With the COVID-19 pandemic, in-person fellowship interviews were curtailed, leading candidates to seek information from other resources. Our main purposes were (1) to determine what information recent participants in the match needed to evaluate programs and (2) to assess which of these were available online.A focus group of ten recent graduates/applicants identified information that was important in choosing a fellowship program. In August 2020 and December 2021, websites belonging to the American Pediatric Surgical Association (APSA) and individual programs were assessed.Recent applicants identified 55 pieces of information considered important to their decision making. Of 57 pediatric surgery fellowships, 98% were listed on APSA’s website. Program descriptions on APSA’s website listed on average 60% of program information desired by applicants. All listed fellowship director, accreditation status, faculty list, and current fellow(s). Other descriptors frequently noted were alumni (95%), graduate’s board performance (83%), ECMO exposure (77%), and curriculum (70%). Information desired but less frequently available were fellow case logs (63%), trauma center designation (53%), burn center designation (40%), research opportunities (30%), candidate interview assistance (25%), and supplemental fellowships (12%). There were 7% of program descriptions that were not updated for at least a year.APSA and individual program websites were complimentary. Websites often lacked data that applicants sought to inform their rank list. To best adapt to the evolving virtual interview paradigm, we suggest reporting key information on a central APSA website with more nuanced information available via links to program specific websites.The online version contains supplementary material available at 10.1007/s44186-023-00104-w. Recent data show that more than 80% of graduating general surgery residents plan to enter a fellowship . Only thPrior to mandatory remote interviews developed in response to the COVID-19 pandemic, a survey of general surgery residents applying to fellowship showed that 62% could have eliminated interviewing at potential programs if their websites had been more useful and informative . Most peOur purposes for this study were to (1) identify what specific program characteristics applicants considered important to make informed decisions for their rank list, (2) determine what proportion of the above-mentioned program characteristics were readily available to applicants online. We additionally analyzed how information available on pediatric surgery fellowship program websites compared to that available on websites for other specialty fellowships.Institutional Review Board approval was waived because neither participants nor institutions were identified in this study. Pediatric surgery fellowship programs and frequency of match participation were identified using websites for the National Resident Matching Program (NRMP), Accreditation Council for Graduate Medical Education (ACGME), the Fellowship and Residency Electronic Interactive Database Access (FREIDA), and the Electronic Residency Application Service (ERAS) , 22–24.The senior author first reviewed the published data and developed a list of criteria that potential applicants would possibly consider relevant while deciding the rank order list. A focus group of seven pediatric surgery fellows and recent fellowship graduates was convened . The list of criteria was then distributed to all other members of the focus group for their input. All suggestions were included in the final tally of criteria queried on the individual program/APSA websites.Two independent reviewers (M.A.S. and Y.D.J.) searched for the 55 criteria identified by the focus group by querying the APSA and individual program websites. During the course of our study, the APSA website underwent structural changes with updated program descriptions. Therefore, the program characteristics available online via the APSA website were reviewed twice: in August 2020 and again in December 2021 to check for any interim updates. When programs included links to the Graduate Medical Education (GME) office and affiliate hospitals or surgery departments, these links were followed, as well. A third reviewer (F.A.K.) assessed 10 random programs with < 5% disagreement in data collection noted.The primary outcome was the online availability of information regarding the 55 program characteristics deemed important by the focus group to assist with developing a well-informed rank list during the pediatric surgery fellowship application process. Secondary outcomes included the accessibility of a direct, functional link to the program and a date stamp for the most recent update.t-test. P-values of less than 0.05 were considered statistically significant.Most data points are descriptive in nature and are reported as mean ± standard deviation or number and percentage. Multiple comparisons were made using one-way ANOVA and continuous data was compared using the student’s The focus group created a list of 55 discrete program characteristics considered important for making an informed assessment of the individual training program. These 55 program characteristics were organized into six categories for evaluation Fig. .Fig. 1LiOf the 57 pediatric surgery training programs identified, 54 (94.7%) were listed on APSA’s website during the original review. Between the original and follow up reviews, two more programs were added. Program descriptions contained an average of 33 ± 5.5 potential criteria (60%). All webpages listed the fellowship director, accreditation status, staff surgeons, and current fellow. Differences in information available on the APSA website and individual program websites are detailed for several key criteria in Fig. All programs were grouped by number of fellows and geographic region for comparison. There were no significant differences in the website content available from programs based on number of fellows, department caseloads, hospital type, level I trauma center designation, or designation as a burn or ECMO center Table .Table 1AWhen reviewing reporting of the fellow experience and educational support, programs presented a variety of information. Programs reported information about the following characteristics on the APSA website: alumni 54 (95%), whether the hospital classified as a stand-alone children’s hospital 49 (86%), graduate’s board performance 47 (83%), availability of an ECMO program 44 (77%), curriculum 40 (70%), fellow case logs 36 (63%), and level of trauma center designation 30 (53%). Less than half of programs listed burn center designation 23 (40%), available research opportunities 17 (30%), or candidate interview assistance 14 (25%)  Accreditation status. The accreditation status was listed for the vast majority of programs on the APSA website 55 (95%). Previous fellows or alumni were listed 54 (95%) and alumni placements after fellowship 51 (90%) were frequently listed. While these are important for convincing fellows they will succeed in the program, other key considerations, such as the presence of pediatric surgery subspeciality fellowships (PSSF) 7 (12%), area demographics 17 30%), and health insurance offerings 10 (18%) were not well listed on the APSA website; however, they were more frequently noted on individual program websites . While most programs 49 (89%) on the APSA website were listed as most recently updated in February 2020, only 4 (7%) of programs had been updated in the last 12 months at the time of the review in April 2021. After the APSA website changed vendors, another review in December 2021 revealed only 34 (60.7%) had been updated during the calendar year 2021, including the two programs that were added to the APSA website during that time.The APSA website  outscoreOur paper identified a total of 55 program characteristics that recent applicants to pediatric surgery fellowship deemed critical to making an informed assessment of the individual training program. While some of these program characteristics were readily available online, there was substantial heterogeneity among programs. There remains considerable room for improvement to ensure that accurate and relevant information is made available to pediatric surgery fellowship applicants in the era of virtual interviews.During the fellowship match, both applicants and programs try to find the best fit, hopefully matching applicants to fellowships that they consider desirable and allowing programs to find trainees with characteristics that are important to them , 21. DurThere has been a recent decline in the total number of applicants seeking fellowship training in pediatric surgery , 26, 27.The American Pediatric Surgeons Association (APSA) has an informative website for applicants; however, at the time of the initial review in August 2020, which corresponded to the start of the application cycle for the year, most program data had not been updated in the preceding 6 months . This imThe core characteristics of the program, namely accreditation status, case exposure, and alumni job placement were amongst the most important for applicants. Beres et al.  publisheth out of 18 in order of importance when formulating a rank list [While the main focus of clinical fellowship is to become a competent pediatric surgeon, in a study of pediatric surgery fellowship applicants, 55% of successful applicants had dedicated research time before or during residency . Only 17ank list , 18, 20.It is also helpful for applicants to know about where they (and their families) will be spending their training years. Applicants ranked spousal input in 9th place when developing their rank list . It is hAccurate and updated websites are very helpful to prospective candidates and may attract some of the strongest candidates to consider programs they may not have initially considered the best fit . At a mihow does the information available to pediatric surgery fellowship applicants stand against other surgical subspecialties? The online data was matched to information published by other surgical subspecialty website reviews. While there is still work to be done, we should commend the APSA website for serving as the central repository for this information and containing details on 60% of characteristics of interest. This compares favorably to other surgical subspecialty societies that have websites containing much less.One may ask: ©, LinkedIn©, or Twitter©, which are not captured here.There are several limitations in this study. First, the program characteristics deemed important for making an informed decision on where to rank a program may not be generalizable to the entire cohort of applicants and wider scale surveys of the applicants will be needed to make sure accurate and timely information is made available to the applicants. Second, these data was compared to other surgical subspeciality website reviews for benchmarking; however, some criteria are specific to pediatric surgery thereby making a comparison from other subspecialties challenging. Third, this review does not account for other resources that may be available to an applicant, as programs may directly send applicants additional information or they may have alternative resources, such as InstagramDue to the intense competition and near 100% program fill rate, there may be a perceived minimal incentive to maintain informative up-to-date websites for pediatric surgery fellowships; however, individual programs should see this as an opportunity to attract the best fit applicants. With the changes in application paradigms and applicant rate falling to the lowest since 2012, we should see this as an indicator of change and an opportunity to adapt to the changes in the pediatric surgery fellowship application process.As applicants rely on the APSA website as an invaluable resource for Pediatric Surgery training programs in the era of virtual interviews, the information posted becomes increasingly important but limited by its accuracy. We recommend that the APSA page listing all participating programs should have a standardized checklist with suggested information, and, additionally, provide a functional hyperlink to the individual program sites for supplementary information. The program descriptions on APSA should be updated at least yearly, ideally, prior to each application season. Pediatric surgery training programs should also adapt to this evolving paradigm, updating their detailed individual websites to assist future trainees in one of the most competitive general surgical subspecialty matches.Supplementary file1 (DOCX 13 KB)Supplementary file2 (DOCX 24 KB)Below is the link to the electronic supplementary material.


ARID1A expression in colorectal cancer cell lines.The current study aimed to focus on the role of histone deacetylation in reduced ARID1A expression in colorectal cancer cell lines; however, the histone modification role in ARID1A suppression in colorectal cancer remains unclear.ARID1A, a subunit of the switch/sucrose nonfermentable chromatin remodeling complex, has emerged as a bona fide tumor suppressor and is frequently downregulated and inactivated in multiple human cancers. Epigenetic modifications play an important role in dysregulation of gene expression in cancer. DNA methylation has been reported as an important regulator of The expression levels of ARID1A mRNA were determined using real-time quantitative PCR in colorectal cancer cell lines including HCT116, SW48, HT29, SW742, LS180, and SW480. To evaluate the effect of histone deacetylation on ARID1A expression, all cell lines were treated with trichostatin A (TSA), a histone deacetylase inhibitor. SPSS software (Version 23) and GraphPad Prism (Version 6.01) were applied for data analysis using one-way ANOVA, followed by Tukey’s multiple comparison tests. ARID1A expression in a cell line-dependent manner, suggesting that histone deacetylation is at least one factor contributing to ARID1A downregulation in colorectal cancer. Treatment of colorectal cancer cell lines with TSA increased ARID1A expression and may bring benefits to the colorectal cancer patients with a broader range of genetic backgrounds.Histone deacetylase inhibitors might provide a strategy to restore  The molecular and genomic understanding of colorectal cancer (CRC) has progressed considerable in the last decade; however, CRC is still one of the most common causes of cancer deaths in the world , 2. CRC ARID1A) encoding gene  in cancers of the MSI type (ARID1A expression in tumors (ARID1A in mice led to the spontaneous formation of invasive adenocarcinomas in the colon (ARID1A expression loss was correlated with lymphatic invasion and early onset in T1 CRC (ARID1A significantly enhanced the migration activity of HCT116 human colon cancer cells (Data from The Cancer Genome Atlas project (TCGA) revealed that MSI type -18. Analn tumors , 18. Thehe colon . In addin T1 CRC . Moreoveer cells .ARID1A gene in cancers is attributed to genomic deletion, DNA mutation, DNA methylation, and microRNA-mediated inhibition  or RPMI 1640 , supplemented with 1% penicillin/streptomycin, 10% fetal bovine serum (Gibco-BRL), and 2 mM glutamine, and then incubated in a 37 °C humidified atmosphere with 5% CO2.TSA treatment Cells were plated in T-25 flasks and allowed to attach for 24 h, after which cells were incubated for 24 h in media containing 300 nM TSA . Then, RNA was isolated as described below. DMSO (Sigma-Aldrich) was used as vehicle control. RNA extraction and real-time quantitative PCRTotal RNA was purified from CRC cell lines using Accuzol total RNA extraction reagent , according to the manufacturer’s instructions. Purified RNA quantification and purity was analyzed by a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies). The purified RNA integrity was detected by 0.8% agarose gel electrophoresis visualized by Gel-Red staining. Then, two micrograms of purified RNA were applied for cDNA synthesis by RevertAid First Strand cDNA Synthesis Kit (Fermentas), according to the manufacturer’s instructions.ARID1A and GAPDH as an internal housekeeping gene was determined by real-time quantitative PCR (qPCR) using SYBR Green master mix  on an ABI 7500 Sequence Detection System . Primer sets used for qPCR assay were as follows: ARID1A ; GAPDH . Next, qPCR reactions were performed in triplicate with a 60 °C annealing temperature and a total cycle number of 40. The relative expression of ARID1A mRNA was normalized to GAPDH mRNA levels and determined using formula 2-ΔΔCT. The workflow for investigating the role of histone deacetylation in reduced ARID1A expression in colorectal cancer cell lines is shown schematically in The mRNA analysis of Statistical analysisp-value ≤ 0.05 were considered statistically significant. Statistical analysis was done using SPSS software (Version 23) and GraphPad Prism (Version 6.01). Data reported as mean ± standard deviation (SD) was analyzed using one-way ANOVA, followed by Tukey’s multiple comparison tests. Differences with a ARID1A expression is heterogeneous in CRC cell linesARID1A by real-time quantitative PCR in six CRC cell lines, namely LS180, SW480, SW742, SW48, HCT116, and HT-29/19. ARID1A expression  was highly heterogeneous among cell lines . The mRNA levels of ARID1A were significantly higher in HCT116, HT29, and LS180 cells compared to other cell lines . ARID1A mRNA expression was low in SW742 and SW480 cell lines with no significant difference with SW48 cells.We studied the expression profile of ll lines . The lowARID1A was used as a calibrator (expression level set to 1.0), and expressions in all other cell lines were presented as a fold-change relative to the SW48 cell line. GAPDH was used to normalize ARID1A gene expression values. Mean ± SD of three experiments is reported. . Error bars show standard deviation (SD) of the mean for each triplicate experiment. The SW48 cell line with the lowest expression level of Effects of TSA on the expression of ARID1A mRNAin human CRC cell linesARID1A mRNA expression, CRC cells were treated for 24 h with TSA, an inhibitor of histone deacetylase, and ARID1A expression was determined by real-time quantitative PCR. Treatment of SW48, HCT116, and SW480 cells with TSA had no significant effect on ARID1A expression  deletion of ARID1A alone led to the formation of invasive colorectal adenocarcinomas in mice (ARID1A-deficient colorectal cancer.It has also been shown that the biallelic ( in mice . TherefoARID1A gene downregulation in cancer (ARID1A downregulation in CRC cell lines (ARID1A expression in breast cancer demonstrated that histone modification and promoter hypermethylation are the main causes of ARID1A gene expression loss (ARID1A expression, which indicated the important role of DNA hypermethylation in ARID1A downregulation (ARID1A expression was not restored after 5-aza treatment in SW480 and LS180 cell lines, which indicated the possibility of another mechanism in ARID1A downregulation, like histone modifications in these cell lines (ARID1A expression can be increased in CRC cell lines by blocking histone deacetylation. Treatment with TSA influenced ARID1A expression in a cell line dependent manner. The highest upregulation of the gene was observed in the LS180 and HT29 cell lines and was almost 10-fold and 6-fold change, respectively. No such significant change was observed in the SW480, HCT116, or SW48 cell lines. Treatment of SW742 cells with TSA resulted in an almost threefold upregulation of ARID1A mRNA levels.Interestingly, accumulating evidence indicates that epigenetic modifications such as DNA hypermethylation and histone modifications play significant roles in n cancer , 23, 32.ion loss . In addill lines . Decreasll lines . TherefoARID1A expression in LS180, HT29, and SW742 cells; however, it is still unknown whether this expression increase is due to a direct effect of TSA on the acetylation state of ARID1A genes. There are alternative explanations, e.g., TSA may alter the epigenome of genes that regulate ARID1A transcription, such as a coactivator. Therefore, more experimental studies that examine acetylation levels of histones within regulatory regions of the ARID1A gene after TSA treatment are necessary.The current results suggest that TSA inhibition of histone deacetylation leads to increased ARID1A in CRC cell lines by HDACs pharmaceutical inhibitors seems to be a therapeutic approach for the treatment of ARID1A-deficient colorectal cancer.Clinical efficacy against cancer has been established for HDACs inhibitors, which can be considered as attractive targets for CRC therapy , 29. UprARID1A expression in mRNA levels only, but not protein level, is another limitation of this study, which can be considered in future studies. Moreover, additional studies such as analysis of the ARID1A gene promoter histone modification levels by ChIP- qPCR (ARID1A promoter histone deacetylation in ARID1A gene expression loss in CRC. In addition, the correlation between ARID1A expression and promoter histone modification needs to be assessed in patients with CRC. TSA also affects other epigenetic processes, including the methylation of DNA and histones (ARID1A expression restoration by TSA.Evaluation of cancer cells only, and not normal cells, can be considered as a limitation of this study. Evaluation of IP- qPCR  will be histones , 36. HenARID1A expression in CRC are unknown, according to our cell line-based study, it is likely that epigenetic modifications play a role in ARID1A downregulation. More research is needed to support these findings.Although the precise mechanisms accounting for decreased The authors declare that they have no conflict of interest.


We analyze implementation bottlenecks experienced in the DREAMS program in Namibia that can provide valuable insights and suggest ways to anticipate and overcome these challenges when managing HIV and gender-based violence prevention programs for adolescent girls and young women. Adolescent girls and young women (AGYW)–centered HIV prevention programs, such as DREAMS, must employ layered interventional approaches that address both AGYW health and sociobehavioral issues and must engage stakeholders across multiple sectors, increasing the potential for implementation bottlenecks that can undermine their efficiency and effectiveness.Bottlenecks identified involved challenges with program access and enrollment, outdated and unengaging health education curriculum and lack of safe meeting spaces, stock-outs of HIV commodities, and limitations in supporting gender-based violence (GBV) cases.Solutions include increasing the government role in identifying safe spaces for AGYW, expanding sexual and reproductive health services for AGYW within existing institutions, ensuring a continuous supply of sexual and reproductive health and HIV commodities, and decentralizing GBV care to increase access.These bottlenecks highlight practical implementation issues and higher-level considerations for AGYW-centered HIV/GBV prevention programs.The unique programming needs for HIV/GBV prevention in AGYW require multilevel collaboration and forecasting. Successful program implementation necessitates adequate preparation. We synthesize implementation bottlenecks experienced while implementing the DREAMS  program, an HIV prevention intervention for adolescent girls and young women (AGYW), in Namibia from 2017 to 2019. Bottlenecks were organized into the following 4 AGYW program components.Enrollment was slowed by the time-intensive nature of screening and other baseline data collection requirements, delays in acquiring parental consent, and limited time for after-school activities. Solutions included obtaining advance consent and providing 1-stop service delivery and transportation assistance.We experienced difficulty identifying safe spaces for AGYW to meet. A lack of tailored curricula also impeded activities. Governments, stakeholders, and partners can plan ahead to help DREAMS identify appropriate safe spaces. Curricula should be identified and adapted before implementation.Uneven availability of government-provided commodities  and lack of AGYW-centered PrEP delivery approaches impacted services. Better forecasting of commodity needs and government commitment to supply chain strengthening will help ensure adequate program stock.The availability of only centralized care following gender-based violence (GBV) and the limited number of government social workers to manage GBV cases constrained service provision. Triaging GBV cases—i.e., referring high-risk cases to government social workers and providing DREAMS-specific social services for other cases—can ensure proper caseload management.These bottlenecks highlight practical implementation issues and higher-level considerations for AGYW-centered HIV prevention programs. The critical need for multilayered programming for HIV/GBV prevention in AGYW cannot be addressed simply with additional funds but requires multilevel collaboration and forecasting. The urgency to achieve results must be balanced with the need for adequate implementation preparedness. As a public-private partnership supported by the U.S. President’s Emergency Plan for AIDS Relief (PEPFAR), DREAMS combines multisectoral, layered, evidence-based interventions for AGYW, their sex partners, parents, and the community.,–“Layered” here refers to the provision of multiple interventions or services to each AGYW as well as contextual level interventions not delivered directly to an AGYW but from which she may benefit .Because of the number of concurrent interventions and components, the DREAMS program must include the active engagement of stakeholders across multiple sectors, such as education, health, social welfare, and safety and security sectors, and are implemented in myriad settings including community centers, schools, churches, and health facilities. The program’s inherent complexities increase the possibility of bottlenecks that can hamper the efficiency of implementation and ultimately undermine program effectiveness, acceptance, and long-term sustainability.,Few published studies of AGYW-centered programs examine and report on the role of bottlenecks in implementation, though key organizations have published guidelines asking for more reporting on implementation challenges.–The Namibia DREAMS program launched in 2017 in 2 high HIV prevalence regions, Khomas and Zambezi .21–24 ThThe DREAMS program in Namibia used a safe space model for program and service delivery.DREAMS in Namibia used a safe space model for program and service delivery where AGYW meet in a supportive, judgment-free, safe, and private location.Recruitment was organized by age group. Participants aged 9–14 years were recruited almost entirely from primary schools. Girls aged 15–19 years were recruited from secondary schools, house-to-house engagement, special events, and health facilities. Young women aged 20–24 were recruited from tertiary education settings, house-to-house engagement, special events, and health facilities. In addition to providing classroom space, teachers and school administrators were involved in helping communicate with parents, assisting AGYW who experienced violence, and providing insight into the HIV/GBV education curricula, as many teachers had taught the same content to students. Recruitment was driven by PEPFAR targets for DREAMS , which aimed to reach “saturation” whereby 75% of vulnerable AGYW aged 9–24 years in a district or region complete an age-specific primary package of DREAMS interventions and need-based secondary interventions .27Enrollment procedures included consent, collection of basic demographic information, and an HIV/GBV risk and vulnerability assessment that was introduced 1 year into program implementation. All AGYW provided written consent before participation. Written parental consent was obtained from parents of AGYW aged 9–19 years regardless of setting . To reach the most vulnerable AGYW, and as required by PEPFAR, program staff administered an HIV/GBV risk and vulnerability assessment to each AGYW, with responses determining eligibility for secondary DREAMS interventions.Regardless of risk assessment results, all AGYW received basic HIV/GBV prevention education and health and social services  staff, and over 200 part-time staff . The total implementation cost of DREAMS in the 2 regions over 2 years was approximately $US3.2 million. I-TECH established a separate DREAMS office in each region where staff were organized by team: M&E, social services, and health services. Each regional office oversaw a large team of part-time staff who were entirely community-based. Each region had 2 vehicles.,A monitoring system was designed to capture data required for PEPFAR reporting and to support program quality and continuity of services. Data were collected at enrollment and at each point of service delivery and managed using Research Electronic Data Capture (REDCap).Within 18 months, the DREAMS program under I-TECH enrolled 20,150 participants, approximately 26% of the AGYW population living in the Khomas and Zambezi regions according to the 2011 national census.We report bottlenecks experienced during the implementation of 4 different program components for DREAMS in Namibia: program access, health education, health services, and social services  and thenAlthough gathering AGYW in safe spaces was logistically easier in school settings, it was challenging to screen and enroll hundreds of AGYW at the end of the school day, especially when AGYW had to walk long distances home before dark for their safety . GatheriPreprogram planning with school and community group representatives identified alternative time periods for enrollment of AGYW, such as during the school day or during special school or community events. Programs could also explore one-by-one DREAMS enrollment during door-to-door recruitment in the community, special events, or automatic enrollment when students enroll in school .Program staff and time burdens were addressed through the introduction of self-administered risk/vulnerability assessments for older AGYW aged 20–24 years. In some settings, self-administered online enrollment could also be utilized. DREAMS programs should carefully consider the added value of all preenrollment procedures given the time and effort involved in the field and burden on AGYW .DREAMS programs should carefully consider the added value of all preenrollment procedures given the time and effort involved in the field and burden for AGYW.Electronic written consent, through WhatsApp or a similar platform, or utilizing verbal consent procedures given the low-risk nature of the program, could be explored. Participation of AGYW aged 18 years or older  should not require parental consent since this is the age of majority, even in school settings. In Namibia, this requirement sometimes prevented inclusion of AGYW most in need of services as older adolescents often live in school dormitories away from home, especially in rural areas .This bottleneck was addressed partly through organization of 1-stop DREAMS events where AGYW in this age group could receive all the services available. To further aid with access, the DREAMS program organized transportation for this age group and provided babysitting services in addition to small snacks and drinks. Colleges, universities, and training institutions should plan in advance to allow for DREAMS access for AGYW enrollees aged 20–24 years . Also, tAlthough there were Government of Namibia–approved curricula available for HIV/GBV prevention for 9- to 14-year-olds and 15- to 19-year-olds, neither curriculum had been updated for at least 5 years . UpdatinWith thousands of AGYW at over 100 safe spaces, attendance tracking required for reporting purposes was an especially time- and labor-intensive process. It took up valuable time for all team members, especially peer mentors and M&E teams, and came at the expense of other M&E processes needed for improving program quality .Employment as a peer mentor was often the first formal employment experience for the young women hired into this role. Even though all peer mentors were fully trained to deliver the education sessions, their skill and competence varied widely, requiring considerable ongoing training, support, and supervision to maintain a high teaching standard and ensure ongoing attendance reporting .Many of the challenges in implementing health education related to identifying safe spaces that were private and safe, affordable, and suitable for the required activities. Program staff need to conduct advance strategic planning with ministries, the private sector, nongovernmental organizations, churches, and local authorities to identify an adequate number of appropriate safe spaces for DREAMS meetings .Removing attendance tracking and administrative tasks from peer mentors, or simplifying them with better technology, would enable peer mentors to focus their time and energy on teaching and mentoring AGYW, improving health education delivery, and allowing for expansion of the peer mentoring component. In addition, a frank discussion about the value of the extensive DREAMS M&E is also needed. Simplifying attendance reporting also frees up time for the M&E team to focus on data quality and analysis of data for improving the program .DREAMS programs need considerable advanced planning with appropriate ministries and other stakeholders to be able to identify and/or adapt an acceptable curriculum for each age group . SuccessAlthough PrEP was included in the Namibia 2016 National Guidelines for Antiretroviral Therapy, development of the Government’s PrEP implementation plan launched concurrently with the DREAMS program, leaving DREAMS without an existing national PrEP delivery plan to follow.DREAMS relied on the Namibian government’s commodities such as condoms, HIV test kits, family planning methods, and PrEP, which are distributed to public health facilities by the MOHSS Central Medical Stores. For various reasons, including a severe economic contraction in the country, these commodities were at times unavailable for long periods. Stock-outs not only delayed DREAMS health services but also complicated follow-up services for AGYW, especially family planning and PrEP. Other complications included the refusal of some schools early in the implementation of DREAMS to allow family planning services to be delivered in the school setting. While family planning service delivery in schools was under discussion between stakeholders, the program provided these services to AGYW in community settings only.The DREAMS program introduced innovative AGYW-friendly PrEP services in Namibia. In Khomas, PrEP service delivery successfully transitioned from an initial hybrid community-clinic model where AGYW were referred to government facilities for refills to a wholly community-based model, even though the transition was time-intensive and logistically challenging. In Zambezi region, where PrEP services began after those in Khomas, a community-based PrEP model was successfully implemented from the onset by applying lessons learned in Khomas. DREAMS and other similar programs for AGYW should consider different models for delivering PrEP to AGYW and organize strong support for selected models before launching the service .In both regions, PrEP service delivery for AGYW successfully transitioned from a community-clinic model to a wholly community-based model.Delivering services to thousands of AGYW may well put an additional strain on an already undersupplied health sector. Ongoing coordination and collaboration are needed to anticipate commodity supply requirements and ensure that government forecasting can fully account for the increased demand from DREAMS . It may All DREAMS stakeholders, especially ministries of education, need to understand and communicate support for DREAMS services from national to regional and local levels to avoid possible misunderstandings. Since national school health policy at the time did not explicitly mention SRH services as part of the range of health services to be offered at schools by the MOHSS, PEPFAR and other AGYW program donors need to advocate for national school health policies that include provision of SRH services so that interventions designed to meet the SRH needs of AGYW in school settings can be legally implemented .The Ministry of Gender Equality, Poverty Eradication and Social Welfare, which is responsible for managing GBV cases in Namibia, was often too understaffed to manage the high number of cases identified through DREAMS . While tFinally, the DREAMS program did not have enough private, confidential spaces to provide in-depth psychological counseling to many AGYW. This lack of privacy made it difficult for AGYW to discuss their GBV experiences with program staff. In such cases, the sessions were delayed until a private space could be identified .Over time, the program addressed the issue of the high GBV caseload by having the Government manage only the most time-sensitive GBV cases, such as imminent rape cases. Other GBV cases, including prior rape cases that had already received services by the relevant ministry but where the AGYW needed more care and follow-up, were managed by DREAMS social workers and lay counselors. This triaging of cases helped the program manage the GBV caseload .Multisectoral involvement and coordination are essential to create awareness of and appropriately plan for the expected increase in demand for GBV services under DREAMS. Involving stakeholders early in the planning process for GBV programming could circumvent unnecessary obstacles such as limiting postrape services to difficult-to-reach locations . It is critical that post-GBV care is decentralized from tertiary care centers to more peripheral levels in Namibia to help increase access to postviolence .Multisectoral involvement and coordination is essential to create awareness of and appropriately plan for the expected increase in demand for GBV services under DREAMS.Social workers and other staff should receive training on trauma counseling in anticipation of managing cases of rape, incest, drug addiction, human trafficking, and severe emotional and physical abuse, within the context of the LIVES approach .32 IndeeTo truly take on the individual and group GBV services needed, DREAMS or any AGYW program manager needs to anticipate the considerable staffing, training, and infrastructure needed . This in–Our program results show the substantial reach of the DREAMS program in Namibia and the significant demand for AGYW-specific services. AGYW loved the opportunity to participate in DREAMS because there are often few other fun activities available for preteen girls, teen girls, and young women where they can interact openly and freely with their peers and near-peer mentors. Such demand has been documented in other high HIV prevalence countries.–Previously published articles have reported on DREAMS topics such as HIV/GBV prevalence, characterization of male partners of AGYW, and how to rigorously evaluate DREAMS and AGYW PrEP persistence, but few have reported on the implementation challenges associated with launching and establishing a complex new multisectoral combination prevention program.Anticipating many of the bottlenecks and potential solutions we describe (also summarized in The bottlenecks and solutions described here are not unique to DREAMS and likely apply to other combination prevention programs for AGYW.We do not report here on the number of services AGYW received, as the definition of each service changed considerably over the 2-year implementation period, but this measurement is critical for AGYW programs since they are more likely to benefit if they receive 3 or more services.Effective HIV/GBV prevention programs for AGYW are multicomponent, layered programs that respond to the multidimensionality of localized influences on AGYW health-related behaviors. Given the complex nature of AGYW programs, a variety of implementation bottlenecks are inevitable. These challenges can be overcome with iterative innovation and flexibility and by planning well in advance for contingencies related to probable future difficulties. Programs for AGYW should continue to document implementation challenges and bottlenecks so lessons learned can be widely shared.


The demand for well-trained bioinformaticians to support genomics research continues to rise. Unfortunately, undergraduate training in Kenya does not prepare students for specialization in bioinformatics. Graduates are often unaware of the career opportunities in bioinformatics, and those who are may lack mentors to help them choose a specialization. The Bioinformatics Mentorship and Incubation Program seeks to bridge the gap by laying the foundation for a bioinformatics training pipeline using project-based learning. The program selects six participants through an intensive open recruitment exercise for highly competitive students to join the program for four months. The six interns undergo intensive training within the first one and a half months before being assigned to mini-projects. We track the progress of the interns weekly through code review sessions and a final presentation at the end of the four months. We have trained five cohorts, most of whom have secured master’s scholarships within and outside the country and job opportunities. We demonstrate the benefit of structured mentorship using project-based learning in filling the training gap after undergraduate programs to generate well-trained bioinformaticians who are competitive in graduate programs and bioinformatics jobs. PLOS Computational Biology Methods paper.This is a The field of bioinformatics has grown substantially since the completion of the first draft of the human genome in the 1990s . AlthougThe countries of the global north have incorporated the core competencies of bioinformatics into undergraduate life sciences courses , includiAfrica, regarded as the cradle of humankind , has a hicipe), the International Livestock Research Institute (ILRI), Kenya Medical Research Institute, Wellcome Trust (KEMRI-WT)—play a crucial role in closing this gap through short-term training and workshops, but this is not enough. In the absence of ongoing support, those who receive training cannot continue learning and utilize the skills gained through research projects. Furthermore, short-term training is sustainable because trainees do not retain their bioinformatics skills.Few organizations in Kenya—International Centre of Insect Physiology and Ecology  project led to the demand for bioinformaticians who could analyze genomic data generated from H3Africa projects . The Fogparticipate in structured training and mentorship. The aim is to generate a pool of competitive bioinformatics trainees who can proceed to master’s degree training or support ongoing research.We established the Bioinformatics Incubation and Mentorship Program to fill the gap by creating a foundation for bioinformatics career development. The program recruits six highly motivated students interested in bioinformatics every four months to The mentorship program is based on a project-based learning approach to increase bioinformatics capacity. We modeled the incubation program to impart key bioinformatics competencies to bioinformatics scientists, according to ISCB , with clThe selected interns undergo rigorous training within the first one and a half months. The training covered by different tutors is meant to introduce the field of bioinformatics, guide the interns in designing their purpose road maps, teach key people skills, and introduce them to data management, open science, and reproducible research . In addiicipe. In the last two weeks of the second month, the interns reproduced methods in a paper that would have applied techniques taught in the first one and a half months—sequence analysis, phylogenetics, and NGS—and shared their data. The interns are assessed through a final presentation at the end of the month; the strengths of the intern are evaluated to facilitate appropriate assignments to projects from the second phase. In the second phase, the mini-projects carried out within two months related to ongoing genomics work at icipe. In the first week, the interns present how they would tackle the project, how they would collaborate, and the tools they would use. Weekly code review sessions help track the progress of interns and enable code debugging and documentation using GitHub wikis and GitHub projects.The project-based learning approach is implemented in two phases: reproducing bioinformatics analysis in a published paper and data from ongoing projects at The various fields of bioinformatics are condensed into a progression of self-contained modules taught in logical order . The curUsing seven contact hours per day for six weeks, the course is delivered using both theory sessions in the morning and practical hands-on sessions in the afternoon, which are handled by the local Senior Faculty, the program coordinator, and MSc fellows as teaching assistants. When mentees require them outside those scheduled times, the program coordinator and teaching assistants provide additional practical sessions and office hours. The interns are then tested at the end of each module through regular reading and practical exercises.Once the key concepts have been delivered in the first six weeks, the interns are put into groups of 2 or 3 based on shared interests in readiness for the mini-projects phase. First, the interns are tasked to reanalyze data from published papers and their results compared with those published study. The interns are then assigned projects from ongoing research at the Centre, where they work closely with the scientists to analyze and interpret the data. During this phase, they receive help from the local senior faculty, program coordinator, and MSc fellows through weekly code review sessions. They then make a final presentation to summarize their mini-projects on the last day of the internship.In addition to standard bioinformatics topics , journal club meetings, soft skills sessions, and the information literacy workshop are part of the internship program. These sessions are included because they are crucial to the professional development of the mentees. We also guide interns in job and MSc applications through mock reviews and interviews in the last month. The cover letters, CVs, and motivations of the fellows are reviewed, and feedback is provided based on the skills gained by the interns.Mini-projects focused on open and reproducible genomic analysis .icipe Molecular Biology and Bioinformatics Unit (MBBU). To be selected for the program, interested students from different universities in Kenya submit applications to icipe through a web-accessible form developed in REDCap [The Bioinformatics Incubation and Mentorship Program is designed for highly motivated students interested in bioinformatics recruited through widely advertised calls and direct recommendations from relevant university departments. It is hosted at the n REDCap ,20. The For each round, we shortlist at least 15 applications for an interview. The applications are critically reviewed through an initial long list to ensure completeness and essential qualifications. We are interested in highly motivated students who have completed their undergraduate degrees. The desire to pursue Bioinformatics should not be an afterthought and must have put some effort into writing their motivation letter with clarity on how it fits their career plan. At least two reviewers then score long-listed applications based on whether the applicant is motivated to pursue a career in bioinformatics, the clarity of how the internship fits their career plan, and the quality of the application.The rigorous selection process is vital for success, as we work with highly motivated and curious students. Interview questions aim to reveal what makes the applicant uniquely qualified for the internship based on prior interests, problem-solving skills, curiosity, and how the training aligns with their career goals. The questions were as follows: (1) For this internship, tell us who you are and what makes you uniquely qualified for the position. (2) You are faced with a challenge you have not encountered before; briefly tell us about your thought process and the approach to solving it. (3) What are your expectations from the internship and how does it align with your goals? Three interviewers select six interns to join the program through independent ranking. A summary of the recruitment process is described in icipe and with our collaborators. The internship program has been run successfully for five iterations since October 2020, mentoring 27 students. The program has generated a pool of competitive students who have secured master’s degrees within and outside the country and job opportunities. Some interns continued various ongoing projects at We assess and monitor the progress and impact of the bioinformatics mentorship and incubation program through pre- and post-internship surveys, weekly updates, and code reviews. The interns join the program with the expectations of learning bioinformatics in depth, improving presentation skills, learning to code, creating meaningful friendships and networks, being mentored, interacting with leading scientists, and gaining in-depth knowledge of genomics and genetics. Before the program, the research interests of the interns were more generalized, but after the internship was completed, the interests aligned. Some of the examples are as follows:“Research has always been my interest, and training has shaped me in an interesting field of metagenomics. An added value was that I got to do metagenomics analysis as a mini-project. Yes, my research interests have changed.”“Before the internship, I wasn’t quite sure what I wanted to venture into, but now I am interested in using bioinformatics tools to study infectious and non-infectious diseases.”“Before training, I wanted to be a genomics researcher. Going out, I am still interested in genomics research, but the internship program opened my mind to find a field to specialize in, and that’s what I’m still trying to figure out.”The interns improved their competencies in command line , Git andAs part of the post-internship survey, the interns highlighted their training experience, as shown in In addition to quantitative feedback, the interns reflected on their experience through a blog post published on our program page.We highlight one representative example in the following:“Coding has always fascinated me. Being from an applied bioengineering (genomics-based) background, I actively sought a bridge between the two. The internship allowed me to learn and polish my programming skills under the guidance of a tutor and to see its application in multi-omics data processing of real-life problems. In addition, it has cultivated a self-learning discipline that has played a key role in solidifying and expanding my bioinformatics knowledge. The skills and experience gained during the internship gave me a competitive advantage when applying for bioinformatics opportunities, paving the way to obtain the EANBiT Master’s fellowship. The internship increased my resilience and self-discipline, which still plays a key role in my MSc Bioinformatics journey.”Design and implementation of a project-based learning bioinformatics training curriculum. Using the curriculum, we have mentored and trained 27 interns, generating a pool of fellows to join the bioinformatics training pipeline and contribute to the bioinformatics capacity in the continent.A key component of the mentorship program is to enhance collaboration and reproducible research. All mini-projects have been properly documented on the organization’s GitHub account, enhancing reproducibility. The curriculum and the resources used for the internship program are publicly available on the MBBU GitHub pages and can be used to establish a similar program.To continue mentoring interns beyond the internship period, we launched the Science Journey Seminar series, where interns interact with scientists worldwide through seminars. These seminars highlight paths and journeys of established and upcoming scientists to motivate, enlighten, and guide them on potential career paths in bioinformatics.The fast pace and turnaround of the program meant that we had to continuously design projects that fit the interest of the interns. Sometimes, however, some interns’ interest did not align with the work at the Centre, or that data-rich projects requiring their expertise were not readily available from within the host institute. We addressed this by forging collaboration outside the institute to ensure that the interest of fellows was catered to. The short duration of the mentorship program, though by design, may not allow the completion of some of the projects. In these cases, we have had fellows retained by the projects after the internship.icipe. Mentorship is vital as the research mentor influences the professional growth of the intern [A substantial increase in genomics and proteomics data has catalyzed the demand for well-trained bioinformaticians , but unde intern . The cure intern  and be ee intern . Reproduicipe. The emphasis on project-based learning (PBL) is vital in cementing the knowledge and skills learned, an integral approach yet to be widely implemented in bioinformatics curricula [Structured training and dedicating the last two months of the internship program to a mini-project contributed to the success of the program. The projects span various topics based on the interests of the interns, available data, and ongoing research within urricula . The PBLurricula , as prevurricula . Studenturricula .During the mini-projects, we train the interns to think critically and computationally, as this helps them understand research problems and code debugging. “Confidence in dealing with complexity, persistence in working with difficult problems, tolerance of ambiguity, the ability to deal with open-ended problems, and the ability to communicate and work with others to achieve a common goal” are all skills that computational thinking helps students develop . The proWeekly code review sessions facilitate continuous feedback and help uncover bugs and code issues with a fresh set of eyes. They also provide room for new ideas and easier ways to perform tasks. In addition, they help track the progress of a project and identify gaps and ways to improve the project. Bioinformatics research and practice have significant implications for life sciences, and effective quality assurance techniques such as code reviews and testing are vital to ensuring software quality . We advoThe interns give feedback occasionally by filling out pre-internship surveys, verbal feedback after every taught module, and post-internship surveys. The pre-internship surveys highlight the training needs of the interns and point out areas that require more emphasis during training. It also helps to highlight the research interests of the interns so that we can help them find suitable mentors. Verbal feedback after every taught module is essential in gauging whether the interns understood the topic of interest. The post-internship survey highlights the success of the internship journey and the areas that need improvement.Figs We have established a structured project-based mentorship and incubation program that fills the bioinformatics training gap between undergraduate and graduate programs or job opportunities. The program trainees are highly competitive for MSc programs and job placements within and outside Kenya, demonstrating the benefit of mentorship in filling the transition gap, thus creating a pool of highly motivated trainees for recruitment to graduate school, and supporting genomic data analysis in Africa.


The University of Rhode Island Engaging Generations Cyber-Seniors Program was first implemented in the fall semester of 2015. URI students from 15+ majors have supported the digital competency of older adults in Rhode Island each semester and summer session since its inception. Gradually over time, we modified and expanded this in-person program to meet student, older adult, community partner, and faculty needs. The pandemic led to our program expanding exponentially due to new grant funding opportunities, new partnerships, and student interest. This presentation will address strengths and challenges of implementing this program state-wide in a small, mostly urban state with community partners, mostly involving senior centers. We will describe moving from in-person to phone/virtual and now to a hybrid model. Last, we will explain the program’s efforts to conduct formative and summative evaluation research to assess program output and examine outcomes for students and older adults in the program.


BCR-ABL1 transcript level via real-time quantitative-polymerase-chain reaction (Q-PCR) is a clinical routine for disease monitoring, assessing Tyrosine Kinase Inhibitor therapy efficacy and predicting long-term response in chronic myeloid leukemia (CML) patients. For valid Q-PCR results, each stage of the laboratory procedures need be optimized, including the cell-counting method that represents a critical step in obtaining g an appropriate amount of RNA and reliable Q-PCR results. Traditionally, manual or automated methods are used for the detection and enumeration of white blood cells (WBCs). Here, we compared the performance of the manual counting measurement to the flow cytometry (FC)-based automatic counting assay employing CytoFLEX platform. Methods: We tested five different types of measurements: one manual hemocytometer-based count and four FC-based automatic cell-counting methods, including absolute, based on beads, based on 7-amino actinomycin D, combining and associating beads and 7AAD. The recovery efficiency for each counting method was established considering the quality and quantity of total RNA isolated and the Q-PCR results in matched samples from 90 adults with CML. Results: Our analyses showed no consistent bias between the different types of measurements, with comparable number of WBCs counted for each type of measurement. Similarly, we observed a 100% concordance in the amount of RNA extracted and in the Q-PCR cycle threshold values for both BCR-ABL1 and ABL1 gene transcripts in matched counted specimens from all the investigated groups. Overall, we show that FC-based automatic absolute cell counting has comparable performance to manual measurements and allows accurate cell counts without the use of expensive beads or the addition of the time-consuming intercalator 7AAD. Conclusions: This automatic method can replace the more laborious manual workflow, especially when high-throughput isolations from blood of CML patients are needed.Background: Detection of  The eormation ,5,6. BCR-ABL1 tyrosine kinase inhibitors (TKIs) has significantly improved the outcomes of most CML patients, generating unprecedent rates of complete hematological (CHR), cytogenetic (CCyR) and molecular (MR) responses [Over the past 20 years, the development of esponses ,8,9,10. esponses ,12. In tesponses ,14,15,16esponses ,18,19. OBCR-ABL1 transcript monitoring [BCR-ABL1 oncogene transcript should be performed every 3 months after TKI therapy initiation, then at least every 3–6 months. For valid Q-PCR data, standardization of each stage of the laboratory procedures is mandatory, including the amount of blood collected, the method for measuring total white blood cells (WBCs), and RNA isolations [BCR-ABL1 and ABL1 gene transcripts. In the current clinical practice, the quantitative polymerase chain reaction (Q-PCR) is the “gold standard” for diagnostic nitoring ,21,22,23olations ,25,26,27The considerable burden of work for molecular laboratories necessitates the development of a high-throughput method for a fast, reproducible, and efficient counting assay. Traditionally, manual counting-chambers or automated methods are used for the detection and enumeration of cells. Although the traditional way of counting cells manually, by hemocytometer, is very simple and straightforward, it is often error-prone, time-consuming, and highly subjective. For example, a wrong dilution or wrong samples may result in inappropriate volumes and concentrations of cells. Therefore, an ideal cell counting method should be accurate as well as repeatable, with low variability attributed to operator error . Due to BCR-ABL1 and ABL1 gene transcript levels from matched samples of CML patients.The purpose of this study was to compare the traditional manual counting method to the automatic counting assay based on the CytoFLEX instrument for their ability to measure white blood cells from peripheral blood (PB) of CML patients. In the automatic method, we investigated four different types of measurements: absolute cell-counting, a measurement based on beads, cell counting with the use of 7AAD day, and a combined method including counting beads and 7AAD. Our analyses focused on both qualitative and quantitative parameters, including total amount of WBCs, RNA recovery efficiency, and quantification of The research study was carried out at the “Center of Experimental Oncology and Hematology” of the A.O.U. Policlinico “G. Rodolico-San Marco” of Catania. Between May 2021 and December 2021, peripheral blood samples from 90 adults’ chronic phase CML (CP-CML) patients were collected and analyzed for molecular monitoring of CML in our Molecular Diagnostic laboratory. Blood samples collection was conducted in accordance with the Declaration of Helsinki. All patients gave written informed consent prior to participation.All patients received a TKI  as first-line treatment. Treatment response was assessed according to the 2020 ELN criteria .®, Becton Dickinson, Franklin Lanes, NJ, USA), according to the manufacturer’s instructions. After blood draw, blood samples were stored at room temperature and further processed within 24 h. Total WBC were isolated using the Biomek i-5 Automated Workstations integrated with the CytoFLEX Flow Cytometer , as previously described [Ninety CML patients provided 28 mL of PB within a single blood draw, collected in sterile 4 × 7 mL ethylene-diamin tetra-acetic acid (EDTA) tubes , according to the manufacturers’ instructions  and the automatic (B–E) methods. In the manual methods, samples were diluted and counted using a hemocytometer counting-chamber with a microscope. Next, 1 × 10ructions . RLT lys7 cells were lysed in RLT buffer, as mentioned above. RLT lysates were stored at −80 °C until further processing.In the automatic method, cells were loaded in a 96-well plate and cell-count was carried out in the CytoFLEX Flow Cytometer by four different types of measurements. In the first measurement (B), absolute cell-counting was performed using 200 µL of re-suspended cells in PBS buffer, as previously described . The oth7 WBC lysed in RLT buffer by QIAsymphoy® technology , according to the manufacturers’ instructions, and RNA was eluted in Dnase/Rnase free water, as previously reported [Total RNA from matched samples was extracted from 1 × 10reported .Purified total RNA was quantified with a BioSpectrometer  at wavelengths of A230, A260 and A280 nanometer. RNA purity was calculated by A260/280 ratio (~1.9–2.0) and A260/230 ratio (~2.0–2.2). RNA integrity was then verified by electrophoresis running samples on 1.2% denaturing agarose gels. Total RNA samples were stored at −20 °C until further use.Complementary DNA (cDNA) was synthetized from a total of 1 µg of purified RNA using random hexamer primers  and moloney murine leukemia virus reverse transcriptase  enzyme, as previously reported .BCR-ABL1 and ABL1 gene transcript levels from matched samples were quantified using real-time PCR (Q-PCR), at the Centre of Experimental Oncology and Hematology, as previously reported [BCR-ABL1/ABL1 determination was assessed according to the international scale (IS), and calculated by ratio of BCR-ABL1 and ABL1 transcript levels. This value was expressed as percentage on a log scale and using a conversion factor (CF) calculated every year, as previously described [ABL1 copies [BCR-ABL1 and ABL1 genes.The reported . The BCRescribed . The Q-P1 copies ,33,34. Qt-test was used to compare the difference between matched samples obtained with the five counting methods. Difference in number of white blood cells and Ct values (for BCR-ABL1 and ABL1 genes) were calculated and a p value below 0.05 was considered statistically significant. To evaluate the bias between the mean differences of the methods and to estimate an agreement interval within 95% interval, a Bland-Altman plot was used. For both t-test and Bland-Altman plot, Prism software v. 8.4 was employed.Cell count was performed using the CytoFLEX Flow Cytometer and the data analyzed employing CytExpert program . The 9/L (range 6.20–20.8) and the median of hemoglobin was 12.5 g/dL (range 10.8–14.5). Thirty-four patients showed an e13a2 (b2a2) BCR-ABL1 fusion transcript, 48 subjects showed an e14a2 (b3a2) BCR-ABL1 rearrangement and eight exhibited both e13a2 and e14a2 isoforms. According to ISBCR-ABL1 transcript levels, we selected patients with a molecular response distributed in three groups of 30 subjects each: Group A (10% > ISBCR-ABL1/ABL1 > 1%), Group B (1% ≥ ISBCR-ABL1/ABL1 > 0.1%) and Group C (0.1% ≥ ISBCR-ABL1/ABL1 > 0.01%).Patient characteristics are summarized in 6 (range 1.20 × 106–6.30 × 106) with the Manual protocol, 2.46 × 106 (range 1.20 × 106–6.27 × 106) with the Automatic method, 2.60 × 106 (range 1.26 × 106–6.06 × 106) when we measured the cells with Automatic and Beads measurement, 2.43 × 106 (range 1.22 × 106–6.16 × 106) when we used the fluorescent intercalator 7-AAD solution and, finally, 2.47 × 106 (range 1.20 × 106–6.04 × 106) when we combined Automatic count plus Beads and 7AAD solution  and A260/230 .Next, to evaluate the impact on the downstream analysis for molecular monitoring of CML patients, we compared the quantity and quality of RNA extracted from matched RLT samples provided by five different measurements. We observed that the median of RNA concentration was similar in all used methods  and then we compared Ct value obtained from matched specimens counted by the five different methods. By considering the BCR-ABL1 Q-PCR data, we found similar Ct values in all investigated groups. Specifically, we observed that in Group A the Ct value median was 29.30 (range 25.52–31.01) for samples counted by hemocytometer counting-chamber, 29.28 (range 25.66–31.10) for the automatic method, 29.37 (range 25.89–31.55) using counting beads, 29.24 (range 25.50–31.08) adding only the intercalator 7AAD and 29.34 (range 25.56–31.06) combining the counting beads with 7AAD solution . Overall, we found a 100% concordance. Next, we investigated the  methods A–C. FurtIn this case also, the Bland-Altman plot showed no consistent bias between samples counted by the manual vs the other four methods .BCR-ABL1 oncogene relative to an internal control gene , are the “gold standard” for monitoring the kinetics of disease burden variation. Moreover, molecular detection of BCR-ABL1 transcript by Q-PCR has the advantages of reliability, sensitivity and reproducibility of results.The molecular monitoring of CML patients has become of pivotal importance to predict treatment response and relapse ,27. QuanBCR-ABL1 molecular testing is generally recommended every 3 months as far as a stable deep molecular response is achieved. Currently, more frequent molecular testing is encouraged for CML individuals achieving persistent deep molecular response after TKI discontinuation (a condition defined as TFR) [BCR-ABL1 values should trigger a thorough evaluation regarding compliance or possible relapse [Upon TKI therapy initiation,  as TFR) ,35. More relapse . To achiNowadays, many cell counting assays enable the detection and enumeration of cells, either manually or via an automated process. In order to obtain reliable results, the entire cell counting process should be standardized, as variability of the measurements may introduce unintended error. Despite the easy procedure, the manual method is laborious can process a small number of samples at a time and is less suitable for large-scale studies or in diagnostic practice. Here, we present a comparison between a traditional counting method and our automated system based on the CytoFLEX Flow Cytometer integrated with the semiautomatic Biomek i-5 Workstation. Overall, the automatic counting system determined a hands-on time reduction allowing processing of a large number of samples concurrently (up to 20 specimens in only five minutes). Samples are loaded in a 96-well plate and then a peristaltic pump system permits recording of an unlimited number of patients, without any time delay or dilution influence. Furthermore, the opportunity to manipulate the live cell gate on the CytoFLEX software enabled a more reliable count.Different studies have recommended the addition of counting beads, at a known concentration, to the cells before measurement in order to calculate cell densities through flow cytometry ,38,39. HStaining with the intercalator 7AAD dye has the advantage of discriminating dead from live cell population, even with the limitation of more time for staining. Therefore, we also compared the absolute count to the measurement based on 7AAD solution. Again, no differences were observed among the counting assays, suggesting that the 7AAD intercalator does not provide additional information, either used alone or in combination with counting beads.BCR-ABL1/ABL1 transcript level may influence the molecular outcome for CML patients [ABL1 gene copies to pass minimum quality standards. Furthermore, the amount of the control gene is critical to define the kinetics of the disease burden reduction. In this context, we also compared the Ct values from matched counted samples obtained by Q-PCR assay on BCR-ABL1 and ABL1 genes and observed similar quantitative results as well as recovery efficiency. Likewise, matched samples were correctly classified into the same group of molecular response. These data underline the importance of taking into consideration the counting method used  and readout (BCR-ABL1 and ABL1 transcript levels) when comparing results for diagnostic practice.Several reports have shown that the quality and quantity of the patients ,24. PartIn conclusion, the automatic system based on absolute cell counting allows the obtaining of accurate cell counts without the use of expensive beads or the addition of the time-consuming intercalator 7AAD, and can streamline the entire procedure. Therefore, this measurement can replace the more laborious manual workflow, largely based on an interaction with the users, especially when high-throughput isolations from the blood of CML patients are needed.


Pinus sylvestris) softwood biomass (PWB). The optimization of three process parameters, viz., the incubation time, temperature, and biomass:PIL (BM:PIL) ratio, was performed to determine the best pretreatment conditions for lignin extraction. Under the optimal pretreatment conditions , [N11H(2OH)][LAC] yielded 61% delignification with a lignin recovery of 56%; the cellulose content of the recovered pulp was approximately 45%. Further, the biochemical composition of the recovered lignin and pulp was determined and the recovered lignin was characterized using 1H–13C heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectroscopy, quantitative 31P NMR, gel permeation chromatography (GPC), attenuated total reflectance (ATF)–Fourier transform infrared spectroscopy (ATR-FTIR), and thermal gravimetric analysis (TGA) analysis. Our results reveal that [N11H(2OH)][LAC] is significantly involved in the cleavage of predominant β–O–4’ linkages for the generation of aromatic monomers followed by the in situ depolymerization of PWB lignin. The simultaneous extraction and depolymerization of PWB lignin favors the utilization of recalcitrant pine biomass as feedstock for biorefinery schemes.Lignin-based chemicals and biomaterials will be feasible alternatives to their fossil-fuel-based counterparts once their breakdown into constituents is economically viable. The existing commercial market for lignin remains limited due to its complex heterogenous structure and lack of extraction/depolymerization techniques. Hence, in the present study, a novel low-cost ammonium-based protic ionic liquid (PIL), 2-hydroxyethyl ammonium lactate [N11H(2OH)][LAC], is used for the selective fractionation and improved extraction of lignin from Scots pine ( As a progressive society, the human race has thrived on the unrestrained usage of natural resources, especially fossil resources, to generate fuels and chemicals. However, the extensive exploitation of fossil fuels creates paramount pollution stress over our planet’s environment, which aids in global warming. Therefore, new strategies/techniques need to be designed to identify sustainable alternatives for the production of fuels and building blocks for chemicals from renewable carbon sources . Lignoceβ-O-4) which constitute <50%, followed by β–β and β–5 carbon linkages. The oxidative disruption of inter-unit linkages and the subsequent oxidation of hydroxyl groups results in lignin degradation to generate high-value, low-molecular-weight aromatic compounds [LAC]. The process parameters for the enhanced extraction of lignin are optimized. The biochemical composition of lignin and residual pulp was determined through a fiber analyzer. The surface morphology of the cellulose-rich pulp was characterized using SEM analysis. Further, the depolymerization properties of the extracted lignin were analyzed using GPC. The recovered lignin was characterized by 1H–13C HSQC NMR, quantitative 31P NMR, GPC, FTIR, and TGA analysis to determine the mechanism of fractionation from biomass.The economic sustainability of ionic-liquid-mediated pretreatment depends upon the optimization of the process parameters for the enhanced selective fractionation of biomass. Specifically, the higher biomass-loading ratio intensifies the pretreatment and decreases the capital expenditure of the process . Hence, Pinus sylvestris) softwood biomass was collected from southern Estonia (58°09′28.4″ N 26°44′27.1″ E). The moisture content of the biomass was determined using Kern MLS-50-3D moisture analyzer . Fiber content of the pine biomass was determined using Ankom 200 fiber analyzer , and composition was estimated following Hasanov et al. [LAC] was synthesized by an acid-base neutralization method .1H and 13C NMR spectra of [N11H(2OH)][LAC] was recorded on a Bruker 400 MHz Avance III NMR  spectrophotometer.Briefly, 226 mL of 2-dimethylamino-ethanol  was added dropwise to a solution of 159 mL racemic lactic acid  and incubated in a water bath for 2 h. After entirely adding the base, the reaction continued for 6 h under constant stirring—the excess of amine produced during the neutralization process was eliminated using a high vacuum. After drying under a vacuum for 16 h, the product was obtained in quantitative yield as a slightly yellow, viscous liquid. The 1H NMR : δ/ppm = 6.23 , 3.87 , 3.63 , 2.84 , 2.55 , 1.18 .13C[1H] NMR  δ /ppm = 178.55 , 66.73 (CHOH-CH3), 59.58 , 56.74 , 43.50 , 21.17 (CHOH-CH3).The collected pinewood was debarked, air-dried, ground to 1–2 mm, maintained at <10% moisture, and stored in an air-tight container. The fractionation and extraction of lignin from PWB using [N11H(2OH)][LAC] were adapted according to the methodology of Gschwend et al. . BrieflyThe delignification of the PWB by [N11H(2OH)][LAC] was determined by estimating the residual lignin in the biomass before and after pretreatment on a dry weight basis . Thus, tThe lignin recovery from [N11H(2OH)][LAC] pretreatment was determined by the amount of lignin retrieved from the precipitation relative to the initial lignin content in the raw PWB . The ligSEM images of the biomass pretreated with [N11H(2OH)][LAC] and the untreated pine wood biomass were obtained using SEM Helios NanoLab 650  at the acceleration voltage of 10 KeV, and the images were obtained at various magnifications.−1 interval range with a resolution of 4 cm−1.The surface functionalities of the lignin obtained from the [N11H(2OH)][LAC] pretreatment were measured using FTIR spectroscopy  with the universal attenuated total reflection (ATR) method. The spectra were recorded with an average accumulation of 16 scans in the 4000–600 cm2 gas with a flow rate of 100 mL/min at a heating rate of 10 °C /min [The thermal degradation properties of the [N11H(2OH)][LAC]-extracted lignin were determined using a NETZSCH STA 449 F3 Jupiter simultaneous (TGA and DSC/DTG) thermal analyzer . Approximately 5 mg of the extracted lignin was heated up to 900 °C under N °C /min .The molecular weight distribution of the [N11H(2OH)][LAC]-extracted lignin was analyzed using high-performance liquid chromatography (HPLC)  equipped with LabSolutions GPC software. The HPLC system contained a pump, an auto-sampler, a set of 2 MCX columns , and a pre-column (8 mm × 50 mm) (Polymer Standards Service (PSS), GmbH, Mainz, Germany) with a UV detector (280 nm). The lignin samples were dissolved in 0.1 M NaOH (5 mg/mL), and isocratic flow was maintained with 0.1 M NaOH solution at a flow rate of 0.5 mL/min with an injection volume of 20 µL. The relative molecular weight of the lignin was determined using polystyrene sulfonate sodium salt standards  ranging from 1100 Da to 100,000 Da.31P NMR and HSQC spectra. All of the NMR measurements  for the determination of the subunit abundance, interunit linkages, and phenolic OH content of the lignin were recorded on an Ascend Neo 500 MHz NMR spectrometer ) equipped with a TCI Prodigy cryo-probe head following the procedure of Wang et al. [The characterizations of the functional groups present in the [N11H(2OH)][LAC]-extracted PWB lignin were determined by g et al. .The effects of the operational process parameters viz., the incubation time (0.5–4 h), temperature (150–210 °C), and BM:PIL ratio  on PWB fractionation were investigated to determine the optimal operational conditions for maximal lignin extraction. The percentage of lignin extracted from the biomass, along with those of cellulose and hemicellulose, were determined by the compositional analysis of the recovered biomass obtained after the [N11H(2OH)][LAC] pretreatment, which was related to the biomass composition of untreated (UT) biomass .Initially, the effect of the incubation time on lignin removal was studied by varying the incubation time and keeping the other parameters, i.e., the temperature and BM:PIL ratio, constant . The ini4] required an optimal incubation period of 8 h [LAC] was visualized using SEM . The untThe visible deformities on the surface can be attributed to the removal of lignin and hemicellulose from PWB . FurtherThe ATR-FTIR analysis of the [N11H(2OH)][LAC]-extracted PWB lignin under optimal conditions was compared with the commercial Kraft lignin to determine the changes in surface functional properties. The ATR-FTIR analysis results of the [N11H(2OH)][LAC]-extracted lignin and Kraft alkali lignin are shown in −1 corresponds to the O=H stretching in the hydroxyl group of phenolic and aliphatic groups of lignin [LAC] pretreatment [LAC]-extracted PWB lignin displays improved surface properties due to the simultaneous extraction and functionalization of lignin by pretreatment. The characteristic band at 3439 cmf lignin . The peareatment . Furtherectively . The appectively . Furtheryl units .Thermogravimetric analysis is used to characterize the thermal properties, such as stability and pyrolytic decomposition, at various temperatures, and to identify the potential relationship between degradation and chemical structure. Thus, the thermal stability of [N11H(2OH)][LAC]-extracted PWB lignin was determined using thermogravimetry (TGA) and derivative thermogravimetry (DTG), as shown in 2, and CH4 [LAC]-extracted lignin was divided into three stages. In the first stage, the initial weight loss observed at 120.6 °C is due to the evaporation of absorbed moisture . The sec and CH4 . The las and CH4 . From DT(355 °C) . The rel(355 °C) ,34. Furt(355 °C) .The development of a high-value product from lignin is significantly influenced by the molecular weight and the polydispersity index (PDI) properties. Therefore, tuning these properties improves the application of lignin in various fields . Thus, t−1 and PDI of 3.28 from PWB compared to Kraft lignin (control). The decreased Mw and PDI of PWB obtained after pretreatment confirms the lignin depolymerization through breakage of interunit linkages [LAC]-extracted PWB lignin were characterized by combining the two-dimensional heteronuclear quantum coherence (HSQC) NMR and quantitative 13C NMR . The assc units) . Scheme 2,C5, and C6 represent G units at 110.9/6.91, 114.9/6.86, and 119.2/6.77, respectively, whereas the S units showed a prominent signal at S2,6 with a correlation at 104.1/6.63. The complete cleavage of the β–O–′ interunit linkage represents in situ depolymerization of PWB lignin during [N11H(2OH)][LAC] pretreatment [LAC] pretreatment. Further, the occurrence of stilbene moieties [(β–′5′)β and (β–′1′)α] due to the reverse aldol reaction of β–′5′ displays modification of interunit linkages of recovered PWB lignin [β–O–4′ motifs [2 cond signal [The aromatic region of the HSQC for [N11H(2OH)][LAC]-extracted PWB lignin after 3 h at 180 °C depicts the characteristic peaks for phenylpropanoids viz., guaiacyl (G) and syringyl (S) units. The correlations for Creatment . FurtherB lignin . The acc′ motifs . The pred signal . However31P NMR  in PWB [LAC]-extracted lignin, is determined by 31P NMR  38]. Af. Af31P Nd groups . The pri) in PWB . The com linkage . The relproducts ,41.1H–13C HSQC NMR and quantitative 31P NMR reveals the cleavage of β–O–4′ linkages and a significant increase in hydroxyl groups. Moreover, the in situ depolymerization of [N11H(2OH)][LAC]-treated lignin was confirmed by TGA and GPC analyses. This implies that [N11H(2OH)][LAC] pretreatment is able to convert the recalcitrant softwood biomass (lignin) into depolymerized aromatic units, which can be extended for other softwood biomasses for value-added chemical production.This study aimed at the synthesis of a novel PIL, 2-hydroxyethyl dimethyl ammonium lactate [N11H(2OH)][LAC], for the extraction of lignin from Scots pine biomass. Under optimized process conditions, [N11H(2OH)][LAC] recovered 56.13% of lignin from PWB. The compositional and SEM analyses of the recovered pulp showed selective removals of lignin. Further, the recovered lignin analyzed through


A total of 18 participants completed dietary intake assessments (4-day food diary via Easy Diet DiaryTM) and 14 participants completed an 83-question sports nutrition knowledge assessment (Sports Nutrition Knowledge Questionnaire (SNKQ)) before and after the 8-week program. Sports nutrition knowledge scores improved by 15%  following the program. Male participants reported higher energy intakes before  and after  the program compared to females. Carbohydrate intake at breakfast (p = 0.022), daily intakes of fruit (p = 0.033), dairy (p = 0.01) and calcium (p = 0.029) increased following nutrition education. Irrespective of gender, participants had higher intakes of energy (p < 0.001), carbohydrate (p = 0.001), protein (p = 0.007), and fat (p = 0.007) on heavy training days compared to lighter training days before and after the program with total nutrition knowledge scores negatively correlated with discretionary food intake . A remotely delivered nutrition education program by an accredited sports nutrition professional improved sports nutrition knowledge and subsequent dietary intake of junior elite triathletes, suggesting remote delivery of nutrition education may prove effective when social distancing requirements prevent face-to-face opportunities.Triathlon is a physically demanding sport, requiring athletes to make informed decisions regarding their daily food and fluid intake to align with daily training. With an increase in uptake for online learning, remotely delivered education programs offer an opportunity to improve nutritional knowledge and subsequent dietary intake in athletes. This single-arm observational study aimed to evaluate the effectiveness of a remotely delivered nutrition education program on sports nutrition knowledge and the dietary intake of junior elite triathletes ( Triathlon is a physically demanding sport, involving between 20–30 h of training per week across three disciplines, swimming, cycling, and running . Junior Although the importance of adequate nutrition knowledge for optimal dietary intake is recognized, the recommendations for post-exercise carbohydrate and protein intakes are not well understood among triathletes . Often, While sports nutrition education interventions typically improve nutrition knowledge and eating habits among athletes in team sports , the curn = 9; male n = 12; 18.9 ± 1.6 y) and provided written informed consent, including guardian consent for those aged under 18 years. Participants who failed to complete the dietary intake and nutrition knowledge assessments were excluded from final analysis. Permission to conduct the observational study was granted by the relevant ethics board at Bond University, Australia . Twenty-five triathletes attending the Triathlon Australia Performance Health Program (TAPHP) were approached by the primary researcher (GC) to participate in this study. The TAPHP was a remotely delivered 8-week program that included a medical review and subsequent follow-up, alongside weekly nutrition education sessions. Verbal and written communication of the scope and risks of the study were provided to participants and their guardians prior to their involvement. Twenty-one triathletes were recruited  a remotely delivered 5-week nutrition education series ; and (2) a 30-min individual dietary consultation (Weeks 6–8) delivered by an Accredited Sports Dietitian (MM). The weekly nutrition education topics included: training nutrition, energy availability, hydration, macronutrient  and micronutrient  requirements, supplements, food safety, and travel nutrition. Visual aids, online chat  and short quizzes  were used to engage athletes during sessions. Several practical activities were also used, including meal planning for heavy training days and snack/smoothie preparation. Relevant nutrition factsheets were developed and provided electronically to the athletes immediately after each session. Immediately before and after the program, participants completed an 83-question Sports Nutrition Knowledge Questionnaire (SNKQ) and a 4-day food diary .An online Sports Nutrition Knowledge Questionnaire (SNKQ) was administered to participants immediately before and after the 8-week nutrition education program via Qualtrics Experience Management  Survey Software . The SNKQ, which has been previously validated to assess the nutrition knowledge of athletes , was modParticipants received a score of +1 for a correct answer and 0 if the selected response was incorrect or left blank. The scores for each subsection and total score were calculated based on the number of questions answered correctly with a maximum total score of 83.Dietary intake assessments using a 4-day food diary (two weekdays and two weekend days) in the mobile application Easy Diet Diary™  were completed before and after the nutrition education program. Participants used household measures to estimate intake and were asked to photograph and record timing of meals and snacks to allow for greater accuracy in reporting. Participants were advised to record the timing, duration (mins), distance (km) and sports discipline  of all training sessions during the 4-day collection period. Dietary intake data was tracked in real-time via Easy Diet Diary Connect  and members of the research team (Accredited Sports Dietitians (MM and AB)) prompted participants individually throughout the collection period to minimize recorder error and ensure accurate dietary collection. Participants were sent daily reminders during the recording period and were supported with expert assistance (MM and AB) to ensure recorded food diaries reflected actual dietary intakes.Dietary intake data was entered into FoodWorks™  for final nutrient analysis and cross-checked for entry errors (NR and GC). Energy, macronutrient , and micronutrient breakdowns  were calculated for each participant before and after the nutrition education program. Dietary collection days were categorized as light (up to one single training session ≤ 1 h per day) and heavy (a single session > 1 h or multiple training sessions). Dietary intake was categorized according to meal timing . Dietary quality was assessed by comparing dietary intake to the Australian Guide to Healthy Eating (AGHE) [t-tests were used to determine mean difference between pre- and post-program nutrition knowledge scores and compare dietary intake on light vs. heavy training days, pre- vs. post-eating occasions and weekday vs. weekend intakes. Independent t-tests were employed to compare differences in pre- and post-data (nutrition knowledge and dietary intake) between females and males. One-way ANOVAs were used to assess differences in nutrition knowledge against various participant characteristics such as level of competition, and level of nutrition knowledge at baseline, and reported energy and macronutrient intakes at different meals . Pearson’s correlation  or Spearman’s correlation  was used to determine the relationship between nutrition knowledge and dietary intake. Significance was set at p ≤ 0.05. The effect sizes (Cohen’s D) for nutrition knowledge scores were calculated and were interpreted as small (0.2–0.4), medium (0.5–0.7), and large (≥0.8) [All statistical analyses were conducted using the Statistical Package for Social Sciences , where appropriate, results are reported as mean ± SD  or median and IQR . Categorical data are presented as percentage of total responses n (%). Shapiro–Wilk tests were used to determine normality of data and Wilcoxon Signed Ranks Tests performed for non-normally distributed variables. Paired sample e (≥0.8) . n = 18) completed the 4-day food diary and 14 participants (n = 14) completed the SNKQ before and after the program. The demographic characteristics of recruited participants (n = 18) are summarized in n = 17) did not follow a specific diet, while 1 reported following a paleolithic diet and 2 participants reported allergies to lactose and nuts. Participants reported coaches , sports dietitians , and parents  as the three most common sources of pre-existing nutrition information. Secondary sources of nutrition information were the internet , school or university , doctors or other medical staff , and social media .A total of 18 participants (n = 14) are outlined in p < 0.001; ES = 0.9) and subsection scores for hydration , training nutrition  and nutrients  following the nutrition program. No significant improvement was observed for the supplement subsection (p = 0.19). Females had a significantly higher SNKQ total score  and nutrient subsection score  at baseline compared to males. Following the program, there was no difference in total SNKQ score or rate of improvement between female and male participants (p = 0.397). No differences in pre- or post-program SNKQ total scores were found between the level of competition (p = 0.226) or the number of years competing (p = 0.255). Pre-program, no differences in SNKQ total score were observed between participants who had previously received sports dietitian education and those without prior education (p = 0.639).SNKQ scores of participants before and after the program (p = 0.043), carbohydrate , fat  and iron  intakes compared to females. Similarly, male participants reported higher daily energy  and fat  intakes compared to females following the education program. Calcium intake was significantly higher after the program among both males and females   . No othep < 0.001), carbohydrate , protein  and fat  intakes were higher on heavy compared to light training days both before and after the program. There was no difference in energy, carbohydrate, protein, or fat intake between weekdays and weekends. Energy , protein , and fat  intakes at dinner compared to lunch, before and after the program. Similarly, participants reported higher energy , protein  and fat  intakes at dinner compared to breakfast, before and after the program. There was a significant increase in carbohydrate intake at breakfast  following the program. Participants reported higher energy  of participants met their age-appropriate daily fruit intake recommendations, which increased to 72% (n = 13) after the program . Similarly, milk and alternatives increased from 55% (n = 10) of participants meeting recommended serves, to 61% (n = 11) after the program . There was no change in intake for other food groups following the program. Food group intake was assessed using the AGHE for healthy non-athletic populations, with an additional food group added (sports foods) . At baser = −0.695, p = 0.001). No significant associations were observed between intake from other food groups and sports nutrition knowledge. There was no correlation between nutrient intake  and SNKQ total score before or after the program.SNKQ total score was negatively correlated with discretionary food intake, irrespective of the program  sports nutrition knowledge and subsequent dietary intake of junior elite triathletes improved following the nutrition education program; (2) participants reported higher energy, carbohydrate, protein, and fat intakes on heavy training days compared to light training days, irrespective of the education program; and (3) athletes increased their carbohydrate intakes at breakfast following the program reflecting a favorable change in dietary behavior, which emphasizes appropriate fueling ahead of daily training. Despite the challenges the COVID-19 global pandemic has created for the delivery of effective and meaningful interactions between performance support staff and athletes, the findings from this study suggest that remotely delivered nutrition education programs provide an avenue to align dietary intakes of athletes with daily nutrient requirements. Junior elite triathletes who completed the 8-week nutrition education program in this study improved sports nutrition knowledge scores by 15%. Previous studies investigating the effectiveness of online education delivery approaches have reported similar improvements in athlete nutrition scores ranging from 6.4–25.2% ,17,18,19We devoted 10 of 83 questions within the sports nutrition knowledge questionnaire to hydration and included questions that assessed knowledge of beverage composition, implications of altered hydration status and appropriate fluid intake strategies for triathlon training specific scenarios. Ad libitum fluid intake during exercise is multifactorial  and likeIn this study, higher nutrition knowledge scores were associated with lower discretionary  food intake, which is consistent with previous research that demonstrated that nutrition knowledge scores were negatively correlated with the consumption of carbonated drinks and processed foods in athletes ,29. HeavA novel finding from the present study was that participants aligned their daily dietary intakes with fluctuations in daily training. Elite junior triathletes in this study, reported higher intakes of energy, carbohydrate, protein, and fat on heavy training days compared to light training days before and after the nutrition education program. Similarly, elite adolescent triathletes living in a full-board residential campus have previously been shown to increase relative (9.0 vs. 7.8 g.kg body mass) daily carbohydrate intake during an intensive training week compared to a moderate week . A noticDaily macronutrient intake remained unchanged following the 8-week nutrition education program, however there was an important change in the patterning of intake over the day. While the evening meal (dinner) was higher in energy, carbohydrate, protein, and fat compared to other meals and snacks consumed throughout the day, carbohydrate intake at breakfast increased following the nutrition education program. This change in dietary patterning is important as altering the timing of carbohydrate intake around exercise can optimize the response to daily training . Furthern = 18); however, this study does include almost all elite junior triathletes categorized by Triathlon Australia (18 of 25 categorized athletes). The aim of this study was to determine the effectiveness of a tailored, remotely delivered education program in emerging triathletes, which future studies can build upon amongst other triathlon cohorts  who have been previously shown to lack knowledge of sports nutrition guidelines [One of the perceived limitations of the current study is the small sample size (idelines . As prevIn conclusion, results from this study suggest that a remotely delivered 8-week nutrition education program by an Accredited Sports Dietitian can improve the nutrition knowledge and dietary intakes of junior elite triathletes. Specifically, athletes improved their nutrition knowledge by 15%, which resulted in positive dietary changes that better aligned dietary intake to daily training. The nutrition education program used in this study provides a template for future education programs when social distancing requirements prevent face-to-face opportunities. Future research is warranted to explore long-term changes in sports nutrition knowledge and subsequent dietary behaviors in response to novel education programs that accommodate social distancing requirements, such as online modules or facilitated education sessions, mobile phone applications or social media-based interventions.