Engineering key components in a synthetic eukaryotic signal transduction pathway

Signal transduction underlies how living organisms detect and respond to stimuli. A goal of synthetic biology is to rewire natural signal transduction systems. Bacteria, yeast, and plants sense environmental aspects through conserved histidine kinase (HK) signal transduction systems. HK protein components are typically comprised of multiple, relatively modular, and conserved domains. Phosphate transfer between these components may exhibit considerable cross talk between the otherwise apparently linear pathways, thereby establishing networks that integrate multiple signals. We show that sequence conservation and cross talk can extend across kingdoms and can be exploited to produce a synthetic plant signal transduction system. In response to HK cross talk, heterologously expressed bacterial response regulators, PhoB and OmpR, translocate to the nucleus on HK activation. Using this discovery, combined with modification of PhoB (PhoB-VP64), we produced a key component of a eukaryotic synthetic signal transduction pathway. In response to exogenous cytokinin, PhoB-VP64 translocates to the nucleus, binds a synthetic PlantPho promoter, and activates gene expression. These results show that conserved-signaling components can be used across kingdoms and adapted to produce synthetic eukaryotic signal transduction pathways

Clinically-relevant cutaneous lesions by nitrogen mustard: useful biomarkers of vesicants skin injury in SKH-1 hairless and C57BL/6 mice.

A paucity of clinically applicable biomarkers to screen therapies in laboratory is a limitation in the development of countermeasures against cutaneous injuries by chemical weapon, sulfur mustard (SM), and its analog nitrogen mustard (NM). Consequently, we assessed NM-caused progression of clinical cutaneous lesions; notably, skin injury with NM is comparable to SM. Exposure of SKH-1 hairless and C57BL/6 (haired) mice to NM (3.2 mg) for 12-120 h caused clinical sequelae of toxicity, including microblister formation, edema, erythema, altered pigmentation, wounding, xerosis and scaly dry skin. These toxic effects of NM were similar in both mouse strains, except that wounding and altered pigmentation at 12-24 h and appearance of dry skin at 24 and 72 h post-NM exposure were more pronounced in C57BL/6 compared to SKH-1 mice. Conversely, edema, erythema and microblister formation were more prominent in SKH-1 than C57BL/6 mice at 24-72 h after NM exposure. In addition, 40-60% mortality was observed following 120 h of NM exposure in the both mouse strains. Overall, these toxic effects of NM are comparable to those reported in humans and other animal species with SM, and thus represent clinically-relevant cutaneous injury endpoints in screening and optimization of therapies for skin injuries by vesicating agents

Cutaneous injury-related structural changes and their progression following topical nitrogen mustard exposure in hairless and haired mice.

To identify effective therapies against sulfur mustard (SM)-induced skin injuries, various animals have been used to assess the cutaneous pathology and related histopathological changes of SM injuries. However, these efforts to establish relevant skin injury endpoints for efficacy studies have been limited mainly due to the restricted assess of SM. Therefore, we employed the SM analog nitrogen mustard (NM), a primary vesicating and bifunctional alkylating agent, to establish relevant endpoints for efficient efficacy studies. Our published studies show that NM (3.2 mg) exposure for 12-120 h in both the hairless SKH-1 and haired C57BL/6 mice caused clinical sequelae of toxicity similar to SM exposure in humans. The NM-induced cutaneous pathology-related structural changes were further analyzed in this study and quantified morphometrically (as percent length or area of epidermis or dermis) of skin sections in mice showing these lesions. H&E stained skin sections of both hairless and haired mice showed that NM (12-120 h) exposure caused epidermal histopathological effects such as increased epidermal thickness, epidermal-dermal separation, necrotic/dead epidermis, epidermal denuding, scab formation, parakeratosis (24-120 h), hyperkeratosis (12-120 h), and acanthosis with hyperplasia (72-120 h). Similar NM exposure in both mice caused dermal changes including necrosis, edema, increase in inflammatory cells, and red blood cell extravasation. These NM-induced cutaneous histopathological features are comparable to the reported lesions from SM exposure in humans and animal models. This study advocates the usefulness of these histopathological parameters observed due to NM exposure in screening and optimization of rescue therapies against NM and SM skin injuries

Silibinin attenuates sulfur mustard analog-induced skin injury by targeting multiple pathways connecting oxidative stress and inflammation.

Chemical warfare agent sulfur mustard (HD) inflicts delayed blistering and incapacitating skin injuries. To identify effective countermeasures against HD-induced skin injuries, efficacy studies were carried out employing HD analog 2-chloroethyl ethyl sulfide (CEES)-induced injury biomarkers in skin cells and SKH-1 hairless mouse skin. The data demonstrate strong therapeutic efficacy of silibinin, a natural flavanone, in attenuating CEES-induced skin injury and oxidative stress. In skin cells, silibinin (10 µM) treatment 30 min after 0.35/0.5 mM CEES exposure caused a significant (p<0.05) reversal in CEES-induced decrease in cell viability, apoptotic and necrotic cell death, DNA damage, and an increase in oxidative stress. Silibinin (1 mg) applied topically to mouse skin 30 min post-CEES exposure (2 mg), was effective in reversing CEES-induced increases in skin bi-fold (62%) and epidermal thickness (85%), apoptotic cell death (70%), myeloperoxidase activity (complete reversal), induction of iNOS, COX-2, and MMP-9 protein levels (>90%), and activation of transcription factors NF-κB and AP-1 (complete reversal). Similarly, silibinin treatment was also effective in attenuating CEES-induced oxidative stress measured by 4-hydroxynonenal and 5,5-dimethyl-2-(8-octanoic acid)-1-pyrolline N-oxide protein adduct formation, and 8-oxo-2-deoxyguanosine levels. Since our previous studies implicated oxidative stress, in part, in CEES-induced toxic responses, the reversal of CEES-induced oxidative stress and other toxic effects by silibinin in this study indicate its pleiotropic therapeutic efficacy. Together, these findings support further optimization of silibinin in HD skin toxicity model to develop a novel effective therapy for skin injuries by vesicants