Clinical Tear Fluid Proteomics : A Novel Tool in Glaucoma Research

Tear fluid forms the outermost layer of the ocular surface and its characteristics and composition have been connected to various ocular surface diseases. As tear proteomics enables the non-invasive investigation of protein levels in the tear fluid, it has become an increasingly popular approach in ocular surface and systemic disease studies. Glaucoma, which is a set of multifactorial diseases affecting mainly the optic nerve and retinal ganglion cells, has also been studied using tear proteomics. In this condition, the complete set of pathophysiological changes occurring in the eye is not yet fully understood, and biomarkers for early diagnosis and accurate treatment selection are needed. More in-depth analyses of glaucoma tear proteomics have started to emerge only more recently with the implementation of LC-MS/MS and other modern technologies. The aim of this review was to examine the published data of the tear protein changes occurring during glaucoma, its topical treatment, and surgical interventions.publishedVersionPeer reviewe

Comparison of early changes in tear film protein profiles after small incision lenticule extraction (SMILE) and femtosecond LASIK (FS-LASIK) surgery

Background: Small incision lenticule extraction (SMILE) and femtosecond laser-assisted in situ keratomileusis (LASIK) are widely used surgical methods to correct myopia with comparable efficacy, predictability, and safety. We examined and compared the early changes of tear protein profiles after SMILE and FS-LASIK surgery in order to find possible differences in the initial corneal healing process. Methods: SMILE operations for 26 eyes were made with Visumax femtosecond laser. In FS-LASIK surgery for 30 eyes, the flaps were made with Ziemer FEMTO LDV Z6 femtosecond laser and stromal ablation with Wavelight EX500 excimer laser. Tear samples were collected preoperatively, and 1.5 h and 1 month postoperatively using glass microcapillary tubes. Tear protein identification and quantification were performed with sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS). Results: Immediately (1.5 h) after we found differences in 89 proteins after SMILE and in 123 after FS-LASIK operation compared to preoperative protein levels. Of these differentially expressed proteins, 48 proteins were common for both surgery types. There were, however, quantitative differences between SMILE and FS-LASIK. Upregulated proteins were mostly connected to inflammatory response and migration of the cells connected to immune system. One month after the operation protein expressions levels were returned to baseline levels with both surgical methods. Conclusions: Our study showed that immediate changes in protein profiles after SMILE and FS-LASIK surgeries and differences between the methods are connected to inflammatory process, and the protein levels quickly return to the baseline within 1 month. The differences in protein profiles between the methods are probably associated with the different size of the epithelial wound induced.Peer reviewe

Age-associated changes in human tear proteome

Additional file 1. Analysis results of proteins with unadjusted p-value < 0.05

Proteomics of Deep Cervical Lymph Nodes after Experimental Traumatic Brain Injury

Traumatic brain injury (TBI) damages the glymphatic-lymphatic system. We hypothesized that brain injury associated with trauma results in the enrichment of brain-relevant proteins in deep cervical lymph nodes (DCLNs), the end station of meningeal lymphatic vessels, and that some of these proteins will present mechanistic tissue biomarkers for TBI. Proteomes of rat DCLNs were investigated in the left DCLN (ipsilateral to injury) and right DCLN at 6.5 months after severe TBI induced by lateral fluid percussion injury or after sham operation. DCLN proteomes were identified using sequential window acquisition of all theoretical mass spectra. Group comparisons, together with functional protein annotation analyses, were used to identify regulated protein candidates for further validation and pathway analyses. Validation of a selected candidate was assessed using enzyme-linked immunosorbent assay. Analysis comparing post-TBI animals with sham-operated controls revealed 25 upregulated and 16 downregulated proteins in the ipsilateral DCLN and 20 upregulated and 28 downregulated proteins in the contralateral DCLN of post-TBI animals. Protein class and function analyses highlighted the dysregulation of enzymes and binding proteins. Pathway analysis indicated an increase in autophagy. Biomarker analysis suggested that a subgroup of post-TBI animals had an increase in zonula occludens-1 coexpressed with proteins linked to molecular transport and amyloid precursor protein. We propose here that, after TBI, a subgroup of animals exhibit dysregulation of the TBI-relevant protein interactome in DCLNs, and that DCLNs might thus serve as an interesting biomarker source in future studies aiming to elucidate pathological brain functioning.Peer reviewe

Characterisation and Functional Studies of the DNA Cytosine-5-Methyltransferase 3-like Gene

DNMT3L-GEENIN KARAKTERISOINTI JA TOIMINTA Epigenetiikka tutkii DNA:n emäsjärjestyksestä riippumattomia geenin toimintaan vaikuttavia perinnöllisiä muutoksia. DNA:n metylaatio on epigeneettinen säätelymekanismi, joka vaikuttaa geenien ilmentymiseen, alkiokehitykseen ja syövän syntyyn. Nisäkässoluissa DNA:ta metyloivat neljä erilaista DNA-sytosiini-5-metyylitransferaasi-entsyymiä, DNMT1, DNMT2, DNMT3A ja DNMT3B. Tutkimuksessa tunnistettiin DNMT3-geeniperheeseen kuuluva uusi geeni, DNMT3L (DNA cytosine-5-methyltransferase 3-like) ja selvitettiin Dnmt3L-geenin säätelyyn vaikuttavat promoottorirakenteet. Lisäksi osoitettiin, että DNMT3L estää muiden geenien ilmentymistä sitoutumalla transkription säätelyssä toimivaan histonideasetylaasi 1­proteiniin. Tutkimustulokset ja niiden pohjalta muualla tehdyt jatkotutkimukset ovat auttaneet ymmärtämään epigeneettisen säätelyn toimintamekanismeja.DNA methylation is important in the regulation of gene expression, embryonic development, genomic imprinting and X chromosome inactivation. Abnormal methylation has been associated with carcinogenesis. So far, four separate genes, DNMT1, DNMT2, DNMT3A and DNMT3B encoding DNA cytosine-5-methyltransferases have been cloned from mammalian cells. DNMT1 is a maintenance methyltransferase, copying existing methylation patterns into newly replicated DNA strand. Dnmt3a and Dnmt3b are required for genome-wide de novo methylation and are essential for mammalian development. Gene expression is also regulated through modification of histone proteins that are essential for folding and packing of DNA inside the cells. Usually, histones are modified by acetylation, methylation and phosphorylation. In this thesis we have identified a novel member of the DNMT3 gene family, DNA cytosine-5-methyltransferase 3-like gene (DNMT3L). DNMT3L protein is similar to DNMT3A/Dnmt3a and DNMT3B/Dnmt3b, sharing the greatest similarity with other DNMT3 family members at the zinc finger region in the N-terminal part of the DNMT3L/Dnmt3L. In order to perform functional studies, the mouse Dnmt3L gene was also identified. Human and mouse genes showed high identity with each other and were both strongly expressed in testis and to a lesser extent also in ovary, thymus and fetal tissues. To study the regulation of Dnmt3L, we isolated the Dnmt3L promoter region and identified a minimal promoter area and regulatory elements in it. We demonstrated that Dnmt3L was regulated through Sp1/Sp3 transcription factors and epigenetic chromatin modifications, DNA methylation and histone deacetylation. Since Dnmt3a and Dnmt3b function as transcriptional repressors using zinc finger-domain to interact with histone deacetylase, HDAC1, we tested the repressional capacity of DNMT3L. Our data indicated that DNMT3L represses transcription by binding directly to HDAC1 protein and demonstrated the PHD-like zinc finger as a main repressional domain of DNMT3L

Characterisation and Functional Studies of the DNA Cytosine-5-Methyltransferase 3-like Gene

DNMT3L-GEENIN KARAKTERISOINTI JA TOIMINTA Epigenetiikka tutkii DNA:n emäsjärjestyksestä riippumattomia geenin toimintaan vaikuttavia perinnöllisiä muutoksia. DNA:n metylaatio on epigeneettinen säätelymekanismi, joka vaikuttaa geenien ilmentymiseen, alkiokehitykseen ja syövän syntyyn. Nisäkässoluissa DNA:ta metyloivat neljä erilaista DNA-sytosiini-5-metyylitransferaasi-entsyymiä, DNMT1, DNMT2, DNMT3A ja DNMT3B. Tutkimuksessa tunnistettiin DNMT3-geeniperheeseen kuuluva uusi geeni, DNMT3L (DNA cytosine-5-methyltransferase 3-like) ja selvitettiin Dnmt3L-geenin säätelyyn vaikuttavat promoottorirakenteet. Lisäksi osoitettiin, että DNMT3L estää muiden geenien ilmentymistä sitoutumalla transkription säätelyssä toimivaan histonideasetylaasi 1­proteiniin. Tutkimustulokset ja niiden pohjalta muualla tehdyt jatkotutkimukset ovat auttaneet ymmärtämään epigeneettisen säätelyn toimintamekanismeja.DNA methylation is important in the regulation of gene expression, embryonic development, genomic imprinting and X chromosome inactivation. Abnormal methylation has been associated with carcinogenesis. So far, four separate genes, DNMT1, DNMT2, DNMT3A and DNMT3B encoding DNA cytosine-5-methyltransferases have been cloned from mammalian cells. DNMT1 is a maintenance methyltransferase, copying existing methylation patterns into newly replicated DNA strand. Dnmt3a and Dnmt3b are required for genome-wide de novo methylation and are essential for mammalian development. Gene expression is also regulated through modification of histone proteins that are essential for folding and packing of DNA inside the cells. Usually, histones are modified by acetylation, methylation and phosphorylation. In this thesis we have identified a novel member of the DNMT3 gene family, DNA cytosine-5-methyltransferase 3-like gene (DNMT3L). DNMT3L protein is similar to DNMT3A/Dnmt3a and DNMT3B/Dnmt3b, sharing the greatest similarity with other DNMT3 family members at the zinc finger region in the N-terminal part of the DNMT3L/Dnmt3L. In order to perform functional studies, the mouse Dnmt3L gene was also identified. Human and mouse genes showed high identity with each other and were both strongly expressed in testis and to a lesser extent also in ovary, thymus and fetal tissues. To study the regulation of Dnmt3L, we isolated the Dnmt3L promoter region and identified a minimal promoter area and regulatory elements in it. We demonstrated that Dnmt3L was regulated through Sp1/Sp3 transcription factors and epigenetic chromatin modifications, DNA methylation and histone deacetylation. Since Dnmt3a and Dnmt3b function as transcriptional repressors using zinc finger-domain to interact with histone deacetylase, HDAC1, we tested the repressional capacity of DNMT3L. Our data indicated that DNMT3L represses transcription by binding directly to HDAC1 protein and demonstrated the PHD-like zinc finger as a main repressional domain of DNMT3L

Monimuotoinen kuivasilmäisyys

Peer reviewe

Looking deeper into ocular surface health : an introduction to clinical tear proteomics analysis

Ocular surface diseases are becoming more prevalent worldwide. Reasons for this include the ongoing population ageing and increasing use of digital displays, although ophthalmologists have a wide selection of tools, which can be implemented in the evaluation of the ocular surface health, methods, which enable the in-depth study of biological functions are gaining more interest. These new approaches are needed, since the individual responses to ocular surface diseases and treatments can vary from person to person, and the correlations between clinical signs and symptoms are often low. Modern mass spectrometry (MS) methods can produce information on hundreds of tear proteins, which in turn can provide valuable information on the biological effects occurring on the ocular surface. In this review article, we will provide an overview of the different aspects, which are part of a successful tear proteomics study design and equip readers with a better understanding of the methods most suited for their MS-based tear proteomics study in the field of ophthalmology and ocular surface.publishedVersionPeer reviewe

Epigenetic modifications affect Dnmt3L expression.

Imprinted genes are expressed from a single allele due to differential methylation of maternal or paternal alleles during gametogenesis. Dnmt3L (DNA cytosine-5-methyltransferase 3 like), a member of de novo methyltransferase Dnmt3 protein family, is a regulator of maternal imprinting. In the present study, we have characterized the promoter region of the mouse Dnmt3L gene. Transient transfection assays performed with 5'-deletion promoter constructs indicated a minimal promoter area within 440 bp upstream from the translational start site. Longer promoter constructs showed decreased activity, suggesting the presence of repressor elements within the upstream sequences. According to electrophoretic mobility-shift assays and mutation analysis, the minimal promoter region contained four functional binding sites for the Sp1 (specificity protein 1) family of transcription factors, Sp1 and Sp3. In vitro methylation of Dnmt3L promoter constructs decreased the transcriptional activity significantly, demonstrating down-regulation by cytosine methylation. This was supported by the results from bisulphite sequencing and real-time quantitative reverse transcriptase-PCR analysis of different mouse cell lines and tissues. In testis and embryonic stem cells showing strong Dnmt3L expression, all CpG sites studied were fully unmethylated, whereas non-expressive cell lines and tissues with lesser Dnmt3L expression showed complete or diverse CpG methylation levels. Treatment of Dnmt3L non-expressive cell lines with deacetylase inhibitor trichostatin A and methyltransferase inhibitor 5-aza-2'-deoxycytidine induced the expression of Dnmt3L mRNA. Furthermore, we show that the repressional effect of longer promoter fragments was also relieved by these inhibitors, altogether indicating an epigenetic control for Dnmt3L gene regulation