The Integration of Proteome-Wide PTM Data with Protein Structural and Sequence Features Identifies Phosphorylations that Mediate 14-3-3 Interactions.

14-3-3s are abundant proteins that regulate essentially all aspects of cell biology, including cell cycle, motility, metabolism, and cell death. 14-3-3s work by docking to phosphorylated Ser/Thr residues on a large network of client proteins and modulating client protein function in a variety of ways. In recent years, aided by improvements in proteomics, the discovery of 14-3-3 client proteins has far outpaced our ability to understand the biological impact of individual 14-3-3 interactions. The rate-limiting step in this process is often the identification of the individual phospho-serines/threonines that mediate 14-3-3 binding, which are difficult to distinguish from other phospho-sites by sequence alone. Furthermore, trial-and-error molecular approaches to identify these phosphorylations are costly and can take months or years to identify even a single 14-3-3 docking site phosphorylation. To help overcome this challenge, we used machine learning to analyze predictive features of 14-3-3 binding sites. We found that accounting for intrinsic protein disorder and the unbiased mass spectrometry identification rate of a given phosphorylation significantly improves the identification of 14-3-3 docking site phosphorylations across the proteome. We incorporated these features, coupled with consensus sequence prediction, into a publicly available web app, called "14-3-3 site-finder". We demonstrate the strength of this approach through its ability to identify 14-3-3 binding sites that do not conform to the loose consensus sequence of 14-3-3 docking phosphorylations, which we validate with 14-3-3 client proteins, including TNK1, CHEK1, MAPK7, and others. In addition, by using this approach, we identify a phosphorylation on A-kinase anchor protein-13 (AKAP13) at Ser2467 that dominantly controls its interaction with 14-3-3.

Effect of delay between nuclear medicine scanning and sentinel node biopsy on outcome in patients with cutaneous melanoma.

BACKGROUND: Sentinel lymph node biopsy (SLNB) is an important staging tool for the management of melanoma. A multicentre study was done to validate previous findings that the timing of lymphoscintigraphy influences the accuracy of SLNB and patient outcomes, particularly survival. METHODS: Data were reviewed on patients undergoing SLNB for melanoma at three centres in the UK and Sweden, examining the effect of timing of SLNB after nuclear medicine scanning. Kaplan-Meier survival analysis was used to assess overall (OS), disease-specific (DSS) and progression-free (PFS) survival, stratified by timing of lymphoscintigraphy. Independent risk factors for survival were identified by Cox multivariable regression analysis. RESULTS: A total of 2270 patients were identified. Median follow-up was 56 months. Univariable analysis showed a 4·2 per cent absolute and 35·5 per cent relative benefit in DSS (hazard ratio 1·36, 95 per cent c.i. 1·05 to 1·74; P = 0·018) for 863 patients whose SLNB was performed up to 12 h after lymphoscintigraphy compared with 1407 patients who had surgery after more than 12 h. There were similar OS and PFS benefits (P = 0·036 and P = 0·022 respectively). Multivariable analysis identified timing of lymphoscintigraphy as an independent predictor of OS (P = 0·017) and DSS (P = 0·030). There was an excess of nodal recurrences as first site of recurrence in the group with delayed surgery (4·5 versus 2·5 per cent; P = 0·008). CONCLUSION: Delaying SLNB beyond 12 h after lymphoscintigraphy with 99 Tc-labelled nanocolloid has a significant negative survival impact in patients with melanoma.

ANTECEDENTES: La biopsia de ganglio centinela (sentinel lymph node biopsy, SLNB) es una técnica importante para la estadificación y tratamiento del melanoma. Se realizó un estudio multicéntrico para validar hallazgos previos según los cuales el momento de la linfogammagrafía (lymphoscintigraphy, LS) influye en la precisión de la SLNB y en los resultados de los pacientes, especialmente en la supervivencia. MÉTODOS: Se revisaron los datos de los pacientes a los que se realizó una SLNB por melanoma en 3 centros en el Reino Unido y Suecia, con especial atención al efecto del período entre la inyección el material radioactivo y la SLNB. Se realizó un análisis de supervivencia mediante el método de Kaplan-Meier para la supervivencia específica de la enfermedad (disease-specific survival, DSS), supervivencia global (overall survival, OS) y supervivencia libre de progresión (progression-free survival, PFS), todas ellas estratificadas por el momento de la LS. Los factores de riesgo independientes para la supervivencia se determinaron mediante un análisis de regresión multivariable de Cox. RESULTADOS: Se incluyeron 2.270 pacientes. La mediana de seguimiento fue de 49 meses. El análisis univariado mostró un beneficio absoluto del 4,2% y relativo del 35,5% (cociente de riesgos instantáneos, hazard ratio, HR: 1,36 (i.c. del 95% 1,05-1,74, P = 0.02)) en la DDS para los pacientes a los que la SLNB se realizó < 12 horas después de la LS (n = 863) en comparación con aquellos realizados > 12 horas (n = 1407). Se detectaron beneficios similares para la OS y la PFS (P = 0,04 y P = 0,02, respectivamente). El análisis multivariable identificó el tiempo entre la LS y la SLNB como un factor independiente de OS (P = 0,017) y DSS (P = 0,03). Hubo un aumento en las recidivas ganglionares como primer sitio de recidiva en el grupo de > 12 horas (2,5% versus 4,5%; P = 0,008). CONCLUSIÓN: Estos datos validan nuestra investigación previa y tienen implicaciones significativas para las unidades de melanoma, en el sentido de que retrasar la SLNB más allá de las 12 horas después de realizar la LS con nanocoloides marcados con Tc99 tiene un impacto negativo significativo en la supervivencia de los pacientes y debe evitarse. Se presenta la hipótesis de que la causa subyacente es la migración temporal del trazador que determina una SLNB incorrecta. .

A Rare Fourth Branch of the Marginal Mandibular Nerve.

This report describes a good example of the rare fourth branch of the marginal mandibular nerve. This case emphasizes the need for respecting the variation in the marginal mandibular nerve when carrying out surgery.

Microsurgical Reconstructions for Head and Neck Cancers in Elderly Aged >80 Years: An Analysis of Surgical Outcomes and Quality of Life.

BACKGROUND: The rising incidence of primary head and neck (H&N) cancers in the elderly presents a dilemma regarding the appropriateness of complex surgery in this assumed frail age group. With limited data on surgical morbidity, survival, and patient quality of life (QOL), this analysis aimed to broaden the understanding of safety and effectiveness of microsurgical treatment in very elderly H&N cancer patients. METHODS: A prospective database analysis was used to evaluate surgical outcomes (morbidity, survival, and QOL) in all patients aged 80 years and older undergoing microsurgical reconstruction for cutaneous and intra-oral H&N cancers between 2004 and 2014. Outcomes were assessed for their association with surgical, tumour, and patient variables. Comorbidities were categorized by the ACE27 index and postoperative morbidity by the Clavien-Dindo scoring system. QOL was analyzed using the UW-QOLv4. RESULTS: Of 720 microsurgical reconstructions, 96 patients were identified. Median survival was 25 months. The ACE27 index was the only variable significantly associated with survival with a 5-year survival of 59.2 % in the least comorbid group versus 19.7 % in the most comorbid group (p 0.015). ACE-27 showed influence on socioemotional QoL scores. Physical QOL scores were influenced by tumour and operative factors. Patients were found to value physical QOL over socioemotional. CONCLUSIONS: Microsurgical reconstructions are well tolerated in the very elderly patients and should be considered predominantly based on comorbidity. Tumour stage, flap type, and cancer site should still form part of the preoperative counseling due to their implication on postoperative physical function.

Incidence of infection in 39-month-old ewes with TMEM154 diplotypes "1 1," "1 3," and "3 3" after natural exposure to ovine progressive pneumonia virus.

Production and well-being of sheep and goats in many countries are harmfully impacted by small ruminant lentiviruses (SRLV) that cause incurable, progressive diseases. Susceptibility to ovine progressive pneumonia virus (OPPV), the North American form of SRLV, is influenced by variants of the ovine transmembrane protein 154 gene (TMEM154). The experimental objective was to estimate additive and dominance effects of TMEM154 haplotypes 1 and 3 on susceptibility of breeding ewes to infection after natural exposure to OPPV from birth to 39 mo of age. Sires and dams were heterozygous for TMEM154 haplotypes 1 and 3, producing ewe lambs with diplotypes "1 1," "1 3," and "3 3." These lambs were raised by mature, infected dams to ensure natural, maternal exposure to OPPV. Ewe lambs (n = 108) were kept for breeding and joined an infected flock of ewes to guarantee natural, nonmaternal exposure to OPPV. Ewes were bred to lamb at 1, 2, and 3 yr of age. Serum samples were collected at breeding, 1 mo before lambing and shortly after weaning each year to monitor infection status to 39 mo of age. During the experiment, 9 of the 108 ewes died while uninfected and data collected on these ewes were not analyzed. Infection status of the remaining 99 ewes at 39 mo of age was analyzed using logistic regression procedures. Effects of ewe type of birth, ewe type of rearing, and breed type of dam were not detected (P > 0.10), and the estimated sire variance component was nil. Ewe diplotype affected infection status (P < 0.0001), as did additive (P < 0.0001) and dominance (P < 0.0022) effects. Predicted probabilities of infection for ewes with diplotypes "1 1," "1 3," and "3 3" were 0.10, 0.88, and 0.89, respectively, and confidence intervals for diplotypes "1 3" and "3 3" were distinct from "1 1." Haplotype 3 was completely dominant to haplotype 1 at 39 mo of age. The probability of infection for ewes with either diplotype "1 3" or "3 3" averaged 8.5 times that of ewes with diplotype "1 1." Diplotype "1 3" and "3 3" ewes were highly susceptible to nonmaternal transmission of OPPV, in contrast to diplotype "1 1" ewes. Therefore, the distribution of ewes with diplotypes "1 1," "1 3," and "3 3" within a flock will influence the number of infections caused by each route of transmission. Selection and mating strategies can be implemented to produce sheep that are genetically less susceptible to OPPV infection.

A novel method for predicting antioxidant activity based on amino acid structure.

Epidemiological studies show a positive correlation between oxidative stress and chronic disease development such as heart disease and cancer. While several antioxidant compounds with varying physical and chemical characteristics are able to reduce oxidative stress in biological systems, relatively few studies have been performed to examine the structural characteristics that produce potent antioxidants. We examined 20 essential and non-essential amino acids using the ORAC assay and used a simplest-case amino acid model to gather data to make predictions regarding the antioxidant activity of non-amino acid compounds; we also tested our findings on chalcone and nitrone data from the current literature. We observed that the sp(2)-hybridized carbons were the most consistent predictors of antioxidant activity in all groups. Valence electron to carbon ratio and length of conjugated double bond groups also emerged as important structural characteristics. Further testing may help to elucidate more accurate trends, as well as nonlinear relationships.

Rhodotorula minuta fungemia in a ewe lamb.

An 8-month-old crossbred ewe, normal upon physical examination, was humanely euthanized for tissue collection. After approximately 3 weeks in tissue culture, fungi began budding out of cells obtained from the choroid plexus. After an additional 3 weeks, budding was observed in kidney cell cultures and eventually in monocyte cultures as well. Serum from the lamb was submitted to the Veterinary Diagnostic Laboratory at Colorado State University for fungal diagnosis and was found negative for Aspergillus, Blastomyces, Coccidioidomycosis and Histoplasmosis. DNA was isolated from fungi collected from tissue culture supernatants and used in a set of pan-fungal PCR assays with DNA from Candida acting as a positive control. PCR products were sequenced and BLAST analysis performed. The unknown fungal sequence aligned with 100% identity to Rhodotorula minuta an emerging opportunistic pathogen. Samples were submitted to The Fungal Testing Laboratory at The University of Texas Health Science Center at San Antonio for additional validation. We believe this to be the first report of Rhodotorula fungemia in a sheep in the United States.

Effects of TMEM154 haplotypes 1 and 3 on susceptibility to ovine progressive pneumonia virus following natural exposure in sheep.

Small ruminant lentiviruses (SRLV) adversely affect production and well-being of sheep and goats throughout much of the world. The SRLV, including ovine progressive pneumonia virus (OPPV) in North America, cause lifetime infections, and management procedures to eradicate or reduce disease prevalence are costly. Variants of ovine transmembrane protein 154 gene (TMEM154) affect susceptibility to OPPV. The primary experimental objective was to estimate additive and dominance effects of TMEM154 haplotypes 1 and 3 on susceptibility to OPPV infection following natural exposure. A group of 187 trial lambs was born and raised by mature, infected ewes to ensure natural exposure to OPPV. Parents of trial lambs were heterozygous for haplotypes 1 and 3, producing lambs with diplotypes "1 1," "1 3," and "3 3." A group of 20 sentinel lambs was born and raised by mature, uninfected ewes that were diplotype "1 1." Sentinel lambs had diplotypes "1 1" and "1 3," being sired by the same set of rams as trial lambs. Trial and sentinel lambs were comingled during the experiment. Lambs were weaned at 60 d of age, bled 1 wk after weaning, and thereafter at intervals of 4 or 5 wk until 9 mo of age when OPPV infection status was determined by use of a competitive enzyme-linked immunosorbent assay. Only 1 sentinel lamb became infected. Infection status of trial lambs was analyzed using logistic regression procedures to account for the binary nature of infection status and random effects of sires. Effects of sex, type of birth, type of rearing, age of dam, breed type of dam, and sires were not detected (P>0.20). Infection status was affected by diplotype of lamb (P=0.005), with additive (P=0.002) and dominance (P=0.052) effects identified. Predicted probabilities of infection for lambs with diplotypes "1 1," "1 3," and "3 3" were 0.094, 0.323, and 0.346, respectively. Confidence intervals for probabilities of infection for diplotypes "1 3" and "3 3" were similar, but distinct from diplotype "1 1." These results are consistent with complete dominance of haplotype 3 relative to haplotype 1. The probability of infection at 9 mo of age for lambs with either diplotype "1 3" or "3 3" averaged 3.56 times that of lambs with diplotype "1 1." Genetic susceptibility to OPPV infection can be reduced by selection to increase the frequency of haplotype 1, resulting in a greater proportion of lambs with diplotype "1 1."

Consistent divergence times and allele sharing measured from cross-species application of SNP chips developed for three domestic species.

Recent advances in technology facilitated development of large sets of genetic markers for many taxa, though most often model or domestic organisms. Cross-species application of genomic technologies may allow for rapid marker discovery in wild relatives of taxa with well-developed resources. We investigated returns from cross-species application of three commercially available SNP chips (the OvineSNP50, BovineSNP50 and EquineSNP50 BeadChips) as a function of divergence time between the domestic source species and wild target species. Across all three chips, we observed a consistent linear decrease in call rate (~1.5% per million years), while retention of polymorphisms showed an exponential decay. These results will allow researchers to predict the expected amplification rate and polymorphism of cross-species application for their taxa of interest, as well as provide a resource for estimating divergence times.

Discovery, validation and characterization of 1039 cattle single nucleotide polymorphisms.

We identified approximately 13 000 putative single nucleotide polymorphisms (SNPs) by comparison of repeat-masked BAC-end sequences from the cattle RPCI-42 BAC library with whole-genome shotgun contigs of cattle genome assembly Btau 1.0. Genotyping of a subset of these SNPs was performed on a panel containing 186 DNA samples from 18 cattle breeds including 43 trios. Of 1039 SNPs confirmed as polymorphic in the panel, 998 had minor allele frequency > or =0.25 among unrelated individuals of at least one breed. When Btau 4.0 became available, 974 of these validated SNPs were assigned in silico to known cattle chromosomes, while 41 SNPs were mapped to unassigned sequence scaffolds, yielding one SNP every approximately 3 Mbp on average. Twenty-four SNPs identified in Btau 1.0 were not mapped to Btau 4.0. Of the 1015 SNPs mapped to Btau 4.0, 959 SNPs had nucleotide bases identical in Btau 4.0 and Btau 1.0 contigs, whereas 56 bases were changed, resulting in the loss of the in silico SNP in Btau 4.0. Because these 1039 SNPs were all directly confirmed by genotyping on the multi-breed panel, it is likely that the original polymorphisms were correctly identified. The 1039 validated SNPs identified in this study represent a new and useful resource for genome-wide association studies and applications in animal breeding.

Scrapie resistance in ARQ sheep.

Variation in the ovine prion protein amino acid sequence influences scrapie progression, with sheep homozygous for A(136)R(154)Q(171) considered susceptible. This study examined the association of survival time of scrapie-exposed ARQ sheep with variation elsewhere in the ovine prion gene. Four single nucleotide polymorphism alleles were associated with prolonged survival. One nonsynonymous allele (T112) was associated with an additional 687 days of survival for scrapie-exposed sheep compared to M112 sheep (odds ratio, 42.5; P = 0.00014). The only two sheep homozygous for T112 (TARQ) did not develop scrapie, suggesting that the allelic effect may be additive. These results provide evidence that TARQ sheep are genetically resistant to development of classical scrapie.

Evaluation of associations between prion haplotypes and growth, carcass, and meat quality traits in a Dorset x Romanov sheep population.

There is concern about potential antagonistic correlated responses due to intensive selection for scrapie-resistant haplotypes of the prion (PRNP) gene in sheep. The objective of the present research was to test for associations of PRNP haplotypes for codons 136, 154, and 171 with growth, carcass, and meat quality traits in an F2 Dorset x Romanov population (n = 415) segregating the 2 callipyge alleles. Haplotypes of the 3 PRNP codons were determined for each sheep, and breed of origin of each gamete was predicted by genotyping 6 microsatellite markers flanking the PRNP locus. Twenty-five growth, carcass, and meat quality traits were evaluated. Data were analyzed using a basic model consisting of fixed effects of year, sex, and callipyge genotype, the random effect of sire, and 7 covariates corresponding to the probability that a lamb inherited a specific PRNP haplotype of either Dorset or Romanov origin. A fixed effect of litter size was added to the model for growth traits. The model for carcass traits contained the linear and quadratic effects of chilled carcass weight and the interactions among callipyge genotype and linear and quadratic terms. For meat quality traits, the model contained chilled carcass weight as a covariate and the interaction between callipyge genotype and chilled carcass weight. A contrast between the resistant ARR haplotype and the average effect of other PRNP haplotypes was tested to investigate the effects of potential selection for ARR within each breed of origin (Dorset, ARR vs. ARQ, VRQ, and AHQ; Romanov, ARR vs. ARQ and VRQ). There was limited evidence that selecting for scrapie resistance would cause correlated responses due to linkage disequilibrium. Associations of only 3 traits with PRNP haplotypes were detected in either breed of origin. In Romanov, the ARR haplotype was associated with longer carcasses (P < 0.013), narrower rumps (P = 0.038), and less marbling (P = 0.022) than the average of ARQ and VRQ haplotypes. No significant contrasts were detected for Dorset. This study is the first to account for breed of origin while investigating haplotype associations in an F2 population. This study provided limited evidence of associations between PRNP haplotypes and growth, carcass, and meat quality traits.

Karyotypic analysis of adult pluripotent stem cells.

Three categories of precursor cells have been identified in postnatal mammals: tissue-committed progenitor cells, germ layer lineage-committed stem cells and lineage-uncommitted pluripotent stem cells. Progenitor cells are the immediate precursors of differentiated tissues. Germ layer lineage stem cells can be induced to form multiple cell types belonging to their respective ectodermal, mesodermal, and endodermal embryological lineages. Pluripotent stem cells will form somatic cell types from all three primary germ layer lineages. Progenitor cells demonstrate a finite life span before replicative senescence and cell death occur. Both germ layer lineage stem cells and pluripotent stem cells are telomerase positive and display extensive capabilities for self-renewal. Stem cells which undergo such extensive replication have the potential for undergoing mutations that may subsequently alter cellular functions. Gross mutations in the genome may be visualized as chromosomal aneuploidy and/or chromosomes that appear aberrant. This study was designed to determine whether any gross genomic mutations occurred within the adult pluripotent stem cells. Karyotypic analysis was performed using pluripotent stem cells purified from adult male rats using established procedures. Giemsa Banding was used in conjunction with light microscopy to visualize metaphase chromosome spreads. To date over 800 metaphase spreads have been analyzed. We found that the metaphase spreads averaged 42 chromosomes and concluded that these pluripotent stem cells isolated from adult rats have a normal karyotype.

Operation notes illustrated with digital images.

We would like to report on our experience of illustrating our operation notes with pre-, per- and post-operative digital images.

A rapid screening technique for feline odontoclastic resorptive lesions.

The feline odontoclastic resorptive lesion (FORL) status (presence or absence of odontoclastic resorptive lesions) of 423 clinically healthy cats was determined based on radiographic findings in a series of full mouth radiographs (eight views). This status was compared with the FORL status based on evaluation of only two views, namely the right and left mandibular premolar and molar views. Using the FORL status of the right and left third mandibular premolars (307 and 407) alone correctly predicted overall FORL status in 93.4 per cent of cats. The sensitivity of the new technique (FORL cases correctly diagnosed as positive by the test) was 78.5 per cent, while the negative predictive value (negative FORL cases correctly diagnosed by the test) was 91.3 per cent. Overall FORL status can therefore be confidently diagnosed in nine out of 10 cats by assessing FORL status in just two teeth (307 and 407) using two films, which has benefits for the cat (less anaesthetic time and reduced exposure to radiation) and the owner (reduced cost of screening).

The effects of chronic ethanol consumption on neurotrophins and their receptors in the rat hippocampus and basal forebrain.

Damage to the basal forebrain frequently results in deficits in learning and memory. Mnenonic dysfunction also occurs following prolonged ethanol consumption in humans and in animal models of chronic ethanol intake, accompanied by specific abnormalities in synaptic transmission between the basal forebrain and hippocampus. The integrity of at least some of the reciprocal neuronal connections between these brain regions is influenced by target-derived neurotrophic factors. We used a semiquantitative reverse transcription polymerase chain reaction technique to measure the messenger RNA for neurotrophins BDNF and NGF, and for their receptors trkB, trkA, and the low affinity receptor, p75(NTR) in the hippocampus and basal forebrain of rats after 28 weeks of alcohol consumption without malnutrition. This chronic ethanol treatment (CET) resulted in a marked and selective reduction in basal forebrain trkA mRNA. Western blotting revealed a similar reduction of basal forebrain trkA protein. CET effects on basal forebrain trkA may reflect impaired NGF signaling that could compromise septohippocampal synaptic connections, cholinergic differentiation, and emergent functional abilities dependent on these properties.

Evaluation of single-nucleotide polymorphisms in CAPN1 for association with meat tenderness in cattle.

Micromolar calcium activated neutral protease (CAPN1) was evaluated as a candidate gene for a quantitative trait locus (QTL) on BTA29 affecting meat tenderness by characterization of nucleotide sequence variation in the gene. Single-nucleotide polymorphisms (SNP) were identified by sequencing all 22 exons and 19 of the 21 introns in two sires (Piedmontese x Angus located at the U.S. Meat Animal Research Center in Clay Center, NE; Jersey x Limousin located at AgResearch in New Zealand) of independent resource populations previously shown to be segregating meat tenderness QTL on BTA29. The majority of the 38 SNP were found in introns or were synonymous substitutions in the coding regions, with two exceptions. Exons 14 and 9 contained SNP that were predicted to alter the protein sequence by the substitution of isoleucine for valine in Domain III of the protein, and alanine for glycine in Domain II of the protein. The resource populations were genotyped for these two SNP in addition to six intronic polymorphisms and two silent substitutions. Analysis of genotypes and shear force values in both populations revealed a difference between paternal CAPN1 alleles in which the allele encoding isoleucine at position 530 and glycine at position 316 associated with decreased meat tenderness (increased shear force values) relative to the allele encoding valine at position 530 and alanine at position 316 (P < 0.05). The association of maternal alleles with meat tenderness phenotypes is consistent with the hypothesis of CAPN1 as the gene underlying the QTL effect in two independent resource populations and presents the possibility of using these markers for selective breeding to reduce the numbers of animals with unfavorable meat tenderness traits.

Structure of the genes for porcine endometrial secreted and membrane folate binding proteins.

The endometrium of the pig produces two types of folate binding proteins (FBP) which, based on their sequences, are likely to be membrane (m) and secreted (s) forms. A clone containing both a gene coding for the sFBP cDNA and a gene coding for the mFBP was isolated from a yeast artificial chromosome (YAC) library. Each gene was subcloned and sequenced. The gene for sFBP spanned 4.4 kbp and included 5 exons. The mFBP gene spanned 7.0 kbp and also contained 5 exons. Structures of the genes were very similar for the last three exons, and this similarity was shared with other known FBP/folate receptor (FR) gene sequences. Unexpectedly, portions of introns 3 and 4 of both genes were highly homologous, suggesting the possibility that sequences within these introns served some as yet unknown function. In contrast, the structures of the 5' exons differed between the two genes and other known FBP/FR genes. Comparison of putative promoter regions for the two genes with promoter regions for human FBP/FR genes revealed significant sequence homology between sFBP and human gammaFBP and between mFBP and human alphaFR. These regions of homology may play a role in control of transcription of each gene.

Keratinocyte-driven contraction of reconstructed human skin.

We have previously reported that reconstructed human skin, using deepidermized acellular sterilized dermis and allogeneic keratinocytes and fibroblasts, significantly contracts in vitro. Contracture of split skin grafts in burns injuries remains a serious problem and this in vitro model provides an opportunity to study keratinocyte/mesenchymal cell interactions and cell interactions with extracted normal human dermis. The aim of this study was to investigate the nature of this in vitro contraction and explore several approaches to prevent or reduce contraction. Three different methodologies for sterilization of the dermal matrix were examined: glycerol, ethylene oxide and a combination of glycerol and ethylene oxide. While the nature of the sterilization technique influenced the extent of contraction and thinner dermal matrices contracted proportionately more than thicker matrices, in all cases contraction was driven by the keratinocytes with relatively little influence from the fibroblasts. The contraction of the underlying dermis did not represent any change in tissue mass but rather a reorganization of the dermis which was rapidly reversed (within minutes) when the epidermal layer was removed. Pharmacological approaches to block contraction showed forskolin and mannose-6-phosphate to be ineffective and ascorbic acid-2-phosphate to exacerbate contraction. However, Galardin, a matrix metalloproteinase inhibitor and keratinocyte conditioned media, both inhibited contraction.