Monitoring the stabilities of a mixture of peptides by mass-spectrometry-based techniques.

Biomolecular degradation plays a key role in proteostasis. Typically, proteolytic enzymes degrade proteins into smaller peptides by breaking amino acid bonds between specific residues. Cleavage around proline residues is often missed and requires highly specific enzymes for peptide processing due to the cyclic proline side-chain. However, degradation can occur spontaneously (i.e. in the absence of enzymes). In this study, the influence of the first residue on the stability of a series of penultimate proline containing peptides, with the sequence Xaa-Pro-Gly-Gly (where Xaa is any amino acid), is investigated with mass spectrometry techniques. Peptides were incubated as mixtures at various solution temperatures (70℃ to 90℃) and were periodically sampled over the duration of the experiment. At elevated temperatures, we observe dissociation after the Xaa-Pro motif for all sequences, but at different rates. Transition state thermochemistry was obtained by studying the temperature-dependent kinetics and although all peptides show relatively small differences in the transition state free energies (∼95 kJ/mol), there is significant variability in the transition state entropy and enthalpy. This demonstrates that the side-chain of the first amino acid has a significant influence on the stability of the Xaa-Pro sequence. From these data, we demonstrate the ability to simultaneously measure the dissociation kinetics and relative transition state thermochemistries for a mixture of peptides, which vary only in the identity of the N-terminal amino acid.

Pemphigus Vulgaris Persistently Localized to the Nose with Local and Systemic Response to Topical Steroids.

is missing (Short communication).

The ABCs of Perineal Streptococcal Dermatitis.

Perineal streptococcal dermatitis (PSD) is largely known to be caused by group A ß-hemolytic Streptococcus (GAS). We would like to bring cases of non-GAS PSD to the attention of dermatologists, as there are implications for workup and therapy. We report 3 pediatric cases of PSD: 1 caused by GAS, 1 caused by group B ß-hemolytic Streptococcus (GBS), and 1 associated with group C ß-hemolytic Streptococcus (GCS). GBS and GCS are very rarely reported in pediatric cases of PSD. The literature on non-GAS PSD is reviewed, which additionally revealed several instances of PSD caused by group G ß-hemolytic Streptococcus (GGS) and Staphylococcus aureus. GBS, GCS, GGS, and S aureus are significant causes of PSD to consider, particularly among adult patients, based on our encountered cases and the literature. If using rapid antigen tests to expedite the diagnosis of GAS, we recommend supplementing with a lesional swab for bacterial culture and sensitivity as the rapid antigen test does not detect non-GAS organisms. Therapy should be tailored to the microbiologic cause.

Molecular aspects of prostate cancer with neuroendocrine differentiation.

Neuroendocrine differentiation (NED), which is not uncommon in prostate cancer, is increases in prostate cancer after androgen-deprivation therapy (ADT) and generally appears in castration-resistant prostate cancer (CRPC). Neuroendocrine cells, which are found in normal prostate tissue, are a small subset of cells and have unique function in regulating the growth of prostate cells. Prostate cancer with NED includes different types of tumor, including focal NED, pure neuroendocrine tumor or mixed neuroendocrine-adenocarcinoma. Although more and more studies are carried out on NED in prostate cancer, the molecular components that are involved in NED are still poorly elucidated. We review neuroendocrine cells in normal prostate tissue, NED in prostate cancer, terminology of NED and biomarkers used for detecting NED in routine pathological practice. Some recently reported molecular components which drive NED in prostate cancer are listed in the review.

Piezo1 opposes age-associated cortical bone loss.

As we age, our bones undergo a process of loss, often accompanied by muscle weakness and reduced physical activity. This is exacerbated by decreased responsiveness to mechanical stimulation in aged skeleton, leading to the hypothesis that decreased mechanical stimulation plays an important role in age-related bone loss. Piezo1, a mechanosensitive ion channel, is critical for bone homeostasis and mechanotransduction. Here, we observed a decrease in Piezo1 expression with age in both murine and human cortical bone. Furthermore, loss of Piezo1 in osteoblasts and osteocytes resulted in an increase in age-associated cortical bone loss compared to control mice. The loss of cortical bone was due to an expansion of the endosteal perimeter resulting from increased endocortical resorption. In addition, expression of Tnfrsf11b, encoding anti-osteoclastogenic protein OPG, decreases with Piezo1 in vitro and in vivo in bone cells, suggesting that Piezo1 suppresses osteoclast formation by promoting Tnfrsf11b expression. Our results highlight the importance of Piezo1-mediated mechanical signaling in protecting against age-associated cortical bone loss by inhibiting bone resorption in mice.

MicroRNA-204/211 alters epithelial physiology.

MicroRNA (miRNA) expression in fetal human retinal pigment epithelium (hfRPE), retina, and choroid were pairwise compared to determine those miRNAs that are enriched by 10-fold or more in each tissue compared with both of its neighbors. miRs-184, 187, 200a/200b, 204/211, and 221/222 are enriched in hfRPE by 10- to 754-fold compared with neuroretina or choroid (P<0.05). Five of these miRNAs are enriched in RPE compared with 20 tissues throughout the body and are 10- to 20,000-fold more highly expressed (P<0.005). miR-204 and 211 are the most highly expressed in the RPE. In addition, expression of miR-204/211 is significantly lower in the NCI60 tumor cell line panel compared with that in 13 normal tissues, suggesting the progressive disruption of epithelial barriers and increased proliferation. We demonstrated that TGF-beta receptor 2 (TGF-betaR2) and SNAIL2 are direct targets of miR-204 and that a reduction in miR-204 expression leads to reduced expression of claudins 10, 16, and 19 (message/protein) consistent with our observation that anti-miR-204/211 decreased transepithelial resistance by 80% and reduced cell membrane voltage and conductance. The anti-miR-204-induced decrease in Kir7.1 protein levels suggests a signaling pathway that connects TGF-betaR2 and maintenance of potassium homeostasis. Overall, these data indicate a critical role for miR-204/211 in maintaining epithelial barrier function and cell physiology.

An immigrant with acquired immunodeficiency syndrome presenting with a rash: A case report.

A 58-year-old woman from Zimbabwe, with a history of untreated human immunodeficiency virus, presented with leonine facies and a diffuse rash. The rash occurred in the context of a 1-year history of constitutional symptoms and cognitive decline. Laboratory investigations confirmed that her human immunodeficiency virus had progressed to acquired immunodeficiency syndrome. Through imaging, tissue biopsies, and polymerase chain reaction, a diagnosis of disseminated histoplasmosis was made. Since there was no history of travel and histoplasmosis is not locally endemic, the patient likely contracted this fungal infection more than 7 years ago, while living in Africa. We speculate that the histoplasmosis remained latent until her immune system began to decline. The work-up and management of this rare cutaneous presentation of a systemic disease, which should be added to the list of "great mimickers" in dermatology, are discussed.

LCAT, ApoD, and ApoA1 Expression and Review of Cholesterol Deposition in the Cornea.

Lecithin:cholesterol acyltransferase (LCAT) is an enzyme secreted by the liver and circulates with high-density lipoprotein (HDL) in the blood. The enzyme esterifies plasma cholesterol and increases the capacity of HDL to carry and potentially remove cholesterol from tissues. Cholesterol accumulates within the extracellular connective tissue matrix of the cornea stroma in individuals with genetic deficiency of LCAT. LCAT can be activated by apolipoproteins (Apo) including ApoD and ApoA1. ApoA1 also mediates cellular synthesis of HDL. This study examined the expression of LCAT by epithelial cells, keratocytes, and endothelial cells, the cell types that comprise from anterior to posterior the three layers of the cornea. LCAT and ApoD were immunolocalized to all three cell types within the cornea, while ApoA1 was immunolocalized to keratocytes and endothelium but not epithelium. In situ hybridization was used to detect LCAT, ApoD, and ApoA1 mRNA to learn what cell types within the cornea synthesize these proteins. No corneal cells showed mRNA for ApoA1. Keratocytes and endothelium both showed ApoD mRNA, but epithelium did not. Epithelium and endothelium both showed LCAT mRNA, but despite the presence of LCAT protein in keratocytes, keratocytes did not show LCAT mRNA. RNA sequencing analysis of serum-cultured dedifferentiated keratocytes (commonly referred to as corneal stromal fibroblasts) revealed the presence of both LCAT and ApoD (but not ApoA1) mRNA, which was accompanied by their respective proteins detected by immunolabeling of the cultured keratocytes and Western blot analysis of keratocyte lysates. The results indicate that keratocytes in vivo show both ApoA1 and LCAT proteins, but do not synthesize these proteins. Rather, keratocytes in vivo must take up ApoA1 and LCAT from the corneal interstitial tissue fluid.

Clinical-grade stem cell-derived retinal pigment epithelium patch rescues retinal degeneration in rodents and pigs.

Considerable progress has been made in testing stem cell-derived retinal pigment epithelium (RPE) as a potential therapy for age-related macular degeneration (AMD). However, the recent reports of oncogenic mutations in induced pluripotent stem cells (iPSCs) underlie the need for robust manufacturing and functional validation of clinical-grade iPSC-derived RPE before transplantation. Here, we developed oncogenic mutation-free clinical-grade iPSCs from three AMD patients and differentiated them into clinical-grade iPSC-RPE patches on biodegradable scaffolds. Functional validation of clinical-grade iPSC-RPE patches revealed specific features that distinguished transplantable from nontransplantable patches. Compared to RPE cells in suspension, our biodegradable scaffold approach improved integration and functionality of RPE patches in rats and in a porcine laser-induced RPE injury model that mimics AMD-like eye conditions. Our results suggest that the in vitro and in vivo preclinical functional validation of iPSC-RPE patches developed here might ultimately be useful for evaluation and optimization of autologous iPSC-based therapies.

Mapping of the neonatal Fc receptor in the rodent eye.

PURPOSE: The neonatal Fc receptor (FcRn) has been known to modulate IgG transport and protect against IgG catabolism, resulting in extension of the serum half-life of IgG. The goal of this study was to localize FcRn receptor expression in the rat's eye. METHODS: The cornea, retina, conjunctiva, ciliary body and iris, retinal pigment epithelium and choroid, and lens were dissected from each rat's eye, and total RNA was purified. The first-strand cDNAs were synthesized and subjected to PCR reaction. For control samples, reverse transcriptase was omitted. A monoclonal antibody against the FcRn heavy chain was used to localize the distribution of the FcRn receptor in ocular tissues. Lymphatic vessels and blood vessels were stained with a rabbit anti-mouse lymphatic vessel endothelial receptor-1 polyclonal antibody and a rabbit anti-human von Willebrand factor polyclonal antibody, respectively. RESULTS: RT-PCR demonstrated expression of FcRn RNA in cornea, retina, conjunctiva, ciliary body and iris, and lens but absence of expression in the retinal pigment epithelium and choroid. Immunohistochemistry and double staining confirmed the expression of FcRn receptor to the conjunctival lymphatic vessels but not in the conjunctival blood vessels. In the ciliary body, the FcRn receptor was found to be expressed in both the nonpigmented ciliary epithelium and the ciliary blood vessels. The expression of FcRn receptor was confirmed in the retinal blood vessels, iris blood vessels, optic nerve vascular structures, corneal epithelium and endothelium, and lens epithelium. CONCLUSIONS: The FcRn receptor is expressed in multiple ocular tissues. The blood-ocular barrier showed FcRn receptor expression, indicating that IgG transport from ocular tissues to the blood system may use this receptor. The role of the FcRn receptor in the anterior segment and the conjunctiva remains unclear.

Interleukin-4 Expressed By Neoplastic Cells Provokes an Anti-Metastatic Myeloid Immune Response.

OBJECTIVE: Interleukin-4 (IL-4) can induce macrophages to undergo alternative activation and polarize toward an M2-like or wound healing phenotype. Tumor associated macrophages (TAMs) are thought to assume M2-like properties, and it has been suggested they promote tumor growth and metastasis through effects on the tumor stroma, including extracelluar matrix remodeling and angiogenesis. IL-4 also promotes macrophage survival and formation of multinucleated giant cells, which have enhanced phagocytic behavior. This study was designed to explore the effect of cancer cell derived IL-4 on the tumor immune stroma and metastasis. METHODS: The metastatic mouse mammary carcinoma cell line AC2M2 was transduced with control or IL-4 encoding retroviruses and employed in orthotopic engraftment models. Tumor growth and metastasis were assessed. The cellular composition and biomarker expression of tumors were examined by immunohistochemical staining and flow cytometry; the transcriptome of the immune stroma was analyzed by nanoString based transcript quantitation; and in vivo and in vitro interactions between cancer cells and macrophages were assessed by flow cytometry and co-culture with video-time lapse microscopy, respectively. RESULTS: Unexpectedly, tumors from IL-4 expressing AC2M2 engrafted cells grew at reduced rates, and most surprising, they lost all metastatic potential relative to tumors from control AC2M2 cells. Myeloid cell numbers were not increased in IL-4 expressing tumors, but their expression of the M2 marker arginase I was elevated. Transcriptome analysis revealed an immune signature consistent with IL-4 induced M2 polarization of the tumor microenvironment and a generalized increase in myeloid involvement in the tumor stroma. Flow cytometry analysis indicated enhanced cancer cell phagocytosis by TAMs from IL-4 expressing tumors, and co-culture studies showed that IL-4 expressing cancer cells supported the survival and promoted the in vitro phagocytic behavior of macrophages. CONCLUSIONS: Although M2-like TAMs have been linked to enhanced tumorigenesis, this study shows that IL-4 production by cancer cells is associated with suppressed tumor growth and loss of metastatic potential as well as enhanced phagocytic behavior of TAMs.

Survival of retinal pigment epithelium after exposure to prolonged oxidative injury: a detailed gene expression and cellular analysis.

PURPOSE: To detail, by DNA microarrays and cellular structure labeling, the in vitro responses of retinal pigment epithelial (RPE) cells to a nonlethal dose of the oxidant agent hydroquinone (HQ). METHODS: The viability of growth-quiescent ARPE-19 cells after treatment with HQ was measured by XTT conversion, (3)H-leucine incorporation, trypan blue exclusion, and the presence of DNA laddering. The effect of a nonlethal dose of HQ on the localization of apoptosis-induced factor (AIF) and phosphorylation of stress-activated kinase-2/p38 (SAPK2/p38) was detected by immunocytochemistry. Actin structures were visualized by phalloidin staining. Cell membrane blebbing was detected using GFP-membrane-labeled RPE cells (ARPE-GFP-c'-rRas). Changes in gene expression patterns of RPE cells within 48 hours of prolonged treatment with a nonlethal dose of HQ were evaluated by microarray analysis and confirmed by Northern blotting. RESULTS: The viability of RPE after a prolonged sublethal injury dose of HQ was determined by multiple assays and confirmed by the absence of AIF translocation or DNA laddering. Prolonged exposure (16 hours) of RPE cells to a nonlethal dose of HQ resulted in actin rearrangement into globular aggregates and cell membrane blebbing. Kinetic microarray analysis at several time points over a 48-hour recovery period revealed significant upregulation of genes involved in ameliorating the oxidative stress, chaperone proteins, anti-apoptotic factors, and DNA repair factors, and downregulation of pro-apoptotic genes. Genes involved in extracellular matrix functions were also dysregulated. Recovery of RPE cells after the injury was confirmed by the normalization of gene expression dysregulation back to baseline levels within 48 hours. CONCLUSIONS: RPE cells avoided cell death and recovered from prolonged oxidative injury by activating a host of defense mechanisms while simultaneously triggering genes and cellular responses that may be involved in RPE disease development.

Increased measures of anxiety and weight gain in mice lacking the group III metabotropic glutamate receptor mGluR8.

To study the role of the metabotropic glutamate receptor 8 (mGluR8), mice lacking this receptor were generated by homologous recombination. Homozygous mGluR8-deficient mice are about 8% heavier than their wild-type age-matched controls after reaching 4 weeks of age. This weight difference is not caused by an altered food intake and is not exacerbated by feeding the animals a high-fat diet. Moreover, mGluR8-/- mice are mildly insulin resistant, possibly as a result of being overweight. Behavioral testing revealed a reduced locomotor activity of mGluR8-/- mice compared with wild-type mice during the first 3 days in a novel enclosed environment. However after 3 days, the locomotor activities of wild-type and mGluR8-/- mice were similar, suggesting a reduced exploratory behavior of mGluR8-/- mice in a novel enclosed environment. By contrast, there were no genotype differences in locomotor activity in the open field, plus maze, or in total time spent exploring objects during object recognition tests, indicating that there is a dissociation between effects of mGluR8 deficiency in exploratory activity in a novel safe enclosed environment vs. a more anxiogenic novel open environment. The absence of mGluR8 also leads to increased measures of anxiety in the open field and elevated plus maze. Whether the diverse phenotypic differences observed in mGluR8-/- mice result from the misregulation of a unique neural pathway, possibly in the thalamus or hypothalamus, or whether they are the consequence of multiple developmental and functional alterations in synaptic transmission, remains to be determined.