The ecological effects on populations of non‐game species driven by the annual release and management of tens of millions of gamebirds for recreational shooting are complex and relatively poorly understood. We investigated these effects at a national scale, considering multiple taxa simultaneously. We used records from the UK National Biodiversity Network Atlas to compare animal species and diversity metrics previously suggested to be affected by behaviors of the released birds, or because resources or habitats are influenced by game management or both processes. We contrasted records from 1 km grid squares where gamebirds were reported released in Great Britain, and control squares with similar land cover but where no releases were reported. There were more records overall reported from release grid squares (RGS) compared with controls (CGS), perhaps due to greater reporting effort or greater biological richness. We found fewer foxes in RGS and fewest in grid squares with largest releases, but more carrion crows in RGS. We found no consistent effects for buzzards, ravens, jays, or magpies. There were more rodents and gray squirrels reported from RGS but no differences for reptiles. There were more butterflies but fewer beetles reported from RGS but no consistent patterns for Orthoptera or ground beetles considered common gamebird prey. Farmland and woodland birds exhibited higher abundance, richness, and diversity in RGS when considering absolute records, but woodland bird abundance and richness were lower when correcting for the relative number of records. These nationwide results, despite crude data resolution, reveal diverse effects of gamebird release and management at a national scale and across trophic levels, increasing some non‐game animal populations while decreasing others. This should alert practitioners, opponents, and legislators that a focus on single taxa effects, either positive or negative, may obscure the simultaneous changes in other taxa. The release of gamebirds in the GB is accompanied by changes in populations of non‐game species in those areas where release and management occur. These changes include both increases and decreases and span a wide range of taxa and trophic levels. The ecological effects of the release and subsequent management of gamebirds in GB are complex and concentrating on changes in single taxa may miss other, simultaneous, effects. We also look within RGS for relationships between the numbers of gamebirds reported as released and biodiversity records. This allows us to ask, at a national scale, how does the presence and scale of gamebird release and management relate to populations of non‐game fauna in the UK?To better understand the ecological effects of gamebird release and management on non‐game animal populations, we should explore relatively fine‐scale spatial associations between gamebird release and management with records of taxa of interest across a large area. Here, we do this by combining official records of gamebird release with a national database of UK biodiversity records. We analyze datasets at the level of 1 km22.12 grid square contained >1 release site, we combined Usual Stock Numbers for all sites in the grid square. This left us with 3284 grid squares where gamebirds were reported as being held for release which we defined as Release Grid Squares (RGS)  and duck (no distinction by species) and numbers held for release (as opposed to those registered held for breeding or rearing) as denoted under Livestock Unit Animal Purpose as release for shooting. This included details of Usual Stock Numbers of gamebirds designated as held for release at 3624 sites in the UK. We removed sites where Usual Stock Numbers were zero, grid squares with >50% urban cover (where we assumed that the person completing the Register had mistakenly given their home address rather than the site where the birds were held for release), and sites in Northern Ireland (not included in the Centre for Ecology and Hydrology [CEH] land cover data—see below). When a 1 kmAlthough registration with the Poultry Register is a legal requirement for holdings of ≥50 birds, it appears that compliance is low with the register only accounting for about one‐third of the gamebirds estimated to be released in the UK annually and about half to one‐third of the number of locations compared to the estimated number of shoots releasing gamebirds Madden, . Therefo2.22 of Great Britain  by ensuring that they contained similar land cover patterns to those seen in RGS because it would be uninformative to compare biodiversity measures from RGS which are reported to mainly occur in lowland agricultural areas with CGS in the uplands, urban areas or coastal sites where the species compositions are going to be markedly different. Therefore, we used the CEH land cover data 2015 to examine the landcover measures in grid squares. The aggregated CEH data provides a percentage cover of 10 different habitat types for each 1 kmFirst, we compared RGS with all other grid squares in the UK Appendix . As expeFor RGS, we included data on the number of gamebirds  reported as held for release. This varied across the grid squares from 1 to 255,500 birds/site. The distribution of releases was highly skewed. Most sites  reported releases of less than 3000 birds. These could be classified as small shoots  than CGS (x = 2.14) . We also tested how well the grid square classification reflected the extent of habitat management associated with game releasing and shooting. We randomly selected 100 RGS and CGS and viewed them on Google Earth (with grid squares demarked using http://nearby.org.uk/google.html#9) using the Historic View function with images dating back to 2000. Each grid square was viewed by JRM, blind to the classification, known to RB. The number of game strips in each grid square was counted. Game strips are typically planted along field margins or woodland edges and comprise thin areas of mixes of vegetation that contrast with the crops of grassland in the adjoining field, but they are often not separated from those crops by a hedge  than CGS (0.35/km2) . These results confirm that our sets of grid squares differed in expected ways in terms of the release and management of gamebirds, but also demonstrate that at least some CGS maybe release sites that are not recorded in the Poultry Register, given the presumed relatively low compliance with the register , we used t‐tests assuming unequal variances. All analyses were conducted in R  compared to CGS (x = 329) . We cannot determine whether this difference occurs because, despite an assumed equal sampling effort in RGS and CGS, there is genuinely more biodiversity at release sites, or because there were higher biodiversity sampling efforts made on RGS. We discuss the likelihood of each explanation below, but in our subsequent analyses, we consider both possibilities. We analyzed absolute record data under the assumption that sampling efforts were equal and differences in reported records reflected actual differences in biodiversity amounts. We also analyzed data for each taxa of interest controlling for the total number of records reported for that grid square, meaning that we used proportion values under the assumption that there was differing sampling effort across the grid square types.There were 32.5% more records reported from RGS . This supports Pringle et al.  who founRodents and gray squirrels appear to benefit from gamebird release and management, with higher populations on RGS. This national pattern matches some of the site‐specific findings of Davey  who founThe mix of effects on invertebrate populations that we found resembles the variation in magnitude and direction of effects that others have reported in site‐specific studies. Neumann et al.  reportedSpecies of farmland and woodland birds used as indicators by DEFRA showed higher levels of total abundance, species richness, and Shannon diversity in RGS compared with CGS. This matches some effects on woodland specialist species from site‐specific studies showing (some) such species being more abundant in game‐managed woodland compared to nearby unmanaged areas  is around one‐third of the mean estimate of birds released calculated using a range of other methods Madden, . TherefoHow the differences in populations that we report are perceived and valued is likely to be subjective. For example, high numbers of rodents may be perceived as a source of agricultural pests and considered a negative consequence, or, at least in the UK, local population increases may be set against a general nationwide decline in small mammals ; formal analysis ; methodology (lead); project administration (lead); supervision (lead); visualization ; writing – original draft (lead); writing – review and editing (lead). Rosie Buckley: Data curation ; formal analysis ; visualization ; writing – original draft (supporting); writing – review and editing (supporting). Sophia Ratcliffe: Data curation ; writing – original draft (supporting); writing – review and editing (supporting).The APHAPR2020 dataset was obtained in conjunction with Dave Stone of Natural England. RB was funded by an internship from the Department of Psychology, University of Exeter. JRM conducted this research while supported by the Department of Psychology, University of Exeter. The NBN Atlas is funded by the National Biodiversity Network, with major contributions from Natural England, NatureScot, Northern Ireland Environment Agency, and Natural Resources Wales.Appendix S1Click here for additional data file.Appendix S2Click here for additional data file.Appendix S3Click here for additional data file.Appendix S4Click here for additional data file.Appendix S5Click here for additional data file.Appendix S6Click here for additional data file.


To the editor,Vertigan et al.We sought to assess the clinical characteristics and airway inflammatory processes associated with work‐related dysphonia in a cohort of subjects with sensitizer‐induced occupational asthma (OA) ascertained by a positive specific inhalation challenge (SIC). This retrospective study included 341 subjects identified among the multicenter European network for the PHenotyping of OCcupational ASthma (E‐PHOCAS)2‐agonist once or more a day); OA caused by a low‐ versus a high‐molecular‐weight agent; as well as eosinophil and neutrophil sputum cell counts  and a higher sputum neutrophil count  were significantly associated with a higher likelihood of work‐related dysphonia (Table p = 0.057). Dysphonia showed a negative association with increased sputum eosinophil counts .Forty‐nine 14.4%) subjects experienced dysphonia while exposed at their workplace. The baseline clinical features and sputum cell counts of the subjects with and without dysphonia as well as the univariate associations with dysphonia are detailed in Table 4.4% subjDysphonia is a main symptom of worked‐associated irritable larynx syndrome (WILS) which has been defined as neuronal sensitization by a workplace trigger bringing about laryngeal dysfunction.To our knowledge, our study is the first to describe a relationship between neutrophilic inflammation and work related dysphonia.We acknowledge the limitations inherent to the retrospective cross‐sectional design of this study. The presence of dysphonia was not objectively documented through direct visualization of inappropriate laryngeal movement. In addition, dysphonia was not assessed during the SIC procedure implying that it was not possible to ascertain that the agent inducing the positive SIC response was also the cause of dysphonia at work.Despite their inherent limitations, our findings suggest that airway neutrophilic inflammation could be involved in the development of work‐related laryngeal dysfunction. This study highlights the need for further prospective studies using validated questionnaires, laryngoscopy, and induced sputum analysis in order to explore the association between laryngeal dysfunction and neutrophilic airway inflammation.All E‐PHOCAS investigators contributed to the conception and design of the study. All authors contributed to the acquisition, analysis and interpretation of the data. They provided input into the drafting of the manuscript, critical feedback, and final approval for submission of the manuscript for publication. NM is the guarantor of the final content of the manuscript.The authors have no conflicts of interest to declare.Association d'Aide aux Insuffisants Respiratoires d'Alsace (ADIRAL); Fondation Mont‐GodinneSupporting Information S1Click here for additional data file.


All participants visited the lab four times over the putative 28‐day menstrual cycle. At each visit, we performed blood draws to measure plasma estrogen (estradiol) levels, and tests of postural sway using a force plate. During late follicular and mid‐luteal phase, estradiol levels were lower in participants using oral contraceptives , reflecting expected consequences of oral contraceptive use. Despite these differences, postural sway was not significantly different between participants who were using oral contraceptives and participants who were not . Overall, we found no significant effects of the estimated menstrual cycle phase—or absolute levels of estradiol—on postural sway.When people stand still, they exhibit a phenomenon called postural sway, or spontaneous movement of the body's center of pressure, which is related to balance control. In general females show less sway than males, but this difference only begins to appear around puberty, pointing to different levels of sex hormones as one potential mechanism for sway sex differences. In this study, we followed cohorts of young females using oral contraceptives ( Estrogen may change the mechanical properties of connective tissues, which could affect balance. We followed two cohorts of female participants—one naturally cycling and one on oral contraceptive—over four visits during the menstrual cycle to test postural sway and plasma estradiol level. We found no difference in balance over the course of the menstrual cycle in either cohort or between cohorts, despite phasic differences in estrogen levels between cohorts. In addition to its wide‐ranging effects on bone Cutler, , estrogeOne important motor ability is balance or our ability to maintain an upright posture without falling over. Postural sway—a measure of spontaneous shifts of a person's center of mass as they stand stationary—is consistently lower in females compared to males, but this difference does not appear until around puberty and is not accounted for by other likely covariates like height or weight . These participants were part of a larger study investigating the effects of oral contraceptives on bone health. All participants provided written informed consent. We conducted this study in accordance with procedures approved by the San Diego State Institutional Review Board's Human Research Protection Program.We recruited healthy young female participants who were either using oral contraceptives or not using oral contraceptives to participate in this study. Participants were required to be between 18 and 25 years old, have a BMI between 18 and 32 kg/m2.2g at 4°C and subsequently stored the samples at −80°C until analysis.Following an initial lab visit for familiarization and screening, we scheduled each participant to make four visits for blood collection and balance testing. For participants who were not using oral contraceptives, these visits corresponded to menstrual cycle days 2, 4, 11, and 21 . For participants using oral contraceptives, these visits corresponded to pill pack days 21 (start of placebo pills), 24, 3, and 13. We chose these intervals for participants on oral contraceptives to coincide with the start of the estrogen‐free pill phase of the oral contraceptives (Day 21) and to match the time between visits in the naturally cycling cohort. Participants always visited the laboratory in the same order: their first testing visit corresponded to cycle day 2 —participants contacted the study group to schedule their first test as soon as menses began—and the last visit was Day 21 . We asked participants to fast for 10 h before their scheduled visits and we instructed oral contraceptive users who consumed their pill in the morning to wait until after the blood draws. We centrifuged blood samples immediately after blood draws for 15 min at 4000 × 2.3We evaluated participants' balance using the BtrackS Balance Test . The BtrackS system incorporates a standardized force plate and data collection protocol to quantify postural sway during quiet standing. Postural sway results from the natural, spontaneous motion of a person's center of mass as they stand stationary and reflects the combination of neural outputs and sensory inputs that govern balance. Balance is interpreted as inversely related to postural sway: a person who displays more postural sway (meaning their center of mass moves more) is considered to have worse balance.The BBT testing protocol involves recording postural sway during three, 20 s trials following one familiarization trial. During these trials, participants are instructed to stand as still as possible on the BtrackS force plate with their eyes closed and feet shoulder width apart. The BtrackS force plate records the center of pressure (the projection of the center of mass onto the ground) at a rate of 25 Hz. Results from the BBT have been validated against laboratory‐quality force plates . These ultra‐sensitive ELISA kits have a sensitivity of 5.14 pmol/L with a range of 5.14–734.21 pmol/L. For samples that exceeded the upper limit of the ultra‐sensitive ELISA kits, we then used standard ELISA kits (ALPCO) which have a sensitivity of 36.71 pmol/L with a range of up to 11,747 pmol/L. To further analyze participants' hormone profiles, we also measured serum progesterone levels. The ELISA kits (ALPCO) we used to measure plasma progesterone levels have a sensitivity of 0.32 nmol/L and a range of 0.95–191 nmol/L.repository.We imposed additional inclusion criteria on participants who were not using oral contraceptives to ensure they showed a typical hormonal profile during the menstrual cycle. We only included participants who (1) showed peak progesterone levels at Visit 4 , and (2) showed an increase in estradiol levels from Visit 1 (early follicular phase) to Visit 3 (late follicular phase). We imposed these criteria to ensure a participants showed a relatively regular menstrual cycle; it has been suggested that a major source of variability in previous literature investigating the menstrual cycle is the inclusion of females with atypical menstrual cycles in supposedly eumenorrheic groups  for additional interpretive value; unlike large p‐values, small Bayes Factors can be interpreted as evidence for the null hypothesis  to perform our statistical analyses. We analyzed the effect of the day of the menstrual cycle on hormone levels and postural sway using linear mixed‐model analysis. We constructed the linear mixed‐effects models using the Linear Mixed Models workflow in JASP with Visit number (4 levels) and Contraceptive status (2 levels) treated as fixed effects and individual participants as random factors for which we estimated random intercepts but not slopes. We performed the same analyses in a Bayesian framework with the Bayesian ANOVA workflow in JASP treating individual participants as random factors and default assumptions. We performed repeated‐measures correlations using the  Vallat,  for Pyth3Fifty‐eight of the participants completed full sets of both balance tests and blood draws . This mainly occurred because some participants had not yet fully healed from their first blood draw when they returned for their second blood draw (2 days later) and were therefore unable to undergo that blood draw. After imposing additional constraints on hormonal profile in participants who were not using oral contraceptives , indicating that estradiol levels were higher during Days 11 and 21 than Days 2 and 4 for participants not using oral contraceptives. In contrast, estradiol levels did not vary significantly over the course of the menstrual cycle for participants using oral contraceptives, so estradiol levels in the cohort using oral contraceptives were lower than in the cohort not using oral contraceptives during Days 11 and 21. These results are shown in Figure First, we confirmed that oral contraceptives altered estradiol. We found a significant interaction between day of menstrual cycle and oral contraceptive use  or of contraceptive use , additionally, there was no significant interaction between contraceptive use and day of menstrual cycle. Across days, the mean difference between participants who used oral contraceptives and those who did not was 2.09 cm ; day‐by‐day means and standard deviations (as well as mean differences and 95% CI) for postural sway are shown in Table We next investigated whether postural sway varied over the course of the menstrual cycle for the two cohorts of female participants. We again used a linear mixed‐effects model to investigate the effect of day of the menstrual cycle and contraceptive use on postural sway. This analysis did not show any significant effect of day of the menstrual cycle  or for participants not using oral contraceptives . These data are shown in Figure Finally, we directly tested whether estrogen level was associated with postural sway using repeated‐measures correlation . We found no significant differences in postural sway between participants using oral contraceptives (who had lower levels of estradiol) and participants who were not using oral contraceptives, but a larger sample size might be required to elucidate this point further (BF10 ≈ 1). When we compared estradiol levels to participant sway across all participants, we found no evidence that varying levels of estradiol affect postural sway in people using oral contraceptives.Our measures of balance do not provide evidence that postural sway changes over the course of the menstrual cycle . Additionally, we did not record participants' levels of physical activity, which could also affect menstrual or balance outcomes.Postural control is difficult to define, and postural sway is not a direct measure of balance. It is possible that our measure of sway did not capture some aspect of balance that is associated with changes in hormone levels. Additionally, in the context of sports injuries, the series of events that lead to an injury (like a rapid change in direction) may not be closely linked with the factors measured via postural sway. Because of this, it is possible that there are balance‐ or injury‐related measures that would be sensitive to changes in hormone level but which we did not capture using postural sway.4.4While previous studies have investigated the effects of the menstrual cycle on balance, our study makes the following important contributions. First, we quantified balance using an easily understood measure  which is reported in common physical units and can be compared to normative data from more than 16,000 people (Goble & Baweja, Sasha Reschechtko drafted the manuscript. Sasha Reschechtko, Mark Kern, and Shirin Hooshmand edited and revised the manuscript. Mark Kern and Shirin Hooshmand designed the research. Thuy Ngoc Nguyen, Kristine Giltvedt, Mark Kern, and Shirin Hooshmand collected the data. Sasha Reschechtko and Thuy Ngoc Nguyen analyzed the data. Sasha Reschechtko and Thuy Ngoc Nguyen interpreted the findings.Data collection for this study was supported by the California Prune Board Grant G00012885 to M.K. and S.H.The authors declare no conflicts of interest, financial or otherwise.This study including all experimental procedures was approved by the San Diego State Institutional Review Board’s Human Research Protection Program.


To test the hypothesis that infant night waking is an adaptation to increase interbirth intervals (IBIs)  by exhausting the mother, we made an initial attempt at investigating whether maternal sleep disturbance is associated with longer IBIs. We also explored whether postpartum depression symptoms mediated the association between maternal sleep disturbance and IBI length. We used retrospective self-reports from 729 mothers living in Finland. We conducted structural regressions separately for the mother’s two first children at two different age intervals (0–1 and 1–3 years). Infant night waking was associated with maternal sleep disturbance (β  =  .78–.84) and maternal sleep disturbance was associated with postpartum depression symptoms (β  =  .69–.81). Postpartum depression symptoms were also associated with longer IBIs for the first child (β  =  .23–.28). This result supports the notion that postpartum depression in and of itself could be viewed as adaptive for the offspring’s fitness, and not just as an unintentional byproduct of the mother’s sleep disturbance. Contrary to our prediction, maternal sleep disturbance was, however, associated with shorter IBIs for the first child (β  =  −.22 to −.30) when including postpartum depression symptoms in the model. We discuss the potential role of social support as an explanation for this unexpected result. According to the mother-offspring conflict theory, what is the ideal amount of maternal investment from the perspective of the infant is higher than from the perspective of the mother . The intOne way mother-offspring conflict might express itself is in the length of interbirth intervals . The optimal IBI differs from the standpoint of the mother’s and the offspring’s fitness, with the mother benefitting more from shorter intervals than the offspring . This isPrevious studies have suggested that infant night waking to breastfeed could be an adaptive strategy by the offspring to lengthen the IBI by suppressing the mother’s fertility . A recenIt is unsurprising that frequent infant night waking affects maternal sleep. For instance, a recent German population-based large-scale longitudinal study showed that parental sleep satisfaction declines postpartum, with maternal sleep being especially affected . StudiesBased on the premise that the infant benefits more from longer IBIs than the mother and that infant night waking is an adaptation to increase IBIs by exhausting the mother, we examined whether maternal sleep disturbance is associated with longer IBIs and postpartum depression symptoms. We were specifically interested in testing whether more maternal sleep disturbance was associated with longer IBIs. We also explored whether this association would be mediated by postpartum depression symptoms. In line with the substantial literature on maternal sleep, we also expected that more infant night waking would be associated with more maternal sleep disturbance and that more maternal sleep disturbance would be associated with more postpartum depression symptoms.The current study was granted ethical permission by the Board for Research Ethics at Åbo Akademi University before data collection began. All participants gave their informed consent prior to participating in the study. They were informed that participation is voluntary and that they can terminate their participation at any point during the survey.SD  =  9.2).We collected data in April 2020. We recruited mothers between ages 18–60 with at least two biological children. Mothers of twins were excluded from the study. The participants were recruited through a Facebook advertisement targeting female adults living in Finland. After completing the survey, participants were invited to a separate questionnaire where they could fill out their e-mail address to participate in a lottery of a 100€ gift card to a multi-brand online shop. The completion rate for those who started the survey was 64.0%. Participants who did not respond to all items in the questionnaire were excluded from the statistical analyses. The final sample consisted of 729 mothers, aged 22 to 60 years old, with a mean age of 41.3 years . We assessed the below-mentioned measures separately for two age intervals; the child’s first year and when the child was between 1 and 3 years old. Mothers of three or more children additionally answered the descriptive information and below-mentioned measures for the second IBI .Infant night waking. To measure the frequency of infant night waking, we used three self-made questions: “How many times on average did your child wake up per night?”, “How well did your child sleep?” and “How difficult was it to get your child to sleep through the night?”. The items were rated on a 5-point Likert-type scale (1–5), with the anchors 1  =  hardly ever/very well/very easy; 5  =  more than five times per night/very poorly/very hard .Maternal sleep disturbance. To measure the level of maternal sleep disturbance, we used the Insomnia Severity Index .Descriptive statistics of the sample are presented in n  =  729), 433 (59.4%) had two children and 296 (40.6%) had three or more. The mean for the IBI between the first and second child was 39.9 months (SD  =  29.1) and for the IBI between the second and third child 46.4 months (SD  =  33.5).Of the participating mothers . For the first child at the second age interval (1–3 years), the associations were in the opposite direction: IBI was significantly and negatively associated with all maternal sleep disturbance items (r  =  -.03– -.16), indicating that mothers with more sleeping problems had shorter IBIs. For the second child at the second age interval, some associations between IBI and maternal sleep disturbance were positive, and some were negative.Correlations for individual items are presented in Supplementary Online Figure 1. For both children at the first age interval (0–1 years), IBI was significantly and positively correlated with all but one of the maternal sleep disturbance items, indicating that mothers with more sleeping problems had longer IBIs. The effects, however, were small , Maternal Sleep Disturbance (eight indicators), and Postpartum Depression Symptoms (seven indicators). For the measurement model, responses for both children at both age intervals were included. Our initial model showed suboptimal fit , so we adjusted the model by adding five residual correlations, all within scales. The residual correlations increased the χ2 fit with at least 50 points. Two of the residual correlations were between EPDS items . Three of the residual correlations were between ISI items . After the above-mentioned adjustments, our final model showed adequate fit .Structural regressions. The results of the four structural regressions are summarized in The results for the structural regressions showed that infant night waking was associated with maternal sleep disturbance for both children at both age intervals. Maternal sleep disturbance was also associated with postpartum depression symptoms for both children at both age intervals. Interestingly, postpartum depression symptoms were associated with longer IBIs for the first child during both age intervals but were not significantly associated with longer IBIs for the second child at either age interval. Finally, maternal sleep disturbance was associated with shorter IBIs for the first child at both age intervals, but not significantly associated with IBIs for the second child at either age interval. The total effect of maternal sleep disturbance  was not statistically significant in any of the regressions. The indirect effect  was statistically significant and positive for the first child during the first year, but not for the first child at the second age interval or the second child at either age interval.Based on the premise that infant night waking is an adaptation to increase IBIs by exhausting the mother , the preCompletely in line with our expectations based on previous research, we found that infant night waking was associated with more maternal sleep disturbance and that maternal sleep disturbance was associated with more postpartum depression symptoms.shorter IBIs for the first child at both age intervals. This indicates that first-time mothers who suffer from more severe sleep problems, but not from symptoms of postpartum depression, have a shorter IBI between their first and second child. These results contradict our prediction that postpartum depression symptoms could be an unintentional byproduct of the infant’s behavior to exhaust their mother. A potential reason for this association could be received social support. Previous research has theorized that postpartum depression signals the need for additional social support  there were weak positive correlations between IBI length and the maternal sleep disturbance items during the first year postpartum, indicating that mothers with more sleeping problems had slightly longer IBIs. However, in the structural regression analysis, we found that maternal sleep disturbance—when controlled for postpartum depression symptoms—was associated with  support . It is pPostpartum depression symptoms predicted longer IBIs for the first child at both age intervals. These results support the notion that postpartum depression in and of itself could be viewed as adaptive from the offspring’s fitness’ point of view, since more severe postpartum depression symptoms are associated with longer IBIs. Furthermore, the indirect effect of maternal sleep disturbance on IBIs via postpartum depression symptoms indicates that postpartum depression symptoms indeed mediate part of the association between maternal sleep disturbance and IBIs for the first child during the first age interval. However, the total effect of maternal sleep disturbance  on IBIs was not significant for any of the regressions. That is, accounting for the association between postpartum depression symptoms and longer IBIs, maternal sleep disturbance did not predict shorter IBIs any longer .Finally, the results imply that there is a difference between mothers of two and mothers of three or more children. For both the associations between maternal sleep disturbance and IBIs as well as for postpartum depression symptoms and IBIs, there were no significant associations for the second child at either age interval. The average age to give birth for first-time mothers in Finland was 29.3 years in 2018 . This isThe present study is one of the first to study the relationship between maternal sleep and IBI length. However, there are several aspects that need to be considered before drawing strong conclusions from the associations found. One limitation of the current study is that data were gathered retrospectively, as it increases both under- and overreporting symptoms and behaviors. The high, positive within-subject cross-time correlations could be a sign that it was hard for the mothers to differentiate between their experiences during the two age intervals. As being able to measure an IBI per definition requires that some time has passed since the maternal sleep disturbance and postpartum depression of interest, a longitudinal design could minimize retrospective bias. Moreover, one study inclusion criterion was having at least two children, excluding any mothers that decided not to have additional children after the first child due to severe postpartum depression or sleeping difficulties. All mothers who had an IBI shorter than three years were also excluded from the analyses for the second age interval (1–3 years), skewing the results in favor of longer IBIs. The sample size for these analyses was also smaller, resulting in issues with statistical power.Finally, the participants in our study were in large part recruited via a social media ad targeting women living in Finland. The Nordic countries stand out with generous social support and parental leave policies —possiblyContrary to our initial hypothesis, we did not find support for the notion that infant night waking is an adaptive strategy in order to tire the mother, making her less likely of conceiving and gestating an additional child, thus lengthening the IBI. Interestingly, the results indicate that postpartum depression symptoms could themselves be adaptive from the offspring’s fitness’ point of view, by being associated with longer IBIs. In other words, if infant night waking was indeed an adaptive strategy to extend the IBI by exhausting the mother, it seems like maternal sleeping difficulties alone are not sufficient to result in longer IBIs. Future research should investigate the role of maternal social support to shed light on potential explanations as to why mothers who suffer from sleep disturbance but not from postpartum depression have shorter IBIs. Furthermore, investigation into the difference between mothers of two and mothers of three or more children is needed to understand why some of our results were significant only for the first IBI.Click here for additional data file.Supplemental material, sj-docx-1-evp-10.1177_14747049211046162 for The Mother–Offspring Conflict: The Association Between Maternal Sleep, Postpartum Depression, and Interbirth Interval Length by Annika Gunst, Elin Sjöström, My Sundén and Jan Antfolk in Evolutionary PsychologyClick here for additional data file.Supplemental material, sj-docx-2-evp-10.1177_14747049211046162 for The Mother–Offspring Conflict: The Association Between Maternal Sleep, Postpartum Depression, and Interbirth Interval Length by Annika Gunst, Elin Sjöström, My Sundén and Jan Antfolk in Evolutionary Psychology


DNA is matter in becoming. DNA changes as it travels across forensic sites and domains. New formations occur as humans, technologies and DNA interact. Understanding DNA as in‐formation is of particular relevance vis‐à‐vis the increase of algorithmic technologies in the forensic sciences and the rendering of DNA into (big) data. The concept can help identifying, acknowledging and communicating those moments of techno‐scientific interaction that require discretion and methodical decisions. It can assist in tracing what form DNA will take and what consequences this may have.Traces are fundamental vectors of information. This is the first of seven forensic principles formulated by the 2022 Sydney declaration. To better understand the Crime Scene Investigation > From Traces to Intelligence and EvidenceForensic Biology > Ethical and Social ImplicationsForensic Biology > Forensic DNA TechnologiesThis article is categorized under: Digital forensic technologies change the ways in which DNA is processed as information. Understanding DNA as in‐formation can help us to assess the consequences of this shift. “View of the cell nucleus” by istockphoto. Besides the many DNA databases for scientific and criminalistic research, private databases are on the rise, too. Many of them are tied to ancestry research, such as The growth of DNA databases, their accessibility and their integration with work processes can be of great assistance to crime solving. These shifts in DNA collection and storage may even render it possible to crowdsource some aspects of crime solving ; funding acquisition (lead); methodology (lead); writing – original draft (lead); writing – review and editing (lead).This project has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (Grant agreement No. 947681).Privacy implications of the new "omic" technologies in law enforcementNone


Community ecology is built on theories that represent the strength of interactions between species as pairwise links. Higher‐order interactions (HOIs) occur when a species changes the pairwise interaction between a focal pair. Recent theoretical work has highlighted the stabilizing role of HOIs for large, simulated communities, yet it remains unclear how important higher‐order effects are in real communities. Here, we used experimental communities of aquatic protists to examine the relationship between HOIs and stability (as measured by the persistence of a species in a community). We cultured a focal pair of consumers in the presence of additional competitors and a predator and collected time series data of their abundances. We then fitted competition models with and without HOIs to measure interaction strength between the focal pair across different community compositions. We used survival analysis to measure the persistence of individual species. We found evidence that additional species positively affected persistence of the focal species and that HOIs were present in most of our communities. However, persistence was only linked to HOIs for one of the focal species. Our results vindicate community ecology theory positing that species interactions may deviate from assumptions of pairwise interactions, opening avenues to consider possible consequences for coexistence and stability. This study explores the relationship between higher‐order interactions (HOIs) and community stability in experimental communities of aquatic protists. We found evidence that additional species positively affected the persistence of the focal species and that HOIs were present in most of our communities. Our results vindicate community ecology theory positing that species interactions may deviate from assumptions of pairwise interactions, opening avenues to consider possible consequences for coexistence and community stability. Species interactions, such as competition and predation, are important processes determining community structure and ecosystem stability and hence are important drivers of diversity  abundance by consuming limpets , which dislodge or consume young acorn barnacles. An example of an interaction modification, or interspecific HOI, is barnacles altering the bird–limpet interaction by changing the ability of birds to find limpets due to the similar color of L. digitalis and barnacles shells. HOIs are most commonly described in terms of how a third species impacts the interaction between a focal species pair. However, intraspecific HOIs are also important to consider and represent the cumulative impacts of interactions among the individuals of each “competitor species”  on the focal species  decomposing the protozoan pellet medium  constitute the intermediate consumers in our experimental microcosms. Both C. striatum (length: 50–100 μm) and D. campylum (35–90 μm) are consumed by the top predator Spathidium sp. (40–300 μm), which cannot survive only on bacteria as prey    because their pairwise trophic interactions are well documented by previous studies  alone to investigate the direct effects between the two focal species (CD) (2) two prey and Paramecium (CDP). (3) two prey and Spirostomum (CDS). These communities were also grown in the presence of the top predator Spathidium   two prey (ies (CD)  two preyTo start the experiment, we took out 20 or 30 mL of the bacterized PPM medium  and replaced it with 10 mL of the stock culture of each consumer species at their carrying capacity. Therefore, the starting densities of all consumers were 10% of their carrying capacities. In the predation treatments, 4 days after the introduction of the consumer species, ten predator individuals were pipetted from the maintenance plates to the microcosms. To assure establishment in each microcosm, another ten individuals of predators were added after 8 days. The experiments were conducted at 15°C with no light, and suitable growing conditions for ciliates  were taken using 25× magnification on a stereomicroscope (Leica M205 C) mounted with a digital CMOS camera  with dark field illumination. We took three subsamples for each microcosm to get a precise estimate of abundance and processed the videos with the R package BEMOVI (version 1.0.2)  classifier on 350 to 400 randomly chosen and manually labeled trajectories of each species across the community compositions and time with the R package e1071 .To understand the effect of community composition on the abundance of the species, we calculated the mean abundance over the entire 53‐day experiment for each species in each of the six communities. Mean abundance of each of the five species was analyzed with two‐way ANOVA . First, we fitted the Lotka–Volterra model with no HOIs:We estimated the effect of species Pfister, . The perλi is the intrinsic population growth rate and αij represents both the intra‐ and interspecific interaction coefficients. To test for intra‐ and interspecific HOIs, we then added interaction terms to the additive model LV, resulting in the interactive LV model :To test whether the shape of the density dependence deviates from the linear form of the additive Lotka–Volterra model, we fitted the Ricker model with nonlinear density dependence. First, we fitted the additive model without HOIs:i on itself and the per capita effects of species j on species i. Interaction terms describe the mediating effects that the density of species j can have on the effect of species i (and vice versa) on the population growth rate of the focal species , accounting for right‐censoring, that is, the possibility of missing future extinctions since we only recorded the abundance for 53 days. We tested the effect of community composition on the Kaplan–Meier estimate of the focal species with log‐rank tests, followed by pairwise tests of community composition in case of a significant community wide effect with the survminer package  and Dexiostoma  and Colpidium  than Paramecium  and Dexiostoma . In contrast, when Colpidium was present with Dexiostoma and Spirostomum, the interactive Ricker model was best supported, suggesting interspecific HOIs. For the per capita population growth rate of Dexiostoma, the interactive LV model only including intraspecific HOIs (ΔAIC = 0.59 but fewer parameters) was most supported when only Colpidium was present, suggesting that Colpidium modified the effect of Dexiostoma on itself. When a third species was present, in both cases the additive Ricker model was best supported, suggesting that neither intra‐ nor interspecific HOIs were operating. Since all the best supported models were either the interactive LV or Ricker models, all intra‐ and interspecific interactions were nonlinear.Model selection revealed that HOIs influenced the per capita population growth rates of es Table . For Col3.3Colpidium survival was contingent on the community composition , since adding Paramecium to the community increased Dexiostoma survival , with Paramecium increasing the survival of Spathidium  of Spathidium is furnished with a rod‐like tip (toxicyst) to paralyze or kill other microorganisms for easier consumption ; data curation ; formal analysis ; investigation ; methodology ; validation (supporting); visualization ; writing – original draft ; writing – review and editing . Kimberley Lemmen: Conceptualization (supporting); formal analysis (supporting); investigation ; methodology ; writing – original draft (supporting); writing – review and editing . Jake Alexander: Conceptualization (supporting); investigation (supporting); methodology (supporting); supervision (supporting); writing – original draft (supporting); writing – review and editing (supporting). Frank Pennekamp: Conceptualization ; data curation ; formal analysis ; funding acquisition (lead); investigation ; methodology ; project administration (lead); resources (lead); supervision (lead); validation (lead); visualization ; writing – original draft ; writing – review and editing .None.https://doi.org/10.5281/zenodo.7896204].This article has earned Open Data and Open Materials badges. Data and materials are available at [Appendix S1Click here for additional data file.


The use of endocardial pacing in patients with univentricular hearts and intracardiac shunts is limited, primarily due to the increased risk of thromboembolism. However, when accompanied by proper long‐term anticoagulation therapy, it may be safer than epicardial interventions in selected patients at high risk for surgery.We report transvenous endocardial pacing through the atrial septal defect in a patient with tricuspid atresia, transposition of the great arteries, severe pulmonary hypertension, and complete heart block. This study is among the first reported cases using this pacing method in a patient with a univentricular heart and intracardiac shunt. Dual‐Chamber Endocardial Pacing for a Univentricular Heart with Tricuspid Atresia. His electrocardiogram revealed a complete heart block with a right bundle branch block (Figure A 42‐year‐old man from Iraq with frequent episodes of syncope since 1 month ago was admitted to the Tehran heart center hospital. He had a history of tricuspid atresia type IIc without reparative surgery. On his admission, the patient had fatigue. On physical examination, he had nail clubbing and cyanosis with an OThe risk of the surgery for the epicardial lead placement was high due to pulmonary hypertension; on the other hand, the transvenous pacing would increase the risk of systemic thromboembolism. We discussed the risk associated with each procedure with the patient and recommended the endocardial route, which he accepted. Thus, endocardial pacemaker implantation was scheduled for the patient. After prep and drape, local anesthesia and light sedation were conducted. The left subclavian vein access was gained, and a dual‐chamber rate‐modulated permanent pacemaker was implanted using a peel‐away introducer. Atrial lead was inserted in the right atrial auricle. Ventricular lead was advanced through the atrial septal defect and mitral valve and was placed in the common ventricle. The final position of the leads is indicated in Figure 3Epicardial pacing has been a regular option for patients with complex congenital heart disease. Epicardial leads may be placed during cardiac surgery for underlying congenital heart disease with a plan to replace them in the future with transvenous leads.Endocardial ventricular pacing is technically possible in patients with univentricular hearts without previous reconstructive surgery, but the higher risk for thromboembolism limits this procedure.Our report explains a technique including the insertion of an endocardial ventricular lead through the intra‐atrial septum. This method is similar to the one employed in patients with normal cardiac anatomy who undergo transseptal puncture. In a similar study, Scott et al. reported endocardial ventricular pacing in a univentricular patient with tricuspid atresia and intracardiac shunt using the same method we used. The patient had no thromboembolic events or complications related to the procedure at the 1‐year follow‐up.Therefore, transvenous endocardial pacing may be the better option for high‐risk patients for surgery. This case was a candidate for a Fontan procedure with epicardial lead placement, but this was a high‐risk procedure considering his chronic high pulmonary artery pressure. As a result, we deemed that transvenous lead placement with a tight anticoagulant regimen has a lower risk.Only a few studies have compared the outcomes of dual‐chamber pacing versus single‐chamber ventricular pacing in patients with single ventricle physiology. Nevertheless, dual‐chamber pacemakers are generally considered in these patients because of AV synchrony, which enhances cardiac output and the capacity to interfere if atrial arrhythmia or sick sinus syndrome occurs.The bilateral perirenal fluid accumulation was a rare finding in the present case. It has been reported in cases with intracardiac shunt and pulmonary hypertension.In conclusion, endocardial pacing, in the presence of intracardiac shunts, has the risk of systemic thromboembolism. Nevertheless, when accompanied by proper anticoagulation therapy and tight control of INR at 2–3, it may be a safer alternative for epicardial interventions in patients at high risk for surgery.Ahmad Yamini‐Sharif: Conceptualization; supervision; writing – review and editing. Ramin Yaghoobian: Writing – original draft; writing – review and editing. Homa Ghaderian: Supervision; writing – review and editing. Najme‐Sadat Moosavi: Writing – review and editing.The authors declare that they have no conflicts of interest.Ethical approval for this case report was not required.Written informed consent was obtained from the patient for the publication of this report.


Balloon‐assisted endoscopy enables stable endoscopic maneuverability. Balloon‐assisted endoscopic submucosal dissection (BA‐ESD) is useful in the treatment of proximal colorectal tumors where scope maneuverability is poor. Herein, we reported a case in which BA‐ESD was successfully performed using a long colonoscope with a guidewire, although the lesion could not be reached using the balloon‐assisted endoscopy technique with a therapeutic colonoscopy.A 50‐year‐old man underwent a colonoscopy that revealed a tumor in the ascending colon. BA‐ESD was performed using a conventional therapeutic endoscope due to excessive intestinal elongation and poor endoscopic maneuverability. However, the transverse colon loop could not be reduced, and the total colonoscopy failed despite using balloon‐assisted endoscopy. The scope was then changed from a conventional colonoscope to a long colonoscope, inserted into the terminal ileum, and the loop was reduced. After the guidewire was placed at the terminal ileum and the long colonoscope was removed, a therapeutic colonoscopy with an overtube was inserted into the ascending colon without reforming the colonic loop, allowing safe BA‐ESD. Endoscopic submucosal dissection (ESD) for proximal colorectal tumors is at times difficult because of the formation of intestinal loops during long procedures, leading to paradoxical movement. The efficacy of balloon‐assisted ESD (BA‐ESD) as an approach for proximal colorectal tumors has been reported.A 50‐year‐old man presented to our institution with a positive fecal immunochemical test for colorectal cancer. Colonoscopy was performed using a magnified colonoscope  by an endoscopist with 14 years of experience, and cecal intubation was performed for 10 min. Colonoscopy revealed a 50 mm‐sized lesion in the ascending colon Figure . AccordiThe procedural time from the mucosal incision to the complete resection was 260 min. The resected specimen had a diameter of 77 × 48 mm, and histopathology revealed intramucosal tubular adenocarcinoma with negative horizontal and vertical margins and no lymphovascular invasion Figure . Based oHere, we reported a successful case of BA‐ESD for a colonoscopically difficult proximal colorectal tumor using a simple method with a long colonoscope and guidewire. This technique can be applied as a rescue procedure for patients in whom it is difficult to reach the lesion even with BAE.The usefulness of the BAE method has been reported in cases with difficult cecal intubation. There are two types of BAE, including single BAE in which a balloon is attached to the overtube, and double BAE in which balloons are attached to the scope tip and overtube. Their cecal intubation rates were as high as 92.9%–98%In this study, a long colonoscope was more effective for difficult insertion cases with transverse colonic loops. The long colonoscope was inserted into the terminal ileum, which allowed the transverse colon loop to be reduced, and the guidewire was subsequently inserted into the ileum. The rigidity of the guidewire enabled deep insertion of the therapeutic colonoscope with an overtube without excessive intestinal elongation and loop reformation. A similar technique, endoscopic retrograde ileography, in which an overtube is inserted into the terminal ileum with a guidewire placed at the terminal ileum, has been reported to be useful in evaluating the terminal ileum in Crohn's disease.An important finding from our case was that the method using a long colonoscope and guidewire may be an alternative technique for accessing the cecum in cases with difficult insertion, even when using BAE. Using this method, the therapeutic colonoscope and overtube can be inserted without intestinal loop reformation, and ESD can be safely performed under stable maneuverability.In conclusion, we present the first case of BA‐ESD for a colonoscopically difficult proximal colorectal tumor using a long colonoscope and guidewire. A simple technique using a long colonoscope and guidewire are useful when cecal intubation cannot be achieved using BAE with a therapeutic colonoscope and an overtube.None.


Transbronchial lung cryobiopsy (TBLB) is routinely used to diagnose the interstitial lung disease (ILD). These results are consistent with those of surgical lung biopsy. Fluoroscopy is also used to confirm the final position of the cryoprobe; however, it can increase radiation exposure for both patients and medical care personnel. Probe‐based confocal laser endomicroscopy (pCLE) is a novel optical imaging technique that allows real‐time imaging at the cellular level in vivo. pCLE technology can also be used to identify malignancy, acute rejection in lung transplantation, amiodarone lung, and pulmonary alveolar proteinosis and visualize elastin fibres in the alveolar compartment.The aim of this study is to investigate the ability of pCLE to distinguish fibrotic pulmonary issues from normal lung disease and the safety and feasibility of CLE‐guided bronchoscopy and transbronchial lung cryobiopsy (TBLC) in patients with interstitial lung disease (ILD).pCLE images from 17 ILD patients were obtained during TBLB. These images were then compared with histology results to assess the correspondence rate.pCLE imaging of the alveolar structures was performed. Key characteristics were visible, which could potentially influence the diagnostic rate (fibrotic areas) and the complication rate (blood vessel and pleura).pCLE may reduce complications and increase the diagnostic yield. It is a potential guidance tool for cryobiopsy in the patients with ILD without fluoroscopy. Probe‐based confocal laser endomicroscopy (pCLE) may reduce complications and increase diagnostic yield. Thus, it can be a potential guidance tool for cryobiopsy in patients with ILD without fluoroscopy. Non‐smoking (for at least 3 months) patients with ILD confirmed by high‐resolution enhanced CT scan requiring histological examination were selected for inclusion in this study between October 2018 and April 2020. All patients were aged between 18 and 80 years old. Written informed consent was obtained from all participants.9/L) that cannot be corrected after anticoagulant therapy; ILD in acute aggravated phase; severe impaired lung function (FVC < 50% predicted and DLCO < 35% predicted); hypoxaemia while breathing room air (SpO2 < 90%), diffusing capacity for carbon monoxide less than 40% predicted.Exclusion criteria included the following: severe hypertension or arrhythmia; recent myocardial infarction or a history of unstable angina; severe heart, lung, liver, or kidney dysfunction, or extreme systemic failure; severe pulmonary hypertension (systolic pulmonary artery pressure > 50 mmHg assessed by echocardiography); severe superior vena cava syndrome; coagulopathy  or severe thrombocytopenia  was performed on the other lobe of the ipsilateral lung to reduce interference with pCLE images.pCLE probe  was introduced into the working channel of the bronchoscope and slowly proceeding to the alveolar compartment until the pleura was reached. The distance from the secondary carina to the target position was recorded by a marking ring  and a freezing time of 3 to 6 s.The samples were fixed in 10% buffered formalin, embedded in paraffin and then sliced into 4‐μm‐thick sections for Elastica van Gieson staining. Histology images of the lung biopsies were assessed by an experienced pulmonary pathologist. A final diagnosis was determined based on patient history, laboratory results, high‐resolution CT scan, BAL, and histology from the lung biopsies after an ILD multidisciplinary discussion session.3n = 17). Sixteen patients were confirmed definitely diagnosed with ILD, including 13 nonspecific interstitial pneumonia (NSIP), one chronic hypersensitivity pneumonitis, one bronchiolitis obliterans organizing pneumonia (BOOP), and one pneumoconiosis. One patient had an inconclusive pathologic diagnosis. The diagnosis rate reached 94.1%. The complication rate was 17.6% (one case of moderate bleeding and two cases of pneumothorax without chest tube).High‐resolution computed tomography (HRCT), pCLE imaging, and TBLC were collected in 17 ILD patients. Clinical characteristics and final diagnosis of the patients are show in Table On the pCLE imaging, the alveolar cavity and the elastin fibres within the alveolar septum could be seen. The adjacent blood vessels were clearly visible  patient due to suspected dermatomyositis  patient due to suspected dermatomyositis in ICU. The case was excluded because TBLC was unavailable. From our observation, we noticed densely packed fibres and alveolar structures absent, which is different of the pattern of acute cardiac failure or infectious pneumoniaTo our knowledge, there is only one previous study that assess pCLE in combination with fluoroscopy as a guidance tool for cryobiopsy in ILD.There are several limitations in this study. First, it was a retrospective study. Second, the results were limited by the small sample size. ILD is a heterogeneous disease; larger prospective studies should be performed to assess in future investigations. In addition, fluoroscopy as a basic radiology measure should be recommend due to the consideration of potential life‐threatening side effects of TBLC especially haemorrhage and tension pneumothorax.In conclusion, our findings suggest that pCLE is the potential tool to help identify biopsy areas and might reduce complication and improve diagnostic yield. Further investigations with a prospective design and a larger number of patients are required to confirm the value of TBLC guide by pCLE in ILD.Yu Zheng and Liyan Zhang contributed equally to this work.The authors have no conflicts of interest to declare.All subjects have given their written informed consent and that the study protocol was approved by the institute's committee of Ren Ji Hospital on human research (2018113).


Many biocatalytic processes inside cells employ substrate channeling to control the diffusion of intermediates for improved efficiency of enzymatic cascade reactions. This inspirational mechanism offers a strategy for increasing efficiency of multistep biocatalysis, especially where the intermediates are expensive cofactors that require continuous regeneration. However, it is challenging to achieve substrate channeling artificially in vitro due to fast diffusion of small molecules. By mimicking some naturally occurring metabolons, nanoreactors are developed using P22 virus‐like particles (VLPs), which enhance the efficiency of nicotinamide adenine dinucleotide (NAD)‐dependent multistep biocatalysis by substrate channeling. In this design, NAD‐dependent enzyme partners are coencapsulated inside the VLPs, while the cofactor is covalently tethered to the capsid interior through swing arms. The crowded environment inside the VLPs induces colocalization of the enzymes and the immobilized NAD, which shuttles between the enzymes for in situ regeneration without diffusing into the bulk solution. The modularity of the nanoreactors allows to tune their composition and consequently their overall activity, and also remodel them for different reactions by altering enzyme partners. Given the plasticity and versatility, P22 VLPs possess great potential for developing functional materials capable of multistep biotransformations with advantageous properties, including enhanced efficiency and economical usage of enzyme cofactors. Functionally coupled enzymes and their cofactor (nicotinamide adenine dinucleotide) are colocalized inside P22 virus‐like particles. The resultant modular nanoreactors induce substrate channeling, enhancing the efficiency of cascade reactions and the utility of the cofactor. Many cofactors are not present in the high quantities needed for each biochemical turnover in vivo, but rather a small pool of these molecules are shuttled as metabolic intermediates between enzymes and are continuously regenerated to complete mass and energy transfer. To economically utilize cofactor‐dependent multistep biotransformations in vitro, biocatalytic materials are required with properties that include enhanced efficiency as well as regeneration and recycling of costly cofactors.The diversity of biocatalysis inside cells provides inspiration and a massive database for catalytic engineering, including different chemistry, ideal selectivity, and efficiency under mild conditions.1, 2 This process is defined as substrate channeling, which prevents intermediates from diffusing into bulk environment and thus increases their local concentration around the active sites. As a result, multistep reactions can proceed at high overall rates even with only trace quantities of intermediates. With substrate channeling, metabolic flux inside cells is elevated toward desired pathways (even when enzyme concentration is very low) while toxic or labile metabolic intermediates are diminished and/or segregated from bulk environment, which assists overall cell function. It was believed that colocalization of sequential enzymes would automatically lead to kinetic advantages. However, theoretical analyses demonstrate that proximity between enzymes alone does not necessarily induce substrate channeling, since most enzymes operate at rates much slower than the diffusion rate of small molecules even in a crowded space such as intracellular environment. This perspective has been verified experimentally by us and others by packing enzymes of metabolic pathways inside compartments, with the degree of crowding close to levels found in intracellular environments and crystal packing. Computational modeling also suggests a mechanism whereby orientation rather than just proximity between enzymes is required to induce channeling and there is minimal effect of channeling when the active sites are over 1 nm apart, which is hard to achieve even in the solid state. This issue presents a technical challenge in designing artificial metabolons with enhanced activities.Some naturally occurring enzyme complexes, recognized as metabolons, can achieve direct passing of intermediates from one enzymatic active site to the subsequent one.10 Thi For example, some metabolons comprise clearly defined noncatalytic structures that are solely for controlling the spatial range of intermediate diffusion. In carboxysomes, the noncatalytic capsid proteins form a compartment with a cage‐like architecture and selective permeability, which not only defines a space for colocalizing an enzyme couple noncovalently but also limits the diffusion of the intermediate (CO2) inside the compartment to maintain a high local concentration  or by limiting the space for diffusion .9 For s Figure .[18]Salmonella typhimurium bacteriophage P22, we have designed biomimetic, modular metabolons capable of enzymatic cascade reactions with enhanced efficiency. This is achieved by substrate channeling between functionally coupled enzyme partners and concomitant in situ regeneration of their shared cofactor, nicotinamide adenine dinucleotide (NAD). P22 VLP is a 56 nm, T = 7, icosahedral capsid, which results from self‐assembly of 420 copies of coat proteins (CPs) directed by ≈100–300 copies of scaffold proteins (SPs). Programmed encapsulation of cargo proteins has been demonstrated by genetic fusion of cargo proteins to truncated SP (SPt), with no changes in the behavior of VLP assembly both in vivo (recombinant coexpression) and in vitro (with purified proteins). By encapsulating enzymes, we have successfully constructed P22 VLP nanoreactors capable of a variety of biochemical reactions. Previous work shows simple enzyme encapsulation inside P22 VLPs fails to induce significant kinetic advantage in multistep cascade reactions. In this work, we studied multistep NAD‐dependent hydride transfer reactions catalyzed by functionally coupled enzymes , from Pyrococcus furiosus, was selected as a model reaction of hydride acceptance due to the potential industrial utility of alcohol dehydrogenases and their alcohol products. The dense packing of proteins inside P22 VLPs resulting from enzyme encapsulation (biomacromolecular concentration ≈300 mg mL−1) resembles the architecture of carboxysomes, inducing colocalization of the enzymes and CP. Highly porous P22 capsids show no barrier to small molecule diffusion, different from the molecular discrimination exhibited by carboxysomes. By mimicking the swing arm mechanism in acetyl‐CoA carboxylase, an NAD cofactor is covalently tethered to the interior side of the noncatalytic CP subunits to control its diffusion, inducing channeling of the cofactor between the two enzymes. Since the high proximity between the enzyme couple and their cofactors is maintained by noncovalent SP–CP interactions, this artificial metabolon can be disassembled and tailored for other redox reactions by recycling NAD‐labeled CP subunits. This plasticity of the system was additionally demonstrated by reduction of carbon–carbon double bonds catalyzed by an ene reductase enzyme.Using virus‐like particles (VLPs) derived from ns (SPs).21 Pros Figure . An engi22.1 with an NAD‐maleimide derivative, given that the only endogenous cysteine (C405) of wild‐type CP is not addressable. NAD has been reported to exhibit plasticity for modification on its adenine moiety with some decrease in its activity as an enzyme cofactor. Based on an established method, the adenine moiety of NAD was functionalized to incorporate a maleimide group to allow conjugation with thiols  with NAD‐maleimide  and transmission electron microscopy (TEM).  The results show that AdhD and PtDH coassembled with NAD‐CP to form VLP nanoreactors with high enzyme packing density, as seen in reported P22 VLP systems. The particles were intact and homogenous with a size and morphology similar to wild‐type VLP in the procapsid (PC) morphology.To obtain the designed nanoreactors conceptualized in Figure 37 wers Figure . NAD‐lab2.2 This implies that the NAD immobilized on the VLP inner surface is likely to only sample the volume within about 4 nm from the capsid surface (probably even less when considering the linker conformation). Previous cryo‐electron microscopy (cryo‐EM) data show that the encapsulated cargo proteins are anchored to the PC shell via CP‐SP interactions and form a more than 10 nm thick shell, which is probably beyond the region where the immobilized NAD can access. This limited access of the NAD might reduce the availability of the cofactor to the active sites of the encapsulated enzymes, resulting in lower utility level of the immobilized NAD and consequently reaction efficiency. Therefore, we employed two different CP variants to investigate the effects of the swing arm length on nanoreactor activity  was designed with a long extension of N‐terminus, which, from the cryo‐EM structure, is likely located inside the capsid , containing Gly‐Ser repeats and a part of SP. CPN‐ext has similar behavior to the wild‐type with respect to particle formation and characteristics  and CPN‐ext  segments, which can serve as a flexible “swing arm” after NAD immobilization; the net length of this PEG linker is estimated to be about 4 nm when it is fully extended.38 This Figure . The in N‐ext and NAD‐CPS39C) catalyzed phosphate formation, suggesting immobilized NAD shuttles between the encapsulated enzymes to assist the two‐step hydride transfer, in contrast to the enzyme‐encapsulated particles assembled from unlabeled CP. To better understand the kinetic behavior of NAD‐CP particles, the unlabeled CP particles were supplemented with unmodified NAD+ that can freely diffuse in solution and through the capsid shell.  In the early stages (0–60 min) of the reaction, a lag phase in phosphate production was observed for NAD+‐supplemented unlabeled CP particles before reaching the steady‐state rate, while for NAD‐CPN‐ext particles the lag phase was minimal and the phosphate production rate remained almost constant through the kinetic progression. This stark contrast is indicative of substrate channeling in the multistep cascade reaction catalyzed by the NAD‐CP particles.Kinetic studies on the two‐step reaction, catalyzed by the encapsulated AdhD and PtDH, were performed by monitoring phosphate production Figure . The kinarticles.9+‐supplemented unlabeled CP particles, which became more prominent with increasing NAD+ concentration  and increased with the increasing NADH:NAD+ ratio until reaching steady state. At the end of the burst phase, it is likely that, due to decreased NAD+ concentration, the initial fast reduction of supplemented NAD+ slowed down, and the phosphate production gradually started to reflect the overall cascade reaction. Simultaneously, the NADH:NAD+ ratio was still lower than the value required in the steady state  before the lag phase for free NADn Figure . The bur−6m  was calculated Figure . In the + Figure , i.e., tN‐ext particles , we were able to confirm the synthesis of 2,3‐butanediol , and was recycled over 200 times, demonstrating the robustness of the system.To investigate whether the biomimetic VLP nanoreactors can be used as functional biocatalytic materials, we also monitored 2,3‐butanediol, the product of the AdhD reaction Figure 4ai, whicFigure 42 Usi and a lot of input proteins remain unassembled or form aggregates. Recycling NAD‐CP from the unassembled components allows for a new round of in vitro assembly, circumventing the drawback of low assembly yield.In our design, proximity is introduced between functionally coupled enzymes and their cofactor using the noncovalent interactions between CP and SP. The modular feature of P22 allows us to easily disassemble the particles and recycle NAD‐CP for remaking active nanoreactors after the encapsulated enzymes are beyond their lifetime , or for remodeling the nanoreactors for other enzymatic chemistry. The initial particle assembly yield is only 10%–20% using the current (unoptimized) in vitro assembly protocol,37 andN‐ext recovered and recycled from the nanoreactors for ketone reduction described above, as well as the unassembled components after the initial in vitro assembly  NAD+ , the ability to induce biomacromolecular proximity , high porosity , and morphological homogeneity . The colocalization of enzymes and cofactor in the VLP by noncovalent interactions between the cofactor‐CP module and the enzyme‐SP modules (rather than irreversibly immobilization of cofactors to enzymes or enzyme complexes) lends itself to modular recycling of the components. P22 nanoreactors capable of various biochemical reactions can be readily designed and made by genetic fusion of SP to different enzymes. These features make the system highly versatile for tailoring to the needs of tunable composition as well as a range of chemistries.Here we demonstrate biomimetic nanoreactors capable of multistep enzymatic reactions with enhanced efficiency, which is induced by substrate channeling realized by colocalization of functionally coupled enzymes with their shared cofactor inside P22 VLPs. As protein‐based compartments, P22 VLP nanoreactors possess advantageous properties,19 inc shown in Figure Substrate channeling is the mechanism behind the increased multistep efficiency in our designed nanoreactors. The diffusion of the reaction intermediates . This biased comparison therefore underestimated the effect of NAD immobilization on the efficiency of the multistep reaction, and thus immobilization itself leads to an even more significant enhancement of the multistep efficiency than that observed in the experiments. The compromised activity of the immobilized NAD due to the structural modification can potentially be compensated for by raising the amount immobilized on the VLP  participate in the multistep reaction. Using the P22 VLP platform, this work achieves increased efficiency of multistep enzymatic reactions by controlling the diffusion and subsequent in situ regeneration of an NAD cofactor. This system could potentially be applied to a wide variety of NAD‐dependent cascade reactions, as suggested by the successful turnover of two different types of NADH‐consuming oxidoreductases, AdhD and ER. We have also previously reported that P22 VLP nanoreactors enhance enzyme stability against proteolysis and thermal denaturation, which prolongs the lifetime of the biocatalysts. Moreover, simple methods have been developed to assemble higher‐order structures of P22 VLP nanoreactors to fabricate heterogenous catalysts, with benefits of tuned efficiency and substrate selectivity, as well as storage, reusability and downstream separation in catalytic handling. Given the versatile utility of P22 VLP nanoreactors and potential accommodation of the swing arm‐based strategy to other cofactors , the prospect of more complex functional materials can be expected for completing a broad range of biochemical pathways with enhanced multistep efficiency and more economical usage of enzyme cofactors, as well as other properties of interest such as heterogenous catalysis.P22 VLP shows great potential in the development of functional biomaterials, especially in the area of biocatalysis.44 UsiThe authors declare no conflict of interest.Supporting informationClick here for additional data file.


The objective of this work is to compare our outcomes using thulium laser enucleation of prostate (ThuLEP) to the single‐port robot‐assisted simple prostatectomy (SP RASP) in the surgical management of benign prostatic hyperplasia (BPH).A retrospective cohort study was conducted from January 2017 through December 2021 of men who underwent SP RASP and ThuLEP performed by a single surgeon with an enucleation experience of >300 cases and extensive robotic experience. The primary outcome was changed in International Prostate Symptom Score (IPSS) postoperatively. Secondary outcomes were operative time, length of stay (LOS), change in post‐void residuals (PVR), de novo stress‐ or urge‐urinary incontinence , and rate of complications.p = 0.2956). SP RASP had a longer operative time . There was no difference in LOS . There was no difference in change in PVR . SP RASP patients had significantly less postoperative SUI than ThuLEP , while there was no difference in UUI between both groups . There was no difference in 30‐day complication rate , although there were three ThuLEP patients with Clavien–Dindo Class III or higher complication.One hundred two patients underwent surgery during the study period: 33 RASP and 69 ThuLEP. There was no difference in preoperative characteristics, including age and body mass index, between both groups. Changes in IPSS scores postoperatively were not significant between SP RASP versus ThuLEP (−17 vs. −14, There was no difference in change in IPSS scores between the two groups. ThuLEP is associated with shorter postoperative catheter days and decreased operative times. Hospital LOS was equivalent. SP RASP demonstrates significantly improved continence rates. Though SP RASP is within the initial learning curve at our institution, early results demonstrate the role for this modality alongside ThuLEP in the treatment of large gland BPH. All surgeries were performed by a single surgeon with an enucleation experience of over 300 cases along with extensive robotic experience. All patients who underwent either ThuLEP or SP RASP for benign prostatic enlargement or urinary retention due to prostatic obstruction with a minimum of 1 month follow‐up were eligible for inclusion in the study. Patients were excluded if they were lost to follow‐up after surgery. This study was performed in alignment with the guidelines on Strengthening the Reporting of Observational Studies in Epidemiology (STROBE).The ThuLEP and SP RASP surgeries were respectively performed in two different hospitals with similar catchment populations. This was due to the resource capabilities at each hospital. The operative teams were well trained in their respective hospitals. The surgeon operated at both hospitals with full privileges. Patients at both institutions with indications for surgery were offered choices of surgical approach and decided based on shared decision making. This study received approval from the institutional review board (IRB) from both institutions.2.2The primary outcome was change in International Prostate Symptom Score (IPSS) postoperatively. Secondary outcomes were operative time, length of stay (LOS), change in post‐void residuals (PVR), de novo stress‐ or urge‐urinary incontinence , and 30‐day complications by Clavien–Dindo classification.2.3α) of 0.05 was used.Using SAS® Studio 2022 , Wilcoxon‐Mann–Whitney test for hypothesis tests on continuous variables was performed. Chi‐square or Fisher exact tests were used for categorical variables. A statistical significance level , and the arms are tucked. An 18Fr Foley is placed on the field and the bladder emptied. A flexible cystoscope is introduced per the urethra with COA wound protector component of the SP access port is placed into the bladder and a 5‐mm assistant port is placed to the right side of the bladder under palpation for utilization of rigid suction. The SP access port is attached to the wound protector component, and then the robot is docked using the floating technique with a 5‐mm AirSeal inserted through the side port for insufflation. The robot is then docked.Ureteral orifices are visualized. The dissection line is marked circumferentially. We then begin to enucleate the prostate by developing a plane with electrocautery along the anterior bladder neck at the level of the surgical plane between peripheral part and adenoma. Once this is well established, we proceed to develop the lateral aspect of the plane bilaterally, with care taken to avoid the ureteral orifices (UOs). We then focus on the posterior plane. The surgical plane is developed from the bladder neck to the apex. The enucleation plane is further developed circumferentially until the prostate apex is reached. We then transect the urethral mucosa cold, releasing the prostate adenoma. Pressure is then dropped to 8 mmHg, and haemostasis is obtained.A posterior reconstruction is performed using a 3‐0 V‐Loc suture, incorporating the urethra, posterior peripheral zone, and the bladder neck. The bladder neck reconstruction is performed using a 3‐0 double armed Stratafix suture, with the goal of collapsing the prostatic fossa created from the enucleation. A 20 Fr 3‐way haematuria catheter is placed, and the balloon is inflated with 20‐cc sterile water.The cystotomy is closed in two layers with running 2‐0 vicryl for the mucosa and the 0 vicryl stay sutures for the second layer. Patients are discharged the same day, and the Foley catheter is routinely removed on postoperative day (POD) 5.3.2The patient is placed in the high dorsal lithotomy position and a 24Fr sheath resectoscope with a visual obturator is used initially. The obturator is removed and a 30° lens is inserted. Bilateral ureteral orifices are identified.A 1000‐μm thulium fibre  is used, with coagulation and vaporization settings adjusted for the prostate at 30 W. Settings can also be adjusted to a pulse or continuous mode, with the pulse setting providing shallower tissue penetration. Our institution prefers an en‐bloc enucleation technique. Care is taken while initially developing the enucleation plane to perform an early release of the sphincter complex to prevent traction injury. This is performed by defining the enucleation plane circumferentially proximal to the sphincter by marking and releasing the mucosa first. The verumontanum is used as the distal marker for resection and a deeper U‐shaped marking is made just proximal to this. The plane is started at the six o'clock position proximal to the verumontanum, and then carried out laterally at the three and nine o'clock position, and anteriorly at the 12 o'clock in a retrograde fashion. This circumferential dissection releases the sphincter complex and prevents prolonged traction on the sphincter during the blunt dissection required during the enucleation. Following this, the lateral planes are developed partially. The anterior plane is developed until the bladder is entered at the anterior bladder neck. This plane is then carried down and connected with the lateral planes until the entire adenoma is released and then pushed into the bladder.A Piranha morcellator  is used to remove the adenoma. It is crucial to maintain constant bladder pressure and volume during morcellation to aid in visibility and to prevent bladder injuries. Our institution uses the Thermedx FluidSmart Fluid management system . Pressures of 60 mmHg are maintained during the enucleation component, and 130 mmHg during the morcellation. Upon completion of morcellation, a 22Fr 3‐way haematuria catheter is inserted, and balloon filled with 80‐cc saline, and placed on light traction which is released after approximately 1 h. Patients typically are discharged the same day with the catheter and return to the clinic for catheter removal in 3–4 days.4p = 0.3929).A total of 103 patients underwent surgery during the study period: 34 SP RASP and 69 ThuLEP. One patient was lost to follow‐up from the SP RASP group. The patient demographics and preoperative characteristics are summarized in Table p = 0.2956). There were no conversions to open surgery. There was no difference in LOS (p = 0.2904). The final prostate specimen weight was larger in the SP RASP cohort . There were no SP RASP patients postoperatively who developed SUI , while there was no difference in number of patients who developed postoperative UUI . There was no difference in the proportion of patients who had any 30‐day complications . Operative times were significantly longer for SP RASP versus ThuLEP . SP RASP patients had significantly longer catheter time than ThuLEP .Table p = 0.9517) (Table p = 0.1016).There were no significant difference in the proportion of any 30‐day complication between the SP RASP and ThuLEP  adenoma has long been considered the gold standard in the treatment of BPH. However, performing bipolar or monopolar TURP when approaching large or very large glands may be challenging due to longer operative times, bleeding risk, and likelihood of incomplete resection of tissue. Advances in technology have allowed us to revisit old techniques, that is, open simple prostatectomy, but offer less morbid, minimally invasive, and durable responses for all size prostates. To our knowledge, this is the first study comparing outcomes between ThuLEP and the SP RASP.While simple prostatectomy has been utilized for many years, it has evolved from an open approach to the preferred robotic approach. This minimally invasive approach decreased morbidity by reducing blood loss, hospital stay, and pain.Previous studies have demonstrated that both laser enucleation of the prostate and SP RASP provided durable and comparable improvement in symptoms of BPH. A recent systematic review suggested the enucleation be the preferred approach due to decreased EBL, catheter days and length of hospital stay.The utilization of the purpose‐built SP access port system allows for improved use of the “floating dock technique.” This provides increased working surgical space, in particular in small surgical fields with a close surgical target.While ThuLEP may be considered less invasive as compared to SP RASP, as no incision is required, the avoidance of urethral instrumentation during SP RASP should be considered as well. The vigorous movements during enucleation risks formation of urethral and meatal strictures. Incidence of urethral and bladder neck strictures after ThuLEP or HoLEP range from 1 to 5%, and while this risk was significantly greater for smaller glands in these studies, this risk can be reduced with a transvesical approach due to almost complete avoidance of urethral instrumentation.Many laser enucleation scopes commonly used during enucleation are 26 or 28 Fr in size, though our institution utilizes the 24Fr Wolf scope. The 24 Fr scope ideally reduces the urethral trauma secondary to instrumentation as compared with larger scopes. An ongoing clinical trial aims to further evaluate the impact of this reduced diameter scope on vision, flow rates, and bladder injuries.The complication rates between two groups have not been statistically different; however, there were more Clavien III and higher complications in the THULEP group. These were related to postoperative bleeding associated with specimen morcellation and clot retention, which required intraoperative clot evacuation and bleeding control. Both stem from the imprecise nature of laser enucleation and morcellation of the specimen inside of the bladder. Although the number of patients in the SP RASP group is smaller, in our experience, the patients are not brought back to the operating room for clot evacuation and bleeding control as the instruments used for SP RASP allow for a more precise enucleation plane and better directed haemostasis. The authors acknowledge that more long‐term follow‐up is necessary capture any delayed complications.Our study identified a significant difference in rates of SUI, with SP RASP showing decreased rates of incontinence. We attribute this to the ability to perform a bladder neck reconstruction, moving the urethra back in the pelvic space and better control over the apical dissection. The collapse of the fossa obtained with this reconstruction also aids in haemostasis, obviating the need for continuous bladder irrigation and therefore hospital admissions.Our study is not without limitations. Both surgeries were performed by a single surgeon at a high‐volume academic centre. We recognize that our experience may not be generalizable to the community setting. Additionally, our data do not directly compare our outcomes to those from open simple prostatectomies. The retrospective component of our study presents difficulty with variable follow‐up. We did not have a standardized follow‐up protocol or pathway for either surgical cohort. Additions of flow parameters preoperatively and postoperatively would be another objective measurement to assess functional outcomes.6There was no difference in change in IPSS score between SP RASP and ThuLEP. ThuLEP is associated with shorter postoperative catheter days and decreased operative times. Hospital LOS was equivalent. SP RASP demonstrates significantly improved continence rates, likely a consequence of the bladder neck reconstruction and possibly reduced traction injury to the sphincter complex. While previous studies have recommended use of laser enucleation ahead of RASP in the treatment of large glands due to shorter LOS and reduced blood loss, this study adds to the evidence suggesting there is equivalent LOS between the modalities, due to the practice of same day discharge with the SP platform. The use of SP RASP is relatively new at our institution, though early results demonstrate the role for this modality alongside ThuLEP in the treatment of large gland BPH.All contributions have been made with authorship in the manuscript.Dr. Simone Crivellaro is a consultant for Intuitive Surgical, Inc. All other authors have nothing to disclose.