Autoimmune thyroid diseases (AITDs), including Hashimoto’s thyroiditis (HT) and Graves’ disease (GD), are the most common cause of acquired thyroid disorder during childhood and adolescence. Our purpose was to assess the main features of AITDs when they occur in association with genetic syndromes. We conducted a systematic review of the literature, covering the last 20 years, through MEDLINE via PubMed and EMBASE databases, in order to identify studies focused on the relation between AITDs and genetic syndromes in children and adolescents. From the 1654 references initially identified, 90 articles were selected for our final evaluation. Turner syndrome, Down syndrome, Klinefelter syndrome, neurofibromatosis type 1, Noonan syndrome, 22q11.2 deletion syndrome, Prader–Willi syndrome, Williams syndrome and 18q deletion syndrome were evaluated. Our analysis confirmed that AITDs show peculiar phenotypic patterns when they occur in association with some genetic disorders, especially chromosomopathies. To improve clinical practice and healthcare in children and adolescents with genetic syndromes, an accurate screening and monitoring of thyroid function and autoimmunity should be performed. Furthermore, maintaining adequate thyroid hormone levels is important to avoid aggravating growth and cognitive deficits that are not infrequently present in the syndromes analyzed. Autoimmune thyroid diseases (AITDs) are the most common autoimmune diseases, affecting approximately 5% of the general population, with a female gender predominance . AlthougThe etiology of AITDs is complex and multifactorial, since environmental and genetic factors act in synergy to promote thyroid autoimmunity. Genetic factors are responsible for 80% of the risk of developing AITDs, while the remaining 20% is due to environmental factors  . EpidemiSeveral genes have been identified for AITDs: some genes are specific for GD or HT, others are common to both diseases or non-thyroidal autoimmune disorders . The majThe major genes related to AITDs include thyroid-specific genes and immune-regulatory genes:Thyroid-specific genes: Considering the pathogenesis of GD, the thyroid-stimulating hormone receptor (TSHR) gene, located on chromosome 14q31, was thought to be a possible candidate gene of disease. Genome-wide association studies (GWASs) confirm this theory [s theory ,8. Conses theory . Thyroglobulin (TG) accounts for 80% of thyroid protein content and represents an important target in AITDs. Linkage studies demonstrated an association between AITDs and a locus on chromosome 8q, containing the TG gene. Sequencing of the TG gene has shown several SNPs, strongly associated with AITDs, that caused amino acid changes in TG. Some studies have found a statistical interaction between TG gene variants and the HLA-DR variant, containing an arginine at position β74. HLA-DR is a class II HLA gene that plays a critical role in antigen presentation. Considering the statistical interaction between the two genes, some authors have hypothesized that the HLA-DR variant may present pathogenic TG peptides and trigger AITDs. However, the link between TG SNPs and the production of pathogenic TG peptides has not been established to date ,10,11,12Immunoregulatory genes: FOXP3 is a gene localized on the X chromosome and is involved in the control of regulatory T cells (Tregs). Mutations in FOXP3 cause a rare disease characterized by immune dysregulation, polyendocrinopathy and enteropathy (IPEX syndrome) [yndrome) . Severalyndrome) ,15. Other genes that seem to be associated with AITDs, in particular with GD, are CD25 and CD40 genes, located on chromosome 10p15 and 22q11, respectively . CD40, aCTLA-4 and PTPN22, located on chromosome 2q33 and 1p13, respectively, are negative regulators of T cell activation and polymorphisms of these genes are associated with AITDs ,19,20,21HLA-DR, in particular the DRb1-Arg74 HLA variant, is the main susceptibility gene for GD. Some reports suggest that HLA-DR can also predispose to HT, but the results are less conclusive ,23.AITDs are the most common thyroid disease in the pediatric population, they usually occur during puberty, are more prevalent in females and show familiar clustering ,25. SpecThe main features of GD presentation are related to hyperthyroidism, whereas HT at diagnosis shows variable degrees of thyroid function impairment. Euthyroidism is by far the most common initial pattern (about 52% of patients), followed by overt hypothyroidism (22.2%), subclinical hypothyroidism  and hyperthyroidism (about 6.5%) . ConversA systematic research was carried out, covering the last 20 years (from January 2000 to October 2020), according to the PRISMA statement , throughThe inclusion criteria were: articles written in English, belonging to the categories of clinical study, clinical trial, clinical trial protocol, multicenter study, randomized controlled trial and observational study, which report an association with AITDs  and confirmed genetic syndromes in a pediatric population (age < 18 years). Due to the rarity of GD and some genetic syndromes, case reports were also included.The exclusion criteria were: articles belonging to the categories of review, systematic review and meta-analysis or which included only a non-pediatric population (age ≥ 18 years) and/or did not evaluate AITDs or confirmed genetic syndromes.From our initial research, all the genetic syndromes most frequently associated with AITDs were subsequently reviewed individually, hence, we focused on: Turner syndrome, Klinefelter syndrome, Down syndrome, 18q deletion syndrome, Prader–Willi syndrome, 22q11.2 deletion syndrome, neurofibromatosis type 1, Noonan syndrome and Williams syndrome. Titles and abstracts of all retrieved articles were screened by one author (C.C.) to identify articles for full-text review. The eligibility of all full-text articles was assessed by all authors.The initial search resulted in 1654 references. Articles that did not report an association between AITDs and genetic syndromes were excluded on the basis of title/abstract (1147 articles). The remaining records were screened for inclusion criteria. After an additional, comprehensive analysis and the exclusion of duplicates, 114 potentially eligible studies were retrieved for full-text screening. For our final evaluation, we selected 90 articles ,121,122,Turner syndrome (TS) is caused by a partial or total monosomy of one X chromosome, with or without cell line mosaicism. It is one of the most common chromosomal abnormalities, affecting one in 2000–2500 live-born females. The TS phenotype includes short stature, gonadal dysgenesis, peculiar facial and skeletal dysmorphisms, lymphedema and renal and cardiovascular anomalies .TS girls are at higher risk of developing autoimmune diseases, and AITDs are the most frequent . Other aSeveral mechanisms have been proposed to explain the increased susceptibility to autoimmune diseases in TS, including haploinsufficiency of X chromosome-related genes. On the X chromosome, there are at least ten genes that are involved in immunoregulatory functions. In particular, haploinsufficiency of the FOXP3 gene, located in the Xp11.23 region, may contribute to increasing the susceptibility of TS girls to AITDs . The higAdditionally, alterations of both humoral and cellular immunity profiles and a lack of Treg immunosuppression have been suggested as possible mechanisms leading to autoimmunity in TS girls. Gawlik et al. evaluated the immunological profile of 37 TS subjects compared with non-TS controls and found lower levels of IgG, lower CD4+ lymphocytes and a significantly lower CD4:CD8 ratio in the TS group ; the samThe association between a specific TS karyotype and autoimmune disease is still controversial. According to some authors, patients with an X isochromosome are more prone to develop autoimmunity, especially AITDs ,100,122 The prevalence rate of AITDs in TS varies widely across studies, probably due to the great difference in age, number and geographical origin of the selected cohorts. However, the prevalence of AITDs in TS girls is higher than in the general population and increases with age ,37. The In TS girls, HT is the most common autoimmune disease. The study by El-Mansoury et al. reveals an annual incidence of HT in TS girls of about 3.2% . Aversa In addition to changes in function, thyroid structural alterations may also be associated with HT . CalcateSeveral reports described the association between GD and TS ,58,60,61To summarise, AITDs in TS, presenting with high prevalence rates, may not be associated with a positive family history of AITDs. With regard to HT, it may have a mild biochemical pattern at presentation, but a progressive deterioration of thyroid function over time and frequent conversion to GD.DS is the most common autosomal aneuploidy, affecting about 1 in 1000 live-born babies . In 95% DS represents the most important genetic cause of intellectual disability, which can be of varying grades, but is usually progressive over time . DS patients typically exhibit facial dysmorphisms, hypotonia, congenital heart defects, gastrointestinal abnormalities, increased risk of hematologic malignancies and immunological disorders. Endocrine disorders such as thyroid dysfunction, low bone mass, diabetes, short stature, infertility and propensity to be overweight/obese are much more prevalent than in the general population ,137. Above all, thyroid dysfunction is the commonest endocrinopathy in DS, ranging from hypothyroidism to hyperthyroidism, either overt or subclinical, of autoimmune or non-autoimmune etiology ,138. In Several mechanisms have been proposed to explain the increased susceptibility to autoimmunity in DS, which may include: (1) partial central tolerance failure due to altered thymic expression of the autoimmune regulator (AIRE) gene, which is located on chromosome 21and involved in immune regulation ,83; (2) AITDs are the most frequently observed autoimmune disorders in DS. The prevalence rates for HT and GD in DS children and adolescents DS are detailed in If compared to the general pediatric population, HT in DS occurs earlier (mean age at diagnosis: 6.5 years), shows no gender predominance, lower frequency of family history for thyroid diseases and higher prevalence of extra-thyroidal autoimmune disorders ,86,93. HT biochemical presentation in DS seems to be more severe, with a lower frequency of euthyroidism and increased prevalence of SH, which is the most common pattern at diagnosis, followed by overt hypothyroidism ,65 Figu.Additionally, the natural history of HT in DS is also unusual, displaying a more severe clinical course and higher rate of conversion to GD over time (about 25% of cases) ,111 Fig. Indeed, DS patients are exposed to a higher risk of thyroid function deterioration over time, which seems to be related to higher baseline TSH levels at diagnosis and to autoimmunity . NeverthIn DS, both HT and GD occur at a younger age than in the general population (between late childhood and early adolescence), without gender preference and frequently in association with other autoimmune disorders ,77,96,97The presenting features of GD at diagnosis seem not to differ in children with or without DS. Instead, the GD clinical course appears to be less severe in DS children and adolescents. According to some authors, GD in DS children shows lower relapse rates during the first methimazole therapy cycle, lower methimazole dosages required to maintain biochemical euthyroidism, higher remission rates after methimazole withdrawal and less need for non-pharmacological treatments ,77,99. However, this is not a consistent finding, since other authors observed a shorter duration of remission and higher relapse rates with medical treatment in DS patients, suggesting that radioactive iodine treatment may be the best option ,142. Finally, DS patients are more prone to fluctuations of thyroid function, between hypo- and hyperthyroidism, within the spectrum of thyroid autoimmunity . As mentGiven the high frequency of thyroid alterations, periodic screening and monitoring of thyroid function and autoimmunity over time are strongly recommended in DS children and adolescents.Klinefelter syndrome (KS) is the most frequent sex chromosome disorder in men, with the 47, XXY karyotype occurring in about 150 in 100,000 men .Various thyroid abnormalities, including a low response of serum TSH to a thyrotropin-releasing hormone (TRH) stimulation test ,144, impMoreover, there were some reports suggesting that males with KS might be at increased risk of certain autoimmune diseases ,146. The-Endocrine organ-specific autoantibodies can be detected in 13% of KS subjects; the frequency progressively increases in those with higher-grade aneuploidies and is higher in children than in adults.-The influence of sex hormones as modulators of the immune response and X-linked gene dosage has been implicated in the pathogenesis of autoimmunity in KS.-In KS patients, measurement of TSH and free T4 (fT4) levels at diagnosis and annually thereafter is suggested.-Screening for thyroid autoantibodies should be considered in KS cases with TSH elevation and/or the presence of goiter or periodically in the absence of suggestive clinical or biochemical signs.In conclusion, the following points regarding AITDs in KS should be highlighted:Williams syndrome (WS) is a multisystem disorder caused by a microdeletion of chromosome 7q11.23. The deletion is 1.5 to 1.8 Mb and contains 26–28 genes, including the ELN gene that code for “elastin”. WS is a rare disorder, with an estimated incidence of 1:7500 live births and affects boys and girls equally. Common clinical features of WS are facial dysmorphisms (elfin face), cognitive impairment with a friendly and social personality , cardiovascular disease , renal and genitourinary tract defects . Morphological and functional anomalies of the thyroid in WS have been reported by several authors, but association with AITDs is rare. Thyroid hypoplasia is common in WS (about 70% of cases), with the left lobe prevalently involved, and the prevalence increases with age. Additionally, thyroid ectopy and hemiagenesis are described. The mechanism underlying this altered development of the thyroid gland is unclear, and it has been hypothesised that the thyroid gland growth impairment may be linked to a defect of some gene mapped in the 7q11.23 region ,150. RecWith regard to thyroid function, SH is a frequent finding in young children with WS (about 30% of cases), while the prevalence rate of hypothyroidism is about 5–10%. Thyroid function impairment is commonly associated with thyroid hypoplasia, suggesting that the reduction of thyroid volume may be the cause ,152. SevAITDs are rarely associated with WS  Table 2Table 2. Although autoimmune manifestations are infrequent, given the possible association between thyroid disorders and WS, a comprehensive evaluation of thyroid morphology and function should be performed in these patients, especially in younger children.Deletion of chromosome 22q11.2 syndrome (22q11DS) is the most common microdeletion syndrome, with an incidence of about one out of 4000 live births. Phenotypic expression is highly variable and more than 100 different phenotypes have been described, including DiGeorge syndrome  . The maiIn 22q11DS, autoimmune diseases occur in about 8% of cases. The most common are juvenile idiopathic arthritis, autoimmune cytopenias, autoimmune skin disorders and AITDs . AdditioAnomalies in thyroid function and structure are frequently associated with 22q11DS. The most common thyroid structural anomalies include: hypoplasia, absence of one lobe, absence of the isthmus and a retroesophageal or retrocarotid extension ,161,162.Children with 22q11DS show a high prevalence of both hypo- and hyperthyroidism  . Choi et al. evaluated the prevalence of endocrine disfunctions in 61 patients with 22q11DS: the most common disease was hypocalcemia (32.8%), followed by overt hypoparathyroidism (13.1%) and short stature (16.4%); AITDs were recorded in 3.28% of patients (1.64% for HT and 1.64% for GD)  Table 2Table 2. Hypothyroidism seems to be related mainly to thyroid morphology anomalies, and more rarely to HT . Several studies described an association between 22q11DS and GD ,108,110.Kawame et al. reported a case of a 27-month-old 22q11DS female with tachycardia, irritability and seizures due to GD, suggesting that, in addition to the early onset, AITDs in 22q11DS may have an atypical presentation . AdditioConsidering these findings, evaluation of thyroid function, structure and autoimmunity may be considered for 22q11DS children and adolescents ,108,163.Deletions of chromosome 18q syndrome (18qDS) occurs in approximately 1:40,000 live births. The phenotype of the disorder varies greatly between individuals and is not correlated to the size of the deletion. The main clinical features are mental retardation, hypotonia, short stature, flat midface, ear anomalies, abnormal genitalia and foot deformities . Autoimmune diseases are described in association with 18qDS, including AITDs, DMT1, juvenile rheumatoid arthritis and pernicious anemia ,166,167.Both HT and GD are reported in 18qDS subjects and can occur at an early age. In fact, Tutunculer et al. described GD in a 1-year-old girl with the syndrome ,115,116.AITDs appear to be common in 18qDS, suggesting that thyroid function screening may be useful in the management of children and adolescents with the syndrome, but further studies are needed to assess the exact incidence of these disorders in this population ,168.Prader–Willi syndrome (PWS) is a rare genetic disorder resulting from the loss of gene expression within the paternal chromosome 15q11-q13. PWS has a prevalence rate of 1/10,000–30,000 and is characterized by endocrine abnormalities due to hypothalamic–pituitary insufficiency and complex physical, behavioral and intellectual difficulties. Individuals with PWS can present several different endocrine disorders, most of them caused by hypothalamic–pituitary insufficiency ,169. TheSimilar to other endocrinopathies, the etiology of hypothyroidism in PWS is thought to be central in origin. The abnormalities of thyroid function are discussed in the literature and published data are discordant. Hypothyroidism has been reported in approximately 20–30% of children with PWS and AITDs are very rare ,170 Tab. In the Neurofibromatosis type 1 (NF1) is an autosomal dominant tumor predisposition syndrome, caused by loss of function mutations of an anti-oncogene suppressor gene called neurofibromin 1 . ApproxiThe number of studies reporting an association between NF1 and autoimmune disorders is small but increasing. AITDs have been scarcely described in the literature . Nanda aNoonan syndrome (NS) is a multisystem genetic disorder, occurring in 1:1000 to 1:2500 live births. The causative mutations alter genes encoding proteins with roles in the RAS-MAPK pathway, leading to pathway dysregulation. The main clinical features of this syndrome are distinctive facial features, developmental delay, learning difficulties, short stature, congenital heart disease, renal anomalies, lymphatic malformations and bleeding difficulties .In the study of Quaio et al., comprising 42 patients with RASopathies, the majority of whom had NS, 17% had SH without thyroid antibodies, 7% were euthyroid with positive thyroid autoantibodies and another 7% presented overt autoimmune hypothyroidism. Six patients (14%) fulfilled the clinical criteria for autoimmune diseases, including systemic lupus erythematosus, CD, primary antiphospholipid syndrome, autoimmune hepatitis, vitiligo and AITDs  Table 2Table 2. Moreover, Lee et al. reported a case of an adolescent with NS, cardiac tamponade and HT with clinical and biochemical hypothyroidism, who underwent pericardial drainage . According to some authors, screening for AITDs seems to be unnecessary in patients with NF1 . Until aIn this section, we report an additional genetic disease, McCune–Albright syndrome (MAS). Despite not being included in the systematic review, it is important to mention this syndrome because it represents a relevant cause of hyperthyroidism in children.MAS is a rare congenital sporadic disorder , caused by a postzygotic somatic activating mutation in the GNAS gene encoding the G-protein α subunit (Gsα). MAS is defined by the triad of polyostotic fibrous dysplasia of bone, café-au-lait skin pigmentation and peripheral precocious puberty. Other multiple endocrinopathies—including hyperthyroidism, growth hormone excess, hypercortisolism and renal phosphate wasting—could be associated with the original triad . Thyroid disease is the second most common endocrinopathy in patients with MAS, with a reported prevalence of about 31% and onset ranging from 1 to 20 years of age ,177. As In conclusion, we must take into account that thyroid disorder with hyperthyroidism, also at a very young age, although not autoimmune, is the second most common endocrinopathy in MAS. For this reason, strict monitoring of thyroid function is recommended every six months in these patients.Our analysis confirms that AITDs show peculiar phenotypic patterns when they occur in association with some genetic disorders, especially chromosomopathies. To improve clinical practice and healthcare in children and adolescents with genetic syndromes, pediatricians and pediatric endocrinologists should be aware of the importance of the accurate screening and monitoring of thyroid function and autoimmunity in all conditions with an increased incidence of AITDs, such as TS, DS, KS and 22q11DS. Additionally, a careful assessment of thyroid function is recommended in children and adolescents with PWS, WS and MAS, due to a high frequency of hypo- or hyperthyroidism, although it is not autoimmune based. Furthermore, maintaining adequate thyroid hormone levels is important in order to not aggravate the growth and cognitive deficits that are not infrequently present in the syndromes analyzed.Alterations in various genes have been suggested to be responsible for the increased prevalence, the earlier onset or the atypical evolution over time of AITDs in some syndromes. Nevertheless, the underlying pathophysiological mechanisms have not yet been fully elucidated. New technological developments, such as next-generation sequencing technologies, can help identify the causative variants of autoimmune diseases and their association with genetic syndromes.


Among the various forms of colorectal carcinomas, primary signet ring cell carcinoma (SRCC) of rectum is infrequent. Primary SRCC with adenoma is even rarer. Due to its low morbidity and lack of obvious manifestations at early stages, it is difficult to make an early diagnosis and perform surgical intervention in time. Herein, we reported a case of primary SRCC with tubular adenoma of rectum and also performed a review of the literature of such cases, in hopes of expanding the general understanding regarding such cases.A 61-year-old male patient presented with rectal bleeding for 1 week.A neoplasm could be palpated through a rectal examination, with a size of 4.0 cm by 3.0 cm, at a distance of 5 cm from the anal edge. Magnetic resonance imaging examination and colonoscopies were performed to confirm the finding, and 4 tissue specimens were obtained for histopathologic biopsy. The result of biopsy was high-grade intraepithelial neoplasia with an adenoma component.Surgical resection was performed, and histopathologic and immunohistochemical staining examination of the resection confirmed the diagnosis of SRCC with tubular adenoma.The patient was discharged from hospital 12 days postsurgery, without any complications. Further chemotherapy and supportive treatments were suggested to him and will be followed at a local hospital.Primary rectal SRCC has a rather low morbidity. Furthermore, a rectal SRCC with adenoma which was presenting in this case is even more rare. Besides lack of clinical characters, delay of diagnosis and treatment frequently occur. So far, a surgical procedure has still been one of the most effective treatments. Considering of metastasis and the poor prognosis, early diagnosis, in-time radical resection, and a comprehensive followed treatment are recommended for a higher 5-year overall survival. The term of SRCC is a descriptive term, denoted by the histologic observation of cell nuclei being pushed to the periphery due to excess intracytoplasmic mucin.–5 SRCC was firstly reported in 1951 by Laufman and Saphir. The majority of SRCC cases are found in stomach, although some cases have also been reported in breast, lung, bladder, pancreas, and colon-rectum.Colorectal carcinoma (CRC) is one of the major causes of cancer mortality worldwide. As such, improvements that allow for more timely diagnosis and earlier surgical intervention may significantly improve the 5-year survival rates and overall outcomes.Since the clinical manifestations typically appear late, colorectal SRCC is frequently detected at an advanced stage. Furthermore, SRCC tends to be more aggressive than carcinomas of other histologic types, leading to poor prognosis for colorectal SRCC cases.However, because of lack of obvious clinical characteristics of rectal SRCC, and poor awareness by the doctors, misdiagnosis of such tumors may occur, and some patients may consequently miss the optimum opportunities for intervention. Here, we reported a case of rectal SRCC with rectal adenoma components and its clinical characteristics, and hope to raise appreciation for the possibility of such rarer cases during clinical diagnosis Fig. .2A written informed consent statement was obtained from the patient following approval from the Institutional Ethics Committee of the Second Hospital of Jilin University.A 61-year-old male patient presented with a complaint of rectal bleeding for more than 1 week, with no abdominal pain or any changes of bowel movement. He denied having any history of recent fever, chills, nausea, vomiting, or weight loss. The patient had a history of smoking for around 25 years, of 20 cigarettes per day. He denied any history of drinking, or other concomitant diseases. His father died from heart disease, and his mother died from stomach carcinoma.A physical examination showed us that his overall condition was relatively normal: blood pressure, 124/84 mm Hg; heart rate, 74 beats per minute; respiratory rate, 17 breaths per minute; oxygen saturation, 100% on room air; and body temperature, 36.6°C. Abdominal examination also had no significant abnormal findings. However, rectal examination (knee-chest position) found that a neoplasm with a smooth surface was located at 5 cm from anal edge. Its size was around 3.0 cm × 3.0 cm. The root of this neoplasm could be moved slightly by palpation. No dark red blood or pus remains were found on the glove when the rectal examination was finished.9 cells/L ; red blood cell count, 4.53 × 1012 cells/L ; platelet count, 208 × 109 cells/L . However, the patient did show slightly increased carcinoembryonic antigen (CEA) levels. A colonoscopy confirmed the presence of a tumor at about 5 cm from the anal edge, with a size of 4.0 cm × 3.0 cm, smooth surface, wide basement, and surrounded by congestive mucosa. Four tissue specimens were obtained from this tumor for pathologic biopsy. The result of this biopsy revealed that tubular adenoma cells were found, together with cells of high-grade intraepithelial neoplasia. A consequent nuclear magnetic resonance imaging (MRI) examination confirmed that tumor was in the rectal cavity, with the location as same as that described during colonoscopy, with no swollen lymph nodes being found around the rectum  for the rectal carcinoma. During the surgery, we found that tumor in the rectum at a distance of about 6 cm distance from anal edge, with no obvious evidence of invasion of the tumor toward the surrounding tissue. Laparoscopic total mesenteric excision was performed, and several enlarged suspected lymph nodes were found during surgery. No complications occurred during or postsurgery. That patient was discharged from hospital 12 days postsurgery.Subsequent pathologic examination of the resected surgical specimen supported the diagnosis of rectal SRCC with tubular adenoma. From the serial sectioning of the surgical specimen, the histology assessment showed scattered foci of signet ring carcinoma cells invading the lamina propria nearby the adenomatous lesion  Fig. . MeanwhiAfter his discharged from hospital, a 6-month follow-up was performed by telephone. He was then asked to perform a blood examination to monitor levels of CEA and CA-199. Both CEA and CA-199 fell in the normal range postsurgery. Neither a computed tomography scan performed 3 months postsurgery or an MRI performed 6 months postsurgery found evidence of potential recurrence. At the same time, the patient also underwent 6 cycles of XELOX chemotherapy. The patient did not report experiencing any serious adverse effects, such as neutropenia, neurotoxicity, or bone marrow suppression.3 Due to the low incidence, SRCC has only been evaluated sporadically in a limited numbers of cases. However, SRCC is almost always reported to be found at an advanced stage and with a poor prognosis, as compared with the more common forms of common adenocarcinoma (CAC) and mucinous adenocarcinoma (MAC).,5,9,10The SRCC is a rather rare subtype of CRC. According to previously published literature, tits incidence ranges from 0.1% to 0.9% among all forms of CRC.3.1; adenoma, typically reported as a combination of adenoma cells and signet ring cells, just as this case–15; atypical epithelium, which is associated with p53-positive intraepithelial signet ring cells; and a combination of these lesions.There has been considerable debate about the histologic origin of SRCC for decades. Four main theories have been proposed: CAC, due to the finding of transition areas between typical CAC and SRCC in many cases Meanwhile, an A:T transversion at the 3rd base position of K-ras codon 61 has also been reported. Additional biomarkers, such as Reg IV and claudin-18, have been reported to be more highly expressed in SRCC as compared to CAC, and both markers have been previously implicated to be involved in gastric cancer. HATH1, MUC2, and SOX2, identified as the key genes involved in controlling mucin secretion in the gastrointestinal tract, have also been reported to be more highly expressed in SRCC, consistent with histologic findings of excess mucin buildup.–20A distinct pattern of Kras mutations and lower Kras mutation frequency has been reported for colorectal SRCC cases as compared with that of CAC.3.2,10,21–24 To confirm this significance, we further analyzing the TNM staging data for cases of SRCC, CAC, and MAC from our hospital over the past 2 years , and larger studies may be necessary to clarify the true utility of surgical resection for such patients.Normally, as with the other subtypes of CRC, a timely surgical resection of colorectal SRCC has been considered to be the most effective treatment with the highest promise of overall survival.4Due to the low morbidity and lack of defining clinical characteristics, rectal SRCC is usually diagnosed at an advanced stage. However, SRCC may be considered as a possibility in cases of hematochezia of unknown cause. Since SRCC is typically more aggressive and has higher risks of metastasis, timely detection and surgical intervention, and comprehensive follow-up treatments are recommended to ensure better patient outcomes.The authors appreciate the contributions of all the surgeons, coworkers, and friends involved in this study and thank the editors and reviewers for their help with this manuscript.Yong-Ping Yang and Tong-Jun Liu conceived of this study, designed it. Yong-Ping Yang and Ling-Yun Yu drafted the manuscript. Yong-Ping Yang, Ling-Yun Yu, Jian Shi, and Jian-Nan Li participated in design of this study, acquired the data and analysis of data. Xin-Yu Wang acquired the data. Yong-Ping Yang and Tong-Jun Liu participated in manuscript preparation and critical revision. All the authors read and approved the manuscript.


The vital signs or laboratory test results of sepsis patients may change before clinical deterioration. This study examined the differences in prognostic performance when systemic inflammatory response syndrome (SIRS), Sequential Organ Failure Assessment (SOFA), quick SOFA (qSOFA) scores, National Early Warning Score (NEWS), and lactate levels were repeatedly measured. Scores were obtained at arrival to triage, 1 h after fluid resuscitation, 1 h after vasopressor prescription, and before leaving the emergency room (ER) in 165 patients with septic shock. The relationships between score changes and in-hospital mortality, mechanical ventilation, admission to the intensive care unit, and mortality within seven days were compared using areas under receiver operating characteristic curve (AUROCs). Scores measured before leaving the ER had the highest AUROCs across all variables . When combined, SIRS + lactate (0.882 [0.804–0.960]), qSOFA + lactate (0.872 [0.808–0.935]), NEWS + lactate (0.909 [0.855–0.963]), and SOFA + lactate (0.885 [0.832–0.939]) showed improved AUROCs. In patients with septic shock, scoring systems show better predictive performances at the timepoints reflecting changes in vital signs and laboratory test results than at the time of arrival, and combining them with lactate values increases their predictive powers. Sepsis accounts for about 30–50% of in-hospital deaths and often does not present with serious symptoms at the time of visit; therefore, 55.9% of cases involve normal blood pressure (BP) and lactate levels under 4 mmol/L . AlthougSepsis was previously identified and defined using the systemic inflammatory response syndrome (SIRS) criteria, though this definition was limited by its poor specificity ,8. SIRS Various score systems were used to predict sepsis patients’ prognosis, mainly using the maximum value, or the initial or last value, over a certain period of time. This study was conducted to find out how comparison of scores before and after use of fluid resuscitation and vasopressor, which are key to early treatment of sepsis patients, affects the accuracy of prognosis predictions.Medical records of adult patients aged 19 and older who visited the ER from January to June 2018 with confirmed sepsis based on bacterial culture  and a mean arterial pressure (MAP) of less than 60 mmHg were analyzed. Blood culture sampling was performed before antibiotic prescription, and sputum, urine, stool, and body fluid cultures were all carried out within 24 h of antibiotic prescription.This retrospective study was approved by the Inje University Haeundae Paik Hospital Institutional Review Boards .2O, urine output ≥0.5 mL/kg/h, and central venous oxygen saturation (ScvO2) of >70%; the items measured repeatedly at certain intervals were vital signs and laboratory test. If the MAP was <60 mmHg, two intravenous lines were secured and 30 mL/kg of fluid was rapidly injected; furthermore, an arterial line was secured and central venous catheterization and Foley catheterization were performed to monitor BP, CVP, and urine output. If the MAP had not recovered even after the initial fluid resuscitation, vasopressor injection was initiated with repeated fluid loading with 500 mL of crystalloid fluid. The time of vasopressor initiation depended on the decision of the emergency medical specialist. The amount of fluid loading in addition to the initial 30 mL/kg of crystalloid fluid was recorded.This emergency center is a secondary medical institution visited by 60,000 patients a year, with eight emergency medical specialists and four emergency medical residents working in shifts 24 h a day, and the treatment for septic shock patients is based on early goal-directed therapy. The goals were set as an MAP >65 mmHg, central venous pressure (CVP) of 8–12 cmHThe NEWS, qSOFA scores, and SIRS scores were obtained four times: at the time of visiting the ER, during fluid resuscitation, at least 1 h after starting vasopressor administration, and before leaving the ER. The results of laboratory tests were replaced with the closest results when they did not match the exact time. The SOFA score was obtained twice: the first laboratory test results within 24 h and the laboratory test results after 24 h; in patients who left the ER within 24 h, the earliest results from the tests conducted after hospitalization were used. The primary outcome was mortality within seven days, and the secondary outcomes were in-hospital mortality, M/V, and ICU admission.2 (pCO2), and bandemia were used when available to calculate the SIRS score, and pO2, bilirubin levels, and creatinine levels were used when available to calculate the SOFA score. A study physician calculated the NEWS and qSOFA, SIRS, and SOFA scores using the vital signs documented in the electronic medical records. Additionally, leukocyte counts, partial pressure of COThe qSOFA score is defined as a (1) systolic BP of ≤100 mmHg; (2) respiratory rate ≥22 breaths/min; and (3) altered mental status [The NEWS is an aggregate score that has been validated as a risk indicator of clinical deterioration and mortality. It includes heart rate, systolic BP, respiratory rate, arterial oxygen saturation, any supplemental oxygen, temperature, and mental status, with each item weighing 0–3 points.2 < 32 mmHg; and leukocyte count >12,000/mm3 or <4000/mm3 or the presence of >10% immature neutrophils. The SIRS criteria are fulfilled with a score of ≥2 points.In the SIRS score, 1 point is assigned to each of the following items: body temperature >38 °C or <36 °C; pulse rate >90 bpm; respiratory rate >20 breaths/min or pCO2/FiO2 ratio derived from arterial blood gas analysis, platelet counts, Glasgow Coma Scale score, bilirubin levels, MAP, and creatinine levels, with each item weighing 0–4 points.The SOFA score includes the PaOhttp://www.R-project.org/). p-values less than 0.05 were considered statistically significant.Descriptive statistics were used to quantify baseline characteristics and patient outcomes. Data are presented as means ± standard deviations (SD) or medians with interquartile ranges (IQR) for continuous variables and frequencies and percentages for categorical variables. The accuracy in predicting outcome measures was assessed by calculating the area under the receiver operating characteristic curve (AUROC). The optimal cutoff point was chosen as the one that maximizes the Youden index (sensitivity + specificity-1). All statistical analyses were carried out using SPSS 21.0 software  and R 3.4.4 , and the median Charlson comorbidity index was 6. The details of initial treatment were as follows: the amount of additional fluid loading within 3 h was 2000 mL, the time to antibiotic prescription was 1.5 h, and the time to vasopressor administration was 3 h. The median SIRS and qSOFA score was 2, the median NEWS was 9, the median SOFA score was 8; and the median lactate level was 2.8 mmol/L.During the study period, 18,574 patients visited the ER: 332 cases of undifferentiated shock, 167 were excluded, and finally, 165 patients were included . The basp = 0.661 and p = 0.342, respectively). The qSOFA and SOFA scores significantly increased by 0.099 and 0.329 over time, respectively . The NEWS significantly decreased by 0.928 over time (p < 0.001).There were no significant increases in the predictive value for mortality within seven days based on AUROCs when examining the maximum values, the differences between the values at ER triage and the maximum values, or the differences between the values at ER triage and the values on leaving the ER. Additionally, there were no increases in the predictive value, based on AUROCs, for in-hospital mortality, M/V, or ICU admission when examining the maximum values, the differences between the values at ER triage and the maximum values, or the differences between the values at ER triage and the values on leaving the ER .The combination of any severity score with lactate levels increased the AUROC at all four timepoints (two timepoints for SOFA) for mortality within seven days, and also increased the predictive value, based on AUROCs, for in-hospital mortality, M/V, or ICU admission .The SIRS score, qSOFA score, NEWS, and SOFA score were measured at triage at the ER, 1 h after fluid resuscitation, 1 h after vasopressor initiation, and before leaving the ER in all patients with septic shock. The lactate level was the best predictor of mortality within seven days, and all four severity scores showed higher AUROCs before leaving the ER than at triage. As an exception, the AUROC of the NEWS was 0.888 before leaving the ER, which was higher than that of the lactate level (0.872). For all scores, the maximum value or the change in values between timepoints did not have a higher AUROC than that before leaving the ER. In other words, the maximum value or change in values between timepoints did not show an improved performance in predicting mortality within seven days compared to individual values. However, when lactate was combined with the severity scores, the AUROCs increased at all four timepoints, with the highest AUROC observed before leaving the ER for NEWS + lactate (0.909), followed by SOFA + lactate (0.885), SIRS + lactate (0.882), and qSOFA + lactate (0.872).The highest AUROCs of severity scores for in-hospital mortality, M/V, and ICU admission were seen before leaving the ER, except for NEWS, which had the highest AUROC at the time of vasopressor initiation (0.822). The maximum AUROC for lactate levels was 0.800, which was higher than that at the time before leaving the ER (0.795). For all scores, there were no increases in the predictive value, based on AUROCs, for in-hospital mortality, M/V, or ICU admission when examining the maximum values, the differences between the values at ER triage and the maximum values, or the differences between the values at ER triage and the values on leaving the ER compared to individual values.When lactate levels were combined with the severity scores, the AUROCs increased at all four timepoints, and the highest AUROC at the time before leaving the ER was observed for qSOFA + lactate, followed by SOFA + lactate, NEWS + lactate, and SIRS + lactate.The target patients were prescribed both antibiotics and vasopressors, including fluid resuscitation. Therefore, rather than determining disposition within a short period of time, it is necessary to measure vital signs repeatedly over the course of treatment, observe the responses to treatment, and repeat laboratory tests. For predicting a poor prognosis, none of the scoring systems were particularly excellent, but performance was improved in all scoring systems on combination with lactate levels. As an ER tends to be overcrowded and lacking in personnel, triage inevitably proceeds with simple tools and preferably, a relatively easy method for the escalation of treatment or determination of disposition.ER clinicians use sepsis screening tools for broader purposes, such as for identifying critically ill patients, initiating critical and time-sensitive interventions, and determining the need for ICU care. Identifying patients with critical illness is paramount in the emergency care of patients with infectious diseases. Highly sensitive prediction tools will allow clinicians to identify nearly all critically ill patients early in their treatment course and will enable the provision of time-sensitive interventions such as fluid resuscitation, early antibiotic administration, and timely vasopressor administration. Highly specific predictive tools allow for more judicious disposition decisions and expenditure of resources, particularly ICU beds . TherefoWhile using a single scoring system or using a system in combination with laboratory test results, the worst value is usually used during ER stay; however, studies have shown that repeated measurements improve performance. Quinten et al. evaluated whether repeated vital sign measurements in the ER can be used to identify patients with sepsis or infection whose conditions will deteriorate within 72 h; the inclusion of repeated MAP measurements resulted in the highest AUC (0.800) . SchulteLevin et al. reported that dynamic vital signs categorized by the MEWS in the ER, specifically abnormalities that failed to normalize, were associated with increased mortality, a higher probability of ICU admission, longer length of hospital stay, and sepsis . ChangesThe limitations of this study are as follows. First, it was performed at a single medical institution with a small number of patients, and thus, it is not fully representative of all patients. Second, there was a potential for selection bias as this was a retrospective study. Third, although a protocol was created and implemented for shock management, the only parameter among the set goals that was repeatedly measured was MAP. As the studied patients had septic shock, there were no missing data on vital signs or lactate levels, except in two cases.NEWS plus lactate was the best for predicting mortality within seven days in sepsis patients admitted to the emergency room, and qSOFA plus lactate and SOFA plus lactate were good for predicting in-hospital mortality, application of mechanical ventilation, and admission to intensive care unit. As for the point of score measurement, the performance of the values measured at the time of leaving the emergency room after all of the initial treatments for sepsis, such as fluid resuscitation, vasopressor, and antibiotics, were performed.


The discrimination of early Alzheimer’s disease (AD) and its prodromal form  from cognitively healthy control (HC) subjects is crucial since the treatment is more effective in the first stages of the dementia. The aim of our study is to evaluate the usefulness of a methodology based on electroencephalography (EEG) to detect AD and MCI. EEG rhythms were recorded from 37 AD patients, 37 MCI subjects and 37 HC subjects. Artifact-free trials were analyzed by means of several spectral and nonlinear features: relative power in the conventional frequency bands, median frequency, individual alpha frequency, spectral entropy, Lempel–Ziv complexity, central tendency measure, sample entropy, fuzzy entropy, and auto-mutual information. Relevance and redundancy analyses were also conducted through the fast correlation-based filter (FCBF) to derive an optimal set of them. The selected features were used to train three different models aimed at classifying the trials: linear discriminant analysis (LDA), quadratic discriminant analysis (QDA) and multi-layer perceptron artificial neural network (MLP). Afterwards, each subject was automatically allocated in a particular group by applying a trial-based majority vote procedure. After feature extraction, the FCBF method selected the optimal set of features: individual alpha frequency, relative power at delta frequency band, and sample entropy. Using the aforementioned set of features, MLP showed the highest diagnostic performance in determining whether a subject is not healthy  and whether a subject does not suffer from AD . Our findings suggest that our methodology can help physicians to discriminate AD, MCI and HC. Dementia due to Alzheimer’s disease (AD) is a progressive neurodegenerative disorder associated with cognitive, behavioral and functional alterations. AD prevalence increases exponentially with age, from 1% in people between 60 and 64 years up to 38% in people over 85 years . In thisSeveral neuroimaging techniques have been used during the last decades with the aim of distinguishing AD and MCI patients from cognitively healthy control (HC) subjects: functional magnetic resonance imaging (fMRI), positron emission tomography (PET), magnetic resonance spectroscopy, electroencephalography (EEG), and magnetoencephalography (MEG), among others . PET andThe abnormalities that AD and MCI elicit in EEG activity have been traditionally analyzed using simple signal processing methods, such as spectral techniques ,14. SpecThe main objective of this study is to evaluate the diagnostic usefulness of an EEG-based methodology by means of different multiclass classifiers: logistic discriminant analysis (LDA), quadratic discriminant analysis (QDA) and multi-layer perceptron neural network (MLP). We hypothesize that the combination of spectral measures and nonlinear methods can be useful to help in AD and MCI diagnosis. For this reason, our proposed methodology is based on both frequency  and time domain (nonlinear features) analyses applied to EEG recordings. However, this exhaustive characterization of EEG may lead to obtaining redundant features sharing similar information. In order to avoid this issue, an automatic feature selection stage based on the fast correlation-based filter (FCBF) is followed . FinallyEEG data were recorded from 111 subjects: 37 AD patients, 37 MCI patients, and 37 elderly HC subjects. Patients with dementia or MCI due to AD were diagnosed according to the clinical National Institute on Aging and Alzheimer’s Association (NIA-AA) criteria, whereas HC were elderly subjects without a cognitive impairment and with no history of neurological or psychiatric disorder . InclusiAll participants and patients’ caregivers were informed about the research background and the study protocol. Moreover, all of them gave their written informed consent to be included in the study. The Ethics Committee at the Río Hortega University Hospital  endorsed the study protocol, according to The Code of Ethics of the World Medical Association (Declaration of Helsinki).®, Natus Medical, Pleasanton, CA, USA). Specifically, EEG activity was acquired from Fp1, Fp2, Fz, F3, F4, F7, F8, Cz, C3, C4, T3, T4, T5, T6, Pz, P3, P4, O1, and O2, at a sampling frequency of 200 Hz. Subjects were asked to stay in a relaxed state, awake, and with closed eyes during EEG acquisition. During the recording procedure, EEG traces were visually monitored in real time, and muscle activity was identified to avoid high-frequency noise. Additionally, independent component analysis (ICA) was performed to minimize the presence of oculographic, cardiographic, and myographic artifacts , with a value of SU = 1, indicating that, when knowing one feature, it is possible to completely predict the other, and a value of SU = 0 indicates that the two variables are independent. Then, a ranking of features is done based on their relevance since the higher the value of SU is, the more relevant the feature is.In the first step, a relevance analysis of the features is done. Thus, SU between each pair of features SU is sequentially estimated beginning from the first-ranked ones. If iX shares more information with jX than with the corresponding group Y, SU SU (with iX being more highly ranked than jX), the feature j is discarded due to redundancy and it is not considered in subsequent comparisons. The optimal features are those not discarded when the algorithm ends.The second step is a redundancy analysis used to discard redundant features. The described AD-MCI-HC diagnosis problem corresponds to a pattern classification task. Specifically, it can be modeled as a three-class classification problem. Bayesian decision theory establishes the rule to make such a decision to minimize the probability of misclassification . We haveK classes using linear hyperplanes as decision surfaces  (step = 0.005). Values of r <0.01 were not considered, since they led to a CTM value close to 0 for every subject, whereas values of r >0.5 were also discarded since they led to CTM values equal to 1 regardless the group. For both SampEn and FuzzyEn, m and r optimal values were obtained by evaluating all the combinations for m = 1, 2 and r ∈  (step = 0.05), where SD is the standard deviation of the time series [FuzzyEn, values of n = 1, 2, 3 were also evaluated to obtain its optimal value [r = 0.075 for CTM; m = 1 and r = 0.1·SD for SampEn; and m = 1, r = 0.1·SD, and n = 3 for FuzzyEn) for which the corresponding CTM, SampEn, and FuzzyEn values showed the lowest p-value  among the three groups. IAF and RP(d)) and a nonlinear one (SampEn).In order to select the optimal value of the different input parameters of each feature, only a training set was used. The optimal value for e series ,46. In tal value . We choshn and u. Both were optimized by cross-validation, leaving all trials for each subject out in every iteration. For each value of u between 0 and 100 (step = 5), we varied the number of neurons in the hidden layer from 1 to 20 (step = 1) in order to compute the k value. This procedure was carried out 30 times to minimize the effect of network random initialization. Then, the k values were averaged [k for trials) were u = 45 and 11 neurons in the hidden layer, as The MLP model was obtained according to the optimal values for averaged . The optk values of 0.3824 with LDA, 0.4118 with QDA and 0.4412 with MLP. These results show that MLP outperformed the discriminant analyses classifiers.Once the models were trained, their diagnostic ability was only evaluated using the test set. The overall accuracy of the models in the three-class classification task was 58.82% with LDA, 60.78% with QDA, and 62.75% with MLP. Additionally, we obtained Se, Sp, Acc, PPV and NPV for each method for HC vs. all and AD vs. all, derived from confusion matrices. MLP showed the highest diagnostic performance when determining whether a subject is not healthy . Furthermore, the network showed the highest diagnostic capability when determining whether a subject does not suffer from AD . LDA and QDA showed similar tendencies although reaching lower diagnostic performance than MLP, as RP values revealed that AD patients reached higher RP values in low frequency bands (q) and lower RP values in high frequency bands  than HC subjects. For the MCI group, a slight slowing of neural oscillations was found in comparison with HC. This increase of slow rhythms in spontaneous EEG activity was also observed by means of MF and IAF. Both spectral parameters were lower for AD patients than for MCI and HC subjects. These findings confirm the trend reported in previous studies: AD and MCI are accompanied by a progressive slow-down of EEG [SE results showed changes in the frequency distribution of the power spectrum. However, the physiological explanations for all of these alterations are not clear. The most extended hypothesis is that a significant cerebral cholinergic deficit underlies cognitive symptoms, as memory loss. A loss of cholinergic innervation of the neocortex might play a critical role in the EEG slowing associated with AD [Our spectral results suggested that AD and MCI elicit a slowing of spontaneous EEG activity. Further inspection of n of EEG ,25. Fina with AD . Analogo with AD .LZC, SampEn, FuzzyEn and higher AMI values for AD patients than for HC subjects. For these measures, MCI subjects showed intermediate values between AD and HC. Previous EEG studies also reported a loss of complexity and irregularity associated with early AD and MCI by means of nonlinear measures [CTM values were higher in AD patients and lower in HC subjects. This result suggests a decrease on variability in AD, as Abásolo et al. previously reported [Regarding the nonlinear parameters that quantify the complexity and irregularity of EEG recordings, our findings showed lower measures ,32,33,34reported . Taking reported . The decPrevious studies explored several EEG features for AD and MCI discrimination from HC, focusing on binary discrimination problems  ,18,19,20Se)—with a positive post-test probability of 84.85% (28 subjects rightly classified out of 33 subjects predicted as AD or MCI)—and only predicting two out of 17 AD patients as HC. In addition, the same model also showed the ability to discard AD in 27 out of the 34 subjects not suffering from it (79.41% Sp), including 15 out of the 17 HC (88.24%). These results highlight the clinical usefulness of our proposal, which might be expressed as a screening strategy similar to: If the MLP model predicts AD, recommend beginning a treatment since most probably  the patient suffers from AD or MCI.If the MLP model predicts HC, do not treat the patient, since most probably  he/she does not suffer from AD; consider a regular evaluation of the subject in the persistence of symptoms in order to minimize the number of AD and MCI missed subjects.If the MLP predicts MCI, conduct a regular evaluation of the patient since doubts arise about the cognitive status of the subject.In contrast to the above-mentioned studies, our MLP single model can be used not only for the three-class classification task but also in binary assessments of healthy vs. cognitively impaired subjects. As derived from Despite the fact that we showed the usefulness of our proposal, some limitations need to be addressed. Although we used a large data sample to train and validate the models , they were obtained from 111 subjects. Hence, analyzing more recordings from different subjects would enhance the generalization ability of our results. Moreover, taking into account the MCI heterogeneity, it would be useful to characterize different subtypes and conduct a longitudinal analysis to characterize subjects with stable MCI and those who progress to AD. Finally, only three classification approaches  have been used in this study. In future research works, the usefulness of other advanced classification methods, such as spiking neural networks and support vector machines, should be evaluated.To sum up, our results show that both AD and MCI elicit changes in the EEG background activity: a slowing of EEG rhythms, alterations in the frequency distribution of the power spectrum, a complexity loss, a regularity increase and a variability decrease. Our proposal has shown that spectral and nonlinear features allows us to characterize the brain abnormalities associated with AD and MCI. In addition, we have shown the high diagnostic ability of different three-class models trained with this EEG information, particularly when predicting AD and HC status. These results highlight the usefulness of our proposal in order to help physicians classify AD, MCI and HC from EEG data.


The expression of glypicans in different hair follicle (HF) compartments and their potential roles during hair shaft growth are still poorly understood. Heparan sulfate proteoglycan (HSPG) distribution in HFs is classically investigated by conventional histology, biochemical analysis, and immunohistochemistry. In this report, a novel approach is proposed to assess hair histology and HSPG distribution changes in HFs at different phases of the hair growth cycle using infrared spectral imaging (IRSI). The distribution of HSPGs in HFs was probed by IRSI using the absorption region relevant to sulfation as a spectral marker. The findings were supported by Western immunoblotting and immunohistochemistry assays focusing on the glypican-1 expression and distribution in HFs. This study demonstrates the capacity of IRSI to identify the different HF tissue structures and to highlight protein, proteoglycan (PG), glycosaminoglycan (GAG), and sulfated GAG distribution in these structures. The comparison between anagen, catagen, and telogen phases shows the qualitative and/or quantitative evolution of GAGs as supported by Western immunoblotting. Thus, IRSI can simultaneously reveal the location of proteins, PGs, GAGs, and sulfated GAGs in HFs in a reagent- and label-free manner. From a dermatological point of view, IRSI shows its potential as a promising technique to study alopecia. A hair follicle (HF) is a real mini-organ that makes hair growth possible. The hair shaft extends under the skin into the HF. Histology of a HF shows that it is organized into two compartments . The firA HF undergoes cyclic changes over the course of its life  allowingThe morphological changes in the HF observed during the hair cycle involve many cell types: keratinocytes, fibroblasts of the DP, endothelial, fat, and immune cells. The presence of these different cell types makes the study of the regulation of the hair cycle complex. It is also known that a fine regulation of growth factors involved in the hair shaft growth is essential for the passage between the different phases of the hair cycle . The mechanisms involved in the regulation of these growth factors are still poorly understood, but it is highly probable that heparan sulfate proteoglycans (HSPGs) are involved because of their capacity to sequester growth factors . HSPGs aPrevious studies have shown modifications in the distribution of various ECM HSPGs  or membr−1, also known as the fingerprint region, shown to be the most specific range for GAG studies [−1, centered at 1248 cm−1, that is specific for GAG sulfation [HSPG distribution, localization, and quantification in HFs are classically investigated by conventional histology, biochemical analysis, and immunohistochemistry. In this report, a novel approach based on infrared spectral imaging (IRSI) is proposed to assess, in the first part, the HSPG distribution in the HF, and in the second part, to compare HSPG variations in HFs at different phases of hair cycle. Fourier-transform infrared (FTIR) spectroscopy is a vibrational method based on the principle of interaction between mid-IR radiation and matter, which is used to analyze pure samples but also more complex systems such as cells, tissues, or biofluids. It is non-invasive, label- and chemical-free, very sensitive, and requires no specific preparation . The spe studies , and theulfation . Based on this knowledge, we propose in this original study to probe the distribution of HSPGs in HFs at different phases of the hair growth cycle by IRSI using sulfation as a spectral marker. IRSI might constitute a promising technique for early diagnosis and prevention in alopecia. The goal was to study HSPG, GAG, and sulfated GAG distribution and variation in HFs without any staining or labeling. It allows investigating these changes during the hair growth cycle. In parallel, our data were supported by Western immunoblotting and immunohistochemistry assays, more specifically by analyzing the GPC1 expression and distribution in HFs at different phases of the cycle. The workflow of the present study is illustrated in Human scalp biopsy samples were obtained from human donors during surgeries after obtaining informed consent under applicable ethics guidelines and regulations and were provided by Alphenyx .Two different methods were used for obtaining human HFs. The first one consists in maintaining the hair follicle in its phase to investigate the three major phases of hair growth cycle  and the second one permits inducing intermediate phases, in particular, to study intermediate anagen stages of the hair growth cycle. HFs were isolated from the human scalp according to Philpott’s method .For all our experiments, four donors were involved with two to three hair samples on the average per donor. For each hair sample, two to three sections were obtained. More precisely, for the first method, 32 anagen A1, 25 anagen A3, 26 catagen C1, 28 catagen C2, 31 telogen T1, and 41 telogen T3 HF sections were analyzed. For the second method, 9 early anagen D0, 18 intermediate anagen D3, and 11 catagen D6 HF sections were analyzed.In the first approach, the HFs were isolated from the scalp at different hair growth cycle phases  and maintained in culture in the William’s E medium  supplemented with 0.5% antibiotics, 2 mM L-glutamine , 10 ng/mL hydrocortisone , 10 µg/mL transferrin , and 10 ng/mL selenite  for one day  and for three days  before analysis. In the second approach, the different phases of the hair cycle  were induced in culture. To do so, early anagen HFs were isolated and selected from the scalp; one part was directly analyzed , while the other part was maintained in culture for three days  and for six days  in the cell culture medium described above, supplemented with 0.5 µg/mL insulin .® O.C.T.™ Compound  and snap-frozen at –80 °C. Seven micron-thick longitudinal sections of HFs were prepared using a cryostat . The sections were then placed on an IR-transparent calcium fluoride (CaF2) window  for IRSI analysis (2 to 4 sections per window). The sections were imaged directly without any staining. For the different approaches described above, HFs were embedded individually in cryoprotective Tissue-TekThe same process described for HF preparation for infrared analysis was used to prepare sections. Sections were placed on glass slides and air-dried; then, they were fixed in acetone for 10 min at –20 °C. After three washes in PBS, the sections were placed in a sheep serum solution . Primary antibody anti-GPC1  was incubated overnight at 4 °C. After several washes with PBS, the secondary antibody coupled with Alexa 488 was applied for 45 min at room temperature and in the dark. The Evans blue counterstain was applied after several washes for 5 min at room temperature. After the final washes, the glass slides were mounted under a coverslip using Fluoprep. The observations were performed using a confocal microscope .−1, spectral resolution of 4 cm−1, and at 16 scans/pixel. IRSI acquisition was performed on whole hair follicles. Prior to this, for each section, a background spectrum was measured using 90 scans in a region of the CaF2 window without sample or optimal cutting temperature (OCT) compound and was automatically removed from each pixel spectrum of the image. A total of four spectral images were also acquired on the OCT compound using the same experimental conditions. All acquisitions were performed using the Spectrum Image 1.7.1 software . All IR images underwent atmospheric correction using the same software. This step reduces the interferences due to molecules present in the sample environment such as carbon dioxide or water vapor.All sections of HFs were imaged in the transmission mode using the Spotlight 400 infrared imaging system . The acquisition parameters were as follows: spatial resolution with a projected pixel size of 6.25 μm × 6.25 µm, spectral range from 4000 to 900 cmPixel spectra were extracted from specific regions of interest of the IR images, smoothed , baseline-corrected (elastic), vector-normalized, and offset-corrected using the OPUS 5.5 software . −1.For spectral processing, principal component analysis (PCA), an unsupervised exploratory method, was used. This method allowed spectral data reduction, replacing original and correlated variables with synthetic and uncorrelated variables called principal components (PCs). These PCs contain the information of interest and are ranked from the highest to lowest variance in the dataset. In this study, PCA was performed on mean-centered spectra to remove redundant information and using the spectral ranges 1800–900 or 1350–1190 cm−1 spectral region using a developed in-house routine in Matlab .All images were preprocessed with the extended multiplicative scatter correction (EMSC) algorithm which included correction of the baseline, variations related to the difference in sample thickness, and vector normalization. Indeed, this step allowed removing the outliers and artifacts that can influence spectral image analysis. All spectral images were preprocessed in the 1800 to 900 cmk-means clustering algorithm, which is one of the most popular unsupervised classification methods [k-means algorithm thus converges to a local minimum. Finally, each class is represented by a color and the cluster image is reconstituted as a false-color map. In order to compare different HF section images, a common k-means was applied using 10 classes in the 1800–800 cm−1 spectral range.Spectral image analysis was performed using the  methods . It aims2 window, dried, and imaged in the transmission mode using a Spotlight 400 infrared imaging system. For each image, the spectra were averaged to obtain the mean representative spectrum of each standard. Each standard spectrum was then in turn correlated pixel by pixel with HF images corrected for atmospheric contribution using the Spectrum Image 1.7.1 software. The resulting correlation maps show the distribution of the standards with a correlation scale ranging from 0 (dark color) to 1 (white color), corresponding to low and high levels of correlation, respectively.The spectral images of HF sections obtained previously were processed to produce correlation maps corresponding to heparan sulfate (HS), GPC1, and hyaluronic acid (HA) distribution. To do so, solutions of standard GAGs  and recombinant human GPC1  were deposited on a CaFTM  six times at 6.0 m/s for 40 s. The obtained lysates were incubated 20 min on ice with vortex-mixing every 5 min. Cell debris were precipitated by centrifugation at 10,000 g for 10 min at 4 °C. Proteins in the supernatant were collected and assayed using the Bradford  technique [HF proteins were extracted from a pool of five HFs and prepared in a radioimmunoprecipitation assay buffer (RIPA) buffer supplemented with 1% protease inhibitor cocktail added (Sigma-Aldrich) using the FastPrep 24echnique .Samples were deposited onto polyacrylamide gels as previously described . The GPCThe present report describes the evolution of GAGs, sulfated GAGs, and, more specifically, the HS-type GAGs and GPC1 distribution in hair follicles at different phases of the hair growth cycle using spectral imaging. IRSI presents the advantage of being label-free, reagent-free, and rapid. The data obtained by this novel approach were compared to the immunohistochemistry and Western immunoblotting data. Anagen, catagen, and telogen HFs were individually isolated and maintained in culture for one day  or three days  before analysis. The white light image in −1 spectral range  succeeded to precisely discriminate the germinative matrix structure (group 1) from the four other groups and, in particular, from the differentiation zone (group 2), in contrast with the overlap observed in −1  spectral range . This zone was chosen because it is rich in HS see . PCA wasange  were analyzed by spectral imaging and Western immunoblotting. Before analysis, the intermediate anagen (D3) and catagen (D6) HFs were maintained in culture for three and six days, respectively. As previously, spectra used for PCA analysis were extracted from the ORS region.−1  changed with the phases. In particular, cleaved and glycanated forms exhibited a noticeable difference observed between D0, D3, and D6 HFs B. IndeedAccording to the different HF phases, differential glycation of GPC1 could be demonstrated by Western immunoblotting. These observed differences may explain the PCA grouping of HF spectra with regard to the different phases based on sulfated GAG absorption.k-means clustering, and correlation mapping, could identify the histological structures and the different phases of the hair growth cycle. In addition, these approaches corroborate immunohistochemical and biochemical analyses of the present report and from the literature.Altogether, the IR spectral images processed by different chemometric approaches such as PCA, This report demonstrates the capacity of IRSI to identify the different tissue structures of the HFs and to show protein, PG, GAG, and sulfated GAG distribution in these structures. In addition, the comparison between the anagen, catagen, telogen phases of the hair growth cycle shows the qualitative and/or quantitative evolution of GAGs as supported by Western immunoblotting. Thus, IRSI can reveal some information on the location of proteins, PGs, GAGs and sulfated GAGs in HFs in a reagent- and label-free manner. In the long term, from a dermatological point of view, IRSI might constitute a promising technique for the early diagnosis and prevention in alopecia.


Papillary thyroid cancer (PTC) patients are staged according to the Tumor-Node-Metastasis staging system (TNM). This work was aimed at comparing the usefulness of the 8th edition of TNM (TNM-8), currently used, and that of the previous one (TNM-7) for predicting disease-free interval (DFI) in a cohort of 1148 patients. Moreover, clinicopathological and molecular factors were statistically evaluated in order to determine which of these was/were the best predictor(s) of DFI. Results obtained from the multivariate analysis indicated that advanced tumor stages were independent risk factors for a lower DFI regardless of TNM, but the statistical model created with the TNM-7 was most accurate. When stage-determining factors were included individually in the multivariate analysis, LN metastases, tall cell variant, and age emerged as independent risk factors for a shorter DFI, with lateral LN metastases being the most relevant. No molecular parameters could improve the prediction of DFI provided by LN metastases. The American Joint Committee on Cancer has revised the Tumor-Node-Metastasis (TNM) staging system for papillary thyroid cancer (PTC) patients. We examined the impact of this new classification (TNM-8) on patient stratification and estimated the prognostic value of clinicopathological features for the disease-free interval (DFI) in a cohort of 1148 PTC patients. Kaplan–Meier analyses showed that all clinicopathological parameters analyzed, except age and multifocality, were associated significantly with DFI. Cox regression identified tall cell PTC variant and stage as independent risk factors for DFI. When the stage was replaced with age, tumor size, and lymph node (LN) metastases in the set of covariates, the lateral LN metastases stood out as the strongest independent predictor of DFI, followed by tall cell variant and age. A noteworthy result emerging from these analyzes is that regression models had lower Akaike and Bayesian information criterions if variables were categorized based on the TNM-7. In addition, we examined data from a different PTC patient cohort, acquired from The Cancer Genome Atlas database, to verify whether the DFI prediction could be enhanced by further clinicopathological and molecular parameters. However, none of these was found to be a significant predictor of DFI in the Cox model.  Accurate staging of patients affected by differentiated thyroid cancer (DTC) is of crucial importance to ensure the appropriate therapeutic strategy and follow-up, and to ensure the patients’ quality of life . To dateWe first evaluated the impact of the new TNM-8 on patients’ risk stratification compared to the previous TNM-7. In this regard, 1113 patients for whom all clinical data required were available, were staged according to both TNM systems. Since in the TNM-8 the patients’ age cutoff has been shifted from 45 to 55 years, the percentage of younger patients (age < 55 years) in our case study increased from 46.4% (516/1113) in TNM-7 to 68.6% (764/1113) in TNM-8. Due to the lack of distant metastases, all these patients were classified as stage I regardless of TNM edition. As expected, among older patients (age > 55 years) we observed a considerable reduction in the relative frequencies of individuals at stage III and IV, with a concomitant increase in those at stage I and II, moving from TNM-7 to TNM-8 . As noteUnivariate analysis was performed to evaluate the association of several clinicopathological parameters, including age at diagnosis, gender, autoimmune thyroid disease (AITD), tumor histology, size (T), lymph node metastases (N), stage, multifocality, capsular, muscle, and vascular invasion, with PTC recurrences. Since some categories were poorly represented, especially among tumor sizes and stages, they were combined with other categories to avoid the inclusion of too small groups in the statistics see . As showThese observations were confirmed by the Kaplan–Meier analysis, reported in We finally created Cox regression models to predict the probability of DFI as a function of different sets of independent variables. Clinical parameters were categorized as in the univariate analysis see . The firp < 0.001). Although in the univariate analysis dichotomous age was not significantly associated with recurrences or DFI, in Cox regression it became a significant predictor both applying the 55-year and 45-year threshold. When the hazard function was generated with T and age categorized according to the TNM-7 edition, the sclerosing and tall cell variants also displayed significant hazard ratios. Moreover, this model had lower AIC and BIC than that resulting from categories based on TNM-8, similarly to what was observed for the Cox regression including stage.We then replaced stage with stage-related parameters, so that the new set of covariates comprised lymph node metastases, tumor size, dichotomized age at diagnosis, gender, histological variants, AITD, multifocality, muscle and vascular invasion see . Capsulap = 0.014), differentiation score (p = 0.045), and lymph node metastases (p = 0.005) associated significantly with recurrences. Nonetheless, adding all the above variables with p < 0.25 in the univariate analysis to lymph node metastases and age in the Cox regression did not improve significantly the prediction of DFI. Considering a subgroup of patients (n = 310) for which lymph node metastases were categorized as N0, N1a, and N1b in the database, N1b displayed the highest hazard ratio and significance, similar to what emerged from the analysis of our patient cohort (p < 0.05) and N1b had a HR of 14.46 .After that, we sought to verify if the DFI prediction could be strengthened by combining lymph node metastases and age with further histological and molecular parameters that were not available for our patients. These data were acquired from a previous study by the Cancer Genome Atlas Research Network for Cancer Genomics ,11,12. St cohort . In partFollicular thyroid cell-derived tumors are the most common endocrine malignancy and the fifth most common cancer in women ,14,15. IIn the present study, as expected, the comparison of the two TNM systems in 1113 PTC patients evidenced a considerable patient downstaging by TNM-8, in agreement with a number of earlier reports ,26,27,28In the framework of The Cancer Genome Atlas (TCGA) project, a comprehensive multiplatform analysis was carried out to determine the genomic landscape of 496 PTC cases, and a reclassification of PTC into molecular subtypes was proposed to improve clinicopathological grading and management of patients . In thisWhen in our regression analysis the TNM stage was replaced by stage-related parameters analyzed as independent variables, lymph node metastasis and age dichotomized in <55 years and ≥55 years emerged as the only significant predictors of DFI. Taking advantage of a PTC cohort of patients derived from the TCGA, we sought to verify whether, besides lymph node metastasis and age, the DFI prediction could be improved by additional histological and molecular parameters not available for our case study. The results obtained demonstrated that only a couple of them (BRAF-RAF score and differentiation score) were significantly associated with recurrences, and none of the parameters selected for the Cox regression impacted significantly on the model. Additionally, in this PTC cohort, lateral lymph node involvement represented the best predictor of DFI, with the N1b category  having the highest HR.131I whole-body scan, or histological analysis following surgical resection of the lesion. The same was applied to AITD patients with positive Tg auto-antibodies with the exclusion of Tg measurements. During the follow-up, we observed 11 persistence  and 55 recurrences, including 54 lymph node metastases and 1 lung metastasis. In addition, we used data previously obtained from a study that described the genomic landscape of 496 PTC, with 396 of which follow-up was available [In this study, we retrospectively analyzed 1148 patients affected by PTC enrolled in the period 1995–2018 at the Umberto I hospital, Department of Surgical Sciences of the Sapienza University of Rome. The case study included 883 females and 265 males with a median age of 47 years range 12–88 years). All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of the Umberto I hospital (Protocol No. 2615). Of the 1148 patients, 1127 patients had a total thyroidectomy, while 21 patients had a lobectomy. Post-surgical RAI was performed on 565 patients, but not on 583 patients including 486 pT1a cases and 97 pT1b cases. Thyroid hormone replacement was applied following international guidelines recommendations ,32. For –88 yearsp-value < 0.25 in the univariate analysis, together with those of known clinical significance. Proportional hazards assumption and absence of multi-collinearity were preliminarily assessed for these covariates. The backward stepwise approach was used for the model selection. Data analyses were done by using the SPSS software for Windows , considering the probability value <0.05 as the threshold limit for statistical significance.To avoid bias in the univariate and multivariate analyses, variable categories containing a small number of events, whose statistical results were unreliable, were merged with larger categories. Thus, in accordance with TNM-7, we divided tumor sizes (T) in T1 + T2 and T3 + T4, and tumor stages (S) in S1 + S2 and S3 + S4. The tumor sizes as defined by the TNM-8 were classified in T1 + T2 + T3a and T3b + T4, and the tumor stages in S1 and S2 + S3 + S4. Univariate analyses were executed for each clinical parameter in relation to the presence/absence of recurrence. Statistical comparison between recurrent and non-recurrent patients was carried out with the Chi-square test or the Fisher exact test for categorical variables, and with the Mann–Whitney test for continuous variables . Where a significance was found in the Chi-square test, the Cramer’s V was used as a post-test to determine the strength of association between variables. Kaplan–Meier curves were created to estimate the DFI in patient groups, and differences were statistically evaluated by the log-rank test. Finally, Cox regression was performed to quantify the hazard ratios of several explanatory variables, both continuous and categorical. The selection of covariates was made by including all parameters with a In conclusion, our study evidenced that lymph node metastases display a predictive value for DFI of PTC patients far higher than other clinical, histological, and molecular parameters currently available. Hence, it would be desirable to try to further improve the prediction of the DFI by employing more in-depth analysis of lymph node characteristics, through both instrumental and molecular investigations.


Transcranial direct current stimulation (tDCS) has strong potential for outpatient clinical use, but feasibility and safety of tDCS has only been evaluated in laboratory and inpatient clinical settings. The objective of this study was to assess feasibility and safety of tDCS for stroke in an outpatient clinical setting. Individuals with stroke in outpatient therapy received tDCS during physical therapy sessions. Feasibility was assessed with screening, enrollment, withdrawal, and adherence numbers, tDCS impressions, and perceived benefits and detriments of tDCS. Acute changes in fatigue and self-reported function and pre-post changes in fatigue were also assessed. Safety was assessed as adverse events and side effects. In total, 85 individuals were screened, and 10 were enrolled. Most exclusions were unrelated to clinical feasibility. In total, 3 participants withdrew, so 7 participants completed 2 sessions/week for 5–6 weeks with 100% adherence. In total, 71% reported positive impressions of tDCS. tDCS setup decreased to 5–7 min at end of study. There was one adverse event unrelated to tDCS. Mild to moderate side effects  were experienced. In total, 86% of participants recounted benefits of tDCS. There were acute improvements in function and energy. Results support the feasibility and safety of tDCS in an outpatient clinical setting. Stroke affects ~800,000 people each year in the United States and is a major cause of long-term disability ,2. IndivTo address hemispheric imbalance and promote recovery, recent investigations have used neuromodulation in combination with motor training to increase the excitability of the lesioned hemisphere and/or decrease the excitability of the non-lesioned hemisphere. One of the most commonly used modalities is transcranial direct current stimulation (tDCS), which involves the application of non-invasive low intensity direct currents to the scalp, altering the membrane potentials that affect neuronal excitability in the underlying cortical tissue . Both tDtDCS also appears to be safe, easy to use, and cost-effective. A recent review of data from 18,000 research sessions found no evidence of serious adverse events and found that tDCS does not cause neuronal damage at levels used in research in a variety of populations . There iIn a research setting, tDCS appears to be efficacious and safe. Considering that tDCS is also tolerable, easy to use, portable, and cost-effective, it has strong potential for use in a clinical setting . SurprisAll participants were recruited from the patient pool at the Shirley Ryan AbilityLab Homewood DayRehab Center, Homewood, IL, USA. Participants were enrolled if they were 18–80 years old, had a clinical history of single unilateral stroke and were scheduled to receive at least 6 weeks of gait training after the commencement of the study. Participants were excluded if they had contraindications to tDCS, including: history of epilepsy or seizures, use of a pacemaker, skin hypersensitivity, contact dermatitis, allodynia and/or hyperalgesia, or any other skin or scalp condition that could potentially be aggravated by tDCS. Participants were also excluded if they were pregnant, had neurological conditions besides stroke, or were unable to perform at least 10 min of walking on a treadmill or stepping on a recumbent stepper. This study was conducted in accordance with the Declaration of Helsinki, the protocol was approved by the Northwestern University IRB (STU00210329), and all participants provided written informed consent. Information such as date, location, and type of stroke, tests of physical function, and activities performed during rehabilitative treatment were extracted from patient records. Deidentified data that underlie study results will be shared by the corresponding author upon reasonable request from qualified investigators.All participants received ongoing rehabilitative treatment at the Homewood DayRehab Center , an outpatient neurorehabilitation center that provides therapy services to ~80 patients per day. Typically, patients undergo treatment 2–5 days per week for 3 h  or 6 h (full day). Each day is split up into 55-min sessions, and each session focuses on one discipline: physical therapy, occupational therapy, or speech therapy. The specific daily and weekly schedule is individualized for each patient.Prior to the start of the study, physical therapist clinicians received ~5 h of instruction, training, and practice in the application of tDCS from research scientists with extensive experience in the use of tDCS. Training included a didactic lecture on the scientific premise and physiological basis of tDCS and hands-on activities to ensure proper landmark identification, electrode application, current administration, and troubleshooting. Training was provided to 5 clinicians, of whom 3 were ultimately involved in study procedures. The other 2 clinicians relocated or had other professional obligations.2, and the total charge was 0.036 C/cm2, which were within safety limits [tDCS was performed twice per week during a physical therapy session that focused on gait training. The application of tDCS was incorporated into the standard time for the treatment session (55 min) and applied during the ~15-min warmup exercise performed at each participant’s comfortable intensity and personalized based on each patient’s baseline self-selected gait speed. Warmup exercise was performed on a recumbent stepper (gait speed ≤0.4 m/s) or a treadmill (gait speed >0.4 m/s). To prepare for tDCS, the vertex was identified as the intersection of the midline from the nasion to the inion and the midline from tragus to tragus. The vertex was marked with indelible marker. The leg representation of the ipsilesional motor cortex was approximated as 1 cm posterior and 1 cm lateral from the vertex and marked with indelible marker . Saline-y limits . tDCS duy limits ,29. tDCSy limits .The Chattanooga Ionto was selected for this study because it allows easy application of tDCS and is low-cost, portable, and adaptable. Users can apply between 0.5 and 4 mA of constant current (regardless of skin impedance) with an automatic 30-s ramp up and down to two different sites simultaneously. The device is available for USD ~500 and is pocket-sized . Users can interface with the device with a variety of reusable or single-use electrode types.After warmup, tDCS equipment was turned off and removed, and high-intensity treadmill walking or overground gait training were performed. For high-intensity exercise, intensity was constantly modulated with the goal of obtaining a Rating of Perceived Exertion  betweenThe number of patients screened and enrolled for the study was recorded, along with reasons for enrollment refusal and study withdrawal. For each participant, the number of sessions and weeks completed and adherence to the use of tDCS was recorded. To assess acute (within-session) changes in fatigue and function, participants completed the Rating-of-Fatigue Scale   and the The number of adverse effects associated with the application of tDCS was actively monitored during the study. In addition, any participant who reported intolerable discomfort or pain related to the application of tDCS or any other study procedures was planned to be removed from the study. Withdrawals and adverse effects were recorded. After completing the last session involving tDCS, participants completed a survey assessing tDCS-related side effects , largelyp-value of 0.05 was accepted as significant.Related-samples Wilcoxon signed-rank tests were performed to compare ROF, PSFS, and VAS-F measures from the start to end of the tDCS session  and from pre to post study (VAS-F). Statistical analyses were performed with IBM SPSS Statistics 22. A In total, 85 individuals were screened between November 2019 to February 2020 for participation in this study after referral to the clinic with a diagnosis of stroke and/or recommendation for study consideration by their treating therapist. Most of these (69%) failed screening because they had history of multiple strokes, another neurological condition, or were greater than 80 years old. Screening identified 10 eligible individuals with stroke who were recruited, provided informed consent, and started the study. Of these, 3 participants withdrew from the study after enrollment because: (1) decreased mood that limited accurate feedback and engagement, (2) aphasia prevented accurate feedback to clinicians, and (3) other undisclosed personal reasons. Thus, data from 7 participants who received tDCS were included in the final analyses. A flow chart is shown in p = 0.02) and average PSFS  increased from the start to the end of each exercise session (p = 0.04). There was no change in VAS-F fatigue or total rating (p ≥ 0.61).Participants completed 10–12 sessions distributed as 2 sessions/week for 5–6 weeks. Attendance and transcranial direct current stimulation (tDCS) application rate for scheduled sessions were 100%. In two participants, sessions were completed for 5 instead of 6 weeks. One participant was removed from the study because of a hypoglycemic event (see Safety section below); the study was discontinued in another participant because of the severe acute respiratory syndrome coronavirus 2  pandemic. Across all participants and sessions, mean (standard deviation) warmup duration was 15.1 (0.2) min and high intensity training duration was 18.2 (1.7) min. Warmup walking/stepping speed was 0.35 (0.22) m/s, and high intensity training speed was 0.55 (0.25) m/s. Warmup RPE was 4.2 (1.1), and high intensity training RPE was 6.3 (1.1) on a scale of 10. Average ROF , brain (n = 2), flexibility (n = 1), talking (n = 1), legs (n = 1), and strength (n = 1). Retrospective, self-reported benefits of tDCS were improvements in walking (n = 3), community walking (n = 1), energy (n = 1), balance (n = 1), standing (n = 1), dressing (n = 1), overall physical function (n = 1), back (n = 1), and arm (n = 1). On average, participants gave an agreement rating of ≥7.8/10 for all perceived benefits of tDCS and gave an agreement rating of 5.1/10 for the perceived detriment of more fatigue . In totaClinicians involved in the study reported that the tDCS setup time decreased from 10 min at the start of the study to 5–7 min at the end of the study. Setup was identified as a task that could be completed by rehab technicians. Clinicians noted that because priming took 15 min, participants were limited to 20–25 min of high intensity gait training. The study clinicians also suggested that it may be beneficial to do some of the high intensity training on a recumbent stepper in combination with tDCS. Finally, the clinicians subjectively noted that participants receiving tDCS had an increased awareness, commitment, and investment in their treatment plan of care.Of the 7 participants, one was removed from the study because of an adverse event. In this individual, the 10th session was stopped during high intensity treadmill walking because the participant had a hypoglycemic event (blood glucose = 60 mg/dL). The participant was transferred to a supine position with lower extremity elevated, and orange juice and a granola bar were provided. The participant’s family reported that the participant had experienced high blood sugar prior to the treatment session, and insulin had been administered. This participant continued with rehabilitative treatment but was withdrawn from the study.In the post-study survey of tDCS-related side effects, five participants reported mild to moderate side effects from tDCS. Reported side effects were mild to moderate tingling (n = 3), mild itching (n = 2), mild to moderate fatigue (n = 2), and mild pinching (n = 1). Focal side effects were felt in the vicinity of the tDCS electrodes. Side effects were reported at the beginning (n = 2), middle (n = 1), and end (n = 1) of the stimulation, or throughout the entirety of stimulation (n = 1). No participants reported that side effects affected their performance, and no pain was reported.In this preliminary study, we found evidence that tDCS is feasible and safe for persons with stroke with other co-morbidities when applied in an outpatient clinical setting. Impressions of tDCS were positive, and participants completed the tDCS sessions without any adverse events. Self-reported benefits of the treatment provide preliminary support for the efficacy of tDCS applied in a clinical setting.Our results support the feasibility of applying tDCS in an outpatient clinical setting. During the eligibility assessment, most potential participants were excluded for reasons related to internal validity , not because of contraindications to tDCS. Overall, 85% (72 out of 85) of the individuals who were screened could have been included in the study if not for these internal validity controls. It is likely that it would be safe for a large portion of the outpatient rehabilitation clinical population to receive tDCS as part of their plan of care . Of the Participants who received tDCS tolerated the treatment well and attended all sessions. Response to warmup and high intensity exercise appeared to be normal. We found evidence of acute reductions in perceived physical function limitation and pre-post improvements in perceived energy. If tDCS improves patient energy levels, this may provide an added benefit for individuals experiencing low energy levels, especially after stroke. However, it is interesting to note that fatigue symptoms did not change from pre to post study. Although fatigue is a common acute side effect of tDCS ,37, and Six out of seven (86%) participants recounted perceived benefits of tDCS, including improved walking. This coincides with a number of publications demonstrating the potential benefits of tDCS for lower limb movement ,20,21,39Clinicians were eager to use tDCS (based on previous knowledge regarding the priming effects on motor recovery), and the two clinicians who did not participate did so for non-tDCS related reasons. Required setup time decreased with experience, eventually only requiring 5–7 min, which can be easily incorporated into the standard treatment. As noted, setup could be performed by rehab technicians to decrease clinician burden and maximize time spent on skilled intervention. As found in this study, greater use of tDCS by clinicians will likely provide insights into ways to optimize this treatment in the outpatient clinical setting.There was one adverse event that took place during this study, which appeared to be unrelated to the administration of tDCS. Study participants noted mild to moderate side effects from tDCS, including tingling, itching, pinching, and fatigue. Most of these side effects were only experienced during current ramp up or ramp down. Consistent with our findings, some of the most commonly reported side effects in the healthy and post-stroke population are tingling and itching ,37. It iOur findings provide preliminary support for feasibility and safety of tDCS in an outpatient clinical setting, but our conclusions are limited by several factors. Our sample size was small (n = 7), which may have limited interpretations of our results. However, our sample size is consistent with other demonstrations of tDCS feasibility in stroke in other settings ,27. AlthIn this study, we found preliminary evidence that the use of tDCS in an outpatient clinical setting is feasible and safe. Further research is needed to determine the efficacy of this treatment, and insights from clinicians can help optimize application of tDCS in the clinic.