Not all anterior cruciate ligament (ACL) injuries are preventable. While some ACL injuries are unavoidable such as those resulting from a tackle, others that occur in non-contact situations like twisting and turning in the absence of external contact might be more preventable. Because ACL injuries commonly occur in team ball-sports that involve jumping, landing and cutting manoeuvres, accurate information about the epidemiology of non-contact ACL injuries in these sports is needed to quantify their extent and burden to guide resource allocation for risk-reduction efforts.To synthesize the evidence on the incidence and proportion of non-contact to total ACL injuries by sex, age, sport, participation level and exposure type in team ball-sports.Six databases  were searched from inception to July 2021. Cohort studies of team ball-sports reporting number of knee injuries as a function of exposure and injury mechanism were included.I2 = 82%; females: 63%, 95% CI 53–71, I2 = 84%; males: 50%, 95% CI 42–58, I2 = 86%). The overall incidence of non-contact ACL injuries was 0.07 per 1000 player-hours , and 0.05 per 1000 player-exposures . Injury incidence was higher in female athletes  than male athletes , and this difference was significant. Injury incidence during competition was higher  than during training  and these differences were significant. Heterogeneity across studies was generally high.Forty-five studies covering 13 team ball-sports were included. The overall proportion of non-contact to total ACL injuries was 55% (95% CI 48–62, This study quantifies several key epidemiological findings for ACL injuries in team ball-sports. Non-contact ACL injuries represented over half of all ACL injuries sustained. The proportion of non-contact to total ACL injuries and injury incidence were higher in female than in male athletes. Injuries mostly occurred in competition settings.The online version contains supplementary material available at 10.1007/s40279-022-01697-w. Anterior cruciate ligament (ACL) injuries commonly occur in team ball-sports –3 but weWe need to better understand the extent of non-contact ACL injuries because they impose a wide-ranging personal, societal and economic burden –14. ACL This review is on ACL injuries only and it forms part of a larger systematic review on the epidemiology of non-contact knee injuries sustained in team ball-sports. Future publications will focus on other non-contact knee injuries like gradual-onset knee injuries. The review was prepared and conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2020 statement , and washttp://www.covidence.org). Bibliographic hand-searches were also performed to supplement the electronic database search.Six electronic databases  were systematically searched from inception to July 2021. Search terms consisted of controlled vocabulary and free text, and were mapped to medical subject where possible to capture records of knee injury  epidemiology  in team ball-sports . The MEDLINE search strategy is provided in Appendix A1. All records were downloaded to EndNote X8  where duplicates were removed, then uploaded to Covidence  the number of ACL injuries as a function of injury mechanism were reported; (ii) they were prospective cohort studies or retrospective cohort studies examining routinely collected data ; (iii) they featured athletes from field and court-based team ball-sports  because ACL injury mechanisms on these surfaces were more comparable , 18, 19;Two authors (LC and MW) independently assessed study quality using a modified six-item Newcastle–Ottawa scale for cohort studies where one star was awarded for each item for a maximum of six stars . A similPublication information , population characteristics , exposure type , number of injuries, exposure, unit of exposure, surveillance information , and severity of injuries were extracted and recorded on a customised spreadsheet by one author (LC), and double-checked by another author (DS). Disagreements were resolved by a third author (MW). Sample populations were classified into three age groups: children (≤ 12 years), adolescents (13–18 years), and adults ≥ 19 years). Participation level was classified into three categories: amateur , intermediate , and elite  [ years. Pmeta  and tidyverse packages. Meta-analyses of incidence were carried out using a random effects Poisson regression model  to produce forest plots with 95% confidence intervals (CIs) [I2 statistic where I2 < 50% was considered as not important, 50–75% as moderate, and > 75% as high heterogeneity [All meta-analyses were performed in R  using the ls (CIs) , 26. Thels (CIs) , 27. Staogeneity . Betweenogeneity , 30. Conogeneity , 32, we The online database and bibliographic hand search yielded 8,015 non-duplicate studies that were screened by title and abstract: 708 potentially eligible studies were identified. Following full-text review of the 708 studies, 45 met the inclusion criteria and were included in this review  from 13 sports  , but all disagreements were subsequently resolved by consensus. Seventeen studies were awarded the maximum six stars and one study scored two stars . Studies were missing from the lower right quadrant in the funnel plot to assess publication bias in studies included in meta-analyses of overall proportion of non-contact to total ACL injuries, and this quadrant represents smaller studies with a high proportion of non-contact to total ACL injuries. Studies were evenly distributed in the funnel plot to assess publication bias in studies included in meta-analyses of overall incidence of non-contact ACL injuries.I2 = 82%)  compared to male athletes  , 58% in basketball , 54% in rugby union , 53% in soccer , and 38% in American football  (Appendix A4-1). We were unable to perform meta-analyses for the other sports because there were less than three included studies (Appendix A4-1). Only the difference between field hockey and American football was significant, as confirmed by meta-regression  (Appendix B1). There were sufficient studies to sub-group by sport and sex for soccer only. In female soccer athletes, the proportion of non-contact to total ACL injuries was 55%  (Appendix A4-2).The overall proportion of non-contact ACL injuries to total ACL injuries was 66% in floorball  and 68% in adolescents  (Appendix A5-1). After sub-grouping by age group and sex, this proportion was 60% in adult female athletes  and 52% in adult male athletes  (Appendix A5-2). There were insufficient studies to investigate injury proportions by sex in adolescents (Appendix A5-3) [The overall proportion of non-contact ACL injuries to total ACL injuries was 55% in adults  , 59, 66.I2 = 16%), 55% in intermediate-level , and 56% in amateur-level athletes  (Appendix A6-1). After sub-grouping by participation level and sex, this proportion was 65% in elite-level female athletes , and 59% in elite-level male athletes  (Appendix A6-2). In intermediate-level athletes, the proportion of non-contact to total ACL injuries in females was 58% , and in males was 50%  (Appendix A6-3). In amateur-level athletes, this proportion was 67% in females , and 48% in males  (Appendix A6-4).The overall proportion of non-contact to total ACL injuries by participation level was 61% in elite-level  in competition and 47% in training settings  (Appendix A7-1). After sub-grouping by exposure type and sex, the proportion of non-contact to total ACL injuries during competition in female athletes was 58% , and 35% in male athletes  (Appendix A7-2). This difference between females and male athletes was significant as confirmed by meta-regression  (Appendix B2). In training settings, this proportion was 68% in female athletes  and 36% in male athletes  (Appendix A7-3).The overall proportion of non-contact to total ACL injuries by exposure type was 42%    Fig. , and 0.07%) Fig. . Figure I2 = 40%) and 0.06 per 1000 player-exposures   and 0.04 per 1000 player-exposures . Only the difference between female and male athletes per 1000 player-hours was significant as confirmed by meta-regression  (Appendix B2).In females, non-contact ACL injury incidence was 0.14 per 1000 player-hours  Figs. , 4. In mI2 = 68%) in rugby union, 0.08 per 1000 player-hours  and 0.05 per 1000 player-exposures  in soccer, 0.17 per 1000 player-hours in floorball , 0.05 per 1000 player-exposures in basketball , and 0.06 per 1000 player-exposures in American football  (Appendices A8-1 and 8-2). There were insufficient studies for field hockey [Overall, non-contact injury incidence was 0.06 per 1000 player-hours  compared to male athletes  (Appendices A8-3 and 8-4). Only the difference between female and male soccer athletes per 1000 player-hours was significant as confirmed by meta-regression  (Appendix B2). Injury incidence was higher in female basketball players  compared to males  and this difference was significant  (Appendices A8-5 and B2).Sub-grouping by sport and sex was only possible for soccer and basketball. In soccer, injury incidence was higher in female athletes  and 0.08 per 1000 player-exposures  (Appendices A9-1 and 9-2). In adolescents, the incidence was 0.19 per 1000 player-hours  and 0.02 per 1000 player-exposures  (Appendices 9-1 and 9-2). Only the difference between adults and adolescents per 1000 player-exposures was significant as confirmed by meta-regression  (Appendix B2).The overall incidence of non-contact ACL injuries in adults was 0.07 per 1000 player-hours  and 0.08 per 1000 player-exposures in female athletes , and 0.05 per 1000 player-hours  and 0.06 per 1000 player-exposures  in male athletes (Appendices A9-3 and 9–4). The differences between adult male and female athletes per 1000 player-hours and player-exposures were significant as confirmed by meta-regression  (Appendix B2). In adolescents, injury incidence was 0.02 per 1000 player-exposures in male athletes , and 0.28 per 1000 player-hours  and 0.03 per 1000 player-exposures for female athletes  (Appendices A9-5 and A9-6). There were insufficient studies of male adolescent athletes to compare incidence per 1000 player-hours between adolescent male and female athletes [After sub-grouping by age group and sex, adult injury incidence was 0.11 per 1000 player-hours , in intermediate-level athletes 0.10 per 1000 player-hours  and 0.10 per 1000 player-exposures , and in amateur-level athletes 0.13 per 1000 player-hours  and 0.03 per 1000 player-exposures  (Appendices A10-1 and 10–2). There were insufficient studies of elite-level cohorts to compare incidence per 1000 player-exposures [β = 1.03, 95% CI 0.26–1.79, p < 0.01) (Appendix B2).Overall, the incidence of non-contact ACL injuries in elite-level athletes was 0.06 per 1000 player-hours  and in male athletes was 0.04 per 1000 player-hours  (Appendix A10-3). There were insufficient studies to perform similar analyses per 1000 player-exposures [β = − 0.91, 95% CI − 1.31 to − 0.52, p < 0.01) (Appendix B2). At the intermediate-level, injury incidence in females was 0.16 per 1000 player-hours , and in male athletes was 0.04 per 1000 player-hours  and 0.08 per 1000 player-exposures  (Appendices A10-4 and A10-5). There were insufficient studies to meta-analyse data from female intermediate-level athletes [β = − 1.32, 95% CI − 1.85 to − 0.80, p < 0.01) (Appendix B2). At the amateur-level, injury incidence in females was 0.27 per 1000 player-hours  and 0.05 per 1000 player-exposures , and in males was 0.10 per 1000 player-hours  and 0.02 per 1000 player-exposures  (Appendices A10-6 and A10-7).After sub-grouping by participation level and sex, the incidence of non-contact ACL injuries in elite-level female athletes was 0.10 per 1000 player-hours  and 0.32 per 1000 player-exposures , and during training was 0.04 per 1000 player-hours  and 0.02 per 1000 player-exposures  (Appendices A11-1 and A11-2).The overall incidence of injury during competition was 0.48 per 1000 player-hours  and in training was 0.07 per 1000 player-hours  (Appendix A11-3). There were insufficient studies to perform meta-analysis by player-exposures in females [I2 = 72%) and 0.34 per 1000 player-exposures, 95% CI 0.12–0.99, I2 = 96%) during competition while for training was 0.02 per 1000 player-hours  and 0.03 per 1000 player-exposures . All competition to training comparisons reported above were significant as confirmed by meta-regression (Appendix B2).After sub-grouping by exposure types and sex, injury incidence in female athletes during competition was 0.67 per 1000 player-hours  compared to elite-level athletes  (Appendix A12). This difference was significant as confirmed by meta-regression  (Appendix B2).There were sufficient studies to perform a meta-analysis by participation level per 1000 player-hours for female soccer athletes. Injury incidence was higher in intermediate-  and contact sports  [Our findings were inconclusive regarding the influence of sport, age group and participation level on non-contact ACL injury epidemiology in team ball-sports. In relation to the influence of sport, Montalvo et alketball) . It is nketball) , 88, 89.ketball) –92. We oketball) , 9, or iketball) , 93. We Lastly, in order to fully establish the extent of an injury problem to inform the development of injury risk-reduction strategies, injury epidemiology studies must report injury mechanisms . We had Firstly, there was substantial heterogeneity among the included studies. This is inevitable in meta-analyses of epidemiological studies and does not invalidate our findings . We atteNon-contact ACL injuries represented over half of all ACL injuries sustained in team-ball sports. The proportion of non-contact to total ACL injuries and injury incidence was higher in females than males in team ball-sports. Injuries mostly occurred in competition settings in team ball-sports. More research is required to fully understand the influence of sport, age group and participation level on injury proportion and incidence in team ball-sports. Our findings have implications for future ACL epidemiological research in sport, and the development and implementation of effective ACL injury risk reduction efforts in team ball-sports.Supplementary file1 (DOCX 4860 KB)Supplementary file2 (DOCX 45 KB)Below is the link to the electronic supplementary material.


We report the experimental observations of Bell inequality violations (BIV) in entangled photons causally separated by a rotating mirror. A Foucault mirror gating geometry is used to causally isolate the entangled photon source and detectors. We report an observed BIV of CHSH- It is commonly accepted that the Aspect  and ZeilWe will now review the isolation experimentally achieved thus far in the current state of the art experiments. We will observe a static line of sight between emitting atoms and observing ones. Bell famously prescribed “randomly setting the polarizers during the flight of the photons” as a way to close the locality loophole. Modern experiments causally isolate the observers between themselves. In the Aspect and Weihs–Zeilinger experiments, we never set the direction of the polarization analyzers during the flight of photons. This is, in fact, mechanically impossible . Insteaduncorrelated laser sources [With regards to Bell’s theorem, such a background field would allow for Bell-type correlations in simple local realistic ways. Such a de-localized background field trivially breaks the isolation assumption of the theorem according to . This is sources .It stands to reason that if field communication is responsible for the Bell effect, then when we remove said field, we can anticipate a loss of the Bell effect (no line of sight? no BIV!). It is simple to test this background influence hypothesis: we introduce a Foucault mirror designed to disrupt the EM line of sight between emitting atoms and polarizers. With this rotating mirror, we remove any EM background field between observers and emitters. How does this affect a Bell–CHSH measure?To test this background field hypothesis, we introduced a spinning Foucault mirror in a classic 2-channel Bell experiment. See An entanglement source commercially available from quTools, GmbH, allows for a classic 2-channel singlet experiment showing Bell–CHSH violations. A singlet state of entangled photons was prepared in a superposition of horizontally and vertically polarized photons. Both photons were sent down 200 m of single-mode fiber into a single rotating mirror  purchased from Cambridge Technologies (model SA34 P/N:1-1-3304-001-00). The geometrical arc described by the beams reflected off each of the rotating mirror facets covered the slits over Alice and Bob. See Time of Aperture:We can adjust the time this window stays open and during which the paths are connected. The time of aperture is determined by the aperture width of the slits Since we used the same mirror for both paths, the shutter effects at both slits were in phase. The slits were illuminated at the same time, and we thus detect coincidences in the APDs. Note that two different mirrors would not be in phase, and we would not detect coincidences in the counters because the APDs would not be iluminated at the same time, or at least within the 20 ns coincidence window programmed in the hardware.Causal Isolation:During the opening time of aperture, photons traveled a distance of 140 m. We introduced 200 m of single-mode fibers between source and mirror as in Degradation:Luminosity and time of aperture are proportional. The shorter the time gate, the shorter the exposure to the light, and the shorter the collection of photons. This is true of single counts. The coincidence count is proportional to the single count, and not a quadratic of the single count. As we have observed, the mirror illuminates the separate individual slits at the same time; thus, the gate effects at distant slits are in sync. This is the reason we detect coincidences in the first place. This yields a linear relationship between coincidences and singles. The floor in our experimental setup was set by the dark count of singles and the dark count of coincidences. With rotation, the photons were spread over Source CharacterizationThe singlet state coming out of the source was characterized by both QuTools and the author, using, respectively, a full tomography and a custom design involving a quarter wave plate in one arm, asWith this source, and without mirror gating, we have routinely observed Bell–CHSH measures up to the Axes of MeasurementThe polarization measures were conducted along the usual angles of Dark counts and detection window:Our APDs had individual dark count rates of Degradation:The results of measurements for determining the luminosity and signal degradation due to the rotating Foucault mirror can be seen in Bell–CHSH observation:We have observed BIV of Bell–CHSH We demonstrated that the AE/OMs Bell experiments close the locality loopholes between observers but do not causally isolate the sources from the observers in classic 4-channel loophole experiments. We introduced a spinning mirror to create such isolation in a 2-channel Bell experiment. By intermittently removing this hypothetical background, we anticipated a loss of the Bell effect (loss of BIV). We reported instead on observed Bell violations.Regarding a hypothetical background mediating an influence from the polarizers to the emitting atoms and which is responsible for the Bell effect, we will now prove that the influence cannot be a traveling wave.To prove this statement, we first observe that the time the gate is open is We therefore conclude that the influence cannot be that of a travelling wave, not even super-luminal, not even an instantaneous wave. This experimentally rules out all super-luminal theories explaining Bell. QED.For discussion, we offer the following logical argument. If a background field is responsible for Bell violations, then the communicating waves are either travelling or standing.1/ There is no background influence, or;2/ There is a background influence, and either:–2a/ It is a traveling local wave, or;–2b/ It is not (standing wave or deBroglie hypothesis).Either:The logical structure of the above is a list enumeration of the possible scenarios: ∄ background As for the physical content, one can argue in scenario 1 that there is no background field; it simply does not exist and something else is responsible for the Bell effect. This is the view espoused in the contextuality approach ,15: neitIn scenario 2, we still consider the existence of a background influence. The conclusion of this paper was to experimentally rule out any travelling wave  or option 2a.Therefore, we are left with option 2b. The Bell effect cannot be due to traveling waves. What form could this wave be? Standing waves come to mind. The author looks to the hydrodynamic walkers for visual inspiration and intuition . A droplAs noted, the above experiment is performed on a commercially available 2-channel static measure setup. It is not a state of the art 4-channel dynamic-choice experiment for the simple reason that the author does not have access to such equipment . It has also been noted that the isolation between the source and the detectors is non-existent in the classic 4-channel experiments. When one considers the source-to-polarizer isolation, there is no loophole closing. The lines of sight and settings are all static. It would strengthen both the conclusions in this paper, as well as the loop-hole closing conclusions of the classic papers, if such a 4-channel dynamic-choice test were to be performed in the presence of this Foucault mirror gate. Based on the present positive result, the author would not expect a different outcome: i.e., we should still observe BIVs in such an enhanced geometry. The author is open to lending his mirror equipment to, and collaborating with, laboratories capable of conducting such experiments.


Cyperus aromaticus (Navua sedge) is a problematic perennial weed in pastures and crops including sugarcane, banana, rice, and fruits and vegetables in tropical climates. It reproduces both via rhizomes and seeds. As a regenerative and storage organ, these rhizomes play an important part in the invasion, establishment, and persistence of this weed. To eliminate their regenerative ability, it is important to understand the regrowth potential with respect to rhizome fragment size and burial depth. This study evaluated the emergence of C. aromaticus from rhizomes in a controlled condition. Three different sizes of rhizome fragments were buried at seven depths of up to 20 cm in two soil types. The experimental measurements included (i) the time for tillers to emerge, (ii) the cumulative emergence of tillers, recorded weekly, and (iii) the number of underground emerging tillers. The cumulative shoot emergence and the number of underground tillers produced were found to be positively correlated with the initial length of the rhizome fragments and negatively correlated with the burial depth. The time for the emergence of the tillers was negatively correlated with the burial depth, and soil type had no significant effect on any of the parameters recorded. There was no emergence recorded from rhizomes buried at 15 cm depth and deeper, irrespective of their size. Our results indicate that the combination of the fragmentation of rhizomes into small pieces and a deep burial, below 15 cm, is an important aspect to control the regeneration of C. aromaticus from rhizomes, if tillage is carried out, and can therefore form a part of an integrated weed management strategy for this troublesome weed. Cyperus aromaticus (Ridley) Mattf. & Kükenth (Navua sedge) is an invasive C4 perennial sedge species, which is found predominantly in tropical environments. It is an aggressive weed that causes significant impacts on livestock grazing industries and sugarcane and banana plantations and also affects a range of native ecosystems in tropical north Queensland in Australia [C. aromaticus is capable of spreading at an alarming rate across larger areas within a short period of time [C. aromaticus plant persists by means of a superficially placed sympodial rhizome system primarily found in the top 5–7 cm of the soil surface . The rhizome is formed by many conical basal swellings joined together at their bases, and from the apex of the conical swelling, the aerial shoot arises. The basal swelling consists of several closely situated nodes , enclosed by scale leaves [ustralia . It can ustralia . If past of time . A signi of time . The C. e leaves . Some ofe leaves . A smallC. aromaticus show that sequential applications of herbicide are required for the control of this species [C. aromaticus in an ecologically sustainable way, simultaneously targeting both the above- and belowground biomasses without the excessive use of herbicides is essential.This dual mode of reproduction implies that the management of this species should target control of both the aboveground and underground systems rather than the aboveground biomass alone . Studies species ,7, and c species . In addi species ,10. HencC. aromaticus.Rhizomes serve two-fold functions as organs for survival and propagation; they are the main reserves of carbohydrate as well as the housing of the dormant buds for survival during unfavourable periods ,12. The Mechanical intervention forms an important part of the integrated weed management system to reduce the reliance on herbicides . In thisC. aromaticus, making understanding the effect of burial depth on the regeneration from rhizomes an important control issue [C. aromaticus to gain knowledge about fragmentation and establishment dynamics under various soil types. To help to systematise our study, we hypothesised that:Regeneration from buried rhizomes and their fragments is an important phenomenon contributing to the rapid increases in population of rhizomatous weeds, such as ol issue . Studiesol issue ,27,28,29ol issue . The purHypothesis (i).Total emergence of tillers is positively correlated with length of rhizome fragment and negatively correlated with burial depth.Hypothesis (ii).Time to emergence of tillers is positively correlated with length of rhizome fragment and negatively correlated with burial depth.Hypothesis (iii).Total emergence and time to emergence are dependent on soil type.The results obtained from the mixed models for the cumulative emergence of tillers are summarised in p ≥ 0.05) in the emergence from any of the rhizome sizes (p < 0.05) (p ≥ 0.05) throughout the experiment, and these depths were significantly higher than the emergence from 9 and 12 cm burial depths (p < 0.05)  that total emergence is positively correlated with the length of the rhizome fragment and negatively correlated with the burial depth . An examme sizes . However < 0.05) . At day  < 0.05) . A decre < 0.05) . The eme < 0.05) . There w < 0.05) .p < 0.001). However, there was no significant effect of the rhizome size on the time to emergence (p = 0.943) (p = 0.622), nor the time to emergence (p = 0.529) was influenced by the soil types (Hypothesis (ii) stating that the time to emergence of the tillers is positively correlated with the length of the rhizome fragment and negatively correlated with the burial depth was partially supported by the statistical analyses. There was a significant negative relationship between the burial depth and the time to emergence (= 0.943) . For theil types .p < 0.001) (p < 0.05). Underground emerging tillers from rhizomes buried at 6, 9, and 12 cm depth were similar in number and significantly higher than those buried at 15 and 20 cm depths. None of the buried rhizomes died.The burial depth and rhizome size individually had significant effects on the number of underground emerging tillers (< 0.001) . The numTussilago farfara (Coltsfoot), Physalis viscosa (Prairie groundcherry), and Cynodon dactylon (Bermudagrass) [C. aromaticus, is their ability to store a large amount of food reserves in their rhizomes [The emergence from the rhizome decreased as the burial depth of the rhizome increased. A decline in emergence with increasing burial depth has also been observed in other rhizomatous species, such as dagrass) ,27,31. Tdagrass) . One of rhizomes ,33,34. Trhizomes .C. aromaticus. At all the burial depths, where emergence was recorded, the total emergence of tillers from small rhizomes was lower than those emerging from large rhizomes. With increasing burial depth, the effects of the rhizome size became stronger. An increase in burial depth is more detrimental to the survival of shorter rhizome fragments compared to larger fragments due to a greater chance of survival of buds on larger rhizomes as there are more buds, whilst bigger rhizomes also have more energy reserves to help tillers emerge from greater burial depths [The hypothesis that the total emergence of tillers from the rhizomes is positively correlated with the rhizome size holds true in the case of l depths . Sproutil depths .In the case of mechanical control, the number of tillers emerging from a rhizome following cultivation is determined by both the lengths to which the rhizomes are broken and the depth to which fragments are buried. Logically, fragmentation by itself will increase the potential for rhizomatous weed spread due to breaking of apical dormancy, but the process of fragmentation can also lead to deep burial of rhizome pieces . Thus, aC. aromaticus were buried, the longer it took them to emerge. This delay in weed emergence time allows the competing crop to establish in the field thus weakening the weed’s ability to compete [The hypothesis that the time to emergence was negatively correlated with the burial depth was supported in this study. The deeper the fragments of  compete . In addi compete .As we determined that none of the rhizomes died in the experiment, the observation that none of the underground emerging tillers present had emerged from the soil, leaves the possibility open that they could have emerged if the experiment continued longer. Hence, it is recommended that future studies of this type continue for a longer duration to check whether the developing underground tillers would eventually emerge, which has implications for the fate of the rhizomes.C. aromaticus rhizomes buried at 15 cm depth and deeper, irrespective of their size in this study. Our results indicate that the combination of fragmentation of rhizomes into small pieces and deep burial, below 15 cm, is an important aspect to control the regeneration of C. aromaticus from rhizomes, if tillage is carried out, and can therefore form a part of an integrated weed management strategy for this troublesome weed. This study on the propagation/sprouting of C. aromaticus from rhizomes of different sizes and at different depths is important in devising integrated management strategies. The information provided here can be applied to other invasive species with vegetative and sexual reproduction, thus helping to control other species.In conclusion, there was no emergence recorded from C. aromaticus were collected from the banks of Nyleeta creek , near Innisfail, Far North Queensland in April 2020. This is a rainforest creek, upstream from most agricultural and other soil disturbance. The rhizomes were dug up from the sandy substrate of the creek banks. The aboveground parts were removed, and then the rhizomes were washed and wrapped in moist paper towels to keep them moist until they were potted in the glasshouse four days later. Size classes were chosen in order to encompass the variation in length observed among the collected rhizomes. The study sample contained 168 rhizomes, which were classified into three length categories: (i) small fragment ; (ii) medium fragments ; and (iii) large fragments .The experiment was conducted between April and July 2020 at the Mount Helen campus of Federation University, Australia . Rhizomes of C. aromaticus rhizomes to soil types, burial depths, and fragment sizes and to expand our knowledge of the conditions affecting the emergence of tillers from rhizomes. The pots were placed randomly in the glasshouse, maintained at day temperatures between 32 °C and 27 °C and a night temperature between 23 °C and 18 °C, and kept at a relative humidity above 80%. The pots were sprinkler-irrigated using an automatic watering system for 10 min daily to avoid any moisture stress.The experiment was conducted on a completely randomised design, using a three-factorial pot approach, where the factors were (i) soil type, (ii) rhizome length, and (iii) burial depth. Four replicates were carried out for each treatment, leading to 2 soil types × 7 depths × 3 rhizome lengths × 4 replicates = 168 pots. The factors were chosen to understand the response of Two types of soils were used for the experiment: (i) clay loam  and (ii) sandy loam . For each of the soil types, rhizomes were buried at seven different depths; 0, 3, 6, 9, 12, 15, and 20 cm. Cylindrical pots measuring 32 cm height and 15 cm diameter were partially filled with the relevant soil; the rhizome was placed on to the surface, and additional soil was added to create the required burial depth. Soil was packed before and after placing the rhizome by tapping on to the bench. Each of the soil type and burial depth variations had three sizes of rhizomes; small, medium, and large as the third factor.C. aromaticus.Cumulative emergence of tillers (including newly emerged tillers and matured tillers) was recorded every seven days from the commencement of the experiment until day 70. At the end of the experiment, rhizomes were carefully removed; their status was checked and recorded as ‘virtually healthy’ or ‘decayed’, and the number of underground emerging tillers was counted. Linear mixed models were conducted using SPSS to investigate the main effects of soil, burial depth, rhizome size, time, and their 2-way interactions on the cumulative emergence of tillers. Time was treated as a random effect to account for the emergence from the same rhizome being measured on several occasions. Time to emergence and the number of underground tillers were analysed using general linear models with soil, burial depth, and rhizome size as the main effect and their 2-way interactions. The significance of the main effects was analysed using Tukey’s post hoc analysis, and significant interactions from the mixed models were analysed by investigating the simple main effects with Bonferroni adjustments. All assumptions were checked by investigating the normality and spread of the residuals.


One month after severe bilateral cervical contusion, rats received daily intensive rehabilitation, NPC grafts, or both rehabilitation and grafts. Notably, only the combination of rehabilitation and grafting significantly improved functional recovery. Moreover, improved functional outcomes were associated with a rehabilitation-induced increase in host corticospinal axon regeneration into grafts. These findings identify a critical and synergistic role of rehabilitation and neural stem cell therapy in driving neural plasticity to support functional recovery after chronic and severe SCI.We reported previously that neural progenitor cell (NPC) grafts form neural relays across sites of subacute spinal cord injury (SCI) and support functional recovery. Here, we examine whether NPC grafts after  Spinal cord injury (SCI) afflicts more than 2 million people worldwide  and lackspared neural circuits that stimulates spared axon sprouting  SCI. Rats underwent a new, more severe cervical injury that substantially compromised bilateral white matter and resulted in persistent 75% loss of skilled grasping; grafting was delayed for 1 full month after injury, a time point at which most humans are admitted to rehabilitation centers after SCI. We found that only the combination of rehabilitation and grafting supports significant recovery of forelimb grasping when both treatments were initiated at a chronic time point of 1 month after injury (P < 0.01) and that rehabilitation enhanced regeneration of injured host corticospinal axons into the lesion site (P < 0.05).To date, it has not been established whether rehabilitation also augments functional outcomes when combined with a proregenerative intervention such as stem cell grafting. Thus, we designed the present experiment to test the hypothesis that forelimb rehabilitation combined with NSC grafting will drive anatomical plasticity of host axons n = 20), (b) NPC transplant alone (n = 21); (c) rehabilitation alone (n = 22); and (d) NPC transplant and rehabilitation (n = 22). Four weeks after lesions, all groups underwent reexposure of the lesion, and 2 groups received implants of E14-derived spinal cord NPCs, while the 2 nongrafted groups received injections into the lesion site of the grafting matrix consisting of fibrinogen/thrombin with BDNF, FGF2, VEGF, and MDL28170 (see Methods). Rehabilitation began the next day for 5 days per week; it consisted of forelimb grasping for food reward pellets for 2 hours per day and overnight (14 h/d) exposure to a floor grid containing food reward pellets accessible only by skilled grasping (https://doi.org/10.1172/jci.insight.158000DS1). Quantitative assessment of forelimb skilled grasping was performed weekly on the Montoya staircase  lesion alone  and 24.9% ± 1.6% cells in the NPC transplant–alone group (n = 14) expressed the neuronal marker NeuN, respectively, a difference that was not statistically significant . The estimated total number of neurons per graft was 260,000 ± 22,000 in the NPC transplant and rehabilitation group compared with 213,000 ± 32,000 in the NPC transplant–alone group, a difference that was not significant . 36.4% ± 1.7% of cells in the NPC transplant and rehabilitation group expressed the astrocyte marker GFAP compared with 36.2% ± 2.2% in the NPC transplant–alone group (Transplanted NPCs differentiated into neurons and glia . 27.9% ±ne group . The oline group . These p2) compared with the NPC transplant–alone group (15.9 ± 5.4/mm2)  compared with the NPC transplant–alone group (20.2 ± 3.8 axons/mm2)  and a group × time interaction . No other group exhibited significant and persistent improvements in pellets eaten relative to their own baseline 1 month after lesion ; no other group comparisons were significant.Four weeks after injury, animals received NPC grafts or sham surgery, and rehabilitation was started the next day in 2 groups . Over the subsequent 12-week period, significant differences among groups were observed using a Poisson loglinear model with autoregressive correlation matrix structure for repeated measures (r lesion . There waccuracy (pellets eaten/pellets displaced) after NPC transplant and rehabilitation using an autoregressive correlation matrix structure for repeated measures . The NPC transplant and rehabilitation group differed significantly from every other group, with a main effect of group  and a group × time interaction . Accuracy improved by approximately 20% and was maintained through all weeks of testing relative to the pretreatment baseline. No other group exhibited significant and persistent improvements in pellet retrieval accuracy relative to their own baseline after lesion .Similarly, significant differences among groups were observed in pellet retrieval r lesion . There wregeneration. Moreover, functional outcomes only improved among animals receiving both NPC grafts and rehabilitation; either treatment alone was not effective in statistically improving motor outcomes. In the context of these long, 4-week delays before application of treatment and a more severe, bilateral cervical lesion model, both NPC grafting and rehabilitation were required to support significant functional improvement.These findings demonstrate for possibly the first time that a proregenerative NPC therapy combined with rehabilitation significantly improved both anatomical and functional outcomes after SCI. While rehabilitation has previously been reported to promote sprouting of spared axons , 13, 19,chronic time point after injury trended toward improving functional outcomes (P = 0.1 compared with lesioned controls), providing evidence of a possible benefit of rehabilitation in chronic SCI. While this study was well powered with n = 20 animals per group, further expansion of group sizes would be required to establish whether there is a statistically significant benefit of rehabilitation alone in chronic SCI. The potential benefit of rehabilitation alone merits further study, because a current dogma in the field is that rehabilitation is most likely to be effective when applied in subacute, but not chronic, phases of injury. A possible mechanism underlying rehabilitation-induced improvement in function after chronic injury in the absence of a NSC graft is axonal sprouting, which could occur either at the level of corticospinal sprouting within the spinal cord, or sprouting of corticospinal projections to the red nucleus and the reticular formation. Rehabilitation could also induce functional improvement through sprouting of noncorticospinal systems, including brain stem motor projections  and intraspinal systems. In this study, we did not detect sprouting of corticospinal projections to the spinal cord rostral to the lesion, and future studies will accordingly focus on sprouting in other locations.We note, however, that the functional outcomes of the NPC transplant and rehabilitation and rehabilitation-alone groups did not differ statistically from each other, even though only the NPC transplant and rehabilitation group reached statistical significance in differing from lesioned controls. In fact, the provision of rehabilitation alone starting at a (16), but spinal circuitry for hind limb locomotion is contained in lumbar spinal cord segments below a complete transection: simple activation of these circuits is sufficient to enable hind limb movement , weighing 150–200 g, were used for this study. Animals had free to access to food and water throughout the study; among animals undergoing rehabilitation, food was freely available through the training tasks and animals were weighed weekly to ensure they did not lose weight. For all surgeries and perfusion animals were deeply anesthetized using a combination (2 mL/kg) of ketamine (25 mg/mL), xylazine (1.3 g/mL), and acepromazine (0.25 mg/mL).Rats underwent training on 3 behavioral tasks prior to placement of spinal cord lesions. The Montoya staircase was used to assess forelimb reaching performance  were administered daily for 7 days. 148 rats survived the first postoperative week. Postlesion forelimb reaching was measured 1, 2, and 3 weeks after lesion. After lesion, at week 3 animals were pseudorandomly divided into 1 of 4 groups matched for food pellet grasping performance on the Montoya staircase, ensuring distribution into 4 groups with similar degrees of deficit (n = 20), (b) NPC transplant alone (n = 21); (c) rehabilitation alone (n = 22); and (d) NPC transplant and rehabilitation (n = 22).A dorsal laminectomy was performed to expose the C6 spinal cord. A bilateral contusion was administered using an Infinite Horizons Impactor (Precision Systems & Instrumentation) set to 200-kilodyne force with 1-second dwell time and an impactor head of 3.5 mm diameter  lesFour weeks after lesions, primary E14 rat spinal cord NPCs were grafted into the lesion site in the NPC transplant–alone and rehabilitation NPC transplant and rehabilitation groups. Cells were grafted in a fibrinogen/thrombin solution containing BDNF (50 μg/mL), FGF2 (10 μg/mL), VEGF (10 μg/mL), and an antiapoptosis small molecule, MDL28170 (50 μM). Previous work determined that this grafting cocktail is necessary to support graft survival in larger lesion cavities . Cells wTwo weeks prior to sacrifice, all animals underwent anterograde tracing of the corticospinal tract using intracortical injections of AAV8 viral vectors expressing membrane-targeted TdTomato . VectorsAs previously described , spinal Animals were transcardially perfused with 4% paraformaldehyde. Blocks of cervical spinal cord 1.5 cm long centered around the C6 lesion were sectioned in the horizontal plane on a cryostat set to 30 μm thickness. In addition, C2 and C8 spinal cord levels were sectioned in the coronal plane on a cryostat set to 30 μm thickness. Sections were labeled for (a) GFP rabbit polyclonal antibody  or GFP chicken polyclonal antibody  to assess graft survival, differentiation, and process outgrowth; (b) RFP rabbit polyclonal antibody  to amplify TdTomato signal for corticospinal tract tracing; (c) NeuN guinea pig polyclonal antibody  to label mature neuronal nuclei; (d) GFAP mouse monoclonal antibody  to label astrocytes; (e) APC mouse monoclonal antibody  to label oligodendrocytes; (f) mouse monoclonal synaptophysin antibody  to label presynaptic proteins; and (g) rabbit polyclonal vGlut1 antibody . After incubation in primary antibodies, sections were incubated with Alexa 488–, 594–, or 647–conjugated donkey secondary antibodies  and were coverslipped with Fluoromount-G (SouthernBiotech).For examination and quantification of GFP-labeled axons, light-level GFP immunolabeling was performed in every sixth section incubated overnight at 4°C with GFP primary antibody  and then with HRP-conjugated secondary antibodies  for 1 hour at room temperature. Diaminobenzidine (0.05%) and nickel chloride (0.04%) were used as chromogens, with reactions sustained for 10 minutes at room temperature. The number of differentiated neurons labeled with NeuN, astrocytes labeled with GFAP, and oligodendrocytes labeled with APC within each NPC graft was quantified in a box of fixed size  at ×200 magnification within the graft. This number was divided by the total number of cells per sample box labeled with DAPI. To estimate total number of cells per graft, we divided the total number of DAPI-stained cells in the sampled box by the sampling fraction  and multiplied this number by the section sampling frequency (1-in-36 sections). The total number of neurons per graft was estimated by multiplying total number of cells per graft by the proportion of neurons among total DAPI-labeled cells. All quantification was conducted by an examiner, with group identity blinded (+ axons within a 1 square millimeter (mm2) area in the intermediate bilateral white matter was quantified, together with all graft-derived axonal profiles in the entire gray matter. Mean values were calculated per group ± SEM.The number of GFP-labeled axons extending caudally from NPC grafts to the C8 spinal cord level was quantified in both white matter and gray matter in 1 coronal section per animal. The number of GFPFor quantification of regenerated corticospinal axons into the NPC graft, a series of 5 to 8 horizontal spinal cord sections spanning the dorsal-ventral interface and 1 cross-sectional spinal cord at C2 level were imaged at ×10 magnification and autostitched using a Zeiss Axion Scan Z1 microscope and the software module Zen slidescan.For each animal, the entire GFP-expressing graft area was sampled and the percentage of the graft occupied by corticospinal axons was measured using ImageJ software . To normalize values for efficiency of corticospinal tracing per animal, the total percentage of CST-labeled axons in grafts was divided by the number of corticospinal axons in the main tract at C2.To quantify corticospinal axon sprouting rostral to the injury, coronal sections at C2 labeled for RFP were imaged at C2 coronal at ×100. Using ImageJ, the entire gray matter region was outlined, and thresholding values on images were chosen such that only RFP-labeled corticospinal axons were measured, and light nonspecific background labeling was not detected. To normalize values for efficiency of corticospinal tracing, the total labeled CST pixels in each animal were divided by the number of corticospinal axons in the main tract of the same section.t test (JMP software), at a designated significance level of P < 0.05. Data are presented as mean ± SEM. JMP Bivaiate fit analysis was used to estimate the correlation between number of neurons in the graft and the number of graft-derived axon profiles at C8 spinal cord gray matter.In all quantification procedures, observers were blind to the nature of the experimental manipulation. Analyses were performed by in-house statisticians using IBM SPSS (v.27). For behavioral studies, generalized estimating equations were used to assess group differences in the number and accuracy of pellets eaten after treatment. This is a robust approach similar to mixed repeated measures 2-way ANOVA but applicable to data that are nonnormal. A Poisson loglinear model (for count outcomes) with autoregressive correlation matrix structure for repeated measures was used for the number of food pellets eaten. A γ distribution with loglink function (for percentage outcomes) with autoregressive correlation matrix structure for repeated measures was used for the accuracy of food pellets eaten. Comparisons between 2 groups were tested by 1-tailed Student’s All experiments were conducted in strict accordance with NIH laboratory animal care and safety guidelines. All animal procedures were approved by the Institutional Animal Care and Use Committee of the Department of Veterans Affairs San Diego Healthcare System .PL, CMDF, and MHT designed the research studies, reviewed the data analysis, and wrote the manuscript. PL, CMDF, LG, ANT, AV, and DY conducted the experiments and acquired the data. JRH and ARF did the statistical analysis.


Managing multiple sclerosis (MS) includes different treatment approaches. Rehabilitation is a key strategy in MS for improving functioning, activity and participation. As part of a larger study on overall patient experiences with different treatment approaches, this study aims to give an overview of different patients’ experiences and perspectives on inpatient rehabilitation in MS.We conducted problem-centered interviews in 50 persons with MS in Germany, of whom most had relapsing–remitting MS. We used the maximum variation sampling method during recruitment. Data were analyzed thematically.As a result of the analysis, three major themes were identified: 1) factors contributing to the decision-making concerning rehabilitation, 2) experience with the rehabilitation setting, 3) benefits of rehabilitation treatments. The treating physicians’ attitude had a major impact on the decision to either opt for rehabilitation or not. Setting goals prior to rehabilitation was given a high priority. Exchanging experiences with other persons with MS presented a major benefit from rehabilitation while for some being separated from regular daily life resulted in a more ambiguous attitude ranging from appreciation of escaping daily hassles to substantial behavioral change management.Patients reported various experiences in the process of decision-making with regard to rehabilitation. Physicians´ advice, goal setting and the selection of the most suitable rehabilitation clinic were considered most relevant.The online version contains supplementary material available at 10.1186/s12913-022-08150-8. Multiple sclerosis (MS) is an autoimmune inflammatory, demyelinating and degenerative disease of the central nervous system (CNS) with secondary axonal degeneration resulting in a wide variety of neurological symptoms . At the Recently, the World Health Organization (WHO) has stressed that rehabilitation is of crucial relevance for medical management in the twenty-first century . SocietiAlthough MS is not a curable disease, rehabilitation as a highly individualized process may help to improve and maintain the ability to perform basic daily functional activities, mobility, and participation in the society  , 10. By In terms of symptomatic treatment, multidisciplinary rehabilitation is the key approach in MS and studies have shown beneficial effects with regard to exercise and physical activity programs in particular , 11. Hown = 590 pwMS indicated substantial barriers with regard to MS-rehabilitation, largely due to the lack of adequate information and missing support when applying for rehabilitation . I was in a wonderful rehab clinic, and it had a special program, a so-called Training Program, which was like being a beginner at school again.” (pwMS 28).A few patients decided upon a rehabilitation stay after the diagnosis had been made to gain a better understanding of the disease and to cope with the stress and anxiety evolving because of having been diagnosed with MS.“And then I was discharged [from hospital], with no advice on what I should do next. All I had was the information on those flyers. The sum of my knowledge when discharged. And then rehab was mentioned. That was at diagnosis time.” (pwMS 42).One patient felt completely left alone after the diagnosis of MS had been made in the hospital.“Because I always felt fit enough already, I did sport, and the limitations imposed on my body weren’t so huge.” (pwMS 40).A common factor for a decision against a rehabilitation program was that some pwMS felt too healthy to consider rehabilitation.When deciding for or against inpatient rehabilitation, pwMS were influenced by their expectations and assumptions regarding the inpatient rehabilitation.“Simply attending rehab with an expectation that then I’ll be better, isn’t workable […] I had set myself the goal of finding out what I could do to help my walking […] most of all for when I was back home, to really consider the question, what can I integrate into my everyday life? It’s no good thinking that a visit to rehab is all it takes.” (pwMS 22).Many patients who decided in favor of rehabilitation expected to gain a better health status, e.g. to regain “normal gait”. Accordingly, some patients emphasized the importance of defining specific goals prior to rehabilitation and of applying rehabilitative techniques in daily living to alleviate symptoms and maintain the achievements obtained by inpatient rehabilitation.“I really put that into practice because I believe it is more effective to do something every day than to go to physio once or twice a week, that this is the way to stay fully charged […]. That is what really swung it for me for residential rehab, rather than doing it from home.” (pwMS 7).Some pwMS explained that participating in rehabilitation gave them the opportunity to fully focus on physical training, thereby increasing the chances of a beneficial effect substantially.“I actually never did rehab. I was always put off by the thought I would see people there who were worse off than me and that this wouldn’t be good for me. That is what has stopped me from going thus far.” (pwMS 2).“Well, you know, going to rehab straight away after that first relapse, seeing other MS patients, the full spectrum from those who are like me, where it’s not visible, to those who are in wheelchairs, that can really pack quite a punch to begin with, it’s quite something to process mentally.” (pwMS 22).The thought of being confronted with the disease in all its facets especially shortly after the diagnosis was a factor in deciding against participating in inpatient rehabilitation. Some participants decided against rehabilitation expecting to be negatively affected when meeting other pwMS supposed to be more disabled than themselves.The professional and private environments of pwMS, such as the workplace and private surroundings, yielded conflicting reactions by either playing a facilitating role or being a barrier to some patients.“It feels good, simply having time away from work, from family, from everyday life, and immersing yourself there where the subject matter is the disease […]. For it’s not through my illness that I want to live. I am living my life and this darned disease is a companion. But I do not want my everyday life to be defined by the disease.” (pwMS 26).Many participants wished to escape from everyday life. Some pwMS wished to obtain a break from their everyday life by finding time to relax on the one hand and to fully concentrate on MS on the other. Subsequently, they wished to be able to focus less on the disease once they have returned to their home.“No, I’ve never been away on rehab. […] I’d say that’s definitely to do with my husband who said: Well now, what am I supposed to do with the children, and so on and so forth, and somehow it never came to be.” (pwMS 8).Others described that incompatibility of inpatient rehabilitation with their personal lives was an important factor to decide against it. One mother explained that she had never been able to realize a longer stay away from her home and children. Here the decision against a rehabilitation strongly points to a gender issue.“After the hospital I actually had a really great physiotherapist and […] both my general practitioner and neurologist were people I trusted very much and I didn’t want to leave [these familiar surroundings]. I had a lot of thinking to do and for me the familiar setting was where I needed to do this.” (pwMS 48).A few pwMS decided against rehabilitation, because they had the opportunity to obtain adequate treatment in their own environment, emphasizing the wish to reconcile rehabilitation and private lives thereby increasing the chance for long-term results.“When I needed help, or when I noticed that I needed some more support, I was sure to go to the clinic. That is ultimately a lot like rehab anyway. You are there and cared for and have the best of therapists.” (pwMS 11).A possible alternative for pwMS who decided against inpatient rehabilitation would be an outpatient setting such as a day unit rehabilitation. Some participants felt that a specialized day unit was very much like an inpatient rehabilitation and therefore didn´t feel the need to apply for an inpatient rehabilitation.“The decision for rehab, yes, the first time that was on the advice of the doctors here, the second time there was a little push from the neurologist […]. It’s possible that this was during a phase when I was teetering on the edge a little and she could see that.” (pwMS 5).PwMS also stressed the importance of communicating with their treating physician with regard to the decision to participate in inpatient rehabilitation. Some pwMS followed the physician’s advice on going to rehabilitation. One interviewee described receiving positive pressure from the treating neurologist to apply for rehabilitation. The citation indicates that patients in the beginnings of a negative spiral may require external guidance to exit this phase.“I knew that rehab away might be a possibility. And then I spoke to my neurologist about it and asked him: Is there anything we can do to stabilize my state of health, because I’m just not managing to do this in day-to-day life. […] And then he recommended it to me, and said, yes, this is something we can do.” (pwMS 22).Other pwMS demonstrated a more active approach by presenting their wish to participate in inpatient rehabilitation when seeing their treating physicians. These pwMS aimed to actively improve their state of health realizing that this objective could not be achieved along with the daily working load, and as a result, the neurologist reacted by recommending rehabilitation. The ensuing citation nicely illustrates the concept of enabling adaptation as the main aspect of rehabilitation.“I was having issues with my walking […]. And so I asked my neurologist, I said, before it gets worse wouldn’t rehab be an option now, to prevent that? And I was told: No, you are too well for that.” (pwMS 50).One pwMS reported that the physician disagreed with their proposal to apply for rehabilitation, claiming that the patient´s health status was too good. This attitude either points to a limited understanding of what can be achieved by rehabilitation or a recollection of bad experiences when these more educational objectives had not been met in rehabilitation.“I attend an MS group with regular meet-ups […] and lots of people there had already been and were really enthusiastic about [rehab residencies]. And that’s when I thought, right, I’m going to do it, too.” (pwMS 21).“You chat about it with people and I also heard: […] “I’ve been in other clinics that were better” and then I researched a bit on the internet to find out what a clinic actually is and what they offer.” (pwMS 22).In Germany, according to the code of social law (§8 SGB IX), each patient in principle has the right to decide upon a certain inpatient rehabilitation facility or outpatient rehabilitation treatment best suited for their individual needs.However, only a few pwMS actively searched for information and recommendations for a suitable rehabilitation facility. The vast majority of pwMS agreed to the recommendation provided by the health insurance. Additional sources of information were other patients’ experience with rehabilitation either shared by pwMS in patient support groups or on the internet.The second theme is characterized by pwMS’ overall positive or negative experiences with inpatient rehabilitation.“Yes, it’s a place where you can discuss things with like-minded people. It’s where you get to know the various forms of MS. […]. And it’s also where you discover that MS does not automatically mean a wheelchair and full-time care, and psychologically, that is really important.” (pwMS 5).Most participants found the personal contact with other pwMS in a rehabilitation facility particularly valuable, especially from a psychological point of view. PwMS described how helpful it was to gain a better understanding of the disease and learn about the spectrum of MS manifestations.“To be honest, your fellow patients are the best therapists. Because we talk a lot to each other, privately, too, not only about the disease, but about everything, for everyday problems still exist for us, too. And that is the best therapy of all.” (pwMS 30).Conversations with other pwMS were described as very helpful not only to cope with MS but also with regard to other life issues.“What I also found really great was simply switching off from the world, so that dream holiday that you always longed to have can actually be found at rehab, and it was a really lovely combination of medical input, sport, therapies of every stripe, shape and hue. […] You are away from home and those constant thoughts of tomorrow I have to go back to work, I must do the laundry, when am I going to cook […] instead you simply switch off and truly leave the everyday behind you.” (pwMS 10).As previously described in the context of influencing factors in the decision-making, escape from everyday life was indeed experienced by pwMS. Some pwMS even reported that the stay in a rehabilitation facility almost felt like a holiday. Moreover, they could fully focus on the treatment of their illness with the help of a multidisciplinary team without being distracted by their daily workload and issues.“The thing is, being at rehab for me is a little like being in a bell-jar, for these circumstances are not those of my everyday life. For example, I can get up and turn my attention immediately to the rehab task at hand […] and that is not real life. […] Everyday life is 1000 times more difficult for me than when I can restore away at rehab and […] move a little in the swimming pool or whatever. That is not the major challenge for me. Rather it is that peacefulness you experience at rehab.” (pwMS 35).“Personally, I like being an inpatient for rehab. And I’ve had the good fortune of being in a really excellent clinic. You feel like you are in a bell-jar. Personally, I felt so cared-for there; the people there were great.” (pwMS 40).There were also pwMS with a more skeptical view on rehabilitation since the almost ideal circumstances were regarded unrealistic when compared to real life. One participant underlined the somewhat artificial setting of inpatient rehabilitation using the image of being under a “bell-jar.” However, these pwMS rather enjoyed these effects of rehabilitation to be able to calm down.“Well, at rehab I also heard a lot of lectures about what you should and shouldn’t eat, but I think for me, the disease already decides so much in my life. So I really don’t feel like being told in addition what I should eat or shouldn’t eat, or when I should be doing sport. That at least I want to decide myself.” (pwMS 10).Five interviewees voiced negative impressions with regard to their stay in rehabilitation facilities, expressing feelings of lost autonomy and annoyance at having to act under orders.“It’s pretty tightly controlled: […] you get given numbers telling you where you have to sit at lunchtime and then that’s checked up on. […] Then in the evening, doors are locked, you can’t go out after 10 pm.” (pwMS 39).One pwMS participating in a psychosomatic rehabilitation clinic felt too tightly controlled and pressed into a scheme to function.“Within the framework of rehab I always found the external control a real trial and burden and that’s why out-patient rehab was a really good option for me.” (pwMS 3).Another pwMS perceived out-patient rehabilitation as a good alternative to the inpatient setting allowing more time to organize for themselves.Inpatient rehabilitation was considered to exert positive effects on their overall well-being, both mentally and physically by pwMS.“I attended […] medical rehab, […] I had sport therapy there and so on … and I entirely regained my ability to walk.” (pwMS 1).In accordance with their expectations and assumptions, many pwMS reported physical benefits. More specifically, pwMS experienced an improvement of balance, spasticity and gait, sometimes even to an extent that their level of activity prior to the relapse or worsening of MS was regained.“Oh yes, simply doing something at last, doing a bit of sport again. […] and then eventually going to bed with that feeling of satisfaction at what I’d achieved that day, for body and soul, and also in terms of friendship and general well-being.” (pwMS 17).Twenty-six pwMS felt mentally and physically empowered to take up new challenges.“If I didn’t have rehabilitation in the year, I am quite sure that I would no longer be able to work on account of the fatigue, the exhaustion syndrome, because at rehabilitation I can let myself go entirely, can truly relax, and refuel the energy-tank, and thus be ready to more or less make it through another year of work.” (pwMS 25).Complementary to being challenged, relaxation and stress management and just letting things flow were considered important aspects of improving well-being. By doing so, pwMS felt prepared to face another year of workload. In fact, many pwMS described participation in a rehabilitation program once a year as a kind of “refueling “, enabling them to carry on with their daily living for the next year.“When brushing my teeth, alternating from the right to the left leg, just that bit of movement. I learned that at rehab, and I do it. These are the little things that I have built into my everyday now.” (pwMS 25).“And in the clinic I learned about occupational therapy […] for everyday life. Things like how to become more adept with the wheelchair; how to empty the washing machine whilst sitting in a wheelchair, how to hang out washing, how to cook meals, all those kinds of things.” (pwMS 37).Participants reported how they applied newly learned techniques derived from balance training, stress management seminars, psychotherapy, mental exercise or nutritional counselling in their everyday life. They described how these measures helped them cope and better engage with some of the difficulties in daily life related to MS.“I stayed at a rehab clinic this year and was particularly eager to discuss nutrition and I had lots of conversations with an assistant nutritional therapist who helped me so much there, who provided me with an incredible number of tips, and that was really like the flicking on of a switch for me.” (pwMS 46).“The time at rehab helped me not only in terms of the disease itself, but also as regards my living environment as a whole, so my perspective on work, on certain personal stories, on problems really changed.” (pwMS 23).Some pwMS reported that rehabilitation even evoked fundamental changes of perspective with regard to MS and their lives almost as “flipping a switch”.Many pwMS became aware of the fact that in order to maintain or even increase treatment effects, exercise and relaxation techniques need to be applied continuously in their private surroundings.To date, only very few attempts have been made to sample pwMS’ experiences with rehabilitation in a systematic way. This study addressed patients´ experiences with rehabilitation in MS demonstrating that the process of decision-making, goal setting and the selection of the most suitable rehabilitation facility for the individual needs were of critical importance.When identifying the influencing factors in the process of decision-making for or against inpatient rehabilitation, the four subthemes ‘physical and psychological aspects’, ‘expectations and assumptions’, ‘environmental aspects’ and ‘information and advice’ emerged. This is in line with the findings of a qualitative study in Norway by Helland et al. . Here, iThe decision for rehabilitation appeared to be easy in the context of a recent diagnosis of MS or a relapse with incomplete remission and the resulting physical impairment. The psychological consequences following the diagnosis of MS can be devastating, and consequently depression is frequent in pwMS . In someThe study results emphasize the critical importance of the treating physician´s attitude and communication regarding rehabilitation in the process of decision-making, as also pointed-out in the qualitative study by Helland . In factIn the process of deciding which inpatient rehabilitation facility might be most suitable for the individual patient, some pwMS sought advice from their social environment or information from other pwMS, which is in line with previous findings , 31. PeeInterviewees in our study expected to improve their health status during a rehabilitation stay. In accordance with recent work, they clarified that personally meaningful and specific goal setting prior to rehabilitation may help to increase motivation and to accept a possibly incomplete recovery . PlayforInpatient rehabilitation also offers the opportunity to escape from everyday life which one pwMS described as “dream holiday”, which was also a finding of the study by Helland . For patA few pwMS felt that inpatient rehabilitation was incompatible with their personal life. Of note, this argument was frequent in female pwMS, who felt unable to relinquish their family duties for a longer period. With regard to rehabilitation, pwMS tend to accept an inpatient setting more easily if their work, family, and social life is less affected .Patients experienced the rehabilitation setting in different ways: Some revealed emotions such as feeling protected and cared for with the ability to fully concentrate on themselves and the disease and to “fuel the tank for another round”. Others felt separated from their private surroundings as well as put into an artificial setting not transferable to their daily life. Sometimes the holistic approach was negatively perceived as pwMS felt overwhelmed by management concepts touching all aspects of life. As mentioned above, setting meaningful goals might help to modify this ambiguity without fully resolving it.With regard to possible achievements of rehabilitation, there were also conflicting results: Some pwMS felt that the mere escape from their daily duties was an achievement, whereas others acquired new behavioral techniques which could be transferred into their daily living, and still others reported that rehabilitation was a turning-point in their treatment history of MS. Of note, psychological achievements of rehabilitation were mentioned most frequently, although they may get lost within months as we recently have shown in a larger controlled multicenter study on a metacognitive intervention .The significance of the data presented here may be limited by the fact that the data are derived from a cohort of pwMS with only mild to moderate disability, showing an interest in sharing their experiences by video files on the internet. Hence, we might have collected a non-representative sample of pwMS characterized by higher openness and extraversion than the “average” pwMS. Only about half of the interviewed patients really had experience with rehabilitation. On the one hand, this can be seen as a limitation as they may represent a subgroup who have managed to cope with the disease and are rather skilled with regard to rehabilitation. On the other hand, our study sample enabled us to gain insight into reasons for deciding against rehabilitation. However, the aspects that were discussed also cover the needs of pwMS in transition to or within a progressive phase . ExperieIn summary, this study indicates that pwMS may lack information about how to gain access to inpatient rehabilitation. They stressed the relevance for early rehabilitation soon after diagnosis for educational purposes. The input from other patients was considered important but ambivalent. The setting and structure of rehabilitation was mostly looked at as protective, but sometimes also as too rigid. A maintenance concept after rehabilitation was a major concern of most participants.Additional file 1. Interview guide.


These results indicate that research data obtained from concentrated NaOH solutions are not appropriate to apply to the crevice environments of SGs.The corrosion behavior and integrity of steam generator (SG) tube materials have frequently been tested in solutions containing sodium hydroxide (NaOH), assuming that NaOH is a typical contaminant concentrated in the crevices of SGs in a pressurized water reactor. The purpose of this study was to investigate the adequacy of using concentrated NaOH solutions to simulate the crevice environments of SGs. The dissolution behavior of magnetite deposit flakes formed in an operating SG was tested in a 0.4 wt.% NaOH solution at 300 °C, and the thermodynamic stability of magnetite was investigated using the potential-pH diagram for an iron–water system. The magnetite deposits were rapidly dissolved in the test solution, which was supported by the fact that magnetite is thermodynamically unstable under the test condition to dissolve to dihypoferrite ions (HFeO In a pressurized water reactor, high-pressure steam to drive a turbine is generated by a classic shell-and-tube type steam generator (SG). Both end sides of a heat-transfer tube in the SG are leak-tight expanded in a thick tubesheet. Tube bundles are supported with horizontal and vertical plates to prevent flow-induced vibration and fretting wear of the tubing. Therefore, these manufacturing processes inevitably create numerous crevices around tubes expanded in the tubesheet and tubes supported by the tube support plates (TSPs). Typical crevice geometries around SG tubing are shown in 3O4) that contains numerous pores [It is well known that aggressive impurity chemicals in the SG feedwater are concentrated into the narrow-heated crevices, thereby inducing corrosion damage of the SG tubing including intergranular attack, pitting, denting, and stress corrosion cracking. On the other hand, corrosion products released due to the flow-accelerated corrosion of iron-based piping materials are transported into the SGs and deposited on the surfaces of the SG materials including tubes, TSPs, and tubesheets. The SG deposits are mainly composed of magnetite FeO4 that cus pores ,6,7. As us pores ,9,10.SG water chemistry is controlled on the all-volatile treatment basis using a pH control agent  and an oxygen scavenger (hydrazine) . The pH 42−), sulfite (SO32−), tetrathionate (S4O62−) and thiosulfate (S2O32−) [2, Na2SiO3) [Against this background, corrosion tests for SG materials have been performed in aqueous solutions containing various contaminants to simulate corrosive crevice environments. The typical contaminants are as follows: sodium, chloride, sulfur, lead, silica, etc. ,14,15,16S2O32−) ,27,28,29(S2O32−) ,31,32, a− ,27,28,Na2SiO3) ,34,35. ANa2SiO3) . In othe2O4), jacobsite (MnFe2O4), and metallic copper. The porosity of the deposits was measured to be approximately 9.8%. The detailed characteristics of the deposits are given elsewhere [The corrosion test of SG deposits was performed using deposit flakes taken from an operating nuclear SG. The deposit flakes were pulled out from the outer surfaces of SG tubes during sludge lancing after operation of fuel cycle 27 in a pressurized water reactor. The SGs of the plant are equipped with nickel-based Alloy 600 tubes (15Cr-9Fe-76Ni in wt.%) as the heat-transfer tubing and have operated under a reducing all-volatile treatment condition with a pH range of 9.0 to 9.6 since the first commercial operation. The deposits were mostly composed of magnetite and contained small amounts of trevorite . Based on the following values of the standard chemical potentials (SHE from Equation (2). Now, the equilibrium potential , T is the absolute temperature (K), and i substance. For the 0.4 wt.% NaOH solution, since magnetite is dissolved, the potentials at which the HFeO2− species (dihypoferrite ion) is stable are in the range of −1.239 to −1.395 VSHE, shown in 2− ions. This is consistent with the immersion test result showing a rapid dissolution of magnetite in 0.4 wt.% NaOH solution. In a similar manner, we can predict that magnetite is no more stable in a caustic solution containing NaOH over 0.1 wt.% under the reducing environments of SGs.The equilibrium of this reaction is expressed by line ① in 627 cal) , Eo is cBased on this work, if caustic environments are formed in heated crevices covered with magnetite deposits and within porous deposits on the tubes, the deposits would be dissolved. In contrast, the deposit inventory of SGs increases during plant operation and is thus removed by sludge lancing and chemical cleaning ,40,41. T2 solutions as accelerated test conditions, which are not relevant to nuclear environments. Similarly, test results in concentrated NaOH solutions may be misleading and not suitable for evaluating the corrosion behavior of SG tubing materials. Consequently, the test parameters should be chosen to closely simulate the conditions encountered in the SG crevices during operation.Corio showed for the first time in the world that intergranular corrosion and stress corrosion cracking of the Alloy 600 SG tube material can occur in deoxygenated pure water , while CThis study investigated the stability of magnetite deposits through immersion experiments in normal and caustic solutions and thermodynamic consideration for an iron–water system at 300 °C. The magnetite deposits formed in a real SG were rapidly dissolved in 0.4 wt.% NaOH solution. This result is consistent with the thermodynamic calculation that magnetite is not stable in a solution containing NaOH over 0.1 wt.% dissolving magnetite to dihypoferrite ions. These results indicate that concentrated NaOH conditions are not applicable in simulating the real crevice environments of SGs. Therefore, the research results from tests conducted in concentrated NaOH solutions may be misleading and not suitable for evaluating the corrosion behavior of SG tubing materials.


Drosophila melanogaster, insulin/insulin-like growth factor (IIS) and mTOR-mediated signaling within adipocytes regulates oogenesis. To facilitate comparative study of nutrient-sensing pathway activity in the fat body, we developed antibodies to assess IIS (anti-FOXO) and mTOR signaling (anti-TOR) across three nymphalid species (Lepidoptera). By optimizing whole-mount fat body immunostaining, we find FOXO nuclear enrichment in adult adipocytes, like that observed inDrosophila. Additionally, we show a previously uncharacterized TOR localization pattern in the fat body.Nutritional stress impacts many insect species that have differing reproductive strategies and life histories, yet it is unclear how nutrient-sensing signaling pathways mediate tissue-specific responses to changes in dietary input. In Drosophila melanogaster. For example, nutrient sensing pathways, like IIS, mTOR, and the amino acid response (AAR) pathway, function within adultDrosophilaadipose tissue to control distinct stages of oogenesis . However, insects as a group that potentially includes 5.5 million species  have diverse life histories. For example, female reproductive strategies are influenced by differences in ovarian dynamics , egg carbon:nitrogen ratios , and ovariole numbers . Insects also vary in distribution of the adipose tissue within the body, which could potentially affect inter-organ communication.Drosophila is therefore an excellent model for one such suite of traits but is not representative of all insects. We must develop additional diverse insect models in order to understand the variety of mechanisms controlling life history responses to dietary stress across insects.Resource allocation is inextricably linked to tradeoffs between life-history traits, allowing organisms to adapt to a dynamic environment . Nutritional control of the tradeoff between survival and reproduction is one of the best-characterized examples of this phenomenon  and has been described for a variety of organisms, including insects. In several insect species, nutrients are reallocated from the energy intensive process of oogenesis to support somatic tissue maintenance during nutrient deprivation . This ensures that organisms survive beyond suboptimal nutritional conditions and do not waste resources on reproduction when the likelihood of offspring survival is low. While it is likely that multiple tissues sense and respond to nutrient input, we are just beginning to understand how inter-organ communication mediates whole organism responses in the heavily studied insect model,Drosophila melanogasterand various butterfly species within the Nymphalidae (Lepidoptera). These species differ in fat body distribution , ovarian dynamics , egg C:N ratio  and completeness of the adult diet .Given that nutrient sensing signaling pathways control the response of insect fat body to changes in dietary input, which in turn affects allocation to egg production and other life history functions , we are interested in comparing the operation of these pathways and their outcomes amongDrosophila.Upon insulin receptor activation, Akt, the main effector kinase, acts on several targets to regulate cellular metabolism, survival, proliferation, and growth as well as reproduction . When phosphorylated by Akt, the transcription factor FOXO is restricted from the nucleus, where it controls expression of genes that mediate the negative effects of reduced IIS . In addition, Akt indirectly activates the mTOR kinase by inhibiting the negative regulatory complex of mTOR, Tsc1/Tsc2 . The mTOR kinase can also be activated by the Ragulator complex which senses amino acid status . In both cases, mTOR activation happens at the lysosome.The response to dietary stress of the IIS pathway has been well characterized inDrosophila, the transcriptomes for our non-model lepidopteran species have very limited similarity to those ofDrosophila melanogaster. Therefore, the likelihood of successful use ofD. melanogasterantibodies in the lepidopteran species is low. In contrast, our identified FOXO and TOR transcriptomes for the three lepidopteran species had high percent similarity (> 95%), indicating likely success in developing an antibody for each protein that would work in all three butterfly species.To gain a better understanding of how IIS functions within lepidopteran fat tissue, we want to visualize changes in localization of FOXO and mTOR. One approach to tracking localization of these components is to use species-specific antibodies for immunocytochemistry. While the required antibodies for FOXO and mTOR are available forAgraulis vanillae andHeliconius charithonia, we first determined the protein sequences for those genes for each species. We downloaded amino acid sequences forBombyx mori(Lepidoptera: Bombycidae) andHeliconius melpomene melpomene for FOXO and TOR from uniprot.org. We then used GeneiousPrime to generate transcriptome sequences for these proteins. We blasted these sequences againstA. vanillae andH. charithoniatranscriptomes to locate the sequences for each gene for these species. Transcriptome data forH. charithoniacame from Catalan et al (2018), and forA. vanillaefrom Hanly et al (2019). Additionally, we used transcriptome and amino acid sequences forSpeyeria mormonia, because we are interested in experiments with that species in the future. These RNA sequences were converted to amino acid sequences for determination of epitopes joint among the species. Epitope identification and antibody production were done by Genscript. Rabbits were used for polyclonal antibody production. We also used GeneiousPrime to compare the similarity of FOXO and TOR RNA sequences among our three species, as well as withDrosophila melanogaster(Diptera: Drosophiladae).In this report, we describe the development of Lepidoptera-specific antibodies for FOXO and mTOR to track changes in localization of FOXO and mTOR. To generate antibodies for FOXO and TOR inDrosophilasince adipocytes in fruit flies from nutrient-rich conditions show low-nearly undetectable levels of FOXO nuclear localization . The majority of mTOR immunoreactivity shows diffuse cytoplasmic localization with slight enrichment at cell-cell junctions . Under nutrient replete conditions, mTOR localizes to lysosomes where it can be activated by amino acids . Thus, future studies will assess if the mTOR staining pattern in butterfly adipocytes colocalizes with lysosomal markers. For both FOXO and mTOR immunolabeling, we are confident that the observed staining patterns are specific since there is no fluorescence in samples incubated without primary antibody . Moreover, the nuclear localization of FOXO is quite different from the cytoplasmic localization of mTOR . There is stronger fluorescence intensity at the lowest antibody concentration for tissue treated with Tween 20 than that treated with Triton X-100 . At higher antibody concentrations, detergent type is not a major factor in immunostaining quality. Taken together, we achieve the best signal-to-noise ratio for both antibodies when using Tween 20 as the detergent and 2.5 μg/mL of anti-FOXO and 5 μg/mL of anti-mTOR .We used a standard whole-mount immunostaining protocol to test immunoreactivity of these antibodies in lepidopteran fat body. We varied antibody concentration  and permeabilization solution detergent (Triton X-100 versus Tween 20). We find that both antibodies show immunoreactivity in lepidopteran fat body at all three concentrations with varying robustness  in all three butterfly species tested. For both antibodies, higher concentrations produced stronger signal . FOXO immunoreactivity showed enrichment in the nucleus with diffuse labeling in the cytoplasm . Since FOXO translocation to the nucleus results from reduced IIS pathway activity, this data suggests that the butterflies from which the fat body originated may have been under nutrient stress. Alternatively, lepidopterans may have a higher baseline of FOXO-dependent gene expression thanDrosophilato other insects that differ in their fat body distribution, mode of ovarian function (e.g. stem cell-supported versus not), and adult diet macronutrient composition?In the future, we will further optimize the immunostaining protocol by modifying additional steps in the protocol, such as fixative type and fixation time. This work demonstrates the feasibility of using these newly developed lepidopteran antibodies to answer questions like how generalizable are nutrient sensing mechanisms inButterfly collectionAgraulis vanillae females were collected in Sunrise, Florida in January 2022.Heliconius charithonia females came from Davie, Florida and south Texas in January 2022 and fall 2021 respectively.AntigensFOXO: MSLPRGGSYQSPWSSQTALSELEGAMGEFEPVGELAEVGFEPQTRARSNTWPQPRPENYVDAEDPGSKKNSNQNLSGAPPLPTSATTKKNSSRRNAWGNLSYADLITQAITSSQEKRLTLSQIYEWMVQNVPYFKDKGDSNSSAGWKNSIRHNLSLHNRFMRVQNEGTGKSSWWMINPDAKPGKSVRRRALSMETSKTEKRRGRVKKKPDALRNGVTADATPSPSSSISESLDIFPDSPIHSSFQLSPDFRQRASSNASSCSRLSPIPSLIPTEPEWSTDYTPSGDFASTSDFSPADYTQDQDLAGSLADSMKLHGADPFLNTYVPTTSSSSSGGSYRFTYGACPRHPHGGCACAPLYPAHPAHPTHSHQHSLDHFVRPPPPADPADIMQTENSQTQMVATSDATLMNGGIMVQTGPMGPTTVMGQIMGALNTGLSEDLNIEALEHSFDCNVDEVIRHELSMDGTLDFNFPQQNTAMAAEAESQFRAPSAPVPTTLSGGGASQTPYTVAPSWVH.TOR: MYMFLLKGHEDLRQDERVMQLFGLVNTLLQADPDTFRRDLVIQRYAVIPLSTNSGLIGWVPHCDTLHSLIKDYREKRKILLNIEHRIMQRMASDLEKLMLMQKVEVFEHALEHTAGDDLAKLLWLKSPSSEVWFERRTNYTRSLAVMSMVGYILGLGDRHPSNIMLDRVTGKFLHIDFGDCFEVAVTRDKFPEKIPFRLTRMLINAMEVTGIEGTYRRTCESVMEVLHRHRDSVMAVLEAFVYDPLLNWRLIDAGRRAGAPDDDGDASPQRPDDAPAETNLNKRALAIVNRVRDKLTGRDFTHIEENSVSVQRQVDLLIQQATSNENLCQCYVGWCPFW.Whole-mount adipose tissue immunostainingοC until mounted on slides.Females were sacrificed by crushing the ventral nerve and their abdomens were immediately dissected in cold phosphate buffered saline (PBS) to obtain fat body. Fat bodies were placed in 1.5ml microfuge tubes that were previously coated with 3% bovine serum albumin (BSA). Fat bodies were fixed in 1ml of 4% paraformaldehyde diluted in cold PBS (PFA) for 30 minutes at room temperature, rocking on a nutator. The PFA was then removed, and samples were rinsed twice with 1ml of PBS containing either 0.1% Triton X-100 (PBTX) or Tween 20 (PBTW) as a detergent. After rinsing, samples were washed three times with 1ml of 0.1% PBTX or PBTW, rocking each time for 15min on a nutator at room temperature. After the last wash, samples were incubated in 1ml of a blocking solution (0.1% PBTX or PBTW in 5% BSA) overnight at room temperature on a nutator. Samples were incubated in the indicated primary antibody concentration, diluted in blocking solution, overnight at room temperature. After washing three times for 15 minutes with PBTX or PBTW, samples were incubated in goat anti-rabbit AlexaFluor 488 secondary antibody diluted 1:250 in blocking solution and protected from light by covering with foil. Samples were then washed three times for 15 minutes with 0.1% PBTX or PBTW, protected from light. Following removal of wash solution, two drops of VectaShield containing DAPI were added. Samples were stored at 41X PBS – made from 10X PBS 3% BSA – made from 30% BSA 4% PFA – made from 16% PFA Triton X-100 - VWR, 0694-1LTween 20 – VWR, 0777-1LBlocking solution – 0.1% PBTX or PBTW and 5% BSA in 1XPBSRabbit anti-FOXO – GenScriptRabbit anti-TOR – GenScriptGoat anti-rabbit IgG AlexaFluor 488 – Invitrogen, A27034