During the initial months of the COVID-19 pandemic, rapidly rising disease prevalence in the United States created a demand for patient-facing information exchanges that addressed questions and concerns about the disease. One approach to managing increased patient volumes during a pandemic involves the implementation of telephone-based triage systems. During a pandemic, telephone triage hotlines can be employed in innovative ways to conserve medical resources and offer useful population-level data about disease symptomatology and risk factor profiles.The aim of this study is to describe and evaluate the COVID-19 telephone triage hotline used by a large academic medical center in the midwestern United States.Michigan Medicine established a telephone hotline to triage inbound patient calls related to COVID-19. For calls received between March 24, 2020, and May 5, 2020, we described total call volume, data reported by callers including COVID-19 risk factors and symptomatology, and distribution of callers to triage algorithm endpoints. We also described symptomatology reported by callers who were directed to the institutional patient portal .A total of 3929 calls  were received by the call center during the study period. The maximum total number of daily calls peaked at 211 on March 24, 2020. Call volumes were the highest from 6 AM to 11 AM and during evening hours. Callers were most often directed to the online patient portal , nursing hotlines , or employee health services . Cough , shortness of breath , upper respiratory infection , and fever  were the most commonly reported symptoms. Immunocompromised state  and age >65 years  were the most commonly reported risk factors.The triage algorithm successfully diverted low-risk patients to suitable algorithm endpoints, while directing high-risk patients onward for immediate assessment. Data collected from hotline calls also enhanced knowledge of symptoms and risk factors that typified community members, demonstrating that pandemic hotlines can aid in the clinical characterization of novel diseases. COVID-19 emerged in December 2019 as a highly contagious viral pneumonia caused by SARS-CoV-2 ,4. The gThe rising prevalence of COVID-19 in the United States  created One approach to managing increased patient volumes during a pandemic involves the implementation of telephone-based triage systems. Triage and evaluation phone lines established during previous pandemics were found to be cost-effective methods for prioritizing acute care resources for symptomatic patients with risk factors for serious disease in overburdened hospital systems -11. A stTo this end, we aimed to evaluate the COVID-19 triage hotline established by Michigan Medicine, the academic medical center affiliated with the University of Michigan, which serves the population of Southeast Michigan and surrounding areas. We characterized the call center’s triage algorithm and described the distribution of all calls fielded over a 6-week interval during the initial surge of the pandemic. We then contextualized hourly and daily call volumes against publicly available internet search trends concerning COVID-19–related inquiries. Lastly, we tabulated symptomatology and risk factor profiles for patients who accessed call center services during the 6-week period to provide perspective on local disease burden. Our results illustrate the utility of telephone-based triage and evaluation hotlines in optimizing health care utilization and surveying disease burden during times of crisis that impact human health.Michigan Medicine established a telephone hotline to triage inbound patient calls related to COVID-19. Operations commenced on March 16, 2020, and accepted calls daily from 6 AM to 12 AM. The hotline was staffed both in person and remotely by call center agents, medical assistants, patient service representatives, and medical student volunteers (herein termed as “agents”). The call center used Aspect Call Center software (Aspect) to receive and manage calls. Call center agents used an algorithm developed by the Michigan Medicine Ambulatory Care Administration to triage incoming calls with screening questions and defined endpoints. Screening questions were updated over the duration of the study to accommodate the changing health landscape of the pandemic. Symptom screening criteria were initially limited to fever, cough, and dyspnea. Criteria were expanded to include upper respiratory symptoms (runny nose and congestion), muscle aches, loss of sense of smell, and diarrhea on April 20, 2020. A positive symptom screen was defined as having at least two symptoms from this list. Risk factor criteria initially included age over 65 years, compromised immune system, chronic respiratory illness, presence of a health care worker in the household, hemodialysis patient, resident of a skilled nursing or long-term care facility, and/or living in communal housing. Risk factor criteria were expanded to include primary caretaker for a vulnerable individual and/or close contact of an individual who tested positive for COVID-19 on April 20, 2020. A positive risk factor screen was defined as having at least one risk factor from this list.. The triage survey directed callers toward various algorithm endpoints: Occupational Health Services, non–Michigan Medicine primary care provider or the Centers for Disease Control and Prevention website, patient portal e-visit questionnaire, and adult or pediatric nursing hotlines. Partially completed Qualtrics survey responses were discarded from downstream analysis. The second survey was completed by a subset of call center workers who were volunteers during evening shifts. This survey was created in Google Forms, an additional online survey platform. The Google Forms survey collected additional data about symptomatology and risk factors that were not included in the Qualtrics survey. Qualtrics and Google Forms survey data were merged to produce a composite data set at the end of the study period. We used an additional data source, the Aspect Unified IP System, which automatically logged all telephone calls to our institution’s call center hotline. We used this source to cross-check data on call volumes extracted from the Qualtrics and Google surveys.We used two surveys to collect data for this analysis. The first was created in Qualtrics, an electronic survey platform. Call center workers filled out a Qualtrics survey when they handled each call to the call center. From these results, we extracted call volume, patient employment status , patient status (Michigan Medicine versus non–Michigan Medicine patient), pediatric or adult, symptom profile, and risk factor dataTo contextualize how call volume was related to public interest in the pandemic and disease prevalence, we accessed publicly available data through two sources: (1) Google Trends and (2) the State of Michigan online coronavirus dashboard . We compIn tandem with the creation of a call center to assess patients, Michigan Medicine created a “Cough, Flu, and COVID-19–like Symptoms” patient portal e-visit questionnaire. Patients calling the triage hotline whose symptoms or risk factor profile did not meet criteria to be transferred to the nursing line were directed to complete this e-visit questionnaire in their patient portal. The questionnaire assessed symptom frequencies, symptom onset and duration, and epidemiologic and demographic risk factors. Once completed, a provider would reach out to the patient to determine appropriate next steps in care.Data were collected between March 24, 2020 (8 days after the triage hotline began operation) and May 5, 2020. Total call volume and the total number of calls directed to each triage algorithm endpoint by date and time of day were calculated to assess hotline workload. Symptom frequency was subsequently overlaid on total call volume for the call center and the total number of calls in which patients screened positive for symptoms. Google Forms survey data and the “Cough, Flu, and COVID-19–like Symptoms” patient portal questionnaire responses were analyzed to determine the frequency of patient-reported symptoms and epidemiologic risk factors among both hotline callers and online portal users. Data representation and statistical analysis was completed using Prism . The study was reviewed and approved by the local institutional review board (HUM00179879).Upon establishing a staffed triage hotline, calls were processed through the screening algorithm, which directed patients to specific algorithm endpoints .r2=0.85) suggests that survey responses provide a reasonable representation of call volume received by the triage hotline.To establish that our survey data provided an accurate representation of our call volume, we compared daily call volume to the number of calls routed to the hotline number through Aspect Unified IP, which automatically logged all calls to the call center hotline A. AlthouA total of 3929 calls  were received by the call center during the study period. Overall, call volume gradually declined over time but showed substantial variation between days throughout the study period B. The diWe were interested to see whether call volume and the fraction of callers experiencing symptoms was reflective of the public interest in the pandemic and locoregional case volume. Google Trends frequencies for COVID-19–related searches for the state of Michigan and the Detroit area were highest in late March and gradually decreased until the end of data collection in early May A. ConfirWe next analyzed Google Forms survey data to determine the frequency of patient-reported symptoms and the distribution of epidemiologic risk factors among callers. These represent the subset of callers who were Michigan Medicine patients and contacted the call center between 6 PM and 12 AM. Cough , shortness of breath , upper respiratory infection , and fever  were the most commonly reported symptoms A. A minoFurther characterization of patient-reported symptoms and epidemiologic risk factors was achieved through “Cough, Flu, and COVID-19–like Symptoms” questionnaire responses. Questionnaire data revealed that nonproductive or productive cough were the most frequently reported symptoms among both adult  and pediatric  patients . Muscle In this study, we illustrated an approach to rapid development and implementation of a telephone-based triage system during the COVID-19 pandemic. Although call volumes fluctuated considerably throughout the study period, we observed a gradual decline in total calls received over time. We noted that referral to the patient portal or transfer to the nursing hotline for further assessment constituted more than 75% of triage decisions. Google Trends data for COVID-19–related terminology and symptoms in Michigan and the Detroit metropolitan area decreased from late March to early May 2020, mirroring the declining trend in call volumes noted at the call center. Callers most frequently reported the following symptoms: cough, shortness of breath, upper respiratory infection, and fever. In addition, the following risk factors for infection were most frequently reported: immunocompromised, age greater than 65 years, and exposure to another individual who tested positive for COVID-19. Analysis of patient questionnaire data revealed that cough was the most frequently reported symptom by adult and pediatric patients, but the frequency of symptoms thereafter differed between these groups. Together, these results demonstrate how patient hotlines and triage systems can be used to properly allocate care and broaden our understanding of disease characteristics during public health crises.Responding effectively to a global pandemic necessitates judicious use of resources and regular assessment of disease burden. Tools with this dual capability are positioned to meaningfully contribute to pandemic response efforts. Specifically, triage and evaluation phone services are well-equipped to manage and survey patient populations when deployed by health systems during pandemics. Characterization of the Michigan Medicine COVID-19 Hotline validated this notion. The call center successfully directed call traffic away from the resource-intensive nursing endpoints while capturing population-level statistics on disease symptomatology and prevalence. This was accomplished by direct triage and surveillance by call center agents as well as questionnaire-based surveillance through a pre-existing online portal. Only 17% of the total call volume was directed to the nursing hotlines, while 21% of calls were directed to the online patient portal. Cough was identified as the most common symptom by triage agents as well as questionnaires, coinciding well with prior reports on COVID-19 symptomatology . Taken tPrior triage hotlines have provided population-level statistics about disease symptomatology and prevalence while directing resource allocation . During Designing the triage algorithm to leverage an online patient portal not only reduced nursing utilization but also unlocked local care capacity without compromising social distancing measures designed to reduce viral transmission. Previous work has demonstrated that call centers can also benefit a community beyond their primary role as a triage service. For instance, South Australia’s COVID-19 Relief Call Centre assisted callers with food insecurity and accessing medical appointments and welfare checks . Call ceNevertheless, a number of operational challenges place constraints on the value of call centers. Hotline staff do not perform physical exams, which are often necessary to properly evaluate the cardiopulmonary complaints that are characteristic of COVID-19. Maintaining social distancing standards and properly sanitizing shared workspaces can also pose a challenge to call center operations during pandemics . To circA number of operational constraints placed limitations on the scope of our study. First, the Google Forms survey designed to screen symptomatology and risk factors was only completed for evening callers. Specific symptoms or risk factors associated with the time frame in which calls were received may have confounded our results. Second, due to the structure of the triage algorithm, we were only able to present symptomatology and risk factor data for a small subset of patients. Further, since the triage algorithm screened callers as positive if they had two symptoms or one risk factor, it is possible that additional symptoms or risk factors were not captured once a caller had screened positive. Third, not all callers had questions or requests that fit perfectly into the triage algorithm. For these calls, call center workers may have skipped the online survey  or filled out the survey with their best approximation of the caller’s needs. Fourth, due to the deidentified nature of our data sets, we were unable to measure caller satisfaction. This would be a useful topic for further research. Finally, this study presents data from one institution, limiting the ability of our data to generate population-level inferences.In summary, our study describes the development and implementation of a COVID-19 patient triage hotline in a single health care system. The triage algorithm successfully diverted low-risk patients to suitable algorithm endpoints, while directing high-risk patients onward for immediate assessment. Data collected from hotline calls also enhanced our knowledge of typical symptoms and risk factors among community members, demonstrating that pandemic hotlines can aid in the clinical characterization of novel diseases. Although future innovation in the areas of triage algorithm design and remote work capabilities can certainly improve the operation of future pandemic hotlines, our work provides critical insight into the role that telephone-based triage systems play in facilitating health care delivery in times of crisis.


Plant virus nanoparticles (VNPs) are inexpensive to produce, safe, biodegradable and efficacious as treatments. The applications of r plant virus nanoparticles range from epitope carriers for vaccines to agents in cancer immunotherapy. Both VNPs and virus-like particles (VLPs) are highly immunogenic and are readily phagocytosed by antigen presenting cells (APCs), which in turn elicit antigen processing and display of pathogenic epitopes on their surfaces. Since the VLPs are composed of multiple copies of their respective capsid proteins, they present repetitive multivalent scaffolds which aid in antigen presentation. Therefore, the VLPs prove to be highly suitable platforms for delivery and presentation of antigenic epitopes, resulting in induction of more robust immune response compared to those of their soluble counterparts. Since the tumor microenvironment poses the challenge of self-antigen tolerance, VLPs are preferrable platforms for delivery and display of self-antigens as well as otherwise weakly immunogenic antigens. These properties, in addition to their diminutive size, enable the VLPs to deliver vaccines to the draining lymph nodes in addition to promoting APC interactions. Furthermore, many plant viral VLPs possess inherent adjuvant properties dispensing with the requirement of additional adjuvants to stimulate immune activity. Some of the highly immunogenic VLPs elicit innate immune activity, which in turn instigate adaptive immunity in tumor micro-environments. Plant viral VLPs are nontoxic, inherently stable, and capable of being mass-produced as well as being modified with antigens and drugs, therefore providing an attractive option for eliciting anti-tumor immunity. The following review explores the use of plant viruses as epitope carrying nanoparticles and as a novel tools in cancer immunotherapy. Nanomedicine is an emerging area of multidisciplinary research that has already shown promise of transforming into a disruptive innovative development . AlreadyNanomedicines includes a wide array of nanomaterials with particle size ranging from 1nm to more than 400 nm and are a remarkably diverse group of materials . They maImmunotherapy in cancer treatment simply refers to a strategy with the objective of galvanizing the immune system of the patient to resist the implanting of cancerous cells. There are several approaches to achieve the desired end. One approach involves the use of drugs known as “Immune checkpoint inhibitors, to block immune checkpoints . The chePlant virus-based nanoparticles (VNPs) have been explored as a unique class of nanocarriers for biomedical applications . In addiin vivo. Tobacco mosaic virus can assemble into VLPs without requiring its RNA genome carry a drug payload on the surface or to a limited extent, within the inner channel of the nanoparticle. Potato virus X, cannot self-assemble in the absence of its RNA genome, and thus can only carry a payload on the outer surface. Cowpea mosaic virus can be made to self-assemble into empty virus like particles in the absence of its RNA genome and can thus carry a payload both inside and outside of its protein shell  and Potato virus X (PVX), or icosahedral shaped, such as Cowpea mosaic virus (CPMV). Different shaped viruses respond differently as nanoparticles in shell . In thisin shell . We discin shell .Viruses are composed of outer protein shells which encapsulate the genomic material. The multiple copies of coat proteins that form the virus outer shell of viruses are collectively known as the capsid . PrimariPlant viruses have been used as virus like particles (VLPs) and virus nanoparticles (VNPs) as epitope display systems for vaccine production. VLPs are a subset of the VNPs but lack any nucleic acid genome, thus making them non-infectious. VNPs and VLPs based on plant viruses are both non-pathogenic to humans and biodegradable . VNPs anTobacco mosaic virus (TMV) is the most well-studied plant virus and was initially characterized in the 19th century. TMV can be easily produced and purified in bulk amounts and can be manipulated genetically due to its relatively simple particle structure and genome organization. The rod-shaped virus particle measures 300 nm in length and 18 nm in diameter and contains a 6.7 kb viral RNA genome that is encapsulated by 2,130 identical copies of the capsid protein assembled in a helical arrangement. At neutral pH and in the absence of RNA, the CP assembles itself into an 18 nm double disk, a 20S aggregate or nano-ring containing two layers of 17 CP molecules which can serve as a nanoscale scaffold.Potato virus X (PVX) is a member of the family Alphaflexiviridae, genus Potexvirus, an important plant pathogen of the family Solanaceae, and specifically infects potato, tomato and tobacco  Massumi. It has in vivo or in vitro, is not possible in the absence of genomic RNA. This reflects the unique connection between virus RNA and CP  belongs to the Comovirus genus. CPMV is an icosahedral shaped virus with a diameter of approximately 27 nm. It is composed of RNA-1 and RNA-2 of 6 and 3.5 kb, respectively packed in 60 copies each of Large and Small coat protein . CPMV isVLPs can act as strong vaccine candidates as they simulate the conformations of native viruses, utilizing their intrinsic immunogenicity without compromising their safety. VLPs evoke effectual immune responses as they are readily internalized by antigen presenting cells (APCs) and are ideal platforms for antigen processing and epitope presentation to immune cells. VLPs are composed of multiple copies of their capsid (coat) proteins, which when assembled appear as repetitive, multivalent molecular scaffolds. As a result, the multiple copies of coat protein can facilitate the multivalent presentation of antigens fused to their surface. Therefore, VLP vaccines afford superior immunogenicity compared to antigens in their soluble states. Additionally, plant viral VLPs and VNPs possess inherent adjuvant properties dispensing with the use of additional adjuvants to evoke strong immune responses .TMV VLPs have been utilized as epitope display systems in a variety of settings, with a first example being a polio vaccine by Staphylococcus aureus protein A fragments on its surface, and proved to be easily functionalized with IgG to be used in biosensing plant viruses. VLPs of Papaya mosaic virus (PapMV), of the Potexvirius family, has been engineered for use as a seasonal flu trivalent vaccine  from Staphylococcus aureus, which has been shown to be able to protect rats against endocarditis  effect depending on the type of tumor , computed tomography (CT), positron emission tomography (PET) and optical imaging, which enable the monitoring of molecular and cellular processes in normal and diseased conditions in living subjects.  is a promising technology for the diagnosis of disease due to its high resolution and deep contrast, however, virus-based nanoparticles have been used to increase sensitivity . TMV can barrier . A bimod barrier .Tobacco Mosaic Virus has been used successfully for CD imaging, targeting atherosclerosis and thrombosis by using an NIR dye as well as a targeting peptide conjugated to TMV [96]. These targeted TMV particles were able to identify atherosclerotic lesions in ApoE−/− mice upon intravenous injection, showing that TMV can be used as a platform to detect at-risk lesions.in vivo imaging. N. benthamiana plants. Significantly, fluorescent PVX could successfully be used for in vivo particle tracking in an HT-29 murine model, for in vitro imaging of HT-29 cells, and for tracing viral infection within plants.VNPs based on PVX have been conjugated to fluorescent reporters as well [135]. As mentioned earlier, the small fluorescent iLOV protein was expressed on the surface of PVX through genetic engineering and served as a fluorescent probe which could be of potential use in vivo imaging of tumors, CPMV-based VNPs have been successfully engineered to target specific tissues  and improved permeability with a retention effect that improves tumor penetration . For in  tissues . These t tissues .Clinical treatment for cancer has been routinely addressed by chemotherapy . RegardlPlant virus VLPs have several attractive features that make them appropriate for targeted administration of therapeutic molecules. The anti-cancer drug doxorubicin (DOX), has been successfully delivered using VNPs and VLPs. TMV- and PVX-derived VLPs and VNPs have been successfully used to deliver DOX . In thisTMV can be used as a carrier of peptides with therapeutic or targeting activity against various cancers. Trastuzumab is a cancer cell inhibiting monoclonal antibody that uses the binding sites of human epidermal growth factor receptor 2 (HER2). Trastuzumab-binding peptides (TBP) are immunogenic in nature and capable of initiating production of HER2-inhibiting antibodies to seize the growth of HER2-carrying cancer cells. TMV particles displaying TBP have been created to activate this immunogenicity . SimilarPlant virus VNPs have been used for targeted administration of platinum-based drugs against cancer. This is important as 50% of chemotherapy treatments involve the use of these platinum-derived drugs. TMV has been demonstrated to efficiently deliver Cisplatin  and Phenin vitro Karpas 299 non-Hodgkin’s lymphoma cell line with an IC50 of 250 nM.Another anti-cancer drug, mitoxanthrone (MTO), is a topoisomerase II inhibitor and has been shown to be encapsulated by TMV . VNPs exHelical plant virus nanoparticles (VLPs) have also been used as combination therapies to augment their immune efficacy. The PVX-DOX (doxorubicin)  combinatin vitro in HCC-38 primary ductal carcinoma, BT-549 ductal carcinoma and the MDA-MB-231 breast cancer cell lines -related apoptosis inducing ligand (TRAIL) on the surface of VNPs. Multivalent display of TRAIL enabled increased recruitment and stimulation of death receptors expressed on cancer cell lines (PVX displaying TNF related apoptosis inducing ligand (TRAIL) was used to promote the recruitment and activation of death receptors ll lines . In vivoll lines . SimilarAn efficient and new drug delivery system has been reported for Non-Hodgkin’s B cell lymphomas (NHL) based on PVX binding affinity towards malignant B cells. PVX loaded with monomethyl auristatin (MMAE) and administered to tissues harboring malignant B cells lead to inhibition of NHL growth in a mouse model . Jobsri PVX nanoparticles are increasingly being used for immunotherapy of tumor microenvironments. The monoclonal antibodies of Herceptin or Trastuzumab can be loaded onto PVX nanofilaments, which successfully induced apoptosis in breast cancer cell lines . PVX wasThe tumor microenvironment poses a great challenge to immune clearance by virtue of being immunosuppressive and favoring immune escape of the tumors through the inhibition of anti-tumor T-cells . CPMV VLThe icosahedral shape of CPMV capsid can be loaded with precise drug cargos to target tumor and cancer cells. CPMV VNPs have also been formulated as slow-release aggregates along with polyamidoamine generation 4 dendrimers (CPMV-G4) , where tCPMV VLPs have been attached to TAAs (tumor associated antigens) using chemical conjugation, genetic fusion and enzyme-mediated ligation techniques. For example, the human epidermal growth factor receptor 2 (HER2) epitope, when conjugated to the icosahedral CPMV, was successfully delivered to the lymphatic system with enhanced uptake and activation of APCs that led to an augmented anti-HER2 immune response. The CPMV HER2 candidate vaccine slowed tumor progression and metastasis in mouse models, enhancing survival . ImportaCancer vaccines against carbohydrate antigens associated with tumors (TACAs) could be useful for diminishing tumor progression. Nevertheless, carbohydrates are weakly immunogenic and therefore, plant viruses used as carriers of these molecules could enhance the immune response to TACAs. CPMV-TACA conjugates targeting the Tn antigen   were demThe chemotherapeutic cyclophosphamide, when used in combination with CPMV VNPs, profoundly elicited tumor cell death, releasing extracellular TAAs and stimulating immune cell invasion in addition to augmenting TAA recognition and antigen presentation  in mousein situ vaccine to stimulate an anti-tumor response and overcome local immunosuppression (The CPMV-DOX conjugate was developed using eighty molecules of the chemotherapeutic drug doxorubicin (DOX), covalently bound to carboxylates at the external surface of the CPMV nanoparticle. This drug delivery vehicle was found to be more cytotoxic than free DOX when used in low concentration, however, CPMV-DOX cytotoxicity is time-delayed at higher concentrations . Cancer pression . CPMV ispression . CPMV napression . Furtherpression .in situ tumor vaccine. Further studies by in vivo ( in vivo .The use of plant virus nanoparticles (VNPs) as drug delivery carriers for the treatment of infectious and chronic diseases including cancer are advantageous when compared with naked drugs . The mosIn the future, plant virus nanoparticles will need to be developed for high throughput manufacturing. This will require the dedication of facilities that can produce many grams of plant virus nanoparticles using tens of thousands of plants . Today,


Extracellular vesicles (EVs) are now understood to be ubiquitous mediators of cellular communication. In this review, we suggest that EVs have evolved into a highly regulated system of communication with complex functions including export of wastes, toxins and nutrients, targeted delivery of immune effectors and vectors of RNA silencing. Eukaryotic EVs come in different shapes and sizes and have been classified according to their biogenesis and size distributions. Small EVs (or exosomes) are released through fusion of endosome-derived multivesicular bodies with the plasma membrane. Medium EVs (or microvesicles) bud off the plasma membrane as a form of exocytosis. Finally, large EVs (or apoptotic bodies) are produced as a result of the apoptotic process. This review considers EV secretion and uptake in four eukaryotic kingdoms, three of which produce cell walls. The impacts cell walls have on EVs in plants and fungi are discussed, as are roles of fungal EVs in virulence. Contributions of plant EVs to development and innate immunity are presented. Compelling cases are sporophytic self-incompatibility and cellular invasion by haustorium-forming filamentous pathogens. The involvement of EVs in all of these eukaryotic processes is reconciled considering their evolutionary history. Within the context of animal extracellular vesicles (EVs) and their interactions, recent developments in the growing fields of plant and fungal EVs suggest complex cross-kingdom communication with microbes for infection/protection. The endomembrane system was discovered in the mid-1940s, aided by newly developed cell fractionation and electron microscopy (EM) techniques, leading the way to understand the traditional secretory pathway with the help of pancreatic exocrine cells Palade . In the Daucus carota) cell suspension cultures  were shown to secrete vesicles, termed lomasomes, that looked and behaved a lot like MVBs , Golgi and plasmalemma  leading to mEV biogenesis are not fully elucidated, but the process does seem to be dependent on an underlying stimulus. There may even be multiple biogenesis pathways depending on the stimulus, and mEV release may occur through either activation of cell death, whether apoptotic or necrotic  release is associated with membrane blebbing, which involves a redistribution of cellular contents, likely due to changes in volume-induced stress during cell death perhaps related to volume stress that occurs as cells die. ROCK-1 (Rho associated kinase 1), an effector of Rho GTPases, is essential for apoptotic membrane blebbing, although not all cells bleb, and is activated during mEV biogenesis  or blebbing of membranes (mEVs), their origin can be tracked by cell-specific protein markers. The rules governing the incorporation of different proteins into EVs are not known. These EVs also carry antigens expressed on the surface of the mother cell  that secrete apoplastic effectors via conventional secretion. IH also form biotrophic interfacial complexes (BICs) that accumulate cytoplasmic effectors via unconventional secretion.Conversely, definitive proof does exist for sEVs biogenesis from MVB in multicellular eukaryotes other than animals. The powdery mildew pathogen Vs Table   and the autophagy regulator Atg7 produce only sEVs, with authors suggesting these to be produced via unconventional secretion that bypasses autophagosomal and ESCRT/MVB pathways  mutant relative to wild-type plants.As in other eukaryotes, production of EVs in plants depends on the secretory pathway and involves the exocyst complex - and t-SNARE (PEN1)-positive putative sEVs were reported in et al. S-locus protein 11/S cysteine-rich ligand is transferred from the pollen coat to the stigmatic papilla cell carrying the corresponding S receptor kinase  to target fungal genes involved in virulence and secretion  found on the surface of macrophages and dendritic cells  also appears to be broadly released from professional antigen presenting cells when in the presence of EVs from C. albicans, Malassezia spp., T. interdigitale and Sporothrix brasiliensis , which can suppress monocytes, neutrophils and T lymphocytes  dissemination by providing protection from enzymatic degradation and opportunities for cell targeting  proteins necessary for silencing are localized  in length, and the presence of 21–24 nt long sRNA in EVs was challenged  has been developed as a novel alternative crop protection strategy against pathogens and pests  produces a primary germ tube after attachment to the barley leaf surface that penetrates the cuticle. This penetration event triggers H2O2 accumulation and formation of a papilla containing host cell wall appositions  contributes to basal penetration resistance of barley against the same pathogen , a subunit of the conserved oligomeric Golgi (COG) complex (HvCOG3) and a MVB/trans-Golgi network-localized ADP ribosylation factor (ARF) GTPase (HvARFA1b/c) are involved in resistance of barley against Bgh  of barley confer durable resistance against powdery mildew pathogens , which together with PUB23 and PUB24 down-regulates pathogen-associated molecular pattern (PAMP)-triggered immunity  DC3000 and obligate biotrophic downy mildew oomycete pathogen Hyaloperonospora arabidopsidis  host. Later stages of colonization by M. oryzae involve vesicle trafficking. Upon penetration of epidermal cells, the fungus forms IH that secrete apoplastic effectors like Bas4 via the conventional secretion pathway. IH form BICs in a newly infected rice cells. BICs accumulate cytoplasmic effectors like Pwl2 that are destined for translocation into host cells via an unconventional secretion pathway; this type of secretion is dependent on exocyst and t-SNARE complexes  species, the immune system stimulates inflammation, attracting cells to control infection. As plants cannot recruit cellular immune effectors, an invading hypha interacts with a solitary plant cell. In the case of biotrophic pathogens, such infection thus triggers programmed cell death of the challenged plant cell, in an attempt to stave off infection. With no cell-mediated immunity, plant EVs with antifungal properties, released into the apoplastic space, take up this role  of Gram-positives suggests that the last common ancestor of all three domains of life produced EVs  and Dawn Arnold (UWE Bristol) for inspirational discussions at BSPP meeting in 2019. John Labavitch (UC Davis) is posthumously acknowledged for his expertise and insights into the dynamics of plant cell walls and their variable pore sizes.


Immune checkpoint inhibitors are now approved for more than 50 indications, and increasing numbers of patients with advanced cancer are receiving immunotherapy. Immune-related adverse events that result from checkpoint inhibitors can affect any organ system. The most common kidney side effect is acute kidney injury, typically caused by acute interstitial nephritis. This review covers the most recent advances in immune checkpoint inhibitor-induced acute kidney injury. The review focuses on the differences between checkpoint inhibitor classes in causing acute kidney injury and differentiating immune checkpoint inhibitor-induced kidney damage from other causes of acute kidney injury. We describe the appropriate use of a kidney biopsy in the diagnosis of acute kidney injury and highlight the need for identification of noninvasive diagnostic and predictive biomarkers of immune checkpoint inhibitor-induced acute kidney injury. In the treatment section, approaches to corticosteroid use and the risks and benefits of rechallenging patients who experience acute kidney injury are debated. We also clarify the long-term adverse effects of immune checkpoint inhibitors on kidney function and the risk of chronic kidney disease in cancer survivors. Since the approval of the first immune checkpoint inhibitor (ipilimumab) for metastatic melanoma by the US Food and Drug Administration in 2011, 6 additional agents have been approved and their clinical use has expanded to more than 19 distinct cancers.,12,,,P = 0.007).,AKI is common in patients with cancer, and cohort studies suggest that AKI in patients receiving ICIs is more commonly caused by non–ICI-related causes than ICI-induced AKI. Studies from different centers from around the world indicate that the overall incidence of AKI in patients receiving ICIs is approximately 17%, while the incidence of ICI-related AKI is around 2%-5%.It should be noted that ICIs are increasingly being combined with conventional chemotherapy, targeted antineoplastic therapy, novel immunotherapies, or radiation. Combination of immunotherapy and traditional chemotherapy, such as triple therapy for lung cancer that includes carboplatin, pembrolizumab (a programmed cell death-1 [PD-1] inhibitor), and pemetrexed, poses important diagnostic challenges, as all 3 of these drugs may lead to AKI. Understanding the chances of toxicity and the specific lesions associated with each agent and performing a detailed work-up, including a kidney biopsy as appropriate, are needed to differentiate between nephrotoxicity associated with chemotherapy and ICI-induced AKI.,,,AIN is not the only kidney manifestation of ICIs. While AIN is the most common pathology seen with ICIs, (affecting >90% of patients who underwent biopsy in a large multicenter series),ICIs may reactivate pre-existing autoimmune diseases.,In addition, distal renal tubular acidosis can be a manifestation of incipient or persistent AIN. In some cases, distal renal tubular acidosis can persist despite improvement of kidney function while tapering corticosteroid therapy.,,,Solid organ transplant recipients are at increased risk of developing and dying from cancer in comparison to the general population.,,Not all ICIs carry the same risk of kidney disease. Cytotoxic T lymphocyte antigen-4 inhibitors (ipilimumab) and PD-1 inhibitors  were the first approved ICI classes. Initially, pooled data from clinical trials showed that the frequency of kidney disease was similar among these 2 classes of ICIs; Cortazar et al,Box 1Note: The decision between confirming an ICI-induced AIN diagnosis through a kidney biopsy versus empiric immunosuppression should weigh the individual’s risk of procedural complications against the side effects of inadvertent steroids in the case of a misdiagnosis.Abbreviations: AIN, acute interstitial nephritis; AKI, acute kidney injury; ANCA: antineutrophil cytoplasmic antibody; BMI, body mass index; ICI, immune checkpoint inhibitor; NSAIDS, nonsteroidal anti-inflammatory drugs; PPI, proton pump inhibitor; RRT, renal replacement therapy; SBP, systolic blood pressure.A kidney biopsy is the gold standard for diagnosing ICI-induced AIN or ICI-associated glomerular disease. However, current guidelines suggest empiric treatment of suspected ICI-induced AIN is acceptable if other causes have been ruled out and glomerular disease is not suspected.18F-fluorodeoxyglucose uptake in the renal cortex may be a valuable adjunct test to confirm ICI-induced AIN, especially in those with a baseline positron emission tomography scan for comparison.,,Currently, validated noninvasive markers for the diagnosis of ICI-induced AKI do not exist, and the use of many of the tools mentioned hereafter are limited to research studies. When biopsies are performed, additional stains to confirm ICI-induced AKI may be used to confirm association with ICIs; positive staining of tubular epithelial cells for PD-L1 has been shown to help differentiate PD-1-related AIN from other AIN.While the occurrence of an immune-related adverse event such as AIN may threaten to derail effective cancer treatment, emerging evidence suggests that immune-related adverse events may be associated with improved overall survival.Current guidelines for ICI-induced AKI are formulated by the Society for Immunotherapy of Cancer per Common Terminology Criteria for Adverse Events (CTCAE version 5.0) .54 For p,,Multiple series have shown that approximately 85% of patients with ICI-induced AKI who receive corticosteroids have full or partial remission . CortazaBox 2Note: Steroid-responsive AIN in the absence of extrarenal immune-related adverse events allows for a quick steroid taper and ICI rechallenge.Abbreviations: AIN, acute interstitial nephritis; AKI, acute kidney injury; ICI, immune checkpoint inhibitor; NSAID, nonsteroidal anti-inflammatory drug; PPI, proton pump inhibitor.Steroid-minimizing strategies that use a lower duration of therapy than current guidelines may be appropriate, particularly in patients who are concurrently receiving another AIN-associated medication, such as PPIs, which can be discontinued at the same time corticosteroids are initiated. In an observational study, Lee et alFinally, it is unclear if patients on ICIs with short half-lives and reduced duration of action such as avelumab may have better kidney outcomes with shorter steroid tapers when compared to those on drugs with longer half-lives , where the duration of checkpoint inhibition may last much longer.In ICI patients experiencing high-grade AKI, the ICI is typically interrupted until AKI resolves, and sometimes permanently discontinued. In the multicenter study by Cortazar et al,2 per year) was common and the incidence of new-onset chronic kidney disease or significant (>30%) estimated glomerular filtration rate decline sustained for >90 days occurred in 20% of survivors who lived at least 5 years.The long-term effects of ICIs on kidney function are not well known. It is well known that AKI is a risk factor for chronic kidney disease and progression to end-stage kidney disease. In a recent study of >2,500 patients treated with ICIs who survived at least 1 year, rapid estimated glomerular filtration rate decline (>3 mL/min/1.73 mManagement of ICI nephrotoxicity is multipronged and should place emphasis on cancer survival and the quality of life of patients. Future research should focus on the identification of sensitive and specific markers that can diagnose and predict ICI-induced AIN, the development of personalized safe and effective immunosuppression protocols, as well as strategies for ICI rechallenge and management of recurrent AKI. Further research is needed to determine the mechanisms of ICI-induced glomerular diseases and determine the best strategies for managing these rarer kidney complications. The long-term risks of ICIs on kidney function need to be better understood as the number of patients being treated with ICIs is rapidly growing; ICIs are now estimated to be indicated in 36% of patients with cancer.


The association between pre-treatment expectations and perceived pain approached significance (p = 0.057) in the relief phase. Our data from an experimental translational placebo intervention in visceral pain support that reported post-treatment medication efficacy is shaped by pre-treatment expectations. The observation that individuals with higher positive expectations reported less pain and higher treatment satisfaction after pain relief may provide first evidence that perceived symptom improvement may facilitate treatment satisfaction. The immediate experience of symptoms within a given psychosocial treatment context may dynamically change perceptions about treatment, with implications for treatment satisfaction, compliance and adherence of patients with conditions of the gut-brain axis.Placebo research has established the pivotal role of treatment expectations in shaping symptom experience and patient-reported treatment outcomes. Perceived treatment efficacy constitutes a relevant yet understudied aspect, especially in the context of the gut-brain axis with visceral pain as key symptom. Using a clinically relevant experimental model of visceral pain, we elucidated effects of pre-treatment expectations on post-treatment perceived treatment efficacy as an indicator of treatment satisfaction in a translational placebo intervention. We implemented positive suggestions regarding intravenous treatment with a spasmolytic drug , herein applied in combination with two series of individually calibrated rectal distensions in healthy volunteers. The first series used distension pressures inducing pain (pain phase). In the second series, pressures were surreptitiously reduced, modeling pain relief (pain relief phase). Using visual analog scales (VAS), expected and perceived treatment efficacy were assessed, along with perceived pain intensity. Manipulation checks supported that the induction of positive pre-treatment expectations and the modeling of pain relief were successful. Generalized Linear Models (GLM) were implemented to assess the role of inter-individual variability in positive pre-treatment expectations in perceived treatment efficacy and pain perception. GLM indicated no association between pre-treatment expectations and perceived treatment efficacy or perceived pain for the pain phase. For the relief phase, pre-treatment expectations ( Numerous studies on placebo effects in acute and chronic pain have demonstrated the pivotal role of treatment expectations arising within the psychosocial treatment context  –23. RepeN = 60 healthy participants who were at inclusion randomized (with a 2:1 randomization) to undergo an established placebo intervention consisting of positive drug-related treatment suggestions  or to receive no drug-related suggestions  prior to experiencing phasic visceral stimuli (details below). All volunteers were recruited de novo for this study and had to be naïve with respect to both the distension model as well as to any prior experimental placebo or nocebo study carried out by our research group. The recruitment and in-depth screening procedures consisted of an initial semi-structured telephone screening (conducted by author LR), followed by a structured personal interview and a brief general medical examination, including a digital rectal exam . Exclusion criteria included a body mass index (BMI) <18 or >30, age <18 or >45 years, any known medical or psychological/psychiatric clinical conditions, and any current medication use . Participants were also screened for self-reported substance abuse, including number of alcoholic drinks/week (>4/week led to exclusion), smoking (>10 cigarettes per week led to exclusion), and use of other recreational drugs (any reported use within past 3 months led to exclusion). Current anxiety or depression symptoms above the published cut-off values  on the Hospital Anxiety and Depression Scale (HADS)  , and free (HADS)  completee (HADS) . Ethics Experiments were conducted within a medical research setting within a clinical MR suite of the University Hospital Essen, Germany. On the study day see . Given hBased on results of thresholding, individualized pressures aiming to create two distinct stimulation intensities for subsequent application during two series of cued phasic rectal distensions were identified. In the first series, six painful visceral stimuli using distension pressures just below the individual pain threshold were implemented (pain phase). The subsequent second series consisted of six distensions at surreptitiously reduced pressures corresponding to just above the sensory threshold (relief phase). All distensions were cued by a visual signal, and the duration of each distension was 18 s. Pauses of complete balloon deflation in-between distensions were 18 s. Visual analog scale (VAS) were accomplished prior to and after each phase for expectation and efficacy ratings, and after each distension for perceived intensity (details below).N = 40 participants in the placebo group received positive information verbally and in writing, including detailed drug-related information. On the study day, before the i.v. drip was started, pertinent aspects of the positive instructions, were repeated verbally (“medication reminder communication”) by the study physician (author NT), especially focusing on the pain-relieving properties of the drug  received more detailed and personalized information .We implemented a previously established translational placebo intervention for the visceral pain modality to induce positive treatment expectations by suggestions, which we have previously applied in studies with healthy volunteers and patients with IBS and IBD –23. It edrug see . In lineN = 20 individuals were truthfully informed about administration of an inert substance. Specifically, this reference group was informed about i.v. administration of saline, and written and verbal instructions contained a specific mention of absence of active drug and lack of saline effects. The infusion bottle was clearly marked as “sodium chloride,” and no injection into this bottle was accomplished. Any verbal communication between physician and participant during the i.v. preparation was kept as neutral and “technical” as possible, making no references to treatment or pain.As a reference group, herein used to confirm that the placebo intervention successfully induced positive treatment expectations, Visual analog scale  ratings were accomplished using an automated response system . Specifically, expected medication efficacy, i.e., positive treatment expectation, was rated on VAS  immediately after the verbal induction of positive treatment expectations by the study physician. After each distension series , perceived treatment efficacy was assessed. Note that all treatment efficacy VAS were specifically phrased to address the expected or the experienced ability of the drug to successfully relieve visceral pain . In addition, each distension was rated on VAS for intensity, and ratings were averaged within each phase for analyses. VAS state tension ratings were accomplished along with efficacy ratings see .t-tests and Chi2-test. Change in subjective distension perception was calculated with repeated measures ANOVA with the between subject factor group  and time . Next, to exclude differences between positively instructed participants who received augmented vs. limited medication reminder communication, distension-related outcomes were compared with independent-samples t-tests. Data were reported as mean ± standard deviation (SD), effect sizes as Cohen’s d. In case that Levene test for homogeneity of variances was significant, we show corrected df. Data were analyzed with SPSS version 27.0 .For initial manipulation/treatment checks, placebo and reference groups were compared with respect to objective and subjective distension-related measures as well as sociodemographic and psychological measures using independent sample N = 40) were performed with RStudio .1 Separate generalized linear models (GLMs) were calculated using pre-treatment expectation ratings as exploratory and (a) treatment efficacy for the pain and pain relief phases and (b) perceived distension intensity as response variables. All outcome models were additionally corrected for the following covariates: Tension (VAS), duration of the informed consent procedure (min), stimulus intensity (mmHg), treatment efficacy , pain intensity . In supplemental analyses, models were re-computed after exclusion of outliers , defined outliers as values 2 SD below or above mean. Statistical testing was performed at alpha < 0.05.To address main research aims, i.e., to assess the role of positive treatment expectations, analyses within all positively instructed participants  and the reference (N = 20) groups  groups .As manipulation check, we initially ascertained differences between experimental phases with respect to objective and subjective distension-related measures. As intended, distension pressures applied within the pain phase were markedly higher than pressures applied within the pain relief phase, consistent with their selection based on individual thresholds . FurtherN = 40) when compared to the reference group who received no positive drug-related suggestions .Subsequently, the overall efficacy of the placebo intervention was tested by comparing positive expectations in the placebo and reference groups (treatment check). The placebo intervention successfully induced positive treatment expectations, as evidenced by overall significantly higher positive treatment expectations in the placebo group (N = 40). To this end, the entire placebo group was analyzed using GLM, irrespective of two slightly different medication reminder communication strategies implemented just prior to placebo administration by the study physician. For the sake of completeness, we provide comparisons of outcome measures for these subgroups  = 6.4, p < 0.001], which we considered as a covariate in GLM analyses. Further, positive treatment expectations were higher in the augmented subgroup , providing us with variability for primary analyses using GLM.Our primary aim was to assess the role of inter-individual variability in positive pre-treatment expectations in perceived treatment efficacy and symptom perception, which is why our strategy capitalized on variability in the whole sample of all positively instructed participants  together with efficacy ratings assessed after the preceding pain phase (p < 0.001) explained 54% of the variance in perceived treatment efficacy   .p = 0.028), but not with treatment expectation (p = 0.057). In this model, objective stimulus intensity (p = 0.008) and tension after the relief phase (p = 0.001) were significant covariates, with the model explaining 52% of the variance in perceived distension intensity  of the variance in perceived treatment efficacy rated after the pain relief phase, in a model that considered a number of other variables as covariates. Besides treatment expectations, perceived treatment efficacy after the preceding pain phase emerged as a significant predictor for treatment efficacy after the relief phase, suggesting that treatment efficacy not only dynamically changes over the course of a single treatment, but also that treatment-related evaluations during an early phase of treatment modulate subsequent evaluations during later treatment phases. This may seem trivial at first glance but is in fact intriguing in its putative implication for clinical treatment settings where patients receive the same treatment for longer periods of time, on multiple occasions, and/or in different doses. For the pain phase, on the other hand, a significant model emerged only after exclusion of outliers, with pre-treatment expectations explaining 29% of the variability in efficacy. Together, these results support that interindividual variability in the level of positive treatment expectations arising from positive drug-related information prior to treatment explains variability in perceived treatment efficacy assessed after treatment, which is remarkable herein given overall rather highly positive pre-treatment expectations in this placebo group. Even within such an “optimistic” group, inter-individual variability in the extent of positive expectations contributed to treatment satisfaction, most strongly after pain relief, where more than 50% of the variability in perceived efficacy could be explained in our models, which were robust to outliers. It will be intriguing to learn from much-needed prospective clinical work about the impact of the presumably much greater variability in pre-treatment expectations in clinical patients, ranging from very positive to very negative, and hence including not only positive (placebo) but also negative (nocebo) effects on perceived treatment efficacy.Interestingly, treatment expectations were unable to explain variability in perceived distension-induced pain intensity during the pain phase. For the pain relief phase, on the other hand, a significant model emerged, with treatment expectations explaining 52% of the variance in perceived distension intensity. While this finding would indicate that pre-treatment expectations shape the experience of visceral stimuli when intensity is distinctly reduced, caution in this interpretation is warranted given that the model was not robust to consideration of outliers.Based on our findings, we speculate that positive expectancy effects may be facilitated by the experience of pain relief, which would be consistent with recently growing appreciation of reward mechanisms in placebo effects . It is aOur experimental findings in acute visceral pain match observations from clinical trials and longitudinal studies in the broader field of acute , 44 and In conclusion, our data from an experimental translational placebo intervention in visceral pain support that pre-treatment expectations shape reported post-treatment medication efficacy. The experience of pain relief may facilitate perceived medication efficacy and by inference treatment satisfaction. Hence, individuals with highly positive expectations may benefit more from a noticeable symptom improvement, and future studies are needed to determine whether the immediate experience of symptoms within a given psychosocial treatment context may dynamically change perceptions about treatment in order to inform and inspire translational studies addressing implications for treatment satisfaction, compliance and adherence in patients with prolonged or spontaneously recurring pain. After all, it has most recently been concluded that “… the patient-physician relationship’s quality is the principal driver of gastroenterology patients’ satisfaction with their care” . EnhanciThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Essen University Hospital Ethics Committee. The patients/participants provided their written informed consent to participate in this study.NT, LR, and AI acquired data. SE, SB, and NT designed the study and acquired funding. NT, SB, AI, and JK-B analyzed the data. SB, JK-B, and SE wrote the first draft of the manuscript. All authors contributed to the interpretation of the data, revised the manuscript for critical content, and approved the final version of the manuscript.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Understanding of cell-type specific transcription factors has promoted progress in methods for cellular reprogramming, such as directly reprogramming somatic cells to induced neurons (iN). Methods for direct reprogramming require neuronal-fate determining gene activation via neuron-specific microRNAs, chemical modulation of key neuronal signaling pathways or overexpression via viral vectors, with some reprogramming strategies requiring a combination of these methods to induce the neuronal-cell fate. These methods have been employed in a multitude of cell types, including fibroblasts, hepatocytes, peripheral blood mononuclear, and T cells. The ability to create iN from skin biopsies and blood samples coupled with recent advancements in artificially inducing age- and disease-associated phenotypes are accelerating the development of disease models for late-onset neurodegenerative disorders. Here, we review how activation of the neuronal transcriptome alters the epigenetic landscape of the donor cell to facilitate reprogramming to neurons. We also discuss the advantages of using DNA binding domains such as CRISPR/dCas9 to overcome epigenetic barriers to induce neuronal-cell fate by activating endogenous neuronal cell-fate determining genes. These iPSC-derived neurons cluster with RNA samples derived from the murine proliferative germinal zone  transcription factor-based strategies which use neurogenesis and neuronal subtype factors to induce reprogramming, (2) microRNA-based repression of competing donor-cell fates, and (3) the utility of small molecule-based reprogramming to activate neurogenic signal transduction pathways.Developments in the cellular reprogramming field have enabled somatic cells to be directly reprogrammed to induced neurons (iN) , and NeuroD1. These transcription factors regulate gene expression networks which establish and specify the neural identity  from differentiating and maturing in mice  to pyramidal neurons rather than employing direct reprogramming strategies to track NeuroD1 activity, which potentially differs when the starting cell is pluripotent  treatment  has been explored with the transcription factor Sox2, coupled with EGF and FGF2 growth factors  as well as T-cells were reprogrammed to iN with the BAM factors and Ngn2, a transcription factor involved in coordinating chromatin accessibility during neurogenesis   and transcription factors  that increased the number of iN by activating signaling cascades associated with cell cycle regulation, cytoskeletal formation and organization, and metabolic regulation. Analysis of global DNA methylation in ZPAK iN derived from young and aged fibroblasts revealed a retention of age-dependent CpG methylation when compared to the donor fibroblast  fibroblasts containing familial mutations in APP and presenilin 1 were reprogrammed to iN using a 7-factor small molecule cocktail. This cocktail was modified from a minimal cocktail  sufficient to differentiate NPCs to neurons by adding forskolin, SP600125, GO6983, Y-27632 (VCRFSGY). While VCRFSGY reprogrammed fibroblasts to Doublecortin (Dcx), Tuj1, and Map2 positive cells, additional molecules were necessary to improve maturation and survival 7 days post-chemical reprogramming. Culturing iN with CHIR99021, Forskolin, Dorsomorphin, BDNF, GDNF, and NT3 for an additional 2 weeks created functionally mature iN capable of generating evoked action potentials. Importantly, VCRFSGY was successful in reducing the fibroblast-cell fate, thereby minimizing the potential for the donor cell network to influence gene expression network interactions in a disease model. Alzheimer's patient-derived fibroblasts reprogrammed to iN with VCRFSGY revealed higher amyloid beta (Aß) production compared to control iN  disease model for Amyotrophic lateral sclerosis (ALS) provided insightful information on studying disease-associated phenotypes Multiple strategies have been employed to create dopaminergic neuronal models for Parkinson's disease (PD)  are a class of transcriptional activators with a DNA binding domain that can be targeted to neuronal genes  facilitates the conversion of mouse fibroblasts and human neonatal, fetal, or adult fibroblasts to neural stem cells with the potential to differentiate into neural progenitor and glial cells. The reprogramming timeline with transcriptional activators appears to be similar to traditional methods, as Zfp521 needed to have activity for 24 days in order for the NSC transcriptome to be established. Interestingly, the addition of a small molecule cocktail was necessary to reprogram adult dermal fibroblasts, suggesting Zfp521 alone is unable to overcome transcriptional and cell-fate barriers  and CRISPR-associated protein (Cas9) technology has been adapted for endogenous gene regulation and epigenetic editing. A nuclease deficient Cas9 (dCas9) which retains its DNA binding affinity can be engineered by inactivating the RuvC and HNH catalytic domains. These dCas9 complexes are directed to genomic regions that are complementary to a guide RNA (gRNA) sequence . RNA-seq results suggest Ezh2 facilitates differentiation of mouse ESCs and reprogramming of fibroblasts to neurons by repressing endodermal and mesodermal cell-fate determining genes  have also been employed to facilitate tunable regulation from neuronal promoters in primary rat neurons  or epigenetic modifier domains, including catalytic domains of the histone acetyltransferase p300 and DNA demethylase TET1  showed rapid modifications to chromatin accessibility within 48 h of transducing MEFs with Ascl1 (Wapinski et al., JC designed the review. JC, JH, and KF contributed to the conceptualization of the review. JC wrote the paper with input from all authors. KF edited and approved the final manuscript. All authors contributed to the article and approved the submitted version.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.


Gluten is a mixture of storage proteins in wheat and occurs in smaller amounts in other cereal grains. It provides favorable structure to bakery products but unfortunately causes disease conditions with increasing prevalence. In the human gastrointestinal tract, gluten is cleaved into proline and gluten rich peptides that are not degraded further. These peptides trigger immune responses that might lead to celiac disease, wheat allergy, and non-celiac gluten sensitivity. The main treatment option is a gluten-free diet. Alternatively, using enzymes or microorganisms with gluten-degrading properties might alleviate the disease. These components can be used during food production or could be introduced into the digestive tract as food supplements. In addition, natural food from the environment is known to enrich the microbial communities in gut and natural environmental microbial communities have high potential to degrade gluten. It remains to be investigated if food and environment-induced changes in the gut microbiome could contribute to the triggering of gluten-related diseases.Wheat proteins, gluten, are incompletely digested in human digestive tract leading to gluten intolerance.• The only efficient treatment of gluten intolerance is life-long gluten-free diet.• Environmental bacteria acquired together with food could be source of gluten-degrading bacteria detoxifying undigested gluten peptides.•  Gluten is a mixture of grain storage proteins in cereal grains. The major gluten source in the human diet is wheat, domesticated approximately 10,000 years ago when hunter-gatherers settled in the Middle East. Since then, wheat has been an integral part of the Western type of diet. The dough made from wheat flour has unique baking qualities which provides it with advantages over other crops in temperate regions. The properties of wheat dough depend on the major grain endosperm storage proteins, which together form the “gluten” protein fraction. The major components of gluten are responsible for providing “viscoelasticity” while making leavened bread, but also unleavened bread, cookies, pasta, and many other food products. As a consequence, the total protein concentration in the grain is the most widely applied criteria for wheat baking quality, and an important factor in the price of wheat Barneix .Despite 10,000 years of coexistence, gluten-rich food is new for humans—we lack the enzymes that can fully degrade gluten proteins. As a consequence, consumption of wheat products may trigger an autoimmune enteropathy, celiac disease (CD), in genetically susceptible individuals. The genetic predisposition depends on the human leukocyte antigen (HLA) type, specifically the DQ2 or DQ8 alleles  derives its three genomes  from three diploid wild ancestors: Triticum urartu (AA), an unknown close relative of Aegilops speltoides (BB), and Ae. tauschii (DD) . This species subsequently hybridized with the D genome donor Ae. tauschii to form modern hexaploid bread wheat (AABBDD). Durum wheat Triticum turgidum L. spp. dicoccum, genome AABB is derived from the ancient T. turgidum (AABB) , in modern wheat, 80–90% of the total protein is gluten  , barley (Hordeum vulgare) (hordein), and oat (Avena sativa) (avenin) albeit in much lower quantities. The sequences within these proteins are similar, but unique bread making properties are provided only by wheat gluten and to a limited extent rye gluten.In addition to wheat, a prolamin fraction also exists in related cereal grains that belong to the family The extensive proline- and glutamine-rich repeated sequences of wheat seed storage proteins are responsible for bread quality, yet unfortunately play a crucial role in triggering hypersensitivity reactions such as celiac disease (CD), wheat allergy (WA), and non-celiac gluten sensitivity (NCGS)  is an autoimmune enteropathy caused by genetic and environmental factors with an estimated worldwide prevalence of about 1% of the global population  introduced six types of α a-gliadins (named 1–6) into the currently used hexaploid wheat (AABBDD)  possess their own sets of toxic and immunogenic peptides (or epitopes) with distinct immunogenicity. However, gliadin peptides are known to be the most toxic and numerous, specifically derived from α- and γ-gliadin: the strongest and most common adaptive response to gluten is directed toward an α2-gliadin fragment of 33 amino acids in length containing six partially overlapping immunodominant CD epitope regions  may be defined as a gluten (wheat)-dependent disorder with symptoms similar to CD  but with mostly normal small intestinal histology and lack of anti-tTG autoantibodies. Prevalence of NGCS is highly variable 0.63–6.0% )  to proteolytic cleavage.Enzymes that cleave the peptide bond are called peptidases or proteases. These enzyme names are often used as synonyms. Here, we call this class of enzymes peptidases. In the digestive tract, peptidases are responsible for cleavage of ingested proteins. Gluten-related health problems arise from the inefficiency of peptidases to degrade the immunogenic epitopes. Most peptidases, even those with low specificity, are unable to hydrolyze peptide bonds formed by proline residues due to the cyclic structure and the special constraints it exerts on the peptide backbone structure. The N-terminal amine of proline lacks a hydrogen, and the side chains are typically in the http://merops.sanger.ac.uk.), a comprehensive database containing information on peptidase sequence, structure, substrates, and inhibitors , or amidst polypeptide chains (endopeptidases EC 3.4.21-24 and EC 3.4.99). Here, we focus on endopeptidases that are more effective at gluten degradation than exopeptidases   and the food-grade Nattokinase (extracted from a dietary food supplement), degrade the immunogenic gliadin-derived 33-mer peptide and the immunodominant epitopes recognized by the R5 (QQPFP and related sequences) and G12 (QPQLPY) antibodies. Nattokinase abolishes the R5 epitopes but is less effective in eliminating the G12 epitopes  and a source of proteins for human alimentary use  with collagenase activity  (cleaves after P) has been combined with the barley cysteine endopeptidase PEP-B2 (cleaves after glutamine) to form a combined enzymatic activity that attacks the immunogenic peptides after the most frequent amino acid residues (P and Q). This formulation, developed under the name Latiglutanase, is expected to provide synergy and create a more active medication . These enzymes have been extensively studied and are already being developed for medicinal claims. POP from the bacterium Pyrococcus furiosus has been shown to cleave larger substrates , Bacteroidetes , Alphaproteobacteria (Sphingomonas capsulata), Deltaproteobacteria (Myxococcus xanthus), Chloroflexi (Sphaerobacter thermophilus), and Euryarchaeota (Pyrococcus furiosus)  Table . All theBased on a bioinformatic analysis, Kaushik and Sowdhamini  showed tLactobacillus casei strains were constructed to deliver POP from M. xanthus to the gut. It was found that the strain that secretes POP into the extracellular medium more effectively degrades the 33-mer oligopeptide compared to the strain that retained POP to the intracellular environment. The probable reason is that the extracellular enzymes can directly attack the peptide and do not require uptake events prior to cleavage  . Although their activities are quite low, the high abundance of Streptococcus sp. could still substantially contribute to the overall gliadin-degrading capacity in the human mouth  and rises in the jejunum (104–107 CFU/ml) and ileum (103–108 CFU/ml) . Therefore, it is possible that these strains are introduced to the gastrointestinal tract together with food. Currently, it is not possible to estimate if these bacteria inhabit the gastrointestinal tract stably, perhaps with specific required dietary substrates, or are constantly introduced with food.Root vegetables are in direct contact with soil and share some of the surrounding microbiome. We have recently shown in an in vitro study that bacteria that originate from root vegetables can degrade gluten and detoxify the toxic immunogenic CD epitopes at 37°C under microoxic conditions  (Caminero et al. In addition, opportunistic pathogens sometimes produce high levels of peptidases that could also degrade gluten (In conclusion, the enzymatic food supplements and probiotics are being developed to alleviate the symptoms of gluten intolerance. In addition, natural bacterial communities from food could also facilitate detoxification of gluten. Complete gluten degradation is a complex process that can be achieved with assistance of various combinations of enzymes produced by a wide range of microorganism, including bacteria. These bacterial communities could facilitate adaptation to our consumption of crop products that were introduced rather recently to humankind.


ECM) fungi. These fungi facilitate plant growth by increasing nutrient and water uptake and include many fruiting body-forming fungi, including some edible mushrooms. Despite their importance for ecosystem functioning and anthropogenic use, diversity and distribution of ECM fungi is severely under-documented in West Africa. We conducted a broad regional sampling across five West African countries using soil eDNA to characterize the ECM as well as the total soil fungal community in gallery forests and savanna woodlands dominated by ECM host tree species. We subsequently sequenced the entire ITS region and much of the LSU region to infer a phylogeny for all detected soil fungal species. Utilizing a long read sequencing approach allows for higher taxonomic resolution by using the full ITS region, while the highly conserved LSU gene allows for a more accurate higher-level assignment of species hypotheses, including species without ITS-based taxonomy assignments. We detect no overall difference in species richness between gallery forests and woodlands. However, additional gallery forest plots and more samples per plot would have been needed to firmly conclude this pattern. Based on both abundance and richness, species from the families Russulaceae and Inocybaceae dominate the ECM fungal soil communities across both vegetation types. The community structure of both total soil fungi and ECM fungi was significantly influenced by vegetation types and showed strong correlation within plots. However, we found no significant difference in fungal community structure between samples collected adjacent to different host tree species within each plot. We conclude that within plots, the fungal community is structured more by the overall ECM host plant community than by the species of the individual host tree that each sample was collected from.Forests and woodlands in the West African Guineo-Sudanian transition zone contain many tree species that form symbiotic interactions with ectomycorrhizal ( ECM) symbiosis is a mutualistic relationship in which fungal hyphae surround and grow between the cortical cells of specialized fine plant roots. The relationship benefits both organisms, with the fungal partner providing nutrients in exchange for carbon from the plant partner. Although only certain groups of fungi and plants can form ECM, this capacity has evolved convergently in around 80 fungal lineages , Uapaca (Phyllanthaceae) and Monotes (Dipterocarpaceae), all of which form symbiotic associations with ECM fungi  and partial large subunit (LSU) of the nuclear ribosomal DNA. This allowed a model-based approach to generating species hypotheses, based on a phylogenetic tree generated from the more conserved LSU region, as well as a combined approach to taxonomic identification involving both similarity-based assignment on the ITS region and reference to the LSU-based phylogenetic tree. We analysed how ECM host tree species community structure, total soil fungal communities and ECM fungal communities varied in two different vegetation types. Our data provide an important baseline resource for future biodiversity studies of soil fungal communities in West Africa.Although woodlands and gallery forests in the Guineo-Sudanian transition zone are known to host many tructure , we suspECM host trees were present, with relatively uniform vegetation and slope. A total of seven locations with nine sites were sampled: Kota Waterfall (KOTA-G and KOTA-W) in Benin, Kou Forest Reserve (KOUF-G) and Niangoloko Forest Reserve (NIAN-W) in Burkina Faso, Farako Forest Reserve (FA01-W and FA15-W) in Mali, Bissandougou Forest Reserve (BISS-W) and Moussaya Forest Reserve (MOUS-W) in Guinea and Kouadianikro Forest Reserve (KDNK-W) in Ivory Coast . Trees were categorized as ECM or non-ECM  and lysed in the field using a portable bead beater .In each plot, soil sample locations were selected according to the protocol used in ITS and LSU regions were amplified from all soil DNA extracts using the primer set ITS1  and LR5  and quantified using Nanodrop 2000C  before pooling the samples at equimolar proportions together with samples from another unpublished study for sequencing at Uppsala Genome Center (Sweden) using two cells on a Sequel system . Sequences were delivered to us as circular consensus sequence FASTQ files. Raw reads are available in the European Nucleotide Archive (samples ERS5551933–ERS5552022).Field lysed samples were returned to Uppsala University (Sweden) for DNA extraction using the Xpedition Soil/Fecal Prep kit following the manufacturer’s protocol. DNA concentration and integrity were verified by 0.8% agarose gel electrophoresis in 0.5% Tris Acetate-EDTA buffer  stained with 1× GelRed . Approximately 1500 bases of the rDNA  and LR5  with PhuASVs) in DADA2 (ASVs into phylogeny-based species hypotheses (SHs) using a Poisson tree process model  due to the tendency of PacBio sequences to include indels. Singleton ASVs are not included in DADA2 denoising output. De novo chimera detection and removal were also performed in DADA2, with a minimum parent overabundance of 3.5 (default 1.5) and allowing detection of chimeras with a single base difference from their parent sequence. After denoising and chimera removal, 1147 ASVs representing 3.6% of initial reads remained in the dataset  of the ribosomal rDNA within each ASV. Taxonomy was assigned to the ITS1 and ITS2 regions separately using SINTAX  was amended to each ASV name  when possible. ASVs were assigned to the narrowest UNITE SH whose inclusivity threshold they satisfied. ASV sequences are deposited in the European Nucleotide Archive (accession numbers LR993318-LR994464).In order to create a phylogenetic tree to assist in grouping PTP) method to generate phylogenetic SHs based on branch length distribution in a ML tree based on the LSU region, including all fungal ASVs (https://species.h-its.org/ptp/) and run for 500,000 generations, with a range of burn-in rates being tested, before settling on 0.15 as the final burn-in rate. Convergence of MCMC chains was examined manually using Tracer  and to detect possible chimeras across ASVs. An SH occurrence table across samples was calculated based on combined read counts for all remaining ASVs that mapped into an SH.We used the Poisson-tree process , where genus-level taxonomy generally allows for reasonably robust assignment of functional guild. For statistical analysis of the ECM community, all SHs that were assigned to the ectomycorrhizal guild with a confidence level of Probable or Highly Probable were included.Functional guilds were assigned to database  via FUNG2/ha for each ECM species and the total basal area for all non-ECM trees at each site. NMDS was calculated using the metaMDS function from vegan  ordination based on Bray-Curtis distances calculated on total basal area in mMean annual temperature and precipitation for each site were extracted from 1970–2000 historical averages in 2.5-minute maps from WorldClim 2.1  using thSpecies accumulation curves and asymptotic diversity estimates were calculated using the iNEXT package  and Kou (KOUF-G), while Kouadianikro (KDNK-W) was initially classified as a woodland. Despite the location of Kouadianikro, several hundred meters from the nearest river on a sloping hillside, the closed canopy and high dominance of large Berliniagrandiflora trees at the site  rendered it the most similar to the gallery forest sites, in particular KOUF-G. B.grandiflora was the most common ECM host tree at all three gallery forest sites  and woodlands . Trees in gallery forests tended to have greater girth than those in woodlands for both ECM host trees  and non-ECM trees . The total basal area of all trees  and the fraction of the total basal area represented by ECM trees  were both greater in gallery forest plots than woodland plots. There was no significant difference in the total number of trees , or the number of ECM trees  between vegetation types , mean annual temperature  or mean annual precipitation . The average girth of SHs based on branch length distribution in a ML tree generated from an alignment of the LSU region of 1,014 fungal ASVs . However, within plots, the effect of host species is not significant . At plot level, ECM host community composition is likely more important for shaping local fungal communities relative to the closest host tree sampled.Ordination analysis based on relative abundance of soil fungal ype Fig.  and hostASVs based on their phylogenetic placement in the tree , the ECM fungal communities in both vegetation types are made up of species in the phylum Basidiomycota  affect the ECM fungal community composition once site-level effects are excluded , which have not been uniformly propagated into database annotations. However, based on their abundance in ECM woodlands, it is probable that at least some of the detected Sebacinaceae taxa are, in fact, ECM. Many fungal species in West Africa lack reference sequences and our dataset thus provides a mycological resource for future analysis of both described and hitherto undescribed or at least unsequenced fungal species of ECM-dominated woodlands and gallery forests in the Guineo-Sudanian transition zone.Our data largely confirms earlier observations that the horaceae , 2017. Ing fungi , many SHECM trees represent unique, ECM species-rich habitats in West Africa. In the present study, soil fungal communities from gallery forests were enriched in Boletaceae relative to woodland sites. This corroborates the findings of KOTA-G) features the most diverse assemblage of Boletaceae in Benin. In contrast, the lack of sequences from Cantharellus  in the present study is striking, and likely due to primer mismatches . In the Guinean and Sudanian ecozones, fruiting bodies of cantharelloid taxa are commonly found associated with gallery forests dominated by B.grandiflora and U.guineensis (De Kesel et al. 2011, Based on fruiting body inventories in Benin, ECM host tree species shapes ECM fungal communities and total soil fungal communities. Our data provides a baseline, albeit incomplete, for phylogenetic placement and taxonomic resolution of environmental sequences from ECM-dominated forests in the Guineo-Sudanian transition zone. Sampling of more samples per site and more sites of the gallery forest is needed for a more complete characterization of the studied ecosystems.Gallery forest and woodlands in the Guineo-Sudanian transition zone harbor partially overlapping and differently structured soil fungal communities. Site-specific composition of


Atherosclerosis is a chronic inflammatory disease characterized by extensive remodeling of medium and large-sized arteries. Inward remodeling (=lumen shrinkage) of the vascular walls is the underlying cause for ischemia in target organs. Therefore, inward remodeling can be considered the predominant feature of atherosclerotic pathology. Outward remodeling (=lumen enlargement) is a physiological response compensating for lumen shrinkage caused by neointimal hyperplasia, but as a pathological response to changes in blood flow, outward remodeling leads to substantial arterial wall thinning. Thinned vascular walls are prone to rupture, and subsequent thrombus formation accounts for the majority of acute cardiovascular events. Pathological remodeling is driven by inflammatory cells which induce vascular smooth muscle cells to switch from quiescent to a proliferative and migratory phenotype. After decades of intensive research, the molecular mechanisms of arterial remodeling are starting to unfold. In this mini-review, we summarize the current knowledge of the epigenetic and transcriptional regulation of vascular smooth muscle cell phenotype switching from the contractile to the synthetic phenotype involved in arterial remodeling and discuss potential therapeutic options. Atherosclerosis (AS) is a chronic multifactorial disorder of medium and large-sized arteries characterized by inflammation and lipid deposition within the arterial wall, leading to slowly progressive plaque formation . ThroughArterial remodeling reflects the structural and functional adaptation of the atherosclerotic vessel wall to biochemical and biomechanical stimuli triggered by disease . In geneIn AS, low-density lipoprotein (LDL) particles enter the subendothelial space of the tunica intima, where they get oxidized  and trigger the activation of endothelial cells . ActivatThroughout lesion progression, reactive changes in the underlying vessel wall trigger arterial remodeling processes, in which the flow-limiting potential of the plaque either may be strengthened  or attenuated  . In geneVascular smooth muscle cells play a pivotal role in the different remodeling processes as they are the most abundant cell type found in arterial vessel walls . Mature Lineage-tracing experiments have shown that VSMCs are derived from various distinct progenitor cells in embryogenesis due to which VSMCs typically exhibit lineage-dependent responses to signaling pathways and can have distinct functional characteristics . BesidesHowever, unlike most cell types, VSMCs are not terminally differentiated and consequently display plasticity in their phenotypes , rangingAlterations in diverse environmental factors modulate the transcriptional regulation of VSMC-specific genes in AS and arterial remodeling . Main trCollectively, the mechanisms of VSMC phenotype switching and its effect on vascular remodeling are orchestrated by diverse transcription factors, and the mechanisms are still not fully understood . A largeEpigenetic mechanisms of gene regulation can be defined as transcriptional memory which alter gene expression without changing the genome . OverallThe role of epigenetics in cardiovascular disease is emerging as a critical linker and player at distinct levels, ranging from pathophysiology to treatment . Likewisvia genome replication without maintenance or inactivation of DNMTs, or by active demethylation facilitated by Ten-eleven translocation (TET) methylcytosine dioxygenases, which catalyze 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) (DNA methylation is mediated by DNA methyltransferases (DNMTs), which, in vertebrates, covalently bind a methyl group predominantly to the cytosine 5′-carbon in the context of a cytidine phosphate guanosine (CpG) dinucleotide . DNA met (5-hmC) . Cytosin (5-hmC) .Genome-wide investigations concerning the level of DNA methylation in atherosclerotic lesions have documented DNA hypomethylation as a general phenomenon as well as a unique DNA hypermethylation profile, which affects different genes and pathways implicated in AS pathogenesis . Moreovein vivo  and lysine (K) residues . TypicalDifferent histone methylation and acetylation changes have been identified to serve a crucial role in the development of AS and VSMC differentiation toward the synthetic phenotype, including significant decrease in H3K9 and H3K27 methylation  with conExperiments of Recent discoveries in molecular biology have revealed that gene expression is primarily regulated not only by proteins but also by ncRNAs. Among those, microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) play a crucial role in the transcriptional regulation of genes at diverse levels . To dateMiRNAs are small RNA molecules with an average length of 22 nucleotides that typically regulate mRNA expression negatively by binding to a complementary sequence often located in the 3′ UTR region of the target mRNA . Table 1LncRNAs are commonly defined as non-protein-coding transcripts larger than 200 nucleotides . ThroughInterestingly, the most significant cardiovascular disease related genomic locus Chr9p21.3 includes ANRIL lncRNA, which promotes VSMC phenotype switching when overexpressed through possibly acting as a molecular scaffold to promote WDR5 and HDAC3 complex forming . LncRNA in vitro experiments by Zhuang et al., decreased global 5-mC content and re-established myocardin expression in VSMCs induced by PDGF, thus inhibiting excessive VSMCs dedifferentiation  seems to present a promising approach to VSMC phenotype modulation due to its target specificity. At present, antisense oligonucleotides against lipoprotein small a  appear to form an attractive way for the treatment of AS . ExtraceSY-H devised the topic and the principal conceptual ideas. MZ and EA mainly wrote the manuscript. RM contributed to the design of the figure. All authors discussed and commented on the manuscript.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Gangliocytic paragangliomas (GP) are rare tumors encountered exclusively in the second portion of the duodenum. Duodenal gangliocytic paraganglioma (DGP) belongs to a subclass of neuroendocrine neoplasms, characterized with unique histologic features of carcinoid tumor, paraganglioma and ganglioneuromas. According to the recent World Health Organization classification of gastrointestinal neuroendocrine tumors (NETs), there is a debate to classify them either as low-grade NETs or as an independent entity. There are a few reports of regional lymph node (LN) metastasis that could argue DGP as a true neoplasm. However, majority of them had a benign course, raising the question of whether long-term follow-up is required. We report a case of a retroperitoneal LN involvement by metastatic GP and additionally performed a systematic review of the literature to determine the optimal follow-up, since no guidelines exist for this rare entity. Gangliocytic paragangliomas (GP) are rare tumors encountered exclusively in the second portion of the duodenum . DuodenaA 59-year-old female was incidentally found to have a 2.5 cm mass in the descending portion of the duodenum near the ampulla, during her work up for shoulder pain. Endoscopic ultrasound (EUS) with fine needle aspiration (FNA) biopsy revealed atypical cells. Endoscopic retrograde cholangiopancreatography (ERCP) revealed both common bile duct and pancreatic duct dilation. Due to concern for malignant mass causing compression of the ampulla and common bile duct (CBD) ductal dilation, pancreaticoduodenectomy and lymph node dissection for excision of a periampullary and ampullary mass was done. Final pathology was consistent with GP. Microscopically, the tumor was present in the submucosa and invaded the lamina propria and muscularis propria. It was composed of three cell types: epithelioid cells, surrounding spindle-shaped sustentacular cells and scattered ganglion cells . MetastaGPs are rare tumors with unclear origin, which predominantly affect patients in the third to ninth decade of life (median sixth decade). Though most often they occur sporadically, association with type I neurofibromatosis have been reported. These tumors are characterized by its triphasic cellular differentiation: epithelioid neuroendocrine cells, spindle cells with Schwann cell differentiation and ganglion cells . These aet al. [They clinically manifest either in the form of abdominal pain, GI obstruction, and or as an incidental finding. Findings on ultrasonography include submucosal location of a hypoechoic or isoechoic lesion. On imaging with computed tomography and magnetic resonance imaging, these tumors are solid and homogeneous in appearance. Surgical resection remains the standard of care . The useet al.  reportedet al. [In our review of literature of DGP with lymph node metastasis , there het al.  reportedOur case report and review suggest that even with lymph node involvement, there is prolonged disease-free survival. In conclusion, the presence of lymph node metastases does not seem to influence the prognosis but rather the treatment modality. However, no consensus regarding optimal follow-up of DGP with lymph node metastasis exists and further data and reports relevant to these cases are necessary for the determination of the appropriate strategy and surveillance following surgical excision of these tumors.