Osteoarthritis is a progressive multifactorial condition of the musculoskeletal system with major symptoms including pain, loss of function, damage of articular cartilage and other tissues in the affected area. Knee osteoarthritis imposes major individual and social burden, especially with the cost and complexity of surgical interventions. Mesenchymal stem/stromal cells have been indicated as a treatment for degenerative musculoskeletal conditions given their capacity to differentiate into tissues of the musculoskeletal system.A systematic search will be conducted in Medline, Embase, Cochrane Library, Scopus and relevant trial databases of English, Japanese, Korean, German, French, Italian, Spanish and Portuguese language papers published or in press to June 2018, with no restrictions on publication year applied. References will be screened and assessed for eligibility by two independent reviewers as per PRISMA guidelines. Cohort, cross-sectional or case controlled studies will be included for the analysis. Data extraction will be conducted using a predefined template and quality of evidence assessed. Statistical summaries and meta-analyses will be performed as necessary.Results will be published in relevant peer-reviewed scientific journals and presented at national or international conferences by the investigators.CRD42018091763.The protocol was registered on the PROSPERO international prospective register of systematic reviews prior to commencement, The online version of this article (10.1186/s13018-019-1070-8) contains supplementary material, which is available to authorized users. Osteoarthritis (OA) is a progressive condition affecting the articular cartilage and underlying subchondral bone, leading to significant pain and limitations in movement . Knee OAMSCs can be obtained from the stroma of various tissues, including bone marrow, umbilical cord blood, adipose tissue, peripheral blood and synovium, and expanded in culture to increase yield and enhance desired functional properties . The optThere are a large number of preclinical studies reporting a beneficial effect of MSCs on cartilage degeneration and injury, ranging from mouse , 15, rabConsidering the aforementioned lack of clarity regarding cell source, methodological factors, clinical translation and outcome measurement, a systematic review is required to synthesize and evaluate the quality of the available evidence regarding the safety and efficacy of mesenchymal stem/stromal cells for knee OA. The primary objective of this review is to establish in patients or animal models of knee osteoarthritis treated with culture-expanded mesenchymal stem/stromal cells from adipose tissue, bone marrow or synovium, with or without adjunct nonoperative therapies, the clinical, structural and functional outcomes of treatment, as well as the incidence and severity of adverse events. The secondary objective of this review is to identify study, measurement and other methodological characteristics associated with treatment outcomes.The protocol was registered on the PROSPERO international prospective register of systematic reviews, registration number CRD42018091763. The systematic review follows the Preferred Reporting Items for Systematic Review and Meta-analysis (PRISMA) statement  and protRelevant characteristics for included studies were determined using the PICOS  framework for formulating the research question and defining eligibility criteria for the literature search . CharactAll animal models of knee osteoarthritis will be considered for review, without exclusions relative to specimen sex, activity level or age. Studies will be excluded where knee (‘stifle joint’) osteoarthritis is secondary to another condition under examination . Clinical studies involving patients diagnosed with radiographic evidenced osteoarthritis will be considered for review, without exclusions relative to sex or activity level. Articles will be excluded from analysis if they include paediatric cases (aged under 18 years at diagnosis).Studies will be included if they involve the use of culture-expanded, mesenchymal stem/stromal cells from any source delivered by intra-articular injection. Studies will be excluded if they report the delivery of cells during surgical procedures or include other cell populations in the injected concentrate.Comparators considered will include placebos, conventional non-operative therapies including steroid injections, exercise and NSAIDs and cases unaffected by knee osteoarthritis.For preclinical research, studies including outcomes relevant to human osteoarthritis including histological appearance of cartilage and bone, results of noninvasive imaging and measurements of pain and function will be included. Biochemical analyses with unclear relevance to human OA will not be included in the review. For clinical research, studies reporting any outcomes relevant to the efficacy and safety of MSC injection will be included in the analysis. Particular attention will be paid to validated measures of patient-reported outcomes.Observational studies  or randomized controlled trials (RCTs) comparing outcomes of culture-expanded MSC treatment with other modalities at any follow up period will be included. Systematic reviews will be used to source additional primary materials but will not be included in the analysis. The results of meta-analyses will be included as a study in the analysis if they meet the remaining inclusion criteria. English, Japanese, Korean, German, French, Spanish, Italian and Portuguese language papers in publication will be included, with no restrictions on publication year.A systematic search will be conducted in Pubmed, Medline, Embase via Ovid SP, Cochrane Library and Scopus via EBSCO and relevant clinical trials databases of English language papers in publication as of June 2018, with no restrictions on publication year applied.  Secondary searching of reference lists of key articles and grey literature will be undertaken in order to identify any additional studies potentially missed in electronic search. Active researchers in the field will be contacted to ensure relevant references have been captured.Anatomical region: knee OR tibia OR femur OR patella OR tibiofemoral OR patellofemoralPathology: osteoarthrit* OR arthros* OR gonarthrosis OR arthrit* OR degenerat*Intervention: (mesenchym* OR stem OR strom*) AND (adipos* OR ‘bone marrow’ OR umbilic* OR MSC) OR allogen* OR autologous AND (cultur* OR ‘culture expanded’)To permit the search to return other primary studies that were not included to the published reviews, medical subject headings (MeSH) terms and keywords such as systematic review, review and meta-analysis will be excluded. The following are the main key domains: 1) anatomical region, (2) pathology and (3) intervention  histopathology initiative guidelines for specific animal models –37, and For clinical studies, outcomes considered for analysis will include clinically relevant outcomes, such as mortality, morbidity and adverse events, classified as per the US Department of Health and Human Services Common Terminology Criteria for Adverse Events . StructuThe Systematic Review Centre for Laboratory Animal Experimentation (SYRCLE) risk of bias tool will be applied to pre-clinical  studies . This isData synthesis and meta-analysis will be performed separately for clinical and pre-clinical studies, following the guidelines published by Shamseer et al.  and HooiThe revised and validated methodological index for non-randomized studies (MINORS) criteria  will be The results of this review will be published in relevant scientific journals or presented at national or international conferences (‘publications’) by the Investigators.Protocol amendments and updates will be documented via PROSPERO online register. The nature of the changes made will be recorded, dated and accessible along with the most recent version within the record audit trail under the systematic review protocol registration number CRD42018091763.Additional file 1:Screening and core dataset (CDS) template for Systematic Review. (XLSX 466 kb)


Following definitive chemoradiation for anal squamous cell carcinoma (ASCC), patients face a variety of chronic issues including: bowel dysfunction, accelerated bone loss, sexual dysfunction, and psychosocial distress. The increasing incidence of this disease, high cure rates, and significant long-term sequelae warrant increased focus on optimal survivorship care following definitive chemoradiation. In order to establish our survivorship care model for ASCC patients, a multi-disciplinary team of experts performed a comprehensive literature review and summarized best practices for the multi-disciplinary management of this unique patient population. We reviewed principle domains of our survivorship approach: (1) management of chronic toxicities; (2) sexual health; (3) HIV management in affected patients; (4) psychosocial wellbeing; and (5) surveillance for disease recurrence and survivorship care delivery. We provide recommendations for the optimization of survivorship care for ASCC patients can through a multi-disciplinary approach that supports physical and psychological wellness. Anal squamous cell carcinoma (ASCC) is a rare cancer, with only 8580 cases diagnosed in the United States annually. However, the incidence has steadily increased at a rate of 2.2% per year over the last 10 years . AccordiIntensive radiation with concurrent 5-flurouracil and mitomycin is the standard treatment for locoregional ASCC –12. The Cure rates exceed 80% for the 90% for patients diagnosed with locoregional disease , and theThe University of California, San Francisco (UCSF) is home to the Anal Neoplasia Clinic, which was founded in 1991 to address increased rates of anal dysplasia, a precursor of ASCC, observed in people living with HIV (PLWH) . This clIn order to establish our survivorship care model for ASCC patients, a multi-disciplinary team of experts in the Gastrointestinal Oncology Survivorship practice at our institution performed a comprehensive literature review of the survivorship issues and summarized best practices for the management of this unique patient population. Herein, we describe our multi-disciplinary approach to caring for patients with ASCC following curative treatment. The principle components of this approach include: (1) management of chronic toxicities; (2) sexual health; (3) HIV management in affected patients; (4) psychosocial wellbeing; and (5) surveillance for disease recurrence and survivorship care delivery.CRT for ASCC involves a variable amount of radiation delivered to the rectum, large and small intestines. While guidelines exist governing the maximum recommended dose for bowel, tumor stage, bulk, proximity to bowel, and variations in patient anatomy result in significant heterogeneity in bowel exposure to radiation. Not unexpectedly, patients commonly experience bowel dysfunction following CRT with variable onset, degree and duration, ranging from months to years. Long-term toxicity data from clinical trials of chemotherapy with 3D radiation and IMRT report moderate or higher gastrointestinal toxicity rates of approximately 10%  and 8–10Among cancer patients of various tumor types, approximately half of patients who receive treatment with pelvic radiation report their quality of life following treatment is compromised by gastrointestinal symptoms > 3 months after completion of treatment –27. ThesDiarrhea is the most cited gastrointestinal concern that impacts quality of life following treatment for ASCC , 29–35. In a patient who presents with diarrhea and hematochezia after CRT for ASCC, colonoscopy or high-resolution anoscopy (HRA) and/or CT or MRI may be warranted to differentiate chronic radiation enteritis or proctitis from other causes. In the setting of chronic radiation enteritis, biopsies demonstrate increased friability and telangiectasias, consistent with ischemic injury, and CT/MRI demonstrate bowel inflammation with hyperenhancement and bowel thickening . The preWhile radiation enteritis involves damage to the intestines, late proctitis is a manifestation of ischemic radiation injury to the rectum and may present as diarrhea, bleeding, tenesmus, and/or urgency years after radiation exposure , 45. AftGiven the variable latency of late radiation bowel dysfunction, a new presentation of inflammatory bowel disease, infectious gastroenteritis, or acute proctitis secondary to sexually transmitted infection should also be considered. Other causes of bowel dysfunction related to pelvic radiation include bacterial overgrowth and fibrosis. If bacterial overgrowth is suspected due to history of chronic malabsorptive diarrhea in the absence of other identified causes, carbohydrate breath test may aid in diagnosis but is limited by low sensitivity . PotentiAs a key component to curative treatment for ASCC, external beam radiotherapy targets the primary tumor and involved nodes, as well as areas at high risk for subclinical or microscopic disease. Innovations over the last two decades in radiotherapy delivery have moved from 2D or 3D techniques to highly conformal IMRT. This allows personalized delivery with maximum dosing to key areas (gross or visible tumor) and elective dosing to at-risk areas. Notably, this customized sculpting keeps high-dose radiation away from critical bone regions , such asPelvic insufficiency fractures (PIFs) are the most commonly discussed chronic bone toxicity of pelvic radiation , 68; howPIFs are not uncommon among ASCC patients following CRT; however, the complications from these fractures are usually self-limited. The clinical presentation of PIF rarely requires hospitalization or extensive intervention beyond pain management , 86, 87.Radiation treatment to the pelvis and anus can affect pelvic floor muscles, fascia, and nerves, resulting in pelvic floor dysfunction. Specifically, significant pelvic floor muscle weakness is common after pelvic radiation , and myoImpairments in pelvic floor muscle strength, coordination and tone caused by radiation can be successfully treated with neuromuscular re-education, biofeedback, and electrical stimulation. Internal and/or external manual treatments, such as myofascial release, connective tissue manipulation, and joint mobilization to the pelvis, spine and hips, may be used to treat pelvic pain. Vaginal and/or anal dilation therapy is commonly incorporated as a component of pelvic rehabilitation programs to treat canal stenosis and dyspareunia. Finally, due to the impact of deconditioning and fatigue following cancer treatment, patients may benefit from education on cardiovascular exercise and activity pacing; posture training; and core muscle strengthening. Treatment plans are individualized based on examination findings and patient goals and typically range from four to twelve sessions.More than half of women with ASCC will endure some form of sexual dysfunction after CRT, including effects on desire, arousal, orgasm and/or pain , 92. A sAdditionally, pelvic radiation has short term consequences including inflammation, erythema, and desquamation that can cause significant vaginal pain and dyspareunia. Over time, however, the long-term effects of pelvic radiotherapy will result in decreased oxygenation of the tissue, leading to fibrosis, stenosis and adhesions that can leave the vagina shorter, narrower, and less elastic . StudiesThe mainstays of prevention of sexual health toxicities following treatment for ASCC have focused on vaginal dilator therapy. The premise is that by manually dilating the vagina with a phallic shaped device, women will be able to prevent many of long-term toxicities that lead to vaginal shortening, fibrosis, and stenosis which cause dyspareunia. Unfortunately, despite dilators being promoted in a majority of survivorship programs, the data supporting their use is extremely limited. Indeed, a 2014 Cochrane review found “there is no reliable evidence to show that routine, regular vaginal dilation during radiotherapy treatment prevents stenosis or improves quality of life” . A signiWhile the majority of literature focuses on vaginal dilator therapy to prevent local radiation toxicity, recently there is a new focus on ovarian suppression at the time of treatment to prevent menopause induced by CRT. Despite mixed early data on ovarian suppression during chemotherapy , a recenAlthough data on sexual function in male survivors of ASCC is sparse, studies consistently demonstrate that male survivors have significantly impaired sexual function when compared to age and gender-matched controls , 34 and While it has been hypothesized that sexual dysfunction in males following treatment for ASCC is due to tumor fibrosis and pelvic radiation, a correlation has not been found between radiation dose and sexual dysfunction . ED assoPhosphodiesterase type-5 inhibitors (PDE5is) are the general first line approach for treatment of ED, and some data supports the efficacy of PDE5is to treat ED associated with pelvic radiotherapy in cancer survivors –112. PDEPDE5i use in patients with cardiovascular disease requires consideration of cardiovascular risk and concurrent medications or substance use . Patients with high-risk cardiovascular disease should have their cardiovascular status stabilized prior to resuming sexual activity . OptionsIn addition to ED, cross-sectional studies of ASCC survivors note that fecal incontinence is a prominent treatment sequela. For patients who practice anal receptive intercourse (ARI), fecal incontinence might impede sexual function; however, the effect of ASCC treatment on ARI has not been previously studied. Consideration should be given to provide advice regarding safer sex practices, including lubricant usage, and screening for sexually transmitted infections.Infection with HIV is one of the strongest risk factors for ASCC –117. ForCurrent standards dictate that the HIV viral load of patients stable on ART should be monitored at least every 6 months to ensure that it remains undetectable . WhetherPLWH who are not on ART are at risk for infections associated with depleted cell-mediated immunity. Guidelines recommend antimicrobial prophylaxis against such opportunistic infections based on the number of remaining CD4 positive T-lymphocytes (CD4+ T-cells) in circulation . These gNCCN guidelines recommend routine screening to assess the level and nature of emotional distress in all cancer survivors . These gEven in the absence of a clinical event that would trigger distress, many patients will be affected by fear of cancer recurrence, which can persist even when risk of recurrence is low, and is associated with poorer quality of life , 127. AtBecause of the unique risk factors and treatments associated with their disease, ASCC patients may perceive social stigma and experience shame. An ASCC survivor must contend with societal views on HPV and on sexual behavior. As providers, we aim to demonstrate sensitivity and immediate responsiveness to any patient expressions of fear of stigma or of shame/guilt.PLWH and men who have sex with men (MSM) already face stigma, which may be compounded by a diagnosis of ASCC. Patients from sexual and/or gender minorities may be less likely to share information with medical providers because of concerns about stigma or discrimination . As mediThe short and long-term sequelae of definitive CRT are complex and require a multidisciplinary approach. At our institution, care of ASCC patients after successful completion of treatment is delivered in the context of a multidisciplinary survivorship clinic, with representation of clinicians from oncology, surgery, radiation oncology, anal dysplasia, primary care, infectious diseases, gynecology, nutrition, psycho-oncology, social work, urology, and other disciplines. Transgender individuals may also benefit from endocrinology consultation or specialists in transgender health to optimize safe hormone management. In a complex treatment paradigm requiring multiple health care providers, the oncology survivorship provider serves as a navigator, prioritizing subspecialist referrals and synthesizing recommendations from a variety of disciplines Table .Provision of a Survivorship Care Plan to the patient and primary care provider with clearly documented surveillance guidelines ;Ensuring appropriate surveillance is performed;Evaluation and management of acute and long-term toxicities; andPromotion of risk-reduction strategies for cancer survivors including exercise , maintenThe goals of survivorship care for patients with ASCC following completion of definitive CRT are summarized in Fig. Patients should be seen at frequent intervals post completion of CRT until resolution of acute side-effects of treatment. Acute toxicities secondary to definitive CRT have been documented to include: myelosuppression (50%), skin toxicity (65%), bowel dysfunction (17%), constipation (3%), fecal incontinence (9%), proctitis (17%), and fatigue (25%) . With rePer NCCN guidelines, surveillance should include digital rectal exam and inguinal node palpation every three to 6 months for 5 years, proctoscopy or anoscopy every six to 12 months for 3 years, and CT scans of the chest, abdomen, and pelvis annually for 3years Table  141]. A. A141]. The relationship between HPV and ASCC supports the use of HRA as surveillance modality for disease recurrence. The UCSF Anal Neoplasia Clinic employs an alternative screening model in which primary care providers perform HRA and anal cytology screening of our survivor population to evaluate for local recurrence. Most patients are able to undergo surveillance in the clinic, with HRA with biopsies of abnormal lesions performed at three or 4 month intervals  into survivorship care is imperative to ensure that patients resume age-appropriate health care maintenance and to ensure that the PCP is able to assume complete care for the patient. Survivorship care can be transitioned to the PCP during or after the surveillance period is complete.In summary, a growing population of ASCC survivors faces a complex burden of physical and psychosocial sequelae. Our approaches to a range of chronic toxicities, sexual health, HIV management in affected patients, psychosocial wellbeing, and surveillance for disease recurrence are reviewed here. This review provides a rigorous and comprehensive summary of the evidence that informs our multi-disciplinary practice, accompanied by expert opinion. In the absence of prospective studies for this rare disease, we believe this review will serve as a resource for oncology care providers, PCP’s, and subspecialists who encounter ASCC survivors in clinical practice. While we acknowledge that every setting may not have the comprehensive services available that has been developed for these patients at our institution, we provide references to link providers to knowledge and resources to facilitate appropriate referrals. Moreover, the emotional and social issues experienced by ASCC survivors may be ameliorated by provider awareness and ability to normalize the experiences of this unique patient group.Our ongoing research is directed at evaluating the impact of this multi-disciplinary approach in a cohort of ASCC survivors. Future clinical trials and observational studies should collect patient reported outcome measures in addition to acute and chronic toxicities. Recent development of a new, validated quality of life questionnaire specific to anal cancer (EORTC QLQ-ANL27) may facilitate future capture of sub-clinical late effects that cause suffering and functional impairment . In addi


Metastasis is still poorly understood and thus further research must be conducted to provide insight into the driving factors. Novel research has revealed the significance of the microenvironment in the delegation of metastasis, expanding the field of cancer metastasis to cells and cell environments surrounding the migrated tumor cells. Research on hepatic metastasis is an ever-growing domain of this field, as several primary tumors can metastasize to the liver. The two features within the liver that promote metastasis—cellular and acellular—are found in the current interpretation of liver microenvironment. Novel findings of both are included in this review. Different hypotheses detailing the methods by which metastasis can occur must be included to understand the significance of the microenvironment, as well as a brief overview of the methods that can be used during research. This review aims to highlight the importance of liver microenvironment on the development or potential regression of hepatic metastasis through discussing both acellular and cellular components of liver microenvironment and their interaction with metastasis. Organ microenvironments are an undeniable component for metastasis, enabling proliferation of cancer cells to organ systems external to the primary tumor. Migration of cancer cells requires compatibility with the destination, particularly with the microenvironment. Hepatic metastases can arise from the primary cancers in different locations within the human body. Both the acellular-such as extracellular matrix (ECM) proteins (i.e. collagen) and the cellular components of the liver such as Kupffer cells (KCs), hepatic stellate cells (HSCs), and liver sinusoidal endothelial cells (LSECs) contribute to the metastatic ability of tumors of different origins. Common incidences of liver metastasis occur in colorectal cancers, as these cells can take advantage of both the proximity (as a great amount of venous drainage is to the liver) and highly vascular nature of the liver . A statiThe ECM is the major acellular component contributing to the liver microenvironment, and consequently the microenvironment of the tumor. The matrix of biological tissue serves as a framework by which cells organize, intertwined with the local vasculature. The ECM also contains domains that allow proteins to bind, which are important for cell–cell communication as well as specific function. ECM proteins can interact with growth factors, such as hepatocyte growth factor (HGF) which promotes cell migration and vascular endothelial growth factor (VEGF) which can enable metastasis progression through angiogenesis . The majThough the liver microenvironment can promote metastasis, the liver will initially respond in ways to react to inflammation or damage. This premetastatic niche can protect the liver, using cellular signaling to promote immune responses. Both cellular and acellular provide these dueling roles, such as KCs and cytokines, though eventually the prometastatic elements outweigh the opposition as metastasis occurs . Table 1The major acellular components that support or are involved in metastatic niche formation are as follows; the cell-adhesion molecule carcinoembryonic antigen (CEA) and other cell adhesion molecules (CAMs), CXC motif-chemokines (CXCLs), VEGF, MAPK, and NF-κB, Citrullinated proteins/PAD, Spermine pullulan, matrix metalloproteinases, and collagen proteins Table .Abdul-Wahid et al. reported that the CEA, which can be expressed on the surface of colon circulating tumor cells (CTCs), contributes to the subsequent attachment to fibronectin of the liver ECM, resulting in increased levels of metastasis . This isIt is known that CXC motif-chemokines (CXCL) 1, 2, 3, and 5 contribute to colorectal carcinoma, but novel exploration expanded this understanding to metastasis to the liver. Knock-down of Interleukin-8 , which can be produced by macrophages, was shown to inhibit tumor growth in colorectal liver metastasis. This was associated with the reduction in cell proliferation and viability in samples deficient in CXCL8 .VEGF expression levels were also decreased during knock-down of CXCL8, suggesting a mechanism for the decreased proliferation . A studyRecently, ECM proteins were shown to have a greater incidence of citrullinated proteins, catalyzed by peptidylarginine deiminase proteins (PAD), which is specifically seen in liver metastases. Citrullination occurs on the arginine, producing a neutrally charged product. It was suggested in earlier studies that citrullination of certain proteins, which are prevalent and detectable in tumor cells, contribute to the progression of the tumor . A more Aureobasidium pullulans, can be utilized to polarize macrophages towards M1, which can ultimately inhibit metastasis by increasing inflammation [Polysaccharide spermine modified pullulan (SP), which is produced by the fungus ammation .In an experiment involving the treatment of murine models with chemotherapy, periostin and matrix-metalloproteinase 2 (MMP-2) were increased when murine models were treated with the drug cisplatin. Therefore, they concluded that cisplatin may induce liver metastasis of murine melanoma cells via increase in MMPs, which in turn can manipulate the extracellular environment by protein turnover . The stuObservational data show colorectal liver metastasis (CRLM) patients have elevated levels of type I collagen in the urine and plasma . This reCellular components of the liver are as follows: bone-marrow-derived macrophages such as tumor-associated macrophages (TAMs), Kupffer cells (KCs), liver sinusoidal endothelial cells (LSECs), immune cells like tumor-associated neutrophils (TANs), myeloid-derived suppressor cells (MDSC), fibroblasts, hepatocytes, and hepatic stellate cells (HSCs)  Fig. 1,,1.Fig. 1Macrophages can either be resident like KCs, or monocyte-derived, both of which can undergo M1 to M2 repolarization . This trLiver sinusoidal endothelial cells (LSECs) can provide both anti-metastatic and pro-metastatic functions. For example, LSECs can undergo apoptosis or release toxins to aid in elimination of tumors at the site of metastasis, or these cells can increase angiogenesis or EMT to promote metastasis .Different immune cells, such as dendritic cells, tumor-associated neutrophils (TAN), myeloid-derived suppressor cells (MDSC), and TAMs are all associated with different cytokines/chemokines or other factors that contribute to metastasis, summarized in Table As mentioned, chemokines can contribute to metastasis by immune system suppression. The behavior of macrophages and other immune cells towards cancer is dependent on these cell-signal molecules. SDF-1 (CXCL12), or stromal cell derived factor, is known to allow for TAM movement, and can be secreted directly from cancer cells . SDF-1 pCancer stem cells (CSC) are thought to be a portion of the population of a tumor, and can be differentiated based on their surroundings. The tumor microenvironment is crucial to this differentiation. CSCs can interact with both acellular and cellular components of the microenvironment. The review by Lau et al. describes the connections between CSC pathways and ECM changes, hypoxia, growth factors, etc., which all contribute to metastasis . The devRecent studies have shown that pancreatic ductal adenocarcinoma (PDAC)-derived exosomes stimulate liver pre-metastatic niche formation (PMN). Costa-Vista et al. showed that uptake of PDAC-derived exosomes by Kupffer cells caused TGF-β secretion and upregulation of fibronectin production by HSCs . They foIt is assumed that EMT is a primary method of metastasis, as the transition to mesenchymal cells allows for migration to the bloodstream, which acts as a highway for tumor cells. The reverse, mesenchymal-to-epithelial transition (MET), can halt tumor cells at a certain location. Individual cells detected in patient serum are known as circulating or disseminated tumor cells (DTCs), and it is thought that EMT plays a role in tumor cell dissemination . The patThe tumor and organ microenvironment is not as easily manipulated in laboratory settings; in vitro is not primarily involved in the components surrounding individual cells. This barrier prompted the exploration into new methods of visualization and manipulation. Both two-dimensional (2D) and three-dimensional (3D) models can be utilized, and have their respective advantages and disadvantages . In vitrUltimately, the microenvironment of the liver is crucial to the development of hepatic metastases. Different growth factors, cell types, posttranslational modifications, signaling molecules, etc. are all necessary to understand, including their interactions, when deciphering the elements of metastatic outgrowth. The accumulation of observational and experimental studies contribute to the knowledge required to battle cancer metastasis, and the methods to understand this metastasis in laboratory settings continues to progress.


Autism spectrum disorder (ASD) is a neurodevelopmental disorder that is characterized by impaired social interaction, communication and restricted and repetitive behavior. Few studies have focused on the effect of facial emotion recognition on bullying involvement among individuals with ASD. The aim of this study was to examine the association between facial emotion recognition and different types of bullying involvement in adolescents with high-functioning ASD. We recruited 138 adolescents aged 11 to 18 years with high-functioning ASD. The adolescents’ experiences of bullying involvement were measured using the Chinese version of the School Bullying Experience Questionnaire. Their facial emotion recognition was measured using the Facial Emotion Recognition Task . Logistic regression analysis was used to examine the association between facial emotion recognition and different types of bullying involvement. After controlling for the effects of age, gender, depression, anxiety, inattention, hyperactivity/impulsivity and opposition, we observed that bullying perpetrators performed significantly better on rating the intensity of emotion in the Facial Emotion Recognition Task; bullying victims performed significantly worse on ranking the intensity of facial emotion. The results of this study support the different deficits of facial emotion recognition in various types of bullying involvement among adolescents with high-functioning ASD. The different directions of association between bully involvement and facial emotion recognition must be considered when developing prevention and intervention programs. School bullying is a critical problem concerning the psychological health of adolescents. Bullying behaviors include physical attacks , verbal attacks , and intentional actions that cause the victim to experience social isolation . DiffereAutism spectrum disorder (ASD) is a neurodevelopmental disorder associated with impediments in social interaction and communication and a tendency to demonstrate restricted interests and repetitive behaviors . VariousResearch has proposed possible etiologies to explain why adolescents with ASD are more likely to be involved in bullying, including having fewer friends , tendingSeveral studies have indicated that the social interaction impairment of individuals with ASD might be partially caused by their inability to recognize facial emotions ,20,21,22Dyck et al.  reportedThe aim of this study was to examine the correlation between facial emotion recognition and bullying involvement in adolescents with high-functioning ASD. Previous studies regarding teenagers’ bullying have indicated that bullying roles and behaviors are diverse. Different bullying behaviors involve teenagers’ skills, status, and social behaviors . Sutton ASD is often comorbid with attention-deficit hyperactivity disorder (ADHD), depression, and anxiety. Previous studies have discovered that ADHD ,27, deprA total of 210 adolescents with ASD visited the child psychiatry outpatient clinic of a university hospital in southern Taiwan during the period between October 2015 and July 2017. Adolescents with high-functioning ASD from were enrolled from this group for this study. The inclusion criteria were as follows: (1) having an age of 11–18 years; (2) having a diagnosis of ASD ascertained by a certified child psychiatrist according to the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition ; and . This scale was composed of four 4 item subscales for evaluating the severity of victimization by verbal and relational bullying , victimization by physical bullying and snatching of belongings , perpetration of verbal and relational bullying , perpetration of physical bullying and snatching of belongings . Participants who answered 2 or 3 on any item among items 1 to 8 and items 9 to 16 were identified as bullying victims and perpetrators, respectively. The C-SBEQ has excellent reliability and validity ,37. In tThe C-CES-D consists of a self-report questionnaire with 20 items investigating symptoms related to depression such as emotions, appetite, sleep, sadness, loneliness, worthlessness , fatigue, and social withdrawal . The C-CES-D was edited by Chien and Cheng (1985) and is outstanding in sensitivity and specificity . Total sThe MASC-T consists of 39 items measuring multidimensional anxiety symptoms, including physical symptoms (tense/restless and somatic/autonomic), harm avoidance (perfectionism and anxious coping), social anxiety (humiliation/rejection and public performance fears) and separation anxiety, and panic in children and adolescents aged 8 to 19 years . A higheThe 26 item SNAP-IV assesses the severity of inattention , hyperactivity/impulsivity , and oppositional defiant symptoms  . The parParticipants with ASD referred by outpatient clinics were individually provided with an explanation of the purpose, procedure, and privacy policy of this study by the research assistant before the participants signed a consent form. The participants’ parents were asked to respond to the questionnaire for sociodemographic data and SNAP-IV; adolescents were asked to respond to the C-SBEQ, C-CES-D, MASC-T, and C-FERT. The overall response process required approximately 35–45 min. Data analysis was performed using SPSS 22.0 statistical software .In the initial exploration of the data we first divided the participants into four groups, namely pure perpetrators, pure victims, perpetrators/victims, and neutrals . The rest-test. Logistic regression analysis was used to examine the associations between facial emotion recognition and being bullying perpetrators and victims by controlling for the effects of other factors. A two-tailed p value of <0.05 was considered statistically significant.Finally, all participants were divided into the perpetrator group or victim group and the nonperpetrator group or nonvictim group to further analyze the differences between both groups. Differences in demographic data, depression, anxiety, inattention, hyperactivity/impulsivity, opposition, and facial emotion recognition between bullying perpetrators and nonperpetrators and between bullying victims and nonvictims were examined using a chi-square test and In total, 138 adolescents with high-functioning ASD completed this study. Among them, 41 (29.71%) were classified as bullying perpetrators and 24 (17.39%) were classified as victims. This study is the first to examine the association between the ability of facial emotion recognition and bullying perpetration and victimization in adolescents with high-functioning ASD. The study found that bullying perpetrators with high-functioning ASD had a greater ability to correctly rate the intensity of facial emotion than nonperpetrators did. Because perpetrators with high-functioning ASD may have a greater ability to recognize the expressions of others, they recognized how to target weaker people and successfully bully them. By contrast, victims with high-functioning ASD had a lower ability to rank the intensity of facial emotion than nonvictims did. Poor ability to recognize the intensity of others’ facial emotions may make adolescents with ASD less sensitive to others’ emotional changes and increase their possibility to be considered impolite, thus subsequently increasing their risk of being bullied.Previous studies investigating the relationship between bullying involvement and the ability to recognize facial emotion have focused primarily on adolescents with typical neurodevelopment rather than adolescents with ASD. Most of these studies on general children and adolescents have discovered that victims had a lower ability to correctly recognize others’ facial emotions ,44; howePrevious interventions against school bullying have often used universal bullying prevention programs  as the criterion standard . Other rThe present study adjusted for the effects of age, gender, depression, anxiety, inattention, hyperactivity/impulsivity and opposition when examining the relationship between the ability of facial emotion recognition and bullying involvement in adolescents with high-functioning ASD. The study also used the facial emotion pictures of Taiwanese rather than Western people to reduce ASD participants’ unfamiliarity. Despite these adjustments, this study has some limitations that should be addressed. First, the cross-sectional research design of this study limited our ability to draw conclusions regarding the causal relationships between facial emotion recognition and bullying involvement. Second, this study evaluated bullying involvement based on adolescents’ self-reports. Future studies are required to identify whether the results differ based on bullying involvement reported by adolescents’ parents and teachers. Third, this study recruited the participants from outpatient clinics; therefore, many exhibited emotional or behavioral disturbances. Determining whether the results of the present study can be generalized to all adolescents with ASD requires further study. Fourth, the C-FERT used in this study was a random test of pictures of facial expressions. In addition, whether difficulties in specific types of facial emotion recognition exist in relation to bullying perpetration and victimization warrants further examination. Moreover, the small number of participants who were both bullying perpetrators and victims (perpetrator-victims) limited the possibility of this study to examine whether the perpetrator-victims possess unique patterns of facial emotion recognition. Further study should be conducted to realize the role of facial emotion recognition ability in relation to bullying involvement among adolescents with high-functioning ASD when bullying involvement is divided into four categories.This study on adolescents with high-functioning ASD revealed that bullying perpetrators possessed a better ability to recognize facial emotion than nonperpetrators did, whereas bullying victims had worse ability to recognize facial emotion than nonvictims did. The ability to recognize facial emotion should be considered when developing prevention and intervention programs for bullying involvement in adolescents with ASD.


The numbers and strengths of synapses in the brain change throughout development, and even into adulthood, as synaptic inputs are added, eliminated, and refined in response to ongoing neural activity. A number of experimental techniques can assess these changes, including single-cell electrophysiological recording which offers measurements of synaptic inputs with high temporal resolution. Coupled with electrical stimulation, photoactivatable opsins, and caged compounds, to facilitate fine spatiotemporal control over release of neurotransmitters, electrophysiological recordings allow for precise dissection of presynaptic and postsynaptic mechanisms of action. Here, we discuss the strengths and pitfalls of various techniques commonly used to analyze synapses, including miniature excitatory/inhibitory (E/I) postsynaptic currents, evoked release, and optogenetic stimulation. Together, these techniques can provide multiple lines of convergent evidence to generate meaningful insight into the emergence of circuit connectivity and maturation. A full understanding of potential caveats and alternative explanations for findings is essential to avoid data misinterpretation. A fundamental basis of information transfer in the nervous system is the release of neurotransmitter from the presynaptic terminal into the synaptic cleft to activate receptors on the postsynaptic neuron. These receptors can activate downstream signaling processes and initiate ionic flux through receptor pores. This in turn can alter the transmembrane electrical potential, which propagates to the cell body and axon hillock to evoke an action potential (AP) in the postsynaptic neuron. Temporal and spatial summation play pivotal roles in regulating neuronal output, through the filtering and integration of synaptic events with a range of amplitudes and durations  and voltage probes have provided methods for cell-type specific cellular, and even synaptic, interrogation , limiting its utility for understanding complex network-level interactions  depends on the release probability (Pr) from a pool of releasable quanta (N) of defined quantal amplitude (Q). Therefore, the strength of an evoked postsynaptic response was modeled as:The discovery by Katz and colleagues that spontaneous miniature end-plate potentials (MEPPs) recorded from the frog neuromuscular junction were similar in shape and amplitude to minimally evoked end-plate potentials in the presence of high MgAlthough the precise definitions for these parameters may somewhat differ, this model provides a basis for understanding how changes in synaptic strength are measured. Indeed, these canonical electrophysiological approaches are still applied today and continue to provide insights into quantal parameters at the resolution of an individual synapse.However, synapses in the central nervous system (CNS) are fundamentally distinct from peripheral neuromuscular junction synapses. The frog neuromuscular junction characteristically exhibits a low probability of release from defined inputs and high signal-to-noise ratio of quantal events that summate in a linear fashion . While various recipes have been used, ASCF generally contains high levels of Na+ and lower levels of other monovalent and divalent cations and anions. By replacing the endogenous constituents of the extracellular cerebrospinal fluid with known quantities, while using the internal ionic concentrations in the recording electrode to approximate transmembrane ionic gradients, the specific ion species that pass through synaptic receptor channels can be readily identified. Levels of extracellular Ca2+ are critical regulators of synaptic responses, and therefore altering Ca2+ concentrations can have a major impact on the release probability and degree of presynaptic vesicular mobilization, with higher concentrations resulting in increased presynaptic glutamate release and recorded postsynaptic current amplitude  can be recorded while leaving network activity intact. Therefore, sPSCs provide information about the overall synaptic drive and activity of an intact neural circuit in the absence of exogenous artificial stimulation. Used in conjunction with miniature postsynaptic current recordings (mPSCs) in which AP generation is pharmacologically blocked, sPSCs allow for the dissection of overall network activity compared to stochastic spontaneous release and can inform whether alterations following an experimental condition result in changes in network properties or individual synaptic probabilities.+ and K+, with roughly equal permeability, producing a depolarizing current that reverses near 0 mV membrane potential. In contrast, activation of ionotropic inhibitory GABAA receptors passes Cl−, and results in a current that reverses at the equilibrium potential for Cl−, usually near −65 mV. Consequently, by clamping the voltage of a cell at −65 mV, the driving force for the inhibitory component is effectively eliminated leaving only excitatory currents while depolarizing the cell to 0 mV can mute glutamatergic currents to reveal outward GABA responses. Comparison of the respective currents at each of these potentials allows for the calculation of the excitatory/inhibitory (E/I) ratio, which reflects the interplay of network activity that converges on an individual neuron. The balance of excitation vs. inhibition reflects the relative number of excitatory and inhibitory synapses and in healthy neurons, these levels of transmission are assumed to covary proportionally , attention deficit hyperactivity disorder (ADHD), and schizophrenia, this balance is not maintained during synaptic pruning or the removal of aberrant synapses, for example, leading to a state of hyperexcitability that is characteristic of patients diagnosed with autism  or miniature inhibitory post-synaptic currents (mIPSCs). While AP-generated presynaptic responses strongly activate high-threshold P/Q- (Cav2.2) and N-type (Cav2.1) voltage-gated Ca2+ channels, stochastic Ca2+ influx for mPSC release is primarily mediated by low-threshold T-type Ca2+ channels including Cav3.1 and Cav3.2 , a selective and potent blocker of voltage-gated Na Südhof, . The fre Südhof, . Stochassitivity .Pr and the pool of releasable quanta. To delineate the contribution of these presynaptic parameters, use-dependent pharmacological blockers may be employed to estimate the rate of spontaneous release that relies solely on Pr by measuring the decay of recorded currents  and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR) are two types of ionotropic glutamatergic neurotransmitter receptors. The conductance pore of the NMDAR can be blocked by extracellular Mg2+ fluctuations that engaged presynaptic mechanisms of vesicular release similar to evoked release  and activates the fast Ca2+ sensor, synaptotagmin-1, albeit with lower efficacy than Ca2+. Further, Sr2+ is extruded from presynaptic terminals less efficiently, resulting in a slowing of vesicular mobilization machinery , it is thought that residual Ca2+ left over from the first stimulus will transiently increase the release probability upon the second stimulus, termed short-term plasticity . Consequently, changes in PPR have been interpreted to reflect presynaptic changes in Pr, although there are a variety of alternative mechanisms that may contribute to alterations in PPR  signaling pathway in CA1 pyramidal neurons in response to Schaffer collateral stimulation, while simultaneously resulting in significant potentiation of synaptic responses  and decay kinetics (20–30 ms). In contrast, NMDA receptors, which are typically quiescent at resting potential and activate only upon depolarization-mediated removal of Mg2+ ionophore blockade, exhibit a slow rise (~20 ms) to maximal current, exhibiting a permeability for Ca2+, and a slow bi-exponential decay kinetic of 40–200 ms. Kainate receptors, which are active near rest potentials, show rapid rise-time accompanied by a slow decay constant that is sensitive to interactions with Neto1 auxiliary proteins  induction, highlighting a functional heterogeneity of LTP expression at different synaptic connections . Assuming that AMPARs and NMDARs are localized to all synapses, 1/CV2 should be equal for both receptor subtypes due to trial-by-trial variability of Q released from the presynaptic terminal. However, following LTP induction, 1/CV2 for AMPAR-mediated events is consistently decreased compared to NMDAR-mediated components  of synapses capable of responding  ester to the dye, making it membrane permeant. AM-coupled dye could be injected directly into brain tissue to load hundreds of cells simultaneously. The AM ester would then be cleaved off the dye by intracellular esterases, trapping the activated fluorescent dye inside the cells (Garaschuk et al., 2+ chelation, beneficial for relatively slow, laser-scanning microscopy approaches, many of these indicators offer relatively low temporal resolution of cellular excitation compared to more traditional electrophysiological methods. Multiple variants, with specifications for fluorescence change, spatial resolution, and response kinetics are constantly being developed. Recently, a set of four-color, spectrally-resolved Ca2+ indicators, XCaMPs that exhibit a large fluorescence signal change with more rapid kinetics has been reported (Inoue et al., Live imaging of intracellular Cas Tsien, . A wide Optical stimulation employing light-activated actuators or inhibitors can elicit synaptic release or block synaptic input with precise spatial and temporal control. Early versions were based on neuronal ion channels modified to use light to gate conductances and depolarize neurons, but achievable time constants were slow and lacked specificity (Zemelman et al., in vivo and in vitro, faces most of the same issues that concern electrical stimulation, as well as additional concerns that include toxic light exposure and perturbation of normal cellular function. Variability in the level of ChR2 expression can reduce the utility of optogenetics as a tool to study synaptic transmission. Excitation of ChR2 in high-expressing neurons induces a large Ca2+ transient that travels throughout to the neuron to trigger APs with relatively short delays and high reliability, whereas low-expressing neurons routinely require extended blue light activation to elicit neuronal firing, that can result in phototoxicity and deleterious effects on cell health (Wade et al., 2+, likely due to temporally-extended depolarization-mediated activation of voltage-gated Ca2+ channels (Zhang and Oertner, 2+ suggests that their saturation could alter Pr from the presynaptic terminal. These important characteristics of ChR2 require particular attention when using optogenetic tools to study synaptic transmission.Although extremely powerful, the use of optogenetics in measuring synaptic transmission, both Optical stimulation can also impose artificial parameters on network activity. Due to light-mediated saturation of ChR2 currents in presynaptic inputs, a large number of presynaptic axons may be activated simultaneously by diffuse illumination. This mass excitation can impose non-physiological synchronous input on the postsynaptic neuron (Yizhar et al., Natronomas pharaonis (NpHR; Zhang and Oertner, − homeostasis and reversal potential, as the neuron is unable to clear the anionic charge through Cl− transporters. Consequently, at the offset of a light stimulus, neurons expressing NpHR will show a period of rebound excitation, releasing previously-silenced transmitter onto the postsynaptic neuron (Raimondo et al., Halorubrum sodomense strain TP009, termed ArchT (Chow et al., Conversely, neuronal silencing using inhibitory opsins has been used to reduce firing in a defined population of neurons. Effective optogenetic silencing is possible using the chloride pump red-light sensitive halorhodopsin derived from In summary, these new neuroscientific tools to investigate synaptic transmission in the developing and mature nervous system will undoubtedly play critical roles in further understanding network connectivity and synaptic transmission; however, it is critical to understand the utility and limitations of any new tool. A carefully-planned combination of these optical tools alongside electrophysiological validation and calibration is best to ascertain how genetically-defined groups of neurons interact at the synaptic level.Multiple electrophysiological techniques can be used to interrogate synaptic function in the developing and mature brain, and the emergence of new optical tools for both manipulation and measurement has allowed for unparalleled resolution of cellular processes underlying synaptic transmission. It is clear that understanding the contribution of pre- and postsynaptic mechanisms to synaptic plasticity must involve a number of diverse approaches to decipher how the brain changes individual synapses. Traditional interpretations of miniature EPSCs and paired-pulse protocols suggest that changes in these measures can reveal changes at presynaptic terminals. While alteration in AMPAR-to-NMDAR ratio and photo-uncaging have been understood to reflect postsynaptic changes, multiple studies in recent years have provided evidence that it is naïve to conclude that a phenomenon identified using a limited number of traditional approaches is purely pre- or postsynaptic.Implementation of multiple electrophysiological methods, coupled with imaging techniques, is enormously beneficial for dissecting pre- and post-synaptic contributions to synaptic transmission and plasticity. As an illustrative example, we have recently demonstrated that the chemotropic guidance cue, netrin-1, is released from dendrites following NMDAR activation, and contributes to synaptic plasticity in the adult hippocampus (Glasgow et al., Recent technological developments have provided neuroscientists with an unprecedented tool-set to investigate synaptic transmission. The use of optical tools, in conjunction with classic electrophysiological methods, has provided new insights to traditional interpretation of synaptic function. By combining multiple approaches as described in this review article, convergent lines of evidence can be used to attribute changes in synaptic transmission to the pre- or postsynaptic compartment.Recent data confirms that synaptic transmission is an exceedingly complex phenomenon, subject to modifications in signaling at both pre- and post-synaptic sites. The development and refinement of whole-cell patch clamp electrophysiological techniques have greatly improved our understanding of how changes in the relative strength of synapses can contribute to various important functions mediated by the nervous system. Genetically encoded optical reporters and actuators have added powerful cell-type specificity to this analysis. However, interpretation of electrophysiological data requires careful attention to a number of parameters, including voltage-dependence, ionic flux, and experimental conditions. When possible, multiple experimental techniques should be employed to evaluate all possible loci of action. Together, convergent lines of evidence can reveal novel effector sites, and lead to re-evaluation of traditional interpretations and conclusions.All authors contributed to the writing of this review article. The initial draft was written by SG, RM and JM in consultation with ER and TK. It was edited for accuracy and clarity by TK and ER. The summary figure was created by JM with feedback from the other authors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The purpose of this review is to provide a comprehensive overview of the findings of clinical electrophysiology studies aimed to investigate changes in information processing of migraine with aura patients.Abnormalities in alpha rhythm power and symmetry, the presence of slowing, and increased information flow in a wide range of frequency bands often characterize the spontaneous EEG activity of MA. Higher grand-average cortical response amplitudes, an increased interhemispheric response asymmetry, and lack of amplitude habituation were less consistently demonstrated in response to any kind of sensory stimulation in MA patients. Studies with single-pulse and repetitive transcranial magnetic stimulation (TMS) have reported abnormal cortical responsivity manifesting as greater motor evoked potential (MEP) amplitude, lower threshold for phosphenes production, and paradoxical effects in response to both depressing or enhancing repetitive TMS methodologies. Studies of the trigeminal system in MA are sparse and the few available showed lack of blink reflex habituation and abnormal findings on SFEMG reflecting subclinical, probably inherited, dysfunctions of neuromuscular transmission. The limited studies that were able to investigate patients during the aura revealed suppression of evoked potentials, desynchronization in extrastriate areas and in the temporal lobe, and large variations in direct current potentials with magnetoelectroencephalography. Contrary to what has been observed in the most common forms of migraine, patients with familial hemiplegic migraine show greater habituation in response to visual and trigeminal stimuli, as well as a higher motor threshold and a lower MEP amplitude than healthy subjects.Since most of the electrophysiological abnormalities mentioned above were more frequently present and had a greater amplitude in migraine with aura than in migraine without aura, neurophysiological techniques have been shown to be of great help in the search for the pathophysiological basis of migraine aura. During the last 50 years, researchers dedicated their projects to the understanding of neurophysiological peculiarities of the migraine brain which might predispose to the recurrence of migraine attacks. This implies that most of the possible electrophysiological signatures of these subtle underlying factors were detected between migraine attacks, fluctuating depending on the distance from the last or the next attack. Moreover, even though amongst the migraineurs those who experience aura (MA) exhibit more pronounced clinical manifestations, these patients have been less frequently studied from a neurophysiological point of view. This is, by the way, due to its lower prevalence in comparison to the commonest migraine without aura (MO) and because of the short duration of the aura phase. In fact, focal neurological symptoms which precede or accompany the headache phase (when presents), last no more than 60 min with visual - the most common aura symptom - followed by sensory and aphasic auras , 2. HoweThe electrocortical phenomenon of cortical spreading depression (CSD) has been implicated in the genesis of migraine aura: it is a wave of neuronal hyperactivity followed by a wave of hypoactivity which often spreads postero-anteriorly and can reach the parietal and/or temporal lobes travelling at a speed of approx. 3 mm/min . After tTo better understand aura-related changes in sensory processing, several independent research groups have dedicated to the study of electrocortical signals during different phases of the migraine cycle using different sensory stimuli, or single or repetitive neuromodulatory techniques delivered over the scalp. Interestingly, none of the published studies assessed patients suffering exclusively from migraine with aura, at least with respect to the most common episodic forms of migraine. This has happened not only because patients suffering exclusively from migraine attacks preceded by aura are difficult to find, but also because for many authors the two conditions of MO and MA are variable clinical manifestations of substantially the same genetic disorder .The purpSeveral decades passed since the pioneering electroencephalographic studies emphasizing abnormal electrocortical activities in migraine . During Enhanced PD of EEG during intermittent photic stimulation using fast Fourier transform analysis on steady-state visual evoked potentials (SS-VEPs), the so-called H response, was more prevalent in migraine patients than in healthy controls. Researchers observed that the fundamental components of the EEG spectra were increased equally in both MA and MO , 14, prespontaneous electroencephalographic activity showed alpha rhythm and peak frequency asymmetries over the posterior regions, increased power of alpha rhythm [During the interictal period of MA patients, quantitative analysis of a rhythm , and wida rhythm  and theta rhythm , 19 totaa rhythm  or unilaa rhythm , mostly a rhythm . MA patia rhythm , 22. In a rhythm . When usa rhythm . Using ma rhythm . It is ia rhythm –27.In summary, resting electric and magnetic activity may help to better differentiate MA from MO patients than PD.With the help of cortical evoked potentials, higher cortical response amplitudes, an increased interhemispheric response asymmetry, and a deficit of response amplitude decrement were demonstrated by using different types of sensory stimuli and techniques in most of the MA patients.Because in most cases the aura is visual, most of the published studies investigated visual evoked potentials (VEPs) to search for cerebral signatures associated with migraine aura. By analysing the evoked responses in a classical way of averaging a large quantity of trials, mainly increased amplitudes of steady-state (SS) or transient VEPs have been discovered in MA patients during attack-free intervals.In some reports the grand-average of VEP N75-P100 and/or P100-N145 amplitudes has been found greater in MA patients than in controls –33 and/oDecreased amplitude of the prerolandic component (N20) of somatosensory evoked potentials (SSEPs) in both MO and MA patients has been found in one study , but ampMost of the researchers who recorded short-latency brainstem auditory evoked potentials (BAEP) were not able to find any interictal abnormalities in migraine, probably because they pooled patients with different migraine phenotypes (MO and MA or different MA subtypes) in different proportions in a single group (see Table 5 in ). HigherTo sum up, using EPs and ERPs, researchers found that the frequently reported increase in grand-average neural response to any kind of sensory stimuli in MA group is conceivably due to deficient short-term and long-term adaptive processes to external stimuli.Asymmetric neural activities in steady-state VEP amplitude, transient VEP P100 amplitude distribution and in N70 components were detected by some, both related , 54, 55 Similar to the results of VEP and SSEP studies, in one study mean interhemispheric asymmetries of all BAEP peak latencies (except peak IV and VI) were significantly increased in MO and MA patients as compared to those of the control group, despite the fact that the MA group included hemiplegic, and brainstem migraine . This daAnalysing discrete blocks of small amounts of traces, authors found that during repetitive and stereotyped stimulus presentation, VEP amplitudes augmented progressively instead of diminishing (i.e. they lacked habituation) equally in MO and, sometimes even more so, in MA patients between attacks , 61–63. Since in MA patients, different aura phenotypes may be underpinned by different pathophysiological mechanisms, we studied VEP amplitude and habituation in a subgroup of MA with exclusively visual auras and another with visual aura followed by somatosensory and/or dysphasic complex neurological auras . We founIn accordance with VEP studies, a significant habituation deficit has been detected interictally in MA recording SSEPs  and audiStudies with single-pulse and repetitive transcranial magnetic stimulation (TMS) have reported abnormal cortical responsivity revealed as greater motor evoked potential (MEP) amplitude, lower threshold for phosphenes production, and paradoxical effects in response to both depressing or enhancing repetitive TMS (rTMS) methodologies, predominantly in migraine with aura. Magneto-phosphenes measurements of MA patients were significantly lower - revealing higher excitability levels - than healthy controls measurements in most of –77, but In sum, both the paradoxical rTMS response and habituation deficit point to altered synaptic plasticity mechanisms, which prevent the immediate and longer-lasting cortical changes that reflect adaptation to repeated stimulations, i.e. learning and memory. Further studies are needed to verify whether these aberrant ways of responding of the cortex to neuromodulation are related to abnormal thalamic control  or to a Even though brainstem trigeminal nuclei are well knowingly deeply involved in the pathophysiology of migraine without aura, the studies of the trigeminal system in MA are still sparse.Perrotta et al.  studied With the scope to correlate interictal neurophysiological abnormalities of migraine, especially with aura, with a specific genotype, researchers recorded single-fibre electromyography (SFEMG) to explore neuromuscular transmission, as a surrogate biomarker of presynaptic P/Q Ca2+ channels function, in a wide range of migraine aura subtypes. Abnormal finding on SFEMG reflecting subclinical disfunctions of neuromuscular transmission have been detected in patients suffering from MA in between attacks. Patients with unilateral sensorimotor symptoms and/or visual scotoma, other aura symptoms such as sensory/motor disturbances, and/or aphasia, and/or vertigo had noticeable abnormal SFEMG , 95. TheIn one pilot study, the mild single endplate abnormalities detected by SFEMG in 3 MA patients disappeared during acetazolamide treatment in parallel with clinical improvement .So far, few studies during the transient phase of migraine aura have been performed.During visual aura and/or early headache phase, either mild asymmetry of slow waves in the fronto-temporo-occipital areas contralateral to the visual field defect disappearing during the headache phase –101 or nIn a MA patient spontaneously experiencing a scintillating scotoma in the right hemifield, MEG recording showed alpha rhythm event-related desynchronization in the contralateral extra-striate and temporal cortex for the duration of the focal visual symptoms, and gamma band desynchronization peaking 10 min following the aura  and abnoDuring the visual aura the hemisphere contralateral to the field defect showed suppression or complete abolition of the first three components of the flash VEPs  and of tChen and colleagues  showed tElectroencephalographic abnormalities during acute attacks of hemiplegic migraine are often described. During long-lasting attacks of hemiplegic migraine, unilateral or bilateral delta EEG activity – sometimes spreading postero-anteriorly  – and reN = 5; FHM-2 N = 4) and in a group of seven healthy controls [Adult and adolescent patients with brainstem aura (previously termed basilar type migraine) with disturbed consciousness may have severe clinically relevant EEG-slowing or generalized spike and wave complexes that may last for several days –127. Hancontrols . Contrarcontrols .In one study, a group of ten patients with FHM showed basically higher resting motor threshold, longer central conduction time, and lower MEP amplitude on the ictally paretic side than on the non-affected side, while MEP amplitude were significantly increased in a group of MA .Quantitative EEG rather consistently reported enhanced interictal photic driving, so called “H-response”, as well as excess of slow and hyper synchronized alpha rhythmic activity.Less consistently, EP and ERP studies showed many cases of cortical hyper-reactivity to sensory stimuli, including cognitive ones. When present, this heightened cortical response in MA was even more pronounced than in MO.Both lack of sensory habituation, of cortical inhibition, and paradoxical responses obtained after non-invasive brain neuromodulation, such as increased or decreased responses respectively to inhibiting or activating TMS, are commonly observed in MA. Like in MO, interictal abnormal cortical information processing in MA may depends on the time evolved since the last attack.Because the aura has numerous and varied clinical features, it may not be a single entity, but correspond to a spectrum of clinical subtypes that probably differ from a pathophysiological point of view. In fact, neurophysiological patterns distinguish between patients who experience pure visual auras from those with prolonged, somatosensory, dysphasic or motor, auras.Few researchers were able indeed to study patients during an aura. From a functional point of view, they detected unilateral disturbances of cortical electrogenesis – which might reflect an underlying metabolic abnormality  –, desynFew reports in FHM support the concept that various pathophysiological aspects differ between FHM and MO/MA, including cortical and brain stem responsiveness.There is no common agreement yet about what causes and where the cascade of events that lead to the focal neurological symptoms of migraine aura starts and, in most but not all the cases, about its link to the headache phase. However, experimental evidences point towards sequential activation of first-order or second-order trigeminovascular nociceptors via CSD waves . More liWe hypothesized that the neurophysiological pattern which characterizes MA patients of an abnormal cortical rhythmic activity, an increased cortical responsivity, and deficient lateral inhibition may be ascribed to a “thalamo-cortical dysrhythmia” (TCD) , that isIn summary, there are few neurophysiological features peculiar to the brain of patients with migraine with aura, such as frequent detection of an increase in amplitude to evoked potentials and peculiar abnormalities of functional connectivity at the EEG during resting-state. However, in most cases all the electrophysiological abnormalities – even those in common with MO – are more pronounced the more numerous and intense focal neurological symptoms characterize the aura. Intuitively we can say that most of the neurophysiological characteristics are certainly common to migraine patients with and without aura because most patients with migraine with aura also experience migraine attacks without aura . On the 215O-PET scanning [Whatever the peculiar physiological characteristics of the migraineur with aura brain may be, taken alone it is not sufficient to explain all features of the migraine attack. In many patients some migraine-related symptoms may also be present during the intercritical period, and premonitory symptoms, associated with hypothalamic, brain stem and various cortical activations revealed on Hscanning  may occuSupplementary studies are needed to clarify the exact relation between the electrocortical phenomena found outside the aura phase and during the aura itself. Studies correlating aura frequency and duration of the disorder with thalamic/thalamocortical activity in MA are necessary to test whether an abnormal cross-talk between the cortex and the thalamus – the latter area activated by CSD in animal models  –, could


N = 65 non-clinical females to focus on the nose of a photographed face at pre- and post-test. In between, participants gazed at the nose of either the same (relevant gazing) or another face (irrelevant gazing). We found increasing dissociation after gazing in both conditions and a differentially stronger decrease of attractiveness ratings in the relevant gazing condition. Our findings support the hypothesized effect of gazing rituals on attractiveness evaluation in cognitive-behavioral models for BDD.Rituals, such as gazing at faces, are common in body dysmorphic disorder (BDD) and appear in cognitive-behavioral models as a maintaining factor. Rituals are also common in obsessive-compulsive disorder (OCD). In contrast to OCD, the proposed associations between rituals and intrusive thoughts/appearance preoccupation have not been empirically investigated for BDD. We examined if the assumed effect of gazing rituals on attractiveness ratings exists and if it is associated with dissociation. In an experiment, we asked  Individuals with body dysmorphic disorder (BDD) are preoccupied with perceived defects or flaws in their appearance, which are mostly related to the face and not or only slightly noticeable to others. Individuals with BDD experience different levels of insight concerning their beliefs about the perceived defects, ranging from good insight to delusional beliefs . RitualsCognitive-behavioral models of BDD include gazing rituals as a relevant component –8 and a As BDD and OCD share common features, such as intrusive thoughts and rituals ,11, it iFor OCD, effects of rituals have been empirically investigated for different kinds of rituals. The findings imply that OC rituals follow a paradoxical pattern of increasing uncertainty, although they are performed with the intention to reduce that very same uncertainty. For example, our colleagues ,14 showeFor the purpose of our current study, the findings of van den Hout and colleagues  are of pThe results of previous research indicate abnormalities regarding visual perception and appearance evaluation in BDD: based on self-report questionnaires, our colleagues found that individuals with (vs. without) BDD spend significantly more time on looking at others’ specific feature(s) . The autUp to now, the study findings on differences in attractiveness ratings between individuals with and without BDD are inconsistent. One study showed that individuals with (vs. without) BDD did not differ in their attractiveness ratings of other people’s faces . In contn = 16), they found that appearance-dissatisfied women rated unfamiliar faces significantly more attractive after gazing then pre-gazing, and in contrast rated their own face significantly more unattractive after mirror gazing. In contrast, appearance-satisfied women showed no changes in attractiveness ratings for the unfamiliar face and significantly higher attractiveness self-ratings after mirror gazing .4.There were no significant main or interaction effects on the two factors [In this experimental study, we investigated effects of a BDD-like gazing ritual in non-clinical females to evaluate potential associations between such rituals and the preoccupation with appearance, as proposed in cognitive-behavioral models for BDD . We examAs hypothesized, 10 min gazing led to higher levels of dissociation. We found this increase in dissociation from pre- to post-gazing across both conditions (relevant and irrelevant gazing).Our finding on dissociation replicates the effect of increasing dissociation after staring at objects , althougBF of our findings neither speaks in favor of nor against the interaction effect, it remains unclear, if  changing visual input during gazing influences dissociative experiences. However, as gazing rituals seem to cause dissociation, the question emerges, if this causes further negative consequences, for example with regard to the appearance/attractiveness preoccupation.Considering that the Gazing was followed by lower attractiveness ratings of the presented face. This effect was even stronger if the rated face was the same than during the 10 min gazing period (relevant gazing). Our results suggest that gazing at a face per se lowers attractiveness ratings. However, gazing at a face most strongly decreases the attractiveness rating of the very same face. This finding might be especially helpful in explaining the severe effects of gazing at oneself, e.g. during mirror gazing, on the self-evaluation of attractiveness.Remarkably, participants’ confidence in their ratings did not change after gazing, that is participants irrespective of condition felt equally confident in their attractiveness evaluation pre- and post-gazing. This finding suggests that even though the decrease in attractiveness ratings after gazing is not based on any changes in objective matters (they rated the same photograph), the subjective devaluation of the rated face nonetheless feels real and reliable for the individual. In conclusion, individuals may believe in their devaluation of appearance after gazing which is likely due to a misperception (probably based on dissociation).However, one study revealed contrary effects with higher (rather than lower) attractiveness ratings after 3.5 min gazing at a facial photograph . There wOur results on attractiveness ratings indicate that gazing might not only, as BDD models assume ,7, lead BFs. On the one hand, these findings may seem surprising, because van den Hout and colleagues found an increase in perceptual uncertainty (main effect), especially during relevant staring at objects [We did not find any significant effects on uncertainty of perception nor evidence for those effects as concluded from the corresponding  objects . Thus, tprevent harm, inconclusive perceptions of potentially dangerous objects (e.g. stoves) are most likely to trigger uncertainty and thus fan the fear of making mistakes. In BDD, gazing (at faces) is used to investigate attractiveness (other than harm prevention). Inconclusive perceptions of faces may therefore most likely impact attractiveness evaluation and have thus the potential to fan preoccupation with appearance. Based on this hypothesis, study findings may follow the same pattern: Staring rituals and gazing rituals both lead to an increase, rather than a decrease, in preoccupation with pathological fears. This assumption is in line with current BDD models, which do address preoccupation with appearance instead of uncertainty [On the other hand, the inconsistent findings in OCD-related staring vs. BDD-related gazing may become consistent if we propose that the core construct ‘uncertainty’ in OC-related rituals corresponds to the ‘evaluation of attractiveness’ in BDD-related rituals. Since staring (at objects) in OCD is used to become certain about one’s perception to ertainty ,7.accurate view of the feature. This finding might correspond to the concept of insight. Individuals with BDD often show a lack of insight, that is they are convinced of their accurate and undistorted view of the perceived flaw or defect [Further, the unaffected confidence in participants’ attractiveness ratings pre- to post-gazing implies that gazing at appearance-related stimuli does not lead to doubts about an r defect ,42. Compr defect . In corrr defect , but didWe conclude from our findings of an increase in dissociation after gazing and a decrease of attractiveness ratings, most strongly after relevant gazing, that gazing rituals might have a causal effect on the evaluation of attractiveness. This effect might be explainable by dissociative experiences during gazing.One could argue that dissociation during gazing rituals might impair the accurate processing of the stimulus, for example because it is perceived “unreal or dreamlike”, and therefore prevents a valid memory representation of the stimulus. An inaccurate memory representation of the stimulus might affect the evaluation of other stimulus-relevant dimensions, for example the evaluation of attractiveness in the context of facial stimuli. In this case, gazing at faces increases perceptual inaccuracy, but unlike OC-staring this inaccuracy does not impact the subjective certainty about one’s perception.The hypothesized negative effect of gazing and dissociation on the attractiveness evaluation of an observed body part might have important implications for the therapeutic work with individuals with BDD. In this case, one may reduce gazing at others’ as well as at oneself as part of ritual prevention. Provided that this negative effect similarly occurs in gazing at own features, mirror exposure tasks may be conducted in a way that prevents gazing and, thus, dissociation. One treatment manual recommends perceptual retraining as one component in the treatment of BDD . Even thThere are possible study limitations that need to be addressed. We focused on BDD-like gazing at others, which is one of the most frequent rituals in BDD . EspeciaWe examined a non-clinical sample and did not use stress induction pre-gazing to imitate distressing emotions, which are known to trigger engaging in gazing rituals . Note thIn conclusion, this is the first study to investigate the experimental effects of gazing on dissociation and the evaluation of attractiveness. Our findings provide empirical support for the proposed associations in cognitive-behavioral models of BDD  and offeS1 FigHigher values indicate a) higher dissociation, b) higher attractiveness ratings, c) lower reliability and clarity of perception, i.e. higher uncertainty of perception. Error bars indicate 95% confidence intervals.(DOCX)Click here for additional data file.


Prolonged fasting promotes stress resistance, but its effects on longevity are poorly understood. Calorie restriction or major dietary composition changes can have profound effects on healthy aging but the inability of many subjects to adhere to chronic and extreme diets together with the potential of adverse effects limit their application. Fasting-mimicking diets (FMDs) are effective in increasing health and lifespan, possibly by inducing stem cell-based regeneration, or as therapies in mouse models of a variety of diseases. FMDs reduce cancer incidence/progression, modulate the immune response, reduce immuno-senescence, ameliorate or reverse disease progression of multiple sclerosis, Type I and Type II diabetes, and reverse inflammatory bowel disease pathology. In a randomized clinical trial, markers/risk factors for metabolic syndrome and other age-related diseases were favorably impacted after completion of 3 FMD cycles. These effects were larger in participants at risk for age-related diseases. Conclusions: We conclude that the FMD was safe and feasible in rodent and human studies. Larger studies on patients with diagnosed diseases are necessary to determine its impact on diabetes and cardiovascular disease treatment.