Magnifying Endoscopy for Esophageal Ectopic Sebaceous Glands

Ectopic sebaceous glands are found very rarely in the esophagus; heretofore, several cases have been reported. The sebaceous gland is originally a source of an endodermal origin; however, there have been controversies regarding whether the origin of the esophageal ectopic sebaceous gland is ectodermal or endodermal. Ectopic sebaceous glands of the esophagus usually do not cause symptoms; thus, they are often found incidentally on endoscopy for routine health screening. Endoscopic findings are characterized by single or multiple yellow patches or nodular lesions of various sizes, sometimes with small central openings. We report two cases of esophageal ectopic sebaceous glands found incidentally during endoscopy with magnifying endoscopic findings. The lesions were in the mid-esophagus and lower esophagus, respectively, and both endoscopic findings were similar as multiple yellowish patches or plaques. Magnifying endoscopy revealed the openings of the excretory ducts surrounded by circular microvessels in both cases.

Lineage-specific Expression of miR-200 Family in Human Embryonic Stem Cells during In Vitro Differentiation

Although microRNAs have emerged as key regulators in diverse cellular processes, the roles of microRNAs are poorly understood in human embryonic stem cells (hESCs) during differentiation into specialized cell types. In this study, we used a microRNA array with 799 human microRNA probes to examine the expression profiles of microRNAs in hESCs during differentiation into endodermal and mesodermal lineages in vitro. Among the microRNAs analyzed, 7 and 20 microRNAs were enriched in the developmental process of hESCs into mesodermal and endodermal lineages, respectively. In particular, the expression levels of miR-200 family, which is known to regulate the epithelial to mesenchymal transition (EMT), gradually increased in hESCs during differentiation into hepatocytes while they gradually decreased during differentiation into vascular endothelial cells. Downregulation of ZEB1, a direct target of miR-200 family, and E-CADHERIN, a target protein of ZEB1, was observed in hESCs during differentiation into endodermal and mesodermal lineages, respectively. These results indicate that miR-200 family has an important role in determining the cell fate between endodermal and mesodermal lineages from the pluripotent state.

Generation of Insulin-Expressing Cells in Mouse Small Intestine by Pdx1, MafA, and BETA2/NeuroD

BACKGROUND: To develop surrogate insulin-producing cells for diabetes therapy, adult stem cells have been identified in various tissues and studied for their conversion into β-cells. Pancreatic progenitor cells are derived from the endodermal epithelium and formed in a manner similar to gut progenitor cells. Here, we generated insulin-producing cells from the intestinal epithelial cells that induced many of the specific pancreatic transcription factors using adenoviral vectors carrying three genes: PMB (pancreatic and duodenal homeobox 1 [Pdx1], V-maf musculoaponeurotic fibrosarcoma oncogene homolog A [MafA], and BETA2/NeuroD). METHODS: By direct injection into the intestine through the cranial mesenteric artery, adenoviruses (Ad) were successfully delivered to the entire intestine. After virus injection, we could confirm that the small intestine of the mouse was appropriately infected with the Ad-Pdx1 and triple Ad-PMB. RESULTS: Four weeks after the injection, insulin mRNA was expressed in the small intestine, and the insulin gene expression was induced in Ad-Pdx1 and Ad-PMB compared to control Ad-green fluorescent protein. In addition, the conversion of intestinal cells into insulin-expressing cells was detected in parts of the crypts and villi located in the small intestine. CONCLUSION: These data indicated that PMB facilitate the differentiation of mouse intestinal cells into insulin-expressing cells. In conclusion, the small intestine is an accessible and abundant source of surrogate insulin-producing cells.

Induced Pluripotent Stem (iPS) Cells in Dentistry: A Review

iPS cells are derived from somatic cells via transduction and expression of selective transcription factors. Both viral-integrating (like retroviral) and non-integrating (like, mRNA or protein-based) techniques are available for the production of iPS cells. In the field of dentistry, iPS cells have been derived from stem cells of apical papilla, dental pulp stem cells, and stem cells from exfoliated deciduous teeth, gingival and periodontal ligament fibroblasts, and buccal mucosa fibroblasts. iPS cells have the potential to differentiate into all derivatives of the 3 primary germ layers i.e. ectoderm, endoderm, and mesoderm. They are autogeneically accessible, and can produce patient-specific or disease-specific cell lines without the issue of ethical controversy. They have been successfully tested to produce mesenchymal stem cells-like cells, neural crest-like cells, ameloblasts-like cells, odontoblasts-like cells, and osteoprogenitor cells. These cells can aid in regeneration of periodontal ligament, alveolar bone, cementum, dentin-pulp complex, as well as possible Biotooth formation. However certain key issues like, epigenetic memory of iPS cells, viral-transduction, tumorgenesis and teratoma formation need to be overcome, before they can be successfully used in clinical practice. The article discusses the sources, pros and cons, and current applications of iPS cells in dentistry with an emphasis on encountered challenges and their solutions.

Understanding of Human Embryo Development for Teratogen Counselling

Human embryology is the study of development from a single cell to a baby in 9 months. Implantation occurs at the end of the first week of development. The second week of development is known as the week of 2's. Gastrulation, the most characteristic event occurring in the third week, establishes three germ layers composed of ectoderm, mesoderm, and endoderm. The three germ layers and neural crest cells lead to the development of their own tissues and organs during the embryonic period, which extends from the third to the eighth week. Major congenital malformations occur in the embryonic period. The fetal period, from the third month to the day of birth, is the time for maturation of tissues and organs, and growth of the body. Because of the close relationship between embryology and congenital abnormalities, knowledge of human development is essential to assess the effects on the embryo when the mother has been exposed to teratogens. This paper briefly reviews the normal embryonic development and associated congenital malformation.

Ginseng conduces degeneration of differentiating tissues in developing albino mice

The current study was conducted to evaluate its effects on developing skin, heart and intestines, derivatives of ectoderm, mesoderm and endoderm respectively, when given during pregnancy to albino mice. Experimental Randomized controlled trial study. This study was conducted at Anatomy Department, University of Health Sciences, Lahore from Jan 2011 to July 2011. Twelve pregnant albino mice were divided into two groups of 6 each; group A was given distill water, Human therapeutic dose [HTD][780mg/kg/day] was dissolved in 0.1ml of distilled water and was administered to the animals of group B for entire length of pregnancy. Fetuses were delivered and dissected on 18[th] day of gestation. Tissue samples comprising, skin, heart and small intestine derivatives of ectoderm, mesoderm and endoderm respectively, were removed and processed for light microscopic study. In the current study, the difference between dead and alive fetuses, when compared between groups was found to be statistically significant [p value < 0.05]. In addition, the histological examination of the above tissues revealed extensive cell death resulting into loss of normal architecture of skin, heart and small intestine. Cells showed pyknotic nuclei and scanty cytoplasm, indicating process of apoptosis, which when compared between groups was found to be statistically significant [p<0.05]. It is suggested, therefore, that further investigations and monitoring of additional tissues for the effects of Ginsenosides during pregnancy are warranted

La reactividad constitucional del sujeto: una piedra de rosetta interdisciplinaria / A reatividade constitucional do sujeito. Uma pedra de roseta interdisciplinar / Subject’s constitutional reactivitya: interdisciplinary rosetta stone

Aprender a identificar de manera integral, mixta y dinámica la constitución de un sujeto significa, por un lado, ayudarlo a autoconocerse en su manera de reaccionar (por eso se habla de reactividad constitucional y no más de biotipos o rasgos), de desarrollarse (madurar o destruirse), de relacionarse y aceptar a los demás tal como son; por otro lado, sirve para personalizar diagnóstico, terapia y pronóstico. La historia del constitucionalismo y un esquema sinóptico interdisciplinario (filosofía griega, teología, medicina hipocrática, galénica, homeopática, china, ayurvédica y holística, biología, embriología, bioquímica, fisiopatología, neurología, endocrinología, psicología y espiritualidad) muestra una precisa correspondencia y un denominador común sobre la base de la teoría constitucional embriológica que habla de endoblasto, mesoblasto y ectoblasto. Una coherencia que dura por más de 24 siglos en occidente y pasa también las barreras culturales (oriente y occidente) y paradigmáticas (biomedicina y homeopatía), no puede ser casual.

To learn to identify in an integral, mixed and dynamic way the subject’s constitution means, on one hand, to help him to a self-knowledgement of his reactive way (for this, we speak of constitutional reactivity and no more of biotipe or trait), to develop himself (to mature or to destroy), to join and accept the others like they are; on the other way, it serve to personalize diagnosis, therapy and prognosis. The constitution history and a synoptic interdisciplinary diagram (Greek philosophy, theology, hippocratic, galenic, homeopatic, chinese, ayurvedic and holistic medicines, biology, embriology, biochemistry, physiopathology, neurology, endocrinology, psychology and spirituality) shows a precise link and a common denominator according to an embriologic constitutional theory, that speaks about endoblast, mesoblast and ectoblast. A coherence thas lasts for more than 24 centuries in occident and goes through even the cultural borders (eastern and western world) and paradigmatic logics (biomedicine and homeopaty) cannot be casual.

Aprender a identificar de maneira integral, mista e dinâmica a constituição de um sujeito significa, por um lado, ajudá-lo a se autoconhecer na sua maneira de reagir (por isso se fala de reatividade constitucional e não mais de biotipos ou traços), de desenvolver-se (amadurecer ou destruir-se), de relacionar-se e aceitar os demais taisl como são; por outro lado, serve para personalizar diagnóstico, terapia e prognóstico. A história do constitucionalismo e um esquema sinóptico interdisciplinar (filosofia grega, teologia, medicina hipocrática, galênica, homeopática, chinesa, ayurvédica e holística, biologia, embriologia, bioquímica, fisiopatologia, neurologia, endocrinologia, psicologia e espiritualidade) mostra uma precisa correspondência e um denominador comum sobre a base da teoria constitucional embriológica que fala de endoblasto, mesoblasto e ectoblasto. Uma coerência que dura por mais de 24 séculos no ocidente e ultrapassa também as barreiras culturais (oriente e ocidente) e paradigmáticas (biomedicina e homeopatia), não pode ser casual.

Bone marrow stem/progenitor cell mobilization in C57BL/6J and BALB/c mice / 한국실험동물학회지

Bone marrow (BM) has been considered as a reservoir of stem/progenitor cells which are able to differentiate into ectodermal, endodermal, and mesodermal origins in vitro as well as in vivo. Following adequate stimulation, such as granulocyte stimulating factor (G-CSF) or AMD3100, BM resident stem/progenitor cells (BMSPCs) can be mobilized to peripheral blood. Several host-related factors are known to participate in this mobilization process. In fact, a significant number of donors are resistant to G-CSF induced mobilization protocols. AMD3100 is currently used in combination with G-CSF. However, information regarding host-related factors which may influence the AMD3100 directed mobilization is extremely limited. In this study, we were to get some more knowledge on the host-related factors that affect the efficiency of AMD3100 induced mobilization by employing in vivo mobilization experiments. As a result, we found that C57BL/6J mice are more sensitive to AMD3100 but less sensitive to G-CSF which promotes the proliferation of BMSPCs. We excluded S1P as one of the host related factor which influences AMD3100 directed mobilization because pre-treatment of S1P receptor antagonist FTY720 did not inhibit BMSPC mobilization. Further in vitro experiments revealed that BALB/c mice, compared to C57BL/6J mice, have less BMSPCs which migrate in response to host related factors such as sphingosine-1-phosphate (S1P) and to CXCL12. We conclude that AMD3100-directed mobilization depends on the number of BMSPCs rather than on the host-related factors. These results suggest that the combination of AMD3100 and G-CSF is co-operative and is optimal for the mobilization of BMSPCs.

Dinâmica da inversão do saco vitelino em preás (Galea spixii Wagler, 1831) / Dynamics of yolk sac inversion in galea (Galea spixii Wagler, 1831)

O objetivo deste trabalho foi estudar o período de inversão do saco vitelino bem como a dinâmica resultante deste processo na gestação inicial em preás, utilizando-se microscopia de luz, microscopia eletrônica de varredura e de transmissão. No décimo segundo dia de gestação observou-se o desenvolvimento dos endodermas parietal e visceral delimitando a cavidade do saco vitelino. O endoderma parietal foi evidenciado revestindo a superfície fetal da placenta corioalantoidea bem como contornando o espaço delimitado pela decídua capsular. Estes endodermas apresentaram formato prismático e encontraram-se separados do trofoblasto por uma desenvolvida membrana de Reichert. Já o endoderma visceral continha vasos vitelínicos e possuía vilosidades apenas em determinadas áreas. No décimo quarto dia de gestação verificou-se a inversão do saco vitelino, caracterizada pela degeneração do endoderma parietal e trofoblasto mural, associado ao desaparecimento gradual da membrana de Reichert. Como consequência deste fenômeno, o endoderma visceral passou a constituir uma interface com o epitélio uterino. Após a inversão, o endoderma parietal que permaneceu íntegro foi aquele que se apoiava na superfície da placenta corioalantóidea, apresentando células em formato colunar alto e característica de epitélio pseudoestratificado. O endoderma visceral apresentou numerosas vilosidades apicais principalmente em regiões próximas a placenta corioalantóidea. Com o contínuo desenvolvimento do embrião e placenta corioalantóidea, observou-se o surgimento de importante área de aposição entre os endodermas visceral e parietal. A inversão do saco vitelino representou uma disposição anatômica favorável ao desenvolvimento embrionário, além de ser uma característica evolutiva nesta espécie de roedor.

The aim of this study was to study the time of yolk sac inversion as well as the dynamics resulting from this process in galea throughout pregnancy. For this, conventional histological techniques, scanning electron microscopy and transmission electron microscopy were used. Parietal and visceral endoderm delimiting the yolk sac cavity was observed at 12 days of pregnancy. The parietal endoderm was coating the fetal surface of the chorioallantoic placenta as well as delimiting the decidua capsularis area. This endoderm had prismatic format and were apart from the trophoblast by an enlarged Reichert's membrane. The visceral endoderm had vitelline vessels and there were villi only in certain areas. At 14 days of pregnancy the yolk sac inversion was characterized by the degeneration of parietal endoderm and mural trophoblast, and also the gradual disappearance of the Reichert's membrane. So it made the visceral endoderm establish an interface with the uterine epithelium. After the inversion, the parietal endoderm which remained intact was the one that rested on the chorioallantoic placenta surface. It presented cells with high columnar format and pseudostratified epithelium featured. The visceral endoderm presented many apical villi, especially in areas close to the chorioallantoic placenta. The continued development of the embryo and chorioallantoic placenta evidenced the emergence of an important apposition area between visceral and parietal endoderm. The yolk sac inversion represented an anatomical arrangement in favor of the embryo development as well as an evolutionary trait in this rodent species.

Sonic hedgehog inhibition induces mouse embryonic stem cells to differentiate toward definitive endoderm.

In the experimental group (shh inhibited group), there were significant decreases in the expression of Oct4, Nanog, Shh, GATA4, Brachyury and Goosecoid, while increases were observed for TAT and Pdx1. The expression of Sox17 did not differ between two control and experimental groups. In experimental group, the amount of GSC positive cells was somehow lower but it seems that there was no difference for Sox17. Shh inhibition induces ESCs to differentiate toward definitive endoderm by committing mesendodermal lineages.

Primary rectal mature teratoma: a case report / 동물의과학연구지

This report describes a rare case of primary rectal mature teratoma in a 56-year-old woman. She was referred to the outpatient clinic with a large pedunculated rectal mass, which was found during a regular health check-up. Polypectomy was performed and microscopic findings showed various structures derived from all three germ cell layers. Epidermis, hair follicles, sebaceous glands, eccrine glands, and apocrine sweat glands, with some scattered melanophages and lymphocytes were present as ectodermal derivatives. Smooth muscle fibers, blood vessels, and fibrous and adipose tissues were found as mesodermal derivatives. In addition, thyroid follicles, mucinous glands, and bronchial respiratory epithelium with peribronchial glands were detected as endodermal derivatives. She is healthy and has shown no evidence of recurrence or distant metastasis for 25 months post-surgical resection. Primary rectal teratomas are generally benign and primarily affect women. Therefore, minimally invasive surgical procedures, such as endoscopic polypectomy for a pedunculated polyp and segmentectomy for a larger mass, are satisfactory in most cases. Induction of primary rectal teratomas has been suggested to occur mainly by errors in a single germ cell after the end of meiosis I; in addition, it has also been suggested that the difference in gender incidence may be associated with differences in sex chromosomes between males and females rather than with anatomical proximity between ovary and rectum.

Desarrollo del Aparato Digestivo / Digestive System Development

El aparato digestivo deriva del endodermo y el mesodermo, que forman su epitelio y la musculatura lisa respectivamente. Al igual que en el resto de los sistemas, existe un interacción epitelio-mesenquimática mediada por moléculas como Hedgehog, BMP y FoxF1 que determinan el crecimiento intestinal en sus ejes principales. Los genes Hox, junto con el resto de las moléculas, participan en la regionalización del sistema digestivo. En sus inicios lo denominaremos intestino primitivo, formado por un tubo endodérmico que deriva del saco vitelino; dividiéndose en intestino anterior, medio y posterior. En esta revisión veremos cómo estos 3 segmentos darán origen a las diferentes estructuras del sistema digestivo en los vertebrados.

The digestive system is derived from the endoderm and mesoderm, which form its epithelium and smooth muscle, respectively. As in the other systems, there is an epithelial-mesenchymal interactions mediated by molecules such as Hedgehog, BMP and FoxF1, determining intestinal growth in the main axes. The Hox genes, together the rest of the molecules, involved in the regionalization of the digestive system. In the beginning we call it primitive gut, consisting of a tube derived of endodermal yolk sac, divided into foregut, midgut and hindgut. In this review we will see how these 3 segments give rise to different structures of the digestive system in vertebrates.

A Case of Ectopic Thyroid Tissue Diagnosed by Fine Needle Aspiration in the Lateral Neck

Ectopic thyroid glands generally occur in the midline as a result of abnormal median migration and their presence in lateral to the midline is rare. Embryologically, the thyroid gland is derived from two anlages: a large median endodermal anlage and two lateral anlages. The median anlage produces most of the thyroid parenchyma, whereas the lateral anlage is derived from the fourth pharyngeal pouch and contributes 1-30% of the thyroid weight. In rare cases, failure of the lateral anlage to fuse with the median anlage can result in lateral ectopic thyroid gland. For many years, lateral, aberrant thyroid tissue in adults was a term used almost exclusively for metastatic thyroid carcinoma. However, aberrant, benign ectopic thyroid tissue rarely occurs. We present a 47-year-old man who had incidentally detected mass on the right lateral neck. He was clinically in a euthyroid status and the thyroid function test results were normal as well. Neck ultrasonography revealed a mild diffuse goiter and a 1.22 x 0.65 cm sized ovoid mass like lesion was located in the right level IV of the neck. The result of fine needle aspiration cytology was adenomatous goiter without lymphoid tissue or any malignancy. We rarely report aberrant, benign ectopic thyroid presence as a lateral neck mass.

A Case of Ectopic Sebaceous Glands in the Esophagus

Most ectopic sebaceous glands have been reported in the organs of ectodermal origin such as the lips, oral cavity, salivary glands, nipples, palms & soles, and genitals. Ectopic sebaceous glands in the esophagus are extremely rare conditions, because esophagus is an organ of endodermal origin. Whether the histogenesis of these lesions are embryological misplacement or acquired metaplasia remains unclear. We report a case of ectopic sebaceous glands in the esophagus diagnosed by endoscopic biopsy, with a brief review of the histogenesis. This case was followed up after 1 year. There were no significant changes, but the lesions had increased slightly in number compared with the last examination. When the number of lesions increase as in our case, acquired metaplasia is the most likely cause.

A Case of Ectopic Sebaceous Glands in the Esophagus

Most ectopic sebaceous glands have been reported in the organs of ectodermal origin such as the lips, oral cavity, salivary glands, nipples, palms & soles, and genitals. Ectopic sebaceous glands in the esophagus are extremely rare conditions, because esophagus is an organ of endodermal origin. Whether the histogenesis of these lesions are embryological misplacement or acquired metaplasia remains unclear. We report a case of ectopic sebaceous glands in the esophagus diagnosed by endoscopic biopsy, with a brief review of the histogenesis. This case was followed up after 1 year. There were no significant changes, but the lesions had increased slightly in number compared with the last examination. When the number of lesions increase as in our case, acquired metaplasia is the most likely cause.

A Case of Complete Agenesis of the Dorsal Pancreas with Left Renal Agenesis and Absence of the Left Vertebral Pedicle in T12 / 대한내과학회지

Agenesis of the dorsal pancreas is a rare congenital anomaly that arises from the failure of the dorsal pancreatic bud of endodermal cells to form the body and tail of the pancreas and can manifest as diabetes. A 24-year-old man, who had been treated with insulin for 7 years, presented with epigastric pain, vomiting, and watery diarrhea. Abdominal computed tomography showed only the head of the pancreas without visualization of the pancreatic body and tail. Left renal agenesis and absence of the left vertebral pedicle in T12 were also observed. The duct of Santorini and the duct in the body and tail were not visible in magnetic resonance cholangiopancreatography. The associated anomalies reported here are very rare globally. We report a case of complete agenesis of the dorsal pancreas with multiple congenital abnormalities and diabetes mellitus.

Heterotopic Gastrointestinal Cyst of the Tongue: Case Report

Therapeutic potential of adipose tissue-derived stem cells for liver failure according to the transplantation routes / 대한외과학회지

PURPOSE: Even though adipose tissue-derived stem cells (ADSCs) have been spotlighted as a possible alternative for liver transplantation in an experimental setting, the mechanism by which ADSCs improve liver dysfunction remains poorly characterized. The objective of this study was to evaluate the therapeutic ability of undifferentiated ADSCs, and find a few clues on how ADSCs alleviate liver damage by comparing the transplantation routes. METHODS: In vitro generated human ADSCs were checked for surface markers and stage-specific genes for characterization. Afterwards, they were transplanted into C57BL/6 mice with CCl4-induced liver injury. The transplantations were made via tail vein, portal vein, and direct liver parenchymal injection. At 1 and 3 post-transplantation days, serum biochemical parameters and/or liver specimens were evaluated. RESULTS: We have shown here that ADSCs have the characteristics of mesenchymal stem cells, and belong to endodermal and/or early hepatic differentiation stage. After transplantation into the mice with acute liver failure, markers of liver injury, such as alanineaminotransferase, aspartateaminotransferase, as well as ammonia, decreased. Of these transplantation routes, transplantation via tail vein rendered the most prominent reduction in the biochemical parameters. CONCLUSION: Undifferentiated ADSCs have the ability to improve hepatic function in mice with acute liver injury. Moreover, our transplantation route study supports the theory that ADSCs in systemic circulation can exert endocrine or paracrine effects to ameliorate the injured liver.

Reactive oxygen species enhance differentiation of human embryonic stem cells into mesendodermal lineage

Recently, reactive oxygen species (ROS) have been studied as a regulator of differentiation into specific cell types in embryonic stem cells (ESCs). However, ROS role in human ESCs (hESCs) is unknown because mouse ESCs have been used mainly for most studies. Herein we suggest that ROS generation may play a critical role in differentiation of hESCs; ROS enhances differentiation of hESCs into bi-potent mesendodermal cell lineage via ROS-involved signaling pathways. In ROS-inducing conditions, expression of pluripotency markers (Oct4, Tra 1-60, Nanog, and Sox2) of hESCs was decreased, while expression of mesodermal and endodermal markers was increased. Moreover, these differentiation events of hESCs in ROS-inducing conditions were decreased by free radical scavenger treatment. hESC-derived embryoid bodies (EBs) also showed similar differentiation patterns by ROS induction. In ROS-related signaling pathway, some of the MAPKs family members in hESCs were also affected by ROS induction. p38 MAPK and AKT (protein kinases B, PKB) were inactivated significantly by buthionine sulfoximine (BSO) treatment. JNK and ERK phosphorylation levels were increased at early time of BSO treatment but not at late time point. Moreover, MAPKs family-specific inhibitors could prevent the mesendodermal differentiation of hESCs by ROS induction. Our results demonstrate that stemness and differentiation of hESCs can be regulated by environmental factors such as ROS.

Cowden Syndrome Accompanied by Multiple Sclerotic Fibroma on the Face / 대한피부과학회지

Cowden syndrome (CS) is a rare genodermatosis that is characterized by multiple hamartomatous tumors of an ectodermal, mesodermal and endodermal origin. CS is associated with an increased risk of malignancy and especially breast cancer, thyroid cancer, uterine cancer and renal cell carcinoma. The characteristic features of the disease are mucocutaneous findings, including multiple facial trichilemmomas, oral mucosal papillomatosis and acral keratoses. Multiple sclerotic fibroma is also known to be a clue to diagnose CS. We herein emphasize that multiple sclerotic fibromas of the skin are an important marker of CS, which may also be helpful for making an early diagnosis.