A rare castration-resistant progenitor cell population is highly enriched in Pten-null prostate tumours.

Castration-resistant prostate cancer is a lethal disease. The cell type(s) that survive androgen deprivation remain poorly described, despite global efforts to understand the various mechanisms of therapy resistance. We recently identified in wild-type (WT) mouse prostates a rare population of luminal progenitor cells that we called LSCmed according to their FACS profile (Lin- /Sca-1+ /CD49fmed ). Here, we investigated the prevalence and castration resistance of LSCmed in various mouse models of prostate tumourigenesis (Pb-PRL, Ptenpc-/- , and Hi-Myc mice). LSCmed prevalence is low (∼8%, similar to WT) in Hi-Myc mice, where prostatic androgen receptor signalling is unaltered, but is significantly higher in the two other models, where androgen receptor signalling is decreased, rising up to more than 80% in Ptenpc-/- prostates. LSCmed tolerate androgen deprivation and persist or are enriched 2-3 weeks after castration. The tumour-initiating properties of LSCmed from Ptenpc-/- mice were demonstrated by regeneration of tumours in vivo. Transcriptomic analysis revealed that LSCmed represent a unique cell entity as their gene expression profile is different from luminal and basal/stem cells, but shares markers of each. Their intrinsic androgen signalling is markedly decreased, explaining why LSCmed tolerate androgen deprivation. This also illuminates why Ptenpc-/- tumours are castration-resistant since LSCmed represent the most prevalent cell type in this model. We validated CK4 as a specific marker for LSCmed on sorted cells and prostate tissues by immunostaining, allowing for the detection of LSCmed in various mouse prostate specimens. In castrated Ptenpc-/- prostates, there was significant proliferation of CK4+ cells, further demonstrating their key role in castration-resistant prostate cancer progression. Taken together, this study identifies LSCmed as a probable source of prostate cancer relapse after androgen deprivation and as a new therapeutic target for the prevention of castrate-resistant prostate cancer. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Keratin 4 regulates the development of human white sponge nevus.

BACKGROUND: The aim of this study was to investigate the roles of keratin 4 (KRT4) gene in the development of human white sponge nevus (WSN). METHODS: Transgenic mice were created using the microinjection method with pcDNA3.1 vectors expressing KRT4 wild-type (WT) gene and E520K mutation. Polymerase chain reaction (PCR) and Western blotting were used to identify the genotype of transgenic founders and their filial generations. Expression of KRT4 in mouse oral mucosa was characterized by immunohistochemistry (IHC), and the whole epithelium layer of transgenic mice was observed using transmission electron microscope (TEM). RESULTS: The positive rate of KRT4 transgenic mice in F1 generation was 45.5%. Expression level of KRT4 protein was significantly higher in 2-month-old transgenic mice than WT mice. Furthermore, all the epithelial lamina of 3-month-old transgenic mice showed reduced staining of KRT4. The surface and spinous layers were full of hyalocytes and bubble cells, which are similar to the clinical symptoms of WSN. For the ultrastructure, both tonofilaments and Odland bodies increased. CONCLUSIONS: Our study indicated the mutated KRT4 gene may play important roles in the pathogenesis of WSN.

The alcohol-sensitive period during early octavolateral organ development in zebrafish (Danio rerio).

Fetal alcohol exposure can cause Fetal Alcohol Spectrum Disorders (FASD), completely preventable developmental disabilities characterized by permanent birth defects. However, specific gestational timing when developing organs are most sensitive to alcohol exposure is unclear. In this study, we examined the temporal effects of embryonic alcohol exposure on octavolateral organs in zebrafish (Danio rerio), including inner ears and lateral line neuromasts that function in hearing, balance, and hydrodynamic detection, respectively. To determine an alcohol-sensitive period in the first 24 hours post fertilization (hpf), Et(krt4:EGFP)sqet4 zebrafish that express green fluorescent protein in sensory hair cells were treated in 2% alcohol for 2, 3, and 5-hours. Octavolateral organs of control and alcohol-exposed larvae were examined at 3, 5, and 7 days post fertilization (dpf). Using confocal and light microscopy, we found that alcohol-exposed larvae had significantly smaller otic vesicles and saccular otoliths than control larvae at 3 dpf. Only alcohol-exposed larvae from 12-17 hpf had smaller otic vesicles at 5 dpf, smaller saccular otoliths at 7 dpf and fewer saccular hair cells, neuromasts and hair cells per neuromast at 3 dpf. In addition, auditory function was assessed by microphonic potential recordings from inner ear hair cells in response to 200-Hz stimulation. Hearing sensitivity was reduced for alcohol-exposed larvae from 7-12 and 12-17 hpf. Our results show that 12-17 hpf is an alcohol-sensitive time window when morphology and function of zebrafish octavolateral organs are most vulnerable to alcohol exposure. This study implies that embryonic alcohol exposure timing during early development can influence severity of hearing deficits. © 2017 Wiley Periodicals, Inc.

A unique lineage gives rise to the meibomian gland.

PURPOSE: To identify the lineage that contributes to the morphogenesis of the meibomian gland. METHODS: To examine which cell lineage gives rise to the meibomian gland, the expression of Pax6 as well as that of various cytokeratin markers, including keratin 14 (Krt14), Krt15, Krt4, and Krt10, was examined with immunofluorescent staining of C57BL/6J mouse eyelids from P2 to P11 pups and adult mice. RESULTS: Pax6 was localized to the cytoplasm within the acinar region of the meibomian glands during morphogenesis but was absent in the fully developed gland. Keratin 14 was expressed throughout the gland at all stages whereas keratin 15 was absent at all stages. Keratin 4, a marker of mucosal lineage, was present throughout the gland and was colocalized with keratin 10 (epidermal lineage marker) in the developing duct at P4. This colocalization region decreased as the gland developed becoming restricted to the central duct near the opening to the acini in the fully developed gland. CONCLUSIONS: We identified a unique cell lineage that expresses markers characteristic of mucosal and epidermal epithelia during meibomian gland morphogenesis. This unique group of cells was located in the central duct with a concentration near the ductule orifice. The expression of these cells reduced during meibomian gland morphogenesis and may play a role in the development and homeostasis of the gland.

Reconstruction of conjunctival epithelium-like tissue using a temperature-responsive culture dish.

PURPOSE: To study the feasibility of engineering conjunctival epithelial cell sheets on a temperature-responsive culture dish for ocular surface reconstruction. METHODS: Rabbit conjunctival epithelial cells (rCjECs) were cultured in DMEM/F-12 (1:1) medium. The morphology and phenotype of the rCjECs were confirmed with phalloidin staining, periodic acid-Schiff (PAS) staining, and immunocytochemistry. The rCjECs cultured on a temperature-responsive culture dish for 10 days produced confluent conjunctival epithelial cell sheets. Then, the phenotype, structure, and function of the conjunctival epithelial cell sheets were examined. RESULTS: The conjunctival epithelial cells were compact, uniform, and cobblestone shape. All cultured conjunctival epithelial cells were harvested as intact cell sheets by reducing the culture temperature to 20 °C. Conjunctival epithelial cells were stratified in four to five cell layers similar to the conjunctival epithelium. CCK-8 analysis, 5-bromo-2'-deoxyuridine (BrdU) staining, and the live and dead viability assay confirmed that viable proliferation cells were retained in the cell sheets. Immunohistochemistry for CK4, CK19, and MUC5AC showed the cell sheets still maintained characteristics of the conjunctival epithelium. CONCLUSIONS: A temperature-responsive culture dish enables fabrication of viable conjunctival epithelial cell sheets with goblet cells and proliferative cells. Conjunctival epithelial cell sheets will be promising for reconstruction of the conjunctival epithelium.

Latent transforming growth factor-ß binding proteins (LTBP-1 and LTBP-2) and gingiva keratinization.

OBJECTIVE: Transforming growth factor-beta (TGF-ß) proteins are involved in epithelial keratinization. The major function of latent TGF-ß binding proteins (LTBPs) is modulating TGF-ß activity. However, whether LTBP-1 and LTBP-2 play roles in gingiva keratinization remains unclear. MATERIALS AND METHODS: Human keratinized gingiva and non-keratinized alveolar mucosa were processed for LTBP-1, LTBP-2, cytokeratin-1 (K1), cytokeratin-4 (K4), and TGF-ß immunohistochemical (IHC) staining. Porcine heterotopically transplanted connective tissues and newly grown epithelia were harvested for IHC staining. The expression levels of LTBP-1 and LTBP-2 were compared between differentiated and undifferentiated human normal oral keratinocytes (hNOK). The expression of LTBP-1 and LTBP-2 was knocked down in a cell line (OEC-M1) to evaluate the effects on the expression of K1, K4, and involucrin (INV). RESULTS: In human and porcine specimens, LTBP-2 expression patterns distinguished keratinized and non-keratinized oral epithelia. Western blotting results showed that K1, LTBP-1, and INV proteins were upregulated in differentiated hNOK. In OEC-M1 cells, LTBP-2 knockdown resulted in upregulated the expression of K1 and INV and downregulated the expression of K4. LTBP-1 knockdown resulted in opposite effects. CONCLUSION: The expression patterns of LTBP-2 differ in keratinized gingiva and non-keratinized mucosa. LTBP-1 and LTBP-2 are involved in the keratinization of oral epithelium; however, the underlying mechanism remains to be elucidated.

[Morphology, immunohistochemistry and hTERC gene in-situ hybridization in Barrett's esophagus].

OBJECTIVE: To study the clinicopathologic features and differential diagnosis of proximal gastric mucosa and mucosa of Barrett's esophagus (BE) in biopsy specimens. METHOD: Thirty-eight cases of Barrett's esophagus (diagnosed using WHO criteria) and 44 cases of proximal gastric mucosa were studied by immunohistochemistry (for CK7, CK20, CK4, CK8, S-100 protein, MUC6, COX2 and bcl-2) and fluorescence in-situ hybridization (FISH) (for hTERC gene). The pathologic features were analyzed. RESULTS: The differences in expression of CK7, CK20, MUC6, COX2 and bcl-2 between BE and proximal gastric mucosa with intestinal metaplasia were not statistically significant (P > 0.05). There was however a statistically significant difference in expression of S-100 protein (P < 0.05). The expression of CK7/CK4 and CK7/CK8 in BE showed positive correlation (P < 0.05). However, such correlation was not demonstrated in proximal gastric mucosa (P > 0.05). The results of hTERC gene expression by FISH showed a statistically significant difference between the two groups: 57.9% (22/38) in BE and 13.6% (6/44) in proximal gastric mucosa (P < 0.05). CONCLUSIONS: The significance of CK7 and CK20 expression is uncertain in the differential diagnosis between BE and proximal gastric mucosa. On the other hand, positivity for CK7/CK4/CK8 may support the diagnosis of BE and play a role in distinguishing between the two groups. S-100 protein expression and detection of hTERC gene amplification also contribute to the diagnosis of BE.

Splice site m6A methylation prevents binding of DGCR8 to suppress KRT4 pre-mRNA splicing in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is the 11th most prevalent tumor worldwide. Despite advantages of therapeutic approaches, the 5-year survival rate of patients with OSCC is less than 50%. It is urgent to elucidate mechanisms underlying OSCC progression for developing novel treatment strategies. Our recent study has revealed that Keratin 4 (KRT4) suppresses OSCC development, which is downregulated in OSCC. Nevertheless, the mechanism downregulating KRT4 in OSCC remains unknown. In this study, touchdown PCR was utilized to detect KRT4 pre-mRNA splicing, while m6A RNA methylation was identified by methylated RNA immunoprecipitation (MeRIP). Besides, RNA immunoprecipitation (RIP) was used to determine RNA-protein interaction. Herein, this study indicated that intron splicing of KRT4 pre-mRNA was suppressed in OSCC. Mechanistically, m6A methylation of exon-intron boundaries prevented intron splicing of KRT4 pre-mRNA in OSCC. Besides, m6A methylation suppressed the binding of splice factor DGCR8 microprocessor complex subunit (DGCR8) to exon-intron boundaries in KRT4 pre-mRNA to prohibit intron splicing of KRT4 pre-mRNA in OSCC. These findings revealed the mechanism downregulating KRT4 in OSCC and provided potential therapeutic targets for OSCC.

Down-regulation of keratin 4 and keratin 13 expression in oral squamous cell carcinoma and epithelial dysplasia: a clue for histopathogenesis.

AIMS: This study aimed to identify relevant keratin subtypes that may associate with the pathogenesis of oral epithelial neoplasms. METHODS AND RESULTS: Expression of all the keratin subtypes was examined by cDNA microarray analysis of 43 oral squamous cell carcinoma (OSCC) cases. Immunohistochemical expression of the major keratins was examined in 100 OSCC and oral epithelial dysplasia (OED) cases. Many changes in keratin expression were observed and, significantly, consistent down-regulation of keratin 4 (K4) and K13 expression was observed. Aberrant expression of K4 and K13 was associated with morphological changes in the affected oral epithelium. Experiments with cell cultures transfected with various keratin subtypes suggested that alterations in keratin subtype expression can cause changes in cell shape and movement. CONCLUSIONS: Aberrant expression of K4 and K13, which are the dominant pair of differentiation-related keratins in oral keratinocytes, indicates dysregulation of epithelial differentiation in OSCC and OED. These keratins, especially K4, may be useful for pathological diagnosis. We propose that the aberrant expression of K4 and K13 and concomitant up-regulation of the other keratins may be one of the causative factors for morphological alterations in the affected epithelium.

Keratins 2 and 4/13 in reconstituted human skin are reciprocally regulated by retinoids binding to nuclear receptor RARalpha.

Disorders of keratinization are often treated with vitamin A derivatives (retinoids) which affect keratinocyte differentiation, including keratin (KRT) gene expression. In vivo, suprabasal keratinocytes normally express only keratin (K) 1, K2 and K10, but after topical application of all-trans retinoic acid (ATRA), the granular cells will additionally express K4 and K13, i.e. keratins normally present in oral mucosa and in cultured epidermal keratinocytes. To learn more about the retinoid regulation of keratin expression under in vivo-like conditions, we cultured keratinocytes on de-epidermized dermis in only 0.5% serum. These cells produce a normal-looking epidermis that expresses high mRNA levels of KRT1, KRT2 and KRT10, but minimal amounts of KRT4 and KRT13. Addition of ATRA to the medium for 48 h caused a dose-dependent increase in KRT4/KRT13 and a down-regulation of KRT2 mRNA. An increase in K4 protein was also found. The response was greater than the up-regulation of another retinoid-regulated gene, CRABPII. By studying 10 retinoids with different affinities for the retinoic acid receptors (RAR) and retinoid X receptors (RXR) isoforms, the reciprocal expression of KRT2 and KRT4/KRT13 could be connected with agonists for RARalpha. Two of these agonists, CD336/Am580 and CD2081, altered the expression profile with similar potency as the pan-RAR agonists ATRA and CD367. Co-addition of a pan-RAR antagonist (CD3106/AGN193109) markedly inhibited the induction of KRT4/KRT13 expression, whereas the down-regulation of KRT2 was less affected. In conclusion, RARalpha agonists elicit a reciprocal modulation of KRT2 and KRT4/KRT13 expression in human epidermis, but whether or not the keratin genes also possess RARalpha-specific regulatory elements is still unclear.

The role of post-transcriptional RNA processing and plasmid vector sequences on transient transgene expression in zebrafish.

A tissue-specific transgenic model was employed to test the effects of intron and vector sequences on transgene expression in zebrafish after microinjection. In this model, the 2.3 kb promoter taken from the 5' upstream region of the transcription initiation site of keratin 4 (krt4) was used to drive the enhanced green fluorescence protein (EGFP) reporter gene in a transgenic vector. For assaying the strength of EGFP expression, the effects of including an intron before the EGFP coding region or using different forms of DNA, including circular plasmid, linear full-length plasmid, and the linear transgene coding region without any prokaryotic vector sequence, were tested. After microinjection, the transgene expression was analyzed using transient assays. Consequently, further comparative analysis supported by Fisher's exact test was performed based on the data generated by analyzing the strength of the transgene expression. It was shown that inclusion of an intron in the construct increases the transgene expression in a transient transgenic zebrafish assay. Furthermore, the circular plasmid containing the transgene produced the strongest EGFP expression.

CD24 and CK4 are upregulated by SIM2, and are predictive biomarkers for chemoradiotherapy and surgery in esophageal cancer.

Definitive chemoradiotherapy (CRT) is a less invasive therapy compared with surgery for some types of cancer; however, the 5­year survival rate of patients with stages II­III esophageal squamous cell carcinoma (ESCC) is only 37%. Therefore, prediction of CRT responders is necessary. Unfortunately, no definitive biomarker exists that is useful to predict survival outcome following CRT. From our previous microarray study, CD24 and keratin 4 (KRT4), which encodes cytokeratin 4 (CK4), were overexpressed in the favorable prognostic epithelial subtype with SIM bHLH transcription factor 2 (SIM2) expression. This study investigated the association between their mRNA and protein expression levels, and clinicopathological characteristics, and also investigated the functions of CD24 in SIM2­mediated tumor differentiation and CRT sensitivity. High CD24 and KRT4 mRNA expression was associated with a favorable prognosis following CRT. Multivariate analyses revealed that high CD24 and CK4 protein expression, as determined by immunohistochemistry, and differentiated type were independent factors for predicting a favorable prognosis in response to CRT. Notably, in cases with low CD24 or CK4, surgery was suggested to be a good therapeutic modality compared with CRT. CD24 and KRT4 were expressed preferentially in differentiated layers of the normal esophageal mucosa, and their mRNA expression in 3D cultured ESCC cells was induced by SIM2 transfection, thus suggesting that CD24 and KRT4 were downstream differentiation markers of SIM2. Furthermore, CD24 small interfering RNA increased the mRNA expression levels of superoxide dismutase 2 and enhanced H2O2 resistance, thus indicating the involvement of CD24 in the radiosensitivity of patients with ESCC; however, it had no effect on cisplatin sensitivity. In conclusion, the two markers CD24 and CK4 may be considered predictive biomarkers for definitive CRT.

Repeated exposure to acid and bile selectively induces colonic phenotype expression in a heterogeneous Barrett's epithelial cell line.

Barrett's epithelium is a precancerous, specialized columnar metaplasia in the distal esophagus. We demonstrate the changes in cellular phenotype in a non-neoplastic Barrett's cell line (BAR-T), following exposure to acid and bile salt, the two important components of gastroesophageal refluxate. Cell phenotypes in BAR-T cell line were quantified by fluorescence-activated cell sorting (FACS) using monoclonal antibodies against markers: cytokeratin 8/18 (CK8/18) for columnar, CK4 for squamous, mAbDas-1 for colonic epithelial cell phenotype and p75NTR for esophageal progenitors. Cells were exposed for 5 min each day to 200 microM glycochenodeoxycholic acid at pH 4, pH 6 and pH 7.4 or only to acid (pH 4) for up to 6 weeks. The BAR-T cell line comprised 35+/-5.2% CK8/18, 32+/-3.5% mAbDas-1, 9.5+/-3% CK4 and 4+/-2.5% p75NTR-positive cells. Single exposure to acid and or bile did not change cell phenotypes. However, chronic treatment for at least 2 weeks significantly enhanced (P<0.05) the expression of colonic phenotype and CK8/18-positive cells, as evidenced by FACS analysis. Bile salt at pH 4 and bile salt followed by acid (pH 4) in succession were the strongest stimulators (P<0.01) for induction of colonic phenotype cells. Squamous (CK4(+)) phenotype did not change by the treatments. Cox-2 expression was induced after acute treatment and increased to twofold during chronic treatment, particularly in response to acidic pH. We conclude that BAR-T cells can be utilized as an 'in vitro' model to study the effect of environmental factors and their influence on the cellular phenotype and molecular changes in the pathogenesis of esophageal cancer.

A case of bilateral corneal epithelial dysplasia characterized by laser confocal biomicroscopy and cytokeratin immunofluorescence.

PURPOSE: To describe a case of bilateral corneal epithelial dysplasia in which each lesion was characterized by both laser confocal biomicroscopy and cytokeratin immunofluorescence. METHODS: A 52-year-old Japanese woman with bilateral corneal epithelial dysplasia was treated by corneal epithelial debridement. We observed the affected area with laser confocal biomicroscopy before and after treatment and examined the immunofluorescence of cytokeratins to examine the characteristics of the abnormal epithelial cells. RESULTS: Laser confocal biomicroscopy revealed the atypical epithelial cells in all layers of the corneal epithelium as well as the reconstituted normal structure of the corneal epithelium after epithelial debridement. Immunofluorescence of cytokeratin 12 (K12) and K4 revealed the presence of four types of cells (those positive for one, both or neither of these cytokeratins) in each lesion. CONCLUSION: Cells expressing both K12 and K4 probably represented dysplastic cells that had invaded the cornea via the limbus and adopted characteristics of corneal epithelial cells. Cells lacking both K12 and K4 were probably either undifferentiated cells or epithelial cells in which cytokeratin expression had not been initiated.

Short-term Efficacy of Topical Immunosuppressive Agents on the Survival of Cultivated Allo-Conjunctival Equivalents.

PURPOSE: To investigate the short-term efficacy of topical immunosuppressive agents on the survival of cultivated allo-conjunctival equivalents. METHODS: Twenty-five eyes of New Zealand white rabbits were included. Temporal conjunctivae were trephined to a diameter of 7.5 mm, and then cultured allo-conjunctival epithelial cells on amniotic membrane were transplanted onto them. Various immunosuppressants including steroid, cyclosporine, and rapamycin were applied topically four times a day for a week. Epithelial defects and graft edema were graded daily. Numbers of inflammatory cells were measured in H&E. PKH26 and cytokeratin 4 and 7 were immunostained. RESULTS: Earlier epithelialization was observed in 1% steroid-treated eyes and defects persisted significantly in 0.5% CsA applied eyes. In histology, PKH26 positive cells considered as donor cells were only found in 1% steroid or 0.01% rapamycin applied eyes. 1% steroid- or 0.01% rapamycin-applied eyes both showed positive staining for keratin-4 and -7. Inflammatory cells were less found in 1% steroid or 0.01% rapamycin treated eyes. CONCLUSIONS: Topical steroid or rapamycin can help to suppress acute inflammation and enhance the acute survival of transplanted conjunctival cells.

Experimental infection of dromedaries with Middle East respiratory syndrome-Coronavirus is accompanied by massive ciliary loss and depletion of the cell surface receptor dipeptidyl peptidase 4.

Middle East respiratory syndrome (MERS) represents an important respiratory disease accompanied by lethal outcome in one-third of human patients. Recent data indicate that dromedaries represent an important source of infection, although information regarding viral cell tropism and pathogenesis is sparse. In the current study, tissues of eight dromedaries receiving inoculation of MERS-Coronavirus (MERS-CoV) after recombinant Modified-Vaccinia-Virus-Ankara (MVA-S)-vaccination (n = 4), MVA-vaccination (mock vaccination, n = 2) and PBS application (mock vaccination, n = 2), respectively, were investigated. Tissues were analyzed by histology, immunohistochemistry, immunofluorescence, and scanning electron microscopy. MERS-CoV infection in mock-vaccinated dromedaries revealed high numbers of MERS-CoV-nucleocapsid positive cells, T cells, and macrophages within nasal turbinates and trachea at day four post infection. Double immunolabeling demonstrated cytokeratin (CK) 18 expressing epithelial cells to be the prevailing target cell of MERS-CoV, while CK5/6 and CK14 expressing cells did not co-localize with virus. In addition, virus was occasionally detected in macrophages. The acute disease was further accompanied by ciliary loss along with a lack of dipeptidyl peptidase 4 (DPP4), known to mediate virus entry. DPP4 was mainly expressed by human lymphocytes and dromedary monocytes, but overall the expression level was lower in dromedaries. The present study underlines significant species-specific manifestations of MERS and highlights ciliary loss as an important finding in dromedaries. The obtained results promote a better understanding of coronavirus infections, which pose major health challenges.

Differences in the expression of genes between normal tissue and squamous cell carcinomas of head and neck using cancer-related gene cDNA microarray.

CONCLUSION: This study clearly showed the molecular characteristics of head and neck squamous cell carcinoma (HNSCC) on the basis of gene expression patterns. OBJECTIVE: cDNA microarray has recently been shown to have the ability to represent the expression patterns of large numbers of genes from a small amount of tissue, potentially enabling definition of groups of patients with similar biological behavior of cancer. Although gene expression profiling using this technique has proven helpful for predicting the prognosis in various cancers, little is known regarding HNSCC. The aim of this study was to investigate the differences in the expression of various genes between normal tissue and cancers of patients with HNSCC by cDNA microarray. PATIENTS AND METHODS: We extracted mRNA from 17 HNSCC patients and used cDNA microarray analysis to investigate the gene expression patterns. The present study was not designed to perform an inclusive search for genes but rather to focus on cancer-related genes. RESULTS: Seven independent genes were found to be up-regulated in cancer tissues: matrix metalloproteinase-1, -3, and -10, interleukin-8, cadherin 3, hexabrachion, and interferon gamma-inducible protein 10. Hyaluronic acid-binding protein 2, keratin 4, and keratin 13 were categorized as down-regulated. The hierarchical clustering and dendrogram for 17 cancer samples and 425 genes could be grouped into three clusters.

Structure of KRT4 binding domain of Srr-1 from Streptococcus agalactiae reveals a novel ß-sheet complementation.

The serine rich repeat protein-1 (Srr-1) is an adhesive protein of Streptococcus agalactiae. It is the first bacterial protein identified to interact with human keratin 4 (K4 or KRT4). Within Srr-1, the residues 311-641 constitute the non-repeat ligand binding region (Srr-1-BR(311-641)). The C-terminal part of Srr-1-BR(311-641), comprising of residues 485-642 (termed Srr-1-K4BD), have been identified to bind to K4. Here we report the crystal structure of recombinant Srr-1-K4BD(485-642) and its possible mode of interaction with K4 through docking studies. The dimeric structure of Srr-1-K4BD(485-642) reveals a novel two way "slide lock" parallel ß-sheet complementation where the C-terminal strand of one monomer is positioned anti-parallel to the N-terminal strand of the adjacent monomer and this arrangement is not seen so far in any of the homologous structures. The dimerization of Srr-1-K4BD(485-642) observed both in the crystal structure and in solution suggests that similar domain association could also be possible in in vivo and we propose this association would likely generate a new binding site for another host molecule. It is likely that the adhesin can recognize multiple ligands using its ligand binding sub-domains through their intra and inter domain association with one another.

Development of a composite measure of product adherence, protocol compliance, and semen exposure using DNA and protein biomarkers for topical HIV prevention studies.

BACKGROUND: Poor and inconsistent use of study products has hindered clinical HIV prevention studies. It is important to be able to monitor product adherence and protocol compliance in order to determine microbicide efficacy and safety more accurately. Current methods for monitoring adherence are subjective, non-specific, or invasive. Herein, we present a composite, objective measure of product adherence and protocol compliance to assess vaginal insertion, semen exposure and drug expulsion utilizing DNA, protein, and drug isolated directly from returned, vaginally used gel applicators. METHODS: DNA, vaginal cells, and residual tenofovir were isolated from vaginally inserted applicators. Vaginal and semen biomarkers were amplified using a multiplex PCR to determine vaginal insertion. Vaginal cells were fixed followed by cytokeratin 4 immunocytochemistry to confirm DNA assessment of vaginal insertion. Tenofovir was extracted and quantitated through LC-MS/MS. RESULTS: DNA isolated from vaginally inserted applicators were positive for vaginal bacteria DNA and the control eukaryotic gene, amelogenin, while manually handled, "sham", applicators were negative for both. Semen exposure was independently determined by simultaneous amplification of one or both Y-chromosomal genes, SRY and TSPY4. Vaginal insertion determination by DNA analysis was further confirmed by positive cytokeratin 4 (CK4) immunocytochemistry of vaginal cells remaining on the gel applicators. On the contrary, sham applicators provided very few cells when swabbed, and they were all negative for CK4. CK4 was not found in epidermal cells from the hand. Drug expulsion was detected through quantitation of residual gel present on the surface of returned applicators. Sham applicators had no detectable tenofovir. CONCLUSION: Utilizing a composite, triple marker based panel of DNA, protein, and drug present on the surface of returned vaginal gel applicators, it is possible to determine, objectively and non-invasively, product adherence, protocol compliance, and semen exposure in microbicide trials.