Faculty Bibliography

Human skin is a remarkably plastic organ that sustains insult and injury throughout life. Its ability to expeditiously repair wounds is paramount to survival and is thought to be regulated by wound components such as differentiated cells, stem cells, cytokine networks, extracellular matrix, and mechanical forces. These intrinsic regenerative pathways are integrated across different skin compartments and are being elucidated on the cellular and molecular levels. Recent advances in bioengineering and nanotechnology have allowed researchers to manipulate these microenvironments in increasingly precise spatial and temporal scales, recapitulating key homeostatic cues that may drive regeneration. The ultimate goal is to translate these bench achievements into viable bedside therapies that address the growing global burden of acute and chronic wounds. In this review, we highlight current concepts in cutaneous wound repair and propose that many of these evolving paradigms may underlie regenerative processes across diverse organ systems.

Protein function often depends on the exchange between conformational substates. Allosteric ligand binding or distal mutations can stabilize specific active-site conformations and consequently alter protein function. Observing alternative conformations at low levels of electron density, in addition to comparison of independently determined X-ray crystal structures, can provide mechanistic insights into conformational dynamics. Here we report a new algorithm, CONTACT, that identifies contact networks of conformationally heterogeneous residues directly from high-resolution X-ray crystallography data. Contact networks determined for Escherichia coli dihydrofolate reductase (ecDHFR) predict the observed long-range pattern of NMR chemical shift perturbations of an allosteric mutation. A comparison of contact networks in wild-type and mutant ecDHFR suggests that mutations that alter optimized contact networks of coordinated motions can impair catalytic function. CONTACT-guided mutagenesis can exploit the structure-dynamics-function relationship in protein engineering and design.

In response to different environmental stresses, eIF2alpha phosphorylation represses global translation coincident with preferential translation of ATF4, a master regulator controlling the transcription of key genes essential for adaptative functions. Here, we establish that the eIF2alpha/ATF4 pathway directs an autophagy gene transcriptional program in response to amino acid starvation or endoplasmic reticulum stress. The eIF2alpha-kinases GCN2 and PERK and the transcription factors ATF4 and CHOP are also required to increase the transcription of a set of genes implicated in the formation, elongation and function of the autophagosome. We also identify three classes of autophagy genes according to their dependence on ATF4 and CHOP and the binding of these factors to specific promoter cis elements. Furthermore, different combinations of CHOP and ATF4 bindings to target promoters allow the trigger of a differential transcriptional response according to the stress intensity. Overall, this study reveals a novel regulatory role of the eIF2alpha-ATF4 pathway in the fine-tuning of the autophagy gene transcription program in response to stresses.

BACKGROUND: Functional dyspepsia-epigastric pain syndrome (FD-EPS) is characterized pathophysiologically by visceral hypersensitivity, but the effect of the temperature stimulation on gastric function has been seldom studied. AIM: The purpose of this study was to investigate the effects of liquid nutrients at different temperatures on the gastric accommodation, sensitivity, and gastric-wall compliance of healthy subjects (HS) and FD-EPS patients. METHODS: Ten FD-EPS patients (Roma III criteria) and ten HS were recruited into the study. Intragastric pressure (IGP) and gastric perfusion were measured and compared following the administration of liquid nutrients at 37 degrees C on day 1 and at 8 degrees C on day 2. RESULTS: Seven patients developed abdominal discomfort or abdominal pain after being given cold liquid nutrient. The administration of liquid nutrient at 8 degrees C resulted in an increase of IGP in HS (P=0.044), a significant decrease in gastric perfusion (P<0.0001), a marked increase in IGP (P=0.015), and a dramatic reduction in gastric wall compliance (P=0.012) in patients compared to the effects of liquid nutrient at 37 degrees C. In addition, IGP in patients was lower than that in HS at 37 degrees C liquid nutrient (P=0.036), and the gastric perfusion volume in patients at maximal satiety was also significantly reduced at 8 degrees C liquid nutrient compared with HS (P=0.017). CONCLUSIONS: Cold stimulation can increase the IGP in HS and FD-EPS patients, elevate the visceral sensitivity and reduce the gastric volume of FD-EPS patients. FD-EPS patients who are sensitive to cold may develop epigastric discomfort or pain.

Activation of the human embryonic stem cell (hESC) signature genes has been observed in various epithelial cancers. In this study, we found that the hESC signature is selectively induced in the airway basal stem/progenitor cell population of healthy smokers (BC-S), with a pattern similar to that activated in all major types of human lung cancer. We further identified a subset of 6 BC-S hESC genes, whose coherent overexpression in lung adenocarcinoma (AdCa) was associated with reduced lung function, poorer differentiation grade, more advanced tumor stage, remarkably shorter survival, and higher frequency of TP53 mutations. BC-S shared with hESC and a considerable subset of lung carcinomas a common TP53 inactivation molecular pattern which strongly correlated with the BC-S hESC gene expression. These data provide transcriptome-based evidence that smoking-induced reprogramming of airway BC toward the hESC-like phenotype might represent a common early molecular event in the development of aggressive lung carcinomas in humans.

BACKGROUND: Many breast cancer patients are plagued by the disabling complication of upper limb lymphedema after axillary surgery. Conservative treatments using massage and compression therapy do not offer a lasting relief, as they fail to address the chronic transformation of edema into excess adipose tissue. Liposuction to address the adipose nature of the lymphedema has provided an opportunity for a detailed analysis of the stromal fraction of lymphedema-associated fat to clarify the molecular mechanisms for this adipogenic transformation. METHODS: Adipose-derived stem cells were harvested from human lipoaspirate of the upper extremity from age-matched patients with lymphedema (n = 3) or subcutaneous adipose tissue from control patients undergoing cosmetic procedures (n = 3). Immediately after harvest, adipose-derived stem cells were analyzed using single-cell transcriptional profiling techniques. Osteogenic, adipogenic, and vasculogenic gene expression and differentiation were assessed by quantitative real-time polymerase chain reaction and standard in vitro differentiation assays. RESULTS: Differential transcriptional clusters of adipose-derived stem cells were found between lymphedema and subcutaneous fat. Interestingly, lymphedema-associated stem cells had a much higher adipogenic gene expression and enhanced ability to undergo adipogenic differentiation. Conversely, they had lower vasculogenic gene expression and diminished capability to form tubules in vitro, whereas the osteogenic differentiation capacity was not significantly altered. CONCLUSIONS: Adipose-derived stem cells from extremities affected by lymphedema appear to exhibit transcriptional profiles similar to those of abdominal adipose-derived stem cells; however, their adipogenic differentiation potential is strongly increased and their vasculogenic capacity is compromised. These results suggest that the underlying pathophysiology of lymphedema drives adipose-derived stem cells toward adipogenic differentiation.

Myeloperoxidase (MPO) and paraoxonase 1 (PON1) are high-density lipoprotein-associated (HDL-associated) proteins mechanistically linked to inflammation, oxidant stress, and atherosclerosis. MPO is a source of ROS during inflammation and can oxidize apolipoprotein A1 (APOA1) of HDL, impairing its atheroprotective functions. In contrast, PON1 fosters systemic antioxidant effects and promotes some of the atheroprotective properties attributed to HDL. Here, we demonstrate that MPO, PON1, and HDL bind to one another, forming a ternary complex, wherein PON1 partially inhibits MPO activity, while MPO inactivates PON1. MPO oxidizes PON1 on tyrosine 71 (Tyr71), a modified residue found in human atheroma that is critical for HDL binding and PON1 function. Acute inflammation model studies with transgenic and knockout mice for either PON1 or MPO confirmed that MPO and PON1 reciprocally modulate each other's function in vivo. Further structure and function studies identified critical contact sites between APOA1 within HDL, PON1, and MPO, and proteomics studies of HDL recovered from acute coronary syndrome (ACS) subjects revealed enhanced chlorotyrosine content, site-specific PON1 methionine oxidation, and reduced PON1 activity. HDL thus serves as a scaffold upon which MPO and PON1 interact during inflammation, whereupon PON1 binding partially inhibits MPO activity, and MPO promotes site-specific oxidative modification and impairment of PON1 and APOA1 function.

The regulation of the metabolism of cholesterol has been one of the most studied biological processes since its first isolation from gallstones in 1784. High levels of plasma low-density lipoprotein (LDL) cholesterol and reduced levels of plasma high-density lipoprotein (HDL) cholesterol are widely recognized as major risk factors of cardiovascular disease. An imbalance in the production of reactive oxygen species can oxidize LDL particles, increasing the levels of the highly proatherogenic oxidized LDL. Furthermore, under pathological scenarios, numerous molecules can function as pro-oxidants, such as iron or (high levels of) glucose. In addition to the classical mechanisms regulating lipid homeostasis, recent studies have demonstrated the important role of microRNAs (miRNAs) as regulators of lipoprotein metabolism, oxidative derivatives of lipoprotein, and redox balance. Here, we summarize recent findings in the field, highlighting the contributions of some miRNAs to lipid- and oxidative-associated pathologies. We also discuss how therapeutic intervention of miRNAs may be a promising strategy to decrease LDL, increase HDL, and ameliorate lipid- and oxidative-related disorders, including atherosclerosis, nonalcoholic fatty liver disease, and metabolic syndrome.

Multiple reaction monitoring (MRM) mass spectrometry coupled with stable isotope dilution (SID) and liquid chromatography (LC) is increasingly used in biological and clinical studies for precise and reproducible quantification of peptides and proteins in complex sample matrices. Robust LC-SID-MRM-MS-based assays that can be replicated across laboratories and ultimately in clinical laboratory settings require standardized protocols to demonstrate that the analysis platforms are performing adequately. We developed a system suitability protocol (SSP), which employs a predigested mixture of six proteins, to facilitate performance evaluation of LC-SID-MRM-MS instrument platforms, configured with nanoflow-LC systems interfaced to triple quadrupole mass spectrometers. The SSP was designed for use with low multiplex analyses as well as high multiplex approaches when software-driven scheduling of data acquisition is required. Performance was assessed by monitoring of a range of chromatographic and mass spectrometric metrics including peak width, chromatographic resolution, peak capacity, and the variability in peak area and analyte retention time (RT) stability. The SSP, which was evaluated in 11 laboratories on a total of 15 different instruments, enabled early diagnoses of LC and MS anomalies that indicated suboptimal LC-MRM-MS performance. The observed range in variation of each of the metrics scrutinized serves to define the criteria for optimized LC-SID-MRM-MS platforms for routine use, with pass/fail criteria for system suitability performance measures defined as peak area coefficient of variation <0.15, peak width coefficient of variation <0.15, standard deviation of RT <0.15 min (9 s), and the RT drift <0.5min (30 s). The deleterious effect of a marginally performing LC-SID-MRM-MS system on the limit of quantification (LOQ) in targeted quantitative assays illustrates the use and need for a SSP to establish robust and reliable system performance. Use of a SSP helps to ensure that analyte quantification measurements can be replicated with good precision within and across multiple laboratories and should facilitate more widespread use of MRM-MS technology by the basic biomedical and clinical laboratory research communities.