IntroductionThe cross-sectional area (CSA) and length of an individual's hamstring tendons are both variable, making it challenging for the operating surgeon to generate an ideal size graft during arthroscopic anterior cruciate ligament (ACL) reconstruction surgery. If we can predict the hamstring graft diameter using MRI , this information, if obtained before surgery rather than after the harvesting of the hamstring tendons, may influence the graft choice and allow us to successfully perform ACL reconstruction with sufficient graft diameter.AimsThe aims of this study were to determine the reliability and accuracy of 3T MRI in predicting quadruple hamstring graft diameter for ACL reconstruction, to determine the statistical correlation between the CSA of hamstring tendons on MRI and intraoperative quadruple hamstring graft diameter, and to find out the minimum CSA of hamstring tendons (ST+GR) required for an 8 mm quadruple hamstring graft diameter.MethodsThis prospective diagnostic study included 50 patients diagnosed with complete ACL rupture. On MRI, we assessed the CSA of the semitendinosus and gracilis tendons at two levels: one at the joint line and the other at the level where the anteroposterior (AP) diameter of the medial femoral condyle is longest. The quadruple ST+GR graft was passed through 0.5 mm increments of holes in the block  or Smith & Nephew, London, United Kingdom) intraoperatively, and the diameter of the hole that permitted smooth passage of the whole graft was taken as the quadruple graft diameter. The cutoff CSA required for a graft of sufficient size was calculated using simple logistic regression analysis. The correlation between CSA measurements on MRI and intraoperative quadruple hamstring graft diameter was determined using Pearson's rank correlation coefficient.Results2, the minimum CSA was 14.45 mm2, and the maximum CSA was 23.8 mm2. Pearson's correlation between the intraoperative quadruple hamstring graft diameter (mm) and ST+GR CSA on MRI was 0.838. The minimum ST+GR CSA required for an 8 mm quadruple hamstring graft diameter is 17.5 mm2.The mean ST+GR CSA on MRI was 18.9 mmConclusionA strong statistical correlation between ST+GR CSA on MRI and intraoperative quadruple hamstring graft diameter was found . Thus, MRI is a reliable radiological investigation that can be used to predict the quadruple hamstring graft diameter. This method can help orthopedic surgeons successfully perform ACL reconstruction surgery without any graft complications.  The anterior cruciate ligament (ACL) is crucial for stabilizing the knee joint. The ACL prevents excessive forward tibial movement and internal tibial rotation -2. ACL rGraft reconstruction is now the most popular and recommended method for restoring ACL function and preserving knee stability. It can be an autograft or an allograft . The graIn recent years, hamstring tendons, peroneus longus, patellar tendon, and quadriceps tendon have been the most widely utilized grafts globally . In receIn this prospective study, we aimed to perform a preoperative assessment of hamstring tendons (semitendinosus and gracilis) using magnetic resonance imaging. The measurements were validated and correlated with those documented during an ACL tear's surgical reconstruction. Based on the observation and results of this present prospective study, we were able to determine the ability of MRI to predict autografts of sufficient size in arthroscopic ACL reconstructions. We hypothesized that musculoskeletal radiologists and orthopedic surgeons, with combined efforts, would be able to estimate the graft size based on MRI findings.This prospective diagnostic study was approved by the Institutional Ethics Sub-Committee (IESC) of Dr. D.Y. Patil Medical College and Research Centre in Pune . We conducted this study on 50 patients admitted to the department of orthopedics, DPU, Pune, diagnosed with primary complete ACL rupture, and informed written consent was taken from all 50 patients. This study was conducted between June 2019 and July 2021. Patients who were all fit for surgery were scheduled for arthroscopic reconstructive surgery of the ACL rupture after pre-anesthesia check-ups.Inclusion criteriaPatients who have been diagnosed with a primary complete ACL tear and are more than 18 years of age but less than 60 years of age were included in this study.Exclusion criteria Patients who were medically unfit for surgery, patients who had failed primary ACL reconstruction surgery, patients who had multi-ligament injuries to the knee joint in addition to the ACL tear, and patients who had semitendinosus and/or gracilis tendon injuries were all excluded from the study.Preoperative MRI assessment of hamstring tendonsPrior to surgery, the semitendinosus and gracilis tendons were assessed using MRI. All the scans were conducted by a single musculoskeletal radiologist and measurements were recorded. The hamstring tendons were analyzed preoperatively on multiple views of MRI for all patients in the study, and the CSA was documented before surgery by orthopedic surgeons involved in the study. The radiologist who participated in this study was not informed of the predicted values for quadruple graft size. The following were the imaging specifications for the MRI system used in this study. The model used was MAGNETOM Siemens Vida , magnetic strength: 3 Tesla, planes: axial and sagittal; and the sequences were: Proton density/T2 weighted with fat saturation.The freehand area of interest tool in the picture archiving and communication system (PACS) was used for MRI measurements. In this study, we used sagittal and axial views on MRI to measure the CSA of the hamstring tendons (ST+GR), one at the joint line Figures -1B and tOperative procedureAn orthopedic surgeon harvested the semitendinosus and gracilis from the injured lower limb using a standard short linear incision of 3 cm length at the distal attachment of the tendons. The muscle fibers were removed and clipped after the tendons were harvested as part of the graft preparation. The lengths of the gracilis and semitendinosus were measured and documented intraoperatively using a sterilized standard metric stainless steel scale. To construct a quadruple hamstring graft, the ends of harvested tendons were stitched using polyester sutures (ethibond or fiber wire), and both tendon grafts were folded once in the center. In 0.5 mm increments, the graft was passed through the holes of the block  or Smith & Nephew ), and the diameter of the hole that facilitated seamless passage of the complete graft was taken as the quadruple graft diameter . Quantitative data were presented as mean and standard deviation (SD). Pearson's rank correlation coefficient was used to determine the relationship between the two-scale variables. A two-tailed p-value of less than 0.005 was considered significant. A simple logistic regression analysis was used to determine the cutoff values for the ST+GR cross-sectional areas on MRI to prepare a quadruple hamstring graft with a diameter of 8 mm.This prospective research study included 50 patients with a mean age of 37.86 years (SD = 10.99 years), with the highest being 58 years and the lowest being 18 years. There were 40 (80%) males and 10 (20%) females in the study  patients were injured on the right side while the remaining 24 (48%) were injured on the left side  patients were injured due to road traffic incidents while the remaining 16 (32%) were injured while participating in sports  was 18.9 mmThe mean intraoperative quadruple hamstring graft diameter was 8.64 mm, the minimum was 6.5 mm, and the maximum was 10.5 mm. The mean intraoperative semitendinosus graft length was 29.74 cm, the minimum was 23 cm, and the maximum was 40 cm. The mean intraoperative gracilis graft length was 27.36 cm, the minimum was 21 cm, and the maximum was 33 cm  had a quadruple hamstring graft diameter of less than 8 mm, 30 patients (60% of the study population) had a quadruple hamstring graft diameter of between 8 mm and 9 mm, and 13 patients (26% of the study population) had a quadruple hamstring graft diameter of more than 9 mm  and ST+GR CSA on MRI was 0.838. According to this study's statistical results, MRI has a high statistical significance  for predicting intraoperative quadruple hamstring graft diameter Figure . As asseRevision ACL surgeries after primary ACL reconstruction are reported to account for around 5% . AccordiMany studies have been done to predict quadruple hamstring graft size using anthropometric equations and parameters, and many have analyzed the correlation between anthropometric parameters  and quadruple hamstring graft diameter. Several authors have used radiological modalities, such as MRI, USG, and 3D CT, to determine the hamstring tendon graft size prior to the surgery. We found that imaging modalities like MRI and USG had a better correlation with intraoperative graft measurements than anthropometric parameters. The most important finding of this prospective study was that we found a very strong statistical relationship between the CSA of the ST and GR tendons on MRI and the intraoperative quadruple hamstring graft diameter. Pearson’s correlation between the intraoperative quadruple hamstring graft diameter (mm) and ST+GR CSA on MRI was 0.838.2, and its probability was 88%.Bickel et al. reported that the only level was used to measure CSA of ST and GR  . Their s2. Hollangel et al. reported a strong statistical correlation between the combined CSA of semitendinosus (ST) + gracilis (GR) on MRI and intraoperative quadruple hamstring graft diameter (p < 0.001) and the overall interclass correlation coefficient (ICC) was 0.96 [2. Hollangel et al. used two levels on MRI to measure the CSA of hamstring tendons; one at the point where the medial femoral condyle is widest and the other at the joint line [Hanna et al. reported a strong statistical correlation between the combined CSA of semitendinosus (ST) + gracilis (GR) on MRI and intraoperative quadruple hamstring graft diameter (p < 0.001), and the overall interclass correlation coefficient (ICC) was 0.977 . In theiwas 0.96 . In theiint line . Erquiciint line . A studyint line . In the int line . Further2 = 0.36-0.41) between anthropometric parameters and intraoperative hamstring graft diameter [2. According to the data, although there is no significant difference in cutoff values among various studies, variability does exist. For example, Erquicia et al. reported a cutoff value of 25.5 mm2 for an 8 mm graft [2 for a 7.5 mm graft diameter [2 and 17.5 mm2 for 1.5 T MRI and 3 T MRI groups, respectively [2. Wernecke et al. recommended a CSA of 27 mm2 of ST+GR on MRI for reliably predicting the graft diameter for double-bundle ACL reconstruction (p = 0.06) [Tuman et al. conducted a retrospective study, which included 106 study samples with a female to male ratio of 55:51 . They stdiameter . Pinheirdiameter .The mindiameter . Hollangectively . Hanna eLimitationsStandardized graft sizing equipment was used to measure the diameter and length of the tendon, and these measurements may not be accurate to micrometers. In this prospective study, a single musculoskeletal radiologist measured all the cross-sectional areas on MRI using the freehand region tool. Thus, in this study, inter-observer variation was not considered, and the efficacy of the freehand region tool has not been standardized yet.This study showed that ST+GR CSA on MRI and the intraoperative quadruple hamstring graft diameter had a strong statistical relationship. We conclude that MRI is a reliable radiological investigation that can be used to predict the quadruple hamstring graft diameter. Further, preoperative assessment of autografts provides better preoperative planning and graft selection options for ACL reconstruction. This method can assist orthopedic surgeons in performing ACL reconstruction surgery successfully and without graft complications. In this study, intraoperative quadruple hamstring grafts were correlated with MRI measurements, and it would be interesting to see whether MRI prediction works for other multistranded grafts as well.


We are pleased to present this Editorial to the readership of Resuscitation Plus following the journal’s successful launch in 2020. Resuscitation Plus, is a companion title to the well-established journal Resuscitation.At the time of going to press, Resuscitation Plus has accepted 188 articles from around the world . The journal is pleased to have been indexed in the Directory of Open Access Journals, the Emerging Sources Citation Index, Scopus and PubMed Central. The journal is supported by an excellent editorial board of existing and emerging resuscitation leaders see .Table 2EArticles published in the journal have been downloaded 170,635 times. The journals most downloaded articles include a randomised controlled manikin trial evaluating suction-based airway devices,The journal is pleased to have established a collaboration with the International Liaison Committee on Resuscitation (ILCOR).The European Resuscitation Council,Social media is an important tool to further the reach and impact of scientific research. The journal has launched a Twitter profile to assist with dissemination. The PlumX tool indicates that articles published in the journal have been shared on Facebook or Twitter close to 4000 times. The recent appointment of Tommaso Scquizzato as social media editor will drive forward our communication via social media platforms. Follow the journal on Twitter at @Resus_Plus, retweet to your colleagues and peers, share your articles published in the journal, and join the conversation online with other experts in the fields of cardiac arrest and CPR.Whilst it can take several years for a journal to receive an impact factor, we are optimistic the journal will receive one in the near future. Bibliometric scores of course provide just one lens for assessing a journal’s success. We prefer to judge the success of Resuscitation Plus through the quality of articles published and their impact on resuscitation practice and patient outcomes. With the journal’s desire to support the next generation of resuscitation scientists, we particularly welcome submissions from those early in their research careers. We hope the mentorship programme, being developed with the European Resuscitation Council will help accelerate that aspiration.The journal wishes to extend its international research and we particularly welcome articles from low and middle income countries who may benefit from the Research4Life subsidy programme (www.research4life.org). The journal’s reviewers are critical to the journal’s success and we celebrate and acknowledge their contributions. As a small gesture of thanks, the journal is pleased to waive article processing fees for the journal’s most active reviewers.As Resuscitation Plus continues on the journey to becoming the number one choice for researchers who wish to share methodologically sound, open access articles related to resuscitation, we look forward to the number and type of submissions continuing to grow.The editors receive support from Elsevier. All authors have volunteer roles with the International Liaison Committee on Resuscitation and their regional / national resuscitation councils.


Adopting a configurational perspective, this study explored the pathways for small and medium-sized enterprises (SMEs) to achieve high levels of radical innovation. On the basis of dynamic capabilities theory, six causal conditions for radical innovation were identified at both external and internal levels—that is, environmental turbulence  and absorptive capacity . The results of a fuzzy-set qualitative comparative analysis (fsQCA) of 82 Chinese SMEs identified four solutions for high radical innovation. The six causal conditions interacted interdependently and different combinations of these conditions were equally effective pathways for SMEs to achieve radical innovation. Hence, SMEs could generate radical innovation through flexibly allocating resources and capabilities based on the environmental circumstances. By using the fsQCA method, this study contributes to the related literature with an investigation of the complex causal relationship between environmental turbulence, absorptive capacity, and SMEs’ radical innovation. The results resolve some prior contradictory findings and provide new insights for future research. Other theoretical contributions, practical implications, and directions for future research are also discussed. Small and medium-sized enterprises (SMEs) are important contributors to job creation and economic growth worldwide . In the Dynamic capabilities theory  states tAccording to dynamic capabilities theory, absorptive capacity, defined as firms’ ability to “identify, assimilate, and exploit knowledge from the environment” , p. 569,In addition, SMEs’ resource constraints increase their vulnerability to external changes . Thus, tThe fuzzy-set qualitative comparative analysis (fsQCA) method , 2000 isstatus quo, obtain a competitive advantage, and guarantee growth  as the outcome. The truth table contains 2 outcome . We iden outcome . The tru outcome . Frequen outcome . Consist outcome . As reco outcome .The truth table analysis makes counterfactual reasoning about logical remainders and provides three types of solutions: complex, parsimonious, and intermediate solutions , 2008. IThis study identified four pathways that were sufficient for SMEs to achieve high radical innovation. The overall consistency was 0.806 and the overall coverage was 0.540. Solution 1 includes the presence of technological turbulence, R&D intensity, transformative learning, and exploitative learning as a configuration for high radical innovation. The outcome would not be affected whether market turbulence and explorative learning were present or absent. This solution highlights the importance of developing and maintaining the firm’s own core competence. As mentioned, the firm’s knowledge base is its most unique resource for standing out from others and obtaining competitive advantages . AlthougSolution 2 indicates the configuration of the presence of technological and market turbulence, R&D intensity, exploratory learning, and transformative learning, with market turbulence, R&D intensity, and transformative learning as the core conditions. In this solution, exploitative learning is indifferent, which shows that when both technology and customer needs change rapidly, internal exploitation is less valued. Instead, the need for exploratory learning increases, which echoes previous research into dynamic capabilities . In addiSolutions 3a and 3b indicate two pathways to high radical innovation under stable environments, where technological and market turbulence are both absent. These pathways share the core conditions of the absence of market turbulence and the presence of exploitative learning. These two solutions show that in a relatively stable environment, SMEs can capitalize on exploitative learning to achieve radical innovation . ExploitWe also conducted a sufficiency analysis for the absence of radical innovation . The frequency and PRI consistency thresholds were still set at 2 and 0.70, respectively. Considering the consistency distribution, the consistency threshold was set at 0.80. This study has examined the relationship between environmental turbulence, absorptive capacity, and SMEs’ radical innovation, detected several configurations for SMEs to achieve high radical innovation, and made some theoretical contributions as followed. First, we contribute to the research literature on SMEs and radical innovation. We examined how SMEs achieve radical innovation from a configurational perspective and identified several equally effective pathways. In comparison with traditional variance-based methods, such as multiple regression and structural equation modeling, which emphasize the “net effect” between variables, fsQCA focuses on the complex causal relationships through configurational comparative analysis . Based oSecond, we resolve some previously conflicting findings by revealing multiple pathways to high radical innovation. Among these pathways, the underlying conditions can be present, absent, or indifferent, showing that there exist alternative explanations for SMEs to achieve radical innovation. This causal asymmetry of fsQCA is particularly useful for understanding the heterogeneity of business entities and their different ways of surviving and achieving success in turbulent business environments. Previous research has adopted different perspectives and approaches to hypothesize and test the relationship between environmental turbulence, absorptive capacity, and SMEs’ radical innovation and has reported contradictory findings e.g., . HoweverThird, this study also contributes to dynamic capabilities theory and the absorptive capacity literature. As mentioned earlier, few research studies examine the complementary effects of the two dimensions of absorptive capacity see . MoreoveThis study provides several practical implications for SMEs. First, every firm should develop its own core competence. While some SMEs gain a foothold in the market through imitative innovation, this is not a long-term option. SMEs can only fully take advantage of explorative learning in a stable business environment where trends can easily be recognized. To compete and succeed in today’s dynamic environment, SMEs should invest more resources into their R&D and the establishment and maintenance of their own knowledge base. Second, explorative or exploitative learning can both lead to high radical innovation. This depends on the SMEs’ careful evaluations and choices because explorative learning is neither easy nor costless. When the external environment changes rapidly, especially when technological and market turbulence is at high levels, the potentially valuable external knowledge becomes difficult to recognize and assimilate. Therefore, SMEs should balance exploration and exploitation based on their internal knowledge base and external environmental circumstances. Finally, the transformative learning process should not be ignored because turning nascent technology into marketable products or services is an extensive process. Thus, firms should focus on maintaining their knowledge base over time so that related knowledge can be reactivated and applied when needed.Some limitations of this study should be addressed in future research. First, we used the snowball sampling method, which may limit the generalizability of the findings. However, as indicated by Drawing on the dynamic capabilities theory, this study has conducted a fuzzy-set qualitative comparative analysis of the relationship between environmental turbulence, absorptive capacity, and SMEs’ radical innovation. The results indicated that the identified causal conditions interacted in a complex way and that different combinations of these conditions can equivalently lead to high radical innovation. Thus, SMEs could achieve radical innovation through flexibly allocating their limited resources to R&D intensity and the three learning processes based on their environmental circumstances. SMEs should prioritize investment in R&D and transformative learning to store technological and market knowledge so that they can later respond quickly to changes. SMEs should also decide whether to explore or exploit depending on the environmental conditions.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Hubei University. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The standard surgical treatment for ACL tear is ACL reconstruction. There is a debate of a choice between autograft or hybrid graft for treating ACL reconstruction. The purpose of this paper is to compare both case scenarios.  A lot of libraries were searched like PubMed, Cochrane, and EMBASE Library for clinical trials which were then compared and analyzed via meta-analysis. The systematic review and meta-analysis were performed as per PRISMA guidelines, and RevMan software was used to perform the meta-analysis.  We analyzed 6 studies where patients of both autograft and hybrid graft were studied. The study outcomes, graft failures, graft diameters, reoperations, and so on were compared via forest plot and funnel plot. No significant difference was noted in both cases.  In this meta-analysis, the performance of both autograft and hybrid graft was similar. Though the diameters were larger in hybrid, other factors also had an influence like graft failures, reoperations, and age at reconstruction which must be further investigated in detail. Anterior cruciate ligament reconstruction (ACL/ACLR) is one of the most recommended procedures by surgeons in case of sports injury. Every year more than 100,000 patients require ACL reconstruction (ACLR) in US. Even though due to better tendon, bone healing chances, autologous graft reconstruction is a popular choice. However, many evidences have shown that 8 mm autografts have no guarantee of good prognosis mainly in young patients .Knee ioint is comprised of multiple articulations which in turn is responsible for handling multiple physical activities and responding to loads. ACL is a ligament which connects femur to tibia and hence gets torn due to pivot movements leading to knee instability.The main aim of the surgery is to recreate the anatomy of the ACL thus stabilizing the knee joint and hence preventing rupturing of the graft due to technical flaws in the procedures. This surgery is a treatment option for the young athletes or for those professionals who demand stability in the progressively degenerating knee conditions with long instabilities. This treatment resolves and fulfills the occupational demands for those who pursue sports as a profession –5.Commonly opted graft options are semitendinosus-gracilis autograft, various allografts, bone-patellar tendone-bone autograft, and distal quadriceps autograft , 7.A very popular choice of graft is semitendinosus-gracilis autograft which has been proven in many research studies as it has shown high patient satisfaction, knee laxity, patient outcomes, and various other functionality tests when compared against autografts , 8, 9.In this paper, we have performed a systematic review and meta-analysis on anterior cruciate ligament reconstruction with hybrid graft in comparison to autograft so as to analyze the risk benefit and success failure cases and get a comparative analysis of both treatments.A search of data was performed using Ovid of Medline (1950 to May 2021), EMBASE (1974 to May 2021), AMED (1985 to May 2021), and CINAHL (1982 to May 2021) using MeSH terms in order to identify the papers of randomized and nonrandomized clinical trials thus successfully comparing the outcomes of hybrid graft versus autograft in anterior cruciate ligament reconstruction. The PRISMA for selecting the studies is shown in An attempt was done to contact each of the corresponding authors of the papers included in the study after thorough screening. This was done to avoid any type of omitted citations. Trials included in the study were on basis of type of grafting, postoperative rehabilitation, gender, surgery type, and case complexity. Papers were scrutinized on basis of relevancy of publication which was not a part of initial search strategy.(1) Single case reports, incomplete datasets, guidelines, editorials, and review papers were excluded from the study. (2) Cases who were below 16 years of case were not considered in the study. (3) Incomplete details like where time range of surgery was not specifically mentioned were not included. (4) Papers mentioning ACL repair were excluded. (5) Non-English papers were excluded from the study. (6) Studies without any control group were excluded. (7) Full version of text missing was excluded. (8) Nonclear outcome was excluded.Investigators selected the articles on the basis of the inclusion criteria as follows: (1) patients with ACLR; (2) nonclear outcome was excluded; (3) reoperation rate, graft failure percentages, postoperative knee scores, complications in surgery, and outcomes reported; and (4) on the basis of study design: RCTs  and non-RCTs.Five meta-analyses were performed using RevMan 5.4.1 , graft failures, measures of stability, and knee scores between the hybrid grafts and autografts groups.


Brain Communications! The volume of our published papers has steadily increased, despite the difficulties, many researchers have faced during 2020. Thus, in 2021, we are moving to having more issues per year. Since Volume 3, Issue 1 opened in January, we have continued to have growing submissions and have had the privilege to publish fascinating and important work. A couple of original research articles to highlight are: Kalyanaraman and team’s discovery of a pentanucleotide expansion in intron 4 of the SAMD12 gene as a causal mutation for autosomal dominant cortical tremor, myoclonus and epilepsy in a unique ethnic group in South IndiaWelcome to Volume 3, Issue 2, of Brain Communications authors, reviewers and readers. I hope this community will continue to grow. Please join the discussions online with our @BrainComms twitter account, or do get in touch if you have any ideas for letters to the editor, scientific commentaries on our papers, or field potential articles about advances in translational neuroscience and career development of early career researchers in the field. At the first Brain conference in March, I got the chance to briefly introduce our journal to the >700 participants before hearing some inspiring scientific talks, teaching talks and perspectives on the history of the journal Brain by Alastair Compston. Hopefully many attendees will be inspired to engage with us after hearing these talks and seeing the excellent posters at the conference.In our last issue, we received a letter to the editorBrain who have officially started their tenure running the journal. They have implemented some formatting changes which you will soon see in our papers as we will keep the style consistent in the family.Finally, I would like to welcome Prof Masud Husain’s new team at our sister journal The cover image for this issue comes from Balestrini et al.The authors report no competing interests.


Further, our research extends existing ideas on sport-specific adaptation by showing that tendon stiffness seemed to respond more to exercise activities that included an aerial phase such as running and jumping. Interestingly, the comparison of twin pairs revealed a high variation of Achilles tendon stiffness (305.4–889.8 N/m), and tendon adaptation was only revealed when we controlled for genetic variance. Those results offer new insights into the impact of genetic variation on individual Achilles tendon stiffness, which should be addressed more closely in future studies.Achilles tendon adaptation is a key aspect of exercise performance and injury risk prevention. However, much debate exists about the adaptation of the Achilles tendon in response to exercise activities. Most published research is currently limited to elite athletes and selected exercise activities. Also, existing studies on tendon adaptation do not control for genetic variation. Our explorative cross-sectional study investigated the effects of regular recreational exercise activities on Achilles tendon mechanical properties in 40 identical twin pairs. Using a handheld oscillation device to determine Achilles tendon mechanical properties, we found that the Achilles tendon appears to adapt to regular recreational exercise at comparatively low intensities by increasing its stiffness. Active twins showed a 28% greater Achilles tendon stiffness than their inactive twin ( For example, An essential characteristic of running and jumping movements is a repetitive aerial phase, during which the athlete lifts both feet off the ground and then freely falls under gravity before touching the ground again . With toAlthough there is evidence for enhanced adaptations in runners and jumpers, the generalizability of the existing research is problematic because those studies are often limited to elite athletes. Additionally, those studies do not control for genetic variation, even though there is evidence that the genome of elite athletes varies in some alleles compared to non-elite athletes  participated in this explorative cross-sectional study. All participants  were required to be healthy, with no injuries of the lower limbs within the last 6 months. Each participant gave written informed consent to participate in the study. The study was approved by the local ethics committee of the Faculty of Behavioral and Social Sciences and conducted according to the Declaration of Helsinki.All participants answered a questionnaire, including questions about regular physical activity , kind of physical activity , weekly training load (“How many hours do you spend exercising each week?”), and, if they remembered, total years of training (“For how many years have you participated in this sport regularly?”). Participants were considered physically active if they followed a regular training regime of 60 min/week for at least 1 year, regardless of intensity. The different exercise activities were divided into sports with and without an aerial phase. Sports with an aerial phase had to be characterized by regular movements with both feet off the ground. Three investigators  evaluated all sports independently and agreed with their decision on regular aerial phases. Sports without an aerial phase include bouldering, cycling, hiking, horse riding, nordic walking, pilates, resistance training (excluding plyometric training), speed skating, swimming, water gymnastics, weightlifting, and yoga. Sports with an aerial phase include indiaca, running, soccer, tennis, and trampoline.Once the questionnaire was answered, we determined and marked the middle of the free Achilles tendon in the twin pairs. Free Achilles tendon length is defined as the distance from the most distal insertion of the soleus muscle in the Achilles tendon to the distal insertion of the tendon at the calcaneus . Accordi®, Myoton AS, Tallin, Estonia). The device applies an external mechanical impulse to the surface of a tendon and, thereby, compresses the underlying tissue. An accelerometer then registers the response of the tendon in the form of a damped oscillation curve. Consequently, the amplitude and frequency of the sinusoidal curve are used as a surrogate measure of tendon stiffness , each separated by 1 s to allow for the vibrations to dissipate before the next impulse began. The mean of the five consecutive impulses was used to calculate stiffness. It should be noted that tendon stiffness derived from oscillation-based measurements differ quantitatively from stiffness values derived from ultrasound supported dynamometry.t-test of unrelated data (Welch’s t-test) . This analysis aimed to control for genetic predisposition. In addition to that, absolute differences in Achilles tendon stiffness were calculated for those twin pairs, in which both twins were physically inactive, and those twin pairs, in which twin A was regularly physically active, while twin B was not. The Welch’s t-test was applied to compare the two twin pairs. Finally, we plotted Achilles tendon stiffness of twin A against Achilles tendon stiffness of twin B for inactive twins pairs and those twin pairs, in which twin A was regularly physically active while twin B was not. Pearson correlation coefficients (r) were calculated to establish relationships between stiffness measurements of twin A and twin B. Correlations below 0.4 were qualitatively interpreted as weak, between 0.4 and 0.75 as moderate, and above 0.75 as strong  with and (2) without controlling for genetics. First, the unequal variance  t-test)  was appltiffness . Anothers strong .t-test. For all comparisons, we performed a power analysis to determine the effect size (Cohen’s d) of the results   . All staAcross all twin pairs , 17 diffactive = 25: 619.1 ± 121.5 N/m vs. ninactive = 8: 605.0 ± 46.1 N/m) (one active = 7), Achilles tendon stiffness was significantly higher in the active (636.0 ± 115.5 N/m) compared to the inactive twin (496.8 ± 142.7 N/m), on average by 28.0% (p < 0.01) (r = 0.83) and those twin pairs, in which twin A was regularly physically active while twin B was not (r = 0.79)  and inactive twin pairs (both twins inactive) did not reveal any significant differences (n6.1 N/m) . There w < 0.01) . Cohen’s = 0.79) . Notablyinactive = 8: 30.5 ± 26.1 N/m vs. none active = 7: 139.2 ± 86.6 N/m, p = 0.02)   . Cohen’s < 0.01) . Cohen’sThe present study was primarily designed to determine the effect of regular recreational exercise activity on Achilles tendon stiffness. To control for genetic variation, this study was conducted as an identical twin study. We identified a large variability in tendon stiffness among twin pairs, which seems at least partially genetically predetermined. When controlling for genetic preposition, the results of our study indicate that Achilles tendon stiffness was greatest in those individuals who regularly engaged in recreational exercise activities. Moreover, we found differences in Achilles tendon stiffness between individuals who engaged in an exercise activity with an aerial phase and those without an aerial phase. These findings add novel insights to the current understanding of tendon adaptation.PIEZO1, which regulates the mechanosensitive function of tenocytes, can affect tendon properties. Similar results of a potential genetic predisposition have also been found for bone properties . This observation may support the hypothesis that Achilles tendon stiffness is partially genetically predetermined. Further, it lets us speculate about interindividual variation in Achilles tendon adaptation in response to mechanical loading. In this regard, data from genetic analyses indicate a significant interindividual variation in collagen fibril assembly and vulnerability to develop symptoms of chronic Achilles tendinopathy . Relatedoperties  and muscoperties . While cAnother important factor to consider when investigating tendon adaptation to exercise appears to be the presence of an aerial phase during movement. Our findings suggest that sports that include an aerial phase affect Achilles tendon stiffness more than sports without an aerial phase. Achilles tendon differences were greatest among twin pairs in which at least one twin participated in a sport with an aerial phase. In contrast, differences in Achilles tendon stiffness were relatively small when both twins participated in a sport with no aerial phase. This finding is consistent with data obtained by Interestingly, our findings on the impact of an aerial phase on Achilles tendon adaptation contrasts with an argument by Another finding that adds to the existing literature on exercise-related Achilles tendon adaptation refers to the activity level and response to habitual exercise. Currently, most research is limited to elite athletes e.g., . In our To build further confidence in our results more twin pairs with different athletic profiles  would help. Our study shows that most identical twins share a very similar lifestyle, including similar exercise activities. However, we did not collect data on exercise activities during childhood and adolescence. It is reasonable to speculate that exercise activities during childhood and adolescence might have affected tendon properties in adult individuals. Research on the life-long turnover of human tendon tissue indicates that the tendon core is formed during height growth and is essentially not renewed after that . Thus, eBeing limited to the handheld oscillation device for stiffness measurements, this study does not provide information about morphological adaptation, such as the tendons’ cross-sectional area or other relevant material properties, such as the elastic modulus. Those properties should be assessed to develop a more comprehensive understanding of tendon adaptation. That said, a study similar to this one could be carried out using ultrasound and dynamometry. Also, ultrasound would be a more precise method to determine free Achilles tendon length. Previous research indicates considerable variation in the length of the free Achilles tendon length , but alsNotwithstanding these limitations, our study provides first evidence that the individual Achilles tendon stiffness is partially determined by genetic variation. In addition, the study has also indicates that exercise activities at a recreational level can trigger Achilles tendon adaptation if stimulated regularly for years. Our classification of exercise activities revealed that activities with a regular aerial phase seem to have the greatest impact on Achilles tendon adaptation. This finding extends an existing idea on sport-specific tendon adaptation presented by The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethics committee of the Faculty of Behavioral and Social Sciences, Chemnitz University of Technology. The patients/participants provided their written informed consent to participate in this study.FS and KL conceived the experiment, interpreted the data, and drafted the manuscript. NK and KL performed the experiments and substantially contributed to data analysis. FS analyzed the data. NK made important intellectual contributions during revision. All authors approved the final version of the manuscript and agreed to be accountable for the content of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The first WHO International Standard and International Reference Panel for anti-SARS-CoV-2 immunoglobulin were established by the WHO Expert Committee on Biological Standardization in December, 2020. The WHO International Antibody Standards are intended to serve as global reference reagents, against which national reference preparations or secondary standards can be calibrated. Calibration will facilitate comparison of results of assays  conducted in different countries. Use of these standards is expected to contribute to better understanding of the immune response, and particularly of the correlates of protection. This Personal View provides some technical details of the WHO Antibody Standards for SARS-CoV-2, focusing specifically on the use of these standards for the evaluation of the immune response to COVID-19 vaccines, rather than other applications . The explanation with regard to why rapid adoption of the standards is crucial is also included, as well as how funders, journals, regulators, and ethics committees could drive adoption in the interest of public health. Developing, licensing, and rolling out vaccines against an emerging pathogen that is declared a global public health emergency presents many challenges, including accelerated time frames for evaluating safety and efficacy of candidate vaccines. Regulatory processes must be in place for rapid evaluation of submissions and, after careful benefit–risk assessment, the use of vaccines for which a full regulatory package might not yet be available—as in the COVID-19 pandemic declared in 2020.A broad range of COVID-19 candidate vaccines are being developed via technologies and platforms that include viral vectors, protein subunits, nucleic acids, live attenuated strains, and inactivated virus preparations.2One of the important elements of regulatory evaluation is the measurement of the vaccine-induced immune response, for which guidance is available in the WHO guidelines on the clinical evaluation of vaccines.This Personal View aims to raise awareness of the need for standardisation of quantitative assays for the evaluation of clinical trials of vaccines intended for use in public health emergencies. The first step towards standardisation is the availability of a WHO International Standard to serve as a reference point for several assays of defined specificity.WHO has a unique role of supporting regulatory authorities in its 194 Member States. One of WHO's core functions is to set norms and standards and promote and monitor their implementation. The WHO Expert Committee on Biological Standardization has been active in establishing WHO standards for biologicals for over 70 years.WHO standards, both written and measurement , are based on scientific evidence and provide the basis for establishing and updating national regulatory requirements. Their development is supported by WHO collaborating centres, national regulatory authorities in many countries, pharmacopoeias, manufacturers' associations, and academia. The role of the international recommendations or guidelines for biological substances is to ensure the availability of vaccines of assured quality, safety, and efficacy for use in international immunisation programmes. Furthermore, these documents serve as a benchmark for the global acceptability of products and as a basis for defining national regulatory requirements for licensing and for post-licensure evaluation.The development of measurement standards involves elaborate collaborative studies in numerous laboratories worldwide. As examples of the measurement standards for COVID-19, the first WHO International Standard for anti-SARS-CoV-2 immunoglobulin with an assigned unitage of 250 International Units (IU) per ampoule  and the first WHO International Reference Panel for anti-SARS-CoV-2 immunoglobulin were established by the WHO Expert Committee on Biological Standardization on Dec 10, 2020.WHO initiatives are also closely linked to the standardisation of vaccines. Strengthening of national regulatory authorities is an important element in assuring the quality of vaccines worldwide. Prequalification of vaccines is a key mechanism through which WHO ensures the safety and efficacy of vaccines to be supplied by UNICEF. The safety of vaccines and the issues discussed by the WHO Global Advisory Committee on Vaccine Safety are also important, as are WHO activities related to immunisation policy led by the Strategic Advisory Group of Experts on Immunization.In 2019–20, a new type of coronavirus was isolated from human respiratory epithelial cells.The S glycoprotein of SARS-CoV-2 is responsible for the virus attachment and entry to the target cells, initiating the infection process while antibodies against it neutralise infectivity. Immune responses against the S protein are key to host protection during SARS-CoV-2 infection, and it is therefore considered the most attractive target for vaccine and therapeutic development.Several groups are conducting, or have completed, clinical trials to evaluate the efficacy of their candidate vaccines against SARS-CoV-2; comparison of results between studies, or even between different assays in the same study, has proven difficult. The AstraZeneca–Oxford University vaccine ChAdOx1 nCoV-19 (AZD1222) was the subject of a phase 1 clinical trial, for which the data have been published.This approach would also be useful for comparing data from different trials. An adenovirus-vectored vaccine produced by CanSino Biologics  was used in a clinical trial at two dosage levels.A third study involved the vaccine from the Gamaleya Institute in Russia.It is impossible to say whether these three vaccines induce similar responses. There is no reason why the results from these three studies should agree in numerical detail. The ELISAs are formatted differently with different antigens, and the output is in arbitrary in-house units. The formats of the neutralisation assays also differ, and the GMT and seroconversion rates cannot be compared with confidence. In this situation, the inclusion of a single reference antibody preparation would potentially make it possible to compare the different products and trials. In the absence of such a reference, it is clear that all three vaccines induce an immune response, but it is impossible to judge which, if any, is inducing higher antibody responses. Such a comparison would facilitate the defining of protective antibody titres and the possible establishment of efficacy and potency thresholds.The WHO International Standard for anti-SARS-CoV-2 immunoglobulin facilitates comparison between clinical trials by expressing the results of neutralisation assays from different laboratories relative to the International Standard, which has an assigned potency in arbitrary units (IU). An arbitrary unit was chosen instead of a physical measurement  because polyclonal immune sera are complex mixtures of molecules with different specificities, but with a common biological effect  which forms the basis for the range of relevant assay systems. Convalescent plasma or serum from patients are the preferred candidate standard because these samples most closely represent a clinical sample that is analysed in the assay. These samples are consistently able to reduce inter-assay variability when used as a calibrant for a range of tests, as in the case of many other viruses, including MERS-CoV.26The WHO International Standard for SARS-CoV-2 immunoglobulin was characterised in an international collaborative study launched in July, 2020, which has since been completed;The potency of the International Standard was assigned specifically for neutralisation assays, which have a clear functional biological readout. However, although the collaborative study results clearly showed less inter-laboratory variation when ELISA titres were reported relative to the International Standard, the WHO Expert Committee on Biological Standardization requested further data in order to assign an IU value to the International Standard for antigen-specific antibody binding assays, work which is ongoing. Meanwhile, the International Standard is available as a reference reagent for the harmonisation of related binding assays using an arbitrary binding antibody unit specific for each viral antigen .8The challenge in the production of high-order reference materials is that the time taken to produce them does not correspond to immediate needs.Following the establishment of the International Standard, it is now important to ensure the correct use of the antibody standard in vaccine clinical trials to assist in the interpretation of results by providing the basis for expression of antibody titres in IU. This approach is an opportunity for exploring and possibly defining correlates of protection in IU/mL. The International Standard also permits data sets across a range of assays to be compared by reference to the IU. This comparison is especially important because many vaccine candidates are being developed; following approval of the first SARS-CoV-2 vaccines, non-inferiority clinical trial design should be considered. In these cases, the role of the International Standard would be particularly important in efforts to establish correlates of protection. In future, regulatory authorities might consider using immunological data to extend licensure indications to include additional groups .The contribution of cellular immunity, which acts together with antibodies in the immune response to SARS-CoV-2, must not be overlooked. Work on variant SARS-CoV-2 strains seems to suggest that protection against moderate and severe disease can be provided by cellular responses in the absence of neutralising antibodies , although infection per se by the new variant might be compromised. Preliminary data have shown that the International Standard for SARS-CoV-2 is still able to neutralise the new variants B.1.1.7, B.1.351, and P.1, although at higher concentrations than the early 2020 isolates. As new variants emerge, the ability of the International Standard to serve as a calibrant  requires constant monitoring.All vaccine developers should report antibody results from clinical trials relative to the WHO International Standard. Although early clinical trial results might have not been reported in IU/mL because of the urgency for submitting the data for fast regulatory approval, moving forward, all vaccine developers should now calibrate their assays against the International Standard and report results in IU. Regulatory authorities should assist by requiring that data submitted to them include reporting in IU. Other incentives could be considered and include requirements from sponsors of clinical trials and funders of the vaccine development. Ethics committees should also introduce such a requirement because comparability of results greatly adds to the public health value of data. Pharmacopeial monographs would also be helpful in emphasising the importance of reporting data in IU. As with compliance with clinical trial registration, a highly impactful requirement would be if medical journals required use of the IU for publication. We believe it is time to consider such policy levers to increase usage of IU as a unique measurement system for expressing results from clinical trials in the common language of the scientific community.The comparability of results from different immunogenicity studies has long been considered an optional extra. Any risk–benefit assessment of the appropriate use of the WHO International Standard would confirm the need for a common language for communicating the message regarding the clinical performance of COVID-19 vaccines.We declare no competing interests.


The following people completed reviews for Physiological Reports in 2021. We thank these reviewers for their valuable service to the journal.


Resistant starch, microbiome, and precision modulation. Mounting evidence has positioned the gut microbiome as a nexus of health. Modulating its phylogenetic composition and function has become an attractive therapeutic prospect. Resistant starches (granular amylase-resistant α-glycans) are available as physicochemically and morphologically distinguishable products. Attempts to leverage resistant starch as microbiome-modifying interventions in clinical studies have yielded remarkable inter-individual variation. Consequently, their utility as a potential therapy likely depends predominantly on the selected resistant starch and the subject’s baseline microbiome. The purpose of this review is to detail i) the heterogeneity of resistant starches, ii) how resistant starch is sequentially degraded and fermented by specialized gut microbes, and iii) how resistant starch interventions yield variable effects on the gut microbiome. A prebiotic is defined as “a substrate that is selectively utilized by host microorganisms conferring a health benefit”.3 insulin resistance,4 metabolic syndrome,5 kidney disease,6 and schizophrenia,7 and may be especially relevant for illnesses characterized by dysregulated epithelial integrity and immune function, like inflammatory bowel disease.8Resistant starch (RS) is among the recent foci of non-digestible carbohydrate therapies. Boosting gut butyrate production has been the objective of several RS intervention studies associated with aging,9 However, the variable effects of RS on the gut microbiome are striking, whereby RS supplementation may increase butyrate production in one person and lower it in another.11 Such variability underscores the need for precision nutrition to confer health benefits. Predicting the most optimal RS for individual demands understanding the complex structures of RS granules, the enzymatic machinery required to degrade RS, and the individual baseline microbiome. Hence, the goal of this review is to:Describe the physicochemical and morphological heterogeneity of starch, and explain how these properties confer resistance to host and bacterial hydrolysis;Detail the hierarchy of gut bacteria known to degrade and ferment resistant starch; andHighlight the importance of precision resistant starch interventions.Upon reaching the colon, RS acts as a communal resource that is degraded and fermented by a hierarchy of specialized gut microbes. RS selectively feeds upstream keystone species, which produce substrates that are cross-fed upon by butyrogenic bacteria (recently reviewed in ref. 9).12 Starch that reaches the large intestine without being fully digested is termed resistant starch (RS). Resistance depends on several physicochemical features, including physical encasement in non-digestible material (type 1), native supramolecular structure and morphology (type 2), retrogradation via hydrothermal-cycling (type 3), chemical modifications (predominantly ester cross-linking) (type 4), and amylose-lipid complexes formed during cooking (type 5). Types 2, 3, and 4 RS are the primary RS types used in human studies and will be the focus of this review. Extrinsic factors, such as host amylase gene copy number,13 oro-cecal transit time,14 and amylase inhibition,15 further complicate starch bioavailability. Hence, starch digestibility should be considered as a kinetic property (slower to faster) affected by host-specific factors, rather than as a binary trait (resistant or nonresistant).12In humans, digestible starch is susceptible to hydrolysis by salivary and pancreatic α-amylases, which hydrolyze α-1,4-glycosidic bonds.16 Conversely, amylopectin forms much larger molecules  with dense networks of short chains (mean DP 15–18) branching from longer chains (mean DP 48 to 60).16 The intra- and intermolecular interactions of amylose and amylopectin impart starch granules with a complex hierarchical structure  and branching . Amylose possesses a DP below 6,300 glucose subunits, almost entirely (>99.3%) bonded by α-1,4-glycosidic linkages.tructure .Figure 117 Alternating amorphous and crystalline lamellae are thought to form 20–500 nm intermediary structures called “blocklets”19 with amorphous amylose forming an intermolecular matrix or “glue” between blocklets.20 Recent in silico modeling proposes that the blocklet architecture follows phyllotaxic rules, whereby interlocking crystalline platelets form ellipsoid fractals.21 Depending on the amylopectin branching density and chain lengths, blocklets might be more amorphous (“defective blocklets”) or crystalline .22 Blocklets arrange into arrays of alternating crystalline and semi-crystalline concentric rings, disrupted by amorphous channels and veins.23 Foresti and colleagues elegantly demonstrated that amorphous layers are preferentially degraded by soluble α-amylase, leaving behind a crystalline skeleton.24 Overall, blocklet type and arrangement are associated with surface smoothness, porosity, and resistance to hydrolysis.Starch’s supramolecular structure begins with pairs of amylopectin branch chains and amylose chains intertwining to form crystalline double helices. At amylopectin junction points , amorphous regions are thermodynamically favored over helices. Helices can either be dense and orthorhombic (A-type) or open and hexagonal (B-type).26 enriched in B-type crystallites,25 possess longer amylopectin branch chains,25 and have a smoother surface texture with fewer pores.25–27 Huang and colleagues showed that smaller, densely packed blocklets form a resilient shell on the surface of potato starch granules, while the interior is composed of larger, loosely packed blocklets.28 The surface porosity, crystallinity, and RS content of corn starch granules correlate with amylose content; high-amylose varieties exhibit less porous surfaces, higher proportions of B-type crystallites, longer amylopectin side chains, and higher resistance to hydrolysis than varieties with no amylose (i.e. “waxy” corn starch).29 Intriguingly, corn starch resistance peaks with an amylose content of 68%,30 suggesting that both amylose and amylopectin are required to confer resistance to hydrolysis.Across botanical sources, native starch granules vary by size, degree, and type of crystallinity; surface porosity and texture; relative amylose and amylopectin content; and amylopectin branch chain length and density. As a result, starch digestion rates can vary remarkably. For instance, starch granules from tubers tend to be among the most hydrolysis-resistant native starches, possibly because they are larger,31 amorphous regions absorb water, the granule swells, and amylose leaches through surface pores.19 The hydrogen bonds stabilizing the helices become disrupted, causing amylose and amylopectin to unwind and dissociate.32 Upon cooling, new helices are formed, but the degree to which crystallinity increases depends on the amylose content, cooling temperature, and number of repeated cycles. Retrogradation occurs more rapidly for amylose (over the span of minutes to hours) than amylopectin (over days), due to amylopectin’s complex branching networks.33 Colder retrogradation conditions promote the formation of B-type crystallites over A-type,35 and additional cycles of retrogradation are understood to further increase starch crystallinity and resistance to hydrolysis.36 It is thought that the formation of smaller blocklets during retrogradation is favored in starches with higher amylose content and longer amylopectin chains.37 It seems that low surface porosity, enriched B-type crystallinity, and small blocklets formed during retrogradation could confer resistance to hydrolysis analogous to the intrinsic features seen in potato starch.Retrogradation occurs when starch undergoes gelatinization followed by a thermodynamically driven reconfiguration of amylose and amylopectin into a new ordered state. First, when starch is heated in the presence of water,38 Higher concentrations of STMP and STPP (10–12% w/w) increase the degree of cross-linking and resistance to hydrolysis.39 However, lower concentrations (5%) may increase the digestibility of high-amylose corn starch (HACS), likely because the imparted cross-linking is not sufficient to compensate for the gelatinization that occurs during heat treatment.40 Cross-linking can also be induced through specific heat and acid treatment, which forms esters between glucose hydroxyl groups.41 Cross-linking is restricted to the granule surface,42 roughening its texture,43 but leaving the overall morphology intact.44Chemical treatments that introduce cross-links strengthen starch structural networks and generally increase resistance to hydrolysis. Treatment with sodium trimetaphosphate (STMP) and sodium tripolyphosphate (STPP) creates phosphoester bridges between glucose residues in amylose and amylopectin.45 In agreement, cross-linking wheat and high-amylose corn starches has been independently shown to increase RS content more than cross-linked regular corn starch.46The reported effect of cross-linking on the RS content depends on the starch’s botanical source and amylose content. Shin and colleagues found that cross-linking potato, high-amylose corn, and wheat starches results in higher RS content than cross-linking regular corn and rice starches, but precise values depend on the RS measurement method used.47 acetylation (reviewed in ref. 48),48 and octenyl succinylation.49 While these modifications increase resistance to varying degrees,50 they are generally intended to alter rheological properties,42 or encapsulate drugs for delivery to the colon.49 Furthermore, USA federal regulations require that esterification with these agents in food products do not exceed 0.1% to 2.5%,51 indicating an upper limit to the resistance of chemically modified starches in commercial products.Other starch modifications include hydroxypropylation (reviewed in ref. 47),52 cell-derived lipids can form complexes at the granule surface,53 or glucose molecules can be phosphorylated during glycan synthesis.54 In fact, around 0.5% of native potato starch glucose residues are phosphorylated,54 up to 5.5 times that of rice starch.55 These phosphate monoesters might inhibit exo-acting hydrolases,46 or sterically hinder helix packing and reduce overall crystallinity,56 particularly during retrogradation.32Summary: The available lines of evidence converge to suggest that a starch’s surface microstructure is the principal factor affecting its digestibility. Relative amylose content, amylopectin branch chain density, and crystallinity appear to influence the size, type, and packing density of blocklets, which then determine granule surface texture and porosity. Retrogradation and cross-linking modify starch surface crystallinity and intermolecular networks, respectively, thereby increasing resistance to hydrolysis. It remains unclear whether blocklets simply affect surface area and integrity, or constitute “discrete structures”57 that complement amylase active sites. Moreover, these questions tie into whether different bacteria preferentially degrade certain starches more than others based on binding site availability or recognition of discrete microstructures.Some starch modifications occurring during food processing or found in nature can affect its digestibility. Proteins can fortify food matrices,Primary degraders grow on RS in monoculture. They penetrate the outer surfaces of intact RS granules, exposing pores and deeper concentric matrices while liberating oligosaccharides and generating metabolites like lactate and acetate.59Secondary degraders grow on starch in monoculture, but degrade intact RS poorly or not at all. Instead, they may adhere to abrasions and pores on RS before participating in its degradation, and opportunistically utilize solubilized oligosaccharides produced by other RS degraders. Cross-feeders do not grow on starch in monoculture. They utilize the by-products generated by upstream degraders, helping to maintain stoichiometric equilibrium and thermodynamically favorable (i.e. unconstrained) fermentation.60 Most of the metabolites generated are acidic, which may further stabilize the ecosystem.61 Together, the subsystem of microbes involved in RS degradation and fermentation participates in a complex network of cross-feeding interactions.62 In maintaining microbiome homeostasis, the RS nutrient web expands the scope of what could be considered a “beneficial” gut microbe to a cluster of metabolically interconnected microbes.RS is degraded by the colon’s complex ecosystem of microbes, triggering a cascading web of metabolic interactions. 63 Likewise, Cerquiera and colleagues define non-primary degrader, starch-active bacteria as “secondary starch scavengers”.9 We advance that secondary degraders can be delineated by their ability to grow on starch in monoculture, while a tertiary category (cross-feeders) comprises starch-inactive microbes that play a unique and important role in transforming upstream metabolites. Lastly, some secondary degraders show a limited ability to degrade RS in vitro,65 or toggle their starch activity in the presence of fitter degraders.66 Therefore, the “secondary degrader” designation is a fluid concept, but nonetheless useful for the purpose of characterizing individualized responses to RS.Caveats with this model’s classification scheme can be raised. Baxter and colleagues consider secondary degraders and cross-feeders (described below) as butyrogenic “secondary fermenters”.67 CAZymes contain recognition and catalytic domains.68 Endo-acting α-amylases, which hydrolyze internal α-1,4-glycosidic bonds within starch molecules, primarily belong to the family of 13 glycoside hydrolases (GH13).69 Other GH families include exo-acting α-glucosidases and the starch debranching enzymes: limit dextrinase, pullulanase, and isoamylase (reviewed in ref. 70).70 Alas, most microbial GHs have not been studied in the context of the human gut.71 This has left a deficit in our understanding of precise GH ligand specificities in relation to RS microstructures.Microbial degradation of RS is initiated at the granule surface, which primary and secondary degraders must first recognize and adhere to. Generally, these bacteria possess polysaccharide utilization loci (PULs) encoding transport proteins and modular Carbohydrate-Active Enzymes (CAZymes).72 Eighty-eight CBM families have been characterized based on amino acid sequence similarity, of which 15 possess starch-binding activities .73 Early protein crystallization studies showed that CBMs 20, 25, 26, and 34 each recognize glucose residues exposed in starch helical structures.73 CBM20 possesses two separate starch surface binding sites with higher specificity for parallel amylose helices than amorphous coils, and an ability to disrupt the structure of shorter helices.74 Amylases possessing repeated homogenous (e.g. two CBM25 copies)76 or heterogeneous (e.g. CBMs 25 and 26)77 CBMs confer higher avidity and thus tighter adsorption to corn starch granules than those with single CBMs. Of note, most studies investigating CBM ligands use cyclodextrins to infer potential α-glucan helix binding, rather than RS.Starch-active GHs possess one or more carbohydrate-binding modules (CBMs), which are contiguous protein domains that facilitate substrate-specific enzyme docking and enhance catalytic efficiency (reviewed in refs. 69 and 72).78–80 For instance, CBM74 enhances surface pore formation by GHs with higher binding affinity to potato starch granules than wheat and waxy corn.78 MaAmyA, a GH13 produced by Microbacterium aurum B8.A, uses duplicate CBM25 domains to form pores on wheat, but not potato starch granules.79 AmyP, a GH13 found in marine bacteria, exhibits 10-fold higher hydrolytic activity on raw rice compared to potato and wheat starches.80 This remarkable difference may simply be due to rice starch’s higher intrinsic digestibility,26 but may also be explained by specificity imparted by its starch-binding domain, CBM69.81 At present, there is no obvious pattern by which the copy number of GH13’s or CBM family domains can predict whether a bacterial strain can degrade starch or RS  that associate via calcium-dependent cohesion–dockerin interactions.83 Across five different R. bromii strains, there are 17 conserved GH13-containing amylases (Amy1-17) that enable hydrolysis of both α-1,4- and α-1,6-glycosidic bonds.85 In addition to liberating more starch by-products  than it requires to grow,65R. bromii generates acetate, ethanol, formate, and propanol.59 Unlike other notable Ruminococcaceae family members, R. bromii has not been shown to generate butyrate.86In vitro, R. bromii strains L2-63, L2-36, 5AMG, YE282, and ATCC 27255 show amylolytic activity on retrograded (Novelose 330) and high-amylose (Hi-Maize 958) corn starches.84 In strain L2-63, these activities are higher than those on potato starch.83 Intriguingly, R. bromii L2-63 grows better on native potato starch than on cross-linked potato starch (Versafibe 1490), while it grows better on cross-linked high amylose corn starch  than on native HACS.64 Considering that the integrity of native starch reaching the colon depends on host factors, these in vitro observations may not reflect R. bromii’s substrate preference in the colon. This is supported by R. bromii’s response to RS in vivo, contradicting its preferences seen in vitro (discussed in Section 3).Bifidobacteria possess a multi-modular carbohydrate-utilization system (reviewed in ref. 9)9 enriched with GHs with specificity for a broad range of glycan substrates.87 CBMs 25, 26, and 74 are thought to work synergistically to enable Bifidobacteria to not only dock starch,9 but also agglutinate starch granules into clusters.85 This behavior is thought to sequester granules away from competing starch-degraders.88 Within the Bifidobacteria genus, only B. pseudocatenulatum strains M115 and DSM 20438,89 and B. adolescentis have been shown to digest RS in humans.65B. adolescentis comprises 0.25% to 1.4% of the fecal microbiota in European individuals.91B. adolescentis have been isolated from humans.92–94 Of the strains whose activity on starch has been studied in vitro, two (P2P3 and L2-32)93 utilize RS, while four do not .95 Nonetheless, strains that can ferment RS generate lactate, acetate, and formate.58B. adolescentis uses more of its starch degradation by-products than R. bromii, including glucose, signaling a more competitive relationship with other RS guild members.65 Intriguingly, Li and colleagues found that an unspecified strain of B. adolescentis produces butyrate,96 in contrast with prior reports that the species is not butyrogenic.58Fifteen strains of B. adolescentis shows strain-specific preferences for different RS. Strain P2P3 utilizes HACS (Hi-Maize 958) to a significantly higher degree than cross-linked corn starch (Versafibe 2470), cross-linked potato starch (Versafibe 1490), retrograded corn starch (Novelose 330), and most intriguingly, a different HACS (Hi-Maize 260).93 Strain VTT E-001561 exhibits preferential binding  to HACS (Hylon VII), and lowest binding to potato starch.94 These results require careful interpretation because potato starch granules have smaller surface area-to-volume ratios (and thus less binding area) than corn starch. Furthermore, strain IVS-1 shows preferential growth on HACS (Hi-Maize 260), lesser but considerable growth on native potato starch and cross-linked tapioca starch (Versafibe 3490), and little growth on cross-linked corn (Versafibe 2470) and potato (Versafibe 1490) starches.64 The putatively butyrogenic B. adolescentis strain described earlier will adhere to and grow better on partially hydrolyzed than non-hydrolyzed retrograded tuber starch granules, likely because of its rougher yet more crystalline surface.96 Together, these studies seem to indicate B. adolescentis has a nuanced preference for HACS over other types of RS in vitro; however, this finding has not yet been recapitulated by in vivo microbiome studies.B. choerinum FMB-1, isolated from bovine rumen fluid, degrades cross-linked potato starch (Versafibe 1490) and HACS (Hi-Maize 958), but exerts negligible activity on regular (Hi-Maize 260), retrograded (Novelose 330), and cross-linked HACS (Versafibe 2470) starches.93B. pseudocatenulatum strain DSM 20026,97 but not NCIMB 8811, grows on potato starch alone,95 and M115 grows on retrograded corn starch (Novelose 330).89 However, Novelose 330 is approximately half digestible starch by weight,98 thereby detracting the likelihood that this strain is a true primary degrader.R. bromii as compared to RS responders.5 In a follow-up in vitro study, Flint et al. cultured stools from RS non-responders with spiked-in R. bromii L2-63, which boosted RS-fermentation to levels comparable to RS responders.65 This work was seminal for developing a model whereby primary degraders (e.g. R. bromii) “unlock” RS for other community members to degrade and ferment.Primary degraders are thought to be necessary for RS degradation in the human gut. Work by the Flint laboratory revealed that individuals consuming RS whose butyrate-production did not increase (considered to be “RS non-responders”) harbored nearly undetectable levels of Secondary degraders possess extracellular amylases to degrade regular starch, but their contribution to initiating RS degradation is negligible compared to that of primary degraders. Instead, they may require primary degraders to erode smooth RS granule surfaces before adhering to RS and/or scavenging for “substrate spillover” (i.e. excess oligosaccharides generated by primary degraders).58 Conceivably, it is advantageous for secondary degraders to position themselves near primary degraders docked to RS. Among secondary degraders, preferences for certain starches over others have not been greatly elucidated by metaproteomic, co-culture, or clinical studies.Eubacterium rectale is a prominent member of the butyrate-producing Clostridium cluster XIVa, which is implicated in maintaining gut homeostasis.99E. rectale L1-86 is equipped with 11 GH13s, of which two have been deeply characterized: Amy13K and Amy13B.100 Amy13K contains duplicate CBM26 domains and additional CBM41, 82, and 83 domains, enabling E. rectale to bind to certain starch granules (discussed below).101In vitro, Amy13K hydrolyzes amylopectin with twice the activity than amylose, releasing maltotetraose and maltopentaose.100 These solubilized substrates, liberated either by E. rectale or primary degraders, may then be further hydrolyzed by Amy13B, which does not bind larger substrates.100 Finally, malto-oligosaccharides of DP less than 8 are transported into the cell via EUR_01830, EUR_31480, and EUR_01240 ABC-transporters.100E. rectale exhibits RS preferences. E. rectale DSM 17629 shows detectable growth on cross-linked corn starch (Versafibe 2470), but not cross-linked potato (Versafibe 1490) or tapioca (Versafibe 3490) starches.64 Furthermore, it can bind and hydrolyze regular and high-amylose corn starches ,65 but binds potato and cross-linked wheat starch (Fibersym) granules less efficiently.101 Human fecal inocula cultured in a batch-fermentation system revealed that E. rectale responds equally to A-type and B-type retrograded HACS.35 In this study, E. rectale’s growth lagged behind Bifidobacteria, potentially indicating a reliance on primary degraders to erode RS first.35 In agreement with its behavior in vitro, in vivo microbiome studies show that the relative abundance of E. rectale is increased in response to regular (Hi-Maize 260), retrograded (Novelose 330), and cross-linked (Versafibe 2470) HACS  HACS .Figure 2E. rectale enrichment is positively associated with increased butyrate following potato starch intervention.11 It interacts synergistically with R. bromii in co-culture,65 and these species consistently co-enrich in RS intervention studies.103 Considering the apparent reliance upon R. bromii in initiating starch degradation,65 it may ultimately be that any potential preference E. rectale (and other secondary degraders) has for different RS is overshadowed by that of its associated primary degrader, which then dictates overall RS fermentation.E. rectale, other starch-degrading Lachnospiraceae include butyrogenic species belonging to the Roseburia and Butyrivibrio genera. R. faecis M72/1 grows on amylopectin,104 but negligibly on amylose,67 and its amylolytic machinery has not been characterized. R. inulinovorans A2-194 uses Amy13A, a GH13 containing CBM41, 48, 82, and 83 domains,105 to grow on amylopectin.106 This strain upregulates amylase and flagella expression when provided with amylopectin.107 Both R. inulinivorans A2-194 and R. intestinalis L1-952 and L1-82 grow poorly on HACS compared to more readily digestible rice, corn, and waxy potato and corn starches, and B. fibrisolvens 16/4 shows the same general preferences.108B. fibrisolvens’ Amy13B contains two tandem CBM26 and CBM83 domains,101 with higher activity against rice starch than Amy13A from R. inulinivorans.108 Lastly, B. crossotus-related taxa have been found to adhere to HACS (Hylon VII).102n addition to B. thetaiotaomicron is endowed with 88 different polysaccharide utilization loci (PULs) encoding 226 GHs,109 enabling it to utilize at least 32 different types of glycans.110B. thetaiotaomicron also possesses an intricate starch-utilization system (Sus). The first PUL to be described,111 the sus operon encodes independent intracellular (SusR), periplasmic (SusAB), transmembrane (SusC), and cell wall-anchored (SusDEFG) proteins with coordinated action, briefly described in their order of action here (thoroughly reviewed in ref. 112).112 SusD is a CBM-analog necessary for growth on starch granules,113 and binds the endo-regions of α-glucan helices.114 SusG is a GH13 α-amylase that contains CBM58 and a starch surface-binding site, with a twofold higher activity on soluble potato starch than amylopectin and strict hydrolytic-specificity for α-1,4-glycosidic bonds.115 SusG cooperates with SusE and F to bind soluble and granular starch.113 Eliminating all SusEFG starch-binding sites slows growth on amylopectin derived from corn more than potato.113 While not required for cell growth,116 SusE assists in capturing DP 7–18 malto-oligosaccharides,117 and both SusE and SusF bind wound and unwound helices.116 SusC contributes to starch binding and imports malto-oligosaccharides into the cell,118 which are degraded into maltose and glucose by periplasmic SusA and B.111 Lastly, constitutively expressed SusR is activated by intracellular maltose to rapidly upregulate sus gene expression.119B. thetaiotaomicron generates lactate, acetate, and propionate, and while it does not produce butyrate,120 its metabolites drive butyrate production by other bacteria via cross-feeding interactions.121 Most of B. thetaiotaomicron’s GHs localize to the periplasm or outer membrane, thereby contributing to the soluble glycan pool for community use.122B. thetaiotaomicron may also use SusE and SusF to sequester starch from its competitors.116 Furthermore, when B. thetaiotaomicron and E. rectale co-colonize gnotobiotic mice, B. thetaiotaomicron upregulates its starch and host-glycan degradation pathways, while E. rectale downregulates its GH expression and increases expression of transport proteins, relying instead on butyrate-producing pathways to generate energy.66 Interestingly, when provided both pectic galactan and amylopectin, B. thetaiotaomicron will downregulate Sus expression and upregulate expression of other PULs, indicating a substrate preference for non-starch polysaccharides.123 Indeed, starch availability and interspecies competition may relegate secondary degraders to cross-feeders, or vice versa.B. thetaiotaomicron can utilize less than 20% of raw (Hi-Maize 958) and retrograded (Novelose 330) HACS after a 72-hour incubation, but cannot utilize a different type of HACS (Hi-Maize 240).65 However, B. thetaiotaomicron grows with these starches better if co-cultured with R. bromii or B. adolescentis, or if the starches are autoclaved or boiled.65 Hence, while B. thetaiotaomicron prefers other glycans, it is equipped with a sophisticated system to access starch, and can also influence the activity of other RS guild members.Bifidobacteria beyond those mentioned above show strain-specific potential to be secondary degraders .102Several egraders . These sB. cuniculi and B. magnum both grow better on starch when co-cultured than in monocultures.87 In contrast, only B. thermacidophilum subsp. porcinum’s growth on starch is improved when co-cultured with B. longum subsp. suis. Indeed, B. longum subsp. suis growth is unchanged, suggesting porcinum outsources starch degradation to suis.87 While these two pairs of Bifidobacteria were isolated from rabbit and pig feces, respectively, they serve to illustrate that relationships between secondary degraders range from mutualistic to commensal.Lactobacillus amylovorus uses AmyA, an α-amylase containing five tandem CBM26 repeats, to bind to raw corn starch.126Parabacteroides distasonis ATCC 8503 grows on native and cross-linked corn (Versafibe 2470) and potato (Versafibe 1490) starches in culture without a distinct preference, but achieves stationary phase after 4 days rather than 12 to 24 hours as seen with B. adolescentis and R. bromii.64 Two uncharacterized species belonging to Ruminococcaceae and Clostridiaceae have been inferred to be RS degraders that are markedly enriched in subjects consuming either HACS (Hi-Maize 260) or potato starch.63 Lastly, a sequenced taxon most closely related to Ruminoclostridium [Eubacterium] siraeum increased in relative abundance in one subject fed potato starch whose R. bromii and B. adolescentis did not enrich.11Other potential starch-degraders have been identified but studied less extensively than those listed above. R. hominis A2-183 does not grow on starch in monoculture, but it does grow in co-culture with B. adolescentis L2-32, by utilizing substrate spillover and/or the acetate and lactate produced by B. adolescentis.58 Furthermore, R. hominis A2-183 is unable to degrade amylopectin or amylose,67 suggesting that it degrades smaller malto-oligosaccharide fractions. The R. hominis genome encodes fewer GH13s (8) than R. intestinalis (12 to 13) and R. inulinivorans (10 to 12), and it does not encode GH13s possessing CBM26 or CBM41 domains.67 Considering the high prevalence of these CBMs across known starch degraders  are depleted in health conditions associated with low butyrate production, like inflammatory bowel disease.132Describing all known gut bacteria capable of utilizing these substrates exceeds the scope of this review, but one other example is noteworthy. in vitro) and intervention (in vivo) studies. Batch fermentations involve inoculating fecal material into a pH-, temperature-, and anaerobically maintained system that attempts to replicate the gut microenvironment.133–135 By administering RS to these cultures, temporal changes occurring in microbial populations can be readily monitored. While used less frequently, continuous flow fermentations involve cycling gases and substrates in and out of the system to further maintain physiologically relevant conditions.136Our nascent understanding of how individual bacteria respond to different RS can be complemented by monitoring their activity in the full ecosystem of the gut microbiome. Two common approaches are fermentation  that have not been robustly shown to recapitulate human physiology.in vitro microbiome studies include eliminating upstream host variables, predicting bacterial responses in different biogeographical regions of the colon by tweaking media pH, and monitoring changes in bacterial populations over time. For example, Lesmes and colleagues devised a three-compartment continuous-flow fermentation system with pH ranges corresponding to the proximal (pH 5.5), transverse (pH 6.2), and distal (pH 7.1) colon.35 Using stools obtained from three healthy volunteers, they found that Bifidobacteria grew equally well with B-type crystallite-enriched HACS across all pH ranges, but grew most poorly with A-type HACS at pH 5.5. Growth curves indicated that total bacteria declined over the first 10 hours of culture, followed by a compensatory climb for 14 hours, and that E. rectale’s growth lagged 5 hours behind that of Bifidobacteria when grown with HACS, consistent with their putative roles in the RS guild. Of note, B-type HACS induced a fivefold higher increase in butyrate compared to A-type after 24 hours of culture.The advantages of R. bromii enrichment is inversely proportional to the degree of RS cross-linking.40 Regular HACS significantly enriches R. bromii after 4 hours followed by a depletion in the species after 12 hours of incubation, whereas HACS cross-linked in a 12% w/w STMP/STPP solution yields a less pronounced but stable enrichment over 4–24 hours. This work also indicated that RS can potentially become too resistant to hydrolysis even for microbes specialized to degrade it. Furthermore, enrichment of R. faecis and an unclassified Clostridiales member lagged behind R. bromii, parallel to the relationship seen between Bifidobacteria and E. rectale in Lesmes and colleagues’ work.35By incubating stool with HACS cross-linked under different concentrations of STPP/STMP, Wang and colleagues showed that RapidAIM,134 which they employed to assess type 2, 3, and 4 RS’ in stool slurries collected from six healthy volunteers.135 While no effect was seen in response to cross-linked wheat starch, regular HACS (Hi-Maize 260) and retrograded HACS (Novelose 330) enriched E. rectale, R. faecis, Roseburia, and Lachnospiraceae. The retrograded HACS also enriched Bifidobacterium, Subdoligranulum variabile, and Ruminococcaceae.Li and colleagues introduced a novel high-throughput stool fermentation assay, called Supplemental Table 1). While acknowledging that these studies’ methodologies and experimental designs differ, patterns have emerged to show that RS guild members respond to RS in vivo according to RS type , Baxter and colleagues identified several co-associations between primary degraders and butyrogenic taxa. Hi-Maize 260 significantly enriched Bacteroides acidifaciens and Bacteroides ovatus, and Christensenella minuta and Ruminococcus lactaris.138 Of note, F. prausnitzii did not correlate with any of these species, yet butyrate production was significantly increased following RS intervention, indicating potentially novel butyrogenic RS guild members.After 12 weeks of a cross-linked wheat starch (Fibersym) intervention, positive co-associations between 18 pairs of bacteria were observed, including co-enrichments of 64 These co-abundance response groups (CARGs), containing 3 to 18 taxa each, were constructed by hierarchical clustering of Spearman’s correlation distances. Then, each CARGs’ constitutive taxa were summed together into a single entity for downstream statistical analyses. They found significant correlations among CARG1  and CARG7  with cross-linked tapioca starch (Versafibe 3490), and CARG3  with cross-linked corn starch (Versafibe 2470).64 Employing CARG-based analyses in this way enables the detection of underlying ecological guilds, boosting otherwise weak signals of individual taxa. Guild-based approaches have been proposed by others.139 Interestingly, Deehan et al. also reported a significant co-exclusion between R. bromii and an unannotated Ruminococcus taxon (OTU27), suggesting a potential novel primary degrader that competes for R. bromii’s niche.Deehan and colleagues identified 55 taxa that were affected by dose-dependent cross-linked RS intake, which they further categorized into seven distinct co-abundance response groups (CARGs).2 Among the 16 studies listed in Supplemental Table 1, seven reported the butyrate concentrations in fecal samples, and potato starch consistently leads to significant increases in butyrate concentrations.63 Cross-linked wheat starch (Fibersym) was also found to significantly increase fecal butyrate.138 In contrast, various HACS led to no significant changes between groups.141 Cross-linked HACS (Versafibe 2470) led to significant increases in butyrate concentrations, plateauing at a dose of 35 g RS per day, while no significant changes were seen with any dose of cross-linked potato (Versafibe 1490) or tapioca (Versafibe 2490) starches.64 In the cross-linked HACS (Versafibe 2470) group of this study, R. bromii was significantly negatively associated with the proportion of butyrate relative to acetate and propionate, yet there was a significant positive association in the cross-linked potato starch group. Lastly, retrograded HACS (Novelose 330) significantly decreased butyrate in fecal samples from British males.142 Since it is estimated that 95% of short-chain fatty acids are absorbed by the host epithelium, fecal butyrate concentration has been implicated as a low-fidelity proxy for intestinal butyrate production.143 Thus, the discrepant and counter-intuitive changes in butyrate concentrations may be explained by methodological limitations, or nuanced RS guild dynamics and underlying effects seen at the individual, but not group level, described below.Butyrate is a fermentation end-product known for its immunomodulatory bioactivities.in vivo studies report bacterial responses for each subject, showcasing how R. bromii, B. adolescentis, and E. rectale, may increase, remain unchanged, or even decrease in response to different RS depending on the individual.144 Based on the in vitro behavior of RS guild members, we would expect to see these taxa more consistently enriched following RS interventions. This discrepancy may be explained by biological factors and methodological limitations.Several 65 functional subgroup variability,145 metabolic flux sensitivities,136 and baseline diet variability.10 First, Ze and colleagues demonstrated that R. bromii can be necessary for RS degradation in vivo.65 Second, if keystone RS degraders are present in an individual, they may lack downstream subgroups required for converting RS into butyrate.145 For instance, possessing lactate-producing B. adolescentis, but lacking lactate-utilizing butyrogens  may result in a poor butyrate response to RS; likewise, if one possesses net acetate-producing R. bromii but lacks acetate-utilizing butyrogens (e.g. F. prausnitzii). Third, the dominating metabolites produced by the RS guild may exert feedback on the global microbiome structure. For instance, without sufficient microbial lactate utilization, lactate produced by B. adolescentis may accumulate, lower luminal pH,59 stress pH-sensitive microbes, and destabilize the microbiome.136 So far, these insights point to the potential utility of an RS guild member probiotic, synbiotic, or consortium which may enable RS non-responders to benefit from RS therapies. Lastly, McOrist and colleagues observed that individuals with the highest baseline butyrate production tended to decrease butyrate production following a dietary intervention high in RS.10 They commented that these individuals’ pre-treatment diets and microbiomes were likely configured optimally to generate butyrate, and exchanging their dietary patterns for the intervention diet may have disrupted their butyrogenic system. These biological factors underscore that interventions ought to be personalized where butyrate-production is a primary endpoint.Biological factors that may explain inter-individual variability include the presence/absence of keystone RS guild members,R. bromii and B. adolescentis) from their starch-inactive phylogenetic relatives  may increase, but appear to decrease simply because cross-feeders increase in relative abundance to a greater extent.146 While compositionally sensitive techniques like the centered log ratio transformation can help mitigate this technical artifact,146 microbiome responses can also be evaluated by monitoring beta-diversity . Plotting fecal microbiota compositions in two-dimensional space reveals multidirectional changes across individuals before and after RS interventions, signaling that changes are driven by different taxa communities.144 Together, these limitations illustrate the necessity of sufficiently powering RS interventions where microbiome composition is the primary endpoint, collecting critical baseline data and employing appropriate statistical techniques.Methodological limitations that may contribute to inter-individual variability include poor taxonomic resolution, underpowered sample sizes, and compositionally insensitive statistical techniques. First, 16S amplicon sequencing and genus-level qPCR primers used in several of these studies were unable to resolve and differentiate RS- and starch-degrading species and strains , are certain combinations of CAZymes necessary for effective penetration of RS, or does metabolic feedback select for certain RS guild members over others?147 but dietary records are rarely collected.To what degree do host factors (e.g. amylase gene copy number) influence RS digestion prior to entry into the colon? Incorporating these data may improve RS-selection for individuals. Habitual dietary RS intake may also contribute to variable responses,How do other kingdoms (e.g. fungi) contribute to RS degradation in the gut?148Lastly, butyrate production is not the sole indicator of microbiome functionality. Can RS restore gut dysbiosis in other ways, such as by regulating microbial bile acid or tryptophan metabolism?At present, there are several knowledge deficits surrounding these factors, which once elucidated, may enable data-driven RS-selection based on individual microbiome features:Click here for additional data file.