ameliorating SM-induced respiratory injury was further evaluated in a rat model. Anesthetized rats (N = 8/group) were intratracheally exposed to SM by vapor inhalation. For the drug treatment groups, rats were orally given 10, 20, or 40 mg/kg roxithromycin one hr prior to exposure and every twenty-four hr thereafter. After one, three, or seven days of treatment, sections of the lung were examined and scored for histopathological parameters. Treatment with roxithromycin ameliorated many of the symptoms caused by SM in some animals. In particular, treatment at 40 mg/kg for three days showed significant improvements (p < .05) over the untreated group. When the evaluation was focused on trachea, treatment with roxithromycin for three days showed a trend of dose-dependent protection; moreover, the groups treated with 20 or 40 mg/kg of roxithromycin were statistically different (p < .001 and p < .05, respectively) from the untreated group. These results suggest that roxithromycin protects against some damages associated with SM injury in the lung, particularly in the upper respiratory tract.

Enterococcus faecalis Is Associated with Anastomotic Leak in Patients Undergoing Colorectal Surgery.

Background: Anastomotic leak is among the most dreaded complications in patients undergoing colorectal surgery. We have discovered that in rodents, collagenase-producing bacteria, particularly Enterococcus faecalis, promotes anastomotic leak by degrading healing anastomotic tissue. Yet, it is unclear if these organisms play a role in humans. Patients and Methods: Patients undergoing colorectal resection at the University of Chicago from July 2014 through June 2019 who developed a post-operative infection were stratified into infections that resulted from an anastomotic leak, a Hartmann pouch stump leak, or a deep infection without an associated staple line leak. Results: Forty-two patients had available culture data. Of these patients, 19 were found to have an anastomotic leak, 7 had a stump leak, and 16 had a deep infection that was not associated with a staple line. Enterococcus faecalis was identified in 24% of all infections and was associated with the development of anastomotic leak (p = 0.029). When the organisms were classified into their known ability to produce collagenase, 74% of patients with an anastomotic leak were colonized with collagenase-producing organisms, compared with only 28% of patients with a deep infection or stump leak (p = 0.022). Antibiotic-resistant organisms were more common in patients with anastomotic leak (p = 0.01). Conclusions: Collagenase-producing and antibiotic-resistant organisms are more prevalent in anastomotic leak infections compared with other deep or organ/space infections. This lends evidence to a bacterial driven pathogenesis of leak and suggests that targeting these organisms may be a novel strategy to reduce this complication.

Inflammatory effects of inhaled sulfur mustard in rat lung.

Inhalation of sulfur mustard (SM), a bifunctional alkylating agent that causes severe lung damage, is a significant threat to both military and civilian populations. The mechanisms mediating its cytotoxic effects are unknown and were investigated in the present studies. Male rats Crl:CD(SD) were anesthetized, and then intratracheally intubated and exposed to 0.7-1.4mg/kg SM by vapor inhalation. Animals were euthanized 6, 24, 48h or 7days post-exposure and bronchoalveolar lavage fluid (BAL) and lung tissue collected. Exposure of rats to SM resulted in rapid pulmonary toxicity, including focal ulceration and detachment of the trachea and bronchial epithelia from underlying mucosa, thickening of alveolar septal walls and increased numbers of inflammatory cells in the tissue. There was also evidence of autophagy and apoptosis in the tissue. This was correlated with increased BAL protein content, a marker of injury to the alveolar epithelial lining. SM exposure also resulted in increased expression of markers of inflammation including cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNFα), inducible nitric oxide synthase (iNOS), and matrix metalloproteinase-9 (MMP-9), each of which has been implicated in pulmonary toxicity. Whereas COX-2, TNFα and iNOS were mainly localized in alveolar regions, MMP-9 was prominent in bronchial epithelium. In contrast, expression of the anti-oxidant hemeoxygenase, and the anti-inflammatory collectin, surfactant protein-D, decreased in the lung after SM exposure. These data demonstrate that SM-induced oxidative stress and injury are associated with the generation of cytotoxic inflammatory proteins which may contribute to the pathogenic response to this vesicant.

Protective role of PI3-kinase/Akt/eNOS signaling in mechanical stress through inhibition of p38 mitogen-activated protein kinase in mouse lung.

AIM: To test the hypothesis that PI3K/Akt/eNOS signaling has a protective role in a murine model of ventilation associated lung injury (VALI) through down-regulation of p38 MAPK signaling. METHODS: Male C57BL/J6 (wild-type, WT) or eNOS knockout mice (eNOS(-/-)) were exposed to mechanical ventilation (MV) with low (LV(T), 7 mL/kg) and high tidal volume (HV(T), 20 mL/kg) for 0-4 h. A subset of WT mice was administered the specific inhibitors of PI3K (100 nmol/L Wortmannin [Wort], ip) or of p38 MAPK (SB203580, 2 mg/kg, ip) 1 h before MV. Cultured type II alveolar epithelial cells C10 were exposed to 18% cyclic stretch for 2 h with or without 20 nmol/L Wort pretreatment. At the end of the experiment, the capillary leakage in vivo was assessed by extravasation of Evans blue dye (EBD), wet/dry weight ratio and lung lavage protein concentration. The lung tissue and cell lysate were also collected for protein and histological review. RESULTS: MV decreased PI3K/Akt phosphorylation and eNOS expression but increased phospho-p38 MAPK expression along with a lung leakage of EBD. Inhibitions of phospho-Akt by Wort worsen the lung edema, whereas inhibition of p38 MAPK kinase restored activation of Akt together with alleviated capillary leakage. eNOS(-/-) mice showed an exacerbated lung edema and injury. The stretched C10 cells demonstrated that Wort diminished the activation of Akt, but potentiated phosphorylation of MAPK p38. CONCLUSION: Our results indicate that PI-3K/Akt/eNOS pathway has significant protective effects in VALI by preventing capillary leakage, and that there is a cross-talk between PI3K/Akt and p38 MAPK pathways in vascular barrier dysfunction resulting from VALI.

Oral immunization with bacteriophage MS2-L2 VLPs protects against oral and genital infection with multiple HPV types associated with head & neck cancers and cervical cancer.

Human papillomaviruses (HPVs) are the most common sexually transmitted infections. HPVs are transmitted through anogenital sex or oral sex. Anogenital transmission/infection is associated with anogenital cancers and genital warts while oral transmission/infection is associated with head and neck cancers (HNCs) including recurrent respiratory papillomatosis. Current HPV vaccines protect against HPV types associated with ∼90% of cervical cancers and are expected to protect against a percentage of HNCs. However, only a few studies have assessed the efficacy of current vaccines against oral HPV infections. We had previously developed a mixed MS2-L2 candidate HPV vaccine based on bacteriophage MS2 virus-like particles (VLPs). The mixed MS2-L2 VLPs consisted of a mixture of two MS2-L2 VLPs displaying: i) a concatemer of L2 peptide (epitope 20-31) from HPV31 & L2 peptide (epitope 17-31) from HPV16 and ii) a consensus L2 peptide representing epitope 69-86. The mixed MS2-L2 VLPs neutralized/protected mice against six HPV types associated with ∼87% of cervical cancer. Here, we show that the mixed MS2-L2 VLPs can protect mice against additional HPV types; at the genital region, the VLPs protect against HPV53, 56, 11 and at the oral region, the VLPs protect against HPV16, 35, 39, 52, and 58. Thus, mixed MS2-L2 VLPs protect against eleven oncogenic HPV types associated with ∼95% of cervical cancer. The VLPs also have the potential to protect, orally, against the same oncogenic HPVs, associated with ∼99% of HNCs, including HPV11, which is associated with up to 32% of recurrent respiratory papillomatosis. Moreover, mixed MS2-L2 VLPs are thermostable at room temperature for up to 60 days after spray-freeze drying and they are protective against oral HPV infection.

Synthesis and Molecular Properties of Nerve Agent Reactivator HLö-7 Dimethanesulfonate.

The threat of a deliberate release of chemical nerve agents has underscored the need to continually improve field effective treatments for these types of poisonings. The oxime containing HLö-7 is a potential second-generation therapeutic reactivator. A synthetic process for HLö-7 is detailed with improvements to the DIBAL reduction and ion exchange steps. HLö-7 was visualized for the first time within the active site of human acetylcholinesterase and its relative ex vivo potency confirmed against various nerve agents using a phrenic nerve hemidiaphragm assay.

Editor's Highlight: Pulmonary Vascular Thrombosis in Rats Exposed to Inhaled Sulfur Mustard.

Sulfur mustard (SM) is a chemical warfare agent. When inhaled, SM causes significant injury to the respiratory tract. Although the mechanism involved in acute airway injury after SM inhalation has been well described previously, the mechanism of SM's contribution to distal lung vascular injury is not well understood. We hypothesized that acute inhalation of vaporized SM causes activated systemic coagulation with subsequent pulmonary vascular thrombi formation after SM inhalation exposure. Sprague Dawley rats inhaled SM ethanolic vapor (3.8 mg/kg). Barium/gelatin CT pulmonary angiograms were performed to assess for pulmonary vascular thrombi burden. Lung immunohistochemistry was performed for common procoagulant markers including fibrin(ogen), von Willebrand factor, and CD42d in control and SM-exposed lungs. Additionally, systemic levels of d-dimer and platelet aggregometry after adenosine diphosphate- and thrombin-stimulation were measured in plasma after SM exposure. In SM-exposed lungs, chest CT angiography demonstrated a significant decrease in the distal pulmonary vessel density assessed at 6 h postexposure. Immunohistochemistry also demonstrated increased intravascular fibrin(ogen), vascular von Willebrand factor, and platelet CD42d in the distal pulmonary vessels (<200 µm diameter). Circulating d-dimer levels were significantly increased (p < .001) at 6, 9, and 12 h after SM inhalation versus controls. Platelet aggregation was also increased in both adenosine diphosphate - (p < .01) and thrombin- (p < .001) stimulated platelet-rich plasma after SM inhalation. Significant pulmonary vascular thrombi formation was evident in distal pulmonary arterioles following SM inhalation in rats assessed by CT angiography and immunohistochemistry. Enhanced systemic platelet aggregation and activated systemic coagulation with subsequent thrombi formation likely contributed to pulmonary vessel occlusion.

Calmodulin mediates sulfur mustard toxicity in human keratinocytes.

Sulfur mustard (SM) causes blisters in the skin through a series of cellular changes that we are beginning to identify. We earlier demonstrated that SM toxicity is the result of induction of both death receptor and mitochondrial pathways of apoptosis in human keratinocytes (KC). Because of its importance in apoptosis in the skin, we tested whether calmodulin (CaM) mediates the mitochondrial apoptotic pathway induced by SM. Of the three human CaM genes, the predominant form expressed in KC was CaM1. RT-PCR and immunoblot analysis revealed upregulation of CaM expression following SM treatment. To delineate the potential role of CaM1 in the regulation of SM-induced apoptosis, retroviral vectors expressing CaM1 RNA in the antisense (AS) orientation were used to transduce and derive stable CaM1 AS cells, which were then exposed to SM and subjected to immunoblot analysis for expression of apoptotic markers. Proteolytic activation of executioner caspases-3, -6, -7, and the upstream caspase-9, as well as caspase-mediated PARP cleavage were markedly inhibited by CaM1 AS expression. CaM1 AS depletion attenuated SM-induced, but not Fas-induced, proteolytic processing and activation of caspase-3. Whereas control KC exhibited a marked increase in apoptotic nuclear fragmentation after SM, CaM1 AS cells exhibited normal nuclear morphology up to 48h after SM, indicating that suppression of apoptosis in CaM1 AS cells increases survival and does not shift to a necrotic death. CaM has been shown to activate the phosphatase calcineurin, which can induce apoptosis by Bad dephosphorylation. Interestingly, whereas SM-treated CaM1-depleted KC expressed the phosphorylated non-apoptotic sequestered form of Bad, Bad was present in the hypophosphorylated apoptotic form in SM-exposed control KC. To determine if pharmacological CaM inhibitors could attenuate SM-induced apoptosis via Bad dephosphorylation, KC were pretreated with the CaM-specific antagonist W-13 or its less active structural analogue W-12. Following SM exposure, KC exhibited Bad dephosphorylation, which was inhibited in the presence of W-13, but not with W-12. Consequently, W-13 but not W-12 markedly suppressed SM-induced proteolytic processing and activation of caspase-3, as well as apoptotic nuclear fragmentation. Finally, while the CaM antagonist W-13 and the calcineurin inhibitor cyclosporin A attenuated SM-induced caspase-3 activation, inhibitors for CaM-dependent protein kinase II (KN62 and KN93) did not. These results indicate that CaM, calcineurin, and Bad also play a role in SM-induced apoptosis, and may therefore be targets for therapeutic intervention to reduce SM injury.