Recent insights in allorecognition and graft rejection mechanisms revealed a more complex picture than originally considered, involving multiple pathways of both adaptive and innate immune response, supplied by efficient inflammatory synergies. Current pillars of transplant monitoring are serum creatinine, proteinuria, and drug blood levels, which are considered as traditional markers, due to consolidated experience, low cost, and widespread availability. The most diffuse immunological biomarkers are donor-specific antibodies, which are included in routine post-transplant monitoring in many centers, although with some reproducibility issues and interpretation difficulties. Confirmed abnormalities in these traditional biomarkers raise the suspicion for rejection and guide the indication for graft biopsy, which is still considered the gold standard for rejection monitoring. Rapidly evolving new “omic” technologies have led to the identification of several novel biomarkers, which may change the landscape of transplant monitoring should their potential be confirmed. Among them, urinary chemokines and measurement of cell-free DNA of donor origin are perhaps the most promising. However, at the moment, these approaches remain highly expensive and cost-prohibitive in most settings, with limited clinical applicability; approachable costs upon technology investments would speed their integration. In addition, transcriptomics, metabolomics, proteomics, and the study of blood and urinary extracellular vesicles have the potential for early identification of subclinical rejection with high sensitivity and specificity, good reproducibility, and for gaining predictive value in an affordable cost setting. In the near future, information derived from these new biomarkers is expected to integrate traditional tools in routine use, allowing identification of rejection prior to clinical manifestations and timely therapeutic intervention. This review will discuss traditional, novel, and invasive and non-invasive biomarkers, underlining their strengths, limitations, and present or future applications in children.The online version contains supplementary material available at 10.1007/s00467-022-05872-z. The current view of allorecognition and graft rejection mechanisms is that of a highly complex and articulated phenomenon, where inflammatory processes synergize with adaptive immune responses resulting in T or B cell activation. This complexity is incompletely captured by the traditional laboratory tools used for monitoring graft health, and therefore, rejection monitoring remains an unmet clinical need.Current pillars of transplant monitoring include traditional markers, such as serum creatinine, proteinuria, and drug blood levels, implemented by immunological biomarkers such as donor specific antibodies (DSA) . PerturbThe limits of these markers and the invasiveness of repeated biopsies, particularly in children, could be overcome by integration with the information provided by rapidly evolving novel biomarkers, which are currently used in limited clinical settings, particularly in children .In the pediatric setting, overall results of kidney transplantation remain unsatisfactory in the long run, in particular when passing through the risky years of adolescence. A recent study highlighted the excess of late acute rejection episodes in the NAPRTCS cohort , pointinThe basic definition of a biomarker is that of a defined characteristic, objectively measured and evaluated as an indicator of normal or pathogenic biological processes or responses to an exposure or intervention, which can be derived from molecular, histologic, radiographic, or physiologic characteristics.A number of biomarkers can be identified with diagnostic, monitoring, response, predictive, prognostic, safety, and susceptibility/risk values. The NIH-FDA Biomarker Working Group has recently defined the characteristics of an ideal biomarker in the BEST Resource.  allows excluding confidently the disease, while the positive predictive value (PPV) confirms the disease status. A monitoring and predictive biomarker should change significantly in response to treatment, while a prognostic biomarker should predict a clinical outcome regardless of treatment, meaning that it can be used to determine the risk of a bad prognosis.Laboratory tests considering biomarkers should possess a number of requirements, including characteristics of robust reproducibility and interpretability, as well as external validation confirming good performance metrics.One further step is the use of a biomarker as a surrogate end point, as a tool for early detection of a certain condition. In the field of transplantation, a certain value could be considered a surrogate end point of rejection and be the threshold to proceed with biopsy or directly to treatment .Creatinine is the most commonly employed measurement to determine kidney function. While widely available and cheap, it is neither specific nor sensitive and is often a late indicator of subclinical rejection. This is particularly true in children receiving large grafts, as demonstrated by the frequent finding of subclinical histological rejection evident in protocol biopsies, with no modification in serum creatinine .Among other kidney function tests, proteinuria is routinely measured and is considered as being among the standard of care. Its performance as a biomarker for rejection is, however, low, in children: albuminuria and proteinuria are nonspecific signs of allograft injury with high sensitivity for graft loss but low specificity. In the large adult cohort described in , early dAt present, tissue biopsy remains the gold standard for assaying the health of the graft, although arguments on specificity and sensitivity of histology lesions and their predictive value, particularly when performed early after transplant, are reported in different studies. Protocol surveillance biopsies possess the advantage of an unbiased longitudinal approach, but represent invasive procedures, particularly in small children: are risky mainly for occurrence of adverse events, such as bleeding and artero-venous fistula, time-consuming, subject to pathology interpretation, and generally limited to the first 12–24 months after transplantation. The role of protocol biopsies as modifiers of long-term allograft survival in children remains debated, due to different induction and maintenance immunosuppressive regimens, timing of biopsy, and policy for management of subclinical rejection . At the A recent survey in adult centers in the USA  reportedIn the Canadian PROBE multicenter study , adjunctAltogether, these reasons argue for the need to find additional noninvasive or minimally invasive monitoring systems that can be performed easily, diffusely, and longitudinally over time to continuously patrol subclinical rejection, evaluate evolution of the immunological phenomena upon treatment, and to allow personalized optimization of therapy until a successful resolution is confidently reached.Drug level monitoring is generally accepted as a biomarker of proper immunosuppressive drug use. It is routinely performed for tacrolimus, cyclosporin, everolimus, and sirolimus. Mycophenolic acid single sample trough concentration is not a good surrogate for overall exposure of the drug. These limits are overcome by mycophenolic acid area under the curve (AUC) estimation, which proved to be an effective tool, although less practical, particularly in children, due to the need for at least 2 or 3 concentration samplings for mycophenolate mofetil and 3 to 4 samplings for mycophenolic acid assay . SteroidFluctuations in tacrolimus blood levels in individual patients at a fixed dose over time are defined as intra-patient variability (IPV). High tacrolimus IPV in adult studies correlates with development of DSA, allograft dysfunction, rejection, transplant glomerulopathy, and late graft loss (reviewed in Kuypers) .In the few pediatric studies, tac IPV correlates with de novo DSA development. However, in children, the correlation with rejection, function decline, and graft loss is weaker likely due to biases in defining cut-off values, cohort numerosity, and methodological differences , 14 det.Future perspectives should advocate expert systems to estimate drug exposure , novel tDonor-specific antibodies (DSA) are antibodies developed by the transplant recipient against specific HLA antigens present on the donor kidney. Rejection mediated by DSA may be acute if the graft is exposed to rapid increases in high-titer DSA, which may be generated in sensitized recipients or which may represent de novo responses in non-sensitized patients who are non-adherent to immunosuppressive therapy. On the other hand, chronic rejection mediated by DSA is associated with a slower appearance of antibodies, which may be high or low titer and transient or persistent .These antibodies may damage the kidney by causing multi-lamination of the peritubular capillary basement membrane or arteriopathy manifesting as intimal fibrosis . It is wFor these reasons, dnDSA represent an established biomarker predictive of late acute antibody-mediated rejection, chronic antibody-mediated rejection, transplant glomerulopathy, and graft loss , demonstDSA can be detected in the serum of kidney transplant recipients years prior to clinical graft dysfunction. For these reasons, it is important to monitor DSA routinely in the follow-up of transplant recipients, even though homogeneous protocols are lacking .The clinical relevance of dnDSA relies on characteristics of the antibody itself, such as the IgG subclass, influencing the capability to bind complement and recruit effector cells through Fc receptor binding. DSA of IgG3 subclass bind C1q more efficiently, activate the classical pathway of complement cascade, and proceed more frequently to acute antibody-mediated rejection, while DSAs of IgG4 subclass are unable to bind complement and act mainly through the Fc receptor to amplify alloresponses .In general, dnDSA formation has been associated with lower 10‐year graft survival, including in pediatric studies .DSAs are generally measured by the single antigen Luminex technology, where the mean fluorescence intensity (MFI) is a proxy of the intensity of binding of the antibody contained in the patient serum to the beads. Consistently, higher MFI levels have been associated with impaired graft function, even though exceptions occur .While there is intense investigation around therapeutic strategies aimed at reducing DSA levels , little Some firm points concerning the role of DSA in chronic allograft damage can be made:The finding of non-donor-specific antibodies (NDSA), i.e., antibodies which are directed against HLA specificities other than the donor mismatched antigens, is a relatively common finding in post-transplant monitoring. While pre-transplant NDSAs have been demonstrated to have a detrimental role in graft outcome, the clinical significance of de novo NDSA (dnNDSA) is much more controversial . Some stThe observation that a significant subset of patients with histological features of antibody-mediated rejection (AMR) in the graft biopsy lacks evidence of dnDSA  was the MICA-Ab) or B (MICB), or against autoantigens like endothelin-1 type A receptor (ETAR-Ab), perlecan, agrin, or vimentin, among others. Antibodies against angiotensin II type 1 receptor (AT1R-Ab) are among the most studied, including in pediatric patients ) and 18S ribosomal RNA) was prospectively analyzed in 485 patients in the first year post-transplantation. A three-gene signature  formerly known as CXCL10, 18S rRNA) was identified as able to discriminate ACR [CD3ε, CD105, CD14, CD46, and 18S rRNA) further distinguished ACR from AMR [Urine mRNA transcripts were analyzed in several studies using different panels and approaches: in the CTOT-04 study, a set of urinary mRNAs  as urinary biomarkers of rejection. MiRNAs are endogenous, single-stranded molecules of non-coding nucleotides able to repress the expression of target genes through the post-transcriptional degradation of mRNA and inhibition of protein expression. MiRNAs therefore retain an immunomodulant activity in innate and adaptive immune response. Lorenzen et al. studied a panel of 3 deregulated miRNAs and noted that levels of miR-10b and -210 were decreased and miR-10a increased in patients with acute renal allograft rejection. MiR-210 was predictive of GFR decline 1 year after transplantation and was able to differentiate patients with ACR from urinary tract infection .Other miRNAs, including miR-142-3p, miR-210-3p, and miR-155-5p, were reported by Millàn et al.  to be deCXCL-9 and CXCL-10 chemokine levels using a multivariable model evidenced that miR-155-5p, miR-615-3p, and CXCL-9 levels were independent predictors of rejection. Levels of miR-155-5p, miR-126-3p, miR-21-5p, miR-25-3p, and miR-615-3p were significantly different between rejection and no-rejection urine; CXCL-9 and CXCL-10 protein levels were significantly elevated in urine from recipients with rejection, and the combination of these biomarkers produced a model with better diagnostic performance than the single biomarkers [More recently, a combined approach of miRNAs  and urinomarkers   cohort demonstrated that reatment . More inCXCL10 mRNA [The VIRTUUS prospective pediatric multicenter study is ongoing and aims to validate a urinary mRNA and metabolomics profile as diagnostic and prognostic biomarker of ACR using a urinary cell mRNA 3-gene signature including L10 mRNA   is a urinary proteomic profile of 79 proteins able to distinguish with good performance chronic active ABMR and was validated also in an independent cohort , 65. It Another recent study  describeUrinary proteomic biomarkers are indeed very promising. However, until now, they have yet to reach widespread diagnostic use, which will likely rely on technology improvements and assay standardization Fig. .A large number of blood biomarkers have been studied using different methodologies, but only a few have reached clinical practice with assays available beyond the investigational context.The term cell-free DNA (cfDNA) defines DNA fragments approximately 100–200 bp long, which are generated and released into the circulation by cells undergoing apoptosis. While this is a physiologic process, the amount of cfDNA may increase in conditions of cellular damage. In the field of transplantation, cellular injury of the graft derived from immune cell attack should result in a net increase of the amount of cfDNA of donor origin. In line with the notion that cfDNA is a marker of cellular injury, a rise in cfDNA may also be reflective of other causes of allograft injury, i.e., infection and acute tubular injury. Quantification of cfDNA of donor origin may therefore represent an effective and minimally invasive way to monitor rejection. In general, a positive correlation between high levels of donor-derived cfDNA (dd-cfDNA) and development of acute rejection was reported for lung, liver, heart, and kidney, including in pediatric recipients .Even though this marker is promising, several areas of uncertainty remain. The first one concerns how to technically detect cfDNA: the principle behind dd-cfDNA quantification relies on the genetic differences between donor and recipient. The most accurate and sensitive methods are based on the analysis of panels of dozens or hundreds of single nucleotide polymorphisms (SNPs) performed using next-generation sequencing (NGS) approaches, which are quantitative. However, these methods are presently characterized by a long turnaround time of analysis and high costs, therefore limiting its application in the routine management of transplanted patients.Given its high specificity and sensitivity in detecting donor DNA in a recipient’s blood, droplet digital PCR (ddPCR) is a valid alternative to NGS, providing a quantitative approach with contained costs .The second area of uncertainty concerns how to quantify cfDNA and how to define an actionable threshold. The most common way to quantify cfDNA of the donor is as a percentage of the total. While this is a convenient method, it may be troublesome in the presence of infections, which can cause damage to the recipient affected cells and tissues, thereby increasing the total amount of recipient cfDNA and causing an underestimation of cfDNA of donor origin. The issue of the threshold remains perhaps the most critical point in the transfer of the assay in a clinical setting. In general, thresholds are dependent on the “total transplanted mass.” Consistently, the percentage of dd-cfDNA in liver transplanted patients is the highest, followed by lung, kidney, and heart.The third area of uncertainty concerns how to use cfDNA: the issue of the value of cfDNA to monitor transplant rejection remains debatable . In geneLastly, it has to be noted that the use of cell-free DNA in children poses additional problems, linked to the possibility of obtaining enough material from liquid biopsies and to optimizing cut-offs for this population. Recent data, however, have confirmed that in heart transplant recipients, children behave similarly to adults, with cfDNA being a very accurate predictor of rejection episodes , 71. TheIFNG, IP-10, ITGA4, MARCH8, RORc, SEMA7A, WDR40A) predicted rejection 7 weeks in advance of traditional markers. Furthermore, resolution of the rejection episode in response to therapy resulted in the return of gene expression levels to baseline values. Another study identified a 17-gene peripheral blood signature in patients with subclinical TCMR who received a protocol biopsy 3 months post-transplant within the GoCAR study (Genomics of Chronic Allograft Rejection) [Peripheral blood gene expression profiling has also been used in independent studies to highlight a minimum set of genes that can predict rejection. In the Kidney Allograft Immune Biomarkers of Rejection Episodes (KALIBRE) study, Christakoudi and colleagues  studied jection) . The tarCXCL10, FCGR1A, FCGR1B, GBP1, GBP4, IL15, KLRC1, TIMP1) was defined in a cohort of 49 patients with ABMR. This panel, validated in an independent cohort, proved to be of good diagnostic accuracy for times of stable graft function and of graft dysfunction, in the first and subsequent years. The 8-gene assay showed a good correlation with microvascular inflammation and transplant glomerulopathy, but not with the histological grade of the TCMR lesions [In the multicenter prospective BIOMARGIN study, an 8-gene assay ( lesions .The k-SORT (Kidney Solid Organ Response Test) 17-gene signature was defined in the multicenter Assessment of Acute Rejection in Renal Transplant (AART) study, also including a cohort of children: this assay proved predictive with a 93% positive predictive value, of both TCMR and ABMR 3 months before biopsy in the discovery and the validation cohort . In anotThe CTOT-08 prospective multicenter trial enrolled adults on a surveillance biopsy protocol and used a 57-gene expression profile signature that efficiently discriminated between stable grafts without histological signs of rejection and subclinical rejection at 24 months with good accuracy. Thanks to the elevated negative predictive value for subclinical acute rejection (NPV 88%), this assay would potentially allow avoiding unnecessary biopsies in patients with negative results . These dThe identification of blood proteins with biomarker significance requires a high resolving fractionating method, to be able to fish informative proteins at extremely low concentrations and overcome the confounding prevailing presence of plasma proteins deriving from normal tissue homeostasis.Cibrik et al.  identifiExtracellular vesicles (EVs) derived from plasma have been investigated so far only in a few non-cross-validated studies  due to tgp130, SH2D1B, TNFα, and CCL4) significantly higher in the ABMR than TCMR subjects [In a small cohort study of 28 adults, plasma density of EVs with surface expression of C4d/CD144, as marker of endothelial injury, was associated with ABMR, correlated to biopsy severity and was modulated by rejection treatment . Anothersubjects . While EThe assessment of virus-specific T cells (Tvis) is a functional marker of immunosuppression that was prospectively investigated in a pediatric phase 2 randomized multicenter study. Serial assessment of CD4 Tvis against adenovirus, cytomegalovirus, and herpes symplex virus allowed personalized steering of immunosuppressive therapy with a net reduction of the exposure to unnecessary overimmunosuppression, reduced infection events, and comparable kidney function .Another approach, leading to conceptually similar information, is the quantitative assay of torque teno virus (TTV) load, which allows one to simplify the complex laboratory technique used for Tvis analysis. TTV is a non-pathogenic virus present in the majority of individuals, with increasing replication in parallel to the increase of immunosuppression status. TTV load can represent a direct biomarker of the immune status, being potentially useful to steer drug dosage to target the proper balance between rejection and excess infection, with good performance . Two ranAllorecognition is a complex phenomenon that cannot be thoroughly captured by the traditional biomarkers in use, which measure organ function, but generally fail to predict rejection before it becomes clinically evident. Recent experimental approaches have highlighted novel possibilities that will allow us to monitor graft health, modulate levels of immunosuppression, and ultimately to eliminate or reduce the need for protocol biopsies.Even if these approaches have been successful in several clinical trials, they still need to reach wide clinical application. This is particularly true in the pediatric setting, where the small patient numbers often preclude systematic testing of novel assays to diagnose rejection. Large and validated studies, addressing also the pediatric population, are therefore needed to take advantage of these highly versatile tools non-invasively and longitudinally over the long run.Supplementary file1 (DOCX 15 KB)Supplementary file2 (DOCX 46 KB)Below is the link to the electronic supplementary material.


We at Biomarkers Journal had opportunity to report “a clue of persoanlized immunotherapy” . In the


Social media (SoMe) is increasingly important in surgical education and may be necessary in the current learning environment. Whilst expanding in use and applications, few studies detail the impact of SoMe on measurable outcomes. The goal of this study was to quantify the impact of a dedicated SoMe strategy on engagement metrics for surgical research.A retrospective review of a peer-reviewed surgical journal’s Twitter microblog platform  was performed from 6/2015 to 4/2021. A formal SoMe strategy was introduced in September 2018. Data were stratified into 2 time periods: pre-intervention (6-2015 to 9-2018) and post-SoMe intervention (9-2018 to 4-2021). The main outcome was the impact of the SoMe strategy on user engagement with the Twitter platform, journal, and traditional journal metrics. Twitter Analytics and Twitonomy were used to analyse engagement.p < 0.01) was associated with significant engagement growth, including new monthly followers  and interactions with posted articles . Article downloads increased twenty-fold post-SoMe intervention , with significant increases in traditional journal metrics of new subscribers (+11%), article submissions (+24%), and impact factor (+0.9) .From conception to analysis, the microblog published 1198 original tweets, generating 5 million impressions and 231,000 engagements. Increased account activity (increased tweets published per month—5.51 vs 28.79; SoMe directly impacts traditional journal metrics in surgical research. By examining the patterns of user engagement between SoMe and journal sites, the growing beneficial impact of a structured social media strategy and SoMe as an educational tool is demonstrated. Social media (SoMe) is a collective term for online-based applications that facilitate seamless connection and communication between users . SoMe ofWhilst increasing in use, little work to date has measured if SoMe increases the reach and impact of surgical research published by journals . JournalColorectal Disease. Secondly, we aimed to quantify the influence of social media on evidence-based medicine and surgical education. Our hypothesis was that a dedicated SoMe strategy would translate to increased traffic to the journal’s surgical research that was measurable, generalisable, and reproducible.The primary aim of this study was to evaluate the impact of a dedicated SoMe strategy on engagement metrics with surgical research published within one international journal—Colorectal Disease journal from June 2015 to April 2021 was performed. Analysis was stratified into 2 time periods: firstly, pre-intervention  and post-intervention , following the implementation of a targeted SoMe strategy. The SoMe strategy consisted of daily posting, consistent branding, and a defined weekly tweet structure, under the leadership of dedicated SoMe editors and team members. User engagement was defined as SoMe and website interactions, journal traffic, and article downloads, which were assessed using Altmetrics, Twitonomy, Twitter Analytics, and univariate analysis. The main outcome measures were to identify changes in user engagement with the journal before and after the implementation of the SoMe strategy.A retrospective review of user engagement metrics for the SoMe microblog platform (Twitter®) for the Twitter Analytics and Twitonomy (Digonomy Pty Ltd) are online analytics tools which monitor and analyse patterns of user engagement with tweets. The definitions used by these SoMe analytical tools are summarised in Table The outcomes are described in 3 parts: firstly, changes in account activity ; secondly, engagement with followers ; and lastly, account growth (mean number of new account followers per month). New monthly follower data is only available from July 2016 using Twitter Analytics, although the total number of followers from June 2015 to June 2016 is available.n) and percentages (%) for occurrences. Comparative analysis was performed using Student’s paired t-tests. Statistical significance was set at an alpha level < 0.05.Statistical analysis was performed using descriptive statistics, with frequency , generating almost 5 million impressions and over 230,000 engagements. At the time of writing, the total number of tweets published by the @ColorectalDis handle (inclusive of retweets without comment) was 3409 tweets. Mean monthly comparisons between the 2 phases are demonstrated in Table From 25th June 2015 to the time of writing (30th April 2021), the p < 0.01) were observed. Similarly, the total number of tweet impressions increased from 269,152 in Phase 1 to 4,721,198 impressions for tweets posted in Phase 2, with an average of 1251.87 impressions per tweet pre-intervention versus 4802.85 impressions per tweet post-intervention. The total number of interactions with published articles was significantly higher post-SoMe than pre-SoMe .Significant increases in the total and mean monthly tweets posted before (215 vs 5.51) and after the SoMe strategy . The engagement rate in Phase 1 was 4.06%, rising to 5.01% in Phase 2. The total number of URL link clicks relating to published tweets also increased between the 2 phases, with 3533 URL clicks in Phase 1. This increased ninefold to 30,863 URL clicks in Phase 2.p < 0.01) new followers per month in Phase 2. This is also reflected by an increase in new subscribers . At the time of writing, the account has 12,283 followers. Article downloads increased nearly 20-fold . Article submissions increased by 24% during this timeframe (p < 0.01), as did the journal’s impact factor by 0.9 (p < 0.01).Trends in increasing activity and engagement were also accompanied by significant differences in the number of followers, with a monthly average of 38 new followers per month in Phase 1 versus 213 (Increased tweet activity corresponded to specific events such as the Association of Coloproctology of Great Britain and Ireland (ACPGBI) Annual Meetings, weekly ‘takeovers’ by six junior editors, and themed ‘tweetchats’ which used the hashtag #colorectaldischat. Some examples of approaches to SoMe engagement used by @ColorectalDis are listed in Table Colorectal Disease and increased their presence within the colorectal surgery community. For the foreseeable future, a designated multimodal SoMe approach is likely to remain important in maintaining journal relevance and being representative of and of interest to the journal’s readership.This study highlights the advantages that a dedicated social media strategy has for surgical education and journal engagement with its stakeholders. In just 2.5 years, the benefits of a motivated core group without commercial SoMe experience or specific training have rapidly increased the reach of authors’ work within Whilst Twitter is not necessarily the most popular global social media platform in terms of crude user numbers, the platform and its interface have a well-established clinician and patient base , 18. AltWith huge increases in the @ColorectalDis follower base and higher engagement rates, the journal may be considered an ‘influencer’ account within colorectal academia . ‘InflueColorectal Disease is a peer-reviewed journal, caution must be considered regarding tweet content, as it is a digital and public representation of the journal. This places increased responsibility on those managing professional and educational SoMe accounts to post responsibly. Guidance on the ethical usage of SoMe in the UK is provided by the GMC [As with all SoMe platforms, caution must be exercised with their usage. Whilst  the GMC , which a the GMC , 32. Thi the GMC . Other c the GMC . A furth the GMC , 36.n = 2270)); therefore, the map may not necessarily be a true representation of where @ColorectalDis followers are based. Whilst the editorial committee (including junior editors) encompasses a wide variety of surgeons and surgeons-in-training from across the world with differing subspecialty interests, there is always room for increasing diversity of thought and collaboration which will in turn reduce the likelihood of an ‘echo chamber’ effect. A further limitation is that the impact factor of a journal may also be influenced by the publication of articles of greater interest to the wider public and readership or by greater overall numbers of submissions to journals. With the wider adoption of social media, other surgical Twitter accounts have seen an increase in followers. However, we did not have access to other surgical accounts to assess their metrics. It is not possible to confirm the causality of whether the increase in @ColorectalDis metrics was due to the new strategy or due to the increasing popularity and engagement with #SoMe, including other social media platforms.Limitations within this study include an inherent English-language bias. Whilst efforts are made for the journal to be of interest to a global audience, the distribution map of followers has clearly demonstrated a predisposition to English-speaking countries, despite the increasing presence of Spanish-speaking (amongst others) surgeons using Twitter . A distrThe relationship between surgical academia and social media has changed drastically in the last 10 years. Guideline updates, surgical innovation, the sharing of ideas, and networking amongst global peers are now only ‘clicks’ (or tweets) away. For journals to remain relevant to their readership, a designated social media strategy is necessary. With increasing popularity, Twitter remains the ideal SoMe platform for journals to engage regularly with their stakeholders, and SoMe is likely to bear increasing importance in determining and influencing a journal’s impact factor. This study has demonstrated that a dedicated SoMe strategy has increased access to the journal’s content, increased user engagement and number of article downloads, and increased traffic to the journal’s website, providing further evidence that SoMe activity may advance surgical research and education, thus leading to a paradigm shift towards alternative academic metrics.


When handling large wounds, zone of injury is a key concept in reconstructive microsurgery, as it pertains to the selection of recipient vessels. Historically, surgeons have avoided placing microvascular anastomosis within widely traumatized, inflamed, or radiated fields. The harvest of vein grafts facilitates reconstruction in complex cases by extending arterial and/or venous pedicle length. To illustrate the utility and fidelity of these techniques, this paper reviews the indications and outcomes for vein grafting in ten consecutive patients at a single tertiary referral center hospital. The case series presented is unique in three aspects. First, there are two cases of successful coaptation of the flap artery to the side of the arterial limb of an arteriovenous loop. Second, there is a large proportion of cases where vein grafts were used to elongate the venous pedicle. In these 10 cases, the mean vein graft length was 37 cm. We observed zero flap failures and zero amputations. Although limited in sample size, these case data support the efficacy and reliability of long segment vein grafting in complex cases in referral centers. Microsurgery is the technical means through which free tissue transfer is accomplished. In large cancer and trauma referral centers, free tissue transfer involves using skin, muscle, and/or bone to restore massive soft tissue defects in the head and neck, trunk, and extremities. Naturally, executing a free flap requires meticulous planning, including careful flap and recipient vessel selection . “Zone oThe following manuscript describes a case series of ten consecutive patients in a single tertiary referral center in which free tissue transfer was used to restore traumatic or oncologic defects. Descriptive details of the procedures, employed techniques, length of vein grafts used, and outcomes are provided as evidence of the efficacy, reliability, and reproducibility of large segment vein grafting in even the most intricate reconstructive cases. To bolster the significance of these findings, the outcomes from our case series are juxtaposed against the existing literature on long vein graft free flap tissue transfers ,4,5,6,7.Ten consecutive patient charts at a single institution  over a two-year period were studied to elucidate details about the nature of the problems requiring reconstruction, the technical details of the operation, the size of the wounds, and the length of the vein grafts used. Complications such as hematoma, flap loss, microvascular thrombosis, re-operation, and time to soft tissue healing were recorded and tabulated. Institutional review board approval was obtained for the recording and publication of these data (ID: 1052396). All patient photos were de-identified to the fullest extent possible. Written consent for photograph release and for publication of these data were obtained. Vein grafts were used when flaps could not be inset using standard pedicle lengths, especially in cases involving heavily traumatized, scarred, or radiated fields. Consent was obtained from patients pre-operatively. Sonographic vein mapping was performed in one case using vascular lab services. Perioperative chemoprophylaxis was routinely administered. Non-constrictive and well-padded splinting was employed in cases involving extremities to avoid motion when long vein grafts spanned joint soft tissues. Vein grafts used were typically inset under wide subcutaneous tunnels to allow coaptation to recipient vessels. Arteriovenous (AV) loops were allowed flowing for at least 30 min before division, enabling the correction of vasospasm prior to flap coaptation. Multiple types of AV looping techniques exist. Type 1 construct consisted of a flap vein sewn or coupled end to end to a vein graft, and the downstream limb of the vein graft was then sewn or coupled end to end to the recipient vein. There was a total of seven Type 1 vein grafts in seven patients. All seven cases were for efferent connections.Type 2 and Type 3 constructs comprised the arteriovenous loop vein graft group. Type 2 involved creation of a traditional AV loop, in which an end-to-end connection was formed between the vein graft and the recipient artery and vein. This loop was then divided in the same surgery (one-stage AV loop) or in a separate procedure (two-stage), creating afferent and efferent vein segments which were then connected end to end to flap vessels. Type 3 involved a size-mismatched AV loop. In this type, a standard AV loop was created as in Type 2. Unlike Type 2, however, the flap artery was sewn end to side, rather than end to end, to the afferent vein graft stump at the time of loop division. Two AV loop cases  were connected to free flaps in a single stage, while the remaining case (Type 3) was performed in two stages to allow scheduling of a definitive free flap. In the two-stage AV loop, the arteriovenous fistula was left in place under skin flaps and then divided in a separate procedure six days later to allow for OR free flap scheduling.Type 4 consisted of a Y shaped connection to recipient vessels, using vein graft branch points to supercharge flow to or from the flap. Type 4 vein graft case referenced in The diagrams in This case series demonstrates the feasibility of massive flap reconstructions in heavily traumatized wounds using vein grafts as well as the reliability of these techniques across multiple surgeons. Three different surgeons at the same institution were involved in the care of these patients, and excellent outcomes were achieved in terms of flap viability, limb salvage, and amputation-free postoperative courses. Despite the increase in operative time required for vein graft harvest and vascular anastomosis, long segment vein grafting led to successful outcomes in this particular series. All patients exhibited dependable soft tissue healing and experienced successful follow-up, resulting in a 100% salvage rate and a 0% need for post-flap revision amputation. These outcomes compare favorably to the success rates reported in the established literature. Notably, Bost et al., Henn et al., Momeni et al., and Brumberg et al. all reported complex cases involving interpositional vein grafts and arteriovenous (AV) loops, with flap failure rates ranging from 3% to 20.3% ,5,6,7. MTakebacks or unplanned operations occurred in both acute and delayed post-operative phases. In the immediate post-operative period, venous thrombosis presented in the form of an expanding hematoma, which serves as a sentinel sign of venous compromise . The exiThe outcomes and procedures in this case series are comparable to those in the established literature for vein or AV loop grafts for free tissue transfer. Bos et al. and Hen et al. presented a series of single- and two-stage AV loops with flap failure rates of 6%, and vein graft thrombectomy rates were reported to be higher for delayed two-stage AV loop cases ,4. Lin eIn addition to reporting the reliability of these techniques, this case series demonstrates the versatility of vein grafts and highlights the feasibility of an end to side anastomosis to a stump on a vein graft. Four different vein graft constructs were implemented in this case series , and thePerhaps a common but under-reported practice among surgeons, arterial or venous supercharging can be accomplished using vein graft branch points , Type 4 In As acknowledged by multiple authors in the reconstructive literature, the thigh is an excellent reconstructive tissue bank . The thiEven though saphenous veins were preferred as a source of grafts in this case series (90% of patients), deep vein or arterial grafts can be used when superficial veins are unusable or unavailable. The thigh tissue bank is also interesting because it can provide superficial, deep arterial, or venous grafts to elongate flap pedicle length . As showSeveral shortcomings of this data set should be acknowledged. The small sample size allows for the possibility that the reliability of vein grafting is not as high as reported in this case series of ten patients. However, the technical adaptability and remarkable success rates achieved within this cohort of patients showcases the efficacy of these methodologies, which, in terms of outcomes and intricacy, are on par with the literature on vein graft free flap procedures. Lin et al. reported an average vein graft length extension of 26–32 cm for elongating arterial pedicles, whereas in this study, the mean extension reached 37 cm . Meanwhi


Correction to: Journal of Clinical Immunology10.1007/s10875-023-01517-4Due to typesetting mistake, the bottom panel of Fig. 3B (GAPDH) was left blank rather than showing the control bands.The original version has been corrected.


After the U.S. Preventive Task Force recommended against screening for prostate cancer (PCa), the early detection of PCa has moved towards clinically significant PCa (csPCa), reducing the number of unnecessary prostate biopsies and the overdetection of insignificant PCa. This paradigm change is a result of the spread of multiparametric magnetic resonance imaging (mpMRI), which has a high negative predictive value for csPCa and allows guided biopsies to be performed on detected lesions with a prostate imaging‐report and data system (PI‐RADS) score ≥3.p = 0.037, Figure p = 0.037; Figure p < 0.001; Figure p = 0.028).We retrospectively analysed a multicentre dataset of 2881 correlative men with suspected PCa, based on PSA > 3.0 ng/ml and/or abnormal DRE. After pre‐biopsy 3‐Tesla mpMRI, those with lesions with a PI‐RADS v.2 score ≥ 3 were scheduled for 2‐ to 4‐core transrectal ultrasound (TRUS) cognitively guided biopsies and 12‐core TRUS systematic biopsies. Those with a PI‐RADS v.2 score <3 were scheduled for only 12‐core TRUS systematic biopsies. The data were collected between 2016 and 2021 at three academic centres in the metropolitan area of Barcelona. The project was approved by the Vall d'Hebron ethics committee (PRAG‐02/2021). CsPCa, defined as an International Society of Uropathology grade group of 2 or higher, was detected in 1119 men (38.8%). Within a subset of 302 men (10.5%) with serum PSA > 10 ng/ml and abnormal DRE on initial assessment, 264 cases of csPCa (87.4% of the subset) were detected using the systematic biopsies described above.This study shows how calculations of PSAD made using DRE‐based prostate volume category estimates are slightly less accurate than those made using PSAD‐MRI. However, categorisation using PSA levels and DRE followed by the use of PSAD‐DRE is effective in reducing the demand for mpMRI exams. The proposed approach is in line with the EAU recommendation to promote risk‐based organised models to improve the early detection of csPCa, and 20% of mpMRI exams could be avoided while missing less than 4% of csPCa cases. A recent study found a good correlation between DRE prostate volume assessments and MRI assessments.The authors have no conflict of interest to declare.J. M. conceptualised the idea. M. T., M. C., J. M. A., P. S. and E. T. developed the concept. J. M. wrote the first draft of the manuscript. All authors were involved in editing, critical review and final approval of the manuscript.


Current clinical practice suggests using patellar and quadriceps tendon autografts with a 10 mm diameter for ACL reconstruction. This can be problematic for patients with smaller body frames. Our study objective was to determine the minimum diameter required for these grafts. We hypothesize that given the strength and stiffness of these respective tissues, they can withstand a significant decrease in diameter before demonstrating mechanical strength unviable for recreating the knee's stability.We created a finite element model of the human knee with boundary conditions characteristic of the Lachman test, a passive accessory movement test of the knee performed to identify the integrity of the anterior cruciate ligament (ACL). The Mechanical properties of the model's grafts were directly obtained from cadaveric testing and the literature. Our model estimated the forces required to displace the tibia from the femur with varying graft diameters.The 7 mm diameter patellar and quadriceps tendon grafts could withstand 55–60 N of force before induced tibial displacement. However, grafts of 5.34- and 3.76-mm diameters could only withstand upwards of 47 N and 40 N, respectively. Additionally, at a graft diameter of 3.76 mm, the patellar tendon experienced 234% greater stiffness than the quadriceps tendon, with similar excesses of stiffness demonstrated for the 5.34- and 7-mm diameter grafts.The patellar tendon provided a stronger graft for knee reconstruction at all diameter sizes. Additionally, it experienced higher maximum stress, meaning it dissociates force better across the graft than the quadriceps tendon. Significantly lower amounts of force were required to displace the tibia for the patellar and quadriceps tendon grafts at 3.76- and 5.34-mm graft diameters. Based on this point, we conclude that grafts below the 7 mm diameter have a higher chance of failure regardless of graft selection. The ACL originates at the medial side of the lateral femoral condyle and runs an oblique course through the intercondylar fossa. It is the primary restraint to anterior tibial translation and secondary restraint to internal rotation of the weight-bearing and non-weight-bearing knee . An anteOne main limitation of the literature regarding this topic is the need for more data for the quadriceps tendon graft compared to the other graft sources. Additionally, a 2022 systematic review noted a need for studies investigating the effects of diameter on graft selection . ConsideThe extensor apparatus was harvested from fresh-frozen, non-irradiated human knees from male and female donors aged 70–90 without any musculoskeletal pathology. Seven quadriceps tendon-patella-patellar ligament-tibial tuberosity complexes were obtained. Each complex was further divided into two complexes (i) a segment of the quadriceps tendon with the proximal half of the patella attached (QT-P); and (ii) the distal half of the patella, the patellar ligament, and a distal insertion of the tibia (P-PL-T) by performing a transverse cut at the midline of the patella using an oscillating saw following a methodology already used by Staubli . The expUsing a digital caliper, the exposed tendons and ligaments were marked in the thinner cross-section to measure thickness and width with an initial load of 5 N. The initial length of the tendons and ligaments was an average of 38.5 mm, measured between cemented extremities for the patellar ligament and between the cemented extremity and the suture for the quadriceps tendon. The width was measured in this minor position, and the two adjacent sections were spaced along one-third of the tendon length. The thickness was measured at three equidistant points on these reference positions. The cross-sectional area was evaluated as the width multiplication and the tissue's average thickness. After testing the entire segment, lateral portions were removed to have two remaining sequential samples of two-thirds and one-third in width. On the central one-third, measurements were taken until failure was reached, defined as either separating the tendon complex from the metal housing or separating the housing container from the arm of the machine.Stress was determined by dividing the force by the corresponding cross-sectional area measurement see . In addiThe effective mechanical behavior of the graft and the composition of both material characterization and tensioning are evaluated in a computer-simulated model that best approximates the knee biomechanics. This model was based on the Lachman Test, the most widely used clinical test to determine if the ACL has been torn . When buGiven the limitations of using cadavers, many labs report various values for the biomechanical properties of the individual grafts being tested . To balaIn choosing diameters to test in our experiment, we followed guidelines in the literature, stating that appropriate graft sizes for ACL reconstruction need to restore up to 85%–115% of the native ACL total cross-sectional area . We chosThe knee bone Geometry was reconstructed from CT scan data using a BrightSpeed  scanner with a slice thickness of 0.625 mm and pixel size of 0.426 mm. The CT images were then imported to Mimics . The tunnels shown in  for the E = 10 MPa and ν = 0.45 . The cross-sectional area of the patellar tendon was reduced by 10.64% of its initial cross-sectional area at L1 (first-third of the length) and approximately 34.61% at L2 (second third of the length). For the quadriceps tendon, the reduction in cross-sectional area for L1 was 9.77%.During mechanical testing, no specimen slippage from the fixation device occurred, and no slippage from the fixation device was observed. Patellar ligaments were generally smaller than quadriceps tendons . In the graft size of 3.76 mm, the patellar tendon graft (893.4 MPa) was 234% stiffer than the quadriceps graft (263.4 MPa), and the force increment was limited to 5.1% (R2 = 0.946). For graft sizes of 5.34 mm and 7.00 mm, the force increment was evaluated at 8.1% (R2 = 0.928) and 9.8% (R2 = 0.909), respectively.In the cases where the quadricep graft sizes were reduced to 5.34 mm and 3.76 mm, the forces were reduced by 27% and 13%. The ANOVA single factor revealed a significant difference between the three graft sizes  as an alternative to the patellar tendon. The maximum stress for the quadriceps tendon with a graft size of 3.76 mm was 43.8 MPa . IncreasWe found that within our model, the patellar tendon outperformed the quadriceps at all graft diameters. When averaging the values for graft diameter and Young's Modulus of the respective graft sources, the patellar tendon required a 10.7% greater force relative to the native ACL to induce tibial displacement compared to 5.1% for the quadriceps. This suggests that the patellar tendon yields more robust grafts for ACL reconstruction at smaller graft diameters. Additionally, the maximum stress for the patellar tendon is at least double that of the quadriceps tendon and about 3–4 times that of the native ACL. This is visualized in On the other hand, the patellar tendon has its inherent flaws. The patellar tendon was 234% stiffer than the quadriceps tendon. Additionally, past research found that the Young Modulus evaluated for the patellar tendon is approximately two times greater than that of the quadriceps tendon see  15). Th. Th15). Westermann et al. conducted a similar study using a simulated Lachman model to test graft diameters between 5 and 9 mm . HoweverRegarding the graft size of 3.76 mm, we were not surprised by the results collected. We anticipated this graft dimension would not be suitable for ACL reconstruction but chose to include it in our model to validate the diameter at which the grafts fail. Although this graft size is unsuitable for ACL reconstruction, we aimed to understand how small the graft's diameter can be before it is incompatible with ACL reconstruction, making this data point extremely valuable.Furthermore, using smaller graft diameters within the experimental design allows the model to better estimate how the total area of the grafts affect their biomechanical properties, while maintaining core constants such as width and length. This allowed us to model smaller graft sizes in the context of understating if smaller grafts are suitable for patients with smaller body frames, while only changing a single dimensional variable, namely diameter, which was already the variable under investigation within our study. A more favorable approach to investigating total area would be to adjust the width and diameters of the grafts while keep these variables within ranges that are commonly employed in surgical practice, however this was out of the scope of the research question. We were able to still gauge the importance of area on the strength of smaller grafts by using graft diameters smaller than those commonly used within clinical practice while also using these graft diameters to formally investigate the extent of which diameter affects ACL graft selection.Within our study, a few limitations can be identified. Foremost, the cadavers utilized were of a significant age, between 70 and 90. This may have impacted the mechanical properties of experimental characterization. Additionally, the Young's Modulus values found in our study were more significant than those reported in the literature compared to younger cadavers. This finding runs contrary to what has been established in the literature, as it has been well described that the quadriceps undergoes degeneration as the individual ages and should have yielded a weaker response to stress as compared to the quadriceps of younger cadavers tested by other laboratories . The difNext, we used a finite element model to conduct our experiment. We chose to use a model for several key reasons. First, using a model rules out any errors regarding preparing the grafts at their proper diameters. Additionally, variables such as diameter can be easily manipulated within a model, and various results, such as maximum stress and strain across different elements of the reconstructed knee, can be obtained from a single test. This is not attainable when using cadavers. The limitations faced with cadavers are both a lack of accessibility to adequate quantities of cadavers and many inherent flaws that can affect the data collected. These include variables such as how the cadavers were stored, how old the cadavers are, and how the test is designed and carried out. Finally, using a model of the knee in its working environment allowed us to test how diameter affects the mechanics of the joint, something that has yet to be done in prior studies investigating the effects of diameter on ACL grafts.Currently, no established experimental methodologies exist to validate such interactions between grafts and condyles. Consequently, the conclusions regarding the experimental testing of the tissues have been appropriately separated from the results obtained from the computer model, which overlooks the ACL-bone interaction , 46 entiWhile finite element modeling has its benefits, it is limited in its application and accuracy when compared to research with cadaveric models. Further testing with cadaveric models is highly recommended to conclude what has been found in this study. It would be beneficial to test the functionality of these grafts within cadavers at various diameters with 1 mm increments, starting at 7 mm and going up to 10 mm, to generate finite data to guide surgeons when considering matching functional graft sizes to their patients.When designing ACL grafts of a smaller area for use with patients that require smaller than traditional grafts, we found that the patellar tendon provided a stronger graft for knee reconstruction at all diameters. This makes the patellar tendon favorable for use as a graft source in patients requiring smaller grafts. This is further corroborated by the point that the quadriceps tendon undergoes a significantly greater degree of degeneration over time as compared the patellar tendon. For traditional graft sizes, this would not be a factor causing surgeons to favor the patellar tendon over the quadriceps tendon, however when maximally confining graft dimensions in the context of our research question, this element becomes increasingly important to consider.Assuming that a greater emphasis is placed on the utilization of smaller grafts in target populations, it will be increasingly important to choose a graft source that is stronger and retains a greater functional area as the graft ages. Even more, we conclude based on our model that grafts below 7 mm diameter are unsuitable for reconstruction of the knee. Finally, the interaction we discovered between the condyles and the graft paves the way for future investigations into how graft dimensions can be tailored to the morphometric characteristics of the knee.


Previous research has found that individuals may travel outside their home countries in pursuit of alternative cancer therapies (ACT). The goal of this study is to compare individuals in the United States who propose plans for travel abroad for ACT, compared with individuals who seek ACT domestically.Clinical and treatment data were extracted from campaign descriptions of 615 GoFundMe® campaigns fundraising for individuals in the United States seeking ACT between 2011 and 2019. We examined treatment modalities, treatment location, fundraising metrics, and online engagement within campaign profiles. Clinical and demographic differences between those who proposed international travel and those who sought ACT domestically were examined using two‐sided Fisher's exact tests. Differences in financial and social engagement data were examined using two‐sided Mann–Whitney tests.p < 0.001), raised more money , had more donors , and were shared more times  compared to campaigns that did not. The median financial shortfall was greater for campaigns pursuing treatments internationally .Of the total 615 campaigns, 237 (38.5%) mentioned plans to travel internationally for ACT, with the majority (81.9%) pursuing travel to Mexico. Campaigns that proposed international treatment requested more money ($35,000 vs. $22,650, Campaigns proposing international travel for ACT requested and received more money, were shared more online, and had more donors. However, there was significantly more unmet financial need among this group, highlighting potential financial toxicity on patients and families. We used publicly available online crowdfunding data to better understand the financial implications associated with pursuing ACT outside of the United States and determine whether the campaigns for individuals traveling internationally for ACT differed clinically, financially, or in regards to social media engagement compared with those who did not propose international travel. Understanding the clinical and financial implications of fundraising for ACT will help clinicians target these issues in cancer patient education.2The study was a cross‐sectional analysis of GoFundMe® campaigns created between 2011 and 2019 to help individuals with cancer pay for ACT. GoFundMe® is an online crowdfunding platform launched in 2010 that allows individuals to fundraise online for specific causes, including covering medical expenses. The site receives over $140 million donations each month.2.1Custom Python web scraping code was developed and used to search GoFundMe® for English‐language campaigns for beneficiaries living in the United States using the term “alternative cancer” covering campaigns published between 2011 and 2019 .The demographic, clinical and treatment characteristics of the campaigns were described. Two‐sided Fisher's exact tests were used to examine associations between patient/campaign characteristics and plans for international travel. Two‐sided Mann–Whitney U tests were used to examine differences between the two groups in the campaign money requested, campaign money received, number of donors, and page shares. Kruskal–Wallis H tests were used to compare median campaign characteristics among treatment modalities. Statistical analyses were performed using Stata, version 16.1 (StataCorp), and RStudio, version 3.6.0 (R Foundation), and 3Two hundred and thirty‐seven (38.5%) of the 615 campaigns mentioned plans to travel internationally for ACT (Table p < 0.001), hyperbaric oxygen therapy , and pharmacologic and biologic treatments  significantly more represented among campaigns with plans for travel outside the United States , traditional and folk remedies (p = 0.32), mind–body therapies (p = 0.16), or manual healing ; the international travel campaigns raised 29.7% of their fundraising goal (IQR: 10.0% to 57.6%), and domestic campaigns raised 29.9% of their fundraising goal  met or exceeded their fundraising goal; of those, 22 mentioned plans for international travel and 30 did not. The median difference between the fundraising goal and the actual amount donated was significantly larger for international campaigns than domestic campaigns (p = 0.003); the median campaign for international treatments had a financial shortfall of ‐$22,640 , and the median campaign for domestic treatments had a shortfall of ‐$13,436  and a significantly higher number of online page shares through the 10 forms of social media platforms linked with GoFundMe® .Given the substantial out‐of‐pocket cost of ACT, it is understandable that many individuals would seek help from others through online crowdfunding websites. Online crowdfunding encourages compelling personal narratives that convince both the beneficiary's real‐world community and strangers of the merit of their treatment plan and to donate to their cause.The social media engagement data from this study also gives cause for concern due to the spread of misinformation.The significantly larger number of unique donations to campaigns for alternative international treatments is interesting on several levels. First, it highlights that the financial cost associated with pursuing travel for ACT is borne not only by the individual but by their support network. Second, it did not lead to these campaigns reaching a significantly higher percent of their fundraising goal. Rather, since campaigns for international treatments were requesting significantly more money, the gap between their goal and the amount received was larger. In other words, although campaigns mentioning international travel received more money and a greater number of donations, they were ultimately less likely to secure the requested funds. This could strain a cancer patient's personal funds and create or amplify economic hardship. Previous reports have shown that patients seeking online crowdfunding for medical expenses frequently express an increased underlying financial need and request additional financial support including funds for rent payments, lost wages, food, and other urgent financial needs.In general, ACT is not covered by medical insurance, making it potentially financially toxic.Each campaign included a body of text which campaign organizers used to explain their decision to decline conventional treatment in favor of ACT and frequently included references to their associated sources which advocated for ACT. The term “cancer misinformation” has previously been defined and includes recommendations to forego conventional cancer treatments, which is inherent in use of ACT.4.1The primary limitation of the study is that it relied on campaign texts written for the purpose of soliciting financial support for individuals seeking ACT. Information content was inconsistent across campaigns, and there is the possibility of misclassification bias since some campaigns may have been for individuals seeking ACT internationally but did not specify travel plans in their campaign. Additionally, the analysis did not determine whether the international travel plans as described in the campaigns were carried out. GoFundMe® campaign organizers have the option to delete an active or concluded campaign. It is possible that a campaign's success, failure, or conclusion could influence the choice to delete a posted campaign and consequently remove the campaign from our sample. The study is limited to GoFundMe® campaigns created between 2011–2019 and does not include campaigns created after COVID‐19 travel restrictions were lifted, which could limit generalizability to new and future campaigns. All campaigns came from the United States giving the data a US‐centric bias, and the inclusion criteria of English language limits generalizability among non‐English speaking populations. The term “alternative cancer” was used to potentially exclude any patients who are likely using non‐traditional therapies or mind–body therapies with their conventional cancer treatments, or otherwise defined as complementary medicine users. In the effort to exclude those receiving complementary medicines, we may not have evaluated all users of alternative cancer therapies. However, this highly specific search criteria resulted in the largest qualitative study, known to date, of patients traveling internationally for alternative cancer therapies.5Campaigns for patients seeking international travel for ACT received significantly more money, had significantly more donors, and were shared significantly more often on GoFundMe® than those seeking ACT domestically. ACT remains an attractive mode of cancer treatment, as evidenced both by the number of crowdfunding campaigns existing to support them and the amount of money donated to them. The data presented in this study demonstrate the potential financial toxicity that results for those seeking ACT, particularly those traveling internationally. This information can help researchers and cancer providers to target these issues for future research and cancer patient education.John Peterson: Formal analysis ; methodology ; writing – original draft . Trevor Wilson: Formal analysis ; methodology ; writing – original draft . Melissa Watt: Methodology ; writing – review and editing . Joshua D Gruhl: Software . Sydney Davis: Methodology . Jaxon Olsen: Methodology . Matthew W. Parsons: Methodology . Benjamin H Kann: Writing – review and editing . Briony Swire‐Thompson: Writing – review and editing . Angela Fagerlin: Writing – review and editing . Echo L Warner: Writing – review and editing . Andy King: Writing – review and editing . Fumiko Chino: Writing – review and editing . Skyler Johnson: Conceptualization (lead); methodology ; supervision (lead); writing – review and editing .Dr. Fumiko Chino is funded in part through the NIH/NCI Support Grant P30 CA008748.The authors have no conflicts of interest to disclose.Among individuals fundraising online for alternative cancer therapies, those who mention plans to travel internationally appear to carry significantly greater financial risk. This group may be negatively impacted by not only the ineffectiveness of unproven or disproven cancer treatments but the potential financial toxicity on cancer patients and their families.Appendix S1.Click here for additional data file.Table S1.Click here for additional data file.


Lemnaceae plants have great potential for different types of techniques to valorize their biomass for use in biofuel production. For this reason, scientific interest in this group of plants has increased in recent years. The aim of this study was to evaluate the effects of salt stress on the growth and development of S. polyrrhiza and the valorization of biomass for biofuel and energy production in a circular economy. Plants were grown in a variety of culture media, including standard 'Z' medium, tap water, 1% digestate from a biogas plant in Piaszczyna , Poland) and supplemented with different concentrations of NaCl (from 25 to 100 mM). Plants were cultured under phytotron conditions at 24 °C. After 10 days of culture, plant growth, fresh and dry biomass, as well as physio-chemical parameters such as chlorophyll content index, gas exchange parameters , chlorophyll fluorescence measurements were analyzed. After 10 days of the experiment, the percentage starch content of Spirodela shoot segments was determined. S. polyrrhiza was shown to have a high starch storage capacity under certain unfavorable growth conditions, such as salt stress and nutrient deficiency. In the W2 (50 mM NaCl) series, compared to the control (Control2), starch levels were 76% higher in shoots and 30% lower in roots. The analysis of the individual growth and development parameters of S. polyrrhiza plants in the experiment carried out indicates new possibilities for the use of this group of plants in biofuel and bioethanol production.Considering the main objectives of a circular economy,  Using different pre-treatment techniques we can valorize specific biomass for specific purposes–for example the torrefaction process can significantly increase the amount of carbon in solid biofuel4.The increasing demand for alternative energy sources has caused a significant development of technologies for obtaining renewable fuels derived from biological raw materials. Reduction of greenhouse gas emissions, biodegradability, diversification in fuel sectors, development of the agricultural products market and sustainable development are the main reasons for increasing the expenditure on obtaining renewable energy from liquid fuels such as bioethanol and biodiesel. On the other hand, bio-waste treatment processes, including wastewater treatment plants, biogas plants and composting, should be adopted to prevent bio-waste from going to landfills, protect the environment and comply with European environmental policy5. Starch is an insoluble polysaccharide, a polymer of glucose residues synthesized in chloroplasts of higher plants, acting as a storage of energy generated during photosynthesis6. This sugar is produced during the day in plant organs characterized by high photosynthesis intensity, and then used at night to support continuous metabolism, therefore starch is considered as one of the basic factors regulating plant growth7. In addition, starch plays a key role in plant responses to abiotic stress caused by water scarcity, high environmental salinity or adverse extreme temperatures. Under unfavorable conditions, many biochemical processes are limited, including photosynthesis, which mobilizes starch storage and then its break-down to allow normal plant growth. These sugars also have an initiating function and along the signaling pathways together with abscisic acid (ABA) enabling plant reaction to stress8. Starch degradation in response to stress often correlates with tolerance to these conditions9. Gonzalez-Cruz and Pastenes (2012)10 proved that a drought-resistant variety of bean (Phaseolus vulgaris) degraded more starch than a drought-sensitive one11. In addition, Cuellar-Ortiz et al.12 reported that increased accumulation of carbohydrates occurred only in the drought-resistant soybean (Glycine max). It was shown that various factors, including deficiency of macro- and microelements, salt stress, pH, temperature and photoperiod might increase the carbohydrate content in plant cells14. These factors reduced photosynthesis and increased production of stored compounds that can ensure plant survival due to increased tolerance of extreme environmental conditions13.Starch next to cellulose is one of the most common biopolymers on earth. It is not only one of the main elements of the everyday carbohydrate diet of every person, but above all it is a very important substrate used for food, paper, pharmaceutical, and textiles. In recent years it is used in the energy industry for the production of fuels such as bioethanolLemnaceae aquatic plants can be an ideal source of starch for the energy industry16. Lemnaceae are common monocotyledonous aquatic plants also called water duckweed, the most popular are: Spirodela, Landoltia, Lemna, Wolffiella and Wolffia18. These macrophytes are precursors of new generation crops, their cultivation is relatively easy and cost effective19. Fast growth and high accumulation of starch are two key features of the duckweed potential as a raw material for biofuel production. Duckweed grows faster than most other plants and under favorable conditions it can double its biomass within 23–24 hours20. According to Ma et al. (2018) and Cheng et al. (2017) the starch content in Lemnaceae varies depending on the species and growth conditions, from 3 to 60% of dry matter22. The macrophytes can produce starch in an amount of about 28 t ha−1 year−1, compared to corn starch production of about 5.0 t ha−1 year−123. The ethanol yield from 1 ha of duckweed is about 50% higher than in the case of corn-based ethanol16. High starch storage by plants (Lemna minor)  was caused by lack of nutrients or lack of light. The collected duckweed biomass (from wastewater culture) contained 20.3% (w/w) total glucan, 32.3% (w/w) protein, trace hemicellulose and undetectable amounts of lignin25.Research to date has shown that 26. In plant cells, osmotic stress induced by NaCl is responsible for the formation of reactive oxygen species (ROS), the resulting oxidative stress leads to degradation of many cellular macromolecules28. However, sodium chloride is the simplest and cheapest stress factor to be used for the effective production of biomass and biofuels on an industrial scale29. Carbohydrates play a key role in the carbon economy of plants, in photosynthesis, homeostasis and lipid metabolism30. Reduction of carbohydrate content in plant tissues may be caused by decrease in photosynthesis intensity, inhibition of cell division and osmotic imbalance which occur during severe salt stress31. This stress triggers intensive starch storage in various parts of Lemnaceae plants, which makes this plant very good raw material for the production of biofuels32.Sodium chloride (NaCl) is the most common salt on earth, both of natural and anthropogenic origin. Stress caused by salinity of the environment limits the growth and development of terrestrial plants and degrades freshwater floraSpirodela polyrrhiza is one of the most popular species of duckweed, inhabiting fresh water ecosystems. Great potential for the production and accumulation of starch under various growth conditions is characteristic of this plant33. The literature data indicate that macrophytes are involved in phytoremediation, as they take up heavy metals from polluted waters and improve the overall quality of water reservoirs34. Wendeou et al. (2013) and Romanowska-Duda et al. (2019) pointed out that high expansion of Spirodela sp. in contaminated water bodies means that macrophytes, due to their phytoremediation properties, can be successfully used in the process of water purification and biomass production on an industrial scale.35. Basar et al. (2020) explored the impacts of varying doses of cellulase and β-glucosidase enzymes on the production of ethanol and methane from switchgrass. Achieved an increase in energy potential up to 90.6%36. Currently, corn is the most popular raw material used in this process, but it is the basic source of food in the world and is mainly grown for the production of feed and food. According to Chang et al. (2012), water duckweed may be an alternative solution replacing corn in the bioethanol production process. It is simply and quickly hydrolyzed and fermented to ethanol with a yield much higher than that of corn37. After enzymatic hydrolysis, the ethanol yield obtained from duckweed reached 6.42 × 103 L/ha−1, about 50% more than in the case of corn-based ethanol production, which makes Lemnaceae a competitive source of starch for the production of ethanol fuel16. When the macrophytes were subjected to so-called food stress (for 5–10 days) 31.0–45.8% starch content was observed in their dry matter, and up to 94.7% of starch can be converted into ethanol using existing corn starch conversion technologies38. In order to increase the amount of carbon content, we can use steam for obtaining better quality of bioethanol. Superheated steam production technology with simultaneous pressure and temperature control of SHS, which contributes to the development of a thermochemical conversion of biomass that is cheaper in industrial application. Precise control affects the safety of the process, i.e. limiting potential fire or explosion. The torrefaction methods used so far are not able to recover valuable by-products of the entire process39. Therefore, thanks to the use of SHS for torrefaction processes, it is possible to recover by-products and at the same time valuable products, such as, for example, formic acid and acetic acid. This method, thanks to the cessation of the combustion of products and the optimization of obtaining plant, forest and agricultural biomass, leads to a positive impact on the quality of environmental performance40. Economic profitability depends on the use of low-temperature waste heat for pre-drying the biomass and the use of steam instead of other gases such as fossil fuels or gases generated during the torrefaction process, the so-called "Torgas"44.Starch is the best raw material for the production of bioethanol due to the relatively simple conversion process47.Reactor design of the torrefaction process: Lack of economically/environmentally efficient technological options for reactor heating and pre-drying of biomass, use of “torgas” in combination with fossil fuels. Current reactor technologies are inefficient, as they require long residence time of biomass in the reactor Innovative Superheated steam (SHS)-powered torrefaction reactor: improving process control of temperature, torgas production and residence time, reducing residence time and emissions, increasing the recovery rate of by-products from condensate, improving thermal efficiency (heat recovery during water re-condensation), and allowing the use of semi-dry biomass residues (moisture content up to 60%)49.By-products from the torrefaction process: Limited knowledge of valorization options, in particular for the wastewater resulting from the condensate, which is rich in volatile organic compounds. Valorization of the condensate by-product for biofuel production  and for recovery of bioproductsS. polyrrhiza and its possible use for the production of bioethanol.The purpose of the experiment was to examine the impact of NaCl on the starch storage process in S. polyrrhiza, derived from the collection of in vitro cultures of the Department of Plant Ecophysiology of the Faculty of Bioscience of the University of Lodz, Poland. Plant samples for testing were collected and evaluated according to methodologies developed on the basis of national standards and previous research, which are in force at University of Lodz in Poland and have been used in previous studies and international publication19. Test were conducted in accordance with OECD 221 Assess the toxicity of substances to freshwater aquatic plants of the genus Lemna (duckweed).Plant collection, culture, exposure conditions and experiment variants. The experiment was conducted for 10 days under laboratory conditions on the model water plant Spirodela sp. were grown in 250 mL Erlenmeyer flasks with 100 mL liquid medium.The plants were cultured in the presence of different concentrations of NaCl and in control variants. Morphological observation was carried out daily throughout the experiment. There were three control variants (I) standard "Z" liquid medium (pH 6.4), (II) tap water (pH 7.0) and (III) tap water supplemented with 1% post-fermentation effluent from a biogas plant in Piaszczyna, Poland (pH 8.1). Medium “Z” is a standard medium for growing in vitro culture plants containing all necessary micro and macro elements. Plants were cultivated for 10 days in a phytotron room at 24 °C, under constant lighting with PHILIPS MASTER TL-D lamps with a power of 2 × 18W/840. The pH of the liquid medium "Z" was determined using a Seven Compact™ S210 pH meter. The experimental medium was prepared on the basis of previous results from a macrophyte culture obtained at the Department of Plant Ecophysiology at the University of Lodz. The experiment was carried out according to the following experimental variants:Control 1–100 mL standard "Z" mediumControl 2–100 mL of tap waterControl 3–99 mL of tap water + 1 mL of fermentation effluent from a biogas plant Control series:Different variants of medium supplemented with NaCl:Figures 2O/m−2 s−1), transpiration (mmolH2O/m−2 s−1), stomatal conductivity (mmolH2O/m−2 s−1) and intercellular CO2 concentration (µmolH2O/m−2 s−1) were marked using a TPS-2 camera . Chlorophyll fluorescence was determined using a specialized Handy PEA fluorimeter from Hansatech Instruments Ltd.On the last day of the experiment, a number of physicochemical analyses were carried out: the index of chlorophyll content in plant leaves was measured using the Minolta SPAD-502 apparatus, Japan; gas exchange parameters i.e. net photosynthesis (mmolHThe starch contents in shoot and root tissues (in fresh biomass) collected from the research variants containing increasing NaCl concentrations in the range of 25-100 mM were determined using the specialized Starch Assay Kit from Sigma-Aldrich, product Code STA20. The calculation of the percentage of starch in the samples was made in accordance with the equation indicated by the manufacturer:Equation  Formula p values ≤ 0.05 were considered significant.Statistical analyses were performed with the Statistica 12 program. The data are expressed as mean ± standard deviation (SD) of 10 independent experiments. The normality of the data distribution was assessed by Kołmogorov-Smirnov test. The homogenicity of variances was determined using Levene’a test. Differences between samples were analyzed using one way parametric analysis of variance (ANOVA) with the post-hoc Dunnett’s test S. polyrrhiza plants grown on the medium supplemented with various concentrations of NaCl and leachate after methane fermentation. RGR (Relative Growth Rate) is shown in Fig. Daily observation of morphological features, counting of new shoot segments, physical and chemical analyses performed on the last day indicated the varied sensitivity of Equation The series run on "Z" medium supplemented with 75 mM NaCl (Z3), with plant growth 7.5% higher than the control (Control1) was the most effective. In the variant with 1% post-fermentation effluent, the number of macrophyte fronds was higher by 15% (O1–25 mM NaCl) and 7% (O2–50 mM NaCl) compared to the control series (Control2). The beneficial effect of NaCl was also observed in the series with tap water alone, where the plants grown on the medium supplemented with 25 mM NaCl (W1) and 50 mM (W2) showed 3–5% better growth compared to Control3.Lemnaceae shoots (fronds) was obtained in the variant with "Z" standard medium supplemented with 75 mM NaCl (Z3). In the other variants the values were similar to the control samples.Similar results were obtained when analyzing the chlorophyll content index of Fig. S. polyrrhiza plants. In the series of macrophytes grown on "Z" standard medium, the most favorable effect and value of net photosynthesis : O1 and O4 variants (25 mM and 100 mM NaCl) higher by 3.5% and O2 and O3 variants (50 and 75 mM NaCl) higher by 10%.The intensity of the transpiration process observed in all variants grown on “Z” medium Fig. II remain2O/m2s1. This parameter was different in the series carried out on tap water with the addition of NaCl, variant W1 and W2 (25 and 50 mM NaCl) remained at a similar level as control (Control2) while W3 and W4 (75 and 100 mM NaCl ) were higher than the controls by 7 and 6.5% respectively. In the series with 1% leachate, the values of stomatal conductivity were 5.5% higher O1 (25 mM) and 4.5% lower O2, O3 and O4 .The stomatal conductivity Fig. III in th2 was determined in the series with tap water supplemented with: 25 mM NaCl (W1), 50 mM NaCl (W2), 75 mM NaCl (W3). The values obtained were quite similar and ranged from: 671–721 compared to the control (691), with variant W1 having a higher value than control 2. It should also be noted that all experimental variants containing 'Z' medium had the lowest CO2 content values compared to the series containing tap water and 1% leachate, indicating a more efficient photosynthetic process  were characterized by a high concentration of starch. In the W2 series (50 mM NaCl) compared to control (Control2) the starch level in the shoots was higher by 76% and in the roots lower by 30%. The starch contents in the other variants with tap water compared to the control (Control2) were as follows in the shoots: W1 (25 mM NaCl) higher by 14%; W3 (75 mM NaCl) higher by 3%; W4 (NaCl) higher by 10%; in the roots: W1 (25 mM NaCl) lower by 32%; W3 (75 mM NaCl) was lower by 56%; W4 (NaCl) higher by 7%.The starch content Fig.  in LemnaThe macrophytes grown on the medium with 1% post-fermentation effluent supplemented with NaCl compared to the control (Control3) were characterized by varying levels starch content in the shoots compared to the control for the O1 series (25 mM NaCl) was 34% lower; for O2 (50 mM NaCl) 61% lower; for O3 (75 mM NaCl) 42% lower; for O4 (100 mM NaCl) 51% lower. Starch content in the roots for the O1 series (25 mM NaCl) was 37% lower for O2 (50 mM NaCl) 69% lower; for O3 (75 mM NaCl) 27% lower; for O4 (100 mM NaCl) 10% higher.Spirodela plants growing on "Z" standard medium with the addition of various concentrations of NaCl showed high sensitivity to salt stress, which was reflected in the level of starch production and storage in shoot segments. Compared to control (Control 1), the starch level in the shoots was lower and was as follows: for the Z1 series (25 mM NaCl) it was 42% lower; for Z2 (50 mM NaCl) it was 29% lower; for Z3 (75 mM NaCl) it was 21% lower; for Z4 (100 mM NaCl) it was 34% lower, in the roots: for Z1 series (25 mM NaCl) it was 89% lower for Z2 (50 mM NaCl) it was at the same level; for Z3 (75 mM NaCl) it was 11% lower, for Z4 (100 mM NaCl) it was 61% higher.−1 year−1 of water lash, and this was achieved using leachate as a nutrient source, and the physiological parameters determined in the plants were significantly higher than potential energy plants50. Like other authors, we also showed in our study that S. polyrrhiza has a high starch storage capacity under salt stress conditions43. Stelmach et al. (2021) and Wielgosinski et al. (2021) proved that adverse factors such as salt stress, sudden temperature drop, higher daily light intake (DLI) or nutrient deficiency, promoted starch production and accumulation. They proved that the starch content of watercress grown at 5 °C was 114% higher than that of watercress grown at 25 °C, and confirmed that photosynthetic efficiency depends on light availability (increase in DLI) and temperature. The results of their experiment indicated that starch production varied with temperature from 5 °C to 20 °C. The above authors, by changing the culture conditions from nutrient-poor medium to tap water, achieved an increase in starch content by 26.6%51. Similar results were obtained in our work using tap water as a medium supplemented with 50 mM NaCl. We obtained significantly higher starch compactness in shoot members compared to a series of plants grown on water alone. On the other hand, Toyama et al. (2018) studied four species of water cilia  and confirmed their ability to remove nitrogen from the environment52. They cultured these macrophytes for 4 days on municipal waste, animal husbandry waste and after anaerobic digestion. They proved that biomass production and nitrogen removal efficiency from all three types of liquid waste were most effective for S. polyrrhiza. Growth rates of all four species of water lash were higher on nitrogen-rich wastes, i.e. animal waste and anaerobic digestion wastes, than on municipal wastewater. It should also be noted that ethanol and methane production was higher from the biomass of S. polyrrhiza and L. punctata grown on each leachate compared to the other substrates53.The presented results of our research relate to the result of the work of many authors, including Stelmach et al. (2021) and Szufa et al. (2019) who obtained interesting results in obtaining biomass from watercress. The authors of the above work obtained a biomass yield of 39.1–105.9 t haLemnaceae species grown in ponds with leachate has been shown to be comparable to waste biomass (sugarcane), intercrops 47, aquatic plants (water hyacinth and water lettuce)54 and Lemna minor52, Wolffia globosa55 or Wolffia arrhiza56, and microalgae such as Chlamydomonas reinhardtii57 and Chlorella vulgaris59. Our study confirmed that salt stress differentially affected the level of starch in the shoots and roots of S. polyrrhiza and was dependent on the concentration of NaCl.The ethanol production potential of some Lemnaceae strains have been proven to have varying sensitivity to the amount of stored starch, strain optimization and ecotoxicological analyses of individual Lemnaceae strains are needed before they can be used on a large scale in the energy industry.In addition, macrophytes growing in polluted water bodies with high salinity levels can not only produce large amounts of biomass through starch storage, but can also reduce the level of salinity in the environment. Since 16. On the other hand, Sree et al. (2015), in a study of four strains of S. polyrrhiza, showed a slight increase in the starch content of the cells, but in this experiment the level of NaCl used was much higher at 450 mM18.The results obtained in our study confirmed previous literature reports and indicated the relationship between starch accumulation in plants and the effect of salt stress on them. Similar findings were presented by Xu et al. (2011), who analyzed the effects of 10, 20 and 30 mM NaCl on plant growth, as well as on starch content, which was 13.4 and 18.7% higher than in the control sample, respectively60. Yin et al. (2015) analyzed biomass productivity in relation to bioethanol production and showed that it was not the quantity of biomass, but its quality, including plant starch content, light conditions and photoperiod, that was most important. It was shown that a 14-percent increase in grain yields due to fertilization with 120 kg Nxha−1 translated into increased ethanol production of 254.2 Liters per hectare61. S. polyrrhiza, on the other hand, could be a valuable feedstock for bioethanol production in the future due to its rapid biomass growth and high starch storage capacity62. However, the degree of starch production and accumulation depends on the species. In a study conducted by Ma et al. (2018) in 2017, different strains of L. aequinoctialis and S. polyrrhiza isolated from different latitudes were selected to determine their potential for bioethanol production22. The most favourable results were obtained with L. aequinoctialis 6000 plants, where biomass production was 15.38 ± 1.47 gm−2, starch content was 28.68 ± 1.10%, and starch production was 4.39 ± 0.25 gm−2. This strain had the highest starch production after 8 h of exposure, and tap water proved to be the most favorable substrate; interestingly, salt stress in the form of NaCl did not induce starch accumulation in plant cells.Grajek et al. (2008) pointed out that access to low-cost starch and cellulosic feedstocks is crucial for bioethanol production. The development of innovative plant growth technologies for bioethanol production is a priority for the distilling and energy production industryLemnaceae indicated that salt stress had a positive effect reflected in increased starch accumulation in plants, however, some stressors  could inhibit the vegetative development of duckweed more effectively than a decrease in photosynthesis. Photosynthetic low carbohydrate production leads to low utilization by growth-related metabolism17. However, the varying range of plant responses to different concentrations of NaCl causing growth inhibition dependents on the species of plant and may indicate that macrophytes have alternative mechanisms for the use of excess energy.Sree et al. (2016) using 16 varieties of macrophytes S. polyrrhiza strain. First, the duckweed was grown under optimal conditions (nutrient-rich water) to produce a large amount of biomass, and then the plants were transferred to growth-limiting conditions (distilled water), resulting in the accumulation of starch. Sree et al. (2015) proposed an alternative one-step method of limiting the growth and stimulation of starch storage in water duckweed based on the use of salt stress in the range of 50 to 100 mM NaCl. Our results presented in this paper confirm these observations. In our experiment 50 mM NaCl most effectively stimulated starch accumulation in S. polyrrhiza.Xu et al. (2011) used the two-step method to increase the starch content in Lemnaceae to salt stress. The increased starch content observed in our experiment at lower NaCl levels, as in other studies, may be attributed to metabolic modification toward resistance or reduced toxicity of the stress factor. These processes may be related to the adaptive photosynthetic apparatus (mainly PSII), synthesis of various compounds, including glycerol, sugar, other osmoprotectants and specific proteins52. Increased NaCl in the medium also inhibits photosynthesis in Wolffia arrhiza, as evidenced by impaired electron transport and inactive photosystem II reaction centers31.In our laboratory experiments, we obtained 10% starch content (tap water plus a variant of 50 mM NaCl). We suggest that future experiments should take into account the different adaptive strategies of S. polyrrhiza, L. minor and L. gibba18. These observations may lead to the conclusion that the increase in salinity induces osmotic stress26 and activates the antioxidative plant defense system64. Similar effects were observed during starch analysis in plant roots53.In our study, the decrease in the starch content in the roots was observed at the concentration of 100 mM NaCl. Salinity stress-induced inhibition of vegetative growth was reported for several duckweed species, including L. punctata plants were caused by abscisic acid53. ABA supplementation increased the percentage of starch from 2.29% to 46.18% after 14 days, with a total starch level 2.6-fold higher compared to the control group. They showed that endogenous ABA increased biomass efficiency production and promoted the accumulation of starch by duckweed, but also affected the level of other endogenous hormones, it increased zeatin riboside and indole-3-acetic acid, and decreased gibberellin. In addition, abscisic acid regulated the activity of enzymes involved in starch biosynthesis and duckweed catabolism. The activity of enzymes involved in starch biosynthesis increased, while the activity of enzymes catalyzing starch degradation decreased after using ABA. Liu et al. (2018) concluded that ABA might promote biomass and starch accumulation regulating endogenous hormone levels and the activity of key enzymes associated with starch metabolism53.NaCl increased starch accumulation is mainly due to osmotic stress caused by primary stress. Plant responses to osmotic stress are regulated by abscisic acid (ABA). Liu et al. (2018) proved that high biomass growth and starch accumulation in 2/day of dry matter, and its efficiency was documented at 55 tons/ha/dry weight26, so much higher compared to the energy plants used so far.Rapid growth of high-quality duckweed biomass is considered a key step to its use for the production of biofuels. Under ideal conditions, water duckweed could double its biomass in 16–48 h. The rate of growth of water duckweed can reach 12.4 g/mLemnaceae Fig. The experiments showed multidirectional productivity of aquatic plants Lemnaceae an alternative to the plants now used for bioethanol production . Production of energy plants poses many problems including limitation of the area for their growth, harmful effect of adverse weather conditions, high water consumption. The production of the water plants Lemnaceae with the use of biogas plant waste meets the requirements of sustainable technology of renewable energy resources and sustainable recycling economy65.All-year vegetation period, quick and high biomass yield, cost-efficient production and high starch content make water plant Lemnaceae as compared to the other energy plants, from the technological point of view is much more effectively used for bioethanol production. They do not require preliminary heat treatment, which in standard procedures is time consuming and costly. Lemnaceae biomass is produced in bioreactors or in open water bodies. It can be used as a multi-protein supplement of animal feed, as a bioindicator to assess environmental pollution and in phytoremediation during processing of municipal and agricultural waste.The high (up to 70%) starch content and low content of lignin and hemicellulose in Spirodela sp. in response to abiotic stress such as high salinity of the environment was able to increase starch production in comparison with other popular energy plants. The use of duckweed as a source of starch for the production of bioethanol allows to create a wide spectrum of new possibilities for this group of plants. High accumulation of starch with low lignin content increased ability to absorb nutrients such as nitrogen and phosphorus from leachate, increased absorption of CO2 due to intensified photosynthesis make S. polyrrhiza, similarly as other water duckweeds, a promising raw material for the production of biofuels66. Our experiment confirms the effective use of aquatic plants in the production of biofuels, which creates opportunities for the development of innovative, alternative and cost-effective energy sources, and is confirmed by the 2023 review publication.The obtained results indicated that S. polyrrhiza plants as a fuel, especially in view of the benefits that its potential production entails in terms of less environmental contamination, agricultural activation and reduced oil import costs.In recent years, scientific advances in the energy sector have fundamentally contributed to the development of several environmentally friendly technologies and processes, particularly in the production of biofuels including bioethanol. These solutions allow the production of biofuels from plant biomass and the development of clean green technologies. In recent years, there has been a growing interest in the production of bioethanol as a liquid fuel for powering motor vehicles. Ethanol can be produced from products containing monosaccharides as well as polysaccharides including starch and represent promising feedstocks for larger-scale bioethanol production. Technological advances are enabling improved production techniques and cost reductions that will favor the production of bioethanol from