A Novel Approach to Negative Pressure Wound Therapy: Use of High Suction Capillary Device to Improve Wound Healing.

Introduction: Negative Pressure Wound Therapy (NPWT) is a procedure used for nonhealing wounds. In NPWT, a special sealed dressing of large cell foam (>400 µm) or gauze is connected to a pump. Most commonly, negative pressures between -10 and -125 millimeters of mercury (mm Hg) are used.

Significance of the melanocortin 1 receptor in the DNA damage response of human melanocytes to ultraviolet radiation.

Activation of the melanocortin 1 receptor (MC1R) by α-melanocortin (α-MSH) stimulates eumelanin synthesis and enhances repair of ultraviolet radiation (UV)-induced DNA damage. We report on the DNA damage response (DDR) of human melanocytes to UV and its enhancement by α-MSH. α-MSH up-regulated the levels of XPC, the enzyme that recognizes DNA damage sites, enhanced the UV-induced phosphorylation of the DNA damage sensors ataxia telangiectasia and Rad3-related (ATR) and ataxia telangiectasia mutated (ATM) and their respect-ive substrates checkpoint kinases 1 and 2, and increased phosphorylated H2AX (γH2AX) formation.

Oral tungstate (Na2WO4) exposure reduces adaptive immune responses in mice after challenge.

Tungstate (WO²⁻₄) has been identified as a ground water contaminant at military firing ranges and can be absorbed by ingestion. In this study, C57BL6 mice were exposed to sodium tungstate (Na2WO4·2H2O) (0, 2, 62.5, 125, and 200 mg/kg/day) in their drinking water for an initial 28-day screen and in a one-generation (one-gen) model.

The abundance of Rad51 protein in mouse embryonic stem cells is regulated at multiple levels.

DNA double-strand breaks (DSBs) in embryonic stem (ES) cells are repaired primarily by homologous recombination (HR). The mechanism by which HR is regulated in these cells, however, remains enigmatic. To gain insight into such regulatory mechanisms, we have asked how protein levels of Rad51, a key component of HR, are controlled in mouse ES cells and mouse embryo fibroblasts (MEFs).

Defining MC1R regulation in human melanocytes by its agonist α-melanocortin and antagonists agouti signaling protein and β-defensin 3.

The melanocortin 1 receptor (MC1R), a G(s) protein-coupled receptor, has an important role in human pigmentation. We investigated the regulation of expression and activity of the MC1R in primary human melanocyte cultures. Human β-defensin 3 (HBD3) acted as an antagonist for MC1R, inhibiting the α-melanocortin (α-melanocyte-stimulating hormone (α-MSH))-induced increase in the activities of adenylate cyclase and tyrosinase, the rate-limiting enzyme for melanogenesis.

The human DEK oncogene regulates DNA damage response signaling and repair.

The human DEK gene is frequently overexpressed and sometimes amplified in human cancer. Consistent with oncogenic functions, Dek knockout mice are partially resistant to chemically induced papilloma formation. Additionally, DEK knockdown in vitro sensitizes cancer cells to DNA damaging agents and induces cell death via p53-dependent and -independent mechanisms.

The burn wound inflammatory response is influenced by midazolam.

Burn patients requiring hospitalization are often treated for anxiety with benzodiazepines (BDZs). Benzodiazepines are reported to influence immune system function. Immune system alterations are a major cause of burn-induced mortality.

Epidermal keratinocytes from light vs. dark skin exhibit differential degradation of melanosomes.

Modification of skin complexion coloration has traditionally been accomplished by interruption or attenuation of melanogenesis and/or melanosome transfer. Post-transfer modification of pigmented melanosomes provides an attractive and distinct avenue of modulating skin pigmentation. The processing of melanosomes during keratinocyte (KC) terminal differentiation and the degradative variability observed between light and dark skin (LS and DS) remains enigmatic.

Mismatch and base excision repair proficiency in murine embryonic stem cells.

Accumulation of mutations in embryonic stem (ES) cells would be detrimental to an embryo derived from these cells, and would adversely affect multiple organ systems and tissue types. ES cells have evolved multiple mechanisms to preserve genomic integrity that extend beyond those found in differentiated cell types. The present study queried whether mismatch repair (MMR) and base-excision repair (BER) may play a role in the maintenance of murine ES cell genomes.

Lumican is required for neutrophil extravasation following corneal injury and wound healing.

An important aspect of wound healing is the recruitment of neutrophils to the site of infection or tissue injury. Lumican, an extracellular matrix component belonging to the small leucine rich proteoglycan (SLRP) family, is one of the major keratan sulfate proteoglycans (KSPGs) within the corneal stroma. Increasing evidence indicates that lumican can serve as a regulatory molecule for several cellular processes, including cell proliferation and migration.

Melanocortin 1 receptor genotype: an important determinant of the damage response of melanocytes to ultraviolet radiation.

The melanocortin 1 receptor gene is a main determinant of human pigmentation, and a melanoma susceptibility gene, because its variants that are strongly associated with red hair color increase melanoma risk. To test experimentally the association between melanocortin 1 receptor genotype and melanoma susceptibility, we compared the responses of primary human melanocyte cultures naturally expressing different melanocortin 1 receptor variants to α-melanocortin and ultraviolet radiation. We found that expression of 2 red hair variants abolished the response to α-melanocortin and its photoprotective effects, evidenced by lack of functional coupling of the receptor, and absence of reduction in ultraviolet radiation-induced hydrogen peroxide generation or enhancement of repair of DNA photoproducts, respectively.

Mouse embryonic stem cells, but not somatic cells, predominantly use homologous recombination to repair double-strand DNA breaks.

Embryonic stem (ES) cells give rise to all cell types of an organism. Since mutations at this embryonic stage would affect all cells and be detrimental to the overall health of an organism, robust mechanisms must exist to ensure that genomic integrity is maintained. To test this proposition, we compared the capacity of murine ES cells to repair DNA double-strand breaks with that of differentiated cells.

Monoallelic expression of Krt12 gene during corneal-type epithelium differentiation of limbal stem cells.

Purpose: The purpose of this study was to characterize a Krt12-Cre knock-in mouse line for corneal epithelium-specific gene ablation and to analyze the allelic selection of the keratin 12 (Krt12) gene during corneal type-epithelium differentiation.

Methods: Knock-in mice were generated by gene targeting. The authors examined the expression patterns of several reporter genes in the corneas of bitransgenic Krt12cre/+/ROSA(EGFP), Krt12Cre/+/ZEG, and Krt12Cre/+/ZAP mouse lines.

Sodium tungstate (Na2WO4) exposure increases apoptosis in human peripheral blood lymphocytes.

The potential for adverse health effects of using tungsten and its alloys in military munitions are an important concern to both civilians and the US military. The toxicological implications of exposure to tungsten, its alloys, and the soluble tungstate (Na(2)WO(4)) are currently under investigation. To examine tungstate toxicity, a series of experiments to determine its in vitro effects on cells of the immune system were performed.

Mice treated with a benzodiazepine had an improved survival rate following Pseudomonas aeruginosa infection.

Psychological stress has a high incidence after burn injury, therefore, anxiolytic drugs are often prescribed. Unfortunately, to date, no burn study has investigated the effects of anxiolytic drugs on the ability to fight infection. This study was undertaken to determine if psychological stress, anxiety-modulating drugs, or both, alter survival following an infection.

Employing a recombinant HLA-DR3 expression system to dissect major histocompatibility complex II-thyroglobulin peptide dynamism: a genetic, biochemical, and reverse immunological perspective.

Previously, we have shown that statistical synergism between amino acid variants in thyroglobulin (Tg) and specific HLA-DR3 pocket sequence signatures conferred a high risk for autoimmune thyroid disease (AITD). Therefore, we hypothesized that this statistical synergism mirrors a biochemical interaction between Tg peptides and HLA-DR3, which is key to the pathoetiology of AITD. To test this hypothesis, we designed a recombinant HLA-DR3 expression system that was used to express HLA-DR molecules harboring either AITD susceptibility or resistance DR pocket sequences.

Generation of mice with a conditional allele for transforming growth factor beta 1 gene.

Transforming growth factor beta1 (TGFbeta1) is a multifunctional growth factor involved in wound healing, tissue fibrosis, and in the pathogenesis of many syndromic diseases (e.g., Marfan syndrome, Camurati-Engelmann disease) and muscular, neurological, ophthalmic, cardiovascular and immunological disorders, and cancer.

Absence of nucleolar disruption after impairment of 40S ribosome biogenesis reveals an rpL11-translation-dependent mechanism of p53 induction.

Impaired ribosome biogenesis is attributed to nucleolar disruption and diffusion of a subset of 60S ribosomal proteins, particularly ribosomal protein (rp)L11, into the nucleoplasm, where they inhibit MDM2, leading to p53 induction and cell-cycle arrest. Previously, we demonstrated that deletion of the 40S rpS6 gene in mouse liver prevents hepatocytes from re-entering the cell cycle after partial hepatectomy. Here, we show that this response leads to an increase in p53, which is recapitulated in culture by rpS6-siRNA treatment and rescued by the simultaneous depletion of p53.

Intracellular cytokines.

The principal technique described in this unit was developed for detecting cytokines produced by T cells, specifically interferon gamma (IFN-gamma) and interleukin 4 (IL-4). In addition to providing information about which cytokines are being produced, it also helps to phenotypically identify the specific cells producing them. For example, CD4(+) or CD8(+) cells can be subdivided into T(H)1/T(H)2 helper cells or T(C)1/T(C)2 suppressor cells, respectively.

Promiscuous recombination of LoxP alleles during gametogenesis in cornea Cre driver mice.

Purpose: To examine whether promiscuous Cre/LoxP recombination happens during gametogenesis in double transgenic mice carrying LoxP modified alleles and Cre transgene driven by tissue-specific promoter outside the gonads of adult mice.

Methods: Cre driver mice were crossbred with reporter mouse lines (e.g.

Orchestration of the S-phase and DNA damage checkpoint pathways by replication forks from early origins.

The S-phase checkpoint activated at replication forks coordinates DNA replication when forks stall because of DNA damage or low deoxyribonucleotide triphosphate pools. We explore the involvement of replication forks in coordinating the S-phase checkpoint using dun1Delta cells that have a defect in the number of stalled forks formed from early origins and are dependent on the DNA damage Chk1p pathway for survival when replication is stalled. We show that providing additional origins activated in early S phase and establishing a paused fork at a replication fork pause site restores S-phase checkpoint signaling to chk1Delta dun1Delta cells and relieves the reliance on the DNA damage checkpoint pathway.

TGFbeta1 deficiency does not affect the generation and maintenance of CD4+CD25+FOXP3+ putative Treg cells, but causes their numerical inadequacy and loss of regulatory function.

TGFbeta1 is considered to be required for peripheral maintenance of CD4(+)CD25(+)FOXP3(+) T(reg) cells. However, we demonstrate no reduction in the percentage of such T cells in the spleens and thymi of Tgfb1(-/-) mice. Although putative T(reg) cells, characterized as CD4(+)CD25(+)FOXP3(+)CD62L(+) T cells, are increased in Tgfb1(-/-) mice, they may be inadequate to control activated T cells since the ratio of activated T cells:putative T(reg) cells is several-fold higher in Tgfb1(-/-) mice than in control mice.