The state of Washington legalized cannabis for adult use in 2012 and retail stores began to open in 2014. While details of the legal market have been tracked by the state, the total market for cannabis and characteristics of purchasers can only be identified through surveys.Six cross-sectional samples of the Privatization of Spirits in Washington (PSW) surveys were collected between January 2014 and October 2016 with two in each year. Random digit dial procedures were used to recruit a sample aged 18 and older, with 40% of cases from mobile phones. A total of 5492 respondents participated. Analyses of the population-weighted sample utilized purchasing amounts and frequencies, use frequency and related measures to estimate total and mean amounts and expenditures. Sensitivity analyses were conducted for key assumptions.The market for cannabis flower is estimated to have increased from 158 metric tons and $1.23 billion in 2014 to 222 metric tons and $1.7 billion in 2016, with little change from 2014 to 2015. Purchases from legal sources, retail and dispensaries, were estimated at 69% of the total market. Daily or near daily (DND) users accounted for about 83% of sales in 2014, rising to 91% in 2016. The prevalence of past year use rose substantially from 25% in 2014 to 32% in 2016, with DND use rising from 10.2 to 11.3%. Average purchase amounts for DND users rose from 291 g in 2014 to 374 g in 2016, while mean amounts among non-DND users declined from 78 to 28.6 g.The expansion of retail cannabis stores in Washington appears to have led to increased market size in 2016 with all of the increase attributed to DND users. Frequent users may be important to consider in legalization evaluations. With cannabis for adult use becoming legal in an increasing number of states, issues related to the transition from illicit to legal cannabis markets are a significant research priority. Key questions regarding the impacts of cannabis legalization include the degree to which purchasing will shift from the illicit market and the how the market will grow and change. Estimating the size of illicit markets is difficult, but general population surveys offer what is possibly the best opportunity for this in states where cannabis use is legal and where cannabis can be purchased in retail stores. Prior studies have utilized frequency of use data from the National Surveys on Drug Use and Health (NSDUH) series along with data from other sources to estimate use amounts and expenditures , September–October 2014 , March–May 2015 , August–October 2015 , March–April 2016 , and September–December 2016 . At each wave, respondents were selected using a state random probability sample obtained via random digit dial (RDD) of both landline and cell phone samples with about 40% cell phones. Respondent self-identified as adult Washington residents. The AAPOR2 cooperation rates (The American Association for Public OpinionResearch The series of Privatization of Spirits in Washington (PSW) Surveys, conducted between January 2014 and December 2016 by ICF International, was designed to evaluate impacts over time of the privatization of spirits sales and the legalization of marijuana in Washington state. The data analyzed consist of six cross-sectional representative surveys of adults (aged 18 and over), with sample recruitment taking place separately in January–April 2014  marijuana user was determined using the question: “How often have you used marijuana, hash or pot during the last twelve months,” with selection options including “Every day or nearly every day,” “About once a week,” “Once every 2 or 3 weeks,” “Once every month or two,” “less often than that,” and “Never last year.” Respondents were coded as dichotomous PY marijuana users or not. PY use was further classified into Daily or Nearly Daily (DND) users (those who answered “every day or nearly every day”) and non-DND users. Mode of marijuana use was elicited by the question “how do you most commonly consume marijuana” and categorized as smoke, inhalable , edible (food product or beverage), and other product .number of days purchased PY. Marijuana flower amount (in grams) usually purchased was based on the question: “what amount of raw Marijuana do you usually buy”, with selection options ranging from “0.5 g (Nickel Bag)” to “an ounce (28 g),” and including “Other: Specify” where respondents provide the specific amount. To elicit expenditure, purchasers were then asked “about how much this amount cost,” and provided the dollar amount for the usual marijuana flower purchase. Average $ per gram of marijuana flower was derived dividing usual expenditure on usual flower purchase quantity. Cleaning of expenditure data involved editing noticeable typos and adjusting for potential outliers by truncating average $ per gram at the 5th and 95th percentiles. Across six waves there were 1177 PY marijuana users, and 769 reported purchasing at least once PY. Among them, 59 reported “Never” for usual purchase quantity without valid usual expenditure. They were excluded from the final definition of flower purchaser (n = 710). Total quantity (in grams) of marijuana flower purchased PY was derived multiplying the usual grams of marijuana flower purchased by the number of days marijuana was purchased, assuming each trip involved a marijuana flower purchase. In a sensitivity analysis, we adjusted for potential overestimation by accounting for trips purchasing other marijuana products (described below). Fifty-two respondents reported valid usual expenditure but were missing usual purchase quantity. Their usual quantity was imputed by dividing usual expenditure by the sample median $ per gram. For those who reported usual quantity but were missing on expenditure (n = 45 across six waves), their usual expenditure was imputed by multiplying usual quantity by sample median $ per gram as well. Finally, 16 flower purchasers missing both purchase usual quantity and expenditure were assigned the sample median on both measures. Total $ expenditure on marijuana flower PY was then calculated multiplying the usual expenditure on flower by the number of days marijuana was purchased.All PY marijuana users were asked “how often do you usually purchase marijuana” with categorical responses recoded as the PY purchaser of other marijuana products or not and number of days other marijuana products were purchased. Each purchaser was then asked to provide the amount usually bought and how much that amount costs for up to three products. Across six waves there were 391 PY other marijuana product purchasers of whom 240 reported usual expenditure for one product, 64 for two products, and 38 for three products. For those purchasing more than one product, we estimated the usual expenditure assuming that half of the time the products were purchased separately and the other half of the time the products were purchased together. 49 respondents who were other marijuana product purchasers but were missing on usual expenditure were assigned the sample median. Sensitivity analyses also considered the average  and the summation  of costs across products. PY total expenditure on other marijuana products was calculated from the usual expenditure and number of purchase days.All PY marijuana users were asked “how often do you usually purchase marijuana-related products such as hash, oil, edibles, teas or lotions” and coded as store sales  and from the illegal market  in 2016.From Wave 2 (no stores had opened at Wave 1), each marijuana user was asked “Since July 2014, have you purchased marijuana, or other marijuana products from a legal retail store in Washington.” For the Wave 5 and 6 surveys in 2016, three items were added for marijuana users: “Within last 12 months, what proportion of your marijuana purchases were from…”: “a legal store in Washington,” “a medical dispensary in Washington,” and “other sources” with options of “none,” “less than half,” “about half,” “most,” or “all.” These responses were converted to proportions , multiplied by self-reported purchasing quantity and expenditure on marijuana flower to estimate the flower market size from The two cross-sectional waves in each year were combined to generate data for years 2014, 2015, and 2016 separately. Merging each two waves adjusts for potential seasonal fluctuations in marijuana use and purchase behavior, and enhances stability with larger sample size. Sample proportions and means for population prevalence and average estimates are presented. To test trend effects, regressions with survey year as a continuous predictor were estimated. Marijuana market size estimates aggregated individual quantities applying the population weights. All analyses were performed with STATA version 15 survey commands  or individual number of phones (cell sample) Second, the landline and cell sample were combined to reflect the population coverage of landline and cell sample frames. The respondents were weighted to National Health Interview Survey state benchmarks based on their landline/cell usage status. Last, the weighted data were calibrated to reflect population distributions from the American Community Survey, using a raking adjustment for the following dimensions: sex by age, age by race/ethnicity, and education levels. For the combined cross-section data for 2014, 2015, and 2016 separately, the final weighted sample represents all adults (18 and older) residing in the State of Washington in the given year.Several sensitivity analyses were performed. First, to adjust for potential overestimation of the marijuana flower market size, since purchasing frequency of marijuana flower was not specifically asked, the number of days marijuana flower was purchased was re-derived for PY marijuana users whose most commonly used product was not a smoked product . Their flower purchasing frequency was re-estimated by taking the larger value of (1) the difference between the number of days marijuana was purchased and the number of days other marijuana products were purchased or (2) half of the days marijuana was purchased. Furthermore, we re-estimated the market size for other marijuana products, re-calculating the usual expenditure as the average and summation when the respondents reported more than one product was usually purchased.Table Table Table Finally, using the 2016 data only, PY total expenditure on marijuana flower based on store sales  was estimated at $1.24 billion, representing 73% of the $1.70 billion total expenditure from the 2016 PSW surveys ($1.14 billion by DND users and $0.11 billion by non-DND users). Likewise, the total market size for marijuana flower quantity from store sales was estimated at 153 MT, 69% of the 222 MT total quantity based on the 2016 survey . The remaining 27% of expenditures and 31% of quantity purchased from “other sources” was the estimated market share of the illegal market.These 2014–2016 market size estimates and analyses of purchasing behaviors for the state of Washington (age 18 +) build on the small number of population studies addressing marijuana buying and use in a legalized market. This study is the first to provide estimates directly from a representative sample of a state during the early years of retail store openings. Surveys are an important tool for estimating and tracking legal and illegal marijuana use and appear to be more accurate after legalization  users bought 91% of the marijuana flower in 2016 and spent 89% of the dollars spent on other marijuana products. The high proportion of cannabis sold to DND users also suggests that they are the main providers of cannabis to the 25% of users who did not purchase in the past year. Prior studies have also highlighted the importance of DND users: A US study (Davenport and Caulkins There are a number of limitations to this study including the use of self-reported purchasing behavior, which may be mis-remembered or under-reported due to social desirability bias and concerns about reporting illegal activities for illicit market purchases (Kerr et al. Surveys of purchasing behaviors offer a unique and important perspective on marijuana markets in states with legal use enabling tracking of the shift from illicit to legal markets and the expansion of purchasing among users. Surveys are also needed to understand purchaser characteristics and the concentration of activities among certain types of users/purchasers. There is a need for more detailed monitoring surveys such as ours to track behaviors in states adopting retail marijuana markets. These should include more details on products and purchasing patterns than was possible in our PSW surveys, which were designed primarily to capture alcohol use and purchasing. Other marijuana products, particularly concentrates, vape pens, and edibles, have become more popular in recent years increasing the importance of more detailed assessment and tracking (Schauer et al. This study utilized the 2014–2016 PSW surveys to estimate the total market size for cannabis flower and other cannabis products in each year with individually-reported data from within the surveys only, with results similar to those from prior estimates that used multiple sources. The cannabis market was shown to increase over time, particularly in 2016 when the full complement of retail stores were open with an estimated 70% of cannabis purchased legally in that year. Detailed analyses of cannabis purchasing behaviors highlight the importance of DND users who accounted for most of the purchases in each year. These results emphasize the importance of focusing prevention and intervention efforts on 10–11% of the population who are DND users who not only account for the majority of cannabis use but also likely provide most of the cannabis to the 25% of past-year users who did not purchase any themselves.


Organ‐saving treatment for early‐stage rectal cancer can reduce patient‐reported side effects compared to standard total mesorectal excision (TME) and preserve quality of life. An optimal strategy for achieving organ preservation and longer‐term oncological outcomes are unknown; thus there is a need for high quality trials.Save the rectum by watchful waiting or TransAnal surgery following (chemo)Radiotherapy versus Total mesorectal excision for early REctal Cancer (STAR‐TREC) is an international three‐arm multicentre, partially randomized controlled trial incorporating an external pilot. In phase III, patients with cT1‐3b N0 tumours, ≤40 mm in diameter, who prefer organ preservation are randomized 1:1 between mesorectal long‐course chemoradiation versus mesorectal short‐course radiotherapy, with selective transanal microsurgery. Patients preferring radical surgery receive TME. STAR‐TREC aims to recruit 380 patients to organ preservation and 120 to TME surgery. The primary outcome is the rate of organ preservation at 30 months. Secondary clinician‐reported outcomes include acute treatment‐related toxicity, rate of non‐operative management, non‐regrowth pelvic tumour control at 36 months, non‐regrowth disease‐free survival at 36 months and overall survival at 60 months, and patient‐reported toxicity, health‐related quality of life at baseline, 12 and 24 months. Exploratory biomarker research uses circulating tumour DNA to predict response and relapse.Can we STAR‐TREC will prospectively evaluate contrasting therapeutic strategies and implement new measures including a smaller mesorectal target volume, two‐step response assessment and non‐operative management for complete response. The trial will yield important information to guide routine management of patients with early‐stage rectal cancer. Bowel cancer is the third most common tumour with over 42 000 new UK cases annually, 447 000 across Europe and 1.93 million worldwide, of which one‐third are located in the rectum [Surgery alone to remove the rectum, adhering to the principles of TME, is the current standard of care for treatment of early‐stage rectal cancer [The feasibility of an organ‐sparing approach for patients with locally advanced rectal cancer has been demonstrated by non‐operative management or ‘watch and wait’, following complete clinical response (cCR) to preoperative chemoradiotherapy (CRT) [While CRT is commonly used for organ preservation strategies, short‐course radiotherapy (SCRT) may also be considered. The efficacy of preoperative SCRT compared with CRT for prevention of local relapse is supported by two phase III trials in patients with radically excised rectal cancer, where SCRT showed benefits in terms of reduced toxicity [TREC [This is an international multicentre, rolling phase II/III trial. Phase II incorporated three‐way randomization (1:1:1) between (i) TME surgery, (ii) organ preservation via mesorectal SCRT or (iii) organ preservation via mesorectal CRT [An external phase II study was conducted over 2 years, following a 6‐month initial setup, to assess the feasibility of a large, multicentre randomized trial comparing radical surgery versus organ‐saving treatment and selective transanal microsurgery [This is an international hospital‐based study in the UK, Netherlands and Denmark (and planned to open in Belgium and Sweden). Potential candidates are identified in the endoscopy suite following (i) investigation of new bowel symptoms, (ii) personal bowel surveillance or (iii) bowel screening and are referred to a colorectal surgeon or the colorectal cancer multidisciplinary team (MDT), where eligibility is confirmed following review of clinical data Table.All patients must have biopsy proven adenocarcinoma of the rectum, staged as ≤mrT3b, that is, ≤5 mm of mesorectal invasion, Eastern Cooperative Oncology Group (ECOG) performance status 0–1, and the MDT must consider that both TME surgery and organ‐saving therapy are both reasonable and feasible treatments. Major exclusions include primary tumours >40 mm in maximum diameter , mr ≥ N1, and mesorectal fascia threatened by tumour (≤1 mm on MRI). A comprehensive eligibility checklist is provided in Table STAR‐TREC is a hospital‐based study. Candidates will generally be identified in the endoscopy suite following referral (i) for the investigation of new bowel symptoms, (ii) as part of a personal bowel surveillance programme or (iii) through national bowel screening. Subjects will then be referred to either a colorectal surgeon or the colorectal cancer MDT meeting. Recruiting trial sites are likely to act as ‘regional hubs’ for the early rectal cancer service. They will specialize in organ‐saving techniques while being able to provide standard  treatment. Following informed consent, which will be conducted by medically qualified individuals in accordance with good clinical practice standards, confirmation of eligibility and trial entry will be conducted using electronic remote data capture.Standard primary TME surgery may encompass both reconstructive and non‐reconstructive approaches to rectal resection adhering to the principles of TME surgery. The former includes low anterior resection, the latter abdominoperineal excision or low Hartmann's procedure. Surgeons may employ either minimally invasive, open or hybrid approaches to TME surgery. The quality of surgery will be measured using a standardized histopathological assessment that grades whether surgery was performed according to the principles of TME.Local transanal excision is performed either by TEM or an equivalent single‐port transanal technique, to remove the tumour site and underlying muscularis propria en bloc, aiming for a 10 mm margin of normal mucosal tissue. A thin layer of mesorectal fat is also removed; however, the mesorectum is not extensively dissected. The specimen is pinned out in the operating room prior to fixation to facilitate accurate evaluation of margin status. It is recommended that the defect is sutured closed to promote healing.Patients who undergo planned conversion from transanal excision to secondary TME, based upon the presence of designated high‐risk histopathological features in the local excision specimen, are expected to have surgery 8–16 weeks after TEM, allowing time for post‐surgical inflammation to settle.2 twice per day on radiotherapy days only. The protocol includes general guidelines for management of common toxicities related to capecitabine and dose reduction rules based on the relevant product information. SCRT consists of a total dose of 25 Gy in five fractions of 5 Gy, preferably on five consecutive days. Radiotherapy for organ preservation in this group of early cancers is primarily aimed at tumour downstaging and can therefore be restricted to the peritumoural area including the primary tumour and the mesorectum. The treated mesorectal volume now corresponds to the volume that would be resected in a TME resection. Elective irradiation of lateral or presacral lymph nodes cranial to or above the mesorectal volume is thus not indicated. This results in a significant reduction of the irradiated target volume and is expected to result in less toxicity [Long‐course CRT consists of a total dose of 50 Gy in 25 fractions, combined with capecitabine administered at a dose of 825 mg/mThe primary outcome is the proportion of patients with successful organ preservation at 30 months from the start date of CRT. Organ preservation is considered to have failed if (i) the rectum is removed, (ii) the patient develops unequivocal locoregional cancer recurrence or (iii) the patient has a stoma. Analysis of the phase III outcomes will include all the relevant patients recruited during both the phase II and phase III components of the trial (Table Major patient‐ and clinician‐reported secondary outcome measures are presented in Table In the modified phase III design patients can select either (a) TME surgery or (b) organ preservation. Those who prefer organ preservation are randomized 1:1) between (i) organ preservation via mesorectal SCRT or (ii) organ preservation via mesorectal CRT  is not an appropriate criterion because important differences may not be statistically significant [In a comparison of two active treatments, the goal of the analysis is to identify whether either is superior. As the difference between two active treatments may be relatively small, conventional statistical significance  associated with the persistence of tumour tissue to direct or support decisions to escalate medical or surgical therapy. Six sets of blood samples are collected from organ preservation patients explicitly consenting to this optional substudy at baseline, second response assessment, completion of treatment (6 months) and during follow‐up  using vials shipped without processing. Retrospective sensitivity and specificity analysis will evaluate the utility of cftDNA measurement for initial response assessment, that is, partial versus complete response, and also for patient follow‐up, to facilitate early identification of cancer regrowth or relapse. Primary tumour tissue taken before and after radiotherapy will also be collected.Randomization will be achieved by a remote central web‐based service based at the Cancer Research (UK) Clinical Trials Unit (CRCTU), Birmingham. Randomization will use a minimization procedure with the following variables: mriT stage (≤T3a and T3b) and country of recruitment. STAR‐TREC is an open‐label study and neither patients nor site teams are masked to treatment allocation. To avoid any possibility of the treatment allocation becoming too predictable, a random factor was included within the algorithm whereby for a proportion of the allocations true randomization is implemented.Data collection is carried out using patient notes, case report forms and validated quality of life questionnaires . From June 2021 all data except patient questionnaires are entered onto an electronic case report form (by a member of the site staff). Regular data quality checks will be performed as per the Quality Management Plan. All data are handled in accordance with the Data Protection Act and General Data Protection Regulation. Data backups are stored in secure fireproof locations and test restorations are performed on a regular basis. After completion of the trial all essential trial documentation and source documents  are securely retained for at least 25 years.Full details regarding the statistical methods are given in the trials statistical analysis plan (SAP) version 1.0 dated 7 October 2021, authored by V. Homer, S. Gates, S. Bach and L. Navarro‐Nuñez, available on request. An overview is given here. The analytical strategy aims to evaluate whether (a) either organ‐saving strategy is superior in terms of achieving organ preservation, requirement for further surgery, treatment‐related toxicity, HRQoL and other outcomes; (b) either or both organ‐saving strategies lead to an acceptable rate of organ preservation of at least 50%. The primary analyses in STAR‐TREC phase III will be conducted according to the intention to treat principle, where participants are analysed in the treatment group to which they were randomized, regardless of the treatment received.P < 0.05) would be inappropriate, the analysis can provide information that can inform clinical decisions directly about which treatment should be preferred based upon rates of non‐operative management, toxicity profiles and HRQoL.A key advantage of using a Bayesian statistical approach is that it quantifies the probability of benefit, or the probability of benefit of at least a given size, for each treatment arm. Hence, even if differences were relatively small, and conventional frequentist analysis with statistical significance . Other dichotomized outcomes will be analysed similarly. HRQoL (and continuous outcomes) will be analysed using hierarchical repeated measures models to model each patient's trajectory through time, to account for the multiple data points per patient. Analysis of toxicity will evaluate the rate of major treatment‐related complications in each group.Secondary analyses will incorporate the partially randomized standard surgery comparator. This partially randomized TME surgery reference group will provide data relating to the incidence of major treatment‐related toxicity and HRQoL following TME surgery for early‐stage rectal cancer, without the use of radiotherapy. Although the non‐randomized analysis means that conclusions must be treated with extra caution, this analysis will provide valuable additional information for patients with early‐stage rectal cancer who are balancing the risks and benefits of different treatment strategies.In addition to the aforementioned, we will analyse the overall outcome for each patient using a patient‐centred ranked composite outcome analysis which will classify each patient's overall outcome, based on mortality, organ preservation, treatment‐related toxicity, need for surgery and quality of life, into an ordinal scale. The ranking of outcome categories will be determined by consensus among the investigators during the conduct of the trial and will be reviewed  by a sample of clinicians and patients. The ordinal overall outcome measure will be used to compare the randomized organ preservation groups, using ordinal regression models. The main advantages of this approach are that it is more relevant to patients because it considers patients' overall outcome, and it enables all patients’ outcomes to contribute to the analysis [The University of Birmingham is the coordinating sponsor for this international study and is also the National Coordinating Centre (NCC) for the UK. The coordinating sponsor has in force a Public Liability Policy and Clinical Trials Policy which provide cover for claims for negligent harm arising from the design or management of the research. Each country will appoint a National Coordinating Investigator and an NCC who will take national responsibility for the study and manage the trial in accordance with the trial protocol, and their standard policies and procedures. Central and site monitoring activities to ensure compliance with the protocol and applicable regulations will be conducted using a risk‐based approach as documented in the trial risk assessment and quality management plan. The trial management group is composed of representatives from each NCC and the trial team at CRCTU Table . They arThe medical ethics committees of each participating country have approved the study protocol. The UK Research Ethics Committee (REC) approval reference is 16/EM/0186. It is the responsibility of the national coordinating centre to obtain country‐specific approval and the responsibility of the Principal Investigator to obtain local approval.The feasibility of organ preservation using CRT has been proven, both in the setting of locally advanced disease via the international watch and wait registry [To date several relatively small studies evaluating primary organ preservation for early‐stage rectal cancer have reported mixed results. Importantly, there is no evidence that primary organ preservation is oncologically unsafe [Results of the TREC study support further evaluation of SCRT, as an alternative to traditional downstaging CRT, for primary organ preservation in patients with early‐stage rectal cancer in STAR‐TREC. STAR‐TREC also introduces new measures for all patients to further reduce the impact of organ‐preserving treatment, such as smaller mesorectal (only) radiotherapy fields specifically risk‐adapted for early tumours. Moreover, standardized response assessment up to 20 weeks is performed to improve identification of cCR and consequently increase non‐operative management. Lastly, an exploratory biomarker‐driven approach is introduced to determine the feasibility of initiating surgical intervention based upon the presence of cftDNA, thereby avoiding overtreatment of cases where no residual tumour exists. Optimization of the organ preservation rate for each treatment strategy is a key area for improvement as failure of organ preservation most frequently follows conversion to TME based upon the perceived future risk of local relapse in patients with no objective evidence of residual tumour, while salvage TME for local failure is comparatively rare [In summary, STAR‐TREC is an international study to evaluate the effectiveness of two contrasting CRT schedules, optimized for treatment of early‐stage rectal cancer to achieve organ preservation (at 30 months). Key secondary end‐points are the rate of non‐operative management, patient‐reported treatment‐related toxicity, HRQoL and non‐regrowth pelvic tumour control to 36 months. STAR‐TREC also incorporates new exploratory biomarker work to develop objective evidence for escalation of surgical therapy. The trial will yield important information to guide routine management of patients with early‐stage rectal cancer.STAR‐TREC phase III was implemented via protocol version 4.0, 10 October 2019 (copy available upon request).Protocol amendments which may impact the conduct of the study, potential benefit of the patient or may affect patient safety, including changes of study objectives, study design, patient population, sample sizes, study procedures or significant administrative aspects are discussed and agreed by the International Trial Management Group (TMG) composed of the Chief Investigator, NCC representatives (multidisciplinary), patient representative(s) and the CRCTU team , Trial Coordinator, Clinical Trials Monitor). Where required, the independent TSC members are also asked to comment and approve suggested protocol amendments. All substantial amendments are sent to and reviewed by the relevant REC and/or competent authority and trial funders for approval as required; amendments are distributed to sites and implemented following receipt of approvals. Trial registries are updated periodically to ensure that trial information is up to date.The University of Birmingham is the Data Controller for this trial. Personal data recorded on all documents will be regarded as strictly confidential and will be handled and stored in accordance with the General Data Protection Regulation (EU) 2016/679 and the UK Data Protection Act 2018 (GDPR).Requests for access to trial data and translational biobank specimens will be considered on a case‐by‐case basis and approved in writing where appropriate, after formal application to the TMG and TSC. Considerations for approving access are documented in the TMG and TSC terms of reference. Following conclusion of the trial, anonymized data are likely to be added to an open access repository.Patients will revert to local standards of care.The results of this trial will be submitted for publication in peer‐reviewed journals and presentation at relevant open meetings. Closed meetings will be held after the end of each phase of the trial to allow discussion of the main results among the collaborators prior to publication. Publications will conform to the International Committee of Medical Journal Editors guidelines. When manuscripts are submitted, the corresponding author will specify the name of the STAR‐TREC group, and clearly identify the group members who can take credit and responsibility for the work as authors. The results of the trial will also be published on a clinical trials registry, and a lay summary made available on the Cancer Research UK website and via press outlets.SPB, DSM, HdW, KLG‐S, CM, FP, AG, MT, AA, NW, VH, SG, LN‐N, IT each made substantial contributions to the design of this work, drafting and revising of the manuscript, and approval of the final published version; and agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy and integrity of any part of the work are appropriately investigated and resolved. NK contributed to drafting and revising the manuscript.The authors have no relevant conflicts of interest or financial ties to disclose.No conflicts.Appendix S1Click here for additional data file.Appendix S2Click here for additional data file.Appendix S3Click here for additional data file.Appendix S4Click here for additional data file.


At the continental US scale, only 5% of C fixed through photosynthesis remains as net ecosystem exchange (NEE), but ecosystem measurements indicate that only 2% of fixed C remains in grasslands, whereas as much as 30% remains in needleleaf forests. The wet and warm Southeast has the highest gross primary productivity and the relatively wet and cool Midwest has the highest NEE, indicating important spatial mismatches. Newly available satellite and atmospheric data can be combined in innovative ways to identify potential C loss pathways to reconcile these spatial mismatches. Independent datasets compiled from terrestrial and aquatic environments can now be combined to advance C‐cycle science across the land–water interface.Understanding carbon (C) dynamics from ecosystem to global scales remains a challenge. Although expansion of global carbon dioxide (CO Both of these fluxes are an order of magnitude greater than fossil‐fuel emissions  of all organic matter, CO2 represents only a very small fraction of the atmosphere and is therefore measured in ppm (~415 ppm in 2020). Given the abundance of C in the terrestrial biosphere and the massive fluxes of C occurring between the biosphere and the atmosphere, it is no surprise that scientists have developed a myriad of innovative ways for measuring and simulating C‐cycle processes across a range of scales in time and space. For example, chloroplast CO2 fluxes are estimated over millimeters per second, whereas biome CO2 fluxes may be estimated over thousands of kilometers per year. There have been many advances in C‐cycle science over the past 60 years at leaf, plant, ecosystem, and global scales, but both challenges to and opportunities for scientific advancement remain. Progress is necessary, however, especially at the macrosystem scale, where human management and ecological processes are often at odds and create interesting interactions of C dynamics.Carbon (C) is the building block of life. Global photosyn‐thesis generates approximately 100 terawatts (TW) of energy each year by converting solar radiation into stored chemical energy Barber . Photosy2 concentrations . In par2 emitted to the atmosphere from fossil‐fuel and land‐use emissions has been taken up by the terrestrial biosphere  and CH4 (ie XCH4). Although potentially less precise than those relying on surface measurements using infrared gas analyzers, these estimates provide more continuous global coverage, improving characterization of regional flux anomalies and attribution to specific C‐cycle processes  and total ecosystem respiration (TER), and at the ecosystem scale is typically measured using eddy covariance techniques , volatile organic compounds (FVOC), methane (FCH4), dissolved inorganic C (FDIC), dissolved organic C (FDOC), and particulate C (FPC) all represent C loss pathways that may affect the net C balance over time. Although NEE is sometimes used synonymously with NECB, it is an approximation that can, under certain circumstances, leave out quantitatively important non‐respiratory processes that contribute to ecosystem C balance.In this formulation, net ecosystem exchange (NEE) is a measure of the net ecosystem COet al.  concept was first introduced by Schulze et al.  to accou  may provide a useful framework  directly, whereas photosynthetic fluxes and total ecosystem respiration fluxes are estimated, resulting in the potential for systematic biases to occur in these measurements. Eddy covariance methods are inherently challenging in ecosystems with dense canopies , meaning that other loss pathways leading to reduced C (eg CH4 emissions) will not help reconcile these discrepancies of scale.The discrepancy between CEE at the biome scale and the ecosystem scale can also be explained by the lack of measurements of non‐respiratory loss pathways of CO2 to the atmosphere, while eddy flux measurements from central Oregon suggest high CEE.We can also look at the spatial distribution of CEE from the biome to ecosystem scale Figure . At the 2 exchange do not necessarily covary spatially and appear to occupy different climate spaces at regional scales (Figure et al. et al. 2 exchange. In fact, it is impossible in standard eddy covariance approaches for partitioning fluxes to have increases in net exchange without increases in photosynthesis (Reichstein et al. et al. At the continental scale in the US, mean annual primary productivity and net CO2 growth rate and tropical land‐surface temperatures has been identified as an emergent constraint, such that higher surface temperatures diminish NEE (Cox et al. et al. 2 exchange at the local scale, whereas temperature becomes more important at global scales (Jung et al. et al. 2 and solar induced fluorescence during the recent 2015/2016 El Niño event suggest that net tropical C uptake was reduced by different processes in different tropical regions – such as reduced photosynthesis in South America, increased respiration in Africa, and increased fire emissions in Southeast Asia (Liu et al. At the global scale, the relationship between the interannual variability of the atmospheric CO2 observations that have enabled a better understanding of the C cycle at the global scale and a network of eddy covariance measurements of CO2 exchange at the ecosystem scale. However, reconciling differences in net CO2 exchange measured at these different scales continues to be difficult. We are now acquiring data from aircraft and satellites that allow important C‐cycle processes to be resolved at the biome scale. The terrestrial and aquatic ecological research communities are also compiling databases to elucidate important C‐cycle processes that may be merged to provide an integrated understanding of C transport and transformations across watersheds. Collectively, we are identifying missing pieces of the global C puzzle that now make it possible to reconcile and understand processes that help to explain discrepancies in C dynamics across scales.A central goal of ecology at the macrosystem scale is to understand biosphere processes and their complex interactions with climate, land use, and changes in species distribution at regional to continental scales This has also been a central challenge of C‐cycle research because there is a long history of atmospheric COFig S1Click here for additional data file.Panel S1Click here for additional data file.Table S1Click here for additional data file.


In eukaryotic cells, enzymes are compartmentalized into specific organelles so that different reactions and processes can be performed efficiently and with a high degree of control. In this work, we show that these features can be artificially emulated in robust synthetic organelles constructed using an enzyme co‐compartmentalization strategy. We describe an in situ encapsulation approach that allows enzymes to be loaded into silica nanoreactors in well‐defined compositions. The nanoreactors can be combined into integrated systems to produce a desired reaction outcome. We used the selective enzyme co‐compartmentalization and nanoreactor integration to regulate competitive cascade reactions and to modulate the kinetics of sequential reactions involving multiple nanoreactors. Furthermore, we show that the nanoreactors can be efficiently loaded into giant polymer vesicles, resulting in multi‐compartmentalized microreactors. Synthetic silica nano‐organelles designed to regulate kinetics and pathway selection in enzymatic cascade reactions are reported. Like cell organelles, synthetic nano‐organelles can be combined to create integrated systems. Multicompartmentalized synthetic cells were built using synthetic nano‐organelles and giant polymeric vesicles. Compartmentalization is an essential characteristic of life because it enables the spatiotemporal control over multiple biological processes in living cells.The encapsulation of enzymes in polymeric or lipid‐based compartments result in synthetic organelles that mimic organelles found in eukaryotic cells, such as lysosomes and peroxisomes.Here, we describe a strategy for programming the outcome of enzymatic cascade reactions using nanoreactors designed as synthetic organelles. To do that, we quantitatively encapsulated enzymes in semipermeable silica nanocapsules to control the enzyme distribution and the resulting functionality of integrated multi‐organelle systems. Reaction outcomes and performance regulation was achieved by combining different nanoreactors. This strategy allowed us to (i) increase the efficiency of a three‐step sequential reaction and (ii) select distinct pathways in competitive enzymatic reactions. To demonstrate the robustness and versatility of the silica nanoreactors, we built multi‐compartmentalized micro‐reactors with a hybrid nano‐in‐micro architecture using a bottom‐up approach. The cascade reactions between different nanoreactors inside the micro‐reactors mimic the structure and basic function of natural organelles in eukaryotic cells.In the first step, we prepared organelle‐mimicking nanoreactors (NRs) by synthesizing core–shell silica nanocapsules with in situ encapsulated enzymes. Silica nanocapsules possess unique advantages when it comes to enzyme encapsulation efficiency because of their capsular configuration.Cascade reactions between individual nanoreactors were designed to mimic a multi‐step intracellular catalytic process Figure . As a moThe NRs were synthesized by an inverse (water‐in‐oil) miniemulsion polymerization process . In short, the enzymes (GOx or HRP) were first dissolved in sodium phosphate buffer, which was then dispersed in cyclohexane by miniemulsification. The resulting enzyme‐carrying nanodroplets provided a template for the subsequent formation of a silica wall from silica precursors. The polymerization was initiated upon contact of the added precursors with the water phase in the nanodroplets. The (3‐aminopropyl)triethoxysilane precursor catalyzed the rapid condensation of tetramethoxysilane at the water‐oil interface, resulting in a porous silica shell around the nanodroplets.3; HRP: 6.2×4.3×1.2 nm3),Dynamic light scattering  and transmission electron microscopy , which is a substrate for HRP. The maximum reaction velocity (Vmax) of encapsulated GOx was similar to the free GOx, indicating that the enzyme activity was preserved after encapsulation. The substrate affinity (Km) and the turnover number (kcat) of GOx were reduced after encapsulation. For HRP, the kcat and the enzymatic activity were lower in the encapsulated form. The reduced enzymatic activity can be attributed to the additional diffusion barriers offered by the silica shell and the PEG layer present on the shell surface. We assume that the temporary product accumulation within the confined space, due to delayed diffusion of the products, resulted in a stronger inhibition of HRP, leading to the observed reduced turnover number.The encapsulated enzymes remained active and were accessible to external reactants Figure . The semn Figure , S5. GOx2O2. The H2O2 was then used as a reactant by HRP@NRs. Oxidation of the non‐fluorescent probe AmplexTM red generated a highly fluorescent molecule, resorufin. The kinetics of the cascade reactions was monitored by recording the changes in fluorescence intensity of resorufin over time . The reduced kinetics shown by the NRs resulted from the additional diffusion barriers provided by the silica shell and the larger distances between the encapsulated reaction centers.Next, we tested a cascade reaction involving GOx@NRs and the HRP@NRs Figure . Glucosee Figure . GOx@NRsIn eukaryotic cells, organelles can regulate distinct enzymatic pathways to accomplish different tasks required to keep the cell alive. Thanks to the strategic co‐compartmentalization of enzymes and other biological components, cells can accomplish complex tasks with high selectivity and efficiency.2O2, which then becomes a reactant for HRP in the third step. The gluconic acid by‐product created in the second step reduces the pH in the system. A reduction of ca. 4 pH units was observed in mixtures containing GOx@NR and glucose, with the effect being dependent on the initial concentration of glucose . We now discuss how different encapsulation strategies lead to different reaction outcomes and performance.The type 1 cascade reaction takes three steps to be completed Figure . In the First, we encapsulated GOx and HRP in the same nanoreactor and compared it with the separately encapsulated enzymes Figure . By doin2O2 intermediate for immediate use by the co‐encapsulated HRP. When the enzymes are in separate compartments, the reaction intermediates must diffuse from one nanocontainer to the other. This results in the dilution of the intermediate, which explains the slower reaction kinetics observed. The escape of reaction products from NRs was confirmed by the strong fluorescence intensity present in the filtrates after removal of the NRs from the reaction medium . Batch‐to‐batch reproducibility was demonstrated with two individually prepared samples . The effective co‐encapsulation of both GOx and HRP in the same NRs was proved by fluorescently labelling the enzymes and then determining the ratio of their fluorescence intensity before and after encapsulation .The confinement guarantees a high local concentration of the H2O2 produced by GOx also accumulates internally, lowering enzyme activity due to the increased oxidizing stress and acidic environment brought about by the product accumulation  enhanced the overall kinetics, while incompatible pairs (β‐G and GOx) resulted in slower kinetics. These results indicated that the performance of multi‐step cascade reactions can be regulated via the rational distribution of enzymes in different compartments via selective co‐encapsulation.We now discuss the effect of selective enzyme compartmentalization involving three enzymes Figure . On path2O2, which becomes a substrate for two possible pathways. On pathway 1, H2O2 is consumed by HRP@NR to oxidize AmplexTM red and produce resorufin. On pathway 2, the H2O2 is disproportionated by catalase (CAT@NR) to generate oxygen and water. By tuning the ratio of HRP@NR to CAT@NR, the reaction outcome can be switched between the two pathways 14 (PB‐PEO). Amphiphilic copolymers, such as PB‐PEO, can assemble into soft membrane vesicles that are structurally like phospholipid‐based membranes, but more robust due to the higher molecular weight of the polymers.To test if NRs could be used as effective synthetic organelles for synthetic cells, we designed a simple synthetic cell containing GOx@NRs and HRP@NRs as nano‐organelles. The synthetic cells were assembled by encapsulating the NRs into giant polymer vesicles made from the block copolymer poly(butadiene)4 randomly distributed NRs. One advantage of using silica nanoreactors is that the robust silica shell protects the enzymes during the vesicle preparation and later manipulations. This extra stability allows the assembly of robust synthetic cells that can last for days after their preparation. In addition, silica nanoreactors are naturally porous. Therefore, there is no need to account for the lower permeability usually observed when amphiphilic polymers and lipids are used to create soft NRs.The NRs were separately labelled with Cy5 and FITC, making them easy to detect and distinguish by confocal laser scanning microscopy (CLSM). Figure TM red into the external aqueous medium resulted in the formation of the product (resorufin) inside the vesicles confirming that the activity of the NRs is not affected by the encapsulation into the vesicles  should be addressed to the authors.Supporting InformationClick here for additional data file.


Reanalysis of the data for this brain with a focus on the telomerase reverse transcriptase (TERT) promoter revealed a canonical mutation (G > A on chr5:1295250) in a similar fraction of cells in the hippocampus. Concurrent gain of chromosome 7, loss of chromosome 10 and mutation in the TERT promoter are molecular characteristics commonly observed in glioblastoma, a fast‐growing and aggressive brain tumor.The question of which cell type expanded remains open , while it was only ∼2% among younger brains (<40 years of age). As hypermutability was observed in the brains of healthy individuals and in those with neuropsychiatric diseases, no link of hypermutability to neuropsychiatric disease states has been found, nominating age as the primary causal factor. Age is known to be the major factor associated with cancer occurrence. As such, the observed hypermutability carries two major hallmarks of cancers: clonal expansion and age association. This suggests that hypermutability generally represents pre‐cancer or undiagnosed cancer cases, implying a theoretical possibility of cancer monitoring and early detection based on genomics. Association with aging also implies that hypermutability could be relevant in other aging‐associated diseases such as Parkinson's and Alzheimer's. If proven, they theoretically could be diagnosed early before symptoms develop, using hypermutability as a genomic biomarker. However, it is currently unclear how this could be achieved, given that brain tissue is hardly accessible.Among other findings, no significant differences between diseased and normal brains were observed in the proportions of somatic mutations in different categories of genomic annotation . However, in the brains of individuals with autism—as compared to neurotypical ones—somatic single‐nucleotide mutations generated more motifs for transcription factor binding within active enhancers of the developing brain. The most noticeably affected motifs were binding sites for myeloid ecotropic viral integration site (MEIS) homeodomain transcription factors, which are known to be involved in embryonic development.The study made possible by the BSMN revealed the presence of hypermutable brains and insights into the possible role of somatic mutations in autism spectrum disorder. However, despite a significant effort, the studied cohort is still relatively small and at least 100 times smaller than in modern GWAS studies, limiting the discovery of the possible role of somatic mutations in neuropsychiatric diseases. We believe that further studies with larger cohorts will yield more insights.


The introduction of fluids other than breast milk during the first few days of life or later neonatal period has been identified as a risk factor for suboptimal breastfeeding (BF) outcomes in numerous studies using varying study designs. However, the relationship between early introduction of fluids other than breast milk and BF outcomes has not been systematically assessed using only prospective studies that can establish temporality, which is critical for determining whether observed associations are causal. We conducted a systematic review and meta‐analysis of prospective studies to assess if there is a difference in BF outcomes as a result of the introduction of: (a) milk‐based prelacteals, (b) water‐based prelacteals and (c) breast milk substitutes (BMS) between 4 days and 4 weeks postpartum. We searched PubMed, Lilacs, Web of Science and other repositories for original research investigating the relationship between early introduction of prelacteals and/or BMS and BF outcomes. Forty‐eight studies met the inclusion criteria for the systematic review. Of the 39 prelacteal feeding studies, 27 had the prerequisite statistical information for inclusion in the meta‐analysis. Findings from the meta‐analysis showed a relationship between prelacteals and exclusive BF cessation  and any BF cessation  among infants under 6 months old. Nine studies focusing on the introduction of BMS during the neonatal period identified this practice as a statistically significant risk factor for a shorter BF duration. Effective interventions are needed to prevent the introduction of unnecessary milk‐based prelacteals and BMS during the perinatal and neonatal periods to improve BF outcomes. The practice of prelacteal feeding and introduction of breastmilk substitutes (BMS) during the neonatal period is widespread across the globe.In this systematic review and meta‐analysis of prospective studies, the introduction of prelacteals was associated with suboptimal breastfeeding practices including a higher risk of not exclusive breastfeeding and any breastfeeding cessation among infants under 6 months of age.Prospective studies show that the introduction of BMS during the neonatal period is a risk factor for suboptimal breastfeeding practices.Facility and community‐based interventions are needed to prevent the introduction of prelacteals perinatally and BMS during the neonatal period. Findings from the meta‐analysis showed a relationship between prelacteals and lack of exclusive breastfeeding and any breastfeeding cessation among infants under 6 months old. Effective interventions are needed to prevent the introduction of unnecessary milk‐based prelacteals during the perinatal and neonatal periods to improve breastfeeding outcomes. Any breastfeeding was defined as the infant receiving breast milk either directly from the breast or expressed breast milk.This systematic review and meta‐analysis followed the Institute of Medicine guidelines. Before reviewing the literature, the protocol was developed and registered in PROSPERO (ID# CRD42021240669). BF outcomes of interest were grouped into short‐, medium‐ and longer‐term outcomes. The outcomes of this review were EBF duration or prevalence among infants less than 6 months old, duration/prevalence of EBF reported beyond 1 month postpartum and the duration/prevalence of any BF until 1 year postpartum. The short term outcome delayed onset of lactation, that is, milk arrival >3 days after birth, was listed in the original protocol, however, it was decided not to include it because it was an outcome in another systematic review published in this supplement , Web of Science Core Collection , PsycINFO (via Ovid), EMBASE (via Ovid), LILACS , SciELO and Global Index Medicus databases. The searches were run between March and May 2021. We searched for articles containing one or more controlled vocabulary terms or keywords related to both of the following concepts: infant feeding outcomes and prelacteal feeds. The latter concept was operationalized with text words and adjacency statements addressing in‐hospital supplementation and early introduction of BMS, as well as prelacteals per se Table . The sea2.2The records identified from the database searches were screened using Covidence. Studies were included in this review if they: (a) were published in English, Spanish, or Portuguese; (b) used a prospective cohort, quasi‐experimental, or randomized controlled trial (RCT) design; (c) reported BF duration and other BF outcomes. Studies were excluded if they: (a) were cross‐sectional studies, case‐control studies, clinical studies, qualitative studies, reviews, systematic reviews, or meta‐analyses; (b) included women who had a delivery involving serious maternal complications such as severe post‐partum haemorrhaging that prevented them from BF; (c) included women with contraindications to BF such as cancer chemotherapy and taking lithium medications; (d) included newborns without serious medical complications such as asphyxia at birth; (e) focused on premature or very low birth weight babies; (f) did not report prelacteal feeds or early introduction of BMS; (g) did not report any longer‐term BF outcomes  and (h) were not published in the peer‐reviewed literature—for example, technical reports, dissertations, conference abstracts.Titles and abstracts of studies were independently screened for inclusion by two reviewers  and those that did not meet the inclusion criteria were excluded. For standardization and consistency, the reviewers independently reviewed the first 100 titles and abstracts and then met to review the level of agreement and discuss any differences that arose in the operationalization of the inclusion criteria. As the reviewers had a strong agreement in the inclusion/exclusion classification of the first 100 titles and abstracts, the rest of the titles and abstracts were divided between the two reviewers so each of the remaining records could be excluded based on only one screener's opinion. Both reviewers screened all the full texts of studies identified as potentially meeting the inclusion criteria based on the title and abstract. Full texts deemed to be included in the review had to be identified as fulfilling all inclusion criteria by both reviewers. The screening results were compared and any discrepancies were discussed until a consensus was reached. If consensus could not be reached, a third reviewer (R. P. E.) resolved those conflicts.Titles and abstracts identified through citation chaining and experts were searched for in Covidence and any documents that had already been screened in Covidence were removed. The remaining titles, abstracts and full texts of studies were screened by the same reviewers  using the same method as described above.2.3Following the identification of the final set of included studies, one reviewer extracted the data (A. H. F.) including: authors, year, country, timing of assessment, exposure and outcome variables, analyses including control variables and key findings. The data extracted were verified by the second reviewer (E. C. R.).2.4The results of the search and article selection are presented using the PRISMA flow diagram Figure . Forty‐eTo assess the inclusion of the 48 studies in the meta‐analyses, they were all reviewed by two authors . Studies were included in the meta‐analyses if they reported adjusted effect measures to assess the impact of the introduction of prelacteal feeds on BF outcomes of interest. Following a thorough review, 27 of the 48As the length of follow‐up and BF practices assessed varied greatly across studies, outcomes were grouped into five categories: (1) any BF cessation among infants under 6 months of age; (2) EBF cessation among infants under 6 months of age; (3) any BF among infants under 6 months of age; (4) EBF among infants under 6 months of age; and (5) any BF cessation among infants up to 1 year of age.A meta‐analysis was then conducted to test the effect of prelacteal feeds on each of these outcomes Tables .I2 was used to investigate heterogeneity and the funnel plot and Egger test were used to assess the occurrence of publication bias.The included studies were carried out in different world regions, and evaluated different BF outcomes among populations of children of different ages. Hence, we pooled the estimates using both random effects and fixed effects models using Stata 17.0. The findings were similar for random and fixed effects models, hence only the results from random effects models are presented here. The Some studies reported on different effect measures (either hazard ratio or odds ratio). Thus, sensitivity analyses were performed by limiting the models to studies that reported on the same impact measure (Supporting Information Appendix 2.5The Joanna Briggs Institute (JBI) critical appraisal tools for observational, quasi‐experimental and experimental studies was used to assess the quality of each study included in the review  Figure  and the 3.2Prelacteal feeds were also a risk factor for any BF cessation by 6 months post‐partum when the prelacteals were analysed together regardless of type Figure  and the Figure 5(a) Prelacteal feeds and any breastfeeding cessation among infants under 6 months. (b) Prelacteal feeds and any breastfeeding cessation among infants under 6 months. Only milk‐based prelacteals. (c) Prelacteal feeds and any breastfeeding cessation by 1 year.3.3We were able to perform this analysis for any and EBF cessation under 6 months only, as for the other outcomes analysed the same impact measure was reported in the studies (either hazard ratio or odds ratio) .The analysis of funnel plots suggests a small or no publication bias for most outcomes analysed, except for any BF cessation under 6 months and EBF under 6 months  did not clearly describe whether the exposure or outcomes were measured in a valid or reliable manner. Most studies identified confounding factors and many of these used strategies to deal with them through the study design and/or data analysis. All studies used appropriate statistical analyses; however, several studies could have used more powerful analyses to draw conclusions Figures  and 3.4Our findings indicate that milk‐based prelacteal feeds and early introduction of BMS during the neonatal period are risk factors for shorter EBF duration and any BF duration. Prelacteals are broadly classified as water‐based and milk‐based and this distinction is important for understanding the reasons for introducing them as well as the consequences that they may have on BF outcomes. While milk‐based prelacteals are often introduced as breast milk substitutes in response to self‐reported insufficient milk (McKenna & Shankar, Our findings also demonstrated overwhelming evidence that early introduction of BMS is a risk factor for shorter EBF and any BF duration. These findings confirm our hypothesis that the introduction of BMS during the neonatal period, oftentimes as a result of lactation problems related to lack of BF counselling and support during the establishment of the milk supply, is likely to disrupt nursing patterns, milk production and subsequent BF failure as a result (Pérez‐Escamilla et al., Our study had several limitations. First, we could not answer all original questions listed in the protocol by type of prelacteal supplement because only one study included water‐based prelacteal supplements only (Qiu et al., There were also notable strengths. Our findings are based only on results from prospective studies, which are much less likely to be affected by recall bias compared with cross‐sectional and retrospective studies. The included articles represent different world regions, over one‐quarter of them were identified through citation chaining and we reviewed studies in three languages. Furthermore, we conducted sensitivity analyses and found no evidence of publication bias. The findings from the studies not included in the prelacteals meta‐analysis were consistent with the directionality of associations found in the meta‐analysis; that is, they also identified prelacteals as a risk factor for suboptimal BF outcomes. Indeed, 10 of the 12 articles excluded from the meta‐analysis found statistically significant inverse relationships between prelacteal feeds and diverse BF outcomes consistent with the results of the meta‐analysis.Our systematic review and meta‐analysis have important policy implications as previous studies have identified modifiable risk factors, such as caesarean‐section delivery, unsupportive BF maternity practices and health care providers' lack of knowledge about how breast milk supply gets established, which can be addressed through health care facility improvements as well as BF counselling interventions (Kavle et al., Moving forward, experimental interdisciplinary research studies are needed to identify the most promising interventions to address the risk factors for prelacteal feeding and the introduction of BMS during the neonatal period. To optimize the successful implementation of these interventions, it is important to codesign them with the target population and the providers serving them, following a health care systems implementation framework that takes the social determinants of health into account (Boccolini et al., 5In conclusion, this review found that the introduction of BMS during the early and late neonatal periods is a statistically significant risk factor for shorter BF duration. Effective interventions are needed to prevent the introduction of unnecessary milk‐based prelacteals and BMS during the perinatal and neonatal periods to improve BF outcomes.Rafael Pérez‐Escamilla conceptualized and drafted the first version of the systematic review and meta‐analysis protocol and wrote the initial draft of the full manuscript. Amber Hromi‐Fiedler and Elizabeth C. Rhodes reviewed titles, abstracts and full texts of studies, created synthesis tables of studies included in the review and conducted the quality assessment of included studies. They also contributed to conceptualizing and drafting the systematic review and meta‐analysis protocol and manuscript. Rafael Pérez‐Escamilla provided guidance in reaching consensus on inclusion of studies as needed and extracted the data of studies published in Portuguese or Spanish. Paulo A. R. Neves contributed to conceptualizing and drafting the protocol for the systematic review and meta‐analysis and led the meta‐analysis in partnership with Juliana Vaz. They both critically reviewed the manuscript. Sofia Segura‐Pérez critically reviewed the protocol for the systematic review and meta‐analysis and the manuscript draft. Kate Nyhan developed and tested the search strategy, conducted the search and contributed to selecting a quality assessment tool. All authors read and approved the submitted manuscript.The authors declare no conflicts of interest.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.


The Middle East Respiratory Syndrome coronavirus (MERS‐CoV) is still listed on the WHO Research and Development Blueprint of emerging pathogens. Dromedary camels remain the only known animal reservoir of the virus. The animal‐to‐animal as well as the animal‐to‐human transmission in the MERS‐CoV cycles were reported. However, many aspects of these transmission chains are not well studied. One of these directions is the potential roles of various species of arthropods in the transmission of the virus.The main goal of the current work was to study the roles of several species of arthropods in the transmission of MERS‐CoV.Culicoides, Stomoxys, Musca domestica and some Culex species) that shared the habitat and were circulating in this farm during this longitudinal study.To achieve this goal, we identified some MERS‐CoV naturally infected dromedary camel populations. We conducted a longitudinal study among these animals for more than 2 months. This was done by repeated testing of nasal swabs biweekly from some selected animals in this population for the presence of MERS‐CoV‐RNAs by real‐time PCR. During the duration of this study, we collected several species of arthropods (Our results showing, despite the detection of the viral RNAs in some animals throughout this study, none of the examined species of arthropods tested positive for the viral RNA.These results are suggesting that at least the tested species of arthropods may not play roles in the transmission of MERS‐CoV. However, more large‐scale studies are required to explore any potential roles of arthropods in the transmission cycle of MERS‐CoV. • Identification of some index MERS‐CoV naturally infected dromedary camel population.• A longitudinal follow‐up study for MERS‐CoV naturally infected dromedary camels based on repeated testing of nasal swabs from some animals revealed the contentious circulation of the virus in these animals throughout the duration of the study.• The absence of any detectable MERS‐CoV‐RNAs in some collected arthropods shared the habitat with the infected camel population strongly suggests the lack of transmission of MERS‐CoV through the tested species of arthropods. There are 2580 human infections of MERS‐CoV that have been reported to the WHO from 27 countries as of 6 April 2021 WHO, . DromedaHowever, the roles of various species of arthropods in the transmission cycle of MERS‐CoV were never investigated yet.22.1Longitudinal molecular surveillance was conducted among a dromedary camel population for the MERS‐CoV (20 Feb to 20 April 2018). We identified an index MERS‐CoV naturally infected dromedary camel herd to conduct this study. These animals were kept in a 6000 square feet wire fence farm which consists of several animal pens separated by strong metal pipes , the stable fly (Stomoxys), the house fly (Musca domestica) and some mosquitoes particularly (Culex sp) shared the habitat with the dromedary camel farm during the tenure of the current study  for further processing. In the laboratory, we treated each individual whole insect as a separate sample in a separate tube to avoid any type of contamination. We washed the collected insects several times with sterile PBS. Briefly, each inset was placed in a 5 ml sterile tube containing 2 ml of PBS then homogenized tissue lyser by using some copper beads. The homogenized tissue suspensions were clarified by centrifugation at 500 RPM for 5 min at 4°C. The supernatants were collected and stored at (–80°C) for further testing. The extracted RNA concentration was carried out using the NanoDrop machine (Thermo Fisher Scientific/Applied Biosystems). The collected RNAs were stored at (–80°C) for further testing. The collection of mosquitoes was carried out into batches in parallel to animal sampling. Table 2.4We collected nasal swabs from the dromedary camel population for six‐time points biweekly during the duration of this study. Each tube of swab contains 3 ml of the viral transport medium [Kangjian Virus Collection and Preservation System (Cat: KJ502‐28‐1)]. The collected swabs were placed immediately in the ice‐containing tanks provided with a data logger to monitor the temperature. The collected swabs were usually transferred to our laboratory within 30 min of the collection time. Swabs were introduced deep into the nasal orifices of each animal to touch the inner mucous membranes. Special attention was paid that each cotton swab is fully soaked with the nasal secretions per each animal. Each swab was transferred into tubes containing the viral transport medium. The total viral RNAs were extracted from the nasal swabs from dromedary camels by using the Qiagen viral RNA kits . The process of these extractions was carried out as previously described  to test various samples including nasal swabs and tissue suspensions prepared from various species of insects mentioned earlier for the presence of the viral nucleic acids. Several sets of control samples were used including some known negative and positive MERS‐CoV RNA samples. Samples were considered positive only when both targets (ORF1a and UpE) showing positive results. Meanwhile, we considered positive samples when (Ct ≤30).We tested the nasal swabs collected from dromedary camels as well as the suspensions prepared from each species of arthropods by real‐time PCR. Briefly, we used the commercially available real‐time PCR MERS‐CoV kits  and stomoxes touch the mucus membranes of animals and may touch the saliva and other body secretions of dromedary camels. It was also noted that there is always a dramatic increase in the numbers of these arthropods during the summer particularly the Musca domestica. Each region may have its own species of arthropods that infest dromedary camels such as some species of ticks Hyalomma impeltatum (H. impeltatum) from Riyadh, Al‐Qaseem and Jazan regions . The animal experiments and protocols were reviewed and approved by the animal ethics committee of the deanship of scientific research, King Faisal University, Saudi Arabia . All the necessary paperwork for sample collections was obtained.The animal experiments conducted in the current study were conducted as per the instructions of the Animal Ethics protocols and the National Committee of Bio‐Ethics, King Abdul‐Aziz City of Science and Technology, Royal Decree No. M/59 (The authors declare no conflict of interest.https://publons.com/publon/10.1002/vms3.717The peer review history for this article is available at We wish to thank King Abdul‐Aziz City for Science and Technology (KACST), Saudi Arabia, for their generous funding through the MERS‐CoV research grant program (number 20‐0004/24‐1), which is a part of the Targeted Research Program (TRP). The funding body does not have any role in the design of experiments and the execution of the experiments.Maged Hemida: Conceptualization; Data curation; Formal analysis; Funding acquisition; Investigation; Methodology; Project administration; Resources; Software; Supervision; Validation; Visualization; Writing original draft; Writing review & editing. Mohammad Nafi Solaiman Al‐Sabi: Data curation; Formal analysis; Methodology. Mohammed Alhammadi: Conceptualization; Data curation; Methodology. Faisal Almathen: Conceptualization; Data curation; Formal analysis; Methodology. Abdelmohsen Alnaeem: Conceptualization; Formal analysis; Investigation; Methodology


Previous studies also showed that it may affect beneficially the testosterone and cortisol concentration in athletes. Due to the contradiction results between studies, we aimed to conduct this meta‐analysis to assess the HMB supplementation effect on testosterone and cortisol in trained athletes.2 statistic.Scopus, Medline, and Google scholar were systematically searched up to August 2021. The Cochrane Collaboration tool for evaluating the risk of bias was applied for assessing the studies' quality. Random‐effects model, weighted mean difference (WMD), and 95% confidence interval (CI) were used for estimating the overall effect. Between‐study heterogeneity was evaluated applying the chi‐squared and Ip > .05), subgroup analysis based on the exercise type showed a significant decrease in the cortisol concentration in resistance training exercises  and a significant increase in the testosterone concentration in aerobic and anaerobic combined sports .Seven articles were included in the meta‐analysis. Although the meta‐analysis generally showed that HMB consumption did not have any effect on the cortisol and testosterone concentration , we found that HMB consumption led to a significant decrease in cortisol and increase in testosterone concentrations after resistance exercises and aerobic and anaerobic combined activities, respectively. This interest has been motivated by this theory that nutritional supplements may improve performance by increasing anabolic and decreasing catabolic effects, thereby improving body composition and decreasing exercise‐induced muscle damage  supplementation, that it may result in several ergogenic advantages, including anticatabolic   criteria  selected articles based on the inclusion criteria independently. Articles including eligibility criteria in the title and abstract screening were selected to be checked by full manuscript. Parallel clinical trials were selected by applying a control group or crossover design in the systematic review and meta‐analysis. All classified trials were retrieved by either of the authors. According to the data within the full information, we used a form to select the studies eligible for inclusion in the meta‐analysis. Contradictions between the authors were solved by consensus or third researcher (L.Z).2.4SD) of serum hormones' status (cortisol and testosterone) at baseline and after the intervention.M.M and LG extracted the following needed information: first author's name, year and publication country, design of studies, gender and age of subjects, HMB dose and sample size, duration of intervention, and training status. Also, we extracted mean and standard deviation  measure was reported, SE was converted to SD applying this formula: SD=SE×√n (n=number of subjects in each group). Between‐study heterogeneity was assessed using the chi‐squared (χ2) test and quantified applying the I2 statistic, which shows the total variation percentage across studies that is ascribable to heterogeneity rather than to chance. Significant heterogeneity was defined with a p‐value of <.05.For each trial, mean differences and standard deviation were calculated for continuous variables. The differences of mean changes in cortisol and testosterone concentration and their correspondence The random effects model was applied to calculate the weighted mean differences (WMDs) with 95% confidence intervals (CIs) for estimating the overall effect. To evaluate whether the outcomes could have been altered by a single study distinctly, a sensitivity analysis was performed Tobias, . Also, s2.7To assess the overall quality of current meta‐analysis with regard to the HMB supplementation on testosterone and cortisol concentration, we used the NutriGrade scoring system  were published between 2001 and 2017. The duration of these RCTs ranged between 1.5 and 12 weeks. Two studies used a randomized crossover design  and on testosterone .Sensitivity analysis showed that the omission of any of the studies from the meta‐analysis creates no change in the outcomes of the meta‐analysis on testosterone concentration, whereas the results on cortisol concentration were sensitive to omitting 3 studies , and 4.8 for testosterone concentration .4The efficacy of HMB has not been distinctly established regarding hormonal responses; thus, this systematic review and meta‐analysis provided comprehension into the potential benefits conferred by HMB supplementation to help athletes to make aware decisions on its usage and impact. In this systematic review and meta‐analysis of 7 studies (235 participants), we found that HMB consumption led to a significant decrease in cortisol and increase in testosterone concentrations after resistance exercises and aerobic and anaerobic combined activities, respectively. Several probable mechanisms have been suggested for the beneficial effects of HMB supplementation on testosterone and cortisol concentration in athletes.Our results support those of past investigations showing that amino acid consumption may play a role in reducing exercise‐induced increases in cortisol  in athletes. All included studies administered the same doses of HMB for intervention and all participants were male (except in one study (Portal et al., In conclusion, the current systematic review and meta‐analysis outcomes revealed that HMB supplementation may be linked with a decrease in cortisol concentration after resistance exercise. In contrast to cortisol, testosterone concentration significantly increased in aerobic and anaerobic combined training. More investigations are recommended with a variety of doses and exercises, longer time in periods and in both sexes in athletes.None.


Obligate brood parasites depend entirely on other species to raise their offspring. Most avian obligate brood parasites have altricial offspring that require enormous amounts of posthatching parental care, and the large fecundity boost that comes with complete emancipation from parental care likely played a role in the independent evolution of obligate parasitism in several altricial lineages. The evolution of obligate parasitism in the black‐headed duck, however, is puzzling because its self‐feeding precocial offspring should not constrain parental fecundity of a potential brood parasite in the way that altricial offspring do. We used an experimental nest predation study to test the idea that high nest predation rates played a role in the evolution of brood parasitism in this enigmatic duck. Experimental duck eggs in untended nests suffered massive rapid predation, while eggs in tended nests of the three main hosts, all aggressive nest defenders, had very high success, illustrating the benefits of parasitizing these ‘bodyguard’ hosts. We used an experimental nest predation study to test the idea that high nest predation rates coupled with high success by laying in aggressive host nests played a role in the evolution of brood parasitism in the enigmatic black‐headed duck. Experimental duck eggs in untended nests suffered massive rapid predation, while eggs in tended nests of the three main hosts—all aggressive nest defenders—had very high success, illustrating the benefits of parasitizing these ‘bodyguard’ hosts. In our own study of the black‐headed duck, we also noted that the three main hosts used by the ducks—two species of coots and a gull—are all species that aggressively defend their nests against predators. We hypothesized that these aggressive hosts might have higher nesting success than black‐headed duck would, were the ducks to nest on their own in these predator‐rich wetlands  the risk of nest predation in these marshes is extremely high in the absence of aggressive parenting; (2) the main host species are sufficiently aggressive as nest defenders that duck eggs laid in their nests experience high survivorship despite the high potential risk for egg mortality; and (3) the presence of white duck eggs does not lead to an elevated predation risk of those eggs nor of the parasitized host nest.2Fulica armillata), red‐fronted coots (Fulica rufifrons), and brown‐hooded gulls (Larus maculipennis)  are probably the most important avian predator on nests, but other possible avian nest predators that breed in these wetlands include southern caracaras (Caracara plancus) and long‐winged harriers (Circus buffoni). Rarely, we observed the lutrine opossum (Lutreolina crassicaudata), a mammalian predator, preying on both adults and eggs of the hosts at our study area .We conducted the experiments on two wetlands southwest of General Lavalle, Buenos Aires Provence, Argentina in late October and early November 1994  high nest density, (2) low nest density, and (3) nests placed within an active brown‐hooded gull colony. Nest density has been found to affect nest predation rates in experimental nest studies  were alternated along the transect. In the “high‐density” treatment, established on a different part of the same large wetland on 6 November 1994, nests were placed 30 m apart, also in a transect. Finally, the “gull colony” treatment was established in an active brown‐hooded gull colony at Estancia Cari Lauquen on 8 November 1994. To permit all nests to remain within the perimeter of the gull colony, nests were not set in a transect but placed haphazardly within the colony. We did not measure the density of the gull nests, but visual assessment indicated that the gull nests were spaced at lower densities (often 10–20 m apart) than is typical for high‐density colonial gulls nesting on dry land. All nests were checked every one to three days. We checked the high‐ and low‐density nests for a total span of seven and eight days, respectively, and the gull colony nests for a total span of 6 days. The experiments were done on the same large wetland complex but far enough apart so that they would not influence each other. These marshes are interconnected and cover extensive areas. We attempted to separate our experiments spatially  so that one set of experiments did not influence another, but the predator and host communities were the same.2.2The second experiment, with active host nests, was conducted in the same year (1994) at the Estancia Cari Lauquen wetlands. This experiment was undertaken primarily to study natural selection on egg mimicry  nests with chicken eggs painted to match the eggs of hosts simulating an unparasitized nest and 2) nests with one host‐like egg and one white duck‐like egg simulating a parasitized nest  to compare nest survival rates across experiments and treatments. Nests that survived to the end of each experiment were classified as right censored. For the artificial nest experiments, we included experiment and nest treatment as model effects. Partial predation, where a predator only took one of the two eggs on a given day, was common in the artificial nest experiment so we examined nest survival in two ways: (1) considering partial predation as predation  and (2) only counting nests that had lost both eggs as depredated. For the comparison of artificial nests and active host nests, we did not compare the two experiments but instead compared all the treatments in the two experiments combined.33.1p = .0022, high vs. gull p < .0005, low vs. gull p = .036). For survival based on the timing of the loss of any eggs , the high‐ and low‐density coot nest experiments no longer differed but both differed from the gull colony treatments.The survival rates differed across the artificial nest setting treatments Table . The expFor the nests that lost one or both eggs to predators, the most common pattern was asynchronous loss (loss of the two eggs occurred on different days) but synchronous loss was also quite common  Figure . In cont3.2The survival rates of experimental eggs in active real host nests were uniformly high Figure , and the3.3p = .79). Similarly, there was no effect of the presence of a duck‐like egg on nest survival in the active tended host nest experiment  background nest predation rates are very high in the absence of any parental care; (2) the three main hosts used by the ducks are aggressive nest defenders that can maintain high nest survival rates despite the high potential predation risk; and (3) conspicuous white parasite duck eggs do not attract more predators or reduce the benefits of using a bodyguard host.Our experiments revealed that the background rate of nest predation in the absence of attending parents is indeed extremely high—all nests in the low‐ and high‐density artificial nest experiments lost at least one egg within 5 or 6 days, and most lost both eggs. This time frame is a fraction of the incubation period for the duck eggs, roughly 23 days . Interestingly, the eggs disappeared more rapidly in the high‐density plot compared to the low‐density plot as has been shown in several previous studied , an overwater nesting species found in our study area in Argentina, provides the most comparable example of the nesting success expected for a nonparasitic duck in these wetlands. We observed only three pochard nests and did not follow them closely enough to determine predation rates. Weller  Livezey, , all of . Weller  followedAythya americana), combine nesting with parasitism of other species. Facultative parasitism is thought to have been an intermediate stage in the evolution of obligate brood parasitism  , Wyllie  laid on average just eight eggs per season, less than the clutch size of many parental species that raise two broods per season. The fecundity of black‐headed ducks remains to be studied, although genetic analysis of maternity patterns from one year of our study suggests that female black‐headed ducks do in fact have a very low fecundity, at least as assessed on a local scale (within our study site) .Our study focused on the possible role of nest predation and hatching success in the evolution of obligate brood parasitism. We are not arguing that improved hatching success alone drove the evolution of obligate parasitism in the black‐headed duck but instead that it could have been an important contributing factor. We note that increases to other key demographic variables that result from abandoning parental care—fecundity, duckling survival, and adult survival—could all contribute to favor the evolution of obligate parasitism in black‐headed ducks . Foxes do not hunt over water, and accordingly mortality of overwater nesting ducks by foxes is very low ; data curation (lead); formal analysis (lead); funding acquisition (lead); investigation (lead); methodology (lead); project administration (lead); resources ; supervision (lead); writing – original draft (lead); writing – review and editing (lead). Alejandra Carminati: Investigation (lead); methodology . Geneviève Goggin: Investigation (lead); methodology (lead). John M. Eadie: Conceptualization (lead); data curation ; formal analysis ; funding acquisition (lead); investigation (lead); methodology (lead); project administration ; writing – original draft (lead); writing – review and editing (lead).This work was supported by the National Geographic Society (grant #5099‐93), the British Broadcasting Corporation (D. Attenborough's Life of Birds project), the Dennis G. Raveling Endowment of the University of California, Davis (JME), and the Univerity of California, Santa Cruz (BEL).The authors declare that they have no conflict of interest.Appendix S1Click here for additional data file.