Faculty Bibliography

SIGNIFICANCE: Wound repair is a complex biological process that integrates multiple physiologic pathways to restore skin homeostasis after a wide array of gross and anatomical insults. As such, a scientific examination of the wound typically requires broad sampling of numerous factors and is commonly achieved through DNA microarray analysis. CRITICAL ISSUES: In the last several years, it has become increasingly evident that the granularity afforded by such traditional population-based assays may be insufficient to capture the complex relationships in heterogeneous processes such as those associated with wound healing and stem cell biology. RECENT ADVANCES: Several emerging technologies have recently become available that permit high-throughput single-cell gene expression analysis in a manner which provides novel insights into the relationships of complex tissue. The most prominent among these employs microfluidic-based devices to achieve a high-resolution analysis of tissue samples. FUTURE DIRECTIONS: The intrinsically heterogeneous nature of injured tissue, in conjunction with its temporal dynamics, makes wound repair and tissue regeneration an attractive target for high-throughput single-cell analysis. Given the staggering costs associated with chronic and non-healing wounds, the development of predictive and diagnostic tools using this technology would likely be attractive to healthcare providers.

Accumulation of proteins in the endoplasmic reticulum (ER) typically induces stress and initiates the unfolded protein response (UPR) to facilitate recovery. If homeostasis is not restored, apoptosis is induced. However, adaptation to chronic UPR activation can increase resistance to subsequent acute ER stress. We therefore investigated adaptive mechanisms in Oculocutaneous albinism type 2 (Oca2)-null melanocytes where UPR signaling is arrested despite continued tyrosinase accumulation leading to resistance to the chemical ER stressor thapsigargin. Although thapsigargin triggers UPR activation, instead of Perk-mediated phosphorylation of eIF2alpha, in Oca2-null melanocytes, eIF2alpha was rapidly dephosphorylated upon treatment. Dephosphorylation was mediated by the Gadd34-PP1alpha phosphatase complex. Gadd34-complex inhibition blocked eIF2alpha dephosphorylation and significantly increased Oca2-null melanocyte sensitivity to thapsigargin. Thus, Oca2-null melanocytes adapt to acute ER stress by disruption of pro-apoptotic Perk signaling, which promotes cell survival. This is the first study to demonstrate rapid eIF2alpha dephosphorylation as an adaptive mechanism to ER stress.

INTRODUCTION: Normal wound healing mechanisms can be overwhelmed in the setting of complex acute and chronic tissue injury. Biological therapies are designed to augment and/or restore the body's natural wound healing abilities. There are a variety of available and emerging technologies utilizing this approach that have demonstrated the ability to augment wound healing. AREAS COVERED: In this review, the clinical data on launched and emerging biological therapies for wound healing applications are summarized. The methodologies discussed include biological skin equivalents, growth factors/small molecules and stem cell-based therapies. EXPERT OPINION: While many products possess convincing clinical data demonstrating their efficacy in comparison to standard treatment options, more robust, controlled studies are needed to determine the relative value among established and emerging biological therapies. Future bioengineering and stem cell-based approaches are of particular interest due to the simultaneous correction of multiple deficiencies present in the nonhealing wound.

Dopamine and estradiol interact in the regulation of lactotroph cell proliferation and prolactin secretion. Ablation of the dopamine D2 receptor gene (Drd2(-/-)) in mice leads to a sexually dimorphic phenotype of hyperprolactinemia and pituitary hyperplasia, which is stronger in females. TGF-beta1 is a known inhibitor of lactotroph proliferation. TGF-beta1 is regulated by dopamine and estradiol, and it is usually down-regulated in prolactinoma experimental models. To understand the role of TGF-beta1 in the gender-specific development of prolactinomas in Drd2(-/-) mice, we compared the expression of different components of the pituitary TGF-beta1 system, including active cytokine content, latent TGF-beta-binding protein isoforms, and possible local TGF-beta1 activators, in males and females in this model. Furthermore, we evaluated the effects of dopamine and estradiol administration to elucidate their role in TGF-beta1 system regulation. The expression of active TGF-beta1, latent TGF-beta-binding protein isoforms, and several putative TGF-beta1 activators evaluated was higher in male than in female mouse pituitary glands. However, Drd2(-/-) female mice were more sensitive to the decrease in active TGF-beta1 content, as reflected by the down-regulation of TGF-beta1 target genes. Estrogen and dopamine caused differential regulation of several components of the TGF-beta1 system. In particular, we found sex- and genotype- dependent regulation of active TGF-beta1 content and a similar expression pattern for 2 of the putative TGF-beta1 activators, thrombospondin-1 and kallikrein-1, suggesting that these proteins could mediate TGF-beta1 activation elicited by dopamine and estradiol. Our results indicate that (1) the loss of dopaminergic tone affects the pituitary TGF-beta1 system more strongly in females than in males, (2) males express higher levels of pituitary TGF-beta1 system components including active cytokine, and (3) estradiol negatively controls most of the components of the system. Because TGF-beta1 inhibits lactotroph proliferation, we propose that the higher levels of the TGF-beta1 system in males could protect or delay the development of prolactinomas in Drd2(-/-) male mice.

BACKGROUND: The cardiac intercalated disc (ID) has been extensively studied by conventional transmission electron microscopy (EM). Yet, novel methods for tissue preservation (high-pressure freezing), image (3D tomographic EM) and analysis (image segmentation) that greatly improve image quality/resolution, have not been applied to the ID. Recent studies show that, at the ID, the gap junction protein Connexin43 is part of an interactome (a "connexome"). Here, we provide a structural characterization of the connexome. METHODS: Adult mouse ventricular tissue was prepared by high-pressure freezing and freeze substitution and embedded in resin. 200 nm thick sections were imaged with a 200kV electron microscope (FEI TF20). Images were collected at a set magnification of 9.6k on a 4kx4k CCD camera set to 2x binning, giving an effective pixel size of 1.76 nm. Dual-axis tilt series (1 masculine steps, +/-70 masculine per axis) were acquired using SerialEM. Protomo software was used for aligning projection images and reconstructing tomograms. Visualization/segmentation of objects of interest was performed in Amira. RESULTS: In addition to classic ID structures, we observed (a) close proximity between gap junctions and mitochondria of opposing cells; (b) a complex network of tubular structures running perpendicular to the long cell axis; these structures showed a hollow interior, with an estimated inner diameter of ~40 nm and were often adjacent to gap junctions or desmosomes; (c) triads formed by lateral edges of gap junctions and desmosomes, with a rough budding vesicle separating the two structures; (d) budding vesicles of approximately 50 nm interrupting the continuity of one side of the gap junction plaque; (e) vesicular bodies of approx. 65 nm in diameter in the intercellular space, and in proximity to gap junction-containing regions. CONCLUSIONS: We describe the nanometric landscape that surrounds gap junctions. We speculate that the connexome includes a physical association with molecules of the mitochondria, desmosome and microtubular network, and propose that microsomes may pass from one cell to another at the ID. Functional characterization of these structures may lead to novel clues as to the mechanisms of inherited or acquired arrhythmias that involve disruption of the ID.

A total of 135 stomach samples from patients with gastrointestinal diseases and normal controls were examined for Helicobacter pylori infection and Candida colonization. Candida krusei was found in specimens from 20% bleeding, 52% ulcer, and 100% gastritis patients, whereas H. pylori infection rates were 82%, 35% and 30%, respectively, for the same groups of patients. C. krusei was not detected in stomach samples from normal controls.

BACKGROUND: Microorganisms living throughout the body comprise the human "microbiota" and play an important role in health and disease. Recent research suggests that alterations in the skin microbiota may underlie chronic wound pathology. Probiotics are bacteria or yeast that confer a health benefit on the host and may have a role in preventing and treating nonhealing wounds by modulating host-microbe interactions. METHODS: The English literature on skin microbiota, chronic wounds, biofilms, and probiotics is reviewed. RESULTS: Recent evidence indicates that disruption of microbial communities and bacteria-host interactions may contribute to impaired wound healing. Preclinical and human studies highlight the potential of probiotics to prevent or treat various infectious, immune-mediated, and inflammatory diseases. CONCLUSIONS: Advances in molecular sequencing and microbiology have shed light on the importance of the human microbiota in development, health, and disease. Probiotics represent a novel approach to altering the microbial environment with beneficial bacteria. Ongoing challenges include the need for better understanding of therapeutic mechanisms, improved regulation of manufacturing practices, and validation in controlled human trials. Current evidence suggests that probiotic-based therapies have considerable potential to exploit host-microbe relationships and improve clinical outcomes.

Doripenem-colistin exerts synergy against some, but not all, Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae strains in vitro. We determined if doripenem MICs and/or ompK36 porin gene mutations impacted the responses of 23 sequence type 258 (ST258), KPC-2-producing strains to the combination of doripenem (8 mug/ml) and colistin (2 mug/ml) during time-kill assays. The median doripenem and colistin MICs were 32 and 4 mug/ml. Doripenem MICs did not correlate with KPC-2 expression levels. Five and 18 strains had wild-type and mutant ompK36, respectively. The most common mutations were IS5 promoter insertions (n = 7) and insertions encoding glycine and aspartic acid at amino acid (aa) positions 134 and 135 (ins aa134-135 GD; n = 8), which were associated with higher doripenem MICs than other mutations or wild-type ompK36 (all P values 8 mug/ml (P = 0.10 and 0.16). Likewise, doripenem-colistin was more active at 12 and 24 h against the wild type/other mutants than ins aa134-135 GD or IS5 mutants (P = 0.007 and 0.0007). By multivariate analysis, the absence of ins aa134-135 GD or IS5 mutations was the only independent predictor of doripenem-colistin responses at 24 h (P = 0.002). In conclusion, ompK36 genotypes identified ST258 KPC-K. pneumoniae strains that were most likely to respond to doripenem-colistin.

Sonic hedgehog (SHH), a key regulator of embryonic neurogenesis, signals directly to neural stem cells (NSCs) in the subventricular zone (SVZ) and to astrocytes in the adult mouse forebrain. The specific mechanism by which the GLI2 and GLI3 transcriptional activators (GLI2(A) and GLI3(A)) and repressors (GLI2(R) and GLI3(R)) carry out SHH signaling has not been addressed. We found that the majority of slow-cycling NSCs express Gli2 and Gli3, whereas Gli1 is restricted ventrally and all three genes are downregulated when NSCs transition into proliferating progenitors. Surprisingly, whereas conditional ablation of Smo in postnatal glial fibrillary acidic protein-expressing cells results in cell-autonomous loss of NSCs and a progressive reduction in SVZ proliferation, without an increase in glial cell production, removal of Gli2 or Gli3 does not alter adult SVZ neurogenesis. Significantly, removing Gli3 in Smo conditional mutants largely rescues neurogenesis and, conversely, expression of a constitutive GLI3(R) in the absence of normal Gli2 and Gli3 abrogates neurogenesis. Thus unattenuated GLI3(R) is a primary inhibitor of adult SVZ NSC function. Ablation of Gli2 and Gli3 revealed a minor role for GLI2(R) and little requirement for GLI(A) function in stimulating SVZ neurogenesis. Moreover, we found that similar rules of GLI activity apply to SHH signaling in regulating SVZ-derived olfactory bulb interneurons and maintaining cortical astrocyte function. Namely, fewer superficial olfactory bulb interneurons are generated in the absence of Gli2 and Gli3, whereas astrocyte partial gliosis results from an increase in GLI3(R). Thus precise titration of GLI(R) levels by SHH is critical to multiple functions of adult NSCs and astrocytes.