Melanocytes Research Articles

Diagnosis of primary biliary melanoma with distinct imaging features: a case report and literature review

Primary biliary melanoma arises from proliferating melanocytes in the mucosal surface of the bile duct and is extremely rare. Since the vast majority of biliary melanomas represent metastases of cutaneous origin, accurate preoperative diagnosis of melanoma and exclusion of other primary sources are vital in cases involving primary lesions. Although melanomas with pigmented cells have typical signal characteristics, obtaining a non-invasive pre-treatment diagnosis remains difficult, due to their low incidence. Here, the case of a 61-year-old male Asian patient who presented with upper quadrant abdominal pain, swelling and jaundice for 2 weeks, and who was diagnosed with primary biliary melanoma following extensive preoperative blood analyses, computed tomography (CT) and magnetic resonance imaging (MRI), is described. Post-resection immunohistochemistry confirmed the diagnosis and the patient received six chemotherapy cycles of temozolomide and cisplatin, however, progression of multiple liver metastases was observed at the 18-month follow-up CT. The patient continued with pembrolizumab and died 17 months later. The present case of primary biliary melanoma is the first reported diagnosis based on typical MRI features and the exhaustive exclusion of a separate primary origin.

The Suppression Effects of Fat Mass and Obesity Associated Gene on the Hair Follicle-Derived Neural Crest Stem Cells Differentiating into Melanocyte by N6-Methyladenosine Modifying Microphthalmia-Associated Transcription Factor.

Melanocyte (MC), derived from neural crest stem cell (NCSC), are involved in the production of melanin. The mechanism by which NCSC differentiates to MC remains unclear. N6-methyladenosine (m6A) modification was applied to discuss the potential mechanism. NCSCs were isolated from hair follicles of rats, and were obtained for differentiation. Cell viability, tyrosinase secretion and activity, and transcription factors were combined to evaluated the MC differentiation. RT-qPCR was applied to determine mRNA levels, and western blot were used for protein expression detection. Total m6A level was measured using methylated RNA immunoprecipitation (MeRIP) assay, and RNA immunoprecipitation was used to access the protein binding relationship. In current work, NCSCs were successfully differentiated into MCs. Fat mass and obesity associated gene (FTO) was aberrant downregulated in MCs, and elevated FTO suppressed the differentiation progress of NCSCs into MCs. Furthermore, microphthalmia-associated transcription factor (Mitf), a key gene involved in MC synthesis, was enriched by FTO in a m6A modification manner and degraded by FTO. Meanwhile, the suppression functions of FTO in the differentiation of NCSCs into MCs were reversed by elevated Mitf. In short, FTO suppressed the differentiating ability of hair follicle-derived NCSCs into MCs by m6A modifying Mitf.

Melanotic Neuroectodermal Tumour of Infancy.

Melanotic neuroectodermal tumour of infancy (MNTI) is a locally aggressive neoplasm of neural crest origin. It is primarily a paediatric tumour, and 95% of the cases occur in children below 1 year. The tumour mainly affects the head and neck region. It shows a predilection for the craniofacial sites and the most common site affected is anterior maxilla. Microscopically, it is characterized by a biphasic population of neuroblastic cells and pigmented epithelial cells. Although generally considered as a benign tumour, it can invade the adjacent muscle and bone, causing destruction of the involved site. The rapidity in growth and the aggressive clinical behaviour of the tumour can be deceptive and hence lack of familiarity with the clinical characteristics of the tumour may often lead to an erroneous diagnosis of malignancy. The treatment of choice for MNTI is excision, and it is usually curative. Extensive surgery in a child may interfere with the normal growth and development of the facial structures and reconstruction can be very challenging in infants. Hence, early diagnosis is critical for the effective management. Clinical findings, histopathological features, and differential diagnosis of a classic case of melanotic neuroectodermal tumour of infancy are discussed.

Patients with a history of atopy have fewer cutaneous melanomas than those without atopy: a cross-sectional study in 496 patients at risk of skin cancers.

The connection between atopy and skin cancers may be related to the stimulation of protective immune response, for example, through autoreactive immunoglobulin-E (IgE), or to the predisposition to carcinogenesis through chronic inflammation. The aim of this study was to investigate whether a past or present atopic disorder is associated with cutaneous photodamage, pigment cell nevi and skin cancers. For this, adult subjects at risk of any type of skin cancer (aged 21-79 years, 250 males, 246 females, 94 with immunosuppression) were examined for past or present malignancies in skin and extracutaneous site (ECS), photodamage, nevi, past or present atopic disorder in skin or mucus membranes, and possible other cancer-related factors. No association between atopy and photodamage, keratinocyte carcinomas or nevus count was found. Instead, there were fewer subjects with melanoma in 171 atopic (14.6%) than in 325 nonatopic subjects (22.2%) ( P = 0.044), and the investigator-estimated risk class of skin cancers was lower in atopic than nonatopic subjects. In all subjects, the multivariate odds ratio (OR) for melanoma was 0.583 ( P = 0.046; 95% confidence interval, 0.343-0.990) in atopic subjects, but in immunocompetent subjects, the reduced risk was confined to mucus membrane atopy (OR, 0.417; P = 0.020). Also, there were fewer subjects with malignancy in ECS in atopic (8.8%) than nonatopic subjects (15.7%) ( P = 0.031). No association between serum total IgE and skin cancers, photodamage, nevi or malignancies in ECS was found. In conclusion, the atopy, especially mucus membrane atopy, is associated with lower percentages of subjects with a history of melanoma.

Myo/Nog Cells Give Rise to Myofibroblasts During Epiretinal Membrane Formation in a Mouse Model of Proliferative Vitreoretinopathy.

Myo/Nog cells are the source of myofibroblasts in the lens and synthesize muscle proteins in human epiretinal membranes (ERMs). In the current study, we examined the response of Myo/Nog cells during ERM formation in a mouse model of proliferative vitreoretinopathy (PVR). PVR was induced by intravitreal injections of gas and ARPE-19 cells. PVR grade was scored by fundus imaging, optical coherence tomography, and histology. Double label immunofluorescence localization was performed to quantify Myo/Nog cells, myofibroblasts, and leukocytes. Myo/Nog cells, identified by co-labeling with antibodies to brain-specific angiogenesis inhibitor 1 (BAI1) and Noggin, increased throughout the eye with induction of PVR and disease progression. They were present on the inner surface of the retina in grades 1/2 PVR and were the largest subpopulation of cells in grades 3 to 6 ERMs. All α-SMA-positive (+) cells and all but one striated myosin+ cell expressed BAI1 in grades 1 to 6 PVR. Folds and areas of retinal detachment were overlain by Myo/Nog cells containing muscle proteins. Low numbers of CD18, CD68, and CD45+ leukocytes were detected throughout the eye. Small subpopulations of BAI1+ cells expressed leukocyte markers. ARPE-19 cells were found in the vitreous but were rare in ERMs. Pigmented cells lacking Myo/Nog and muscle cell markers were present in ERMs and abundant within the retina by grade 5/6. Myo/Nog cells differentiate into myofibroblasts that appear to contract and produce retinal folds and detachment. Targeting BAI1 for Myo/Nog cell depletion may be a pharmacological approach to preventing and treating PVR.

Surgical decisions in the setting of zonulopathy.

An 85-year-old man with a history of type 2 diabetes, pseudoexfoliation (PXF) in both eyes, and tamsulosin use was referred for the evaluation of a dense cataract in the right eye and a subluxated intraocular lens (IOL) in the left eye. Unfortunately, his surgery in the left eye was complicated by diffuse zonulopathy. The referring surgeon placed a 3-piece IOL in the sulcus. However, the passively fixated 3-piece IOL moved inferiorly causing monocular diplopia for over a year. Because the patient was pleased with the IOL immediately postoperatively, a refixation procedure was performed in the form of sulcus placement with iris suture fixation in the left eye. Fortunately, the iris-fixated IOL in the left eye has remained well centered and stable without cystoid macular edema (CME) or chronic inflammation for over 8 months. The patient is on no ocular medications and has no family history of glaucoma. He now needs cataract surgery in the right eye and is extremely apprehensive because of his difficult course in the left eye. The corrected distance visual acuity is 20/70 in the right eye and 20/25 in the left eye. Intraocular pressures (IOPs) measure 20 mm Hg in the right eye and 14 mm Hg in the left eye by Goldmann tonometry. Pachymetry is 536 µm in the right eye and 543 µm in the left eye. Pupils are round with minimal reactivity and without a relative afferent pupillary defect. Extraocular motility is normal in both eyes, and confrontation visual fields is full in both eyes. Gonioscopy reveals an angle open to the pigmented trabecular meshwork (PTM) in the right eye and the ciliary body in the left eye with 1+ PTM and without peripheral anterior synechia in both eyes. The retinal nerve fiber layer and macular optical coherence tomography are normal in both eyes. On slitlamp examination, pertinent findings include pseudoexfoliative changes at the pupillary margin with poor dilation of 3.5 mm in both eyes; the anterior chamber (AC) is shallow but adequate in the right eye and deep and quiet with rare pigmented cells in the left eye. There is a 5+ nuclear sclerotic cataract with pseudoexfoliative changes on the anterior capsule and no obvious phacodonesis in the right eye and a 3-piece posterior chamber IOL in the sulcus fixated to the iris with 10-0 polypropylene sutures at 6 and 12 o'clock without pseudophacodonesis in the left eye. Dilated fundus examination reveals a cup-to-disc ratio of 0.4 with healthy neuroretinal rims in both eyes, posterior vitreous detachments in both eyes, and no evidence of diabetic retinopathy in both eyes. All other findings are unremarkable. How would you counsel this patient regarding his risk factors for surgery in the right eye? What surgical maneuvers would you use to remove the cataract safely? How would you stabilize the IOL if the capsule bag becomes compromised due to zonulopathy?

What coloration brings: Implications of background adaptation to oxidative stress in anurans

BackgroundUrban development results in habitat destruction, affecting populations of amphibians, the most fragile group of vertebrates. With changes in the environment, these animals become more exposed to light and predators. To enhance their chances of survival, they display plasticity of body coloration. Aside from adaptive benefits, animals exhibiting background matching meet the energetic costs and restrictions of changing body tones. To study the physiological consequences of Hyla arborea tadpole adaptation to background color, we followed oxidative stress parameters after rearing larvae on a constant background (black/white) and after changing the background color.ResultsLarvae cultivated for 20 days on constant substrate color exhibited differences in body coloration but without differences in lipid peroxidation (LPO) concentration between dark and pale individuals, suggesting that coloration investment during this period did not induce higher oxidative damage in darker tadpoles. Prolonged exposure of larvae (37 days) to a dark habitat increased antioxidative system defense and LPO concentrations, compared to animals reared permanently in the white surroundings. The positive correlation of oxidative damage with color intensity of individuals points to the physiological consequences of higher investment in the number of pigment cells necessary for dark pigmentation. In individuals faced with non-matching background and change in body coloration, defense system declined and LPO occurred relative to individuals cultivated in white habitat.ConclusionHere, we have pointed to consequences related to background matching and stress that amphibians experienced during chromatic adaptations. Background color change causes a complex physiological response affecting the antioxidative defense parameters. This investigation elucidates the accompanying cost of amphibiansʼ adjustment to an altered environment.

Acceleration of melanocyte senescence by the proinflammatory cytokines IFNγ and TNFα impairs the repigmentation response of vitiligo patients to narrowband ultraviolet B (NBUVB) phototherapy

Vitiligo is a chronic autoimmune disease characterized by the T helper 1 (Th1) cytokine-driven immune destruction of melanocytes (MCs). Although narrowband ultraviolet B (NBUVB) phototherapy has been proven to be an effective therapeutic option, the repigmentation response to that phototherapy varies greatly in different vitiligo patients. Here, we demonstrate that there is an increase of NBUVB-induced cellular senescence in vitiligo MCs exposed to Th1 cytokine interferon γ (IFNγ) and/or tumor necrosis factor α (TNFα) in lesional vitiligo skin from poor responders who had undergone NBUVB phototherapy. Supplementation with exogenous recombinant human stem cell factor (rhSCF) in the culture medium as well as the lentiviral vector–mediated overexpression of cKIT could prevent the MCs from the IFNγ/TNFα-accelerated cellular senescence. Mechanistic studies indicated that the reduced ratio of membrane-bound KIT (mKIT) to the soluble form of KIT (sKIT) is directly related to the cellular senescence of vitiligo MCs following exposure to IFNγ and TNFα. Furthermore, the matrix metalloprotease 9 (MMP9) inhibitor GM6001 attenuates the production of sKIT via the suppression of cKIT ectodomain shedding. Altogether, our study indicates that the presence of Th1 cytokines IFNγ and/or TNFα in the epidermal milieu might impair the repigmentation response of vitiligo patients to NBUVB phototherapy.

New panel of biomarkers to discriminate between amelanotic and melanotic metastatic melanoma.

Melanoma is a form of skin cancer that can rapidly invade distant organs. A distinctive feature of melanomas is their pigmentation status, as melanin is present in most skin melanomas, whilst many metastatic tumors could become amelanotic. Besides the obvious malfunction of the key genes of the melanin pathway, the amelanotic tumors could bear a characteristic molecular signature accounting for their aggressivity. Using mass spectrometry-based proteomics we report here a distinctive panel of biomarkers for amelanotic aggressive melanoma that differ from the less invasive pigmented cells. The developed method allows the label-free quantification of proteins identified by LC-MS/MS analysis. We found a set of proteins comprising AHNAK, MYOF, ANXA1, CAPN2, ASPH, EPHA2, THBS1, TGM2, ACTN4 along with proteins involved in cell adhesion/migration (integrins, PLEC, FSCN1, FN1) that are highly expressed in amelanotic melanoma. Accompanying the down regulation of pigmentation specific proteins such as tyrosinase and TYRP1, these biomarkers are highly specific for a type of highly invasive melanoma. Interestingly, the LC-MS/MS proteomics analysis in hypoxia revealed that the abundance of this specific set of proteins found in normoxia was rather unaltered in these conditions. These biomarkers could therefore predict a metastatic behaviour for the amelanotic cells in the early stages of the tumor development and thus serve in melanoma prognostic. Applying this algorithm to related databases including melanoma samples published by independent laboratories/public databases we confirm the specificity of the newly found signatures. Overall, we begin to unravel the molecular alterations in the amelanotic melanoma and how basic proteomics offers insights into how to assess the clinical, pathological and misdiagnosis differences between the main subtypes of melanoma.

Melatonin reduces melanogenesis by inhibiting the paracrine effects of keratinocytes.

Keratinocytes regulate melanogenesis in a paracrine manner. Previous studies have shown that melatonin can directly inhibit melanin production in the melanocytes. However, it is unclear whether melatonin can also indirectly regulate melanogenesis through the keratinocytes. In this study, we explored the role of melatonin in regulating keratinocyte-mediated melanogenesis using reconstructed human epidermis (RHE). Melatonin showed an inhibitory effect on melanin synthesis in this model. Furthermore, the conditioned media from melatonin-treated HaCaT cells downregulated melanogenesis-related genes, including MITF, TYR, TYRP1, DCT and RAB27A in the pigment MNT1 cells, and decreased levels of phosphorylated ERK, JNK and p38. RNA sequencing further showed that mitochondrial functions and oxidative stress pathway in the MNT1 cells were inhibited by the conditioned medium from melatonin-treated HaCaT cells. Furthermore, melatonin reduced the secretion of ET-1 and PTGS2 from HaCaT cells by inhibiting the JAK2/STAT3 signalling pathway. In conclusion, melatonin downregulates the paracrine factors ET-1 and PTGS2 in the keratinocytes by inhibiting the JAK2/STAT3 pathway, which reduces melanin production in pigment cells. Thus, melatonin has a potential therapeutic effect on skin pigmentation disorders.

Genetics and Genomics of Melanoma: Current Progress and Future Directions

Melanoma is a form of skin cancer that develops in the skin's pigment cells, known as melanocytes, and can spread via blood and the lymphatic system to nearby tissues or distant organs in the body [...].

Mechanisms underlying the formation of complex color patterns on Nigella orientalis (Ranunculaceae) petals.

Complex color patterns on petals are widespread in flowering plants, yet the mechanisms underlying their formation remain largely unclear. Here, by conducting detailed morphological, anatomical, biochemical, optical, transcriptomic, and functional studies, we investigated the cellular bases, chromogenic substances, reflectance spectra, developmental processes, and underlying mechanisms of complex color pattern formation on Nigella orientalis petals. We found that the complexity of the N. orientalis petals in color pattern is reflected at multiple levels, with the amount and arrangement of different pigmented cells being the key. We also found that biosynthesis of the chromogenic substances of different colors is sequential, so that one color/pattern is superimposed on another. Expression and functional studies further revealed that a pair of R2R3-MYB genes function cooperatively to specify the formation of the eyebrow-like horizontal stripe and the Mohawk haircut-like splatters. Specifically, while NiorMYB113-1 functions to draw a large splatter region, NiorMYB113-2 functions to suppress the production of anthocyanins from the region where a gap will form, thereby forming the highly specialized pattern. Our results provide a detailed portrait for the spatiotemporal dynamics of the coloration of N. orientalis petals and help better understand the mechanisms underlying complex color pattern formation in plants.

Recognizing the Differentiation Degree of Human Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium Cells Using Machine Learning and Deep Learning-Based Approaches

Induced pluripotent stem cells (iPSCs) can be differentiated into mesenchymal stem cells (iPSC-MSCs), retinal ganglion cells (iPSC-RGCs), and retinal pigmental epithelium cells (iPSC-RPEs) to meet the demand of regeneration medicine. Since the production of iPSCs and iPSC-derived cell lineages generally requires massive and time-consuming laboratory work, artificial intelligence (AI)-assisted approach that can facilitate the cell classification and recognize the cell differentiation degree is of critical demand. In this study, we propose the multi-slice tensor model, a modified convolutional neural network (CNN) designed to classify iPSC-derived cells and evaluate the differentiation efficiency of iPSC-RPEs. We removed the fully connected layers and projected the features using principle component analysis (PCA), and subsequently classified iPSC-RPEs according to various differentiation degree. With the assistance of the support vector machine (SVM), this model further showed capabilities to classify iPSCs, iPSC-MSCs, iPSC-RPEs, and iPSC-RGCs with an accuracy of 97.8%. In addition, the proposed model accurately recognized the differentiation of iPSC-RPEs and showed the potential to identify the candidate cells with ideal features and simultaneously exclude cells with immature/abnormal phenotypes. This rapid screening/classification system may facilitate the translation of iPSC-based technologies into clinical uses, such as cell transplantation therapy.

Induced pluripotent stem cells reprogramming overcomes technical limitations for highly pigmented adult melanocyte amplification and integration in 3D skin model.

Understanding pigmentation regulations taking into account the original skin color type is important to address pigmentary disorders. Biological models including adult melanocytes from different phenotypes allow to perform fine-tuned explorative studies and support discovery of treatments adapted to populations' skin color. However, technical challenges arise when trying to not only isolate but also amplify melanocytes from highly pigmented adult skin. To bypass the initial isolation and growth of cutaneous melanocytes, we harvested and expanded fibroblasts from light and dark skin donors and reprogrammed them into iPSC, which were then differentiated into melanocytes. The resulting melanocyte populations displayed high purity, genomic stability, and strong proliferative capacity, the latter being a critical parameter for dark skin cells. The iPSC-derived melanocyte strains expressed lineage-specific markers and could be successfully integrated into reconstructed skin equivalent models, revealing pigmentation status according to the native phenotype. In both monolayer cultures and 3D skin models, the induced melanocytes demonstrated responsiveness to promelanogenic stimuli. The data demonstrate that the iPSC-derived melanocytes with high proliferative capacity maintain their pigmentation genotype and phenotypic properties up to a proper integration into 3D skin equivalents, even for highly pigmented cells.

Proprotein Convertase Furin Regulates Melanogenesis via the Notch Signaling Pathway.

Furin is a calcium-dependent serine protease found in almost all mammals. It plays an important role in embryogenesis, tissue homeostasis, tumors pathogenesis, viral infectious diseases, and neurodegenerative diseases. However, whether furin directly regulates melanin synthesis and transport has rarely been evaluated yet. The present study aimed to investigate furin potential function and mechanisms in melanogenesis. Short hairpin RNAs targeting furin gene (sh-furin RNAs) were used to inhibit furin gene expression in human melanoma cell line MNT-1 cells. Then, intracellular melanin content was measured using a sodium hydroxide method. Extracellular melanin content was measured determining cell culture medium absorbance at 450 nm. Levodopa (L-DOPA) oxidation rate was measured to assess the tyrosinase activity. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting (WB) were performed to measure melanogenesis-related genes and Notch pathway-related genes expression levels. Human primary melanocytes (MCs) were extracted from foreskin tissues and were stimulated with a furin inhibitor. Then, the extracellular and intracellular melanin content, tyrosinase activity and molecules related to melanogenesis and the Notch pathway expression were measured in MCs with or without a furin inhibitor. Additionally, morpholino technology was used to inhibit furin in zebrafish. Zebrafish pigmentary phenotypes in the control group and furin inhibition group were observed with a stereo microscope. Then, MCs number in the tail and head of the zebrafish were counted using Image J software (version 1.53t, National Institute of Health, Bethesda, MD, USA). Meanwhile, melanin content, tyrosinase activity, and molecules related to melanogenesis and the Notch pathway expression levels were measured. Subsequently, valproic acid (VPA), a Notch pathway agonist, was used in MNT-1 melanoma cells treated with or without sh-furin lentiviral vectors for rescue experiments. Furin inhibition enhanced intracellular and extracellular melanin content, and cellular tyrosinase activity in MNT-1 cells and MCs. Additionally, furin inhibition increased melanin synthesis-associated and transport-associated proteins expression levels while inhibiting Notch pathway-relevant proteins. After using VPA to activate the Notch pathway in MNT-1 cells transfected with a sh-furin RNA, the biological effects resulting from furin knockdown were reversed. In addition, the results of in vivo experiments using morpholino to knock down furin gene in zebrafish further confirmed that furin knockdown regulated melanogenesis and impaired the Notch pathway. This study clarified that furin affected the synthesis and transport of melanin via Notch pathway. Notch pathway may be a potential therapeutic target for pigmented skin diseases.

Cytodifferentiation of pinealocytes (I and II) and astrocyte types of mature male sheep epiphysis cerebri with special emphasis on the presence of neuronal and pigmented-like cells.

The epiphysis cerebri (pineal gland) is a small-sized, photo neuroendocrine organ in the brain of most vertebrates. Their effect is through secretion of melatonin, a serotonin-derived hormone which is stimulated by darkness and inhibited by light and modulates the circadian rhythm; light and dark cycle like a biological clock, sleep patterns (sleep-wake cycle), and sexual development. This study aimed to identify and differentiate the different cell types filling the pineal gland parenchyma of mature male sheep. Pineal glands were collected and sliced parasagitally then processed histologically for light and electron microscopic examinations. Two main cell types; pinealocytes and astrocytes were recognized within the gland parenchyma. Pinealocytes were the chief parenchymatus cells that occupied the largest volume of the gland and were classified according to the nuclear pictures (activity status) into two subtypes; pinealocytes I (pale subtype, active) and II (dark subtype, inactive). Astrocyte neuroglial cells had cytoplasmic processes which form a huge supportive framework between the pinealocytes and clarified two types; type I were elongated cells with elongated snake shaped nucleus and type II were smaller in size, with oval nuclei. Another marginal cell type was identified as a neuron-like cell which appeared larger in size than others and distributed sporadically, has eccentric oval nucleus with prominent nucleoli and single, long cytoplasmic process that branched at its terminal forming T-shaped process looks like pseudo unipolar neuron. Moreover, aggregations of pigment granules were markedly observed in the intercellular spaces and also near the blood capillaries. With transmission electron microscope (TEM) a special characteristic feature of pinealocytes; synaptic ribbons were recognized that appeared as bands of electron-dense material with several synaptic spherules; vesicles adjacent to its surface helping in the multivesicular release. The gland parenchyma revealed two main cell types; pinealocytes and astrocytes. Each one was subdivided into two subtypes; I and II. The first one was classified according to their nuclear pictures (activity status) and the second one was according to their shape, size, and cytoplasmic processes. Other cell types were also identified as neuronal and pigmented-like cells in the pineal matrix.

Study of thyroid disorders in vitiligo.

Vitiligo, an acquired sometimes familial depigmentary disorder of the skin and hair that results from selective destruction of melanocytes or pigment cells. It is the single most important non-neo plastic disease that involves both the immune system and melanocytes which are subsequently destroyed and the affected area turns pale and becomes white. The prevalence of the disease is between 1% and 2% in general population. It is a prospective, randomized, and controlled study. Over 90 vitiligo patients attending Dermatology OPD and vitiligo clinic are enrolled in the study. About 35 apparently healthy, age and sex matched individuals are selected to serve as control. A prescribed proforma containing all the demographic data, relevant questionnaire were recorded for each and every case with brief clinical history suggestive of any thyroid disease as well as those referred by the clinicians. P value <0.05 is considered significant. The Quantitative Determination of Thyroglobulin (Tg) Autoantibodies in Human Serum or Plasma by a Microplate Enzyme Immunoassay. Vitiligo group, thirty four (37.78%) patients have clinical hypothyroidism while 9 (10%) patients have clinical hyperthyroidism. This difference in the distribution is statistically significant (P < 0.05) with a Chi-square value of 10.08. The data are entered, analyzed and computed with SPSS version 15 software and well-known statistical test like Chi-square, students' 't' test have been advocated wherever found applicable. P value <0.05 is considered significant. There is increased incidence of autoimmune thyroid diseases among vitiligo patients. The reason being vitiligo usually precedes the onset of thyroid dysfunction.

Light Microscopy and Ultrastructure of Body Wall in Leech Haemadipsa zeylanica.

Leeches are widely distributed worldwide in a diversity of habitats, such as freshwater, seas, desert, and oases. Since literature does not have an elaborate study on histology of leeches, thus emphasis is given to study the histology. The aim of this study is to examine the histology of free-living leeches Haemadipsa zeylanica under light microscopy and extended to ultrastructural study under transmission electron microscopy. Tissue was fixed in 10% formalin, dehydrated with alcohol, infiltrated and embedded in wax, and thin section was cut using a rotary microtome. Sections were stained with hematoxylin and eosin. The epithelial layer of cells in H. zeylanica had a thin layer of cuticles covering it. In light microscopy, the epidermis has three types of epidermal cells, namely, glandular cells (Type I, Type II, and Type III), supporting cells (Type IV), and pigment cells (Type V). When the three glandular cells of the body wall of H. zeylanica epidermis were examined under an electron microscope, three types of distinct electron-dense granules were identified. Type I cells with granules, Type II cells with course granules close to the dermis were identified as pear-shaped secretory cells, and Type III cells with electron-dense granules.

Myelinosome organelles in pathological retinas: ubiquitous presence and dual role in ocular proteostasis maintenance.

The timely and efficient elimination of aberrant proteins and damaged organelles, formed in response to various genetic and environmental stressors, is a vital need for all cells of the body. Recent lines of evidence point out several non-classical strategies employed by ocular tissues to cope with aberrant constituents generated in the retina and in the retinal pigmented epithelium cells exposed to various stressors. Along with conventional strategies relying upon the intracellular degradation of aberrant constituents through ubiquitin-proteasome and/or lysosome-dependent autophagy proteolysis, two non-conventional mechanisms also contribute to proteostasis maintenance in ocular tissues. An exosome-mediated clearing and a myelinosome-driven secretion mechanism do not require intracellular degradation but provide the export of aberrant constituents and "waste proteins" outside of the cells. The current review is centered on the non-degradative myelinosome-driven secretion mechanism, which operates in the retina of transgenic Huntington's disease R6/1 model mice. Myelinosome-driven secretion is supported by rare organelles myelinosomes that are detected not only in degenerative Huntington's disease R6/1 retina but also in various pathological states of the retina and of the retinal pigmented epithelium. The intra-retinal traffic and inter-cellular exchange of myelinosomes was discussed in the context of a dual role of the myelinosome-driven secretion mechanism for proteostasis maintenance in different ocular compartments. Special focus was made on the interplay between degradative and non-degradative strategies in ocular pathophysiology, to delineate potential therapeutic approaches to counteract several vision diseases.

High-efficient CRISPR/Cas9-mediated gene targeting to establish cell models of ciliopathies.

Primary cilia are antenna-like structures developed on the cell surface of mammalian cells during the quiescent G0 phase. Primary cilia in mammalian cells receive extracellular signals for early development and cell tissue homeostasis. Ciliopathies characterized with congenital anomalies such as cerebellar hypoplasia, polycystic kidney and polydactyly are caused by germline mutations of ciliary structure- and function-related genes. Gene knock-out techniques in ciliated cultured cells with the uniformed genetic background are useful to evaluate the pathophysiological roles of ciliopathy-related gene products. Genome editing technology has been applied into the gene knock-out in many types of cultured cell lines. However, the frequency of genome editing varies according to cell species and cycle because of dependency on error-free homology-directed repair (HDR) activity. The human telomerase reverse transcriptase-immortalized retinal pigmented epithelial cell line (hTERT-RPE1) is well known for its suitability in cilia research. However, the efficacy of the HDR-mediated knock-out clone isolation was low. Here, we introduce the clustered regularly interspaced short palindromic repeats-obligate ligation-gated recombination (CRISPR-ObLiGaRe) system, which is a nonhomologous end-joining (NHEJ)-mediated gene targeting method, to generate the knock-out clones effectively even in the lower-HDR activity cell lines including hTERT-RPE1 cell. This CRISPR-ObLiGaRe system is a powerful tool for establishing ciliopathy model cell libraries and identifying each gene function in cilia-related phenotypes.