Glial cell line–derived neurotrophic factor (GDNF) is a 134 amino acid protein belonging in the GDNF family ligands (GFLs). GDNF was originally isolated from rat glial cell lines and identified as a neurotrophic factor with the ability to promote dopamine uptake within midbrain dopaminergic neurons. Since its discovery, the potential neuroprotective effects of GDNF have been researched extensively, and the effect of GDNF on motor neurons will be discussed herein. Similar to other members of the TGF-β superfamily, GDNF is first synthesized as a precursor protein (pro-GDNF). After a series of protein cleavage and processing, the 211 amino acid pro-GDNF is finally converted into the active and mature form of GDNF. GDNF has the ability to trigger receptor tyrosine kinase RET phosphorylation, whose downstream effects have been found to promote neuronal health and survival. The binding of GDNF to its receptors triggers several intracellular signaling pathways which play roles in promoting the development, survival, and maintenance of neuron-neuron and neuron-target tissue interactions. The synthesis and regulation of GDNF have been shown to be altered in many diseases, aging, exercise, and addiction. The neuroprotective effects of GDNF may be used to develop treatments and therapies to ameliorate neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). In this review, we provide a detailed discussion of the general roles of GDNF and its production, delivery, secretion, and neuroprotective effects on motor neurons within the mammalian neuromuscular system. Neurotrophic factors (NTFs) are a class of proteins that promote neuronal survival, control cell proliferation and differentiation, are required for axonal and dendritic elaborations, and regulate synaptic plasticity  activation. GDNF was originally isolated from cultured B49 rat glial cells and found to enhance the survival and differentiation of dopaminergic neurons in primary cultures by promoting dopamine uptake  bonds, dimerizes, and later modified by N-linked glycosylation, and finally undergoes proteolytic processing into its mature form of 134 amino acids. This cleavage is due to a proteolytic consensus sequence located in exon II  that was expressed in the presence of tunicamycin. Tunicamycin is an inhibitor of N-linked glycosylation, and when GDNF was expressed in its presence, the processing of the protein was similar to the mutant’s. This data demonstrates that glycosylation is necessary for proper folding and processing of GDNF protein in mammalian cells. In addition, the mutation at the glycosylation site hindered cleavage of pro-GDNF into mature GDNF . It was concluded that glycosylation is not critical for receptor activation –anchored GFRα1 receptor  and forms a high-affinity complex as shown in Fig. Membrane microdomains are enriched with signaling proteins like the Src family kinases (SFK). This enrichment has led to the reasoning that these microdomains could function as a specialized signaling organelle Anderson . Work frNCAM NCAM isoform. The researchers demonstrated that the NCAM isoform interacts directly with GDNF family ligands along with GFRα receptors and mediates GDNF signaling with no RET presence. GDNF acting via the NCAM pathway promotes Schwann cell migration and axonal growth in cortical and hippocampal neurons  is a genetic disorder that causes alterations in dystrophin leading to muscle degeneration and frailty. A study done by Lie and Weis  examinedNguyen et al. generated a mouse model where muscle fibers make excess quantities of GDNF. The researchers noticed GDNF overexpression caused hyperinnervation at the motor end plates found in muscle fibers. GDNF made in muscle could be acting as a synaptotrophin at the NMJ. Interestingly enough, other neurotrophic factors like NT-3 and NT-4, when overexpressed, did not provoke hyperinnervation of muscle fibers  of post-synaptic end plates significantly increased in the soleus muscle following swimming exercise compared with sedentary control animals. In contrast, the total area of end plates significantly decreased in the EDL muscle after running training, and no differences were noticed after swimming training  is a disease that mainly affects MNs causing loss of voluntary movement. Due to the potential neuroprotective effects of GDNF, many studies have focused on ameliorating neurodegenerative diseases by adding exogenous GDNF. Researchers used human mesenchymal stem cells to deliver GDNF directly into skeletal muscle of familial ALS model rats. The rats showed increased muscular levels of GDNF, retrograde transport of GDNF protein into motor neurons, neuroprotective effects on neuron survival, and function at the neuromuscular junction  carrying the gene for GDNF  which were transplanted to the cortex of an ALS rat model (SOD1G93A). This study is the first to show the transplanted cells survived, eventually migrated, developed into mature astrocytes, and secreted GDNF protein. Results from this study showed MN protection, pathology of the disease was slowed down, and the animals experienced an increased life span. These same cells were also used in the cortex of a primate model (cynomolgus macaques) and also had noticeable GDNF expression, and negative effects relating to behavior in a period over 30 days were not observed (Thomsen et al. GDNF as possible treatment against MN disease. In addition, the successful use of these cells in a nonhuman primate facilitates and opens the door to further explore the hNPCGDNF and potentially use them in clinical trials for humans.Thomsen et al. used human cortical-derived neural progenitor cells modified to secrete GDNF (hNPCAn unfortunate effect of developing an embryonic GDNF knockout mice is renal agenesis causing neonatal death (Moore et al. Research regarding GDNF demonstrated a variety of neuroprotective roles for mammalian neurons and, as discussed in this review, GDNF acts as a potent neurotrophic factor for MNs. Through the research performed in the past few decades, GDNF has been found to aid in establishing new synapses between MN and target tissues, promoting growth, maintenance, and survival of neurons. With advancements in research and medicine, researchers can become more adept at delivering GDNF and other neurotrophic factors as promising candidates for a new way of therapy, preventing or treating neuro-related conditions. Nevertheless, there remains a significant gap in the knowledge of the pathways triggered by the complex of GDNF and GFRα following its activation of RET. Hopefully, future research will address the downstream effects of this intracellular signaling pathway and other pathways which will provide a stronger relationship between GDNF and its neuroprotective properties within MNs. Enhancing our understanding of GDNF and its signaling pathways will allow for the discovery of possible therapeutic approaches for applications of a broad range of neurological disorders such as ALS. Moreover, studying active-dependent GDNF release from skeletal muscle can aid in better creating exercise regimens as additional treatment for maintenance of the neuromuscular system.


Despite a wealth of sport nutrition guidelines for adult athletes, there are currently no nutrition guidelines for youth winter sports athletes. Whilst it may be pragmatic to apply nutrition guidelines for adult athletes to youth winter sports athletes, it is inappropriate. Due to a paucity of research on youth athletes, it is impossible to provide evidence-based guidelines for this population, so careful extrapolation from the theoretical and practical considerations that apply to other athletic groups is necessary. Youth winter sport athletes undergo rapid biological growth and maturation which influences their nutritional requirements. A varied and balanced diet that ensures sufficient energy availability for optimal growth and maturation as well as sporting performance is the cornerstone of youth athlete nutrition and should also allow for youth athletes to meet their micronutrient requirements. In some cases, micronutrient status  should be monitored and optimized if appropriate by a medical professional. Dietary supplement use is prevalent amongst youth athletes, however is often unnecessary. Education of youth athletes, their parents and coaches on best nutritional practices as well as the risks associated with dietary supplements is vital for their long-term athletic development. Further research in youth winter sports athletes across different stages of growth and maturation competing in a variety of sports is urgently required in order to inform nutritional guidelines for this population. Despite a wealth of sport nutrition guidelines for adult athletes  cannot simply be considered “mini adult athletes” despite sometimes competing against adults. Except for the first year of life, adolescence  is the period of greatest growth, maturation, and development across the lifespan  from chronological age–the time interval since birth  their current maturity status and rate of growth and maturation, (2) their current physiological, metabolic, and psychosocial capacities, and (3) their general life and sport demands  to under 16 years  age-groups after which there was no further increase in resting metabolic rate . An overview of the typical training of three Swiss athletes at YOG in Lausanne 2020 is shown in In healthy physically active humans, exercise energy expenditure is the most variable contributor to TEE . Between athletes in this study  may lead to relative energy deficit in sport (RED-S), resulting in impaired growth and maturation of tissues and organs, reduced skeletal bone mineral accrual, increased risk of stress fractures, increased risk of osteoporosis later in life, delayed sexual maturation, disruption of reproductive function  and a less effective immune system  is required.Whilst it is difficult to prescribe energy requirements for youth winter sports athletes at both the population and individual levels, it is advised that youth athletes have sufficient energy availability for growth. Energy availability is the amount of energy left for homeostatic physiological functions and growth once physical activity energy expenditure has been deducted from energy intake and is relative to FFM [energy availability = /FFM]. Chronic low-energy availability  the recommended energy intake value  would underestimate the need for intake to cover actual energy expenditure and might lead to insufficient intake.Total energy expenditure is a better starting point to prescribe dietary intake rather than diet macronutrient composition which is the basis of many sport nutrition recommendations  and ~95% of adult bone mineral content is achieved by the end of adolescence  is a key regulator of calcium homeostasis, with sufficient levels required for calcium absorption Holick, . Therefo3. These decisions should be taken by a medical doctor and individualized to the athlete. Although a blanket approach to vitamin D3 supplementation is not advised, a common approach is to supplement athletes with during winter months even without testing vitamin D status . Nonetheless, the use of dietary supplements such as whey protein, carnitine, branched-chain amino acids, etc. is high amongst youth athletes  and deficiency is generally the only appropriate reason for supplement use in youth athletes. A varied diet that includes sufficient intake of all the essential nutrients should always be the focus. All essential nutrients can be obtained in sufficient quantities from the diet alone with the exception of vitamin D (see section Vitamin D) albeit others may be challenging to obtain in sufficient quantities in certain diets  even though the training session was conducted in a cool environment  may be a risk for youth athletes as their core temperature seems to rise faster than adults under exercise or environmentally induced heat stress  should also allow for youth athletes to meet their micronutrient requirements. In some cases, vitamin D and iron should be monitored and supplemented (if appropriate) under the guidance of a medical professional. In addition to these specific nutritional needs, youth athletes, their parents, and coaches should be educated in sports nutrition. Adolescence is the “prime time” for qualified practitioners to provide athletes, parents, and coaches with information on research-informed nutritional practice. In any case, nutritional considerations for youth athletes should always put growth, maturation and sporting development first and performance second. Further research in youth winter sports athletes across different stages of growth and maturation competing in a variety of sports is now required in order to formulate research-informed nutritional guidelines for this population in keeping with this principle. Key research topics to be addressed include the energy, macro- and micronutrient requirements for youth athletes competing in different winter sports in addition to developing evidence informed educational strategies.MPH and CD were invited to contribute to this special edition. MPH, JLF, VG and CD planned and drafted this manuscript. NP and BK provided critical revision of the manuscript. All authors approved the final version of this 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.


As dietary behaviours developed during early childhood are known to track into adulthood, interventions that aim to improve child nutrition at a population level are recommended. Whilst early childhood education and care (ECEC) is a promising setting for interventions targeting children’s nutrition behaviours, previous interventions have largely used high intensity, face-to-face approaches, limiting their reach, implementation and potential impact at a population level. Web-based modalities represent a promising means of supporting the delivery of childcare-based interventions whilst overcoming challenges of previous approaches; however, the feasibility of using such modalities to support implementation is largely unknown. As such, this study sought to collect feasibility and pilot data to inform the design of a web-based intervention together with health promotion officer support within childcare centres. Child dietary intake will also be assessed to provide an estimate of the impact of the implementation intervention.A superiority cluster randomised controlled trial with repeat cross-sectional data collection employing an effectiveness-implementation type-II hybrid design will be conducted with childcare centres within the Hunter New England region of New South Wales, Australia. Type-II hybrid designs provide the opportunity to assess intervention efficacy whilst piloting the feasibility of the implementation strategies. Centres allocated to the intervention group will receive access to a web-based program together with health promotion officer support to implement targeted healthy eating practices to improve child diet in care. A number of outcomes will be assessed to inform the feasibility to conduct a larger trial, including childcare centre and parent recruitment and consent rates for each component of data collection, uptake of the implementation strategies, acceptability of the intervention and implementation strategies, appropriateness of the implementation strategies and the contextual factors influencing implementation.This study will provide high-quality evidence regarding the potential feasibility of a web-based intervention and the impact of healthy eating practices on child diet in care. Web-based modalities provide a promising approach for population-wide implementation support to childcare centres given their potential reach and consistency with existing infrastructure.ACTRN12619001158156).Prospectively registered with Australian New Zealand Clinical Trial Registry (Supplementary information accompanies this paper at 10.1186/s40814-020-00707-w. Childhood overweight and obesity increases the risk of adult obesity and several other chronic diseases, including cardiovascular disease, type 2 diabetes and specific cancers . InternaEarly childhood education and care (ECEC) is a promising setting for interventions targeting children’s nutrition behaviours. With at least 80% of children in countries including Australia and the U.K. attending formal centre-based childcare (herein referred to as childcare centres) , 9, inclWhilst the implementation of such practices could potentially improve child nutrition, studies show that such practices are not routinely implemented by childcare staff . A 2016 Web-based modalities may be a promising way of supporting the implementation of childcare-based interventions whilst overcoming some of the challenges of previous approaches. As 100% of childcare centres in Australia have access to and use a computer daily for reporting requirements , web-basTo our knowledge, only two randomised controlled trials (RCT) on the impact of a web-based program in childcare centres have been published. A pilot implementation RCT with 31 centres who provided food in care from within the U.S. assessed the impact of the web-based Nutrition and Physical Activity in Child Care (Go-NAPSACC) program on childcare healthy eating environments . The stuGiven the limited existing evidence base, the primary objective of this study is to examine via a type-II hybrid cluster RCT, the potential feasibility of a web-based intervention together with health promotion officer support within childcare centres, whilst assessing the uptake, acceptability and appropriateness of the intervention and implementation strategies, and contextual factors influencing implementation.Examine the potential effects of the implementation strategy employed in the study on childcare centre implementation of recommended practices;Assess the effectiveness of the intervention in increasing child dietary intake of fruit and vegetable servings, and decreasing child dietary intake of sodium (milligrams (mg)), saturated fat (grams (g)) and added sugar (grams (g)) in care; andAssess the effectiveness of the intervention in increasing servings of fruit and vegetables packed within children’s lunchboxes.Secondary objectives are to:A superiority cluster RCT with repeat cross-sectional data collection employing an effectiveness-implementation type-II hybrid design will be conducted . A hybriMunch & Move) [To be eligible, childcare centres must (1) enrol > 20 children per day, (2) have internet access at the centre, (3) not provide meals or snacks to children (i.e. parents or caregivers must be required to provide food packed in lunchboxes), (4) not be currently participating in any other intervention to improve child healthy eating and/or physical activity and (5) not be fully compliant with healthy eating practices targeted by the intervention and specified in the NSW state obesity prevention program (i.e.  & Move) . Mobile A list of potentially eligible centre-based childcare centres located within the HNE region will be provided by the NSW Ministry of Health . EvidencIn order for children to be eligible to participate, they must (1) have prior written consent from a parent or guardian, (2) be between the ages of 2 and 5 years and (3) not have a dietary restriction that requires specialised tailoring of their diet .Approximately 2 weeks prior to data collection, centres will be asked to distribute parent information statements and consent forms via electronic methods, including email and parent communication apps, and child pigeonholes as part of standard communication with parents. The dates of the scheduled site visits will not be disclosed to parents to avoid any changes to parent usual lunchbox packing. Additionally, approximately 1 week prior to the scheduled site visit, and on the day of the scheduled site visits, two RAs trained in recruitment and data collection procedures will be present at the childcare centre to request written consent from parents for child participation in the study.Childcare centres will be randomly allocated following a block randomisation procedure in a 1:1 ratio to either intervention or control using a computerised random number function in Microsoft Excel 2013. Due to the demographic and socioeconomic diversity of the HNE region, randomisation will be stratified by centres with a high number of Aboriginal child enrolments (> 10%) and by centre socioeconomic status (SES), as determined by Socio-Economic Indexes for Areas categorisation using centre postcodes , 36. RanThe intervention will target nominated supervisors and staff within childcare centres and support their implementation of five healthy eating practices. The selection of the targeted practices is broadly consistent with the social ecological framework (SEF) which posits that individual behaviour can be influenced via factors through five nested, hierarchical levels  . Whilst i.)Supporting families to provide healthier foods consistent with dietary guidelines: Childcare centres will be asked to monitor children’s lunchboxes for consistency with dietary guidelines on a daily basis and distribute nutrition-focused messages to parents that promote the packing of healthy lunchboxes at least twice during the intervention period. Messages will offer advice to address parents’ commonly reported barriers to providing healthy foods, including overcoming fussy eating, improving food acceptance, providing healthy foods on a budget and quick and healthy options .ii.)Provision of intentional learning experiences about healthy eating to children: Childcare centre staff will be asked to provide children with intentional learning experiences at least twice per week aimed to support children’s development of healthy eating behaviours . Intentiiii.)Use of feeding practices that support children’s healthy eating: Childcare centre staff will be asked to provide positive reinforcement and encouragement to children to promote healthy eating and trying new foods at every meal and snack occasion. They will also be asked to model healthy food and drink choices at every meal, provide positive comments about healthy foods within children’s lunchboxes and avoid the use of food incentives to encourage desired behaviour , 15, 41.iv.)Staff participation in professional development in healthy eating: Childcare centres will be asked to have at least 50% of staff take part in online training opportunities targeting staff healthy behaviours and practices in the centre , 42. Thiv.)Having a comprehensive written nutrition policy that outlines key healthy eating practices: Childcare centres will be asked to develop or modify their existing nutrition policy to ensure the centre has strategies, procedures and guidelines to enforce the implementation of healthy eating practices to improve child diet . ChildcaSpecifically, childcare centres will be asked to implement the following targeted healthy eating practices within the 6-month intervention period:The Behavioural Change Wheel (BCW)  was usedSpecifically, the implementation strategies incorporated into the web-based program, known as Childcare Electronic Assessment Tool and Support (EATS), include:Childcare EATS includes a self-assessment of the implementation of targeted healthy eating practices. Following the completion of the self-assessment, the web-based program will immediately provide centres with feedback on practice performance. Childcare centres will be encouraged to complete the self-assessment at least twice during the intervention period to self-monitor improvements in practice , 33.Following the completion of the self-assessment, childcare centres will be encouraged to use Childcare EATS to set goals and create an action plan to facilitate improvements in practice . CentresChildcare EATS will house relevant materials developed through consultation with key stakeholders, including childcare centre staff, cultural liaisons and health promotion officers with extensive experience working within the setting. Materials were designed to assist centre’s adoption of targeted practices and include factsheets, email messages and newsletter snippets to facilitate communication with parents regarding children’s lunchbox alignment with guidelines; educational materials to improve staff knowledge of providing a positive healthy eating environment; example activities to demonstrate intentional nutrition learning experiences within the centre; directions to online learning opportunities, including webinars and eLearning modules to support staff professional development in nutrition; and nutrition policy templates .In addition to web-based resources, childcare centres allocated to the intervention will receive support from HPOs within the local health district with experience working with childcare centres. The implementation strategies provided through these HPOs will include:Upon notification of group allocation, the HPO will ask centres to identify and prepare a staff member from the centre who will dedicate themselves to supporting, marketing and driving implementation of the intervention , 51.Centre staff (nominated supervisor and centre champion) will receive one face-to-face training session by an HPO to support implementation of the healthy eating practices and introduce the web-based program at the beginning of the intervention period . This wiCentre nominated supervisors will be asked to show support for implementing targeted healthy eating practices via a memorandum of understanding, which will outline the responsibilities and expected commitment from both the childcare centre and HPO in working to improve the implementation of healthy eating practices to improve child dietary intake in care. The memorandum of understanding will be discussed and agreed upon during the educational outreach visit with centre supervisors.Childcare centre staff will be provided with approximately two telephone calls by an HPO, pending childcare centre needs, within the intervention period , 53. BarChildcare centres allocated to the control group will receive usual care during the intervention period. Usual care includes general support from health promotion officer upon request to implement the state-wide obesity prevention program . SupportFeasibility of the intervention, defined as the extent to which the intervention can be successfully used or carried out within the childcare setting  for a fuChildcare centre use of Childcare EATS will be assessed through data provided via Google Analytics . These aSupervisors and champions of the childcare centres randomised to the intervention group will complete a telephone interview to assess acceptability during follow-up data collection (6 and 12 months). Acceptability will be defined as the perception amongst centre staff that the intervention and implementation strategies are agreeable, palatable or satisfactory . This wiAppropriateness, defined as the perceived fit, relevance or compatibility of the intervention and implementation strategies for the childcare setting , will beRelevant constructs within three of the five domains of the CFIR   and additional data on centre nutrition environments will be assessed with the Environmental and Policy Assessment and Observation (EPAO) tool . Per EPAThe mean servings of fruits and vegetables from all food and beverages consumed whilst in care will be assessed through the measurement of lunchbox foods and beverages across the day. On the days of the site visit, two trained RAs will assess the lunchboxes of participating children. Measurement of lunchbox contents will be conducted on two occasions across the day: prior to the children’s first meal time and after the children’s last meal time. RAs will remove all contents of the lunchbox and remove any lids that inhibit the view of contents. A photo will then be taken of the entire lunchbox contents. RAs will then weigh each food item included in the lunchbox, with strict adherence to safe food handling practices to address occupational health and safety concerns. A written description of the contents will also be captured to enable accurate recordings where ingredients may be easily deciphered via photograph, e.g. sandwiches and mixed meals. The process of photographing, weighing and recording lunchbox contents will be repeated after the children’s last meal. Consumption will be calculated based on foods and beverages present at the first measurement minus foods remaining at the second measurement. Educators will direct children to keep food wastage, including all partially consumed food and beverages, within their lunchboxes. All food wastage will be collected by the research team during the second measurement and factored into child consumption measurements. The weighed plate method with photographs has been previously used by the research team  and has The nutrient output provided by weighed food record data entered into the nutrient analysis database (FoodWorks) following the process described above will be used to measure mean sodium (mg), saturated fat (g) and added sugars (g) from all foods and beverages consumed whilst in care.To determine the impact of the intervention on parent provision of healthy food in lunchboxes, the mean servings of fruit and vegetables packed within children’s lunchboxes will be assessed via observation and measurement of lunchboxes and lunchbox foods and beverages following the same process described above.Operational centre characteristics will be assessed at baseline during a telephone interview with nominated supervisors. Items within the telephone interview have been used previously by the research team , and incChild characteristics, including gender, age, Aboriginal and/or Torres Strait Islander origin, days attending care and parent level of education, will be collected from parents/guardians when providing written consent to participate in the study.As this is a pilot study, a formal sample size calculation for the primary outcome is not required . HoweverThe primary trial end-point will be the 6-month follow-up. Descriptive statistics will be used to describe childcare centre and child characteristics, the feasibility, uptake of implementation strategies, acceptability and appropriateness of the intervention and determinants of implementation. At the centre level, to determine the impact of the intervention on the implementation of healthy eating practices, scores of the EPAO will be compared between intervention and control centres at follow-up, adjusting for baseline, through linear regression analysis. At the child and centre levels, multiple imputations will be performed as part of a sensitivity analysis for missing follow-up data as recommended by White et al. . At the Data obtained from the trial will inform decisions regarding progression to a fully powered implementation trial. Such decisions will be made via majority, from core members of the research team, including a representative from a public health service partnering in the research that intends to adopt the intervention and implementation support strategy if identified as beneficial . The decInterventions targeting the childcare setting are recommended to improve child dietary intake in care due to the potential to reach a large number of children during a crucial developmental period . DespiteAdditional file 1. SPIRIT 2013 Checklist: Recommended items to address in a clinical trial protocol and related documentsAdditional file 2. SPIRIT Figure


Laboratory diagnostic testing service delivery and compliance with international standards for laboratory quality are directly influenced by laboratory workforce competency. Many hospital laboratories in constrained resource settings such as Cambodia struggle to cope with the training needs of laboratory professionals in an environment of competing healthcare development priorities. Resource-limited countries need an adaptable and effective approach to provide laboratory professionals with job-specific quality oversight training to ensure the accuracy, timeliness, and reliability of diagnostic services.Here, we describe the results of an in-service training and mentoring program conducted with the Cambodia Ministry of Health at 12 tertiary-level hospital laboratories to drive improvements in laboratory quality management systems toward ISO 15189 accreditation, which demonstrated significant progress between baseline and outcome audits in a concurrent study. This case study describes the program, and evaluates how the four primary activities, including actionable gap assessments and planning, centralized and in situ training curriculum, in-person mentoring, and remote tele-mentoring via video communication technologies, contributed towards quality improvement in the participating laboratories. We evaluated participant responses to Likert scale and free response questions from program and training evaluation surveys, and we used thematic analysis to develop a model of best practices within the program’s four primary activities to inform future training approaches. Of these activities, participants agreed most highly that in-person visits and planning based on gap assessments contributed to their learning and ability to improve laboratory operations. Tele-mentoring was rated lowest by participants, who were critical of excessive group dialogue and distraction during web-conferencing; however, feedback suggests both in-person and remote mentoring contribute to continuing education, accountability to action, and peer collaboration and problem solving to improve workforce efforts toward improved quality management systems.We recommend here a package of in-service training activities for laboratory quality management system improvement initiatives in resource constrained settings that includes needs-based curricula and personalized action plans for participants; interactive and on-site training workshops; and in-person mentoring, complemented with well managed and regular tele-mentoring that focuses on knowledge retention, accountability to goals, and collaborative problem solving. Our model presents an adaptable approach to human resource development for quality improvement in medical laboratories. Laboratory-based diagnostic testing plays a critical role in clinical decision-making and public health and accurate laboratory data are essential for informed decision-making –3. LaborIn 2016, the first joint external evaluation was conducted to measure Cambodia’s achievements towards meeting the International Health Regulations. This revealed gaps in the areas of LQMS and workforce capacity, and additional investments in these areas were recommended to the Ministry of Health (MoH) . The IntDesign and implementation of laboratory audits and development of action plans with laboratories;Development and implementation of a formal LQMS curriculum and practical application training in situ;In situ mentoring of laboratory staff;Remote training sessions and mentoring through video conferencing technologies.This training and mentoring program was implemented through four primary activities with the objective to educate and mentor staff in the 12 participating laboratories towards implementing a LQMS in compliance with the ISO 15189 standard. These included:In support of a MoH initiative to establish an improved national regulatory process over laboratory quality performance, this program provided training for 12 laboratory staff from participating laboratories to become professional LQMS auditors. This auditor training program was conducted over a period of 4 weeks via tele-conferencing and focused on LQMS principles, emphasizing ISO 15189 requirements (four power-point presentations with Khmer translation) and practice (each auditor trainee performed an audit independently using the CamLQMS checklist under the supervision of experienced auditors). The CamLQMS tool was utilized by the audit team to conduct baseline and exit audits of each laboratory participating in the program. The elements of the CamLQMS checklist specified national standards for LQMS conformity based on ISO 15189 and the Clinical and Laboratory Standard Institute (CLSI) QMS01-A4 guideline  goals anInformation obtained during the audit process informed the development of a competency-based training curriculum for laboratory managers, quality assurance officers (QAOs), and other QI personnel in the laboratory, with training sessions implemented from resulting series of “off-site” and in situ workshops between February 2018 and April 2019. Four off-site workshops were conducted in Phnom Penh and were primarily focused on delivering LQMS and organizational leadership and management theory. These workshops used adult-learning strategies to engage participation and discussion, including dynamic simulation, experiential learning, peer collaboration, and transformative learning through problem solving. In situ sessions were designed for staff to apply LQMS theory using hands-on skills. Each training session included a combination of formal lectures by quality experts, peer discussion, case presentation and reflection, and experiential practice . All traProviding one-on-one follow-up training with laboratories;Ensuring accountability of laboratory management and quality assurance personnel to action plans;Providing on-site technical guidance and collaboration to identify and resolve problems.A team of four Cambodian QI mentors conducted regular site visits to laboratories for individual consultations with each visit designed to guide, support, and engage participants in QI efforts by:Mentors were dispatched a minimum of four times to each laboratory with laboratory specific TA and mentoring following workshops.en masse communication.The program’s tele-mentoring approach was modeled on successful clinical mentoring programs such as Project ECHO™ and used a similar “hub and spoke” structured format to advance knowledge and skills of laboratory staff . Zoom  responded. Results demonstrated that participants agreed most highly that in-person mentoring and CamLQMS audits were valuable , receiving the highest frequencies of “strongly agree” ratings and the highest mean ratings Table . ConversObservations and qualitative analysis uncovered 23 themes regarding best practices, 21 of which were supported by participant feedback. We analyzed 239 individual comments from training evaluations and an additional 39 textual responses from program evaluation surveys. Responses from participants were brief, ranging from 1–68 words in length (median = 13). From these, we identified 147 instances of positive feedback and 99 instances of constructive critique for improvement, from which 123 comments either corroborated best practice themes or introduced novel themes according to the associated primary activity supported (Fig. “Plans > action > results > follow-up—Those process [sic] help to improve my lab quality”. Although not corroborated by participant feedback, we also observed a clear benefit to participants from immediate feedback and corrective action by auditors during audits, and from auditors engaging the attention of both hospital leadership and government stakeholders.Participant feedback indicated the “useful” role of the CamLQMS assessments in their QI progress, which provided a clear entry point for the continuous QI cycle, enabling participants to identify gaps and develop SMART action plans to address deficiencies. As one participant summarized: The audit training program as well was observed to successfully engage selected laboratory staff in the audit process while supporting the MoH initiative to provide trained personnel for regulatory LQMS oversight and auditing. While a separate study would be required to compile and analyze feedback from these additional trainings, improved audit training is a recommended best practice to assure quality metrics.“Every participant should raise, share, [sic] talk about experience”, as one participant suggested.58 comments indicated improved learning due to practical training sessions, 22 of which referred to specific curriculum such as management review and equipment validation as being helpful. Additionally, peer learning and collaboration was remarked highly by participants, who often requested more peer learning activities where participants of different labs could share ideas and experiences, learning from each other and collaborating to troubleshoot and solve practical QI challenges. “This on-site lab training is so important, because we can practice directly and get new experience and skill”. One of the key benefits to the in situ method was the advanced training and improvement opportunity that it gave to higher performing laboratories, which had the opportunity to present their own quality management systems for critical observation by their peer participants from other laboratories. Lower performing laboratories, meanwhile, were able to learn from the best practices of their peers. This practice addressed the need to match training content to benefit all ranges of participant experience. Participants were also able to acquire practical skills through direct hands-on participation during the experience. “I think practice is better than theory”, one participant suggested.Participants expressed considerable preference for in situ training activities, mentioning specifically the practical and experiential learning as important for participant improvement: Finally, although our trainers strived to conduct all training in the local language wherever possible as a best practice, feedback from participants expressed strong critique for translators when information was not translated clearly enough. It was apparent that the training curriculum required further strengthening to meet the language needs of participants.“Mentor can see real situation in my lab and can suggest and help initiate my lab team to do something according to the gap.” Participants also suggested that face-to-face visits from mentors served to improve the professional mentor–mentee working relationship, and further comments remarked that mentor visits had helped engage other important stakeholders such as hospital directors and MoH regulators in laboratory QI. One participant addressed both factors, stating “Regular periodic visits from trainers is important because it makes an important connection between the training institute and the laboratory staff. An in-person trainer visit is also important to increase the credibility of the laboratory staff to the laboratory director.” When mentors visit laboratories, MoH personnel and laboratory directors are invited to meet with mentors and QAO to discuss current and past improvement efforts, garnering support for laboratory improvement efforts; this engagement of leadership is critical.Participant feedback indicated strong value for face-to-face in situ mentoring and appreciated follow-up provided regarding QI plans and holding participants accountable. Participants appreciated the problem-solving suggestions of mentors, identifying and discussing problems on-site with participants and collaborating to overcome challenges to improvement. One participant commented: too much talk with a lot of peoples [sic], difficult to take experience”, “talk for everything”, and “participants not [sic] pay attention during zoom call.” This feedback suggests that improved facilitation structure and efficiency of video conferencing may be a best practice to improve the learning experience of participants, so long as the opportunity for peer learning is not sacrificed.A concurrent study of audit results found a significant correlation between participation time spent in Zoom tele-conferencing and laboratory QI ; howeverAnother concern observed and heard from participants throughout the program, though surprisingly only addressed once through written feedback, is the challenge of unstable internet connections, which often decreased meeting productivity. In some locations, user desktop computers lacked a camera or webcam, which inhibited interactive participation. Assessing and improving technical capacity and internet connectivity should therefore be a necessary component of telementor program planning. Despite these challenges, participants noted improved knowledge from tele-mentoring sessions, appreciated regular feedback from mentors, and again praised the ability to collaborate with both peers and mentors, sharing experience and knowledge. Participants further praised the real-time access to QI mentors being able to ask questions and receive a response immediately, which was an important best practice of the mentors, who set regular open hours for participants to call for guidance. Training in the local language was once again a strong request from participants, and one comment noted the cost effectiveness of tele-mentoring, saving participants time and money required for travel. In addition to participant feedback, several strategies to address these challenges are recommended Table .Table 2RAs demonstrated by measurable improvements in CamLQMS audit scores at the 12 laboratories, this training and mentoring approach made a positive impact in Cambodia and correlates with similar reports in the region , 16. We Our program was designed specifically to utilize peer learning among a set of other dynamic, hands-on, and interactive adult-learning strategies implemented as adaptive drivers of change. Such strategies have been shown to be effective within healthcare professional training , 18, andWe recommend that tele-mentoring activities be designed to reinforce the continuous QI process, be results focused, encourage peer accountability, and collaborative learning and problem solving, and be conducted in the local language, wherever possible. We further recommend that mentors work with smaller groups or individual laboratories as much as possible for best results, maintaining the structure and control of the discussion, and establish time-bound question and answer periods to make the best use of participant time. Lastly, tele-conferencing success is linked to capable hosting and facilitation, reliable computing hardware with audio-visual capability and engaged participants. The importance of this capacity within health systems is visibly apparent during outbreaks of novel communicable diseases such as COVID-19, when physical distancing as a preventive measure hinders in-person training and communication [Because most of these best practice themes are derived first from implementing staff observations, this study may be influenced by positive biases toward the implemented activities and practices, missing potentially useful practices not observed by either staff or participants. Data from program participants too are limited by positive response bias from participants, and is further limited to written feedback only, which is limited to short answer responses. Extensive interviews of participants or key stakeholders may provide additional information. Program evaluation responses were further limited by language barriers among participants, who had varying abilities to respond clearly to the surveys in English, which resulted in some unclear or truncated responses. Feedback from training evaluations, which were responded to in either English or Khmer, contains the potential for translation error and bias as responses were translated into English by mentors rather than professional translators.This study shows how a customizable package of needs-based planning, training, and mentoring can lead to improved laboratory QI via an informed, trained, and empowered workforce. Our model of best practices serves as a guide for future laboratory and health workforce development programs in Cambodia and globally. Participant feedback indicates the usefulness of in situ training and peer learning, and regular contact with experienced laboratory professionals who can provide both theoretical and practical coaching, hold participants accountable to goals, and enable collaboration to identify and solve problems. Support from local QI mentors and tele-mentoring was also associated with improved outcomes. We further recommend that in-person mentoring be maintained to some degree in such programs, due to its perceived value among mentors and participants in the improvement process. The successful blended learning package of activities including the auditor training program suggest that national scale-up would be a worthwhile investment in Cambodia. National entities responsible for health workforce development could model the successful elements of this program such as video conferencing for training and mentoring in order to reach more working professionals, engage more facilities in a national quality improvement effort, and utilize remote technology more fully as part of a staff competency assessment program and become a platform for delivering professional development opportunities in Cambodia.


Unfamiliar face processing is an ability that varies considerably between individuals. Numerous studies have aimed to identify its underlying determinants using controlled experimental procedures. While such tests can isolate variables that influence face processing, they usually involve somewhat unrealistic situations and optimized face images as stimulus material. As a consequence, the extent to which the performance observed under laboratory settings is informative for predicting real-life proficiency remains unclear.n = 252) measures identity matching across substantial age-related changes in facial appearance, while the FICST (n = 218) assesses the ability to process unfamiliar facial identity despite superficial image variations. To determine the predictive value of both tests, a subsample of our cohort (n = 181) also completed a commonly used test of face recognition and two tests of face perception (the long form of the Cambridge Face Memory Test (CFMT+), the Expertise in Facial Comparison Test (EFCT) and the Person Identification Challenge Test (PICT)).We present normative data for two ecologically valid but underused tests of face matching: the Yearbook Test (YBT) and the Facial Identity Card Sorting Test (FICST). The YBT  YBT and FICST performance can predict CFMT+ scores and vice versa; and 2) EFCT and PICT scores neither reliably predict superior performance in ecologically meaningful and challenging tests of face matching, nor in the most commonly used test of face recognition. These findings emphasize the necessity for using challenging and ecologically relevant, and thus highly sensitive, tasks of unfamiliar face processing to identify high-performing individuals in the normal population. How do humans process unfamiliar faces, and how can we reliably identify individuals that are most proficient at it? Motivated by its relevance in applied contexts, recent empirical work has sought to answer these questions. Controlled laboratory tests have been developed to understand the contribution of different variables and differences between observers. However, face processing tests involving face stimuli derived from ideal and highly controlled manipulations may not accurately represent real life. This crucial consideration is often overlooked when laboratory tests are used to predict real-life proficiency. The present study followed the rationale that traditionally used controlled tests should be paired with more realistic and ecologically meaningful ones, in terms of the images used and tasks performed. Testing large and heterogeneous samples, we standardized two less frequently used tests of facial identity matching: the Yearbook Test (YBT) and the Facial Identity Card Sorting Test (FICST). These procedurally simple tests mimic real-life challenges in face perception as they assess unfamiliar facial identity matching across superficial image changes, or substantial age-related changes in facial appearance. Beyond providing normative data, we describe how performance measured by these tests relates to that observed on more commonly used tests of face recognition (the long form of the Cambridge Face Memory Test (CFMT+)) and perception  and the Person Identification Challenge test (PICT)). Our findings suggest that: 1) the YBT and FICST are easy to use and are preferable alternatives to pairwise face-matching tasks which are more prone to speed–accuracy trade-offs and/or ceiling effects; and 2) challenging and ecologically meaningful tests should complement highly controlled measures when aiming to identify individuals with superior face processing abilities for real-life purposes.In our everyday lives, we effortlessly process constantly changing facial information. The commonly accepted notion that it is performed in a highly proficient manner underlies a vast body of research and, for the most part, it coincides with our own personal experiences. However, an increasing number of studies is challenging the idea that face processing is generally highly proficient. Extremely high performance levels are observed in particular for the processing of faces of individuals encountered repeatedly in everyday life—those that are personally familiar to us  designed to capture them in a reliable manner. In the context of unfamiliar face processing, the most commonly assessed processes include face perception and face recognition. These are separate processes that require careful terminological distinction  among image pairs  scenario. This is thought to parallel identity verification at, for example, a passport control point where naturally occurring variation in ambient images is known to affect performance  with previously reported tests of facial identity matching. These tests tap into invariance of facial representations by measuring facial identity matching ability across two dimensions that are pertinent to real life challenges. The YBT  than the EFCT and PICT. Beyond this, we sought to determine the relationship between performance measures provided by the YBT and FICST, as well as the EFCT and PICT, with the most commonly used tool to assess face cognition, the long form of the Cambridge Face Memory Test , the EFCT and PICT were the least challenging. Based on these observations, we advocate for increased use of more challenging measures that involve manipulations that are pertinent to real-life settings, such as the YBT and FICST. Compared to 2AFC scenarios, in these tests time-consuming strategies are less effective for achieving high-performance accuracy. The conditions under which tools are developed  have to be carefully considered in combination with the real-life roles in which they are deployed .A total of 252 observers  completed the aforementioned YBT. Of this cohort, 218 additionally completed the FICST. In addition to the YBT and FICST, 181 observers completed the CFMT+, EFCT, PICT, and Cambridge Face Perception Test (CFPT). Detailed demographic information for each sample is provided in Table Each observer completed the paper-based YBT; if participation was not terminated, additional tests were administered. These included the paper-based FICST, as well as four computer-based tests  which were completed (in randomized order) using the standalone Java applications provided by the authors of the original studies. Performance measures were recorded in keeping with previously adopted procedures, or involved additional measures  as detailed below. Examples of stimulus material presented in each experiment are displayed in Fig. This test of face perception involves the original material reported in Bruck et al. , which dThis face perception test involves a simultaneous card-sorting procedure as reported by Jenkins et al. , and repBased on our experience, we point out three aspects that require careful consideration to ensure the most efficient and informative application of the YBT and FICST. The first pertains to the number of trials contained in the YBT, relative to the number of currently analyzable trials. As mentioned, the authors of this test were able to provide the correct responses for 35 of the total of 40 items. Currently, efforts are underway to determine the most probable correct responses for the missing items using a wisdom of crowd approach  were considered.For the FICST, perfect performance involves separating the 40 images into two piles without inclusion errors. While previous studies considered only the number of piles as an index of performance  determine the nature of subsequent responses. For the CFMT+, the presentation of the to-be-learned faces is fixed, while recognition decisions provided for the test face trio is unlimited. As in this context performance is determined by encoding proficiency and recognition ability, longer decision times will not facilitate recognition performance, and hence no systematic speed–accuracy trade-off is expected.From a procedural perspective, the EFCT and PICT are the least complex compared to the YBT, FICST and CFMT+. Both require binary, perceptually based decisions for face stimuli presented simultaneously without time constraints. In such an experimental context, we reasoned that consideration of RTs is imperative. Under simultaneous matching conditions observers are more likely to display speed–accuracy trade-offs compared to, for example, recognition tasks. Even highly impaired prosopagnosic individuals can achieve accurate performance at the expense of vastly prolonged RTs, which is why it is common practice to take into account RTs in studies of both healthy and impaired individuals  the FICST, EFCT and PICT composite scores.n = 9). We then computed each subsample’s mean performance and retained the value as a reference average. Finally, we extracted the score of each subsample for each respective other test and compared the mean to each test’s reference average.Additionally, in order to determine the predictive value of a given test, we investigated how the top performers on each test scored on the remaining ones. First, and independently for each of the five tests, we identified those participants whose scores were located in the top 5% ; 2) their respective performance on the remaining tests; and 3) the mean and standard deviation for each test based on the normative sample. For the YBT, high-performing individuals identified via the CFMT+ and FICST performed more similarly to the reference score compared to the top performers identified via the EFCT and PICT .In light of the shortcoming of tests currently used to identify individuals with superior face perception, here we aimed to provide normative data for the YBT  a first indication of an observer’s proficiency level; 2) the direction in which further testing should proceed; and 3) potential applied fields in which an individual may be ideally deployed.Overall, the stronger relationship observed between YBT, FICST and CFMT+ is further supported by investigation of test scores of observers identified independently per test as top performers. High-performing individuals identified via the YBT, FICST and CFMT+ scored more similarly across these three tests than individuals identified as top performers in the EFCT and PICT Fig. . This suWe emphasize that in order to screen for potentially superior face processing skills multiple ecologically meaningful and challenging tasks must be employed to assess distinct aspects of face processing  that will include, among other tests, digital versions of the YBT and FICST. This will enable screening of greater numbers of individuals or preselected cohorts, conducting item analyses, and exploiting information related to response times.Independently of these future directions, our results emphasize the need to assess face processing abilities from various angles and considering both test-specific procedural constraints as well as the specific goal(s) of assessment. Complementing highly controlled tests, such as the CFMT+, with more realistic and challenging tasks can provide a more comprehensive and targeted understanding of observers’ abilities, and potentially higher predictive power for performance in applied settings.Additional file 1: Figure S1. CFMT+, EFCT and PICT scores of the top performers identified independently per test. Visualized here are performance levels of individuals identified as the top performers (colored markers) based on the YBT (red circles), FICST (blue crosses), CFMT+ (green squares), EFCT (purple triangles), and PICT (orange asterisks), and their relative location among all observations made (grey dots) for the a CFMT+, b EFCT and c PICT.


We summarize recent advances in strategies that aim to restore optic nerve function and vision in glaucoma through protective, reparative, and regenerative avenues.Neuroprotection relies on identification of early retinal ganglion cell dysfunction, which could prove challenging in the clinic. Cell replacement therapies show promise in restoring lost vision, but some hurdles remain in restoring visual circuitry in the retina and central connections in the brain.Identification and manipulation of intrinsic and extrinsic cellular mechanisms that promote axon regeneration in both resident and transplanted RGCs will drive future advances in vision restoration. Understanding the roles of multiple cell types in the retina that act in concert to promote RGC survival will aid efforts to promote neuronal health and restoration. Effective RGC transplantation, fine tuning axon guidance and growth, and synaptogenesis of transplanted and resident RGCs are still areas that require more research. Glaucoma is treated as a disease of the anterior segment of the eye, since the only modifiable risk factor is intraocular pressure or IOP. Even so, vision loss in the disease arises from degeneration of the 1.5 million or so retinal ganglion cell (RGC) axons that comprise the optic nerve and form the optic projection to the brain [Sensitivity to IOP in glaucoma involves stress conveyed to RGC axons at the optic nerve head, which induces degenerative events in both distal and proximal structures . In the Stress to RGCs in glaucoma progresses in both directions from the nerve head: retrograde towards the RGC body in the retina and anterograde towards central axonal targets in the brain. Work with transgenic mice demonstrates that the proximal (retrograde) and distal (anterograde) programs progressing on either side of this junction are to some extent independent –7. In th−/− mice [Neuroprotective strategies aim to restore homeostasis between stressed RGCs and their environment in the optic projection to promote survival and protect unaffected RGCs from early progression. Animal studies involving either induced  or gene−/− mice –12) elev−/− mice . While c−/− mice , therapiRGC dendrites are dynamic during development, expanding and contracting in response to environmental stimuli, but become stable when cells reach maturity . ShrinkaRGC axons extend a great distance from the retina to multiple termination zones in the brain. In murine models of glaucoma, dysfunctional axon transport is evident early in disease . RGC axoClinicaltrials.gov identifier: NCT02855450) after a previous clinical trial showed that topical delivery was well tolerated by patients [Although preclinical research into the neuroprotective effects of growth factors appears promising, one drawback to recombinant growth factor therapy appears to be the rapid clearance in vivo of exogenous neurotrophins, which are protein-based drugs and are highly sensitive to proteolytic degradation , 38. Advpatients . While nFor patients with severe visual field deficits, indicative of gross RGC degeneration, strategies to replace lost RGCs may be the only way forward to restore vision. Photoreceptor replacement to restore vision provides proof-of-concept that integration of new neurons into the mammalian retina is possible –49, albeIn terms of building new RGCs, there have been significant breakthroughs in the effort to generate pluripotent stems cells in recent years, including the developments of inducible pluripotent stem cells (iPSCs) from human fibroblasts . These cOne major limitation to using iPSC-derived RGCs for transplantation is that generated cells will carry the same genetic susceptibility to degeneration as the donor. For example, iPSC-RGCs derived from a donor carrying a mutation in the optineuron gene implicated in normal-tension glaucoma will carry the same gene , 73. GenMature retinal tissue is an extension of the central nervous system (CNS), which has a very low capacity to regenerate after injury due to both cell-intrinsic and cell-extrinsic factors. Following optic nerve crush, axons with modest sprouting may not extend sufficiently long distances and eventually die . RGCs arWhile early inflammation may benefit optic nerve regeneration, long-term glial scarring and inflammation inhibit axon growth. Reactive astrocytes and oligodendrocytes upregulate genes that inhibit axon regeneration directly or indirectly through activation of receptors on the surface of oligodendrocytes. These factors include chondroitin sulfate proteoglycans (CSPGs), Slit2, and ephrins , 92–94. Late in progression, astrocyte processes fill in optic nerve volumes as glial scaring takes the place of degenerated axons . PioneerDuring early development, RGC axon growth proceeds at a rapid rate to allow the extension of axons from the retina to reach distant targets in the brain. However, as RGCs transition from an embryonic to postnatal state, they reduce their axon growth speed by over 1000-fold . ExternaTargeting cell-intrinsic pathways to reprogram RGCs to a proaxogenic state have been met with more success. Several intrinsic molecular pathways can influence axonal growth including members of the Kruppel-like-factor transcription family. When overexpressed, Klf-4 and Klf-9 suppress axon outgrowth , while oPrompting axon regeneration from mature RGCs is a challenge in itself; once axon generation is initiated, the next hurdle is the correct direction of axon growth and generation of new synapses with targets in the brain. Efforts to regenerate axons have already demonstrated aberrant growth to improper targets , 118. TeAs well as proper guidance to terminals, axons need to establish functional synapses in the diencephalon. Progress in this field is already hampered by limitations in long-distance axon regeneration. Going forward, understanding the process of synaptogenesis and cellular mechanisms involved in RGC development may help to promote synaptic development in regenerated RGCs. For example, in utero, developing RGCs are primed to respond to light stimulation by spontaneous neuronal activity called “retinal waves” . This prWe face many challenges in trying to restore lost vision for patients with advanced glaucoma. The main focus of next generation glaucoma therapies is in the neuroprotection of RGCs to prevent vision loss and in cell replacement therapies to restore vision. Re-wiring the visual system is not a facile goal, the first obstacle lies in identifying windows of restorative opportunity. The earliest window of opportunity is when RGCs first become dysfunctional; in animal models, this is easily identified; however, in humans, it is more challenging to identify early RGC dysfunction. Currently, elevated IOP is the only indicator that RGCs are experiencing increased stress, and it is at this point that our neuroprotective strategies would be most effective. Further research into improved retinal imaging in the clinic would greatly enhance the efficacy of neuroprotective therapy by identifying early RGC dysfunction. It is unlikely that neuroprotective strategies alone will prevent RGC loss, since glaucoma patients often progress with the disease until visual defects occur.Cell replacement therapy has long been a tantalizing prospect for many neurodegenerative diseases. The potential therapeutic value of RGCs derived from retinal organoids and planar systems is vigorously under investigation by numerous groups facilitated by the National Eye Institute’s “Audacious Goals Initiate for Regenerative Medicine.” There are still hurdles to overcome however; for example, defining the most effective delivery system for donor cells into the host retina, examining how the introduction of foreign cells impacts the native retinal environment, and determining if donor RGCs integrate and form functional circuits that restore visual processing. The restoration of vision requires transplanted RGCs to grow axons that extend beyond the optic nerve head, remyelination by oligodendrocytes, and correct guidance to terminals in the brain where new synaptic formation can occur. RGCs do not function in isolation, relying on both cell intrinsic and extrinsic factors during disease to determine cell fate. In fact, RGCs rely on multiple cell types for survival in the optic tract. The novel finding that astrocytes directly act to provide neuroprotection during stress by shuttling metabolic resources further drives home this fact. Therefore, future progress in vision restoration will lie in harnessing the support mechanisms by other cells in the visual system to promote effective RGC transplantation and the formation of functional visual circuits.


GFP transgene in Nicotiana benthamiana. Because biologically meaningful whole plant RNAi has not been observed for endogenous gene products in N. benthamiana or in other plant species tested, under growing conditions including field production, the regulatory risk assessment of foliarly applied dsRNA-based products should not consider exposure scenarios that include systemic response to small RNAs in treated plants.Foliar application of dsRNA to elicit an RNA interference (RNAi) response is currently under consideration as a crop protection strategy. To access the RNAi machinery of a plant, foliarly applied dsRNAs must traverse the plant cuticle, avoid nuclease degradation, and penetrate the cell wall and plasma membrane. Application methods and co-formulants have been identified by Bayer Crop Science researchers and others that can help bypass barriers to dsRNA uptake in plants leading to an RNAi response in greenhouse grown, young plants and cell cultures. However, these advances in dsRNA delivery have yet to yield systemic RNAi silencing of an endogenous gene target required for product concepts such as weed control. Systemic RNAi silencing in plants has only been observed with the  Weed control in crops using herbicides is one of the most important agronomic practices in modern agriculture. In order to work toward the development of biologically based herbicides, foliar application of double-stranded RNA (dsRNA) to plants was evaluated as a method to control weeds  as well Amaranthus palmeri) with 0.5% of the spreading surfactant Silwet L-77. Treated leaf cross-sections were collected at 4 h post application and assessed for the presence of Cy3-siRNA using fluorescence microscopy. A representative image obtained from this study is provided in the The plant cuticle is a lipophilic film generated by and covering the epidermis of leaves, young shoots, and fruit. The primary purpose of the cuticle is to prevent dehydration of plant surfaces. The cuticle is also known to impede the absorption of exogenous water and solutes . We hypoNicotiana benthamiana in the absence of tissue wounding. They accomplished this either biolistically with gold particles (1 μm) and a gene gun or by spraying an aqueous siRNA solution at high pressure (7–8 bar) and close (2–4 cm) to the plant surface. To enhance cuticle penetration of siRNA and achieve robust visual RNAi phenotypes in this and other research , abrasio>0.3% of a super spreading surfactant, such as Silwet L-771, in the siRNA solution (0.1–1.0 mg/mL) at the time of application, which drives uptake of the dsRNA by stomatal flooding. An example of stomatal flooding siRNA delivery and resulting GFP silencing are featured in Successful siRNA delivery resulting in robust visual RNAi endogenous gene and transgene phenotypes in several plant species has been demonstrated using either pipet application  or relatIn vivo nuclease degradation is known to impact the efficiency of applied siRNA to elicit an RNAi response in mammalian systems  and systemic (untreated leaf) silencing of a GFP transgene in 16C cruentus . Attemptcruentus . In reseing RNAi . Distribing RNAi . It is nIn addition to containing sequences that match the target gene, the specific dsRNA size and structure is important for successful local or systemic down-regulation of a plant gene product from topically applied dsRNA.N. benthamiana  in plant cells (unpublished Bayer Crop Science research). These overhangs are particularly sensitive to degradation by RNAses found in plant tissues (unpublished Bayer Crop Science research), and so these structures may not be very long-lived in the environment.in vivo . A weak correlation between target gene expression level and ability to down-regulate that gene with topically applied siRNA suggested that highly expressed genes may be more readily silenced. However, reliable predictors of targets that can be silenced using topical RNA have not yet been identified. Other hypotheses for the limitation on silencing of targets is that secondary structure of the mRNA in vivo , or protin vivo .in vitro assay was developed to identify efficacious siRNAs (esiRNAs) targeting a plant virus gene . Endogenous genes that have been targeted in N. benthamiana included magnesium cheletase subunit H (CHL-H), magnesium cheletase subunit I, GUN4, and phosphoribosylanthranilate transferase .A number of endogenous genes were down-regulated with topically applied siRNAs in Arabidopsis using a 22-nt artificial miRNA. Further work should be done to better understand the role of introns in systemic gene silencing.The presence of introns in most endogenous genes may limit their susceptibility to systemic gene silencing . HoweverN. benthamiana is unclear.Currently, the underlying reason(s) for the limitation of systemic RNAi response by topical dsRNA application to only the GFP transgene and only in 16C N. benthamiana or in other plant species tested, under growing conditions including field testing, the regulatory risk assessment of foliarly applied dsRNA-based products should not consider exposure scenarios that include systemic response to small RNAs in treated plants.Plant tissues have many barriers to entry of foreign and topically applied nucleic acids. Even if a sprayed dsRNA is formulated or applied in a manner that helps to bypass the plant’s physical  and biochemical barriers  and does enter a plant cell, many molecular barriers exist that must be overcome to generate a biologically meaningful RNAi response from the plant. As with all RNAi, the first requirement is that the nucleic acid sequence is complementary to the sequence of a gene product that is expressed in the cell that received the dsRNA; however, our research demonstrated that sequence match alone does not guarantee a biologically meaningful RNAi response. The structure of the dsRNA needs to be efficacious for an RNAi response, including presence of 2 nt 3′ overhangs - which tend to be degraded by plant or microbial nucleases. Finally, even if local down-regulation of a gene product occurs, evidence from this research as well as the literature suggests that a systemic RNAi response would be very unlikely to occur. Therefore because biologically meaningful whole plant RNAi has not been observed for endogenous gene products in The datasets generated for this study will not be made publicly available, the authors are willing to accept requests for data sets referenced in this article. However, since legal intellectual property review may be necessary to release the data sets, the authors cannot commit to release the data sets at this time.MB and JD contributed equally to the research reported and wrote the manuscript. BH and AI contributed to the plant uptake and siRNA testing work reported in this manuscript.The research reported was funded by Bayer Crop Science and the researchers involved in this work were employees of Bayer Crop Science and its predecessors in business.


Various branch architectures are observed in living organisms including plants. Branch formation has traditionally been an area of interest in the field of developmental biology, and theoretical approaches are now commonly used to understand the complex mechanisms involved. In this review article, we provide an overview of theoretical approaches including mathematical models and computer simulations for studying plant branch formation. These approaches cover a wide range of topics. In particular, we focus on the importance of positional information in branch formation, which has been especially revealed by theoretical research in plants including computations of developmental processes. Branch architectures are observed in various plant organs including shoot meristems  is the most important factor for generating complexity in plant morphogenesis, although programmed cell death also contributes to the formation of tubular structures, such as aerenchyma and tracheary elements -mediated polar auxin transport (PAT) is considered to provide various types of positional information in plants. Though many mathematical models have been proposed for the required traits of positional information, the self-organization properties of auxin and different directions of PIN and auxin patterns against auxin flux, that summarized by van Berkel et al. , indicatTo generalize plant branch formation, here we consider situations where disk-like architectures gradually produce branches during their growth processes Fig. a–d. WhenRorippa aquatica, has sequential peripheral complexity from an elliptically shaped simple leaf to a finely dissected leaf , branch positions need to be understood three-dimensionally Honda . In geneIn Fig. pin mutants of A. thaliana  result in phyllotaxis and leaves showing the bifurcation mode in Medicago polymorpha L.  ensures the generation of reproducible and regular divarication arrangements not only in bifurcation but also in monopodial branching. It is considered to be a two-dimensional version of programmed branching observed in the development of murine lungs by Metzger et al. . Divaric


Neuropsychologists quickly incorporated these new techniques in their research methods arsenal and nowadays they are part of the regular curricula in most neuropsychology training programs. In the current Research Topic we sought to gather a group of articles that will showcase the use of various MRI techniques in neuropsychology in both healthy individuals and those affected by different diseases, thus highlighting the specific benefits that MRI can bring in this field.Since its inception as a science, psychology has been explicitly preoccupied with the mind-body relationship, specifically with the role that the nervous system plays in shaping how we perceive the world around us and how we react in our interaction with it. The evidence in this regard are the second and third chapters of William James's 1890 “The Principle of Psychology” (Volume 1) , which eCzekóová et al.; Hlavatá et al.; Liu et al.) and functional  MRI methods, as well as positron emission tomography (PET) , in investigations that range from a case report , to experimental paradigms that involved groups of participants , to reviews and meta-analyses . Likewise, the studies included in this special issue sought to investigate the neurophysiological substrates of various brain functions in both healthy individuals and those afflicted by diseases.This Research Topic includes 12 articles that showcase both anatomical , and cognitive assessments, performed at baseline and at two follow-up evaluations at 6 and 18 months post-injury. Interestingly, the authors also included 13 healthy individuals matched for age and gender, who followed the MRI protocols of the study. The study presents three major findings: an association between structural and metabolic changes within the fronto-thalamic circuitry in the patient, significant differences between the patient and controls involving the anterior thalamic radiation, one of the major fiber tracts in the fronto-thalamic circuitry and—importantly for clinicians—adaptations to bilateral vascular thalamic injury at multiple levels, revealing the metabolic, functional, and microstructural alterations attending to multidomain neuropsychological impairment. Two articles investigated brain structural differences between patients and their healthy counterparts in conjunction with the results of various clinical neuropsychological assessments. Hlavatá et al. report structural differences in cortical areas related to impulse control in Parkinson's disease patients with and without impulse control disorders (ICD) relative to healthy controls. Moreover, patients without ICD had lower volumes and cortical thickness of bilateral inferior frontal gyrus, whereas those with ICD presented higher volumes of right caudal anterior cingulate and rostral middle frontal cortex. Interestingly, patients with ICD performed no worse than healthy controls in various behavioral tasks previously hypothesized as robust impulsivity measures, thus cautioning against quick interpretation of behavioral tests without additional physiological investigations. Czekóová et al. examined the association between gray matter (GM) atrophy in multiple sclerosis (MS) patients and their performance on tasks measuring several fundamental components of social cognition. They report that patients had slower processing speed, poorer perspective taking, and less imitation compared to healthy controls and that these impairments were associated with GM atrophy in putamen, thalami, and anterior insula, predominantly in the left hemisphere. The last paper in this group comes from a new research area in neuroscience dealing with the microbiota-gut-brain axis. Liu et al. conducted a scoping review of recent studies of healthy individuals and patients with diverse neurological disorders that employed a combination of advanced neuroimaging techniques and gut microbiome analyses. The authors report that gut microbiota profile is significantly associated with markers of brain microstructure, but also with those related to functional intrinsic neural activity and brain connectivity at-rest. These findings highlight the need for longitudinal studies that include assessments of the gut microbial community structure and microbial metabolomics in conjunction with neuroimaging and behavioral testing in order to parse out the directionality and causality within the microbiota-gut-brain axis.Regarding the use of anatomical MRI in neuropsychology, we showcase here four articles. One of these articles is a case report by Longarzo et al.). The other is a systematic review of studies that used PET and [18F]-fluorodeoxyglucose (FDG-PET) to investigate changes in brain metabolism in Parkinson's Disease and other α-synuclein pathologies . In this review, Trošt et al. conclude that the neuropsychological data in PD and related α-synucleinopathies correlate with metabolic neuroimaging data, thus suggesting that the latter may be used to derive biomarkers of disease progression.Two articles included PET as imaging technique. One is the case report of the 67-year-old male patient with bilateral thalamic stroke that was already mentioned above  and the other investigating the topological organization of different brain networks in patients with type 2 diabetes mellitus (T2DM), again relative to healthy controls and in association with cognitive dysfunction assessed by neuropsychological tests . In the first article, Fajnerova et al. report altered functional connectivity patterns in OCD patients relative to their healthy counterparts in association with the severity of cognitive and clinical symptoms. In the second article, Qin et al. found changes in topologic properties of several brain networks in T2DM patients relative to healthy controls that were correlated with cognitive performance.The group that used resting-state fMRI (rs-fMRI) contains two articles, one assessing the changes in functional connectivity in patients with obsessive-compulsive disorder (OCD) relative to healthy controls and in conjunction with other variables assessed with neuropsychological tests  and two were meta-analyses of original studies that employed the same neuroimaging technique . Sojka et al. investigated the brain activity of patients with functional movement disorder (FMD) and matched healthy controls during an emotion regulation task. They report increased activation in FMD patients in several brain areas when observing negative pictures, especially in areas associated with self-referential processing in voluntary emotional regulation and lower emotional awareness. Dash et al. examined the effect of age and bilingualism on the brain networks underlying attentional processes. The authors found an increase in brain activity in the frontal and parietal areas in elderly when compared to young bilinguals, as well as a negative correlation between the proficiency level in the second language and activation level in frontal areas, which was observed only in the elderly group, thus providing evidence for age-related neuroplasticity. Gabitov et al. investigated local and long-distance changes in functional connectivity between areas involved in the acquisition of a motor skill during the course of motor learning in a group of healthy individuals. The authors report task-induced changes in functional connectivity that reflect reconfiguration of the intrinsic connectivity patterns within the somatomotor network during learning, changes that cannot be predicted or detected using the traditional brain activation contrasts.Of the remaining five articles, three used task-related fMRI in original investigations , combined with the flexibility of employing various MRI sequences , data analysis techniques  and experimental paradigms  to answer a large diversity of research questions, both fundamental and clinical.OL and MB wrote the editorial article. Both 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.


Every individual needs to make health decisions that meet personal needs as well as the needs of the broader community, such as using facemasks on public transport, observing social distancing advice and self-quarantining when necessary. Such decisions can help to curb transmission and reduce illness, deaths and economic impacts.,Similarly, this global crisis reveals that community needs can be immediate in contrast to the 2030 horizon of the SDGs. Lack of health insurance, reduced access to water during lockdown situations or chronic diseases have suddenly become factors that determine chances of survival. To manage the COVID-19 pandemic, governments have had to balance the need for mitigation, control and eradication. Should such responses entail enforcing states of emergency, or should they involve a mitigation strategy built on the premise of eventually reaching herd immunity, while being sustainable over time?Such issues must be addressed in national strategies and in the individual choices that we all make when we comply with health authorities’ recommendations.