Clinical trials are complicated, time-consuming and costly. From the initial screening, informed consent and recruitment of the participants’ to study completion, the sponsor must undertake a wide array of complex and closely monitored operations, complying with international standards for human subject research and local requirements. Conducting these studies in an underdeveloped country, with limited resources, infrastructure, and experience with regulated clinical trials adds to this complexity. The initial site selection, set up and preparatory activities for the clinical trial are crucial to minimizing the risks to both participants and to successful completion during the subsequent study execution.In this paper, we describe the experience and lessons learned of building clinical trial site capacity in terms of infrastructure and human resource development for a Phase III vaccine clinical trial. We believe that sharing the experience of setting up a clinical trial in a resource-limited country will enable other entities contemplating clinical research in these countries, to prepare and plan ahead, to minimize the impact of barriers, and to contribute to bringing more studies to the countries where people live with the burden of vaccine-preventable, poverty-associated diseases. From the initial screening, informed consent and recruitment of the participants’ to study completion, the sponsor must undertake a wide array of complex and closely monitored operations, complying with international standards for human subject research and local requirements. Conducting these studies in an underdeveloped country, with limited resources and infrastructure, and limited experience with regulated clinical trials adds to this complexity. Despite the burden of infectious diseases, there may not be sufficiently trained and experienced competent sites and personnel capable of executing the clinical trials needed to address these problems. Consequently, these countries are under-represented in research due to lack of commercial viability and research capacity.9 There is little known about the conduct and quality of research in countries that have relatively little clinical research experience.10–13 With more understanding of these settings, there is also a growing realization that many countries in the developing world are not using the enormous research potential offered by their health care services.14 If enhancing clinical trials in developing countries is being considered, then identifying barriers and designing context-appropriate strategies are critical for success.Barriers to conducting clinical trials vary widely between countries and normally are not considered in the early planning phase by sponsors.15–20 To tap the enormous potential and to cultivate the research culture (developing research capacity), in low-resource settings where typhoid fever burden is high, IVI elected to conduct a phase III clinical trial in Nepal, a low-income country where few late-stage clinical trials are conducted. Capacity building in developing countries is one of the mandates of IVI, and the proposed study was a typhoid conjugate vaccine clinical trial, a disease endemic in the local population.22The International Vaccine Institute (IVI), based in Seoul, Republic of Korea has been involved since its inception in developing vaccines for diseases of the most impoverished populations, such as cholera and typhoid. Various modeling studies have estimated that the typhoid burden ranges from 12 million to 21 million cases per year and 129 000 to 145 000 deaths annually worldwide. The disease burden is high in low- and middle-income countries, particularly in Asia and sub-Saharan Africa.23Proposed clinical trial entitled “A phase III multicenter, observer-blinded, randomized, active-controlled, immune non-inferiority and safety study of Diphtheria Toxoid Conjugated Vi-polysaccharide typhoid vaccine compared to Typbar TCV® in healthy 6 months-45 years aged Nepalese participants” was planned with a sample size of 1800 participants divided into 3 age strata, 6 months to less than 2 years, 2 to less than 18 years, and 18 to 45 years, having 600 participants each.2.Understanding the complexities of this project, a blueprint with detailed plan was developed 18 months before the proposed trial start. The plan included activities for regulatory understanding, site identification and capacity building, media sensitivity, risk analysis, and mitigation. All existing IVI internal Standard Operating Procedures (SOPs) on clinical trial conduct were part of this plan, and the first major step was to set up a dedicated team/task force for this purpose. This team was given the responsibility of identifying the challenges and preparing the sites, per study requirements, in coordination with internal and external stakeholders.2.124 As part of this strategy, we organized multiple consultations with WHO PQ team for PQ requirements, Korean Ministry of Food & Drug Standard (KMFDS) for an export only license due to the lack of local endemic typhoid infection, and the Nepal Health Research Council (NHRC) for clinical trial approval and Department of Drug Administration (DDA), Nepal for import permission and local licensure for use in clinical trial. A series of meetings were held with regulatory authorities, outlines of the project were presented and after getting written approvals from the authorities, project preparation activities were subsequently launched in Nepal.The overall aim of the Vi-DT typhoid conjugate vaccine (TCV) development program at IVI is to achieve local licensure of the vaccine in the country of manufacture (South Korea for the vaccine manufactured by SK bioscience) followed by the World Health Organization (WHO) pre-qualification (PQ) which is pivotal to be eligible for purchase by United Nations agencies, like United Nations Children’s Fund (UNICEF), or Gavi, The Vaccine Alliance.2.225 Keeping in mind these limitations, site identification and qualification became the first critical step to ensure smooth study conduct fulfilling the required international quality standards.Conducting a clinical trial in Nepal was a challenge due to lack of well-established clinical trial sites and experienced senior principal investigators with the required infrastructure and research staff experienced in conduct of trials according to the International Committee for Harmonization – Good Clinical Practices (ICH E6 (R2)) guidance.Given the large sample size of 1800, it was decided to set up and engage at least 4 sites for participant recruitment. Factors that were considered included, participant recruitment per week (slow due to lack of experience), follow up visit requirements, geographic representation across the country, urban/semi-urban/rural, social mobilization, community engagement, and awareness.26 This required us to look beyond the conventional site in the Kathmandu valley and throughout various regions in Nepal. We started an extensive search, held discussions with relevant stakeholders and sought recommendations from key opinion leaders to develop a first list of potential sites. After developing the list of potential sites, we contacted the potential site investigators and senior leadership to gauge their interest in participating. A non-disclosure agreement was sent to the Investigators before the study was discussed. Based on telephonic discussions and completion of a site feasibility questionnaire, prospective sites were screened to move to the next stage of site selection. After initial screening discussion, we conducted site qualification visits to assess the ability of the staff and infrastructure of clinical research site to conduct the study in a safe, cooperative, and timely manner. The purpose of the site qualification visit was to investigate the operational, managerial, technical, and clinical capabilities of the participating sites, which helped in identification of gaps to be addressed prior to initiation of recruitment and execution of the phase III trial according to required regulatory, ethical and quality standards. Some of the site evaluation criteria from the SOP for site qualification are listed in At the time of site assessment and selection, there was one site actively involved in a vaccine clinical trial in the Kathmandu valley, and it could not be utilized due to participation in a different typhoid conjugate vaccine study, with the WHO pre-qualified vaccine.During the site qualification visit, Project Lead (PL)/designee discussed the study-specific requirements with the potential site staff, checked and documented various evaluation parameters against the site qualification visit checklist. Multiple photographs were taken during the site visits for discussion with the project team back at IVI headquarters.Based on the data gathered, investigators/sites were shortlisted. A detailed comparative site capabilities table highlighting the strengths and weaknesses of the assessed sites was generated and discussed thoroughly within the project team. During this process, we reached out to a total of 20 sites for feasibility and based on the telephonic discussion and site feasibility questionnaire, 13 sites were shortlisted for further assessment  by IVI staff. Of these 13 sites, 7 sites were initially selected and visited during August 2018 by an external consultant to conduct a thorough gap analysis of site capacities. Finally, four sites were selected for site preparation along with 2 back-up sites Selected sites’ IRBs were contacted through respective site’s PI and availability of IRB SOPs was confirmed. Wherever needed, efforts were made to strengthen the IRBs by inviting IRB members for various training programs. Site IRBs were encouraged for frequent inspections.All the sites and study teams were informed of the final results as per study-specific requirements in writing/e-mails. Entire process of sites’ selection has been summarized in 2.3Understanding the site’s capabilities and willingness to participate in this project was the first crucial step. Thereafter, the project team developed and planned the human resource requirement for each site and a basic team plan was shared and discussed with each site. It was understood that clinical trial sites in LMICs, based in Health Care Facilities or Hospitals, are overburdened by the pressing needs of routine patient care; therefore, staff planning was needed to ensure proper time allocation and resources of each member for the research activities without affecting the ongoing patient care. One critical element was the identification and training of young investigators who could take a leadership role in fostering the scientific and research-oriented culture at the site. Site-specific team requirements were discussed and agreed upon by site authorities, including the time allocation required by the potential site study team.A basic staff team 2.4For the experienced clinical trial sites, a standard training plan is implemented, starting from Investigator meet trainings, site initiation visit trainings, and on-the-spot training, as needed, during routine monitoring visits. In Nepal, however these potential clinical trial sites had no previous experience, and sites were shortlisted based on evaluation criteria mentioned earlier 2.4.1The initial ‘central training sessions’ included three key members from each of the sites, i.e. each site principal investigator, co-investigator and co-ordinator who were invited to Kathmandu for training. A total of three central trainings were conducted per site. The purpose of central trainings was to familiarize the staff with the basic terminology used in clinical research and general principles of clinical research and ethics. Topics covered during the trainings were Good Clinical Practices (GCP), Clinical Development Overview, Pre-clinical requirements, First in Human Studies (FIH), Practical Management of Clinical Trials, and important points for beginners in clinical trials, based on the vast experience of IVI conducting clinical trials for 20 years.The contents of the trainings were adjusted from the first to the third central training, depending on the perceived knowledge level of the participants by means of post-training assessments and feedbacks. The training was made progressively more detailed and study-specific.2.4.2Three ‘site-specific trainings’ spaced roughly 1 month apart, were provided at each site, keeping in mind site-specific requirements and the bigger site teams including all Investigators, Nurses, Lab technicians, Data operators, Field supervisors & Volunteers. Training was conducted in English and local languages. The major focus of these trainings was the operational aspects of clinical trials in addition to the basic training on GCP for all staff.2.4.3Mock drills were planned immediately after each site-specific training. The purpose of mock drills was to simulate the real situation of the clinical trial per the study protocol and study operation manual requirements. Role play with a focus on the informed consent process, case report form, electronic data entry, sample preparation, and handling, sample storage, and safety- reporting were performed several times. In addition, extensive training was done over the site-specific SOPs, which were developed in collaboration with an independent consultant agency, and discussed with the study sites before implementation.2.4.4In spite of the extensive training – including 3 central, 3 site-specific and 3 mock drills, it was important for the key site staff to observe and learn from an ongoing clinical trial. IVI contacted established clinical trial sites in India with which we were familiar and finally settled on the site at Bharti Hospital, Pune, Maharashtra, India for on-site training and observation. This site has successfully conducted multiple clinical trials and is well known for its quality adherence. IVI worked with the principal trainer and team at Bharti Hospital to prepare a week-long extensive training workshop agenda. We identified two key staff from each Nepalese site, and they were invited for hands-on training at Pune. Since participants were invited to observe an ongoing trial, a Non-Disclosure Agreement (NDA) was signed by all the participants before the start of the training workshop. Site principal trainer informed the ongoing study sponsor of this training exercise and necessary permissions were secured. The purpose of this hands-on training was to give participants on-site observation and experience of conducting a clinical trial, and they were expected to observe and understand various aspects of the ongoing clinical trial, including screening, informed consent, recruitment, study visits, etc.At the beginning of the workshop a pre – assessment test was conducted for the trainee participants which was based on General Principles of Clinical Research and Ethics. During the workshop, emphasis was given to the ethical conduct of clinical trials, the voluntary nature of trial participation, volunteer safety and well-being, and documentation. The Trainers at Bharti Hospital were requested to conduct a post-training assessment using a questionnaire based on the study protocol. Assessment results were shared with the sponsor for further development of individual trainees.Activities discussed and observed during the hands-on training are summarized in 2.52.5.1In order to oversee the operational activities during the study conduct with the sponsor, various Contract Research Organizations (CROs) were contacted. Many well-known and global CROs did not participate because they did not have the experience or resources to conduct this kind of study in Nepal. Keeping in mind these challenges, a CRO from India was hired to take care of study monitoring and local project management activities.2.5.2All of the four sites selected were new to the clinical trials. Therefore, in addition to extensive training of the site and the hiring of a CRO for monitoring activities, four additional experts in regulatory activities, laboratory assessment, and clinical trial operational activities were hired: 2 from Nepal and 2 from India who are based in Nepal. This ensured that we had enough support on the ground for crucial capacity-building efforts and management during the study.2.6As the study recruits participants from the local community, social mobilization and community engagement are critical areas for a successful study, and thus a community engagement plan was put in place. The objectives were, to enhance an informed decision process for parents and children invited to participate in the vaccine trial, to provide accurate and up-to-date information regarding the Vi-DT vaccine and the phase III vaccine clinical trial and to establish a mechanism within the trial site’s health network to detect rumors and/or concerns within the community that might enable the project team to respond accordingly in a timely manner.To combat common challenges of fear, distrust, and possible preconceived notions about clinical trials or suspicions of ‘research’ in the community, site staff conducted extensive community outreach in their respective site catchment area. Community support for the study was considered essential and activities ranged from staff going door-to-door and visiting local groups to meetings with the district’s public health division, key opinion leaders, locally elected representatives, caregivers, and physicians. A comprehensive dialogue was established with all the stakeholders, explaining the critical aspects of the proposed study and assuring them that the best ethical, medical, and scientific practices would be followed during the study 2.72.7.1For each clinical trial site, a floor map was developed to help with the efficient use of the space and to fulfil the requirements for a clinical trial site as per ICH-GCP. A minimum of 4 rooms for research activities were set up . Separate space was allotted for a waiting area, document storage under lock & key, serum sample storage, vaccine storage, data entry and long-term archive. We also worked with each site to have a proper participants’ flow from entry to exit at each site. Internet with high-speed Wi-Fi with required band width was ensured at each site to take care of electronic data entry during the study conduct.2.7.2All the required equipment were purchased locally or from India. Equipment/instruments such as refrigerator for IP storage, deep freezer for serum storage etc. were arranged in duplicate, main & back-up, established at separate places, and were calibrated before the study start and checked routinely per SOP. Calibration certificates were issued & maintained in a separate file for quality checks.2.8Recruiting study participants is the most challenging part of any clinical trial and is a major cause of clinical trial delay. To meet the challenge of successful, timely enrollment, site teams were advised to strategize carefully and a study-specific, participant-focused recruitment plan was developed which was specific to each site. A more accurate and realistic assessment of recruitment per week and a true forecasting rate to avoid congestion of study follow-up visits, was determined from various sources of information, such as immunization clinic vaccination records, follow-up OPD visit logs, a well-defined catchment area and available site demographic data. Some sites targeted immunization clinics and follow-up OPDs for participants’ identification with field as back-up, while others targeted field area for participants’ identification as main strategy. Similarly, participants’ retention plan was put in place to ensure minimum dropout, one of the essential component of the plan was frequent contacts with participants by community/field health workers until the study completion.2.9To ensure that the study was conducted according to GCP and bearing in mind the relative lack of experience of the sites, we decided to have the first monitoring visit after 2–3 participants were recruited at each site. The purpose of this early monitoring check was to identify any deviations at an early stage, make timely corrections, and retrain appropriately if required. Study monitoring activities were conducted by CRO, while co-monitoring activities were performed by the sponsor clinical operations group, and to ensure quality, regular quality checks were assigned to senior clinical research officials at IVI. In addition, IVI hired an independent agency to ensure quality check evaluations at predefined time points. The reports from these evaluations were shared with IVI and required corrective and preventive actions were put in place accordingly.2.10Before starting the clinical trial, final site readiness check was performed by an independent consultant from South Africa. The objectives of the site readiness check were; 1) To determine whether all regulatory, central, and local ethics approvals and an executed clinical trial agreement were in place; 2) To determine whether minimum staff requirements were in place to fulfil the relevant roles in the conduct of the trial; 3) To determine whether staff has been adequately trained in GCP and in other aspects of trial conduct; 4) To determine whether there were adequate facilities at site to conduct a large vaccine trial; 5) To determine whether back-up power was in place and temperature of vaccine refrigerators and laboratory freezers were monitored and stable; 6) To determine if the medical emergency trolley was in place and available for use at any time; 7) To determine whether the site staff had good understanding of the participant recruitment and enrollment plan; 8)To determine if the site has final standard operating procedures (SOPs) in place like general operations, obtaining informed consent, medical emergency power failure and natural disaster, quality management etc. and had been trained on those SOPs.During the visit, the following details were checked and verified: organogram of site staff, training records, CVs and job descriptions, investigator site file for all approvals, facility tour of the site, medical emergency trolley, SOPs and study manual of procedure (MOP). Interview with principal investigator and co-investigator was conducted to review and understand the roles and responsibilities of site staff, participant-focused recruitment plan, and participant flow within the site. Furthermore, study staff including physicians and nurses were asked questions around the study procedures on an ad hoc and unplanned manner to ascertain full comprehension of the study protocol.A detailed report was prepared by the consultant and was submitted to the sponsor which served as a final clearance before enrollment.3.27 The Global Forum for Health Research report emphasized that strengthening research capacity in developing countries is one of the most effective and sustainable ways of advancing health and development in these countries, as well as helping correct the gap in health research.28 Conducting more clinical trials in low and middle-income countries (LMIC) will build confidence both in the sites and among sponsors. The capacity building will strengthen institutions and investigators in LMIC; ultimately, these will impact the level and quality of health care provided in these settings.29 In addition, research generated in LMICs help respective governments in policy formulation and implementation of decisions using relevant analyses based upon local data.In 2015, Li et al. reported the findings of their review of the distribution of global clinical trials and concluded that despite an overall increase in clinical trials in developing countries over the last two decades, progress  made was particularly slow and challenging.One of the missions of IVI is to build the research capacity in developing countries, and the Vi-DT phase III clinical trial provided a crucial opportunity to implement that. Several LMICs were evaluated and Nepal was chosen in close consultation with the Bill and Melinda Gates Foundation (BMGF), Seattle, USA and the vaccine manufacturer, SK bioscience, Republic of Korea. The trial was initiated in November 2019 and is ongoing, however, the eruption of COVID-19 globally has resulted in unexpected delays and new challenges, as the trial activities were put on hold during the pandemic. The close collaboration and support of BMGF, manufacturer, site staff, and various government agencies was critical to the progress till date. The typhoid conjugate vaccine Vi-DT should complete phase III in 2020, and regulatory approval and licensure is expected in 2021. Successful examples of the optimal utilization of Product Development Partners (PDPs) such as IVI for Cholera and Typhoid and PATH for Rotavirus vaccine development programs, highlights the importance of PDPs even for manufacturer-sponsored trials in setting-up and executing clinical trials in resource-limited settings.Being new to clinical trials, sites had many weaknesses initially that were addressed through extensive planning, preparation, and training. All the sites were eager to learn and adapt, and this facilitated the site set-up activities. The institutional ethical committees at the sites were at various levels of experience, and the strengthening of site ethical review could contribute further to the enhancement of site research capacity.While preparing clinical trial sites in Nepal, a substantial amount of time; 18 months to be precise, were spent from site identification to eventual readiness for the Vi-DT phase III clinical trial. During the course of site preparation, the sponsor team faced many logistical challenges, for example, difficulty purchasing and calibrating good quality refrigerators, deep freezers and centrifuges. Transportation was another major challenge, due to unpredictable weather conditions leading to local flights delays or cancellations; flight delays varied from 1 to 8 hours depending upon route and season of the year. Absence of a rail network and poor road infrastructure also contributed to the issue. To ensure the smooth execution of the trial, air and road routes were well defined with back-up transport strategies in case of delay or cancellation of flights. For example, prior arrangements were made with World Courier services to supply cold chain boxes capable of maintain required temperatures up to 7 days for serum sample and Investigational Product (IP) transports. Similarly to ensure safety of the IP at sites, 3 sets of IPs, main set, back-up and back-up of back-up set was maintained at 3 different locations.More clinical research should be done in LMICs and sharing the experience of setting up a clinical trial in a resource-limited country will enable entities contemplating clinical research in these countries to prepare and plan, minimizing the impact of barriers to the conduct of GCP trials in under-resourced settings.


Neuronal connectivity provides the basis for brain function. Cortical microcircuits consist mainly of excitatory output neurons or principal cells and a smaller number of diverse inhibitory interneurons. Whereas principal cells (PCs) are generally assumed to sparsely and preferentially innervate their target neurons, the organization of interneuron connectivity is still a matter of debate. A recent paper of Peng and colleagues, published in Science Advances, makes a very stimulating contribution to this discussion .A prevailing view suggests that inhibitory interneurons cover neighboring principal cells (PCs) evenly in a “blanket of inhibition.” In this model, interneurons, belonging to different subtypes, innervate neighboring PCs densely and non-selectively , 8. IndePeng and colleagues give yet another impressive example of such non-random inhibition and at the same time offer an intriguing functional implication such “structured” inhibition may have . Using mTo explore potential functional implications of such polarized connectivity between interneurons and principal cells, the authors designed a presubicular network model Fig.  for the In summary, Peng and colleagues have provided a striking example of structured inhibition and offer an inspiring functional implication such spatially asymmetric connectivity may have to realize specific computations at the level of the microcircuit.


In this study, we shed the light on this route by testing the inhibitory effect of Ocimum basilicum extract on the enzymes α-glucosidase and α-amylase in vitro and in silico. Experimental procedures were performed to test the effect of the O. basilicum methanol extract from aerial parts followed by the in silico docking. 500 μg/mL of the extract led to 70.2% ± 8.6 and 25.4% ± 3.3 inhibition on α-glucosidase and α-amylase activity, respectively. Similarly, the effect of caffeic acid, a major extract ingredient, was also tested, and it caused 42.7% ± 3.0 and 47.1% ± 4.0 inhibition for α-amylase and α-glucosidase, respectively. Docking experiments were performed to predict the phytochemicals responsible for this robust inhibitory activity in the O. basilicum extracts. Several compounds have shown variable levels of inhibition, e.g., caffeic acid, pyroglutamic acid, and uvasol. The results indicated that O. basilicum can be a potent antidiabetic drug.Diabetes mellitus is a metabolic disease that predominates, nowadays. It causes hyperglycemia and consequently major health complications. Type II diabetes is the most common form and is a result of insulin resistance in the target tissues. To treat this disease, several mechanisms have been proposed. The most direct route is via inhibiting the intestinal enzymes, e.g.,  It results from the resistance to insulin or the reduced secretion of insulin. A major consequence of this disorder is the distorted carbohydrate, fat, and protein metabolism and the increased levels of serum glucose. This would result in hyperglycemia and elevated plasma LDL , which wistorted , 5. The istorted . A majoristorted . Prolongistorted .β-pancreatic cells. Even more, some drugs act on enzymes of skeletal muscles and adipose tissues and stimulate the two organs to increase blood glucose uptake [α-glucosidase and α-amylase competitive inhibitors [To treat type I DM, several routes can be followed. These include insulin replacement therapy and the transplantation of pancreatic islets . Type IIe uptake , 10. Anoe uptake . Drugs te uptake . Anti-ine uptake . The roue uptake . Examplehibitors . Digestiin vitro experiments, in animal test models, and in clinical trials. [Several antidiabetic drugs are confined by their limited action and pharmacokinetic influence and have side effects, e.g., biguanides and sulfonylurea. Traditional medicine utilizes herbal-based remedies by about 80% of the world's population. Herbal-derived active compounds or chemically modified herbal phytochemicals are used to produce safer pharmaceutically active drugs , 17. IndOcimum basilicum is among one of the antidiabetic herbs [Ocimum belongs to the family Lamiaceae [Ocimum are used to treat different types of diseases, mainly the species Ocimum basilicum, also known as sweet basil—an herbaceous, perennial plant used in traditional medicine and also an ornamental plant [Aspergillus ochraceus [O. basilicum against species such as P. aeruginosa strains, A. rhizogenes, P. fluorescens, X. campestris, and E. carotovora [E. coli and Staphylococcus aureus [O. basilicum leaves showed an insecticidal activity against larval stages of Culex tritaeniorhynchus, Aedes albopictus, and Anophelessubpictus [O. basilicum contains several active antioxidant compounds, e.g., polyphenoid rosmarinic acid, a derivative of cinnamic acid [Ocimum basilicum aerial parts have robust anti-inflammatory activity against macrophages and human primary chondrocytes [Ocimum basilicum aerial parts have also shown antiplatelet activity through inhibiting ADP-induced platelet aggregation [O. basilicum extract significantly showed antihyperlipidemic effects and could lower both plasma triglycerides (TG) and cholesterol in rats [Ocimum basilicum aerial extracts  reported recently to augment glucose transporter-4 (GLUT4) translocation to the muscle plasma membrane in vitro and thus enhance glucose uptake [ic herbs . The genamiaceae . A numbeal plant , 21. A nal plant , 23. It chraceus  for extrrotovora  and for s aureus . The essubpictus , 28. O. mic acid . Extractdrocytes . Extractregation , anticonregation , and antregation . O. basi in rats . Ocimum e uptake .α-Glucosidase  is a carbohydrate hydrolase that breaks down terminal nonreducing -linkage to release α-glucose residues. Two families of α-glucosidase are examined so far according to the primary structure [α-glucosidase is about 20 kb long, and the structure for the protein it codes for has been resolved [α-glucosidase may result in several disorders, e.g., Pompe disease or glycogen storage disease type II [tructure . The genresolved . The Trpresolved . It was resolved . Deficie type II .α-Amylase  is an enzyme that hydrolyses alpha bonds of polysaccharides, such as starch and glycogen, to produce glucose and maltose. At least, two major forms of amylase are found in humans. Salivary α-amylase breaks starch into maltose and dextrin. The pancreatic α-amylase digests sugars in the diet to small saccharides such as maltose for the uptake in the small intestine on reaching the duodenum. Pancreatic α-amylase was discovered to bind with N-linked oligosaccharides of glycoproteins, which regulates the activities of glycoproteins related to the blood glucose level [α-amylase genes, AMY1 , AMY2A, and AMY2B (pancreatic α-amylase genes), form a cluster on chromosome 1 P21, with a pairwise sequence homology of 93%–94% [se level , 40. Thr 93%–94% , 42.α-amylases: The catalytic domain has an eight-stranded alpha/beta barrel containing the active site, an ∼70-amino acid calcium-binding domain protrudes in the catalytic domain between alpha helix 3 and beta strand 3, and a Greek key beta-barrel domain is the carboxyl terminal. [α-glucosidase and α-amylase in the treatment of T2DM [A number of distinct protein domains make up erminal. –46. Acar of T2DM .O. basilicum extract on α-glucosidase and α-amylase in vitro and in silico.The progress in drug discovery has bloomed hugely nowadays. Chemoinformatics is used to scan hundreds of plausible protein ligands. One method for screening is the ligand-based approach. With the escalating number of resolved crystal structures, modeling for the binding of ligands to the target proteins can be configured. Docking is a structure-based method that acts at the atomic resolution for the screening of robust ligand-protein interactions, including the calculation for ligand position, orientation, inhibition constants, and binding affinities. In this study, we examined the inhibitory effect of methanol μm filter and stored in aliquots at −80°C for further experimental work.The selected plants were extracted according to Kadan et al.  with metα-amylase inhibition assay was performed according to Adisakwattana et al. [α-amylase (4 units/mL) was dissolved in 0.1 M sodium phosphate buffer, pH 6.9.The pancreatic a et al.  using acμL of the enzyme solution at 37°C for 10 min. The reaction was initiated by adding 500 μL of the substrate solution . After 5 min of incubation, the reaction was stopped by adding 1 mL of 96 mM 3,5-dinitrosalicylic acid solution to the reaction mixture. The mixtures were heated at 100°C for 10 min in order to stop the reaction and then cooled to room temperature in a cold water bath. Subsequently, the reaction mixtures were diluted 10 times with distilled water. The absorbance was recorded at 540 nm using a spectrophotometer. The α-amylase inhibitory potential was calculated utilizing the following equation:I (%) is the α-amylase inhibitory percentage.The plant extract, caffeic acid, or acarbose as a positive control of different dilutions was preincubated with 250 α-glucosidase inhibitory activity was performed according to Kim et al. [μL of 0.1 M sodium phosphate buffer, pH 6.9. 15 μL of α-glucosidase (0.1 unit/μL) was added and preincubated at 37°C for 10 min. The reaction mixture was set to 750 μL with distilled water. The reaction was initiated by adding 250 μL of 20 mM p-nitrophenyl α-D-glucopyranoside and further incubated for 10 min. The reaction was terminated by the addition of 100 μL of 0.1 M Na2CO3. The amount of released product (p-nitrophenol) was measured at 405 nm using a spectrometer.The assessment of intestinal m et al. , with a α-glucosidase enzyme inhibitory potential was measured utilizing the following equation:I (%) is the α-glucosidase inhibitory percentage.The intestinal O. basilicum, including caffeic acid. The SMILES structures of the compounds were retrieved from the systemic IUPAC structures [apo forms for the structures of the α-glucosidase enzyme (PDB: 5KZW) and the α-amylase enzyme (PDB: 1C8Q) with the AutoDock program, version 4.2 [apo form in crystal structures. A total of 20 independent docking runs were carried out for each compound against each of the enzymes starting from random positions. The PDB files were extracted and evaluated for the best-ranked fit of the enzyme-ligand interaction for each of the ligands.Input PDB files were prepared for the natural compounds that were extracted from ructures  and thenructures . These csion 4.2 . In eachsion 4.2 . DockingO. basilicum [O. basilicum extract from dried aerial parts was tested herein in vitro and in situ. The potential inhibitory effect of the extract on α-amylase and α-glucosidase was examined as described in the Materials and Methods section. Similarly, caffeic acid (as a major compound in the extract and one that displayed in situ inhibition) inhibitory effect was also tested.We have recently reported the phytochemical analysis of asilicum  with thein vitro antidiabetic activities of O. basilicum methanol extract and for caffeic acid were investigated by the assessment of their pancreatic α-amylase and intestinal α-glucosidase inhibitory effects. Acarbose was used as a standard inhibitory drug. The results revealed that O. basilicum extract inhibited α-glucosidase in a dose-dependent manner and the IC50 value was 160 ± 10 μg/mL  values for the ligand structure upon docking from the reference one. Three compounds showed potent inhibition to both α-glucosidase and α-amylase: beta-sitosterol followed by 4,7-dimethoxyindan-1-one and then caffeic acid (Tables Results for the docking experiments met well with the d Tables  and 3. Wd Tables .α-glucosidase but not α-amylase: E-isoeugenol (Ki = 22.33 μM) followed by linalool (Ki = 78.91 μM), beta-D-glucopyranoside,5-methyl-2-1-methylethyl-phenyl (Ki = 86.74 μM), and finally uvasol (Ki = 118.93 μM) in a descending order with respect to their inhibition constants . Still, the inhibition level of the four common phytochemicals was comparable for the two enzymes. This would justify the higher inhibition levels for α-glucosidase when compared with α-amylase in vitro. The deviations from the reference structure  were highly reasonable for all plausible inhibitors. Of the four common inhibitors, the binding interface for caffeic acid and pyroglutamic acid showed more polar contacts than beta-sitosterol and 4,7-dimethoxyindan-1-one. Thus, more polarity did not contribute to a more stable binding interface and the binding free energy as the hydrophobic contacts did. On the other hand, the four inhibitors unique to α-glucosidase  showed a good hydrophilic interface with the surrounding amino acid residues and the coordinating water molecules.in vivo study and drug identification.Previous works suggested that glucosidase inhibitors and amylase inhibitors are a class of compounds that help controlling diabetes by diminishing the absorption of glucose from the intestine . Some re


B-mediated intracortical inhibition within M1. Although there is strong evidence that intracortical inhibition varies during action stopping, it is still not clear whether differences in the neurophysiological markers of intracortical inhibition contribute to behavioral differences in actual inhibitory capacities. Hence, here we explored the relationship between intracortical inhibition within M1 and behavioral response inhibition. GABABergic-mediated inhibition in M1 was determined by the duration of CSP, while behavioral inhibition was assessed by the SSRT. We found a significant positive correlation between CSP’s duration and SSRT, namely that individuals with greater levels of GABABergic-mediated inhibition seem to perform overall worse in inhibiting behavioral responses. These results support the assumption that individual differences in intracortical inhibition are mirrored by individual differences in action stopping abilities.Inhibitory control is the ability to suppress inappropriate movements and unwanted actions, allowing to regulate impulses and responses. This ability can be measured via the Stop Signal Task, which provides a temporal index of response inhibition, namely the stop signal reaction time (SSRT). At the neural level, Transcranial Magnetic Stimulation (TMS) allows to investigate motor inhibition within the primary motor cortex (M1), such as the cortical silent period (CSP) which is an index of GABA Lacking an efficient inhibitory control may result in a number of different dysfunctional behaviors, as evidenced in several medical and psychiatric conditions2 such as attention-deficit/hyperactivity disorder3, eating disorders4 substance abuse disorders5 and obsessive–compulsive disorder6.Inhibitory control is a central executive function that allows to temporarily withhold or completely suppress inappropriate or unintended responses, even after these are already initiated. This ability plays a pivotal role in everyday life because behaving in a goal-directed manner constantly requires a quick and efficient regulation of our impulses and responses10 (SST). This task allows estimating individuals’ ability to suppress a response already initiated, as it measures the temporal dynamics underlying successful response inhibition13. Performance in this task is highly variable across the normal population and reaches abnormally longer values in clinical conditions2 including attention-deficit hyperactivity disorder3 (ADHD) and obsessive compulsive disorder6 (OCD), as well as Gambling Disorder14. Hence finding biomarkers of response inhibition is desirable.At the behavioral level, one of the most reliable paradigms employed for measuring response inhibition is the Stop Signal Task23. Different TMS-EMG protocols can be used to measure levels of intracortical inhibition within the primary motor cortex (M1). Specifically, intracortical inhibition can be quantified either from the intensity of Short-interval intracortical inhibition  and Long-interval intracortical inhibition , obtained with two different paired-pulses procedures, or from the duration of the cortical silent period , measured following specific single pulse procedures26. The CSP is a cessation in the background voluntary muscle activity induced by a single suprathreshold TMS pulse delivered on M1 during tonic contraction of the target muscle27  has been widely employed to investigate the electrophysiological markers of motor inhibition in the brain cortex M. Specifi31. Interestingly, studies in which TMS was delivered online on participants during the SST revealed that behavioral motor inhibition is deeply influenced by the ongoing electrophysiological modulation of corticospinal motor excitability and inhibition within M132. For example, in go trials, action preparation induces a significant progressive increase in the levels of corticospinal excitability in the contralateral M134 , while during stop trials action inhibition induces both a widespread decrease in corticospinal excitability33  and, concurrently, a significant increase in SICI.Given the complementary nature of these different measures, several works have already tried to employ TMS during the SST to probe the fluctuations in the levels of corticospinal and intracortical inhibition within M1 associated with concurrent action preparation and action stopping35. Recently, quite a few studies42 have investigated whether and to what extent individual levels of resting-state SICI and LICI measured offline might reflect individual differences in the efficiency of the inhibitory process, indexed by the length of the Stop Signal Reaction Time (SSRT). Taken together, these studies support the hypothesis that trait-like individual differences in the neurophysiological markers of intracortical inhibition (and SICI in particular) can predict an individual’s actual behavioral motor inhibition capacities14. Hence, TMS-derived measures of intracortical inhibition might be effectively employed as biomarkers of motor inhibition performance. However, to date, no studies have investigated whether the CSP’s duration, measured offline, might also be considered a viable biomarker of motor inhibition, notwithstanding this being the only TMS-based parameter measured as a time interval. This is the aim and the novelty of the current study.Overall, the ongoing modulation of corticospinal excitability and intracortical inhibition within M1 appears to be critical to the successful restraint and cancellation of actions. Nevertheless, it is still unclear whether individual differences in these neurophysiological markers of intracortical inhibition might be related to actual behavioral individual differences in inhibitory control efficiency9. The average SSRT was 215 ms (SD = 20.6 ms). The table below (Table Raw data were processed via a customized R software (version 3.6.2) for Windows, using the code for the analysis provided by Verbruggen and ColleaguesThe CSP duration was defined as the time elapsed between the offset of the MEP and the time at which the post-stimulus EMG activity reverted to the pre-stimulus level (absolute CSP). The analysis of CSPs was carried out using Signal 6.04 software . Overall, the mean CSP duration was 106 ms (SD = 26 ms). Average MEP amplitude was 0.49 mV (SD = 0.29 mV), while MEP duration was 35 ms (SD = 2 ms). The average rMT was 55% of the maximum stimulator output .p = 0.001; CI = [0.25 0.92], Fig. p = 0.001; CI = [0.34 0.77]). Most importantly, the results of the leave-one-out cross-validation analysis showed a significant correlation between the model-predicted and observed SSRT values (r(25) = 0.51; p = 0.007, Fig. Four Pearson correlations were carried out to look for the relationships between SSRT and the relevant TMS-derived neurophysiological variables . These correlations were tested against a Bonferroni-adjusted alpha level of 0.012 (0.05/4). The linear correlation between the CSP and the SSRT was significant (Pearson r (25) = 0.59; 25. Lengthening of the CSP is observed after disruption of motor attention by sedative drugs such as ethanol or benzodiazepines. Indirect pharmacological evidence supports a largely GABAB-mediated origin of the CSP46. On the other hand, SSRT is a precise index of the duration of the whole chain of processes underlying response inhibition, and so a longer SSRT indicates lower levels of inhibitory control, while a shorter SSRT denotes a better response inhibition. Therefore, our results suggest that CSP might provide a valid trait bio-marker of the quality of action restraint and response inhibition, namely individual inhibitory control capacities.The present study aimed at investigating whether individual differences in the temporal aspect of intracortical inhibition might act as a neurophysiological trait marker reflecting individual response inhibition capacities. Our results revealed a clear relationship between the duration of the cortical silent period (CSP) and the stop signal reaction time (SSRT), obtained from the Stop Signal Task (SST). In particular, individuals with longer CSP performed worse at the Stop Signal Task, as indexed by longer SSRT, compared to individuals with shorter CSP. The duration of CSP is a neurophysiological marker of the levels of intracortical inhibition within M131. Recently, quite a few studies42 have investigated the relationship between offline TMS-derived GABA-ergic inhibitory biomarkers  and behavioral motor-inhibitory efficiency. In particular, in their study Chowdhury and colleagues14 showed a negative correlation between individual GABAA-ergic intracortical motor inhibition (measured via SICI’s amplitude) and SSRT’s length, indicating that subjects with stronger resting state SICI tend to be faster at inhibiting their responses, and so better at action stopping.In general, the relationship between ongoing corticospinal brain activity and behavioral motor functioning has been extensively investigated14. Indeed, intracortical inhibition is not modulated through a single process, but it is the synergic result of the interaction between two functionally distinct neural populations mediating either short-lasting ionotropic GABAA postsynaptic inhibition or long-lasting metabotropic GABAB postsynaptic inhibition. Such two mechanisms are indexed by SICI and LICI/CSP respectively48.Hence, our results complemented those reported by Chowdhury and colleaguesB receptors, but they even selectively suppress SICI via presynaptic GABAB receptors, causing an overall reduction of GABA release51. This GABAB mediated suppression of GABAA effects has been corroborated by converging evidence from in vitro studies52, as well as pharmacological and TMS studies both at rest and during voluntary movement53. Hence, not only SICI and LICI/CSP are mediated by different neural circuits, but the latter can inhibit the first, and so SICI inhibition might result not only from the actual activity of SICI circuits but also as a consequence of increasing GABAB-mediated inhibition51. Given that CSP is regulated by a GABAB-mediated inhibitory network and that longer CSPs are considered an index of upregulated intracortical inhibition54, we hypothesize that stronger GABAB-ergic circuits, indexed by longer CSP, might unbalance intracortical inhibition during action stopping, resulting in a global reduction of SICI-mediated reactive inhibition and so in a longer SSRT. Interestingly, a similar detrimental effect of CSP’s upregulation has been recently associated with eating disorders, and specifically with Binge Eating Disorder55. Keeping this in mind, the negative correlation between SICI’s strength and SSRT found by Chowdhury and Colleagues14 and the positive correlation between CSP and SSRT reported here might originate from the same overall inhibitory mechanism. Indeed, it is entirely plausible that these two correlations reflect the opposite effects of these two distinct neural populations mediating different inhibitory sub-processes during action stopping, and so that stronger levels of GABAB inhibition might unbalance intracortical inhibition diminishing the GABAA-mediated inhibition during action stopping, resulting in worse performance and consequently in a longer SSRT.According to the resulting model of interaction between these different cortical inhibitory systems, LICI and CSP do not only inhibit cortical outputs via postsynaptic GABAOverall, our results suggest that the duration of CSP measured off-task should be considered as a neurophysiological inhibitory biomarker reflecting individual response inhibition capacities, and specifically that individuals with longer CSP performed worse at action stopping (longer SSRT), compared to individuals with shorter CSP (shorter SSRT). Our results also support the idea that TMS-derived biomarkers might provide a reliable methodology to investigate behavioral individual differences in motor inhibition.57 (v3.1.9.6) analysis assuming an effect size (r) of 0.62 (based on Chowdhury and Colleagues14), an acceptable minimum level of significance (α) of 0.05, and an expected power (1-β) of 0.80.Twenty-seven 27  right-handed (self-reported) naïve participants with normal or corrected-to-normal vision took part in the present study. The sample size of 27 was calculated by using G Power softwareDuring the recruitment stage, participants were preliminarily screened for history of neurological disorders and current mental health problems, as well as for hearing and visual difficulties, and completed a questionnaire to check whether they were eligible for a TMS-based study. None of the participants here included reported neither having TMS contraindicators nor having been diagnosed with any psychiatric or neurological disorder, as self-reported. Participants provided written informed consent before taking part in the study. None of the participants reported any negative side effects during or after the TMS procedure. The whole study took place at ITAB  in Chieti and lasted 1 h and 15 min on average. The study was approved by the Ethics Committee of the “G. d’Annunzio” University of Chieti-Pescara and was conducted in accordance with the ethical standards of the 1964 Declaration of Helsinki.The surface EMG signal was recorded from the right First Dorsal Interosseous (FDI) hand muscle using three self-adhesive EMG electrodes connected to a CED Micro 1401 . Before electrode placement, recording areas were wiped using an alcohol swab and a pad with abrasive skin prep. Three electrically conductive adhesive hydrogels surface electrodes were then placed along with the target areas of the right hand. Specifically, the positive electrode was placed over the FDI muscle, the negative electrode was placed on the outer side of the thumb knuckle, and the ground electrode was placed on the ulnar styloid process. EMG raw signals were amplified (by a factor of 1000), digitized at a sampling rate of 8 kHz, and filtered using an analogical online band-pass (20 Hz to 250 Hz) and a 50 Hz notch filter. EMG recordings were then stored on a computer for offline analysis with Signal 6.04 software .4 Magstim stimulators  to induce a posterior-anterior current flow in the brain. The coil was positioned tangentially to the scalp following the orthodox method27, with the handle pointed backward and angled 45 degrees from the midline, perpendicular to the central sulcus. The FDI optimal scalp position for stimulation was identified by maneuvering the coil around the left M1 hand area in steps of 1 cm until eliciting the maximum amplitude motor-evoked potentials (MEPs) in the contralateral FDI muscle using slightly suprathreshold stimuli. Once identified and marked the hotspot on the helmet, coil position was fastened through mechanical support, and its position was constantly monitored by the experimenter. Participants were asked to avoid any head movement throughout the whole TMS session and were also firmly cushioned using ergonomic pads. Afterward, individuals’ resting motor threshold (rMT) was estimated by consistently adjusting the stimulator to find the lowest percentage of the maximum stimulator output necessary to elicit MEPs with a peak-to-peak amplitude of more than 50 μV during muscle relaxation in 5 out of 10 trials27. For each participant, rMT was used to determine the specific intensity of TMS suprathreshold stimulation, which was set at 120% of this individual value. This level of stimulation intensity is considered appropriate for studying CSP58.Before TMS administration, participants wore a hypoallergenic cotton helmet which was used to mark the exact location of the FDI hotspot over the left primary motor cortex (M1). Single-pulse monophasic TMS was delivered over the left primary motor cortex using a double 70 mm Alpha coil connected to BiStim60 and it was determined by averaging the mean peak-to-peak amplitude of the EMG signal (μV) recorded across three trials lasting 3 s each. Before measuring MVC, the experimenter emphasized to the participants the importance of performing at their best and of trying to keep the contraction stable during EMG recording. Once determined participants’ MVC, the level of muscular activation was constantly monitored by the experimenter via online data inspection throughout the whole TMS session. Before the TMS session, each participant took part in a short preliminary training session (never longer than 5 min) to learn how to constantly perform and maintain the appropriate level of FDI contraction (30% MVC) while receiving constant EMG visual feedback displayed on the computer monitor. The TMS session  started only after participants became able to reproduce the adequate level of EMG activity requested without the support of the EMG visual feedback. Each single-pulse TMS stimulation was delivered with an inter-stimulus interval jittered between 8 and 15 s to avoid any habituation effect. Coherently with the short duration of the CSP recording session and with the relatively low level of FDI contraction requested (30% MVC), no participant reported experiencing muscle fatigue at any stage of the recording procedure. Trials were rejected if the participant displayed any pronounced head movement before or during the stimulation. For each trial, CSP duration was first quantified as the time between the offset of the MEP and the return of EMG activity to the pre-stimulus level (± 2 SD) and then double-checked following a standard procedure61. CSPs preliminary inspection, analysis, and offline extraction were all carried out using Signal 6.04 software . CSPs were inspected before processing the Stop Signal Task data, and so the inspector was blinded to the relative behavioral results.Individuals’ CSP was assessed delivering 20 suprathreshold pulses at 120% of rMT while participants were performing an opposition pinch grip at 30% of their FDI’s maximal voluntary isometric contraction (MVC) and maintaining both a static hand posture and a constant level of muscle activity. Individuals’ MVC was used as the reference contraction to normalize the muscular activity between subjects62, a Matlab and Psychtoolbox65 version of the Stop Signal Task. In the beginning, participants were instructed to place their right hand on a specific site of the computer keyboard (the right index finger over the left arrow key and the right ring finger over the right arrow key) and to maintain this position throughout the whole experiment. The task required participants to perform a speeded response go task while discriminating between two different go stimuli, a left-pointing white arrow, and a right-pointing white arrow, responding to both of them as quickly as possible pressing the left arrow key of the computer keyboard with the right index finger and the right arrow key with the right ring finger, respectively . However, in 25% of the trials , the white go arrow would turn blue after a variable delay ), indicating to the participants to withhold their response, whether possible  probability of responding on stop trials < 40% or > 60%; (2) probability of go omissions > 25%; (3) probability of choice errors on go trial > 10%; (4) violation of the independent race model ; (5) either negative SSRT or SSRT < 50 ms. These exclusion criteria were adopted because they help to ensure that participants followed the instruction and got engaged throughout the task66. One participant was excluded for matching exclusion criteria (4) and replaced with another one. In the present study, we used a non-parametric approach for SSRT’s estimation using the integration method with the replacement of go omissions with the maximum goRT9. Overall, the whole task comprised 1 practice block of 32 trials  and 5 experimental blocks of 96 trials  each. Between blocks, participants were reminded about the instructions and provided with block-based feedback on their performance.In the go trials, go stimuli lasted 1 s (maximum RT), while in stop trials the combination of the go, the SSD, and the stop signal lasted 1 s in total, with SSD being initially set at 250 ms. Inter-trial interval lasted 4 s. Crucially, after each trial the SSD automatically varied in steps of 50 ms as a function of participants previous stop performance, decreasing after each unsuccessful stop trial and increasing after each successful stop trial, ensuring an overall successful inhibition rate near 50%, and so a p ≅ 0.50. The task included an initial practice block providing feedback after each trial. Relevant descriptive statistics obtained from the task included: the probability of go omissions, probability of choice errors on go trials, RT on go trials, probability of responding on a stop trial, Stop Signal Delay, Stop Signal Reaction Time, RT of go responses on unsuccessful stop trials. The Stop Signal data collected from each participant were screened according to the following outlier rejection criteriaParticipants took part in either the TMS session or the behavioral task, administered in random order on the same day with a 10 min break between them. The TMS session took place in the TMS/EMG laboratory of ITAB for about 35 min, following the same procedure already described above for each participant. The behavioral task took place in one of the Data Collecting Booths of the TEAMLab of ITAB for about 40 min. During the behavioral task, participants sat on a comfortable chair in front of a computer monitor with a resolution of 1024 horizontal pixels by 768 vertical pixels, at a distance of approximately 56–57 cm. The tasks were administered on Windows 7 using MATLAB R2016b. The computer monitor refresh rate was set to 60 Hz. Once finished the two parts of the experiment, participants were debriefed.To investigate the relationship between behavioral and cortical inhibition we look for a possible correlation between SSRT and CSP.67 using the Robust Correlation toolbox68 and conducted null hypothesis statistical significance testing using the nonparametric percentile bootstrap test , which is more robust against heteroscedasticity compared with the traditional t-test68. Then, we employed a leave-one-out cross-validation analysis69  to test whether participants’ CSP could reliably predict the SSRT. Specifically, at each round of cross-validation, a linear regression model was trained on n-1 subjects' values and tested on the left-out participant. Pearson correlations between observed and predicted SSRT values were used to assess predictive power. All statistical tests were two-tailed. To account for the non-independence of the leave-one-out folds, we conducted a permutation test by randomly shuffling the SSRT scores 5000 times and rerunning the prediction pipeline, to create a null distribution of r values. The p values of the empirical correlation values, based on their corresponding null distribution, were then computed.To control for the specificity of the effect, we run a regression analysis between each of the individual Stop Signal Task significant parameters , acc. ns, miss. ns) and each significant TMS-derived neurophysiological parameter . CSPs inspection and extraction were performed before processing the Stop Signal Task data, and so the inspector was blinded to behavioral results. Moreover, to test the robustness of the relationship we computed skipped parametric (Pearson) correlations


Cassiopea sp.) are mostly sedentary, benthic jellyfish that have invaded estuarine ecosystems around the world. Monitoring the spread of this invasive jellyfish must contend with high spatial and temporal variability in abundance of individuals, especially around their invasion front. Here, we evaluated the utility of drones to survey invasive Cassiopea in a coastal lake on the east coast of Australia. To assess the efficacy of a drone-based methodology, we compared the densities and counts of Cassiopea from drone observations to conventional boat-based observations and evaluated cost and time efficiency of these methods. We showed that there was no significant difference in Cassiopea density measured by drones compared to boat-based methods along the same transects. However, abundance estimates of Cassiopea derived from scaling-up transect densities were over-inflated by 319% for drones and 178% for boats, compared to drone-based counts of the whole site. Although conventional boat-based survey techniques were cost-efficient in the short-term, we recommend doing whole-of-site counts using drones. This is because it provides a time-saving and precise technique for long-term monitoring of the spatio-temporally dynamic invasion front of Cassiopea in coastal lakes and other sheltered marine habitats with relatively clear water.Upside-down jellyfish ( Jellyfish populations are highly variable and many species can grow and reproduce rapidly in response to favourable environmental conditions . EnhanceCassiopea spp.) are found globally in tropical and sub-tropical regions, in shallow, protected habitats such as coral reefs, mangrove forests and seagrass beds [Cassiopea can alter the structure and function of ecosystems through direct impacts, including predation on zooplankton such as fish larvae, and indirect impacts, including displacement of local fish by the release of stinging cells into the water column [Cassiopea stings can also be harmful to humans [Cassiopea are successful invaders because of their high tolerance to environmental variation and their ability to rapidly reproduce sexually and asexually in large numbers, as well as lie dormant as polyps [Cassiopea as they expand their distributional range in the Mediterranean, as well as Hawaiian and Australian waters [Upside-down jellyfish  stage of  in time –31. OwinCatostylus mosaicus [Aurelia sp. [Chironex fleckeri [Cassiopea in aquaculture systems [Cassiopea in their natural environment has not yet been explored. Here, we evaluate the utility and efficiency of drones for estimating the density of Cassiopea medusae by comparing results of drone-based monitoring to a conventional direct observation from a kayak.Remotely piloted aircraft  are becoming increasingly popular for environmental  and wildmosaicus , Aureliaelia sp.  and Chirfleckeri , and to  systems . HoweverCassiopea were first observed in early June 2017 in depths of less than 2 m [Cassiopea in Lake Macquarie appears to be a further southward range-expansion [This study was undertaken at Lake Macquarie , a coastal estuary on the east coast of Australia. The water temperature in the area ranges from 33°C in summer, down to 12°C in winter . In Lakethan 2 m , and subxpansion .2 in area. Within each site, three 50 m transects were randomly marked along the 60 cm depth contour, running parallel to the shore. The start and end of transects were marked with buoys and 1 m PVC pipe for scale. The transects did not go deeper than 60 cm because Cassiopea mostly occur in shallow water environments and can reliably be detected at this depth [Field sampling was undertaken on 27 and 29 May 2019 at five sites  within Lis depth ,29,31,40Cassiopea along the 1 m wide transects. It was important to determine variation in Cassiopea densities as a function of depth and distance from shore, because the depth at which either technique would work was poorly understood, and because drones cannot effectively sample deep water or close to shore due to mangrove cover . This was completed as part of a broader study investigating the population dynamics of Cassiopea within Lake Macquarie between 6 and 9 May 2019 at the same sites around Lake Macquarie with the exception of Mannering Park   and “site” . All statistical analyses were completed in R (V.3.6.3) [Drone footage from each transect was analysed for presence of  0.8.15) . The imarecorded . The couV.3.6.3) . The modV.3.6.3) ,43 of trCassiopea abundance estimates for each of the two surveying techniques was assessed using a multiple linear regression, with drone footage density estimates as the response variable and direct observation density estimates and the secchi depth reading of each site as explanatory factors. Multiple regression assumptions were tested including linearity, independence, normality, homoscedasticity, and multicollinearity.Any potential effect of turbidity on the relationship between Cassiopea was relatively unknown. To overcome these constraints, we first conducted an analysis to determine if there was any evidence that Cassiopea densities were influenced by depth or distance from shore in order to revise the survey area if required. We then restricted the area of interest for the scaled-up estimates to the depth where both techniques would be able to identify Cassiopea reliably. A conservative maximum depth of 60 cm was selected based on the congruence between drone and direct observation counts at this depth in the transect-level analysis . A general linear model was used to determine if there was a relationship between the density of Cassiopea along these transects and their distance from shore and if this was influenced by site. This confirmed there was a significant difference in the density of Cassiopea among the depth of the stratified transects with a greater number of Cassiopea occurring at 15 cm depth compared to 30 or 60 cm, but there was no relationship between density and distance from shore (see Site-level results).In order to determine the variation of Cassiopea habitat, the area sampled by the whole-of-site drone count was compared to Cassiopea densities recorded from the drone and kayak transects multiplied by the area of each site calculated to be equal or less than 60cm. This was calculated by using bathymetry data sourced from Australia’s Integrated Marine Observing System (IMOS) in ArcMap (V.10.7). Transect estimates confined to the area of each site that was equal to or less than 60cm were compared to the number of Cassiopea counted in the whole-of-site drone count using a one-way repeated measure ANOVA, where “method” was the repeated measures factor, having estimates from each method at each of the five sites. To compare the three methods of quantifying the density of Cassiopea for each site, 95% confidence intervals were used, comparing whole-of-site drone count with the scaled-up transect-based estimates using kayak and drone data. The lower- and upper-bound 95% confidence intervals were calculated for the mean of each technique, transect and site, and then scaled according to the area that was ≤ 60 cm depth for that site. Homoscedasticity and normality for the site-level analysis were tested using similar method to those described above.As there was no significant relationship with distance from shore, and due to the shallow nature of Cassiopea compared to visual observations along transects using a kayak, the time taken to complete surveys by each method was recorded.To determine the time-efficiency of drones for a whole-of-site count of Cassiopea density estimates derived from the two survey techniques  or an interaction with method . The mean (± SE) density per m2 of Cassiopea that were detected along transects by drones (0.0613 ± 0.021) was 6.5% greater than visual observations made from a kayak (0.0573 ± 0.024). Across all sites, except for Lake Petite, drones had a higher relative precision .There was no significant difference in Cassiopea density estimated by kayak and drone observations were significantly associated  = 7.770, p < 0.001, 4 = -1.285, p = 0.223).The multiple regression model indicated that Cassiopea occurring along the different depth stratified transects (X2(2) = 7.892, p < 0.05, 2 of Cassiopea found along the 15 cm transect (0.075 ± 0.032) compared to 30 cm  or 60 cm  depth, which did not differ significantly (p = 0.823). Additionally, there was no significant difference in the density of Cassiopea occurring between sites (X2(3) = 0.805, p = 0.848). While most Cassiopea individuals appeared to be less than 15 m from shore, the general linear model was not significant  and kayak transects (100.82 ± 72.41) were substantially larger than those derived from the whole-of-site drone counts across these same sites (36.2 ± 25.43), although these differences were not significant  site-level abundance of  pattern . HoweverCassiopea using visual observations made from a kayak took around 90 minutes, which is almost twice as long as completing a whole-of-site drone count, which would take around 55 minutes , finding that drone footage detected more individuals than visual observations [Cassiopea to be detected than direct observations from the surface using a kayak. However, the extra height biases the drone towards larger individuals and could miss small jellyfish (< 3 cm diameter), which occur at the start of a jellyfish bloom and which are more likely to be detected by closer observations such as those made from the kayak [Cassiopea at Lake Petite detected using direct observations from a kayak, rather than drone, and is why direct observations from a kayak should be considered a proxy for the true value of the number of Cassiopea occurring during the start of a bloom. Monthly or even weekly assessments of Cassiopea would be straightforward using a drone and would allow blooms to be detected as they occur. This is important because of the high rate at which jellyfish populations expand and collapse, allowing successful precautionary methods to be developed to counteract any impacts [The invasive nature of rvations . In our he kayak . This is impacts ,36,45.Cassiopea have patchy distributions within each site, made evident by the large 95% confidence intervals for drone and kayak transects quantifying the density of Cassiopea  allowing for rapid calculations of their numbers, densities, size frequencies and biomass [Cassiopea in aquaculture farms [Cassiopea was already known and a hand-held camera was used to assist density and number calculations, which may not be feasible in natural environments or at larger scales.Despite these limitations, our results indicate that whole-of-site drone counts provide an acceptable benchmark to detect and record the abundance of benthic  tracked . Additio surveys ,50,53,54 biomass . Additiore farms . However-1 and during periods of heavy rainfall. Additionally, the drone often must remain in visual line of site for many civilian applications [Cassiopea were present and amenable to direct observations but not drone survey.Most drones are more subject to favourable weather conditions than direct observation, as they are unable to obtain useable images in winds greater than 30 km hications  and our Cassiopea are most abundant at 15 cm depth compared to 30 cm or 60 cm. This creates potential for the whole-of-site drone count to underestimate the size of the bloom by missing jellyfish at 15 cm depth, which may be around obstacles close to shore. However, we do not believe this was the case because, while Cassiopea were typically found within 15 m of the shoreline, there was no linear relationship between their density and distance from shore. However, if there is a problem of obstacles close to shore restricting drone flight paths, as long as the water is still visible, this could be overcome by using drones that obtain accurate geo-located imagery ) and very high-resolution sensors with a zoom function. This could allow very accurate counts of Cassiopea from drones flying at much higher and safer altitudes (e.g. 50–75 m) than we used in our study. Furthermore, it is possible that the use of multi-spectral or hyperspectral sensors on drones in future research could improve the detectability of jellyfish in more turbid or deeper water [Our results showed that er water . Recent er water ,56.Cassiopea is patchy, hence we recommend five to nine 50 m transects per site (depending on the site’s area) for appropriate precision when making direct visual observations. However, this level of sampling effort is time-consuming and will result in around 135 minutes sampling time, plus the extra time required to unload and load the kayak and associated equipment before and after sampling. A drone, on the other hand, only takes 15 minutes to fly the pre-programmed grid of the whole-site, and, while drones require over twice as much time during the analysis stage in order to review the footage, it is advantageous that the footage can be revisited multiple times, unlike personal observations from a kayak [Cassiopea detected by drones and direct observations demonstrate that direct observations are still a reliable technique and have the added advantage of allowing specimens to be sampled for additional studies such as genetics, morphology, life history, physiology etc. However, when considering purely spatial/temporal density surveys, examining the overall cost and time of the two techniques showed that drones are more efficient as the sampling and analysis cost scale up with time.Survey time and costs are key considerations when deciding between direct and drone-based sampling methods. We showed that the distribution of  a kayak . The linCassiopea recorded in this study was 0.15 individuals per m2, which is relatively low compared to densities of up to 168 individuals per m2 recorded overseas [Cassiopea could be experiencing decreased performance in response to the low winter temperatures experienced at their invasion front in temperate Lake Macquarie, limiting their ability to invade in high densities [Cassiopea recorded in this study should be used as a baseline for future monitoring. Additionally, bell-diameter measurements should be recorded using either technique, as studies have suggested that the benthic coverage of Cassiopea is an important factor for determining the ecological impact the genus [The maximum density of overseas . Cassiopensities . The denhe genus .Cassiopea medusae in shallow waters. The use of transect-based abundance estimates (using drones or kayaks) has the risk of overestimating the number of Cassiopea occurring at larger scales and tends to return estimates with low precision. Future studies should benefit from our whole-of-site drone methods as a benchmark for recording the abundance and distribution of Cassiopea as they expand their range. The efficient and precise nature of drone surveys allows for cost-effective monitoring of spatially dynamic invasions of Cassiopea and should be considered for ongoing monitoring programs.Overall, we demonstrate that drones are a precise, efficient and practical tool for long-term monitoring of the distribution and abundance of sedentary S1 TableSecchi depth reading (cm) for each site was also recorded.(CSV)Click here for additional data file.S2 Table(CSV)Click here for additional data file.S1 File(MP4)Click here for additional data file.


Background: To investigate the accuracy of augmented reality (AR) navigation using the Magic Leap head mounted device (HMD), pedicle screws were minimally invasively placed in four spine phantoms. Methods: AR navigation provided by a combination of a conventional navigation system integrated with the Magic Leap head mounted device (AR-HMD) was used. Forty-eight screws were planned and inserted into Th11-L4 of the phantoms using the AR-HMD and navigated instruments. Postprocedural CT scans were used to grade the technical (deviation from the plan) and clinical (Gertzbein grade) accuracy of the screws. The time for each screw placement was recorded. Results: The mean deviation between navigation plan and screw position was 1.9 ± 0.7 mm (1.9 [0.3–4.1] mm) at the entry point and 1.4 ± 0.8 mm (1.2 [0.1–3.9] mm) at the screw tip. The angular deviation was 3.0 ± 1.4° (2.7 [0.4–6.2]°) and the mean time for screw placement was 130 ± 55 s (108 [58–437] s). The clinical accuracy was 94% according to the Gertzbein grading scale. Conclusion: The combination of an AR-HMD with a conventional navigation system for accurate minimally invasive screw placement is feasible and can exploit the benefits of AR in the perspective of the surgeon with the reliability of a conventional navigation system. An increasing number of pedicle screws are placed using minimally invasive techniques . This haThe traditional image guidance method for pedicle screw placement has been intraoperative fluoroscopy, providing two-dimensional information. The continuous development of three-dimensional imaging technologies for intraoperative use has paved the way for surgical navigation in spine surgery. Navigation improves accuracy and reduces the need for revision surgery ,8. In adAn important feature for all navigation systems is the interface used to present the navigational data. While conventional surgical navigation systems typically use screens to provide information in the standardized perspectives common for radiology, AR-solutions are inherently different and lend themselves to presenting information from the perspective of the surgeon ,15,16,17In this study, we investigate the technical and clinical accuracy of minimally invasive thoracolumbar pedicle screw placement with AR navigation using the Magic Leap head mounted device in combination with a conventional surgical navigation system.The study was conducted in a hybrid OR. The hybrid OR was equipped with a motorized ceiling-mounted C-arm system  for intraoperative Cone beam CT (CBCT) imaging.® 1.0 navigation platform , and used for all experiments.A software prototype designed for AR-navigation using the Magic Leap HMD  was installed on the CurveFour commercially available spine phantoms for minimally invasive surgery training (Th10-Sacrum) were used . The phantoms consisted of X-ray dense bone substitute, elastic foam soft tissue and silicone skin. For each phantom, a high-resolution CT scan was performed preoperatively . At the beginning of each surgery, the phantom was placed on the OR table and a radiolucent reference clamp with a 4-sphere array was attached to the spinous process of Th10 via a small incision. The universal AIR matrix  was placed on the phantom and an intraoperative CBCT scan was performed to allow automatic co-registration. The preoperative CT images were fused with the CBCT images for optimal image quality and used by the software to render a 3D-model of the spine. Due to the properties of the phantom, manufactured with the spine in a prone position and with dense foam resisting intervertebral movement, rigid image fusion was adequate to align pre- and intraoperative images.Each surgeon was fitted with an HMD, which was individually eye calibrated. To align the coordinate systems of the HMD and the navigation system, a custom disposable hybrid marker  was placed in the field of view of both systems. After alignment, the 3D-model was accurately augmented onto the phantom without the need for additional adjustments. Accuracy was checked using the navigational pointer to touch anatomical landmarks which were accessible at the attachment site of the reference. The accuracy of the overlay of the AR image and the dynamic reference frame was confirmed visually: the reflective spheres of the reference frame were overlayed with green circles when correctly aligned .Screw planning was performed as part of the intraoperative workflow. A navigated pointer was used to indicate the desired entry and path for each screw on the augmented 3D model .Each screw placement was assessed and fine-tuned on the touchscreen of the navigation system .The surgeon incised the skin of the phantom with a scalpel and dissected the foam tissue to reach the pedicle entry point, using a minimally invasive, technique. A navigated drill guide and screwdriver  were calibrated to the navigation system and for each screw placement positioned along the planned trajectory. The pedicle was drilled with a 4.5 mm diameter drill bit. The navigated screwdriver was then used to place a 5 mm diameter screw in the pedicle . The timTo assess technical accuracy, the deviation of each placed screw from its planned path was measured at bone entry and at the tip of the screw. Measurements were performed in 3D by fusing the intraoperative scan, including planned screw paths, with the postoperative scan of the placed screws. The angular deviation was calculated based on these data .Three independent reviewers assessed the clinical accuracy according to the Gertzbein grading scale; grade 0 (screw within pedicle), grade 1 (breach < 2 mm), grade 2 (breach 2 to < 4 mm), and grade 3 (breach > 4 mm) . Gertzbep-value less than 0.05 was considered as significant.Accuracy measurements and screw placement times are expressed as mean ± standard deviation and as medians [min-max]. Pearson’s product-moment correlation was performed for calculating r. Statistical analysis was performed using RStudio (RStudio Team (2016). RStudio: Integrated Development for R. RStudio, Inc., Boston, MA, USA) and a A total of 48 pedicle screws were placed. The accuracy was 1.9 ± 0.7 mm (1.9 [0.3–4.1] mm) and 1.4 ± 0.8 mm (1.2 [0.1–3.9] mm) at the entry point  and at tp = 0.12).The mean time for implanting a screw was 130 ± 55 s (108 [58–437] s). The full range of implanting times is shown in Of the 48 pedicle screws placed, 45 were rated to be clinically accurate. The grading of the screws is shown in Compared to the conventional free-hand technique, computer-assisted surgery, including AR and robotic applications, has been shown to improve pedicle screw placement accuracy. Projector based AR has been used to reach high clinical accuracy in a cadaveric model . MonitorThis is the first study using an augmented reality head mounted device (AR-HMD) in conjunction with an established conventional navigation system for minimally invasive spinal pedicle screw placement. The AR-HMD device gives visual guidance for identifying the bone entry point and directing the instruments during minimally invasive procedures. Locating the bone entry point is a key step. The 3D AR model allows for visualization of the instrument position in respect to the deep bony anatomy, simplifying adherence to the planned path.The conventional navigation system provides, according to the manufacturer, an overall accuracy of ≤2.0 mm/2° (mean) and ≤3.00 mm/3° (95th percentile). In this prototypic design, the HMD is aligned to the conventional system. The AR-perspective allows the identification of misalignment, which can be managed with a realignment procedure . The AR-Liebmann et al. performed a study where they used the HoloLens to place pedicle screws in an open lumbar spine model and reported 2.77 ± 1.46 mm mean deviation from the planned path at the entry point and an angular deviation of 3.38°± 1.73° . When MoAccuracy in this study is limited by the intrinsic properties of the HMD-system. In contrast to the solutions studied by Liebmann and Molina, the system evaluated in this study is a combination of outside-in tracking afforded by the underlying conventional reference frame-based navigation system and inside out tracking from the HMD. This combination allows realignment of the AR-view to the conventional tracking when necessary. Of importance, both the technical and clinical accuracy reported in this study reflect the compounded errors of the navigational system and the surgeon’s adherence to the plan. As mentioned above, the target registration error reported for conventional navigation systems is within 1–2 mm. The use of robotic arms may reduce errors related to the surgeon’s ability to maintain the correct position and angle of approach on the bone entry point, thereby increasing the technical accuracy .The use of an AR-HMD has the benefit of placing the navigation information directly in the surgeon’s field of view. Both 2D and 3D models of the spine are visible with the 3D model being aligned with the phantom’s anatomy. Having the same navigation information directly in the surgical field minimizes the risk of attention shift. Attention shift occurs when surgeons must change their focus repeatedly between the surgical field and the screen of the navigation system to follow the navigational cues. This shift of attention has shown negative effects on motor and cognitive tasks during surgery and could be a source of instrumentation error in spinal surgery .However, the use of HMD limits the information to those wearing them. Thus, a screen in the OR with a live navigation feed is beneficial for the rest of the surgical team, especially the OR nurse. In this study, the navigation screen simplified intraoperative communication, giving the OR nurse information about the surgical procedure. This may reduce surgical flow disruption. Lack of communication in the OR has been reported as a major cause of disruption resulting in prolonged operating time ,39.Fluoroscopy is traditionally used for intraoperative guidance when placing screws in minimally invasive procedures. Fluoroscopy assisted screw insertion accuracy is reported to range from 83.9% to 100% but is associated with exposing the surgical team to varying amounts of radiation ,41,42. ULimitations of this study include the small sample size and the fact that plastic torso phantoms were used. There were three screws deemed inaccurate in our study. Analysis of the pre-procedural plan and post-procedural images shows that all three screws were correctly planned but deviated slightly laterally. However, these screws would not necessarily have breached if placed in real bone. The plastic used in our model lacked differentiation between cancellous and cortical bone, meaning the typical resistance offered by cortical bone was absent. Moreover, navigation systems based on dynamic reference frames have been shown to lose accuracy with distance from the reference . This isThese are problems inherent to the study design and future studies on cadavers or animal models, and eventually, clinical trials will allow a better understanding of the efficacy of the system.The combination of an HMD with a conventional navigation system for accurate minimally invasive pedicle screw placement is feasible and can exploit the benefits of AR from the perspective of the surgeon with the reliability of a conventional navigation system.