The molecular mechanisms of probiotic strains in improving ageing bone and muscle of d-galactose-induced ageing rats.

AIM: The aim of this study was to evaluate the molecular mechanisms of Lactobacillus strains in improving ageing of the musculoskeletal system. METHODS AND RESULTS: The anti-ageing mechanism of three probiotics strains Lactobacillus fermentum DR9, Lactobacillus paracasei OFS 0291 and L. helveticus OFS 1515 were evaluated on gastrocnemius muscle and tibia of d-galactose-induced ageing rats. Upon senescence induction, aged rats demonstrated reduced antioxidative genes CAT and SOD expression in both bone and muscle compared to the young rats (P < 0·05). Strain L. fermentum DR9 demonstrated improved expression of SOD in bone and muscle compared to the aged rats (P < 0·05). In the evaluation of myogenesis-related genes, L. paracasei OFS 0291 and L. fermentum DR9 increased the mRNA expression of IGF-1; L. helveticus OFS 1515 and L. fermentum DR9 reduced the expression of MyoD, in contrast to the aged controls (P < 0·05). Protective effects of L. fermentum DR9 on ageing muscle were believed to be contributed by increased AMPK-α2 expression. Among the osteoclastogenesis genes studied, TNF-α expression was highly elevated in tibia of aged rats, while all three probiotics strains ameliorated the expression. Lactobacillus fermentum DR9 also reduced the expression of IL-6 and TRAP in tibia when compared to the aged rats (P < 0·05). All probiotics treatment resulted in declined proinflammatory cytokines IL-1ß in muscle and bone. CONCLUSIONS: Lactobacillus fermentum DR9 appeared to be the strongest strain in modulation of musculoskeletal health during ageing. SIGNIFICANCE AND IMPACT OF THE STUDY: The study demonstrated the protective effects of the bacteria on muscle and bone through antioxidative and anti-inflammatory actions. Therefore, L. fermentum DR9 may serve as a promising targeted anti-ageing therapy.

GENIPAC: A Genomic Information Portal for Head and Neck Cancer Cell Systems.

Head and neck cancer (HNC)-derived cell lines represent fundamental models for studying the biological mechanisms underlying cancer development and precision therapies. However, mining the genomic information of HNC cells from available databases requires knowledge on bioinformatics and computational skill sets. Here, we developed a user-friendly web resource for exploring, visualizing, and analyzing genomics information of commonly used HNC cell lines. We populated the current version of GENIPAC with 44 HNC cell lines from 3 studies: ORL Series, OPC-22, and H Series. Specifically, the mRNA expressions for all the 3 studies were derived with RNA-seq. The copy number alterations analysis of ORL Series was performed on the Genome Wide Human Cytoscan HD array, while copy number alterations for OPC-22 were derived from whole exome sequencing. Mutations from ORL Series and H Series were derived from RNA-seq information, while OPC-22 was based on whole exome sequencing. All genomic information was preprocessed with customized scripts and underwent data validation and correction through data set validator tools provided by cBioPortal. The clinical and genomic information of 44 HNC cell lines are easily assessable in GENIPAC. The functional utility of GENIPAC was demonstrated with some of the genomic alterations that are commonly reported in HNC, such as TP53, EGFR, CCND1, and PIK3CA. We showed that these genomic alterations as reported in The Cancer Genome Atlas database were recapitulated in the HNC cell lines in GENIPAC. Importantly, genomic alterations within pathways could be simultaneously visualized. We developed GENIPAC to create access to genomic information on HNC cell lines. This cancer omics initiative will help the research community to accelerate better understanding of HNC and the development of new precision therapeutic options for HNC treatment. GENIPAC is freely available at http://genipac.cancerresearch.my/ .

High fibrin/fibrinogen degradation product to fibrinogen ratio is associated with 28-day mortality and massive transfusion in severe trauma.

PURPOSE: There is a lack of association between coagulation biomarkers and long-term mortality in severe trauma. We aimed to investigate the association between coagulation biomarkers on admission and outcome of late stage of trauma. METHODS: This retrospective observational study included patients admitted with severe trauma between 2012 and 2015. We used the area under the receiver operating characteristic curve (AUROC) of coagulation biomarkers to determine 28-day mortality. Head Abbreviated Injury Scale scores greater than 3 were defined as traumatic brain injury (TBI). The primary outcome was 28-day mortality and the secondary outcome was massive transfusion. RESULTS: Of the 1266 patients included in the study, 28-day mortality rate was 19.7% (n = 249) and 7.9% (n = 100) of patients received massive transfusion. The AUROC of fibrin/fibrinogen degradation product (FDP) to fibrinogen ratio had a significantly higher prognostic performance than other markers. Multivariate analysis revealed that D-dimer level [odds ratio (OR) 1.033; 95% confidence interval (CI) 1.016-1.051] and FDP/fibrinogen ratio (OR 1.007; 95% CI 1.001-1.013) were independently associated with 28-day mortality. D-dimer (OR 1.028; 95% CI 1.003-1.055) and FDP/fibrinogen ratio (OR 1.035; 95% CI 1.012-1.058) were associated with 28-day mortality in the TBI group. In the non-TBI group, D-dimer was associated with 28-day mortality (OR 1.033; 95% CI 1.008-1.059), but the FDP/fibrinogen ratio was not. FDP/fibrinogen ratio, not D-dimer level, was an independent predictor for massive transfusion (OR 1.005; 95% CI 1.001-1.010). CONCLUSIONS: High FDP/fibrinogen ratio on arrival is a predictor of 28-day mortality and the requirement for massive transfusion in severe trauma.

Comparison of benzene and toluene photodegradation under visible light irradiation by Ba-doped BiFeO3 magnetic nanoparticles with fast sonochemical synthesis.

Bi1-xBaxFeO3 (x = 0.02, 0.04 and 0.07) multiferroic materials with a diameter in the range of 30-40 nm were controllably synthesized by a facile ultrasonic method, with a very short reaction time of 5 min at a low temperature of 30 °C, and the resulting BiFeO3 magnetic nanoparticles (BFO MNPs) exhibited enhanced magnetic and photocatalytic performance. The substitution of Ba2+ ions for Bi3+ ions at the A-site of BFO MNPs, even at only 2%, decreased their particle size and distorted the lattice in the rhombohedral structure of BFO MNPs. Increasing the Ba doping to 7% greatly increased the ferromagnetic properties of BFO MNPs from 3.55 to 6.09 emu g-1. In comparison with pure BFO MNPs, 7% Ba substitution in the Ba-doped BFO MNP samples produced strong absorption in the visible light region, decreasing the band-gap energy from 2.11 to 1.86 eV. Photoluminescence (PL) spectroscopy identified the band-gap emission for BFO MNPs at 587 nm, while for both pure and Ba-doped samples, the other emissions were attributed to the defect states related to oxygen deficiencies inside the band gap. After 50 min of visible light irradiation, Bi1-xBaxFeO3 (x = 7%), with the lowest band gap energy, highest magnetization and smallest particle size, showed almost complete photocatalytic degradation of toluene and benzene (100 mg L-1), with 91 and 81% reduction, respectively, in total organic carbon (TOC). For all irradiation times, the mineralization efficiency of toluene was higher than that of benzene, which demonstrated that toluene is more sensitive to photocatalytic oxidation than is benzene.

Effect of metabolic syndrome on the outcome of corticosteroid injection for carpal tunnel syndrome.

Diffuse peripheral nerve impairment is common in metabolic syndrome: in patients with metabolic syndrome and carpal tunnel syndrome this might affect the outcome of treatment by local corticosteroid injection. A total of 55 consecutive patients with carpal tunnel syndrome and metabolic syndrome treated with corticosteroid injection (10 mg triamcinolone acetonide) were age and sex matched with 55 control patients without metabolic syndrome. Grip strength, perception of touch with Semmes-Weinstein monofilaments and Boston Carpal Tunnel Questionnaires were assessed at the baseline and at 6, 12 and 24 weeks follow-up. The two groups had similar pre-operative grip strength and Boston Carpal Tunnel Questionnaire scores. The Boston Carpal Tunnel Questionnaire symptom and function scores of the metabolic syndrome group were significantly greater than the control group at 12 and 24 weeks follow-up. Except for significantly greater grip strength at the 12-week follow-up in the control group, there were no significant differences in grip strength between the groups. Semmes-Weinstein monofilament sensory index for the control group was significantly greater than that of the metabolic syndrome group throughout the 24-week follow-up. After 24 weeks, five patients (13%) in the control group and 13 patients (27%) in the metabolic syndrome group had had carpal tunnel surgery. Patients with metabolic syndrome are at risk for poor functional outcome and failure of treatment after corticosteroid injection for carpal tunnel syndrome. LEVEL OF EVIDENCE: Treatment benefits III.

Cold plasma inactivation of chronic wound bacteria.

Cold plasma is partly ionized non-thermal plasma generated at atmospheric pressure. It has been recognized as an alternative approach in medicine for sterilization of wounds, promotion of wound healing, topical treatment of skin diseases with microbial involvement and treatment of cancer. Cold plasma used in wound therapy inhibits microbes in chronic wound due to its antiseptic effects, while promoting healing by stimulation of cell proliferation and migration of wound relating skin cells. In this study, two types of plasma systems are employed to generate cold plasma: a parallel plate dielectric barrier discharge and a capillary-guided corona discharge. Parameters such as applied voltage, discharge frequency, treatment time and the flow of the carrier gas influence the cold plasma chemistry and therefore change the composition and concentration of plasma species that react with the target sample. Chronic wound that fails to heal often infected by multidrug resistant organisms makes them recalcitrant to healing. Methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (Pseudomonas aeruginosa) are two common bacteria in infected and clinically non-infected wounds. The efficacies of the cold plasma generated by the two designs on the inactivation of three different isolates of MRSA and four isolates of P. aeruginosa are reported here.

Peroxiredoxin II promotes hepatic tumorigenesis through cooperation with Ras/Forkhead box M1 signaling pathway.

The current study was carried out to define the involvement of Peroxiredoxin (Prx) II in progression of hepatocellular carcinoma (HCC) and the underlying molecular mechanism(s). Expression and function of Prx II in HCC was determined using H-ras(G12V)-transformed HCC cells (H-ras(G12V)-HCC cells) and the tumor livers from H-ras(G12V)-transgenic (Tg) mice and HCC patients. Prx II was upregulated in H-ras(G12V)-HCC cells and H-ras(G12V)-Tg mouse tumor livers, the expression pattern of which highly similar to that of forkhead Box M1 (FoxM1). Moreover, either knockdown of FoxM1 or site-directed mutagenesis of FoxM1-binding site of Prx II promoter significantly reduced Prx II levels in H-ras(G12V)-HCC cells, indicating FoxM1 as a direct transcription factor of Prx II in HCC. Interestingly, the null mutation of Prx II markedly decreased the number and size of tumors in H-ras(G12V)-Tg livers. Consistent with this, knockdown of Prx II in H-ras(G12V)-HCC cells reduced the expression of cyclin D1, cell proliferation, anchorage-independent growth and tumor formation in athymic nude mice, whereas overexpression of Prx II increased or aggravated the tumor phenotypes. Importantly, the expression of Prx II was correlated with that of FoxM1 in HCC patients. The activation of extracellular signal-related kinase (ERK) pathway and the expression of FoxM1 and cyclin D1 were highly dependent on Prx II in H-ras(G12V)-HCC cells and H-ras(G12V)-Tg livers. Prx II is FoxM1-dependently-expressed antioxidant in HCC and function as an enhancer of Ras(G12V) oncogenic potential in hepatic tumorigenesis through activation of ERK/FoxM1/cyclin D1 cascade.

Maternal polycystic ovary syndrome and the risk of autism spectrum disorders in the offspring: a population-based nationwide study in Sweden.

Although many studies indicate the interplay of genetic and environmental factors in the etiology of autism spectrum disorder (ASD), our limited understanding of the underlying mechanisms hampers the development of effective ways of detecting and preventing the disorder. Recent studies support the hypothesis that prenatal androgen exposure contributes to the development of ASD. This would suggest that maternal polycystic ovary syndrome (PCOS), a condition associated with excess androgens, would increase the risk of ASD in the offspring. We conducted a matched case-control study nested within the total population of Sweden (children aged 4-17 who were born in Sweden from 1984 to 2007). The sample consisted of 23 748 ASD cases and 208 796 controls, matched by birth month and year, sex and region of birth. PCOS and ASD were defined from ICD codes through linkage to health-care registers. Maternal PCOS increased the odds of ASD in the offspring by 59%, after adjustment for confounders (odds ratio (OR) 1.59, 95% confidence interval (CI) 1.34-1.88). The odds of offspring ASD were further increased among mothers with both PCOS and obesity, a condition common to PCOS that is related to more severe hyperandrogenemia (OR 2.13, 95% CI 1.46-3.10). Risk estimates did not differ between sexes. In conclusion, children of women with PCOS appear to have a higher risk of developing ASD. This finding awaits confirmation, and exploration of potentially underlying mechanisms, including the role of sex steroids in the etiology of ASD.

Serial transrectal ultrasonography for monitoring the reproductive activity of the Asiatic black bear (Ursus thibetanus ussuricus).

This study evaluated the structural changes in the reproductive tract of Asiatic black bears using serial transrectal ultrasonography. In addition, the ultrasonographic observations were compared with the results of vaginal cytology and hormonal analyses. The collection of blood for hormonal analysis, vaginal cytology and transrectal ultrasonography was performed in two bears (Bears 1 and 2) from June 2011 to August 2013 without mating and in a third bear (Bear 3) from April to December 2012, allowing natural mating. Serial ultrasonographic observations showed cyclic changes in ovarian structures (e.g. emergence of small follicles, growth and ovulation of dominant follicles and corpus luteum (CL) formation) during the reproductive cycles of the three bears. The diameter of the uterine horns remained similar throughout the reproductive cycle in Bears 1 and 2, and it remained similar from April until October, but an enlargement containing foetuses was observed in Bear 3 in December. The ultrasonographic observations were consistent with the data obtained through vaginal cytology and progesterone analysis during the reproductive cycle. An average of 4.0 (±0.4) dominant follicles was observed during the oestrous stage (May-August), during which the superficial cells accounted for >90% of the total vaginal cells. In addition, the detection of an average of 2.6 (±0.2) CL was associated with increased plasma progesterone concentrations (3.0 ± 0.4 ng/ml) between June and December (near hibernation). In conclusion, serial transrectal ultrasonography demonstrated yearly oestrous (ovulation) cycles via follicular dynamics and CL formation on ovaries, accordingly with vaginal cytology and hormonal level in the Asiatic black bear.

Melt-spun shaped fibers with enhanced surface effects: fiber fabrication, characterization and application to woven scaffolds.

Scaffolds with a high surface-area-to-volume ratio (SA:V) are advantageous with regard to the attachment and proliferation of cells in the field of tissue engineering. This paper reports on the development of novel melt-spun fibers with a high SA:V, which enhanced the surface effects of a fiber-based scaffold while maintaining its mechanical strength. The cross-section of the fibers was altered to a non-circular shape, producing a higher SA:V for a similar cross-sectional area. To obtain fibers with non-circular cross-sectional shape, or shaped fibers, three different types of metal spinnerets were fabricated for the melt-spinning process, each with circular, triangular or cruciform capillaries, using deep X-ray lithography followed by nickel electroforming. Using these spinnerets, circular and shaped fibers were manufactured with biodegradable polyester, polycaprolactone. The SA:V increase in the shaped fibers was experimentally investigated under different processing conditions. Tensile tests on the fibers and indentation tests on the woven fiber scaffolds were performed. The tested fibers and scaffolds exhibited similar mechanical characteristics, due to the similar cross-sectional area of the fibers. The degradation of the shaped fibers was notably faster than that of circular fibers, because of the enlarged surface area of the shaped fibers. The woven scaffolds composed of the shaped fibers significantly increased the proliferation of human osteosarcoma MG63 cells. This approach to increase the SA:V in shaped fibers could be useful for the fabrication of programmable, biodegradable fiber-based scaffolds in tissue engineering.

Nutritional and Hormonal Induction of Fatty Liver Syndrome and Effects of Dietary Lipotropic Factors in Egg-type Male Chicks.

This experiment was conducted with male chicks to investigate the influence of hormones and nutrients on the development of fatty liver syndrome (FLS) as well as the effects of dietary lipotropic factors on hepatic fat accumulation and lipogenic enzyme gene expression. A total of two-hundred sixteen 4-wk-old Hy-Line male chicks were divided into six groups and fed an experimental diet (T1, low-energy diet with low levels of lipotropic factors; T2, high-energy diet with low levels of lipotropic factors; T3 and T5, low-energy diet with high levels of lipotropic factors; T4 and T6, high-energy diet with high levels of lipotropic factors) for six weeks. The chicks in T5 and T6 groups were treated with intramuscular injections of estradiol benzoate for three days prior to biopsy and clinical analysis of FLS. Chicks treated with estrogen had significantly greater liver weights than untreated chicks. The abdominal fat contents were increased in chicks consuming high-energy diets as compared to those consuming low-energy diets. Treatment with estrogen significantly increased the concentrations of serum cholesterol, triacylglycerol and phospholipid (p<0.05). The hepatic triacylglycerol levels were tenfold higher in the estrogen treated chicks than in the untreated chicks. There were no significant differences in malondialdehyde levels between the treatment groups. Estrogen treatment dramatically increased the levels of fatty acid synthetase, acetyl-CoA carboxylase and ApoB mRNA. The results indicated that treatment with exogenous estrogen in growing male chicks induced hepatic fat accumulation, which might be partially due to increased lipogenic enzyme gene expression.

The N-terminal region of the dopamine D2 receptor, a rhodopsin-like GPCR, regulates correct integration into the plasma membrane and endocytic routes.

BACKGROUND AND PURPOSE: Functional roles of the N-terminal region of rhodopsin-like GPCR family remain unclear. Using dopamine D(2) and D(3) receptors as a model system, we probed the roles of the N-terminal region in the signalling, intracellular trafficking of receptor proteins, and explored the critical factors that determine the functionality of the N-terminal region. EXPERIMENTAL APPROACH: The N-terminal region of the D(2) receptor was gradually shortened or switched with that of the D(3) receptor or a non-specific sequence (FLAG), or potential N-terminal glycosylation sites were mutated. Effects of these manipulations on surface expression, internalization, post-endocytic behaviours and signalling were determined. KEY RESULTS: Shortening the N-terminal region of the D(2) receptor enhanced receptor internalization and impaired surface expression and signalling; ligand binding, desensitization and down-regulation were not affected but their association with a particular microdomain, caveolae, was disrupted. Replacement of critical residues within the N-terminal region with the FLAG epitope failed to restore surface expression but partially restored the altered internalization and signalling. When the N-terminal regions were switched between D(2) and D(3) receptors, cell surface expression pattern of each receptor was switched. Mutations of potential N-terminal glycosylation sites inhibited surface expression but enhanced internalization of D(2) receptors. CONCLUSIONS AND IMPLICATIONS: Shortening of N-terminus or mutation of glycosylation sites located within the N-terminus enhanced receptor internalization but impaired the surface expression of D(2) receptors. The N-terminal region of the D(2) receptor, in a sequence-specific manner, controls the receptor's conformation and integration into the plasma membrane, which determine its subcellular localization, intracellular trafficking and signalling properties.

In-situ synchrotron radiation photoemission spectroscopy study of the initial atomic layer deposition of Al2O3 film on Si(001) substrate.

In-situ synchrotron radiation photoemission spectroscopy and X-ray photoemission spectroscopy have been used to investigate the initial stages of Al2O3 growth on a Si(001) substrate by atomic layer deposition (ALD). The core level spectra of Si 2p, O 1s, and Al 2p as well as the valence band spectra were measured at every half reaction in the trimethylaluminum (TMA)-H2O ALD process. The line shape changes and binding energy shifts of the core level spectra reveal that Al2O3 is predominantly formed with a small amount of Si oxide in the initial stages without the formation of Al silicate. All core level spectra were alternately shifted toward higher and lower binding energies sides at every half ALD reaction. This can be explained by the band bending effect induced by different chemical species on the surface during the TMA-H2O ALD reaction. The valence band spectra showed that four cycles of ALD reactions were necessary to complete the electronic structure of the Al2O3 film with a valence band offset of 3.73 eV.

Changes in hepatic lipid parameters and hepatic messenger ribonucleic acid expression following estradiol administration in laying hens (Gallus domesticus).

Fatty liver hemorrhagic syndrome (FLHS) is characterized by increased hepatic triacylglycerol content associated with liver hemorrhages and results in a sudden decline in egg production. Genetic, environmental, nutritional, and hormonal factors have all been implicated in the etiology of FLHS, but the exact cause of FLHS is still unknown. Estrogens have been implicated in the development of excess fat content of the liver and in the etiology of FLHS. This study investigated estradiol (E(2)) administration in hens and its effect on lipid metabolism. Hy-Line Brown laying hens were intramuscularly injected with E(2) on a daily basis for 3 wk. The dosages were 0, 0.5, and 1.0 mg/kg of BW, with corn oil injections used as a control. Egg production and quality were measured among the groups, with no significant difference seen in egg production. Liver weights of hens treated with E(2) were greater than those of control hens, but the increase was not statistically significant. Serum glutamic-oxaloacetic transaminase and glutamic-pyruvic transaminase activities and E(2) plasma concentrations increased in a dose-dependent manner, with plasma concentration of E(2) increasing from 6,900 to 19,000 pg/mL. No significant differences in free cholesterol or phospholipids were observed, but there was a significant increase in hepatic triacylglycerol levels. Injection with E(2) showed an increased expression of mRNA for peroxisome proliferator-activated receptor γ (23-fold), but not for peroxisome proliferator-activated receptor α. A statistically significant increase was seen for fatty acid synthase, apolipoprotein B, and adenosine triphosphate citrate lyase, but not for acetyl coenzyme A carboxylase, apolipoprotein VLDL-II, microsomal triglyceride transport protein, or malic enzyme. For proteins involved in the oxidation of E(2), only cytochrome P450 3A37 showed a statistically significant increase. The present results suggest that E(2) upregulates the synthesis of fatty acids and triacylglycerols and the accumulation of hepatic lipids by increasing mRNA expression related to lipid metabolism, and that excess E(2) in the blood leads to activation of E(2) catabolic metabolism (cytochrome P450 3A37)-related mRNA expression.

Allergen-specific immunosuppression by ovalbumin fused with diphtheria toxin in mice sensitized with albumins of different origin.

BACKGROUND: We previously reported that ovalbumin-diphtheria toxin (OVA-DT) fusion protein eliminates mast cells bearing OVA-specific IgE and protects OVA-sensitized mice from fatal anaphylaxis induced by OVA challenge. OBJECTIVE: To prove the specificity of therapeutic effect of OVA-DT to allergy induced by OVA only and not by other allergens such as human serum albumin (HSA), and to examine the cytotoxic effect of OVA-DT on B cells bearing OVA-specific IgE. METHODS: Mice were sensitized with two different antigens, OVA and HSA, and then treated with OVA-DT. The therapeutic effect of OVA-DT on the allergy response to each of allergen was evaluated by anaphylactic test. The effect of OVA-DT on the production of allergen-specific Ig isotypes of the sensitized mice and the cytotoxic effect of OVA-DT on B cells expressing OVA-specific IgE were examined. RESULTS: OVA-DT suppressed only OVA-induced allergy but not HSA-induced allergy in mice sensitized with a mixture of OVA and HSA. The suppression was prolonged even to the mice boosted with the same allergen 14 days after last treatment of OVA-DT. In addition, when the sensitized mice were boosted with the same allergens 14 days after last treatment of OVA-DT, the mice showed to increase the production of OVA-specific IgG2a/IgG3 and decreased that of OVA-specific IgE. OVA-DT targeted B cells bearing OVA-specific IgE, and killed them by DT-mediated cytotoxicity. CONCLUSION: The therapeutic effect of OVA-DT was specific to OVA-induced allergy and the suppression of OVA-induced allergy was continuously shown in the mice boosted with the same allergens. This is considered to be caused by the increase of OVA-specific IgG2a and IgG3, and because of the decrease of OVA-specific IgE by killing of B cells bearing OVA-specific IgE.

In vitro cultured autologous pre-confluent oral keratinocytes for experimental prefabrication of oral mucosa.

The reconstruction of large defects after head and neck cancer resection often requires composite tissue transfer to replace a combination of bone, muscle and mucosa. Thus, tissue engineering techniques may be useful for oral mucosal reconstructive surgery to prefabricate mucosal tissue on the muscle flap in vivo, instead of using conventional skin-bearing composite flaps. The aim of this study was to investigate whether autogenous pre-confluent oral keratinocytes (PCOK) cultured in vitro can create mucosal coverage on muscle in vivo, in a single grafting procedure. In 30 Wistar rats, with a small piece of oral mucosa (2 mm x 5 mm), oral keratinocytes were isolated and then seeded on a hydrophilic PTFE membrane (n = 50) in serum-free culture condition. After 48 h, the membrane, together with the PCOK, was transplanted onto the gracilis muscle to fabricate a mucosal flap in vivo. The wound bed was closed primarily until the time of examination. Biopsies were carried out 1, 2, 3, and 4 weeks, respectively, after transplantation and were evaluated immunohistochemically (AE1/AE3 anti-pancytokeratin, cytokeratin 5/6, collagen IV, laminin, lectin-specific labeling of N-acetylglucosamine oligomeres of endothelial cells) with relation to the following criteria: (1) graft acceptance; (2) inflammatory signs; (3) structural changes and keratinocyte lining; (4) expression of basement membrane components; and (5) vascularization. Ninety-one percent of the grafts showed uniform epithelial layers. The mean number of reconstructed epithelial cell layers was 1.7, 2.0, 1.85 and 2.7 at 1, 2, 3 and 4 weeks, respectively after transplantation (P = 0.342). Collagen IV, laminin and lectin-specific capillaries developed between the neoepithelium and the underlying muscular layer. Only two specimens showed signs of infection 2 weeks after transplantation. In conclusion, this experiment demonstrated that PCOK grafts on muscle in vivo can achieve uniform multi-layered oral epithelial coverage in a short period of time. This technique may be a useful alternative tool for oropharyngeal reconstructive surgery and is also worth considering for further clinical studies.

Increased affinity and stability of an anti-HIV-1 envelope immunotoxin by structure-based mutagenesis.

HIV-infected cells are selectively killed by an immunotoxin in which a truncated form of Pseudomonas exotoxin A is joined to the variable region of a broadly neutralizing antibody (3B3) that recognizes the viral envelope glycoprotein (Env). To improve the efficacy of this molecule, we used three-dimensional structural information and phage selection data to design 23 single and multiple point mutations in the antibody variable region sequences that contact Env. Substituting an aromatic residue for an aspartate in the third complementarity-determining region of V(H) increased the potency of the immunotoxin by approximately 10-fold in a cell-killing assay. Detailed analysis of one such mutant, N31H/Q100eY, revealed both a higher affinity for monomeric and cell surface Env and an increased stability against aggregation compared with the starting immunotoxin. Conversion to a disulfide-linked two-chain format further stabilized the protein. N31H/Q100eY retained the ability to bind to Env from multiple viral isolates, to inhibit Env-mediated cell fusion, and to limit spreading viral infection in peripheral blood mononuclear cells. Such site-directed mutants may increase the utility of immunotoxins for reducing or eradicating persistent HIV-1 infection in humans.

Study of the binding environment of alpha-factor in its G protein-coupled receptor using fluorescence spectroscopy.

Mating in Saccharomyces cerevisiae is induced by the interaction of alpha-factor (W1H2W3L4Q5L6K7P8G9Q10P11M12Y13) with its cognate G protein-coupled receptor (Ste2p). Fifteen fluorescently labeled analogs of alpha-factor in which the 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group was placed at the alphaN-terminus and in side-chains at positions 1, 3, 4, 6, 7, 12 and 13 were synthesized and assayed for biological activity and receptor affinity. Eleven of the analogs retained 6-60% of the biological activity of the alpha-factor, as judged using a growth arrest assay. The binding affinities depended on the position of NBD attachment in the peptide and the distance of the tag from the backbone. Derivatization of the positions 3 and 7 side-chains with the NBD group resulted in analogs with affinities of 17-35% compared with that of alpha-factor. None of the other NBD-containing agonists had sufficient receptor affinity or strong enough emission for fluorescence analysis. The position 3 and 7 analogs were investigated using fluorescence spectroscopy and collisional quenching by KI in the presence of Ste2p in yeast membranes. The results showed that the lambda max of NBD in the position 7 side-chain shifted markedly to the blue (510 nm) when separated by 4 or 6 bonds from the peptide backbone and that this probe was shielded from quenching by KI. In contrast, separation by 3, 5, 10 or more bonds resulted in lambda max ( approximately 540 nm) and collisional quenching constants consistent with increasing degrees of exposure. The NBD group in the position 3 side-chain was also found to be blue shifted (lambda max=520 nm) and shielded from solvent. These results indicate that the position 7 side-chain is likely interacting with a pocket formed by extracellular domains of Ste2p, whereas the side-chain of Trp3 is in a hydrophobic pocket possibly within the transmembrane region of the receptor.

PATE, a gene expressed in prostate cancer, normal prostate, and testis, identified by a functional genomic approach.

To identify target antigens for prostate cancer therapy, we have combined computer-based screening of the human expressed sequence tag database and experimental expression analysis to identify genes that are expressed in normal prostate and prostate cancer but not in essential human tissues. Using this approach, we identified a gene that is expressed specifically in prostate cancer, normal prostate, and testis. The gene has a 1.5-kb transcript that encodes a protein of 14 kDa. We named this gene PATE (expressed in prostate and testis). In situ hybridization shows that PATE mRNA is expressed in the epithelial cells of prostate cancers and in normal prostate. Transfection of the PATE cDNA with a Myc epitope tag into NIH 3T3 cells and subsequent cell fractionation analysis shows that the PATE protein is localized in the membrane fraction of the cell. Analysis of the amino acid sequence of PATE shows that it has structural similarities to a group of proteins known as three-finger toxins, which includes the extracellular domain of the type beta transforming growth factor receptor. Restricted expression of PATE makes it a potential candidate for the immunotherapy of prostate cancer.

Diagnostic and prognostic relevance of expression of human telomerase subunits in oral cancer.

Telomerase activity (TA) is associated with most malignant human tumors but is not detected in normal somatic cells with a few exceptions. Three major subunits (hTR, hTP1 and hTERT) of telomerase have been identified. To investigate the clinical implications of the mRNA detection of these components as useful diagnostic and prognostic markers in oral cancer, we examined TA, hTR, hTP1 and hTERT mRNA expressions in 46 oral squamous cell carcinomas (OSCC) and 15 normal oral mucosal tissues from healthy volunteers using a highly sensitive TRAP assay and RT PCR. In all specimens hTR and hTP1 mRNA were detected regardless whether TA was expressed or not. On the contrary, a significant correlation between hTERT expression and TA was shown indicating that the activity of telomerase could be regulated by the extent of hTERT transcription. In addition, hTERT expression showed close association to malignancies. None of the normal mucosal specimens expressed the hTERT subunit, but 76% (35/46) of the tumor specimens did. None of other clinico-pathological and prognostic parameters showed significant relationship with TA or hTERT expression. These results suggest that the detection of hTERT expression may be another useful diagnostic marker, especially for early detection of OSCC, and for distinguishing healthy tissues from neoplastically transformed ones.