Uveal melanoma (UM) is the most frequent primary ocular cancer in adults, accounting for 5% of all melanomas. Despite effective treatments for the primary tumour, up to 50% of UM patients will develop metastasis, leading to a very poor prognosis and a median overall survival of 6 to 12 months, with no major improvements in the last 30 years. There is no standard oncological treatment available for metastatic UM patients, and BRAF/MEK and immune checkpoint inhibitors show disappointing results when compared to cutaneous melanoma (CM). Recent advances in biology, however, identified specific gene and chromosome alterations, potentially permitting an actively tailored surveillance strategy, and dedicated clinical studies. Being a rare cancer, UM patients have to overcome issues such as identifying referral centres, having access to information, and partnering with oncologists for specific management strategies and research priorities. Here, we describe how the European Rare Adult solid Cancer Network (EURACAN) will help in addressing these challenges and accelerating international collaborations to enhance the development of innovative treatments in UM. Although uveal melanoma (UM) is the most frequent primary ocular tumour in adults, it is a rare cancer with a stable incidence in Europe of approximately 6 per million since the 1970s, and a gradient from Northern Europe, with 8 to 9 per million in Scandinavia, to Southern Europe, with less than 2 per million ,4). PatiIn contrast to cutaneous melanoma (CM), UM does not display any UV mutation signature, and BRAF or NRAS are almost never mutated in primary UM. Moreover, UM is characterized by a very low mutational burden , and by The major challenge in UM is the metastatic risk; the rate of local recurrences is below 5% but correlates with the onset of metastasis . Up to 5The main clinical prognostic factors for metastasis are tumour size, diameter and thickness, extraocular spread and ciliary body extension, and metastatic status, which are all combined in the 7th edition of the American Joint Committee on Cancer staging system . The genSo far, there is no systemic adjuvant treatment available to reduce the risk of metastasis or to improve survival in high-risk patients. Current trials in the adjuvant setting are investigating drugs such as tyrosine-kinase inhibitors (TKIs) or Met inhibitors, histone deacetylase (HDAC) inhibitors, or immunotherapy .Currently, there is no treatment available that increases overall survival in the metastatic setting , and theAlthough extensive prospective data is missing, patients showing a limited extent of metastases seem to benefit from liver-directed therapies . Unfortunately, the subsequent phase III SUMIT trial did not confirm the phase II results [To date, there are no therapies available that directly target the Gαq pathway. Recent UM trials are single drug or combination studies targeting downstream components of the MAPK pathway. The protein kinase C inhibitor (PKC) AEB071 (sotrastaurin) has been evaluated in a phase I study in 153 patients: 4 (3%) had a partial response (PR) and 76 (50%) had stable disease (SD), for a median PFS of 3.5 months . In orde results . Selumet results  and in a results . Other T results .Therapies targeting BRAF or KIT are not indicated in UM, in the absence of the corresponding mutations. As UM patients have been excluded from large prospective trials in melanoma, small published series with anti-CTLA-4 and anti-PD-1 therapies showed low activities, with response rates of 5%, while there was no benefit with regard to progression-free or overall survival . HoweverBased on preclinical results, combination studies are running, with ipilimumab/nivolumab , or ipilimumab/nivolumab and radioembolization (NCT02913417).Recently, novel immune-based approaches have tried to target more specifically the uveal tumour cells. After encouraging preliminary results , IMCgp10Similarly, glembatumumab vedotin, a monoclonal antibody-drug conjugate against the transmembrane glycoprotein NMB that is expressed on the surface of melanocytes, was tested in a phase II study recently (NCT02363283). Binding to the NMB leads to internalization of the conjugate and release of the drug in the cells.Interfering with epigenetic dysregulation represents the most recent approach in UM treatment; trials are ongoing with the HDAC inhibitors vorinostat  and entinostat , and the BRD4 inhibitor PLX51107 (NCT02683395).Basic requirements for optimal management of rare cancers include early diagnosis, referral to a specialized centre, establishment of the therapeutic strategy by a specialist multidisciplinary team (a multidisciplinary tumour board), and access to appropriate treatments at all stages. The major issues in UM are late diagnosis or misdiagnosis, non-expert management, high risk for distant recurrence, and absence of effective treatment in the metastatic setting.Because of limited published studies, insufficient knowledge, and unshared experience, there is a considerable risk for UM patients to be managed outside expert centres. With UM being a rare melanoma, numbers of tumours are unnecessarily checked for BRAF mutational status, and many patients undergo 18F-Fluorodeoxyglucose PETCT and receive immunotherapy as for cutaneous melanoma.To avoid these pitfalls, and compared to frequent cancers, we need to:-expand preclinical research in UM: basic research deciphering UM biology, establishing animal models ,46, reca-arouse scientific interest and attract pharmaceutical companies to allocate resources to UM-dedicated research and clinical trials. Moreover, we should allow specific cohorts of rare cancers with no reference treatment and a dismal prognosis to participate in early phase and molecular-driven clinical trials .-develop training  and information  with adequate and specific communication tools.-act jointly with health authorities in each European Union member state to foster early approval and availability of drugs in such orphan cancers.European Reference Networks (ERNs) are virtual networks launched by the European Commission in 2017 with the support of the EU Health Programme. They involve over 300 healthcare providers within 24 ERNs (four dedicated to rare cancers and predisposing conditions) in 26 member states to share expertise and improve access to care for patients across the European Union, especially for complex or rare medical diseases that require highly specialized healthcare and a concentration of knowledge and resources. ERNs work on a range of thematic issues including bone disorders, childhood cancer, and immunodeficiency.The EUropean Rare Adult solid CAncer Network (EURACAN) is the European Reference Network dedicated to rare adult solid cancers. It aims to improve the quality of care and outcome of patients with rare adult solid cancers in the EU by optimizing diagnostic and therapeutic management, knowledge, research, and communication on all adult solid rare cancers for patients, families, physicians, and all stakeholders.The network gathers 66 health care providers in 17 European countries, identified based on documented expertise, experience treating rare cancers, and endorsement by their member state. It groups all rare solid adult cancers into ten “domains” corresponding to the RARECARE list of rare cancers based on the International Classification of Diseases for Oncology (ICD-O): sarcomas; rare gynaecologic tumours; rare neoplasms of the male genital organs and of the urinary tract; neuroendocrine tumours; rare neoplasms of the digestive tract; rare neoplasms of endocrine organs; rare neoplasms of the head and neck; rare neoplasms of the thorax; rare skin cancers and eye melanoma; rare neoplasms of the brain and spinal cord.EURACAN invited 16 associated partners from major scientific societies and key stakeholders: European Association of Neuro-Oncology (EANO), European CanCer Organisation (ECCO), European Neuroendocrine Tumours Society (ENETS), European Organisation for Research and Treatment of Cancer (EORTC), European Society of Gynaecological Oncology (ESGO), European Society of Medical Oncology (ESMO), European School of Oncology (ESO), European Society of Surgical Oncology (ESSO), European Society for Radiotherapy and Oncology (ESTRO), French Sarcoma Group-Bone Tumour Study Group (GSF-GETO), Joint Action on Rare Cancer (JARC), Organisation of European Cancer Institutes (OECI), French National Network for the Treatment of Rare Peritoneal Surface Malignancies (RENAPE), Network for the management of Thymic tumours and cancer (RYTHMIC), University of Milan (UNIMI), University Claude Bernard Lyon (UCBL) and 13 leading patient advocacy organisations in each domain: Italian Association for Laringectomy (AILAR), Association for Multiple Endocrine Neoplasia Disorders (AMEND), Digestive Cancer Europe (DiCE), European cancer Patient Coalition (ECPC), EUROpean Rare DISease (EURORDIS), International Brain Tumour Alliance (IBTA), International Neuroendocrine Cancer Alliance (INCA), Neuroendocrine tumour (Net) patient foundation, Northern Ireland Rare Disease Partnership (NIRDP), Melanoma Patient Network Europe (MPNE), Salivary Gland Cancer UK (SGC) , Sarcoma PAtient Euro Net (SPAEN), Thyroid Cancer Alliance (TCA) ) to participate in the different network bodies.Groups of experienced specialists gathered in national and international networks can provide collaborative efforts to better understand the biology of rare diseases and to develop innovative treatment approaches. EURACAN provides an infrastructure integrating national networks and European health care providers (HCPs) with a high level of expertise but also European patient advocacy groups (ePAGS) that together act as focal points for research on rare tumours and keys in the dissemination of knowledge and information.Because of the rarity of UM, medical expertise may be difficult to access, and patients may search for cross-border second opinions and appropriate care from country to country. Inappropriate management at early stages may result in an increased risk of metastasis and fatal outcome. Screening by ophthalmologists and referral to expert ophthalmologists is crucial to avoid any late diagnoses.Within the European Reference Network EURACAN, the G9 domain is dedicated to rare skin cancers and uveal melanomas. The objectives of the network for the UM subdomain include the description of the organisation of UM care in each participating country, the development and review of European clinical practice guidelines (CPGs), and the implementation of “roadmaps” for referral and self-referral of patients to expert centres .In the context of the lack of funding for research in rare cancers, EURACAN will also participate in the development of innovative strategies, providing the frame for the initiation and the promotion of novel translational and clinical research programs through international collaborations and associated tools .www.melanomapatientnetworkeu.org).To increase and harmonise the quality of care, a web-based-platform provided by the European Commission and customized to fit all domain needs will be used for multidisciplinary discussions. In parallel, EURACAN will develop medical training programs accessible to physicians and ePAGs representatives and will interact with patient advocacy groups to increase their involvement and assist them in the wide dissemination of communication and educational tools. In 2015, the patient organisation Melanoma Patient Network Europe (MPNE) created a new entity dedicated to rare melanomas and had already begun to organize meetings focusing on UM (Moreover, EURACAN will initiate and promote collaboration with pharmaceutical companies by networking for industry sponsorship for rare cancers, helping to conceptualize clinical trials dedicated to UM with adapted design and consensual endpoints, and facilitating the identification of participating sites in each country and the enrolment of patients in a short period of time. The interaction with health authorities and regulatory agencies could help to successfully drive these efforts to accelerate registration of any active agent as an orphan drug at the European level. Finally, in collaboration with academic societies and international groups, EURACAN provides the integration of clinical care and research programs dedicated to UM patients in order to increase the quality of care for rare cancers in all European countries.The biology of UM differs from that of CM, and this rare melanoma deserves specific means and dedicated funding to develop international collaborations and global integration of research and clinical care, with the aim of ensuring equal access to high quality care and innovation to all UM patients. The ERN EURACAN represents a unique European effort to address this urgent and unmet clinical need.


Type 2 diabetes mellitus and cancer are correlated with changes in insulin signaling, a pathway that is frequently upregulated in neoplastic tissue but impaired in tissues that are classically targeted by insulin in type 2 diabetes mellitus. Many antidiabetes treatments, particularly metformin, enhance insulin signaling, but this pathway can be inhibited by specific cancer treatments. The modulation of cancer growth by metformin and of insulin sensitivity by anticancer drugs is so common that this phenomenon is being studied in hundreds of clinical trials on cancer.Many meta-analyses have consistently shown a moderate but direct effect of body mass index on the incidence of multiple myeloma and lymphoma and the elevated risk of leukemia in adults. Moreover, new epidemiological and preclinical studies indicate metformin as a therapeutic agent in patients with leukemia, lymphomas, and multiple myeloma. In this article, we review current findings on the anticancer activities of metformin and the underlying mechanisms from preclinical and ongoing studies in hematologic malignancies. Based on the increasing incidence of cancer along with increasing rates of obesity and type 2 diabetes mellitus (DM2), much effort has been made to identify the epidemiological and biological connections between these conditions. In the clinic, oncologists are gradually being required to tailor cancer treatments for patients with pre-existing diabetes or obesity, and endocrinologists often need to control diabetes in patients who are undergoing treatments for cancer Despite contradictory reports in the early literature In this review, the association between hematological cancers and diabetes and the epidemiological, clinical, and preclinical data that establish the value of metformin as a potential treatment for hematologic malignancies are summarized.American Diabetes Association (ADA) and American Society of Clinical Oncology (ASCO) presented their consensus on the factors that link diabetes and cancer Although the link between DM2 and carcinogenesis was first reported in 1910 by Maynard and Pearson  Adiposity has been correlated with an increased risk of several cancers, including breast, endometrial, pancreatic, and colon cancer. In addition, obese men and women with esophageal, gallbladder, colorectal, pancreatic, liver, and kidney cancer have an elevated risk of cancer-related deaths  13. NotaMechanistically, obesity—a state of excess nutrient levels—chronically activates signaling pathways that upregulate insulin, insulin-like growth factor (IGF), leptin, and inflammatory cytokines  and increases the risk of neoplastic transformation. Consistent with this model, elevated levels of adiponectin, a marker of reduction in adipose tissue, were recently found to be associated with a lower risk of MM Conversely, studies worldwide have shown that DM2 is associated with increased rates of many cancers and cancer-related mortality  36-47. NAs the incidence of DM2 is increasing worldwide, MM, leukemia, and NHL are being diagnosed more frequently with concomitant diabetes mellitus Early studies have observed that compared to no therapy, metformin monotherapy is correlated with a reduced risk of developing cancer, whereas sulfonylurea therapy is correlated with an increased risk Although an initial meta-analysis failed to detect an elevated cancer risk in association with the use of insulin, the meta-analysis had small sample sizes and short durations of study versus controls In contrast, a recent meta-analysis reported mixed results concerning insulin secretagogues and the risk of cancer, demonstrating a correlation between the administration of sulfonylureas and an increased risk of some cancers but not others Galega officinalis (Leguminosae) (or goat's rue), French lilac, Spanish sainfoin, and false indigo have been recognized for their medicinal value since medieval times in Europe  Although In 1929, several biguanides that reduced serum glucose levels, including dimethyl biguanide , were synthesized. Jean Sterne (1909-1997) was the first to perform studies with galegine. Sterne selected dimethyl biguanide (metformin) for clinical development and proposed the name Glucophage. In contrast to sulfonylureas, metformin fails to induce insulin secretion but impedes the release of glucose by the liver and increases muscle glucose uptake Interestingly, the benefits of metformin in the treatment of diabetes have gained prominence only since 1995. The United Kingdom Prospective Diabetes Study (UKPDS) was a milestone project showing that regardless of glycemic control, metformin reduced the risk of myocardial infarction and all-cause mortality. Consequently, diabetes experts around the world indicated metformin as the first-choice drug for DM2 In 1995, the benefits of metformin in people with diabetes were found to not only be limited to glycemic control, and metformin was shown to reduce the risk of malignancy in patients with diabetes. Evans et al. Two potential accepted antineoplastic mechanisms of metformin have been proposed . First, Despite the importance of LKB1 in the AMPK-dependent antineoplastic effects of metformin, this drug abolishes the increase in tumor growth linked with high-fat diet and hyperinsulinemia, irrespective of LKB1 expression by the tumor. This antineoplastic activity is connected with decreased circulating insulin levels and insulin receptor activation in tumoral tissues, similar to the mechanisms underlying tumor growth repression by dietary control http://clinicaltrials.gov/) on metformin in combination with chemotherapeutics are underway worldwide. These studies are examining the antineoplastic effects of metformin. This drug is used jointly with chemotherapeutic agents in cancers of the digestive  and reproductive systems  and lung, prostate, and breast cancer, and the clinical limitations of metformin in cancer treatment are being determined. Several trials have reported synergistic or additive effects of such combinations Many studies have shown that metformin synergizes with chemotherapeutic drugs at low doses, minimizing the side effects of high doses Cell metabolism is epigenetically regulated in vitro and in vivo, the combination of metformin and dexamethasone synergizes to eliminate MM cells, inhibiting cell proliferation through reduced AKT/mTOR signaling AKT mediates the pathogenesis and progression of MM and resistance to standard treatments. Many upstream signaling pathways converge on AKT to govern survival and proliferative signals and apoptosis suppression. Early data have revealed high expression of AKT in myeloma cell lines and bone marrow aspirates from patients, especially in the advanced stages in vivoNotably, metformin inhibits glucose regulatory protein 78 (GRP78), an essential factor in bortezomib-induced autophagy and pharmacologically increases the anti-myeloma effects of bortezomib. Concomitant treatment with metformin and bortezomib inhibits the effects of the unfolded protein response (UPR) on GRO78, thus impairing autophagosome formation and increasing apoptosis and confirming the impeded growth of xenotransplanted myeloma cells in vitro and in vivo effects of metformin, in combination with the findings of retrospective epidemiological studies in various cancer models, have prompted nearly 200 trials  examining metformin alone or in combination with other antitumoral agents in patients without diabetes. However, few studies are determining the effects of metformin in MM  and acute lymphoblastic leukemia (ALL); consequently, metformin represents a new perspective in hematological cancer therapy. Nevertheless, metformin does not appear to alter the growth, differentiation, or survival of normal CD34+ stem cells Mutations/deletions in PTEN or post-translational inhibition of its lipid phosphatase activity is responsible for much of the chronic stimulation of PI3K/AKT/mTOR signaling in T-ALL Insulin might contribute to the chemoresistance of ALL cells by activating PI3K/AKT/mTOR signaling. IGF-1 signaling increases the proliferation of cell lines The PI3K/AKT/mTOR pathway is also stimulated by upstream oncogenes, such as the protein kinase BCR-ABL, a tin vitro and tumor xenografts In leukemic stem cells and stem cells of solid tumors, metformin is selective toward inducing death When combined with chemotherapy or other drugs, metformin might have additive effects on reducing cell growth and drug efflux through its activity on AMPK or P-glycoprotein Based on these findings, future studies should examine the role of metformin in improving chemotherapy outcomes in ALL patients with or without diabetes. Many trials are testing metformin in patients without diabetes who have solid tumors. Four recent clinical trials  are measuring the clinical and biological outcomes of metformin combined with standard systemic chemotherapy in relapsed ALL , and thein vitro and in vivo activity of metformin in human lymphoma cells, demonstrating that the activity of drug on AMPK inhibits the growth of B and T cell lymphomas by inhibiting mTOR signaling without the involvement of AKT. Moreover, the response of lymphoma to drugs such as doxorubicin and temsirolimus, a mTOR inhibitor, is significantly improves upon coadministration with metformin. In addition to inhibiting the mTOR pathway, metformin activates p53 by suppressing murine double minute X (MDMX), thereby causing apoptosis Shi et al. Rosilio et al. In diffuse large B cell lymphoma (DLBCL) patients with diabetes, metformin, as a first-line chemoimmunotherapeutic agent with rituximab, improved progression-free survival  and overall survival  compared with other antidiabetogenic agents The combined increase in the occurrence of diabetes, obesity, and cancer has created a significant problem in which complex disease pathophysiologies are intertwined. Dissection of the epidemiological and molecular links between these diseases has revealed novel target molecules and new treatment opportunities. In conjunction with interdisciplinary research, epidemiological and preclinical data support that metformin benefits select patients with solid tumors and hematological tumors; however, strict clinical trials are required to pinpoint patients who might benefit from metformin combinations. Thus, we need to assess whether the anticancer effects of metformin depend on metabolic variables, such as diabetes, BMI, insulin resistance, and obesity-related inflammation.Cunha Júnior AD and Pericole FV wrote the initial drafts of the manuscript, and Carvalheira JB revised the manuscript.


Introduction Care Considerations supported by the Centers for Disease Control and Prevention for the management of Duchenne muscular dystrophy were published in 2010, but there has been limited study of implementation in the United States. Methods A questionnaire collecting information about standard care practices and perceived barriers was piloted by 9 clinic directors of facilities within the Muscular Dystrophy Surveillance, Tracking and Research network. Results Six clinic directors completed the questionnaire; 1 adult-only clinic was excluded. Over 80% adherence was found for 30 of 55 recommendations examined. Greatest variability was for initiation of corticosteroids, bone health monitoring, type of pulmonary function testing, and psychosocial management. Barriers included unclear guidelines, inadequate time and funding, family-specific barriers and lack of empirical support for some recommendations. Discussion This pilot study showed implementation of the 2010 Care Considerations, except for recommendations based largely on expert consensus. Complete adherence requires more studies and active promotion. Introduction, 2, 4, 5Duchenne muscular dystrophy (DMD) is an X-linked genetic disorder of the dystrophin (DMD) gene and is well known as a complex, disabling disorder with shortened life expectancy. Advances in the clinical management of DMD have resulted in prolonged survival.1, 8, 10, Survey studies on the provision of care to patients with DMD have been conducted using questionnaire responses from selected medical specialties6MethodsQuestionnaire Development, The pilot questionnaire was developed from existing data items in several programs, including the Canadian Paediatric Neuromuscular Physicians Survey,13The questionnaire was reviewed by representatives of two patient support organizations (JW for MDA and KK for PPMD) for content, breadth, and interpretability. It was piloted by a neuromuscular physician and a nurse practitioner who were involved in DMD care but not associated with the research project. The final questionnaire consisted of 30 multiple-choice questions and four open-ended questions asking about opinions regarding future recommendations, areas of disagreement with current recommendations, and suggestions to improve implementation.https://osf.io/65us2.For reference, the questionnaire can be found at the following link: Questionnaire ImplementationA paper questionnaire was mailed to nine directors of MDA supported clinics located in participating MD STARnet sites  in the fall of 2014. The questionnaire was expected to take 30 minutes to complete. Reminder emails and letters were sent at two and four weeks after the initial email. Responses were entered into a Research Electronic Data Capture (REDCap) database, a secure web application for managing online questionnaires and databases, housed at the University of Iowa. IRB approval was received by the New York State Department of Health Institutional Review Board (IRB number: 03-062/15-0643/15-0644).Statistical AnalysesPercent adherence was calculated by combining response categories that met the minimum recommendation and dividing by the total number of responding clinic directors. SAS® software, Version 9.4 was used for analyses . Responses to individual questions are available from the first author.ResultsQuestionnaires were completed by six clinic directors: two from pediatric clinics, three from mixed pediatric and adult clinics, and one adult only clinic. Questionnaire responses from the adult only clinic director were excluded since the responses pertained only to care of adult patients and the focus of our study was the care provided from the time of diagnosis. Percentages for adherence across all remaining clinics are presented in Table 1. Genetic testing and counseling services were reported as provided by all clinics (Table 1 – Diagnostic Assessment). Access to a multi-disciplinary team of medical providers was reported as available by all but one clinic director . Specialties reported as not available by at least one clinic included: pulmonology; gastroenterology; psychiatry or psychology; physical, occupational or speech therapy; and palliative care clinic or services (data not shown). All clinic directors reported completing at least one recommended assessment every 6 months with variability across the types of assessments completed . Recommended clinical management also varied; all clinic directors recommended stretching, bracing for positioning or standing, adaptive/assistive equipment and wheelchairs, but fewer recommended wrist/hand splints, bracing for walking and communication devices.All clinic directors reported practice patterns consistent with the recommendations, including continued use of corticosteroids unless contraindicated (Table 1 – Corticosteroid Management and Side Effect Monitoring), but showed some variability in recommended age at initiation. Monitoring of side effects occurred at the recommended frequency. Variable adherence was found for the assessment of bone health . Of those who assessed bone health, testing was completed at baseline and annually for high-risk patients (data not shown). Conversely, all directors reported offering bone health interventions consistent with recommended care  and consultations with endocrinologists to assess and treat osteopenia/osteoporosis. All clinic directors reported the use of radiographs to monitor scoliosis with variability in the indications for completing (Table 1 – Orthopedic Assessment).Directors from all clinics reported recommending the use of electrocardiograms and echocardiograms for cardiac monitoring (Table 1 – Cardiac Assessment and Management); cardiac MRIs were not reported by any directors (data not shown). Adherence to frequency of monitoring was variable for patients under the age of 10, but consistent with the Care Considerations for those 10 and over. Holter monitoring was reported as recommended only if the child was symptomatic by all directors (data not shown). Medications were reported as used as treatment of cardiac dysfunction by all clinic directors. ACE inhibitor-angiotensin-converting-enzyme inhibitor was reported as the first medication recommended by all directors, but timing of use varied. Although all clinic directors reported referrals to a cardiac specialist, the timing of referrals varied  (data not shown).Annual monitoring of respiratory function of ambulatory boys ages 6 years and older was reported as recommended by all clinic directors (Table 1 – Pulmonary Assessment and Management). For non-ambulatory boys without respiratory symptoms or use of assisted ventilation, forced vital capacity and peak cough flow measures were reported as occurring at the recommended frequency by the majority (≥3) directors (Table 1). Of those not meeting the recommended frequency, pulmonary testing was reported as not being measured or tested annually (data not shown). The remaining pulmonary tests were reported less frequently as having been measured. Sleep studies were recommended with signs and symptoms and screening for awake or sleep hypoventilation every 6 months by all directors (data not shown). For the management of pulmonary dysfunction, cough assist and non-invasive ventilation were recommended by all clinic directors under multiple respiratory conditions (Table 1). Referrals to pulmonary specialists were not reported as recommended by all clinic directors, and the reasons for referral varied.All clinic directors reported regular monitoring of gastrointestinal and urinary morbidities  and consultation with a nutritionist. The timing of the consultation typically occurred at initiation of steroids, after excessive weight gain, onset of chronic constipation, or when there was evidence of dysphagia (data not shown).Only one clinic reported not completing a formal neuropsychological assessment before entering school (Table 1 – Neurocognitive Assessment); the remaining clinic directors reported either always completing an evaluation or completing if clinically indicated (data not shown). Evaluating for problems with coping, speech and language, learning disability, neurobehavioral disorders, emotional problems, or signs of social isolation at each visit were reported by most clinic directors. Psychotherapy, pharmacological and social interventions were infrequently reported as recommended.Barriers to Implementation of DMD Care ConsiderationsAcross all disease management areas, clinic directors agreed that not all health providers are familiar with each Care Consideration (data not shown). Specifically, primary care and emergency/urgent care providers were reported as having less familiarity or understanding of the Care Considerations. Regarding system barriers, clinic directors agreed there is inadequate time at clinic visits to complete all recommendations, inadequate funding to support ancillary staff and inadequate coordination of care across disciplines and services. Of the potential patient or family barriers queried, all clinic directors agreed that distance to neuromuscular centers, out-of-pocket expenses and access to services/resources in the patient’s community were significant barriers to implementation of care. There was little consistency between clinic directors on whether there is a lack of awareness or familiarity with the content of the Care Considerations by patients and families.Discussion Directors of selected neuromuscular clinics located in the MD STARnet surveillance areas completed a pilot questionnaire describing their recommended clinical management of patients diagnosed with DMD. Their responses were compared to the Care Considerations published in 2010 to evaluate implementation across multiple disease management areas. Each director also provided a perspective about potential provider, system, and patient barriers to implementation of the Care Considerations. Overall, reported clinical practices related to the diagnostic process, access to a multi-disciplinary medical team, timeliness of follow-up by a medical professional specialized in the care of neuromuscular disorders, and recommendation of corticosteroids to all patients and monitoring their side effects were consistent with the Care Considerations and the recently released imperatives for DUCHENNE MD, 16, 18, 19There were several disease management areas for which broad adherence was observed, but variability in patterns of implementation were also evident. For example, although the frequency of respiratory function measurements for forced vital capacity and peak cough flow were consistent with the Care Considerations, other recommended measurements  were either not performed or only completed with signs and symptoms. A survey study of pediatric respirologists and Canadian Paediatric Neuromuscular Group (CPNG) members about respiratory consultations, frequency and reasons for assessing pulmonary function, and interventions showed similar findings.7, 21, 22Research evaluating provider reports of adherence to the Care Considerations is limited. Prior to the release of the Care Considerations, but after the release of guidelines by the American Academy of Neurology, American Thoracic Society, and American Academy of Pediatrics, McMillan et al20Delays in clinical uptake of recommendations may be affected by multiple factors.There are several limitations to the findings from this pilot questionnaire. This study relied on a non-random selection of clinic directors from medical facilities within the MD STARnet surveillance region. Because of the small sample size, we did not examine variability within clinics nor did we examine barriers as potential reasons for observed variability. Specialist practices were summarized by clinic directors and not provided by individual specialists who provided the care. Despite these limitations, this pilot study provides information on the clinical management of DMD patients several years following the release of the Care Considerations, but prior to the release of the updated considerations. In addition to reporting on adherence to specific recommendations, we described patterns of care as provided. This information is important for aligning current practice with further refinement of clinical recommendations. Finally, the questionnaire asked clinic directors about perceived barriers to implementing the Care Considerations, which may provide guidance on potential revisions to recommendations, promote scientific investigations to support the recommendations, and increase visibility of institutional challenges to providing the recommended care.ConclusionThis study showed adherence to many of the assessments and interventions suggested in the Care Considerations. The areas showing less consistency could be classified as derived from expert opinion with little supporting data. Improvement in the implementation of all aspects of recommended care requires continued study into the health impact of the proposed recommendations and continued advocacy for coverage by agencies.Kristin Conway, PhD, Department of Epidemiology, College of Public Health, The University of Iowa, Iowa City, Iowa 52240, USA; Telephone: 319-335-4641 Email: kristin-caspers@uiowa.eduData is available upon request.Deborah Fox, MPH, Director, Congenital Malformations Registry Chief, Birth Defects Registry and Surveillance Section (BDRS) New York State Department of Health, Empire State Plaza, Corning Tower, Rm 1203 Albany, NY 12237, Phone 518-402-7950, Fax 518-402-7959, deb.fox@health.ny.gov, www.health.ny.gov/birthdefectsIRB approval was received by the New York State Department of Health Institutional Review Board (IRB number: 03-062/15-0643/15-0644). No identifiers were collected in the questionnaire. The New York State Department of Health de-identified the questionnaires and assigned random IDs for data entry by the Iowa collaborators. Participants were informed of the purpose of study, which is to inform on clinical practice and future care considerations, but posting of raw data was not disclosed.The authors have declared that no competing interests exist.


Plant probiotic bacteria represent a heterogeneous group of prokaryotes, mostly natural-occurring soil bacteria, which have beneficial traits directly related to enhance plant development. Specifically, this kind of beneficial bacteria, acting as plant symbionts or plant endophytes, enhances plant growth, crop yields and their products quality .Over the last decades, a plethora of studies have focused on the isolation of potential plant probiotic bacteria from soil, rhizosphere or plant tissues. Moreover, their applications and efficiency as single inoculants or as a part of bacterial consortia have been evaluated in several kind of crops, such as cereals, legumes, vegetables and other kind of superior plants, such as trees, ornamentals and aromatic plants.This special issue pretends to cover established and updated studies about plant probiotic bacteria and their benefits for agriculture and forestry, which will have a direct and beneficial impact on human and animal health and also, will minimize the use of chemical fertilizers, contributing to a more sustainable agriculture and forestry.This Special Issue contains 15 publications, 10 reviews and 5 original research articles, which cover most of the current aspects related to the benefits of plant probiotic bacteria in the global agriculture, combining recent basic and applied knowledge about this topic.The content of this Special Issue can be divided in six different parts: (i) Applications of plant probiotic bacteria in agriculture and forestry, with 3 contributions ; (ii) Taxonomy and systematics of Plant Probiotic Bacteria in the “genomics” era, with 1 contribution (Carro and Nouioui); (iii) Effects on yield and quality of crops and fruits, with 4 contributions ; (iv) Effects on biocontrol and bioremediation, with 3 contributions ; (v) plant probiotic bacteria interactions with their hosts and environment, with 3 contributions  and (vi) Technologies and current marketing of PPB-based products, with 1 contribution (Menendez and García-Fraile).The editors acknowledge all the authors for their valuable and straightforward contributions to this Special Issue.


Prescription medicines aim to relieve patients’ suffering but they can be associated with adverse side effects or adverse drug reactions (ADRs). ADRs are an important cause of hospital admissions and a financial burden on healthcare systems across the globe. There is little integrative and collective knowledge on ADR reporting and monitoring in the Norwegian healthcare system. Accordingly, this systematic review aims to investigate the current trends in ADR reporting, monitoring, and handling in the Norwegian healthcare system and describe related interventions. Appropriate keywords, with regard to ADRs in both English and Norwegian languages, were used to retrieve articles published from 2010 to 2019. Six articles met the inclusion criteria. The findings offer a comprehensive picture of ADR reporting and monitoring in the Norwegian healthcare system. Psychotropic medicines were most commonly implicated by patients, while professionals most commonly reported ADRs associated with anticoagulants. The current ADR systems were compiled with the involvement of both patients and healthcare providers to record all types of drugs and ADRs of various severities, and aimed at improving ADR tracking. However, there is a need to improve current initiatives in terms of feedback and quality, and more studies are needed to explore how ADR profiles, and the associated vigilance, can improve the safety of medicines management in Norway.  The prescription and administration of medicines aims to relieve patients’ suffering and ailments. However, they can be associated with adverse side effects or adverse drug reactions (ADRs) that lead to physical and psychological harm, much of which can be prevented. An ADR is defined by the World Health Organization (WHO) as an unintended reaction in the patient to a medicine associated with any dose administered by the healthcare provider . SeriousADRs are an ever increasing and substantial burden on healthcare systems and the leading causes of hospital readmissions and associated co-morbidities. Older people , patientThe safety of medicines cannot be fully established until they are prescribed to patients and related reactions come under surveillance through well-established pharmacovigilance systems . As a glIn Norway, ADRs are estimated to be responsible for 5–10% of all acute internal medicine hospitalizations and cause approximately 1000 deaths per year . PharmacA systematic review was undertaken to ensure the comprehensive coverage of the literature and integrate current knowledge ,29,30 onA pilot search in national and international databases identified appropriate keywords for the search. Next, the Boolean search method was used to retrieve articles describing the condition of ADR reporting and handling in Norway as follows: “Drug-related side effects and adverse reactions” OR “adverse drug event” OR “adverse drug reaction” OR “drug side effects” OR “drug toxicity” OR “side effects of drugs” OR “toxicity, drug” OR “medication side effect” AND Norway OR Norwegian. These keywords were translated to Norwegian and were used for a similar search process in Norwegian research databases. Scientific articles published between 2010 and 2019 in journals in both English and Norwegian languages were retrieved from the online databases of PubMed [including Medline], Embase, Cinahl, Web of Science, Cochrane, Norat, Idunn, and SweMed. Inclusion criteria were a precise focus on ADRs in the Norwegian medicines management context and published in scientific peer-reviewed journals. The authors  performed the systematic review steps independently, but held frequent discussions for sharing results of their search and agreed further review steps. This led to retrieval of 6559 articles . n = 22). Their full texts were obtained from the Norwegian and UK libraries and underwent a careful assessment to select those with a precise focus on the study topic (n = 6). For example, articles with a focus on medication process, quality improvement, multidisciplinary interventions, and prescription and administration of drugs rather than ADRs reporting, monitoring, and handling were excluded. Also, the reference lists of the selected studies was searched manually, but no more articles were identified. Therefore, the number of articles was reduced to six for further evaluation and appraisal with respect to methodological transparency and soundness based on the Enhancing the QUAlity and Transparency of health Research (EQUATOR) tools [Title readings and deletion of duplicates resulted in 137 articles that were shared between the authors for further consideration and bilateral agreement in terms of suitability for inclusion in the next review step. The articles’ abstracts were read by each author  independently and those articles with a possibility of discussion on ADRs in the Norwegian medicines management context were selected (R) tools . The sysR) tools  , female patients reported ADRs in 63% of cases and were most commonly in the age range of 20–29 years (29%) or 30–39 years (23%). Analgesics, including tramadol, codeine, diclofenac, and ibuprofen, were the most commonly mentioned drug group in the ADR reports. There were 1.1 psychotropic drugs per ADR report, and adverse mental and neurological reactions, such as dizziness, anxiety, depression, insomnia, and suicidal thoughts, were the most common suspected adverse reactions reported [The introduction of ADR reporting in Norway by patients was started relatively late in 2010. Only an electronic form was available, and this did not accept reports on unregistered products. This electronic reporting form was very sophisticated and was characterized by completeness checks, data importation directly to a database, presence of free-text and mandatory spaces in the form, and a drop-down list for the selection of co-medication. However, no drop-down lists to select ADRs were included and there was no opportunity for follow-up with patients regarding their ADR reports . In the reported . n = 9629), 58% of healthcare personnel reports were for female patients, most frequently in the age group 0–9 years (17%) and 60–69 years (13%). The number of suspected drugs per ADR report was 1.4, and the most frequently mentioned drug group in the ADR reports was vaccines, where non-specific symptoms and local reactions were the typical suspected adverse reactions [n = 409) on oral anticoagulants, including warfarin, dabigatran, rivaroxaban, and apixaban. Rivaroxaban had the highest number of reports (6.5 reports per 1000 anticoagulant users). The patients were mainly older than 70 years of age (76%); 91 patients experienced cerebral haemorrhages, blood clots, cognitive impairment, headache, and hair loss. The highest number of fatal outcomes were related to rivaroxaban (1.1 deaths/1000 users). Comorbidities, older age, and polypharmacy contributed to increased seriousness of side effects and patient death [In regional centers in Norway only reports from healthcare professionals were assessed for causality, with personalized feedback provided . In the eactions . 291 repnt death . n= 196, 75%). Severe ADRs were reported in 42% of cases including hypersensitivity (n = 71), hepatic damage (n = 56), and interactions with anticoagulant drugs (n = 20). Also, 72% and 20% of the reports specified to 53 years old women and patients with the age ≥70 years [The study of ADR reporting by healthcare professionals on plant-based products including herbal dietary supplements and plant-based drugs e.g., Hypericum perforatum, Ginko biloba, and Valeriana, in the period 2003–2012 showed 260 submitted ADR reports to RELIS, of which 250 reports were related to herbal dietary supplements. There were 10 reports on plant-based drugs, all reported by pharmacists. Overall, doctors most frequently reported ADRs to the RELIS  were combined with dispensing data (n = 800 million) from the Norwegian Prescription Database. Dividing report numbers for each prescribed medicine with the number of users was used to calculate the medicine-specific consumption-adjusted ADR rates to identify medicines with higher risks of safety issues. A high number of ADR reports involved diclofenac, olanzapine, diazepam, warfarin, sildenafil, and methylphenidate. The increased ADR reports and the medicine-specific consumption-adjusted ADR rate for atorvastatin in 2010 reflected a true increase in the number of reports rather than the number of users: 27 different ADRs, with at least one instance, were reported. These included dizziness, nausea, diarrhoea, feeling abnormal, and anxiety in women and aggression, self-injurious behaviors, cerebral infarction, palpitations, attention deficit hyperactivity disorder, hypertension, and vomiting in men. Many of the most commonly used drugs had more than 500,000 users and less than 100 ADR reports; this was due to either underreporting or rarity of serious ADRs. 78% of all ADRs were reported within seven days [ADR reports from 2004–2013 from the EudraVigilance database (ven days . (R)  and drug abuse after its marketing in Norway in September 2004 was selected to assess its safety-related reports. Accordingly, 5427 (26%) of 21,071 parallel questions and answers between pharmacists and healthcare providers in the RELIS database concerned ADRs, which were considered references in 4% of reports in the Norwegian ADRs database. On the other hand, the Norwegian ADRs database was used as a reference in 7% of parallel questions and answers regarding ADRs. Eleven questions and 13 ADR reports concerned Lyrica(R) and different aspects of its abuse: reports described dose escalation problems, craving, and withdrawal reactions, which led to the detection of new drug-safety problems [The RELIS database, as the network of four regional medicine-information and pharmacovigilance centers where pharmacists asked questions and provided feedback to healthcare professionals regarding drug-related questions and ADRs, along with and the Norwegian ADR database (as a reference) were searched in the time period 2003–2012. Lyricaproblems . This review has described the various methods used for reporting, monitoring, and handling ADRs in the Norwegian healthcare system. In general, emphasis was placed on the role of patients, healthcare providers, and innovative systems for ADRs reporting and monitoring. The study findings demonstrated how patients could be involved in ADR reporting and the barriers to patients’ participation in terms of their knowledge, active involvement and participation in medicines management, and feedback on reports. Rather, there was an emphasis on the extent of healthcare providers’ collaboration with the reporting of ADRs along with the use of their knowledge and expertise for the active monitoring of ADRs and medicines adverse side effects. Since low reporting rates of ADRs contribute to major delays in the identification of medicines management issues , patientIt has been reported that patients may not be aware of ADR reporting systems and may be confused about how and where to report. While patients are encouraged to report known ADRs to prevent similar suffering in other patients, they need to increase their familiarity with reporting processes to improve the quality and quantity of reporting . To imprThe study findings showed the use of innovative methods and data collection methods for providing a comprehensive picture of ADRs in the Norwegian healthcare context. Some barriers to the effective implementation of the ADR reporting and monitoring system could be financial constraints, absence of clinical confirmation of patients’ reports, impossibility of sharing patients’ reports with primary healthcare providers, and inability to compare reports from different sources. The development and implementation of protocols for ADR monitoring as an efficient intervention to improve medicines management can contribute to lower reporting costs. Mechanisms for reporting and recording ADRs should not only provide information about the total number of reports, but also their severity, unexpectedness, and the degree of causality attributed to ADRs . VariabiThe results of this systematic review have described initiatives for ADR reporting, monitoring, and handling in the Norwegian healthcare system. The results emphasize the need for data collection systems to provide a comprehensive picture of ADRs. ADR reporting could be improved by: facilitating patients’ participation in reporting ADRs, and sending them feedback on their reports; sharing reports with primary healthcare providers, to enhance healthcare providers’ active collaboration with ADR reporting; implementing existing ADRe profiles for interactive reporting between patients, nurses and healthcare providers ,4; asses


Kazakhstan and the United States have partnered since 2003 to counter the proliferation of weapons of mass destruction. The US Department of Defense (US DoD) has funded threat reduction programs to eliminate biological weapons, secure material in repositories that could be targeted for theft, and enhance surveillance systems to monitor infectious disease outbreaks that would affect national security. The cooperative biological research (CBR) program of the US DoD’s Biological Threat Reduction Program has provided financing, mentorship, infrastructure, and biologic research support to Kazakhstani scientists and research institutes since 2005. The objective of this paper is to provide a historical perspective for the CBR involvement in Kazakhstan, including project chronology, successes and challenges to allow lessons learned to be applied to future CBR endeavors. A project compendium from open source data and interviews with partner country Kazakhstani participants, project collaborators, and stakeholders was developed utilizing studies from 2004 to the present. An earlier project map was used as a basis to determine project linkages and continuations during the evolution of the CBR program. It was determined that consistent and effective networking increases the chances to collaborate especially for competitive funding opportunities. Overall, the CBR program has increased scientific capabilities in Kazakhstan while reducing their risk of biological threats. However, there is still need for increased scientific transparency and an overall strategy to develop a capability-based model to better enhance and sustain future research. Finally, we offer a living perspective that can be applied to further link related studies especially those related to One Health and zoonoses and the assessment of similar capability-building programs. After the Soviet Union breakup, the Nunn-Lugar Cooperative Threat Reduction program began in 1991 which funded work through the United States Department of Defense (US DoD) in several former Soviet Union (FSU) republics, including Kazakhstan, to dismantle, secure, and prevent proliferation of nuclear, chemical, and biological weapons of mass destruction (WMD). Since the early 2000s, the US DoD has funded and implemented a Biological Threat Reduction Program (BTRP) through the Defense Threat Reduction Agency (DTRA) in Kazakhstan.After the BTRP addressed the biosecurity risks resulting from the breakup of the Soviet Union that included accounting for biological WMD and re-training scientists, the BTRP continued to fund the construction of a central reference laboratory (CRL), upgrades of other laboratories, and biological research. BTRP, which now runs programs in over 40 countries, has three lines of work: biosafety and biosecurity (BS&S); biosurveillance (BSV); and cooperative biological research (CBR). As discussed in our earlier manuscripts ,2, KazakKazakhstan has long maintained an infrastructure and tiered network for infectious disease surveillance that was a product of the Russian anti-plague (AP) system designed to combat seasonal epidemics through a multi-sectoral approach that recognized its zoonotic nature since the time of the Tsars ,4. The AThe infrastructure from the AP system also shaped the research culture among Kazakhstan and its neighbors in Central Asia which presented limited work often only in Russian language journals which explains why they are underrepresented in current scientific literature . After tResearch outputs are often measured in bibliometrics such as published and cited peer-reviewed articles. Kazakhstan maintains a high literacy rate among the general population, which reflects their historic scientific record of publications. The 2018 Nature Index ranks the top 50 countries for life sciences according to their research outputs and Kazakhstan is not ranked in the Nature Index, but it is ranked in the second tier of 50 countries at #76 according to the Scimago Institution under agricultural and biological sciences . NationaSince 2005, DTRA BTRP has funded over $20 M in CBR alone in Kazakhstan mainly through single, large integrating contracts where contractors have implemented over 25 studies through their science partners . These sWhile the primary BTRP objective is related to US national security by reducing global health security threats by working with partner countries, CBR enables those partner countries to focus on broader capacity-building for biosurveillance and enhancing BS&S . These cThe objectives for the three lines of BTRP’s work BS&S, BSV, and CBR are often achieved in consensus for capacity-building which is traditionally associated with international development, but are inherently difficult to measure. A major accomplishment of DTRA BTRP funding has been building research networks to better implement the CBR program in Kazakhstan, which can be measured in simple metrics such as conference presentations and peer-reviewed publications, both in the English language, and outcomes such as results from testing suspect samples and operational enhancements. The cooperation between Kazakhstan and the US continues to show intent for reducing biological threats and the CBR program has also increased cooperation among institutes in Kazakhstan that often did not previously communicate their respective infectious disease surveillance results. The overall capacity-building and capability-maturing has been harder to assess and quantify.DTRA BTRP funded several CBR projects and smaller studies which contractors implemented during 2005–2007, 2009–2014, and 2015–2018. In 2005–2007, the initial CBR projects centered on studies involving select agents including zoonoses: anthrax, plague, tularemia, highly pathogenic avian influenza, and brucellosis. The typical CBR projects funded researchers in Kazakhstan and project collaborators in the US and UK who mentored and guided those researchers to develop and test their hypotheses. From 2009–2014, five CBR projects were funded with up to $ 1M each, which covered labor hours, materials, travel, and infrastructure overhead. In parallel, several smaller 12-month studies were funded with approximately $100 k called Threat Agent Detection and Response (TADR) Activity Projects (TAP) and were implemented in parallel to the CBR projects. The TAP studies were intended to support TADR which was the earlier name for the biosurveillance line of work and only funded material and did not fund labor hours. From 2015–2018, the trend for smaller studies continued as TAP studies evolved to Central Reference Laboratory (CRL) Activity Projects (CAP), which were designed and implemented to support studies for the new CRL that DTRA funded and constructed in Almaty, Kazakhstan.CBR has served as a foundation for incorporating standard operating procedures into working practice under multiple research studies, especially related to BS&S to ensure that the work is performed in a safe and secure environment while supporting tasks to enhance infectious disease surveillance for human and animal health. More importantly, growing a quality research program especially in terms of capability maturity, that is the state “as is” and “to be”, requires a strategic vision. The evolution and progression of the Kazakhstan CBR program are linked and illustrated in an iterative fashion . The proCoxiella, and Brucella in milk; TAP-11, A Study of Hantavirus Genotypes Circulating in the West Kazakhstan Region; and CAP-1, Flea-borne disease surveillance) and another CBR project  titled, Epidemiology of CCHF, HFRS (Hantavirus) and Tick-Borne Viral and Rickettsial Diseases in Kazakhstan, and a one-year TAP-2 study (2012) titled, Species Identification of Tick Vectors Associated with Infectious Disease in Kazakhstan, demonstrated the utility of multi-institute cooperation among several project collaborators the success of which led to multiple other projects . KZ-29 wThe multiple institutes involved in KZ-29 included three Kazakhstani institutes: (1) Scientific and Practical Center of Sanitary and Epidemiological Expertise and Monitoring (SPCSEEM), (2) Kazakh Scientific Center of Quarantine and Zoonotic Diseases (KSCQZD), and (3) the Ural’sk Anti-Plague Station (UAPS); and five international collaborating institutions: (1) University of Buffalo, (2) Naval Medical Research Center (NMRC), (3) Public Health England (PHE), (4) University of Florida and (5) United States Army Medical Research Institute of Infectious Diseases (USAMRIID). All of these institutions provided various skill sets and techniques from viral and rickettsial disease research fields, as well as data analysis and mapping. The project was successful, in obtaining many tick and human samples made available by the Kazakhstani scientists. Moreover, during the study a number of Kazakhstani institute staff was trained in modern diagnostic and data management techniques. Infectious disease agents were detected in multiple different areas of the country, several for the first time at specific locations, and the results were written up for presentations at a number of international meetings ,13,14,15Dermacentor (n = 2097), Hyalomma (n = 4), Rhipicephalus (n = 126), and Ixodes (n = 5). After DNA extraction, Dermacentor and Ixodes tick pools were tested using tick genus-specific assays that produced results in concordance with conventional visual identification [Dermacentor and Ixodes assays. A partner country scientist from UAPS presented this data in a poster at the 2013 Conference of Research Workers in Animal Diseases (CRWAD) and demonstrated the utility of a molecular PCR-based method to complement and enhance tick identification [Additionally, some of the smaller TAP and CRL (CAP projects) studies demonstrated higher impact and success than the larger CBR projects because the project activity could be performed with a smaller number of researchers and required less coordination that would be necessary with researchers from various institutes in Kazakhstan. KZ-29 and TAP-2 were performed concurrently and leveraged the same project resources to fulfill both studies. The TAP-2 tick identification study which was designed to apply molecular methods, including using quantitative real-time polymerase chain reaction (qPCR) assays, to confirm the identification of ticks to the level of genus and species when the conventional visual identification was questionable due to inconclusive morphology and life stage presentation. Questing adult ticks were collected by tick drag during spring and summer from 2012–2014 in seven administrative rayons (county level) of West Kazakhstan oblast (province level). The total number of ticks collected (n = 2232) were identified according to visual morphological methods which resulted in four genera: In terms of simple metrics, from 2009–2014, the CBR, TAP, and CAP work resulted in at least 12 peer-reviewed publications, 60 conference presentations, and 30 research collaborator visits to Kazakhstan. Overall, the Kazakhstan CBR projects have resulted in a few hundred conference presentations and several peer-reviewed publications. The benefit of conference presentations is the practice of developing and presenting results while meeting and networking with peers in the scientific community. While the number of peer-reviewed publications is often used to judge scientific merit, publications are often developed well after a project has ended. In addition, the number of citations from these publications also grew over time beyond the time of the initial publications .More recent CBR work has engaged applied science and academic institutes in Kazakhstan to take advantage of their capabilities and resources for next-generation workforce training and tapping into their basic research programs. These resources also include individuals who have been educated through the current system that includes more exposure to US and Western science and technology and better knowledge of the English language associated with major publications. This approach also reinforces similar examples of public–private partnerships where a “triad” is formed when academic and university sectors are engaged . In KazaReducing biological threats through CBR programs is still important even in the absence of biological weapons programs. BTRP has specific objectives for reducing biological threats especially WMD and emerging infectious diseases as it relates to US national security and the research funded is a hybrid of pathogen research and disease surveillance . The cooScientific accomplishments and capability-building were also demonstrated in Kazakhstan through the CBR program despite challenges and limitations. In addition to simple metrics, the nature of the respective collaborations is important to consider as it often influences future outputs such as when publications are made and how further work is proposed and implemented. In Kazakhstan, we also observed that when work is published the impact on citations is not known for some time and often the publication was not the primary reason for collaboration . LimitedAt a 2018 National Academies of Sciences, Engineering, and Medicine workshop called Enhancing Global Health Security through International Biosecurity and Health Engagement Programs, a committee was formed to assist BTRP and provide future guidance. One element related to CBR and capability-building in general is the need to exact expectations, which is important under a highly specific program that may have different views across the funder, implementers, and partner country participants . ExactinContinuing challenges for increasing scientific transparency and incorporating a capability maturity model in order to advance respective research programs were also noted. In addition to the KZ-29 and TAP-2 examples described, the importance and difficulty for increasing scientific transparency via sample material exchange was noted. While the exchange of genomic sequences has been demonstrated, working towards sample material exchange would further increase scientific transparency . BTRP anFor long-term capability-building, the sustainability of CBR program in Kazakhstan needs to reach a maturity level where it can compete and acquire external research funding apart from BTRP; this threshold remains a challenge. When funding a scientific research program, it is difficult to balance immediate needs and manage risk with raising quality and innovation. Similarly, standardized evaluation techniques such as the capability maturity model (CMM) which are often used in business and technology sectors are also good for assessment at a scientific research program level. At the BTRP level, outputs for cost-effectiveness, which could be incorporated into their program lifecycle analysis, and measuring benefits to foreign relations would augment program metrics. A unified top-down and bottom-up approach that is focused on capability maturity also needs to be employed to ensure that funder and recipients agree on expectations and science objectives.Publicly posted funding calls through major national government funders such as BTRP broad area announcements are prime examples of external funding that Kazakhstan can apply for and pursue. While BTRP’s fundamental research scope is on US Biological Select Agents, pathogens of pandemic potential, emerging, and re-emerging infectious diseases, partner country scientists and research collaborators have linked non-Select Agents to research topics through developing hypotheses for co-infection and differential diagnosis.Leveraging existing resources including knowledge of in-country organizational structure, workflow, related networks, and current studies are important to establish buy-in from a top administrative level to the working research level which further reinforces multi-sectoral approach and encourages communication.The degree of collaboration in Kazakhstan is emphasized especially in teamwork to develop strategies and cooperate effectively. Leveraging existing resources while reinforcing networks and maintaining relationships should be frequent and encouraged. The ability to link consecutive projects through effective collaboration and funding pursuits builds greater capability and avoids one-off studies.The CBR program continues to align outputs for long-term sustainability such as encouraging partner countries to establish their own external funding. From our literature review, sustainability and setting policy are two common challenges similar programs also face . For BTROur experience suggests incorporating multi-sectoral partners that better leverage existing resources may offer higher impact especially among similar research studies of small scale and magnitude . For exaOur compendium provides a perspective that can help draw a roadmap for a scientific research program when the project outputs and metrics are incorporated. In addition, aligning program objectives and exacting expectations across participants and stakeholders contribute to the roadmap that ultimately builds that partner country’s capability and capacity necessary for sustainability. Tracking the evolution of program projects and studies, especially collaborations and networks, is emphasized to capture important activities that are often omitted when large programs evolve and institutional memory is lost through turnover of human resources. To advance science through increased transparency and mature capabilities, a multi-sectoral approach, commitment, and vision is required to advocate a strategy and can be implemented through strong leadership.


High prevalence of hepatocellular carcinoma (HCC) and typically poor prognosis of this disease that lead to late stage diagnosis when potentially curative therapies are least effective; therefore, development of an effective and systematic treatment is an urgent requirement.In this review, several current treatments for HCC patients and their advantages or disadvantages were summarized. Moreover, various recent preclinical and clinical studies about the performances of “two efficient agents, sorafenib or natural killer (NK) cells”, against HCC cells were investigated. In addition, the focus this review was on the chemo-immunotherapy approach, correlation between sorafenib and NK cells and their effects on the performance of each other for better suppression of HCC.It was concluded that combinational therapy with sorafenib and NK cells might improve the outcome of applied therapeutic approaches for HCC patients. Finally, it was also concluded that interaction between sorafenib and NK cells is dose and time dependent, therefore, a careful dose and time optimizing is necessary for development of a combinational immune-cell therapy. Hepatocellular carcinoma is the fifth most common malignant tumor and second cause of cancer related death worldwide . DespiteSorafenib which is the first FDA approved drug for treatment of HCC, is a multi-kinase inhibitor that can block proliferation and angiogenesis of tumor cell by inhibiting a wide range of molecular targets including serine/threonine kinases, receptor tyrosine kinases, rapidly accelerated fibro sarcoma (Raf) kinases, vascular endothelial growth factor receptor 2, 3 , platelet-derived growth factor receptor (PDGFR), FLT3, Ret, and c-KIT , 15  and sorafenib in patients with intermediate-stage HCC is well tolerated and efficacious , 23. Amo+, CD3−) is in lung, liver, peripheral blood, spleen, bone marrow, lymph nodes, and thymus. The function of NK cells is very tissue microenvironment dependent; especially in a healthy liver that cytotoxicity and cytokine production of NK cells is higher than that of peripheral NK cells. Furthermore, the proportion of NK cells in the liver (30–50%) is more than peripheral blood NK cells (5–20%), in humans. It has been suggested that progenitors of circulating NK cell in peripheral blood migrate into the liver and differentiate into hepatic NK cells with distinctive properties, such as higher level of cytotoxicity, expression of cytotoxic mediators and cytokine production. There are two subtypes of NK cells, including CD56 bright and CD56 dim subsets, which are categorized based on the levels of CD56 expression. CD56 bright NK cells are induced from NK cell precursors and probably differentiate into CD56 dim NK cells (Approximately 90% of circulating NK cells and 50% of liver NK cells). Moreover, CD56 dim NK cells are more cytotoxic against target cells by releasing lower amounts of cytokines than CD56 bright NK cells [Natural killer cells were identified in 1975 as a subset of lymphocytes with cytoplasmic granules that contribute to the first line of innate immunity in the control of viral infections. The highest frequency of NK cells  class I genes that expressed in normal cells and often down regulated or silent in tumor cells to evade recognition by anti-tumor T cells. These events are known as ‘missing-self’ model and it has been shown the absence of MHC-I expression can be detected by NK cell-inhibiting receptors which result ininhibition of NK cells and prevention of deleterious graft versus host disease (GVHD) event. On the other hand, the activating receptors of NK cells are responsible for identification of induced ligand from DNA damage or cellular stress on the surface of the tumor cells, which lead to cytotoxic activity of NK cells and providing graft vs. leukemia (GvL) effect –50. As rNK cells are the essential components of innate immune system in the liver. They are involved in hepatic immune defense and pathology and perform both the protective and detrimental functions in human liver diseases. Some beneficial functions of these cells are inhibition of viral infection, prevention of liver fibrosis, and cytolytic activity against hepatic stellate cells as well as HCC cells in order to inhibit liver fibrosis and tumor growth, respectively. However, some detrimental roles have also been reported for NK cells, including hepatocellular damage and inhibition of liver regeneration .Inhibition of the frequency or functional impairment of NK cells is correlated with the progression and metastasis of various tumors in human and animal models , 53. It Despite the critical role of NK cells in the hepatic regeneration during viral infection, hepatic fibrosis and hepatocellular carcinoma, the frequency and cytolytic activity of NK cells have been impaired over the progression of liver diseases . Therefohttps://clinicaltrials.gov/, are illustrated in Table Clinical trials cell-based products are commonly classified into four phases. If the cell based therapeutic approach successfully passes through Phases I, II, and III, it will be approved for use in the general population of phase IV that is ‘post-approval’ phase. Clinical trial studies about treatment of HCC patients based on NK cells, alone or combined with other agents that have been registered in the site of In study performed by Kamiya et al.  on immunIn another study conducted by Zhang et al. , it has Krusch et al.  have invSeveral studies have been recently done on the mechanisms of sorafenib plus NK cells efficacy for better suppression of HCC progression Fig. . SprinzlNKG2D is one of the most important activating receptor expressed on the surface of NK cells , 109. MaOther mechanism that has been raised about the correlation of sorafenib and NK cells against HCC cells is targeting androgen receptor (AR) signals. Considering the key role of gender disparity involving AR during initiation and progression of HCC and normal function of liver  and its The majority of studies have shown that NK cells and sorafenib could enhance each other’s performances and compensated the deficiency of one another in the advanced stages of HCC. However, some studies have reported that sorafenib reduced the number of NK cells and inhibited their proliferation as well as reactivity against HCC cells. The paradoxical effect of this combination treatment is time- and dose-dependent. Therefore, careful dose and time optimization is necessary for development of a combinational immunotherapy for HCC. Furthermore, adaptive NK cell therapy or immunotherapeutic approaches activating NK cells along with sorafenib treatment may be a promising strategy to improve the therapeutic efficacy of HCC patients.


With the rapid development of Micro-electro-mechanical Systems (MEMS) fabrication technologies, many microelectrodes with various structures and functions have been designed and fabricated for applications in biomedical research, diagnosis and treatment through electrical stimulation and electrophysiological signal recording. The flexible MEMS microelectrodes exhibit excellent characteristics in many aspects beyond stiff microelectrodes based on silicon or metal, including: lighter weight, smaller volume, better conforming to neural tissue and lower fabrication cost. In this paper, we reviewed the key technologies in flexible MEMS microelectrodes for neural interface in recent years, including: design and fabrication technology, flexible MEMS microelectrodes with fluidic channels and electrode–tissue interface modification technology for performance improvement. Furthermore, the future directions of flexible MEMS microelectrodes for neural interface were described, including transparent and stretchable microelectrodes integrated with multi-functional aspects and next-generation electrode–tissue interface modifications, which facilitated electrode efficacy and safety during implantation. Finally, we predict that the relationships between micro fabrication techniques, and biomedical engineering and nanotechnology represented by flexible MEMS microelectrodes for neural interface, will open a new gate to better understanding the neural system and brain diseases. The function of microelectrodes in neural interfaces can be categorized as neural signal recording and neural stimulating. With the rapid development of micro fabrication technology, biomedical devices can be manufactured in a considerably tiny and structurally diverse manner, which minimizes the damage during and after implantation in both the short and the long term . NowadayOne of the most significant components of the artificial prostheses is the microelectrodes which act as the tissue–machine interface ,7,8. To It is a significant symptom that should be confronted that only electrical interaction between electrodes and muscle or nerve tissue without nutrition factor delivery would eventually lead to denervation-induced skeletal muscle atrophy ,21,22. C2) [In order to upgrade electrode performance of implantable Micro-Electro-Mechanical Systems (MEMS) microelectrodes, the ideal implantable electrode–tissue interface materials should satisfy requirements, including excellent electrochemical performance, good stability and biocompatibility. Therefore, many electrode–tissue interface materials have been developed to meet the practical demands. Nowadays, the most widely used electrode–tissue interface material remains noble metals, such as platinum, gold, iridium, tungsten and their alloys. These metals are chosen to be electrode–tissue interface because of their excellent chemical stability, which implies that they are able to be implanted in tissue without serious erosion. However, these materials are not suitable for microelectrodes with small-area sites due to their low charge injection limits (0.05–0.3 mC/cm2) , and few2) . Some elConducting polymers have also attracted much attention and have been broadly applied in many aspects of research in the biomedical domain for their unique characteristics, which include: low electrochemical impedance, high CSC, favorable plasticity (the performance can be optimized by doping counting ions), electrostriction (the volume can be changed by applying voltage) and biocompatibility. Simultaneously, conducting polymers are capable of meeting the requirements of electrode–tissue interface material. In addition, as shown in With the rapid development of MEMS fabrication technologies, researchers have been developed with various kinds of biomedical microelectrodes applied on electrical stimulation and electrophysiological signal recording for paralysis recovery. Among these many microelectrodes, Michigan neural probes and Utah electrode array are widely applied in central nerve system research as stiff MEMS microelectrodes for neural prosthesis. Many research efforts have been devoted to developing novel flexible MEMS microelectrodes for their excellent multi-functional characteristics, as compared to stiff microelectrodes, including their lighter weight, smaller volume, more effective conforming to neural tissue and lower fabrication cost. For example, Mercanzini et al. of École polytechnique fédérale de Lausanne (EPFL) described the fabrication of a novel, flexible, polyimide neural probe with two layers of platinum electrodes providing mechanical flexibility, high quality electrical characteristics and excellent biocompatibility. Two layers of platinum electrodes were used which greatly reduced the size of the neural probes, thereby limiting the insertion damage . Xu et aFerguson et al. of University of Minnesota used tetrode (four channels microwire electrodes) to record neural signals on freely moving animals in 2009 . As showKim et al. of University of Illinois at Urbana-Champaign developed a thin film microelectrode array based on polyimide for electrocorticogram (ECoG) recording in 2010. The microelectrode array was reinforced by silk fibroin, which acts as a biodegradable substrate, to improve the conformal attachment on the brain tissue surface . As dispTien et al. of Tufts University proposed silk fibroin as a multifunctional material for fabricating brain-penetrating flexible electrodes that combine dynamic mechanical properties with the capacity for local release of sensitive therapeutics . Silk isRui et al. of Shanghai Jiao Tong University developed a flexible 3D microelectrode array with raised hemispherical electrode sites in 2011 . The eleChen et al. of National Tsing Hua University designed a novel three-dimensional flexible microprobe to record neural signals of a lateral giant nerve fiber of the escape circuit of an American crayfish . As showKim and Xu of Wayne State University reported the development of flexible neural probes based on hybrid silicon–parylene structures to overcome the difficulty of implantation of soft probes and decrease the mechanical mismatch by the “local softness” concept . Figure Park et al. of Massachusetts Institute of Technology developed a device composed of an optical waveguide, six electrodes and two microfluidic channels produced via fiber drawing as shown in Xie and Lieber of Harvard University developed a three-dimensional macroporous nanoelectronic brain probe that combined ultra-flexibility and subcellular feature sizes to overcome the dimensional and mechanical mismatch of these probes with the brain tissue which limited their stability in chronic implants and decreased the neuron–device contact . As showWhile the researchers focus on developing MEMS microelectrodes with smaller dimensions, more complex structures and denser electrode sites distribution, they are also devoting much effort to multi-functionalizing the MEMS microelectrodes. The nerve conduction and muscle contraction actions of denervated paralyzed nerve and muscle tissue could be restored by electrical/optical stimulation based on MEMS microelectrodes for the artificial neural system. However, long-term lack of supply of neurotrophic factors would eventually lead to denervation atrophy of nerve and muscle tissue, which would mean irreversible loss of natural conduction of the nerve system and the contraction function of muscle. Therefore, various types of MEMS microelectrodes integrated with micro channels for drug or light delivery have been developed. Based on the inherent electrical/optical stimulation and electrophysiological signal recording properties, the novel MEMS microelectrodes integrate functions of delivering fluidic drugs, nutritional factors and light to target nerve and muscle tissue sites.Metz et al. of EPFL developed flexible MEMS microelectrodes with fluidic channels based on polyimide (PI) in 2004 . As dispZiegler et al. developed flexible MEMS microelectrodes with fluidic channels based on parylene in 2006 . As showGao et al. of the Shanghai Institute of Micro-System and Information Technology (Chinese Academy of Sciences) developed flexible MEMS microelectrodes with fluidic channels based on poly-dimethylsiloxane (PDMS) in 2013 . As showJeong et al. of University of Colorado developed ultrathin and soft neural probes based on PDMS for programmed drug delivery and photostimulation ,47. As iTian et al. of Shanghai Jiao Tong University developed flexible MEMS microelectrodes integrated polyimide micro fluidic channels and parylene micro wire electrodes in 2014 ,49. As sKim et al. of Wayne State University report the development of a novel 3D neural probe coupled simply and robustly to optical fibers using a hollow parylene tube structure . As illuCanales et al. of Massachusetts Institute of Technology present a class of neural probes fabricated from polymer, metal and composite materials by means of a thermal drawing process (TDP) traditionally employed in optical fiber production . They suQ is the flow rate, n is the number of the microchannels, ΔP is the pressure difference between the inlet and the outlet, fR is the flow resistance of the microchannel. Since flexible devices made of polymers have lower ultimate strength than silicon or metal, pressure applied at the inlet should be under a certain threshold to avoid rupture or irreversible deformation in the microchannel. The maximum pressure should also be taken into account at the design stage according to the yield strength of the material and the structure, as well as the allowable strength associated with the fabrication process such as bonding strength between layers. Only after the maximum flow rate and the allowable pressure are checked, the flow resistance can be determined. For uniform microchannels the flow resistance can be estimated by the following formula:η is the dynamic viscosity of the fluid, L is the length of the microchannel, C and A are the section perimeter and area of the microchannel respectively, while D is the diameter of the microchannel for circular microchannels. Equation (2) can be utilized for the dimensional design of the microchannel. For flexible microchannels, the dimensional design can also be affected by some other factors, such as the inflation caused by the applied pressure, bending of the microchannel caused by the insertion procedure and locomotion of the device or the tissue, which would cause change in the flow resistance.Various types of microfluidic neural probes have been reviewed in former studies , includiFunctional interface possesses the ability to combine physical effects, such as electrical , magnetiConducting polymers have also generated much attention, because they possess various distinctive characteristics, including high charge storage capacity, low impedance, excellent plasticity, and volume electrostrictive effect ,65,66. MThe fundamental properties of conducting polymers including surface morphology, electrical stimulating performance, stability and biocompatibility heavily depend on the characteristics of the negatively charged dopants, which are also termed counterions. For instance, mechanically strong macromolecules have the ability to enhance the stability of conducting polymer composites . SimilarCui et al. of University of Michigan doped polystyrenesulfonate (PSS) as negatively charged counter ion into PEDOT to form the PEDOT/PSS composite as electrode–tissue interface . As exhiAsplund et al. of the Royal Institute of Technology doped hyaluronic acid (HA), heparin and fibrinogen as negatively charged counter ions separately into PEDOT to form PEDOT/bio-molecule composites as electrode–tissue interface . As exhiLuo et al. of the University of Pittsburgh doped acidified carbon nanotube (CNT) as negatively charged counter ion separately into PEDOT to form PEDOT/CNT composite as electrode–tissue interface . As exhi4−) as negatively charged counter ion separately into PEDOT to form hollow nanotube structure as electrode–tissue interface [Abidian et al. of the University of Michigan doped perchlorate  as negatively charged counter ion separately into PEDOT to form the PEDOT/GO nanocomposite film as electrode–tissue interface ,85,86. ARyu et al. of the Daegu Gyeongbuk Institute of Science and Technology demonstrated a neural probe structure based on graphene, ZnO nanowires, and conducting polymer that provides flexibility and low impedance performance . As showFlexible microelectrodes create challenges for the fabrication of ultra-small metal patterns for better resolution. Thus, the development of microelectrodes with tiny dimension (sub-micrometer) and high-density electrode sites will be the goal for researchers. However the high-density electrode sites make the electrical connection difficult, which in turn will necessitate integration of the electronic circuit with the implantable electrode. However, integrating a commercial electronic circuit into a soft substrate is a challenge, since the substrate is flexible and sometimes stretchable. Delamination of the metal layer of chronically implanted soft electrodes is another critical problem . TherefoIn recent years, the applications of optogenetics in neuroscience also attracted much attention from neuroscientists. Although electrical stimulation exhibited remarkable efficacy in controlling and exploring the function of discrete brain regions and providing therapeutic solutions, it was unable to target genetically specified neuron types. The disadvantage of electrical stimulation could be overcome with genetically encoded opsins . As a coOther than that, the integration of flexible MEMS microelectrodes for neural interface and other flexible sensors, such as flow rate sensors, will also be promising in future research. Single element or single function devices will not satisfy the requirements anymore. Devices with multiple sensors, which employ electrochemical impedance methods that are compatible with neural probes , will beIn addition, the newly rising nanotechnology and biomedical engineering will open a new gate for the development of flexible MEMS microelectrodes for neural interface. The interdisciplinary research of micro fabrication technology with nanotechnology and biomedical engineering will direct future research ,93,94,95