Background: Exosomes play important roles in angiogenesis, drug resistance, and metastasis of colorectal cancer (CRC), but the underlying mechanism has seldom been reported. Herein, our study aimed to reveal an exosomal miRNA-mRNA network involved in CRC by performing bioinformatical analysis.

Methods: The mRNA and miRNA data of colon adenocarcinoma and rectal adenocarcinoma were downloaded from The Cancer Genome Atlas (TCGA) database, and exosomal miRNAs data were downloaded from the GEO dataset GSE39833. The differential expression analysis was performed using "limma" and "edgeR". Target mRNAs of miRNAs were predicted using FunRich 3.1.3, miRNAtap and multiMiR. The candidate mRNAs and exosomal miRNAs were obtained by intersecting two groups of differentially expressed miRNAs and intersection of the differential expressed mRNAs and the target mRNAs, respectively. Key mRNAs and exosomal miRNAs were identified by the least absolute shrinkage and selection operator regression analysis, and used to construct the exosomal miRNA-mRNA network. The network verified was by receiver operating characteristic curve, GEPIA and LinkedOmics. Functional enrichment analysis was also performed for studied miRNAs and mRNAs.

Results: A total of 6568 differentially expressed mRNAs and 531 differentially expressed miRNAs from TCGA data, and 166 differentially expressed exosomal miRNAs in GSE39833 dataset were identified. Next, 16 key mRNAs and five key exosomal miRNAs were identified from the 5284 candidate mRNAs and 61 candidate exosomal miRNAs, respectively. The exosomal miRNA-mRNA network with high connectivity contained 13 hub mRNAs (CBFB, CDH3, ETV4, FOXQ1, FUT1, GCNT2, GRIN2D, KIAA1549, KRT80, LZTS1, SLC39A10, SPTBN2, and ZSWIM4) and five hub exosomal miRNAs (hsa-miR-126, hsa-miR-139, hsa-miR-141, hsa-miR-29c, and hsa-miR-423). The functional annotation revealed that these hub mRNAs were mainly involved in the regulation of B cell receptor signaling pathway and glycosphingolipid biosynthesis related pathways. All hub mRNAs and hub exosomal miRNAs exhibited high diagnosis value for CRC. Furthermore, the association of the hub mRNAs with overall survival, stages, and MSI phenotype of CRC revealed their important roles in CRC progression.

Conclusion: This study constructed an exosomal miRNA-mRNA network which may play crucial roles in the carcinogenesis and progression of CRC, thus providing potential diagnostic biomarkers and therapeutic targets for CRC.

Keywords: Colorectal cancer; Differential expression analysis; Exosome; Machine learning; MiRNA.

The trophoblast (TR) is the first to differentiate during mammalian embryogenesis and play a pivotal role in the development of the placenta. We used a dual inhibitor system (PD0325901 and CHIR99021) with mixed feeders to successfully obtain bovine trophoblast stem-like (bTS) cells, which were similar in phenotype to mouse trophoblast stem cells (TSCs). The bTS cells that were generated using this system continually proliferated, displayed a normal diploid karyotype, and had no signs of altered morphology or differentiation even after 150 passages. These cells exhibited alkaline phosphatase (AP) activity and expressed pluripotency markers, such as OCT4, NANOG, SOX2, SSEA-1, SSEA-4, TRA-1-60, and TRA-1-81, and TR lineage markers such as CDX2, as determined by both immunofluorescence and reverse transcription-polymerase chain reaction (RT-PCR). Additionally, these cells generated dome-like structures, formed teratomas when injected into NOD-SCID mice, and differentiated into placenta TR cells in vitro. The microarray analysis of bTS cells showed high expression levels of many TR markers, such as TEAD4, EOMES, GATA3, ETS2, TFAP2A, ELF5, SMARCA4 (BRG1), CDH3, MASH2, HSD17B1, CYP11A1, PPARG, ID2, GCM1, HAND1, TDK, PAG, IFN-τ, and THAP11. The expression of many pluripotency markers, such as OCT4, SOX2, NANOG, and GDF3, was lower in bTS cells compared with in vitro-produced blastocysts; however, compared with bovine fetal fibroblasts, the expression of these pluripotency markers was elevated in bTS cells. The DNA methylation status of the promoter regions of OCT4, NANOG, and SOX2 was investigated, which were significantly higher in bTS cells (OCT4 23.90%, NANOG 74.40%, and SOX2 8.50%) compared with blastocysts (OCT4 8.90%, NANOG 34.4%, and SOX2 3.80%). In contrast, two promoter regions of CDX2 were hypomethylated in bTS cells (13.80% and 3.90%) compared with blastocysts (18.80% and 9.10%). The TSC lines that were established in this study may be used either for basic research that is focused on peri-implantation and placenta development or as donor cells for transgenic animal production.

Human esophageal adenocarcinoma (EAC) cell lines and xenografts are powerful tools in the search for genetic alterations because these models are composed of pure human cancer cell populations without admixture of normal human cells. In particular detection of homozygous deletions (HDs) is easier using these pure populations of cancer cells. Identification of HDs could potentially lead to the subsequent identification of new tumor suppressor genes (TSGs) involved in esophageal adenocarcinogenesis. Genome wide single nucleotide polymorphism (SNP) arrays were used to identify HDs in 10 verified EAC cell lines and nine EAC xenografts. In total, 61 HDs (range 1-6 per sample) were detected and confirmed by polymerase chain reaction. Besides HDs observed in common fragile genomic regions (n = 26), and gene deserts (n = 8), 27 HDs were located in gene-containing regions. HDs were noted for known TSGs, including CDKN2A, SMAD4 and CDH3/CDH1. Twenty-two new chromosomal regions were detected harboring potentially new TSGs involved in EAC carcinogenesis. Two of these regions of homozygous loss, encompassing the ITGAV and RUNX1 gene, were detected in multiple samples indicating a potential role in the carcinogenesis of EAC. To exclude culturing artifacts, these last two deletions were confirmed by fluorescent in situ hybridization in the primary tumors of which the involved cell lines and xenografts were derived. In summary, in this report we describe the identification of HDs in a series of verified EAC cell lines and xenografts. The deletions documented here are a step forward identifying the key genes involved in EAC development.

P-cadherin is a cell-cell adhesion molecule codified by the CDH3 gene, which expression is highly associated with undifferentiated cells in normal adult epithelial tissues, as well as with poorly differentiated carcinomas. In breast cancer, P-cadherin is frequently overexpressed in high-grade tumours and is a well-established indicator of aggressive tumour behaviour and poor patient prognosis. However, till now, the mechanisms controlling CDH3 gene activation have been poorly explored. Since we recently described the existence of several CCAAT/Enhancer Binding Protein β (C/EBPβ) transcription factor binding sites at the CDH3 promoter, the aim of this study was to assess if the distinct C/EBPβ isoforms were directly involved in the transcriptional activation of the CDH3 gene in breast cancer cells. DNA-protein interactions, mutation analysis and luciferase reporter assay studies have been performed. We demonstrated that C/EBPβ is co-expressed with P-cadherin in breast cancer cells and all the three isoforms function as transcriptional regulators of the CDH3 gene, directly interacting with specific regions of its promoter. Interestingly, this transcriptional activation was only reflected at the P-cadherin protein level concerning the LIP isoform. Taken together, our data show that CDH3 is a newly defined transcriptional target gene of C/EBPβ isoforms in breast cancer, and we also identified the binding sites that are relevant for this activation.

BACKGROUND

Hypotrichosis with juvenile macular dystrophy (HJMD; OMIM 601553) is a rare autosomal recessive disorder characterized by hypotrichosis with short scalp hair and progressive macular dystrophy leading to blindness between the second and the fourth decades of life. HJMD is caused by mutations in the P-cadherin gene (CDH3), a member of the family of classical cadherins.

METHODS

We analyzed the DNA from members of 2 consanguineous Pakistani families with HJMD for mutations in the P-cadherin gene through direct sequencing.

RESULTS

We identified 2 splice site mutations in the P-cadherin gene in these families. One was a novel mutation, Ivs12-2A->>G and the other a recurrent mutation, Ivs10-1G->>T. A screening assay for the novel mutation ruled out the possibility of a polymorphism. Using haplotype analysis, we determined that the mutation, Ivs10-1G->>T, is a founder mutation in the Pakistani population.

CONCLUSIONS

We identified 2 splice site mutations in the CDH3 gene leading to HJMD, further enriching our understanding of HJMD versus ectodermal dysplasia, ectrodactyly and macular dystrophy syndrome.

Hypotrichosis with juvenile macular dystrophy (HJMD) and ectodermal dysplasia, ectrodactyly and macular dystrophy (EEM) are both caused by mutations in the CDH3 gene. In this report, we describe a family with EEM syndrome caused by a novel CDH3 gene mutation and review the mutation spectrum and limb abnormalities in both EEM and HJMD. A protein structure model showing the localization of different mutations causing both syndromes is presented. The CDH3 gene was sequenced and investigation of the mutations performed using a protein structure model. The conservation score was calculated by ConSurf. We identified a novel CDH3 gene mutation, p.G277V, which resides in a conserved residue located on a β-strand in the second cadherin domain. Review of the data on previously published mutations showed intra-familial and inter-familial variations in the severity of the limb abnormalities. Syndactyly was the most consistent clinical finding present in all the patients regardless of mutation type. The results of our study point to a phenotypic continuum between HJMD and EEM. It is important for genetic counseling to keep in mind the possible clinical/phenotypic overlap between these 2 syndromes and to be aware of the possible risk of limb abnormalities in future pregnancies in families with HJMD syndrome. CDH3 gene mutation screening is recommended in patients with both these syndromes as part of the work-up in order to offer appropriate genetic counseling.

Recessive mutations in CDH3 cause "hypotrichosis with juvenile macular dystrophy," typically recognized by the presence of prominent dermatological features. We report novel phenotypic observations and associated mutations in four patients from three families, including one who did not have frank hypotrichosis.

Retrospective case series (2010-2014).

Four affected individuals from three consanguineous Arabian families were identified. All four subjects (two sisters and two unrelated males; 5, 13, 17, and 26 years old) had homozygous recessive CDH3 mutations not previously associated with the condition (c.307C>T; p.R103 in two sisters, c.1859_1862delCTCT in both unrelated males). Symptomatic visual loss was since birth or early childhood. One male subject did not have frank hypotrichosis, but review of symptoms revealed relatively slow hair growth and an inability to conceive children. None had dental or digital findings, although one female noted slow nail growth. All had a circumscribed central maculopathy with borders that did not respect posterior pole horizontal arterioles (typically extending beyond the major arcades) and associated with polygonal pigment clumping. Recognition of this pattern led us to suspect the diagnosis in the male without frank hypotrichosis. Retinal dysfunction was cone-rod (rather than macular only) by ERG in one patient, who developed severe central macular atrophy and a macular hole.

Ophthalmologists should consider the diagnosis of CDH3-related retinopathy in individuals with such clinical features whether or not there is frank hypotrichosis.

DFNB89 is a novel autosomal recessive nonsyndromic hearing impairment (ARNSHI) locus that was mapped to 16q21-q23.2. Linkage to the region was established by carrying out genome-wide linkage scans in two unrelated, consanguineous Pakistani families segregating ARNSHI. The maximum multipoint LOD score is 9.7 for both families and for each family, a significant maximum LOD score of 6.0 and 3.7 were obtained. The 3-unit support interval and the region of homozygosity for the two families extend from rs717293 (chr16: 62.1 Mb) to rs728929 (chr16: 78.2 Mb) and contain 16.1 Mb of sequence. A total of 146 genes are within the DFNB89 interval. Eight candidate genes, CALB2, CDH1, CDH3, CDH11, HAS3, NOB1, PLEKHG4 and SMPD3, were sequenced, but no potentially causal variants were discovered. DFNB89 is the second ARNSHI locus mapped to chromosome 16.

Cancer genetic heterogeneity offers a wide repertoire of molecular determinants to be screened as therapeutic targets. Here, we identify potential anticancer targets by exploiting negative genetic interactions between genes with driver loss-of-function mutations (recessive cancer genes) and their functionally redundant paralogs. We identify recessive genes with additional copies and experimentally test our predictions on three paralogous pairs. We confirm digenic negative interactions between two cancer genes (SMARCA4 and CDH1) and their corresponding paralogs (SMARCA2 and CDH3). Furthermore, we identify a trigenic negative interaction between the cancer gene DNMT3A, its functional paralog DNMT3B, and a third gene, DNMT1, which encodes the only other human DNA-methylase domain. Although our study does not exclude other causes of synthetic lethality, it suggests that functionally redundant paralogs of cancer genes could be targets in anticancer therapy.

P-cadherin (CDH3), a classical cell adhesion molecule involved in tissue integrity and cell localization, has been implicated in many types of cancer. However, little is known about its function and regulatory mechanism in hepatocellular carcinoma (HCC). Here we report that CDH3 was positively regulated by kr¨uppel-like transcription factor 4 (KLF4), which is a crucial tumor suppressor gene in HCC, at mRNA level in HCC cell lines. Luciferase reporter assay and chromatin immunoprecipitation assay indicated that KLF4 directly bound to CDH3 promoter and transcriptionally activated CDH3 expression. Consistently, CDH3 expression was closely related with KLF4 expression in patients' samples and both proteins exhibited a downregulated expression pattern in cancer samples. Functionally, enforced CDH3 expression suppressed and silenced CDH3 expression promoted HCC cell growth and migration in vitro. Mechanistically, we observed that GSK-3β was regulated by CDH3 and may function as a possible downstream effector of CDH3. Knockdown of GSK-3β showed a similar phenotype with CDH3 silencing. Taken together, these findings establish the KLF4/CDH3/GSK-3β axis as an important regulatory mechanism in HCC development.

We describe the case of a boy with psychomotor delay and dysmorphic features, with a germline 16q22.1 microdeletion identified by array-CGH. The deletion spans 0.24Mb and encompasses three genes (ZFP90, CDH3 and CDH1). The deletion has been demonstrated to be inherited from his mother who was affected by lobular breast cancer (LBC) without any other apparently phenotypic features. We suppose that the microdeletion, in particular ZFP90 which is cerebrally expressed, is causative for the boy's phenotype. Mental retardation in the affected boy can recognize several mechanisms such as variable expressivity, non-penetrance, multifactorial/polygenic inheritance, recessive inheritance, a second rearrangement event and epigenetics. Furthermore, we suggest that the deletion of the CDH1, a tumor suppressor gene, involved in hereditary diffuse gastric cancer (HDGC) and LBC predisposed the mother to the carcinoma.

One of the main challenges in limbal stem cell (LSC) biology and transplantation is the lack of definitive cell surface markers which can be used to identify and enrich viable LSCs. In this study, expression of 361 cell surface proteins was assessed in ex vivo expanded limbal epithelial cells. One marker, CD200 was selected for further characterization based on expression in a small subset of limbal epithelial cells (2.25% ± 0.69%) and reduced expression through consecutive passaging and calcium induced differentiation. CD200 was localized to a small population of cells at the basal layer of the human and mouse limbal epithelium. CD200+ cells were slow cycling and contained the majority of side population (SP) and all the holoclone forming progenitors. CD200+ cells displayed higher expression of LSCs markers including PAX6, WNT7A, CDH3, CK14, CK15, and ABCB5 and lower expression of Ki67 when compared to CD200- . Downregulation of CD200 abrogated the ability of limbal epithelial cells to form holoclones, suggesting an important function for CD200 in the maintenance and/or self-renewal of LSCs. A second marker, CD109, which was expressed in 56.29% ± 13.96% of limbal epithelial cells, was also found to co-localize with ΔNp63 in both human and mouse cornea, albeit more abundantly than CD200. CD109 expression decreased slowly through calcium induced cell differentiation and CD109+ cells were characterized by higher expression of Ki67, when compared to CD109- subpopulation. Together our data suggest that CD200 expression marks a quiescent population of LSCs with holoclone forming potential, while CD109 expression is associated with a proliferative progenitor phenotype. Stem Cells 2018;36:1723-1735.

OBJECTIVE

The increased availability of next generation sequencing has enabled a rapid progress in the discovery of genetic variants associated with vestibular disorders. We have summarized molecular genetics finding in vestibular syndromes during the last 18 months.

RESULTS

Genetic studies continue to shed light on the genetic background of vestibular disorders. Novel genes affecting brain development and otolith biogenesis have been associated with motion sickness. Exome sequencing has made possible to identify three rare single nucleotide variants in PRKCB, DPT and SEMA3D linked with familial Meniere disease. Moreover, superior canal dehiscence syndrome might be related with variants in CDH3 gene, by increasing risk of its development. On the other hand, the association between vestibular schwannoma and enlarged vestibular aqueduct with variants in NF2 and SLC26A4, respectively, seems increasingly clear. Finally, the use of mouse models is allowing further progress in the development gene therapy for hearing and vestibular monogenic disorders.

CONCLUSIONS

Most of episodic or progressive syndromes show familial clustering. A detailed phenotyping with a complete familial history of vestibular symptoms is required to conduct a genetic study. Progress in these studies will allow us to understand diseases mechanisms and improve their current medical treatments.

BACKGROUND

CDH3 on 16q22.1 is responsible for two rare autosomal recessive disorders with hypotrichosis and progressive macular dystrophy: Hypotrichosis with Juvenile Macular Dystrophy and Ectodermal Dysplasia, Ectrodactyly and Macular Dystrophy. We present a new case of Hypotrichosis with Juvenile Macular Dystrophy.

METHODS

A Spanish male born in 1998 from non-consanguineous healthy parents with a suspected diagnosis of Keratosis Follicularis Spinulosa Decalvans and Retinitis Pigmentosa Inversa referred to our Genetics Department (IIS-Fundación Jiménez Díaz). Molecular study of ABCA4 was performed, and a heterozygous missense p.Val2050Leu variant in ABCA4 was found. Clinical revision reclassified this patient as Hypotrichosis with Juvenile Macular Dystrophy. Therefore, further CDH3 sequencing was performed showing a novel maternal missense change p.Val205Met (probably pathogenic by in silico analysis), and a previously reported paternal frameshift c.830del;p.Gly277Alafs*20, thus supporting the clinical diagnosis..

CONCLUSIONS

This is not only the first Spanish case with this clinical and molecular diagnosis, but a new mutation has been described in CDH3. Moreover, this work reflects the importance of joint assessment of clinical signs and evaluation of pedigree for a correct genetic study approach and diagnostic.

OBJECTIVE

Congenital hypotrichosis with juvenile macular dystrophy (HJMD) is a rare disorder presenting in childhood and adolescence with central visual disturbance and sparse scalp hair. Reported retinal imaging is lacking, and whether the condition is progressive remains unclear.

OBJECTIVE

To investigate a series of patients with HJMD due to biallelic mutations in CDH3 and thereby characterize the disorder.

METHODS

Ten patients from 10 families underwent detailed clinical assessment, including serial retinal imaging and electrophysiologic evaluation, at Moorfields Eye Hospital, St James's University Hospital, and Calderdale Royal Infirmary. Patients ranged in age from 3 to 17 years at onset and 5 to 57 years at last assessment. The molecular genetic investigation included bidirectional Sanger sequencing of all exons and intron-exon boundaries of CDH3 and whole-exome sequencing in 2 patients. The study was conducted from June 5, 2013, to January 15, 2016, with final follow-up completed on December 15, 2015.

METHODS

Results of clinical assessment and molecular genetic testing.

RESULTS

All 10 patients (7 male and 3 female) presented with central visual disturbance in childhood and had lifelong sparse scalp hair with normal facial hair. Fundus examination revealed chorioretinal atrophy of the posterior pole contiguous with the disc in all but 1 patient that was associated with marked loss of autofluorescence on fundus autofluorescence imaging. Optical coherence tomography (OCT) demonstrated variable degrees of atrophy of the outer retina, retinal pigment epithelium, and choroid, with outer retinal tubulations frequently observed. One patient had mild disruption of the inner segment ellipsoid band on OCT and additional mild digit abnormalities. Electrophysiologic evaluation in 5 patients demonstrated macular dysfunction with additional mild, generalized retinal dysfunction in 2 patients. Eight patients had more than 1 evaluation; of these, 5 patients showed deterioration of visual acuity over time, 1 patient remained stable, and 2 patients had severe visual loss at presentation that precluded assessment of visual deterioration. The area of atrophy did not progress with time, but retinal thickness decreased on OCT. Electrophysiologic evaluation in 1 patient found deterioration of macular function after 13 years of follow-up, but the mild, generalized photoreceptor dysfunction remained stable. Biallelic mutations were identified in all patients, including 6 novel mutations.

CONCLUSIONS

These results suggest that CDH3-related disease is characterized by a childhood-onset, progressive chorioretinal atrophy confined to the posterior pole. The disease is readily distinguished from other juvenile macular dystrophies by the universally thin and sparse scalp hair. Patients may have additional limb abnormalities.

Although advances have been made in diagnostic tools, the distinction between malignant and benign biliary strictures still remains challenging. Intraductal brush cytology is a convenient and safe method that is used for the diagnosis of biliary stricture, but, low sensitivity limits its usefulness. This study aimed to demonstrate the usefulness of mRNA expression levels of target genes in brush cytology specimens combined with cytology for the diagnosis of malignant biliary stricture. Immunohistochemistry for cadherin 3 (CDH3), p53, insulin-like growth factor II mRNA-binding protein 3 (IGF2BP3), homeobox B7 (HOXB7), and baculoviral inhibitor of apoptosis repeat containing 5 (BIRC5) was performed in 4 benign and 4 malignant bile duct tissues. Through endoscopic or interventional radiologic procedures, brush cytology specimens were prospectively obtained in 21 and 35 paitents with biliary strictures. In the brush cytology specimens, the mRNA expressions levels of 5 genes were determined by real-time polymerase chain reaction. Immunohistochemistry for CDH3, p53, IGF2BP3, HOXB7, and BIRC5 all showed positive staining in malignant tissues in contrast to benign tissues, which were negative. In the brush cytology specimens, the mRNA expression levels of CDH3, IGF2BP3, HOXB7, and BIRC5 were significantly higher in cases of malignant biliary stricture compared with cases of benign stricture (P = 0.006, P < 0.001, P < 0.001, and P = 0.001). The receiver-operating characteristic curves of these 4 mRNAs demonstrated that mRNA expression levels are useful for the prediction of malignant biliary stricture (P = 0.006, P < 0.001, P < 0.001, and P = 0.002). The sensitivity and specificity, respectively, for malignant biliary stricture were 57.1% and 100% for cytology, 57.1% and 64.3% for CDH3, 76.2% and 100% for IGF2BP3, 71.4% and 57.1% for HOXB7, and 76.2% and 64.3% for BIRC5. When cytology was combined with the mRNA levels of CDH3, IGF2BP3, or BIRC5, the sensitivity for malignant biliary stricture improved to 90.5%. The measurement of the mRNA expression levels of CDH3, IGF2BP3, and BIRC5 by real-time polymerase chain reaction combined with cytology was useful for the differentiation of malignant and benign biliary strictures in brush cytology specimens.

Hypotrichosis with juvenile macular dystrophy (HJMD) (MIM 601553) is a rare disorder characterized by the paucity of hair and progressive macular degeneration leading to blindness. We have recently shown that mutations in the CDH3 gene encoding P-cadherin are the proximal cause of HJMD. The present study was performed to establish the histopathology of this disorder. 4 mm punch scalp biopsies from 6 HJMD patients aged 9 to 21 years were studied. All patients had a homozygous missense mutation resulting in a single amino acid substitution at position 503 of P-cadherin amino acid sequence (R503H). The total number of hair follicles varied between 6 and 14 per histologic section. More catagen-telogen hair follicles were observed in five cases, and an increased ratio of vellus hair follicles to terminal hair follicles was observed in two cases. There were no signs of inflammation or scarring. Thus, the most frequent histologic abnormality in HJMD resembles chronic telogen effluvium. This suggests that absence of functional P-cadherin interferes with normal hair cycle.

The aim of the present study was to explore the regulation status of genes in oxidative stress (OS)‑induced endothelial dysfunction and to elucidate the mechanism of action of OS‑associated genes, which induce cavernosal endothelial dysfunction in erectile dysfunction (ED). OS was established in purified cavernosal endothelial cells (CECs) using xanthine/xanthine oxidase and the differentially expressed OS‑associated genes were analyzed using gene microarrays. In addition, an ED rat model was established through bilateral internal iliac artery ligation with hyperlipidemia and was verified by an intracavernosal pressure test. The selected OS‑associated genes were validated in the CECs and ED rat model using reverse transcription‑quantitative polymerase chain reaction. Student's t‑test and one‑way analysis of variance were performed using SBC analysis system. Gene microarray analysis revealed that 13090 (31.92%) genes were expressed in the control group, whereas 12039 (29.35%) genes were expressed in the treated group. The cut‑off value for differential expression was set at 2.0 fold‑change and 2480 genes were found to be differentially expressed compared with the control group. Of these cells, 1454 were upregulated and 1026 were downregulated. Cluster analysis identified relevant cell signaling pathways that were hypothesized to be significant in OS‑associated endothelial dysfunction, including the cytokine‑cytokine receptor interactions, nitrogen metabolism, coagulation cascades and cell adherens. Cxcl12, Tgfbr1, Asns, Bdkrb1 and Cdh3 genes showed a corresponding variation in the CECs and ED rat model compared with the results of the gene microarray analysis. In conclusion, in the present study, the network of differentially expressed genes and OS‑associated signaling pathways identified using gene microarray analysis were validated in the CECs and ED rat model. The results indicated that OS may lead to endothelial dysfunction through certain cell signaling pathways, inducing ED. However, further functional verification is required in order to elucidate the underlying mechanisms of OS‑associated cell signaling pathways in ED.

Adherens junctions (AJs) are one of the major intercellular junctions in various epithelia including the epidermis and the follicular epithelium. AJs connect the cell surface to the actin cytoskeleton and comprise classic transmembrane cadherins, such as P-cadherin, armadillo family proteins, and actin microfilaments. Loss-of-function mutations in CDH3, which encodes P-cadherin, result in two allelic autosomal recessive disorders: hypotrichosis with juvenile macular dystrophy (HJMD) and ectodermal dysplasia, ectrodactyly, and macular dystrophy (EEM) syndromes. Both syndromes feature sparse hair heralding progressive macular dystrophy. EEM syndrome is characterized in addition by ectodermal and limb defects. Recent studies have demonstrated that, together with its involvement in cell-cell adhesion, P-cadherin plays a crucial role in regulating cell signaling, malignant transformation, and other major intercellular processes. Here, we review the roles of P-cadherin in skin and hair biology, with emphasize on human hair growth, cycling and pigmentation.

Hypotrichosis with juvenile macular dystrophy (HJMD) is an autosomal recessive disorder that causes childhood visual impairment. HJMD is caused by mutations in CDH3 which encodes cadherin-3, a protein expressed in retinal pigment epithelium (RPE) cells that may have a key role in intercellular adhesion. We present a case of HJMD and analyse its phenotypic and molecular characteristics to assess the potential for retinal gene therapy as a means of preventing severe visual loss in this condition. Longitudinal in vivo imaging of the retina showed the relative anatomical preservation of the macula, which suggested the presence of a therapeutic window for gene augmentation therapy to preserve visual acuity. The coding sequence of CDH3 fits within the packaging limit of recombinant adeno-associated virus vectors that have been shown to be safe in clinical trials and can efficiently target RPE cells. This report expands the number of reported cases of HJMD and highlights the phenotypic characteristics to consider when selecting candidates for retinal gene therapy.

Nitrophorin 2 (NP2), one of the four NO-storing and NO-releasing proteins found in the saliva of the blood-sucking bug Rhodnius prolixus, has a more ruffled heme and a high preference for a particular heme orientation (B) compared with nitrophorin 1 and nitrophorin 4, which show not a preference (A to B ratio of approximately 1:1), suggesting that it fits more tightly in the β-barrel protein. In this work we have prepared a series of "belt" mutants of NP2(D1A) and (ΔM0)NP2 aimed at reducing the size of aromatic or other residues that surround the heme, and investigated them as the high-spin aqua and low-spin N-methylimidazole complexes. The belt mutants included Y38A, Y38F, F42A, F66A, Y85A, Y85F, Y104A, I120T, and a triple mutant of NP2(D1A), the F42L, L106F, I120T mutant. Although I120 has been mainly considered to be a distal pocket residue, CδH3 of I120 lies directly above the heme 3-methyl, at 2.67 Å, of heme orientation B, or the 2-vinyl of A, and it thus plays a role as a belt mutant, a role that turns out to be extremely important in creating the strong favoring of the B heme orientation [A to B ratio of 1:14 for NP2(D1A) or 1:12 for (ΔM0)NP2]. The results show that the 1D (1)H NMR spectra of the high-spin forms are quite sensitive to changes in the shape of the heme binding cavity. The single mutation I120T eliminates the favorability of the B heme orientation by producing a heme A to B orientation ratio of 1:1, whereas the single mutation F42A reverses the heme orientation from an A to B ratio of 1:14 seen for NP2(D1A) to 10:1 for NP2(D1A,F42A). The most extreme ratio was found for the triple mutant of NP2(D1A), NP2(D1A,F42L,L105F,I120T), in which the A to B ratio is approximately 25:1, a ΔG change of about -3.5 kcal/mol or -14.1 kJ/mol with respect to NP2(D1A). The seating of the heme is modified as well in that mutant and in several others, by rotations of the heme by up to 4° from the seating observed in NP2(D1A), in order to relieve steric interactions between a vinyl β-carbon and a protein side chain, or to fill a cavity created by replacing a large protein side chain by a much smaller one; the latter was observed for all tyrosine to alanine mutants. These relatively small changes in seating have a measurable effect on the NMR spectra of the mutants, but are indeed minor in terms of overall seating and reactivity of the NP2(D1A) protein. The (1)H NMR resonances of the hemin substituents of the low-spin N-methylimidazole complexes of NP2(D1A,F42L,L105F,I120T) as well as NP2(D1A,I120T), NP2(D1A,Y104A), and NP2(D1A,F42A) have been assigned using natural abundance (1)H{(13)C} heteronuclear multiple quantum correlation and (1)H-(1)H nuclear Overhauser effect spectroscopy spectra.