Drosophila melanogaster, an insect with no obvious parental care but which is highly dependent on dietary microbes during offspring development. The process of transmitting microbes involves flies ingesting microbes from their previous environment, storing and vectoring them, and finally depositing them to a new environment. This study revealed that fecal materials of adult flies play a significant role in this process, as they contain viable yeast cells that support larval development. During single patch visits, egg‐laying female flies transmitted more yeast cells than non‐egg‐laying females, suggesting that dietary symbiont transmission is not random, but linked to offspring production. The crop, an extension of the foregut, was identified as an organ capable of storing viable yeast cells during travel between egg‐laying sites. However, the amount of yeast in the crop reduced rapidly during periods of starvation. Although females starved for 24 h deposited a smaller amount of yeast than those starved for 6 h, the yeast inoculum produced still promoted the development of larval offspring. The results of these experiments suggest that female Drosophila fruit flies have the ability to store and regulate the transfer of microorganisms beneficial to their offspring via the shedding of fecal material. We argue that our observation may represent an initial evolutionary stage of maternal care through the manipulation of microbial load, from which more specialized feedbacks of sociality and microbe management may evolve.Parent‐to‐offspring transmission of beneficial microorganisms is intimately interwoven with the evolution of social behaviors. Ancestral stages of complex sociality–microbe vectoring interrelationships may be characterized by high costs of intensive parental care and hence only a weak link between the transmission of microbial symbionts and offspring production. We investigate the relationship between yeast symbiont transmission and egg‐laying, as well as some general factors thought to drive the “farming” of microscopic fungi by the fruit fly  Drosophila melanogaster, an insect with no obvious parental care but which is highly dependent on dietary microbes during offspring development. The results from this research suggest that female Drosophila fruit flies have the ability to store and regulate the transfer of microorganisms beneficial to their offspring via the shedding of fecal material.Parent‐to‐offspring transmission of beneficial microorganisms is intimately interwoven with the evolution of social behaviors. We investigate the relationship between yeast symbiont transmission and egg laying, as well as some general factors thought to manipulate the microbe management of the fruit fly  At the Drosophila‐yeast mutualism as a model of the evolution of mutualism‐sociality feedbacks. Yeast fungi have an important function in the Drosophila life cycle. They provide essential nutrients  in August 2006 . Agar (3 g per 1 L) was added to the juice‐water mixture and was heated to solidify the medium. 750 μL of the raspberry medium were filled in 20 mL plastic cups , which were placed inside a plastic vial ; the vials were sealed with foam stoppers. Individual e Figure . Then, fe Figure  for the 2.1.2D. melanogaster females in Experiment 1, we tested whether they indeed function as supplementary dietary symbionts in the life cycle of the flies. For this, we used axenic larvae that were prepared from freshly laid eggs of D. melanogaster in a vial with an artificial rearing medium. The collected eggs were submerged in 30 mL of 6% sodium hypochlorite solution. A fine brush was used to dislodge the eggs from the medium before transferring the egg suspension to a 50 mL falcon tube. After 5 min, the bleached eggs were filtered through a fine filter mesh, and for the eggs that were still attached to the walls of the falcon tube 70% ethanol was used to release them into the filter. The filtered eggs were washed with 2 L distilled water before being transferred to a chloramphenicol‐glucose agar plate and incubated at 25°C overnight. The following day, the freshly hatched larvae were transferred individually into 2 mL polypropylene micro‐vials filled with 1 mL sterile raspberry medium inoculated with 10 μL of respective yeast solutions (see below) containing 500 fungal cells. The yeasts used for inoculation were cultivated in the liquid yeast medium for 2 days at 25°C in a shaker and consecutively the cells were counted using a hemocytometer to adjust the titer of the cell suspension. The following yeast isolates were used: three isolates (Appendix Hanseniaspora uvarum (isolate P38), Pichia terricola (isolate F13‐1), and Wickerhamomyces pijperi (isolate P51); two isolates that were previously found to be transmitted by drosophilids, Candida californica (isolate PYCC6277), Starmerella bacilaris (isolate PYCC 6281), and Saccharomyces cerevisiae (isolate DSM 70449), commonly known as brewer's yeast. The latter three yeast species are known to positively affect D. melanogaster development  positively correlate with the amount of viable yeast cells it contains, (2) to be higher in mated females, and (3) negatively correlate with progressive fasting. While females empty the crop for their own nutritional purposes during fasting, mated and ad libitum fed females ready to lay eggs could primarily retain a larger number of dietary symbionts in the crop, which are gradually released into the gut to increase the number of yeast cells in the gut and thus in the feces deposited next to the eggs.To test whether the crop has the potential to function as a storage organ for fungal symbionts, and if so, how efficiently it stores viable yeast cells that are then transferred to the next breeding patch, we manipulated the mating and hunger status of D. melanogaster flies were mono‐associated with H. uvarum. Based on previous studies  for oviposition for 5 h and then removed from their vials. The number of eggs laid into the raspberry medium was counted to distinguish the egg‐laying activity between the mated and virgin females. Subsequently, all females were dissected under the stereomicroscope, mature oocytes at stage 14  were removed from the population cage and placed in an individual vial with a sterilized raspberry medium (see Experiment 1). Each female was allowed 2 h to oviposit and was then removed from the vial. The eggs were counted, and the surface of the patches was washed with saline solution with the egg intact for plating. Washing and plating of yeasts were done using the track dilution technique as described in Experiment 1. To also compare the developmental success of larvae that emerged from eggs laid by fasting females a second experiment with the same setting as above was conducted simultaneously, but this time the patches were not washed, and eggs were allowed to develop into the adult stage. Days to emergence and survival were recorded.To investigate how much of the yeast remains in the crop after fasting and to see whether starved females can still support the development of their offspring, 4‐day‐old females, mono‐associated with 2.1.4Drosophila, we explicitly tested to what extent adult fecal material contains viable fungal symbionts. Out of 69 female mono‐associated flies, 35 were mated and the others remained virgins (see Experiment 3). After 4 days, flies were transferred to fresh vials with sterilized raspberry medium (see Experiment 1). The individual flies were given 5 h to interact with the substrate before they were removed. Single clearly visible fecal droplets per patch were observed under the stereomicroscope and were photographed. Images were then used to calculate the area of the fecal droplets . Insignificant terms with p‐values >.05 were eliminated until all terms remaining were significant  was log‐transformed to meet assumptions of normality in the residuals. Additionally, the independent variables of yeast load of a fly, residence time, and the number of eggs oviposited, were log‐transformed to improve the scale and fit of the models. For the survival of larvae to adulthood on different inoculum of yeasts, data were fitted with a generalized linear model with binomial error distribution with a logit‐link function. The yeast transmission by fasting females, yeast load in crops, data were fitted with a generalized linear model with quasi‐Poisson error distribution with a log‐link function to correct for over‐dispersed data. To analyze the proportion of larvae surviving to adulthood with the yeast transmitted by the individual female on different fasting regimes, data were fitted with a generalized linear model with quasi‐binomial error distribution with a logit‐link function to correct for under‐dispersed data. For the size of the crop and the size of the fly, data were analyzed with a generalized linear model with Gamma error distribution with a log‐link function. Minimum adequate models (MAMs) were selected for all datasets, by comparing the 33.1D. melanogaster females during a single patch visit  and the time the flies spent on the fruit patch , that is, the yeast load of flies and their residence time. Flies that laid eggs . However, the number of eggs laid during the patch visit and the flies' body size had no statistical significance on the amount of yeast transferred. The flies' egg‐laying activity is thus positively related to the amount of yeast transmitted when visiting a substrate patch.The amount of yeast fungi transferred by individual t Figure  was incr3.2χ2 = 20.91, df = 6, p < .01). The reference level was the control group that had no yeast inoculum. These findings suggest that the different yeast species found to be transmitted by the flies all positively influenced the survival of the larvae to adulthood.The results of the logistic regression analysis showed that there is a significant effect of yeast inoculation (Treatment) on the successful development of larvae into adult flies . Simultaneously, the mating status of the females significantly influenced the size of the crop , which ultimately influenced the amount of yeasts found in the crops. While virgin females accumulated an average of ~20 eggs in their ovaries and did not lay a single egg, mated females laid numerous eggs throughout the experiment and had an average of only ~5 eggs in their ovaries at the end of the experiment leaving possible space for the crop to expand . Generally, the amount of yeast transferred increased significantly with the increasing number of eggs laid . In Experiment 1, the flies were exposed to the entire microbial community the insects could ingest from the rotting raspberries. In addition, this time we did not record the females' time spent on the patch but set a time limit of 2 h for the flies to interact with the substrate. Despite the different experimental setups, it is remarkable that under certain circumstances not only a general effect of oviposition on the shedding of dietary microbes can be observed but also a positive relationship with the quantity of the reproductive output. The number of eggs laid in the 24‐h fasting treatment was lower possibly due to females laying eggs in the water‐agar on which the flies were kept on during the period of fasting.Mated D. melanogaster females were allowed to hatch and the larvae to develop to the adult stage. The proportion of surviving offspring to adulthood depended significantly on the number of eggs that were oviposited into the same patch  and this could be due to increased competition for food and space in crowded cultures as population density increases .The eggs from a second, similarly treated cohort of h Figure . There w3.5H. uvarum cells providing a route of transfer of maternal microbiota to the next generation in D. melanogaster. Random picking of single fecal droplets from mated and virgin females (see Experiment 4) revealed strong variation in the amount of yeast transferred via these deposits , nor did the number of estimated yeast cells differ between deposits from mated and virgin ones (p = .852). Nevertheless, these data clearly show that the feces of fruit flies can contain copious amounts of viable yeast cells, which can then colonize the developmental habitat of the fly larvae.In the last experiment, we asked whether the fecal deposition during a female's patch visit contains viable s Figure . The dat4Simple forms of insect‐fungus mutualism and primal parental care must have preceded the evolution of advanced mutualism‐sociality feedbacks ; data curation (lead); formal analysis (lead); investigation (lead); methodology (lead); validation (lead); visualization (lead); writing – original draft (lead); writing – review and editing . Marko Rohlfs: Conceptualization ; formal analysis (supporting); methodology (supporting); writing – original draft (supporting); writing – review and editing .University of Bremen funds the research and the publication charge (Golden Open Access).


Pilot and feasibility studies provide information to be used when planning a full trial. A sufficient sample size within the pilot/feasibility study is required so this information can be extracted with suitable precision. This work builds upon previous reviews of pilot and feasibility studies to evaluate whether the target sample size aligns with recent recommendations and whether these targets are being reached.A review of the ISRCTN registry was completed using the keywords “pilot” and “feasibility”. The inclusion criteria were UK-based randomised interventional trials that started between 2013 (end of the previous review) and 2020. Target sample size, actual sample size and key design characteristics were extracted. Descriptive statistics were used to present sample sizes overall and by key characteristics.n = 761), the median target sample size was 30 (IQR 20–50). This was consistent when split by those labelled as a pilot or feasibility study. Slightly larger sample sizes  were shown for those labelled both pilot and feasibility (n = 69). Studies with a continuous outcome (n = 592) had a median target sample size of 30 (IQR 20–43) whereas, in line with recommendations, this was larger for those with binary outcomes . There was no descriptive difference in the target sample size based on funder type. In studies where the achieved sample size was available (n = 301), 173 (57%) did not reach their sample size target; however, the median difference between the target and actual sample sizes was small at just minus four participants (IQR −25–0).In total, 761 studies were included in the review of which 448 (59%) were labelled feasibility studies, 244 (32%) pilot studies and 69 (9%) described as both pilot and feasibility studies. Over all included pilot and feasibility studies (Target sample sizes for pilot and feasibility studies have remained constant since the last review in 2013. Most studies in the review satisfy the earlier and more lenient recommendations however do not satisfy the most recent largest recommendation. Additionally, most studies did not reach their target sample size meaning the information collected may not be sufficient to estimate the required parameters for future definitive randomised controlled trials.The online version contains supplementary material available at 10.1186/s40814-023-01416-w. Pilot and feasibility studies (PAFS) are preliminary studies that are used to assess the utility of undertaking a future definitive randomised controlled trial (RCT) which will test the effect of an intervention . PAFS doThe definitions of pilot and feasibility studies were inconsistent in the literature, but following consensus exercises by Eldridge et al. , it was It has been suggested that PAFS could be an essential prerequisite to completing a definitive study , and theThere is no one guideline for choosing an appropriate size for a PAFS  with difWhitehead et al.  criticisn = 79) in 2013. They found a median sample size target of 30 (IQR 20–45) participants per arm for pilot studies (n = 50) and 36 (IQR 25–50) for feasibility studies (n = 25), although these had large variations. The authors also noted a slight difference in the median target sample sizes for different endpoints (36 (IQR 25–50) for binary, 30 (IQR 20–50) for continuous). These sample sizes satisfy the earlier recommendations of Julious [n = 12), Kieser et al. [n = 10–20) and Sim and Lewis [n = 55 total) as outlined above. However, those by Teare et al. [n = 60 for binary, n = 35 for continuous) were not satisfied.A review by Billingham et al.  was comp Julious  n = 12)r et al.   to evaluate the sample sizes achieved in these studies, finding a median total number of participants of 62.5  and 125.5  for pilot (n = 20) and feasibility (n = 34) studies, respectively. For mostly two-armed studies, the value for pilot studies is consistent with that found by Billingham et al. [The work by Arain et al.  used pilm et al.  as the t IQR 31, 9 and 125m et al. . As one This review aims to gain an up-to-date overview of the target sample sizes used in PAFS. Of particular interest is whether this has changed since the previous review in 2013 as recommendations in 2012 [The International Standard Randomised Controlled Trial Number (ISRCTN) registration website is used by the International Committee of Medical Journal Editors (ICMJE) to verify public trials and shared in the World Health Organization (WHO) trials search system . TherefoThe ISRCTN database was searched for the terms “pilot” and “feasibility” on three separate occasions in September 2019 and July 2020 (for use in specific projects) with the final search completed on the 10th March 2022 to ensure data up to the end of 2020 was included. All identified entries were downloaded directly from the webpage, and duplicates were removed between the two downloads before screening took place. Screening and data collection took place independently by a single researcher (MC/JL/NT), and a 25% sample was checked by a second researcher (NT/JL). General characteristics of the studies as well as sample size information were included in the downloaded records, and a full list of the extracted data can be found in Appendix • Randomised studies containing at least two treatment arms• Interventional studies• Described as either a pilot or feasibility study• Study that started between 2013 and 2020• Study completed within the UKThe inclusion criteria were the following:• A non-parallel groups study including cross-over and factorial designs as these include additional complexity in sample size calculations• Cluster randomised trials and adaptive designs (for reasons described above)• Studies in healthy volunteers• Internal pilots  due to tThe exclusion criteria were the following:1. Whether the study was labelled as a pilot, feasibility or both2. The funder (charity/industry/public/other)3. Endpoint Descriptive statistics on the characteristics of the studies were calculated for the whole dataset as well as split by certain characteristics of interest, namely:The categories for these characteristics were decided by MC and NT after the first extraction of data and used consistently thereafter.Medians and interquartile ranges were used to summarise continuous variables due to the expected skew of the data and frequencies and percentages for categorical variables.The Preferred Reporting Items for Systematic Reviews and Meta-Analyses  checklisThe search of the ISRCTN database yielded 1711 studies with the search term “feasibility” and 1359 studies with the search term “pilot”. Records for each study were downloaded from the ISRCTN webpage, and after eliminating duplicates and removing any studies not meeting the inclusion criteria, 761 studies went on to be analysed. Of those included, 448 (59%) had been labelled as a feasibility studies, 244 (32%) as a pilot study and 69 (9%) had been described as both a pilot and a feasibility study , followed by cancer and circulatory system both with 72/761 (10%) each. The percentages differed slightly depending on the study type with circulatory system  being more prominent than cancer  in pilot studies but feasibility and both pilot and feasibility studies being the opposite. In line with the common condition categories, behavioural interventions were the most prominent intervention type  followed by procedure/surgery representing only 64/761 (8%) of the studies.The largest source of funding for all studies was public funds , and this included government funding programmes such as NIHR and MRC as well as NHS-funded projects. Charity-funded studies were the next most common , finally followed by industry-funded studies , again with this being consistent across study types.n = 761) per arm, and this was consistent for those labelled pilot and feasibility studies separately but was slightly higher  for those labelled as both a pilot and feasibility study.The median sample sizes for all studies and split by the key characteristics of interest are summarised in Table n = 592) than studies using binary endpoints  but when split by funder all medians were the same at 30 participants per arm, aside from those labelled other which was slightly lower.The median sample size per arm was lower for studies using continuous endpoints  and neonatal disease . Similarly, for study types, there were larger median sample sizes for diagnostic studies  and screening studies . Assessing intervention type showed only a reduced median sample size for biological/vaccine studies .Sample size summaries for additional characteristics  can be found in Appendix The plot in Fig. Due to the large maximum value found in the review for the sample size by arm, the twenty largest within the review have been descriptively evaluated further to identify any common characteristics. These had a range of sample sizes per arm from 175 to 1598. The two most common conditions were cancer  and urological and genital diseases  which differs from the summaries of all PAFS. Seven of the twenty (35%) were studies based on GP practices. The most common study type is treatment  as with the characteristics of all PAFS; however, both prevention  and screening  are more prominent. Finally of note is that 10 of the 20 studies (50%) with the largest sample sizes by arm have binary primary endpoints which does not align with the general characteristics of the review.The sample size recommendations outlined in the introduction have been summarised in Table For those with final sample size information, 173/301 (57%) did not reach their target total sample size for the feasibility/pilot study; however, 314/615 (51%) of all completed studies did not have this information included on the ISRCTN registry for this to be evaluated  and feasibility studies  to be consistent to that found by Billingham et al.  for piloThe value of 30 participants per arm satisfies the recommendations outlined by three of the four rules of thumb previously mentioned. Assessing this further found that for the most lenient , which A study of target effect sizes in definitive RCTs published in the Health Technology Assessment journal found the median target effect size was 0.3 ; therefoThere was no distinction in sample size per arm between the main funder types  which all found a median of 30. However, a lower value  was found with those with a funder classed as other. This included investigator-funded and social enterprises; however, there were comparatively very few of these. It was most common for the studies to be publicly funded representing 73% (557/761) of the studies included.Overall, sample sizes of PAFS found in this review were consistent over the years from 2013 until 2018 with then a potential increase in 2019 and 2020. This finding could be a temporary spike or could represent the start of a gradual increase. Further work could assess whether this trend has continued in years post-2020.Over half of the studies which we had information did not reach their sample size target , this is slightly lower than the 63% (245/388) found by Jacques et al.  which coSim and Lewis  recommenThere were a number of limitations to note with this work. Due to the nature of the information available through the ISRCTN registry, some of the information such as condition and intervention type had many responses as “other”, this was the category supplied directly by the study team; however, it could be that they fit into a pre-existing category but the team wanted to add additional detail. This was not checked within this work and the study team response was used. Additionally, the number of studies in 2020 looks low compared with previous years; however, this could be due to a delayed retrospective registration rather than fewer studies. Further research is needed to update the data in the future to assess this properly. The ISRCTN registry only includes details on the total study sample size; therefore, the sample size per arm was calculated by taking this total and dividing it by the number of arms in the study assuming an equal ratio between the arms; however, this may not have always been the case.The search terms used within the review of “pilot” and “feasibility” may have missed some eligible studies that had been described as phase 2 studies. To mitigate this, the search terms were completed on all records within the ISRCTN registration so that if any mention of pilot/feasibility was present this would be identified regardless of the title or study design terminology chosen. However, this does not completely remove the risk of missed eligible studies. This also impacts the generalisability of this review as those that are labelled phase 2 instead of pilot/feasibility may have different sample size characteristics. Additionally, as this review was limited to UK-based studies only, the results found here cannot be generalised internationally. Despite these limitations, we hope that this work provides an overview of a large number of PAFS using an online registry to understand the landscape of sample sizes.Work published since the studies included in this review were designed has stated the need to look beyond setting a sample size for PAFS based on a singular consideration (such as precision of the standard deviation for example) and instead should consider all progression criteria which will ultimately determine the success of the pilot or feasibility study . Future It has previously been stated that all RCTs need to justify the sample size they use, but a formal sample size calculation is not always required . For PAFAdditional file 1.


The purpose of this review was to evaluate data generated by animal models of intervertebral disc (IVD) degeneration published in the last decade and show how this has made invaluable contributions to the identification of molecular events occurring in and contributing to pain generation. IVD degeneration and associated spinal pain is a complex multifactorial process, its complexity poses difficulties in the selection of the most appropriate therapeutic target to focus on of many potential candidates in the formulation of strategies to alleviate pain perception and to effect disc repair and regeneration and the prevention of associated neuropathic and nociceptive pain. Nerve ingrowth and increased numbers of nociceptors and mechanoreceptors in the degenerate IVD are mechanically stimulated in the biomechanically incompetent abnormally loaded degenerate IVD leading to increased generation of low back pain. Maintenance of a healthy IVD is, thus, an important preventative measure that warrants further investigation to preclude the generation of low back pain. Recent studies with growth and differentiation factor 6 in IVD puncture and multi‐level IVD degeneration models and a rat xenograft radiculopathy pain model have shown it has considerable potential in the prevention of further deterioration in degenerate IVDs, has regenerative properties that promote recovery of normal IVD architectural functional organization and inhibits the generation of inflammatory mediators that lead to disc degeneration and the generation of low back pain. Human clinical trials are warranted and eagerly anticipated with this compound to assess its efficacy in the treatment of IVD degeneration and the prevention of the generation of low back pain. Data generated by animal models over the last decade has made invaluable contributions to the identification of molecular events occurring in and contributing to intervertebral disc (IVD) degeneration and pain generation and has identified several potential therapeutic targets. IVD degeneration and associated spinal pain is a complex multifactorial event, its complexity poses difficulties in the selection of the most appropriate therapeutic target of many potential candidates to focus on in the formulation of strategies to effect disc repair and regeneration and the alleviation of associated neuropathic and nociceptive pain. Application of artificial intelligence in facial recognition has improved the assessment of pain in animal models. Deep machine learning and neural networking have also been applied in the analysis of data generated from molecular signaling pathways involved in disc degeneration and pain generation. This methodology has predictive capability, can be automated and offers a superior unbiased assessment of data compared to observer‐based classifications and clinical prognosis data. Of the numerous causes of back pain, a landmark study estimated that in the 632 million, a meta‐analysis reveals 403 Million people (5.5% of world population) have symptomatic disc degeneration.4The IVD is innervated by branches of the sinuvertebral nerve, by nerves derived from the ventral rami of spinal nerves or by nerves derived from gray rami communicantes.Neurons in DRGs can be differentiated on the basis of the pattern of receptors for neurotrophic factors they interact with, including ion channels such as degenerin/epithelial sodium channels (DEG/ENaCs) ; acid‐sensing ion channels (ASIC 1–3) and transient receptor potential (TRP) families .Expression of CGRP ; GFRa1 and GFRa3 ; P2X3 (ATP‐gated ion channel subtype P2X3); SP (substance P); TMP (thiamine monophosphatase); VR1 (vanilloid receptor subtype 1) also differentiates DRG neurons. The Mu receptor interacts with opioid peptides to promote analgesic anti‐inflammatory actions to protect cartilage from posttraumatic degeneration and have also been evaluated for the treatment of chronic LBP.5Acute LBP can result from injury to spinal muscle groups such as the multifidis leading to reduced spinal flexibility, this impacts on neuromuscular control, results in alterations in spinal biomechanics, spinal gait and flexibility effecting spinal ligament, facet and sacroiliac joints and weight loading on the vertebral bodies. Alterations in the expression of connective tissue and collagenous components of the muscle spindle capsule in paraspinal muscles of spinal segments containing degenerate IVDs effects their mechanical properties.6Cases have been reported where degenerate IVDs in patients present with no symptoms of LBP.7An aspect that has emerged in the last decade relevant to the development of prospective animal models of IVDD is the issue of the potentially confounding impact that sex hormones may have on such models. Although the effects of sex hormones on the metabolism of IVD cells was first identified in 1969LBP is a common symptom of premenstrual syndrome, experienced by most women during menstruation and may be exacerbated by premenstrual dysphoric disorder and dysmenorrhea or may be a symptom of endometriosis.Estrogen signals through two classic nuclear receptors, estrogen receptor (ER)‐α and ‐β, and a membrane bound G‐protein‐coupled receptor 30 (GPR30). 17β‐estradiol (E2) enhances cell proliferation and prevents IL‐1β‐induced cell death in IVD cells, but this effect was partially blocked by G36, a GPR30 antagonist and completely abrogated by a combination of ER antagonists ICI 182 780 and G36.Further studies with functional foods and neutraceutical supplements under evaluation for their abilities to alleviate pain may also prove to be a useful non‐drug treatment for post‐menopausal back pain.8IVDD is a pre‐requisite for neural development and receptors with the ability to perceive pain in the mechanically incompetent IVD. The dogmatic view of the IVD for a long time was that it is an immune privileged tissue due to immune cells being excluded from the intact IVD structure. The same cannot be said of the degenerate IVD which can contain defects in the CEP and clefts in the AF which can allow communication with the exterior environment and movement of immune cells into the IVD. The resultant increase in inflammatory cytokine and chemokine mediators, degradative proteases, neurotrophic and angiogenic factors in the degenerate IVD and catabolic events produces a structurally compromised IVD incapable of acting as a weight bearing viscoelastic musculoskeletal supportive tissue and since it is now a mechanosensive structure is also a major source of LBP.V3.2 channel and a high voltage‐activated CaV2.2 channel. The CaV3.2 channel participates in signal conductance along nociceptive neurons while CaV2.2 channels provide synaptic transmission at the dorsal horn of the spinal cord.Thus, while the normal intact healthy IVD is not exposed to the immune systemDuring IVDD, IVD cells secrete increased levels of proinflammatory cytokines,V3.2 channel and a high voltage‐activated CaV2.2 channel. The CaV3.2 channel participates in signal conductance along nociceptive neurons while CaV2.2 channels provide synaptic transmission at the dorsal horn of the spinal cord.Acid‐sensitive ion channels (ASICs) mediate IVDD via various pathways.i subtype have been identified, μ‐, δ, and κ, the μ receptor (Mu) is present in the IVD. μ‐Opioid receptor expression in the IVD, spinal cord and DRGs is associated with pain‐related behavior following IVD herniation in rats.2, N‐MePhe4, Gly‐ol]‐enkephalin) (DAMGO), fentanyl, etorphine, buprenorphin and methadone.Interaction of the 5‐hydroxytryptamine receptor with the inflammatory mediator TNF prolongs pain related behavior emanating from the IVD.9Many animal studies have shown that IVDD significantly contributes to the development of LBP, damage to normal IVD structure compromises its biomechanical competence and the viability of IVD cell populations. The resultant mechanical destabilization and altered cell‐ECM signaling results in the production of inflammatory cytokines and active MMPs which promotes disc degeneration. Degenerative changes in the disc ECM conducive to neovascularization and the ingrowth of nerves and an elevation in the production of inflammatory cytokines and neurotrophic factors all lead to a significant deterioration in the normal cellular microenvironment of the IVD conducive to the production of mechano‐and pain receptors. The appearance of glial fibrillary acidic protein‐immunopositive cells in diseased IVDs is closely associated with nerve ingrowth and suggests that Schwann cells have a role to play in regulating disc innervation and nerve function in the disc.The normal IVD is poorly innervated by sensory and sympathetic perivascular nerve fibers (nociceptive) and postganglionic sympathetic nerve fibers in its outermost annular layer. Upon degeneration, depletion of the IVD aggrecan levels lowers its hydrostatic pressure and the IVD becomes susceptible to the ingrowth of nerves and blood vessels from its periphery. The CS chains of aggrecan in the normal IVD strongly inhibit nerve ingrowth into the IVD furthermore, the high hydrostatic pressure provided by aggrecan‐HA aggregates physically prevents the ingrowth of blood vessels. In‐vitro culture experiments conducted with aggrecan isolated from lumbar IVDs shows this proteoglycan significantly inhibits neuronal growth.While the causes of LBP remain to fully determined, peripheral, spinal, and supraspinal biochemical and electrophysiological studies in animal models indicate that peripheral pro‐inflammatory mediators and neuropeptides sensitize nociceptors in the IVD in a similar manner to how they generate pain in knee OA.TRP channels are also present in the IVD and these are potential sensors and transducers of inflammatory pain. Transient receptor potential ankyrin‐1 (TRPA1) and TRP vanilloid −1, 2, 4  have all been detected in the IVD.9.1Neuropathic pain is associated with inflammation at sites of tissue damage resulting in a cascade of events concentrating and activating innate immune cells by immuno‐stimulatory cytokines, neurotrophic factors, and chemokines.9.2Neuropathic pain is the most difficult type of pain to treat clinically because it is not known how nerve injuries cause chronic pain. A recent study has shown that neurons in the spinal cord which process pain signals can be attacked by activated spinal glia and the perineuronal nets (PNNs) which normally protect these neurons providing neural plasticity become degraded.10Literature reviews in PubMed and Google were used to collect information on the cited studies in Table A number of rodent models have been developed to specifically assess various aspects of IVDD and the development of LBP Table . SeveralA single level annular puncture model of IVDD, rat xenograft radiculopathy model10.1IVD cells have evolved to exist in a weighted environment and biomechanical forces have important regulatory effects on disc cells.http://www.informatics.jax.org) is the primary community database for the laboratory mouse and a key source of gene biological reference data, gene functions, phenotypes, disease models relevant to human biology and disease freely accessible to all researchers.Compared to large animal models of IVDD the husbandry and handling of mice is straight forward and these are a popular and very useful animal model amenable to genetic manipulation.Large animal models of IVDD such as the canine spontaneous and ovine mechanical de‐stabilization models of IVDD have important attributes compared to small IVDD models developed in mice, rats, and rabbits.10.2IVD cells are not subject solely to static compression but as already stated receive a dynamic balance of tension/compression which not only effects nutritive pathways10.3β‐catenin pathway plays a critical role in the pathogenesis of neuropathic pain and may be an appropriate therapeutic target.α and IL‐18 are significantly elevated in neuropathic pain models. Levels of these mediators are significantly lower when the Wnt/β‐catenin pathway is inhibited using a Wnt/β‐catenin pathway inhibitor such as XAV939. XAV939 is a potent, small molecule inhibitor of tankyrase (TNKS) 1 and 2 .Understanding the complexities of neuropathic pain is an important clinical challenge; however, the molecular mechanism remains elusive. Chronic DRG compression models of neuropathic pain suggest the Wnt/11This review has outlined the complexities of IVDD and the multiple IVD receptors and inflammatory mediators and neurotrophins that have roles in the development of nociceptor and mechanoreceptors in the degenerate IVD that produce pain signals transported to the brain by the CNS for interpretation. Inflammation in the IVD has powerful effects on the resident disc cell populations and is the impetus for the ingrowth of nerves and blood vessels into the normal IVD leading to its degeneration. Clearly, in order to prevent events that lead to pain generation, preservation of a healthy functional IVD is important, prevention of inflammation and oxidative conditions also prevents ER stress and mitochondrial dysfunction. Animal models of inflammatory and neuropathic pain indicate that inflammation regulates the resolution of pain by producing pro‐resolving mediators such as resolvin D1.A question has been raised as to whether nutritional intervention can prevent chronic pain development.This study was conceived and written by Ashish D. Diwan and James Melrose. None of the above listed companies had any input into the design, implementation or interpretation of the study.The authors declare no conflicts of interest.


By‐product mutualisms are ubiquitous yet seldom considered in models of mutualism. Most models represent conditional mutualisms that shift between mutualism and antagonism in response to shifts in costs and benefits resulting from changes in environmental quality. However, in by‐product mutualisms, benefits arise as a part of normal life processes that may be costly to produce but incur little‐to‐no additional costs in response to the interaction. Without costs associated with the interaction, they do not have antagonistic alternate states. Here, we present a conceptual model that differs from traditional conditional models in three ways: (1) partners exchange by‐product benefits, (2) interactions do not have alternate antagonistic states, and (3) tradeoffs are allowed among factors that influence environmental quality . We applied this model to bark and ambrosia beetles (Curculionidae: Scolytinae), a diverse group that associates with fungi and that has repeatedly developed two distinct pathways to by‐product mutualism. We used independent axes for each major factor influencing environmental quality in these systems, including those that exhibit tradeoffs . For these symbioses, tradeoffs in these two factors are key to which mutualism pathway is taken. In by‐product mutualisms, benefits arise as a part of normal life processes that do not incur additional costs. They are not conditional where outcomes shift between mutualism and antagonism as environmental context varies. Most models are conditional and a poor fit for by‐product mutualisms. Here, we build on a prior model, removing conditionality and incorporating tradeoffs in environmental quality to support testing hypotheses on by‐product mutualism development and partner fidelity. While tCurrently, many models focus on how mutualisms persist in the face of conflicting interests of the partners. Partners present flexible phenotypes and sanctions or partner choices are exerted upon these phenotypes to weed out cheaters and low return‐on‐investment partners or to enforce reward delivery Akcay, . The mutHere, we take an approach similar to that of Thrall et al.  who incoScolytinae‐fungus mutualisms appear to have arisen as a result of the exchange of by‐products , the basal feeding strategy from which all fungus‐feeding lineages have arisen  associated with the two ends of the spectrum determine the mutualism outcome Figure . On the However, because each niche imposes an opposite set of tradeoffs, the partnerships function in fundamentally different ways. Likewise, the mechanisms that lead to and enforce increased fidelity in each case differ.In both the right and left ends of the spectrum mutualisms with fungi allow Scolytinae to exploit otherwise intractable niches. 4Stephen StillsAnd if you can't be with the one you love honey, love the one you're with—Buck RamYou're my dream come true, my one and only you—Here, we describe in greater detail how differences in biotic complexity  and tradeoffs in the major factors influencing environmental quality  result in different mutualism pathways in our model and a corresponding gradient of partner specialization extending from highly promiscuous to commensal or facultative “friends‐with‐benefits” to fidelity.4.1Ips typographus) leading to high levels of intra‐specific competition with strong negative effects on fitness . A by‐product of nutrient provisioning is that the increase in concentrations of limiting nutrients in the phloem reduces the amount of phloem required by each larva and the high densities of brood resulting from large numbers of mass‐attacking adults can be accommodated. Early entry into the tree reduces exposure to other bark beetles and fungi by allowing them to colonize and capture space in a relatively “empty” tree and to use high‐nutrient quality phloem.All known fungus‐feeding bark beetles are tree‐killing conifer‐colonizing species. These beetles live in the best nutritional resources , but with the tradeoff that they are also those most hostile because of strong tree defenses. To overcome defenses and kill the tree, these beetles must attack Fungus‐feeding beetles and their fungi are specialized to use particular tree species due to adaptation to secondary chemistry. This narrows the potential for inter‐specific competition with other tree‐killing bark beetles and fungi. This also restricts the size of the symbiont pools to which they are exposed, facilitating their ability to develop and maintain stable associations with efficient partners. The evolution of selective mycangia (complex transport structures) by these beetles to facilitate vertical transmission and the reduction of sexual reproduction by the mutualist fungi, most of which are predominantly asexual, has further enforced fidelity among these beetles and their fungal partners.4.4Ambrosia beetles live in sapwood, a substrate notoriously low in nutrients  and the highest biotic complexity, while the greatest specificity occurred on the right and left ends of the spectrum where both environmental quality and biotic complexity were lowest. This makes sense in that mutualists are often required to exploit difficult environments and often require specific mutualists possessing particular traits. Thrall et al. , howeverIn the case of our system, the characteristics of the left and right ends of the spectrum apply several filters  that lower biotic diversity making the formation of consistent associations among high‐functioning partners more likely. Further, vertical transmission of the fungi by the beetles also supports greater consistency of partners for reliable benefit delivery. The environments these mutualisms inhabit are also more predictable and stable than those found in the middle ground. High symbiont reliability, low symbiont richness, and high environmental predictability are expected to reduce context dependency ; writing – review and editing . Diana L. Six: Conceptualization (lead); project administration ; writing – original draft (lead); writing – review and editing .A personal meeting of the authors was enabled through a Mercator fellowship to DS as part of the research unit FOR 5375 funded by the Deutsche Forschungsgemeinschaft —459717468 (sub‐project 2: 500516919). PB acknowledges the support of the DFG in the framework of his Emmy Noether project BI 1956/1–1.


Myeloma of the thyroid cartilage is a rare but important differential diagnosis of a laryngeal mass. Although hoarseness as the initial presenting symptom in multiple myeloma is extremely rare, a clinician should always consider it.Multiple myeloma (MM) is a malignant plasma cell disorder characterized by an uncontrolled proliferation of monoclonal plasma cells. Although the clinical presentation at diagnosis can be quite variable, thyroid cartilage infiltration in MM is rare. Here we discuss a 65‐year‐old Caucasian male presenting to the ENT doctor with continuous hoarseness for 3 months. The initial clinical examination showed a tangible mass at the left lymph node level II–III. Further examination with fiber‐optic laryngoscopy showed a bulging of the aryepiglottic and ventricular fold. Neck and chest CT scan revealed multiple osteolytic bone lesions in addition to the large lesion in the left thyroid cartilage. Laboratory work‐up, PET‐CT scan and biopsy of the thyroid cartilage were performed and eventually all confirmed the presence of a new diagnosis of IgA kappa MM. The patient was referred to the department of hematology to start with chemotherapy. This case report presents a rare case of multiple myeloma in which hoarseness was the initial presenting symptom. The patient reported continuous hoarseness since 3 months which initially started as a tickling cough. There were no periods of complete aphonia. He also complained of sternal pain radiating to the right ribcage, triggered by coughing and sneezing. The intake of solid and liquid substances was preserved and no dysphagia for liquids or solids was reported. The patient showed no weight loss, night sweats or fever. At the time of presentation, the patient had no significant medical history or known allergies and did not take any medication. He had a history of smoking  and alcohol use was limited to two to three units per day. The hoarseness made it impossible to work as he was a professional voice user.On the clinical examination, a tangible mass was discovered in the neck at the left lymph node level II–III. Except for distinct hoarseness, no other abnormalities were observed. To further investigate the pharynx and larynx, a fiber‐optic laryngoscopy was performed. A bulging of the left aryepiglottic fold and ventricular fold was observed. Stroboscopy showed asymmetry of the vocal cords with irregular movement and suboptimal glottal closure. It is unclear whether the hoarseness is due to nerve damage or physical obstruction or a combination of both. Considering there was a subtle mass tangible at palpation and a clear bulging of the aryepiglottic and ventricular fold, a CT scan of neck and chest was performed showing diffuse osteolytic bone lesions of which some had an extraosseous extension Figure . FurtherTo further examine the cause, a more extensive work‐up with laboratory analysis (including protein electrophoresis and immunofixation), PET‐CT scan and an ultrasound‐guided Tru‐Cut biopsy of the right clavicula and the left thyroid cartilage were performed. Lab results showed an M‐peak of 57,3 g/L on protein electrophoresis with IgA protein level of 49.84 g/L, hypercalcemia, renal insufficiency and a mild anemia, all suggestive of MM with high tumor burden Table .Moreover, PET‐CT scan demonstrated multiple intense foci in the skeleton and bilateral in the thyroid cartilage suspicious for a malignancy Figure . A Tru‐CThereafter, the patient was urgently referred to the hematology department to start with chemotherapy after performing a bone marrow biopsy and additional lab analysis. The bone marrow was invaded by an atypical population of plasma cells with kappa light chain restriction, making up 60%–65% of bone marrow cellularity. Cytogenetics showed no high‐risk chromosomal abnormalities and his R‐ISS scoreAs the patient was transplant‐eligible we started with induction chemotherapy consisting of bortezomib, cyclophosphamide and dexamethasone (VCD) once weekly, with the purpose of autologous stem cell transplantation as consolidation after four 28‐day cycles of VCD. It was expected that the dysphonia would get better with treatment.3In most patients with MM the plasma cell proliferation is restricted to the bone marrow, however a subset develops soft‐tissue plasmacytomas, whereby clonal plasma cells escape and are found outside the bone marrow.Although the most common cause of a thyroid cartilage mass is a squamous cell carcinoma, around 80% of EMPs are also located in the head and neck region, making this an uncommon but important differential diagnosis.While hoarseness as the initial presenting symptom in MM is extremely rare, a clinician should always consider it, especially in the presence of systemic symptoms like hypercalcaemia, unexplained renal insufficiency or osteolytic bone lesions.Jules Colebunders: Conceptualization; methodology; writing – original draft. Stig Hellemans: Conceptualization; methodology; writing – original draft. Melek Ahmed: Investigation; supervision; writing – original draft. Sasha Libbrecht: Investigation; supervision; writing – original draft. Sévérine de Bruijn: Supervision; writing – original draft.None of the authors involved in the writing of this case report received financial support from any party in relationship to content of this manuscript that might influence the authors opinion or editorial independence in the present or recent past.The authors have nothing to disclose and indicate no potential conflict of interest.Written informed consent was obtained from the patient to publish this report in accordance with the journal's patient consent policy.


Arctomecon californica (Las Vegas bearpoppy). Arctomecon californica is a relatively short‐lived plant that recruits from seed in sequences of unusually favorable years. We used germination experiments, an in situ seed bank study, and a 15‐year field seed retrieval study to examine factors affecting seed bank persistence. In the germination study, a majority of seeds remained dormant, despite a wide variety of treatments, suggesting that a large proportion of the seed dispersed each year has cue‐nonresponsive dormancy. Our in situ seed bank study showed that seed density varied widely between sites, among transects, and among samples within a transect. The patchiness of seeds in the soil highlights the importance of protecting large areas where A. californica populations are known to have existed in the past. The seed retrieval study provided strong evidence that this species has a long‐lived seed bank in which only a small fraction of seeds (roughly 5%) become nondormant each year, allowing seed banks of this species to last up to 20 years without a seed production event. Whether this impressive life‐history strategy can maintain the species in the face of climate change depends on the future frequency of the well‐timed precipitation that allows for the establishment of new cohorts of adult plants.Seed banks, the collection of viable seeds in the soil, are particularly important determinants of population survival in highly variable environments. Predictions of increased stochasticity in the amount and timing of precipitation in desert environments raise the question of how seed banks of desert species will respond to climate change, and ultimately, whether these species will persist. Here, we present data from our long‐term studies of germination requirements and seed bank dynamics in a rare desert gypsophile perennial,  Arctomecon californica is a short‐lived gypsophile perennial that is found in a stochastic desert environment. We demonstrated through field and laboratory studies that it can survive extended periods of failed recruitment through the maintenance of a persistent seed bank that can last up to 20 years. The seeds have cue‐nonresponsive dormancy that buffers the seed bank against rapid depletion so that populations that have not had actively growing plants for as long as two decades may still have the capacity to reestablish from the seed bank. For perennials, individuals can establish in different years, creating multiple age classes. However, both annual and perennial species can have another pool of individuals that are crucial to population persistence: the collection of dormant seeds that carry over across years to form the persistent seed bank . Previously published research on the seed ecology of A. californica has established that seeds are largely dormant at dispersal  What is the dormancy status of recently produced seeds? (2) Can conditions that seeds are expected to experience in the field allow recently dispersed seeds to break dormancy? (3) Does germination response differ among populations? (4) Is there a persistent field seed bank that remains even after a major germination event, and how is it spatially distributed? (5) What are the patterns of 22.1Arctomecon californica, the Las Vegas bearpoppy, is a rare herbaceous perennial plant of the Mojave Desert  that recruits from seed in sequences of unusually favorable years . It has been thought to depend on a long‐lived seed bank for population persistence through sometimes long periods unfavorable for recruitment and survival . Wide variation among species exists in the conditions required to break morphophysiological dormancy  and St. Thomas Road (STHOM), and also at Rainbow Gardens (RBG) on BLM land in June 1995 . For this reason, we did not include a formal warm moist stratification pretreatment in the 1995 experimental design. Our treatments included the three seed populations , a series of 11 dry storage temperature‐by‐duration treatments, seven moist‐chilling duration‐by‐temperature combinations, and two postchilling incubation temperatures  at 2°C did not result in significant dormancy break for The laboratory seed germination experiment was carried out in two steps: an original experiment (data not shown), and an additional experiment that added a re‐chill treatment as a continuation of the original experiment Figure . The expFor the storage treatments, seeds were stored at 20, 30, 40, and 50°C under unsealed dry conditions for 0–18 weeks. Shorter maximum durations were used at the two warmer temperatures based on our data from other species showing that prolonged storage at constant high temperatures could damage the seeds. Storage duration and storage temperature were therefore not combined in a full factorial design. After each chilling treatment, seeds were incubated at one of the two incubation temperatures for 2 weeks and scored for germination. In the earliest treatments, seeds were then immediately subjected to viability evaluation AOSA, ; viabiliAfter observing essentially no germination in any of the initial treatments, we decided to subject the later treatments to a second chilling experience after the 2‐week germination period. We chose a 12‐week cold stratification period for the re‐chilling treatment. This likely exceeds the maximum chilling duration that would be experienced in the field. After this second 12‐week cold stratification at the same temperatures as the original chilling treatments, the seeds were again incubated at the two original incubation temperatures for 2 weeks and scored for germination and viability. This second round of experimental treatments resulted in some dormancy break as reported below.We have found that long‐term viability in dry storage is usually a necessary but not sufficient indicator of the ability of seeds to persist across years in field seed banks. We evaluated long‐term viability ex post facto for the seed lots used in germination and retrieval studies reported here by performing tetrazolium tests on four replicates of 50 seeds for each seed lot following 27 years of unsealed laboratory storage at room temperature (ca. 20°C). Seeds lose viability gradually over time in dry storage and this is reflected in a gradual diminution in respiration and the resulting red color after tetrazolium staining. As individual seeds lose viability at different rates, this results in a range of embryo color intensity. We took this into account by scoring seeds in three categories; viable (red or dark pink), marginal , or nonviable (white). Seeds in the marginal category would probably not be able to produce seedlings, but the fact that they were still respiring indicates that they maintained viability longer than seeds in the nonviable (white) category.2.3Plots were established in bearpoppy habitat at NLVAT and STHOM in March 2005 Figures  and 5. S2 quadrat. At STHOM, the search protocol was different due to lower seedling densities. The entire area within 2 m on one side of each of the five transects was searched for seedlings. When seedlings were located, 4 dm2 quadrats were designated around them, and these plots were used for both the seedling study and the seed bank study. The transects were laid out in parallel at each site, but spacing and length of transects at each site were constrained by the requirement that there be suitable soils and evidence of bearpoppy occupation within each transect. This resulted in variable inter‐transect spacing and transect length. A paired seed bank sample was taken just outside of each seedling quadrat using a cylindrical soil can 6 cm in diameter and 4 cm in depth. Samples were lifted into labeled paper collection bags with the aid of a mason trowel. Sample size was 247 for NLVAT and 294 for STHOM. Samples were returned to the laboratory for sieving and seed extraction. It was relatively easy to visually identify the 2‐ to 3‐mm‐long jet‐black seeds of A. californica in the white soils where they occur. Seeds in each sample were counted and tested for viability using tetrazolium staining.At NLVAT, 10 transects used for seed bank sampling were established across the study site. Quadrats were located at distances along the transect lines determined by random numbers generated using a stopwatch  and carried out over 15 years  for 4 weeks. Seeds that germinated during this period were scored as germinable . Remaining ungerminated seeds at the end of the incubation period were then evaluated for viability using tetrazolium and were scored as nonviable, viable and dormant, or empty and presumed germinated in the field.2.5Laboratory germination data were analyzed as binomial data using PROC GLIMMIX in SAS  in the trials/events format with germination fraction  as the response variable. This format provides analysis for a binomial response variable using the logit‐link function and the residual degrees of freedom method by default. Predictor variables for analysis as presented here included population, dry storage treatment, and incubation temperature. Initial chilling temperature and duration and re‐chilling temperature had inconsistent minor effects on germination outcomes in preliminary data exploration and were excluded from the present analysis.In situ seed bank density (count) data were analyzed using SAS Proc GLM with population as the main effect and with transect nested within population. Viable seed fraction was analyzed using the same model as described above in SAS Proc GLIMMIX for binomial data. Viable seed proportion was calculated only for samples that had seeds present.Seed retrieval data were first analyzed using SAS Proc REG for each seed population by site combination. For each combination, regression analysis was performed for the variables dormant seed fraction and viable (dormant plus nondormant) seed fraction. This analysis provided the slope of linear decline in seed dormancy and seed viability for each seed population at each site and also an estimate of the number of years to zero seed dormancy or viability, at the point on the time axis when dormancy or viability was projected to be zero.Seed retrieval data were also analyzed using the GLIMMIX procedure in SAS. We used only the first 8 years of data from NLVAT in this analysis to make it directly comparable to the STHOM data set, which included only 8 years as previously explained. Block effects were not significant in this or any other analysis and were not included in the final models. Predictor variables included site (NLVAT and STHOM) and population of origin  as class variables and year as a continuous variable in the analysis of covariance. This enabled us to examine the effects of site, seed population, and their interaction on seed dormancy and viability loss rates. Field germination was estimated as the sum of recently germinated seeds (radicles evident) and empty seed coats. Field germination patterns were examined graphically but were not formally analyzed.33.1In the original factorial experiment, the mean germination percentage across all seed populations and treatments and for each seed population by storage treatment combination was less than 1%, indicating a very high level of primary dormancy for recently produced seeds regardless of high‐temperature after‐ripening or moist chilling. Of 462 total treatment combinations, 428 had zero germination. The maximum germination percentage in any treatment combination was 5% (data not shown).F = 169.05, p < .0001) and storage treatment  highly significant . There were also highly significant differences among transects within sites for total seed density, indicating a patchy distribution of seeds. Seed bank density also varied radically from sample to sample within transects, as evidenced by large error bars. Viable seed fraction was slightly higher for STHOM vs. NLVAT ; however, the buildup of nondormant, viable seeds in the seed bank can be seen over the first 10 years of the study (which were relatively dry), followed by a drop in the number of viable seeds with the mass germination event in 2005, when all nondormant seeds in the packets germinated. It also shows the subsequent buildup of nondormant, viable seeds as seeds lost dormancy without germinating through the end of the study . Studies have shown that some seeds can withstand surprisingly high temperatures  become nondormant each year, allowing seed banks of this species to last up to 20 years without a seed production event. The retrievals allowed us to document that seeds of known age lost dormancy year by year, creating a buildup of nondormant seeds in the seed bank during the series of dry years between 1996 and 2005 . We do not know the mechanism that is producing variation in dormancy duration within populations of A. californica, but a future study looking at dormancy levels among seeds produced at different times during the growing season might provide information.Seeds sometimes have visible heteromorphism that allows for variation in germination behavior leading to bet‐hedging  populations. These sites need to be protected.Our seed bank persistence study provides strong evidence that sites known to have had plants of A. californica seed banks have experienced throughout their history, with seeds produced in favorable years entering the seed bank, then losing dormancy at a slow, constant rate and creating a pool of nondormant seeds. These seeds can then germinate en masse when there is a year of exceptionally favorable winter precipitation. A key take‐home message from this work is that a population of A. californica can persist for at least 20 years without any aboveground plants. Whether this impressive life history strategy can maintain this species in the face of climate change depends on the length of future droughts or, conversely, on the frequency of well‐timed precipitation that allows for germination events followed by the survival of seedlings to adulthood and seed production.The precipitation record Table  shows clA. californica is likely to persist as temperature and precipitation patterns change.Knowledge of seed bank dynamics alone is clearly not sufficient to address the impacts of climate change on plant demography. The seed ecology data sets presented here will be combined with additional data sets addressing recruitment, survival to reproduction, and adult lifespan and reproductive output in our upcoming population viability analysis for this species. We plan to use future climate predictions in conjunction with these demographic data sets to evaluate whether Tara de Queiroz: Conceptualization (supporting); data curation (supporting); funding acquisition (supporting); investigation (supporting); visualization (supporting); writing – original draft (lead); writing – review and editing . Susan E Meyer: Conceptualization (lead); data curation (lead); formal analysis (lead); funding acquisition (lead); investigation (lead); visualization (lead); writing – original draft (supporting); writing – review and editing .The authors declare no conflict of interest.


Canis latrans). Temporally, all species other than cougars (Puma concolor) had diel activity patterns significantly different from that of recreationists, suggesting potential displacement in the temporal niche. Wolves (Canis lupus) and mountain goats (Oreamnos americanus) showed shifts in temporal activity away from people on recreation trails relative to off‐trail areas, providing further evidence of potential displacement. Our results highlight the importance of monitoring spatial and temporal interactions between recreation activities and wildlife communities, in order to ensure the effectiveness of protected areas in an era of increasing human impacts.The management objectives of many protected areas must meet the dual mandates of protecting biodiversity while providing recreational opportunities. It is difficult to balance these mandates because it takes considerable effort to monitor both the status of biodiversity and impacts of recreation. Using detections from 45 camera traps deployed between July 2019 and September 2021, we assessed the potential impacts of recreation on spatial and temporal activity for 8 medium‐ and large‐bodied terrestrial mammals in an isolated alpine protected area: Cathedral Provincial Park, British Columbia, Canada. We hypothesised that some wildlife perceive a level of threat from people, such that they avoid ‘risky times’ or ‘risky places’ associated with human activity. Other species may benefit from associating with people, be it through access to anthropogenic resource subsidies or filtering of competitors/predators that are more human‐averse . Specifically, we predicted that large carnivores would show the greatest segregation from people while mesocarnivores and ungulates would associate spatially with people. We found spatial co‐occurrence between ungulates and recreation, consistent with the human shield hypothesis, but did not see the predicted negative relationship between larger carnivores and humans, except for coyotes ( Protected areas have a dual mandate of providing recreational activities while also protecting wildlife and their habitats. We looked at the effects of human recreation on wildlife use of habitat in space and time, and found responses to recreation varied by species. Within one protected area, we saw that animals may be avoiding humans by shifting their use of habitat in time, more than in space. Cathedral presents a useful case study as it faces common issues of increasing human recreation, limited quantification of human use and poor understanding of the compounding effects of these factors on management efforts. Additionally, Cathedral faces increasing isolation from other high‐quality wildlife habitats due to transportation infrastructure, forestry, agriculture and urban development in the surrounding areas. Given such isolation, the potential for negative impacts of recreation on wildlife within the park takes on added significance .Capitalising on the opportunity for ecological insight derived from camera traps, we sought to investigate the question: We considered two general hypotheses that could explain wildlife responses to recreation: avoidance of risk or attraction to benefits. Firstly, wildlife may perceive risk from humans, similar to the risk perceived by prey from an apex predator  would avoid areas of human recreation due to the risk of persecution under a landscape of fear, either from hunting in portions of the park with less human use or destruction by managers in areas with more human use  would exhibit limited spatial avoidance of humans due to a decreased risk of direct persecution  as compared to larger carnivores. We also predicted that small prey of mesocarnivores  may increase in proximity to humans under the human shield hypothesis where larger predators are repelled, which may further attract mesocarnivores to areas of human use due to increased prey availability. Further, we predicted that mesocarnivores would still minimise risk of persecution by humans under a landscape of fear, resulting in increased cathemeral or nocturnal activity patterns in response to higher human activity , mule deer (Odocoileus hemionus) and mountain goats (Oreamnos americanus) would exhibit a positive spatiotemporal response to humans to benefit from the ‘human shield’ effect, but that habitat specialist ungulates such as mountain goats might display a temporal shift away from mid‐day due to the high overlap between key habitat and human recreation in alpine and sub‐alpine environments at that time  Nation territory Figure . It encok Figure . Biogeock Figure .Pers. Comm.). A small parcel of private land pre‐dating the establishment of the park is centrally located at Quiniscoe Lake in the core area and is operated as a backcountry lodge. Associated with this lodge is a private 4 × 4 access road that provides paid shuttle services for lodge guests and campers. All access to the park is by foot or the private shuttle, as mountain biking and other motorised recreation are not permitted. Hunting is prohibited within the core area of the park  camera traps throughout much of the 330 kmCameras were placed on trees with a minimum diameter > 10 cm, or a large boulder in one case above the treeline, at a height of 0.3–1.0 m above the ground. Camera sets were dependent on site characteristics and distance from the focal feature to provide a consistent detection zone for target animals. Focal features included human trails, game trails and gravel roads. Similar detectability at each camera was confirmed with the camera walk test feature by field personnel moving through the detection zone at different distances and heights . At on‐trail sites, cameras were placed perpendicular to the trail wherever possible to standardise detection probability across locations. Cameras were affixed 3–6 m from the focal target zone of anticipated animal or human travel (e.g. trail). Cameras were set to take one image per trigger, with no delay between successive images and high trigger sensitivity. Once deployed, camera traps were checked every 2 months between June and October for those in the higher use core area or other accessible areas and annually for those in remote areas with limited human access. Camera operability was high, resulting in over 26,000 camera‐days of data collected and a mean of 578 trap nights per camera . The few cases of camera failure were the result of wildlife damage, hardware malfunction, human tampering, or heavy snowfall.We limited this study to medium‐ and large‐bodied mammals, as the decreased detectability of smaller species on camera traps set to capture multiple species decreases the utility of this method for making unbiased inference about smaller animals . We additionally processed data through MegaDetector, a machine learning object detection model to identify humans, animals, vehicles and blank images  algorithm to protect privacy by preventing the identification of individual humans in camera trap images while still allowing the counting of human recreationists  and age class . We also tabulated a group count of the total number of distinguishable individuals within a sequence of related detections, with a sequence  defined as all images of the same species occurring within 5 min of the last image of that given species. Images of commonly confused species  were each reviewed by author MF a second time to ensure misclassification rates were minimised. Where there was uncertainty around the species within an image, an identification of ‘Unknown species’ was assigned.Due to the immense number of human images captured, we used MegaDetector to automatically detect any images containing humans. Using a confidence threshold of 90% and manual visual confirmation, we classified all photos containing at least one human by activity type. Types of human activity included hiking, horseback riding, mountain biking and motorised vehicles; however, the latter only occurred at two sites on the private lodge access road, and horseback riding was only infrequently detected on a single trail skirting the edge of the park. Given the dominance of hiking and minimal detections of other human activities in the park, we constrained our analyses of human activity to focus only on hiking.2.4We used a Bayesian generalised linear mixed‐modelling framework to evaluate spatiotemporal responses in site use by wildlife at the weekly temporal scale. Once images were classified, the number of independent detections per week at each site was summed for each of the focal species Table , servingThe count of detections per site week represents a combination of the number of individuals of a species using that site and their movement patterns relative to the camera. We assumed that animals moving through the camera detection zone were detected with high probability, although some individuals using the surrounding area may not have passed through the detection zone. Nevertheless, we were interested in the variation in detections as a signal of variation in site use rather than sampling error as is assumed in some modelling frameworks (e.g. occupancy). Only weeks with seven active camera days were included. We have no reason to expect unmodeled bias in detections and assumed they were proportional to site use. Further, occupancy may be more sensitive to variations in movement  than detection‐based indices .The environmental covariates described above without human detections or linear features (environment model).All predictors .To parse the effects of recreation, environment or a combination of these predictors we created three candidate models for each species and conducted model selection to discern the best‐supported set of predictors Table . The thrr| < .5; Dormann et al., We modelled the number of independent detections of each species per site week as a negative binomial response and included the camera site as a random intercept to account for potential nonindependence among repeated observations at the same site. All independent variables were standardised by subtracting the mean and dividing by one standard deviation to allow direct comparison of the direction and magnitude of effects on species use of habitat Gelman, . All preR2 values  cross‐validation for model selection by calculating the Leave One Out Information Criterion (LOOIC) for each candidate model , followed by coyote (n = 593) and lynx (n = 419) , and wolves used on‐trail sites significantly more at night than during the day .All species showed relatively low temporal overlap with humans in their diel activity patterns, supporting our prediction of temporal partitioning from people for large carnivores and mesocarnivores but contrary to our prediction of higher temporal overlap between ungulates and people Figure , Table 44Our results are consistent with the hypothesis that human recreation may impact the timing and location of activity by wildlife within a protected area. However, the observed shifts in space and time were highly variable across species. We found that only coyotes displayed patterns of habitat use consistent with strong spatial displacement from recreation on a weekly scale. For two of the three ungulate species, our prediction of a positive spatiotemporal correlation with human recreation was supported, providing partial evidence for the human shield hypothesis, though our prediction that predators would avoid areas of higher human use was not strongly supported  at night  may include reduced fecundity or decreased body condition, leading to negative effects individually and for the population ; data curation (lead); formal analysis (lead); funding acquisition (supporting); investigation (lead); methodology (lead); project administration ; validation (lead); visualization (lead); writing – original draft (lead); writing – review and editing . Adam T. Ford: Conceptualization (supporting); formal analysis (supporting); investigation (supporting); methodology (supporting); writing – original draft (supporting); writing – review and editing . Tara Martin: Conceptualization (supporting); formal analysis (supporting); investigation (supporting); methodology (supporting); writing – original draft (supporting); writing – review and editing . A. Cole Burton: Conceptualization ; data curation (supporting); formal analysis (supporting); funding acquisition (lead); investigation (supporting); methodology (supporting); project administration ; resources (lead); supervision (lead); writing – original draft (supporting); writing – review and editing .The authors have no competing interests or conflicts of interest to declare.Appendix S1Click here for additional data file.


Kaposi's sarcoma (KS) is a rare multifocal angiogenic tumor often seen in immunocompromised setting such as acquired immunodeficiency syndrome (AIDS) or organ transplantation recipients. Pemphigus vulgaris (PV) is a rare blistering disorder with mucocutaneous involvement for which immunosuppressive therapy has long been the core of treatment. Iatrogenic form of KS has been reported infrequently in pemphigus patients as a result of long‐term immunosuppressive therapy.We describe a 39‐year‐old male patient with confirmed diagnosis of PV who developed KS after receiving immunosuppressive agents for his pemphigus. KS was initially localized to the oral cavity with features mimicking exacerbation of his pemphigus.This interesting case of KS suggests that dermatologists visiting patients with pemphigus with discomfort in the oral cavity should have a high degree of awareness and consider other differential diagnoses along with merely an exacerbation of PV. Histopathologic and DIF examinations stablished the diagnosis of PV. His disease had initially been controlled with 60 mg/day prednisolone which tapered to 30 mg/day during 1 month and rituximab (one cycle consisting of one infusion of 500 mg every week for 4 weeks) was added to his therapeutic regimen leading to a considerable clinical improvement. Hence, prednisolone was rapidly tapered to 10 mg/day in 3 months because of his cushingoid appearance and also achieving partial clinical remission. The Pemphigus Disease Area Index (PDAI) score of cutaneous and mucosal lesions from 11 and 10 at the baseline, declined to 0 and 3 at the fifth month of treatment, respectively.The patient had also taken Isoniazide (INH) and vitamin B6 as tuberculosis prophylaxis due to positive skin tuberculin test (induration: 13 mm). Patient was otherwise healthy.By the time, the prednisolone dosage was lowered to 7.5 mg/day and cutaneous PDAI score was 0, but he was still complaining of a persistent odynophagia and dysphonia.On physical examination, the patient had generally good health and no remarkable finding was noted except for mild sore throat and an erythematous nodule with erosive surface at the end of lingual area Figure . AlthougMRI of cervical soft tissue revealed an isosignal 23 × 73 mm mass at oropharyngeal region on epiglottis which was enhanced after contrast injection. Pressure effect on airway was also noted.The patient was referred to the Otolaryngology Department, where the nodule was totally excised after a laryngoscopic evaluation in which a large mass was detected at the base of tongue Figure . HistopaDuring systemic evaluation, no history of opportunistic infections, B symptoms or any sign or symptom regarding visceral involvement of KS was noted.Taking into consideration the clinical complete remission of PV in patient as well as the low values of anti‐Dsg1, 3 , we decided to taper off the prednisolone.One month later, two other nodular violaceous lesions on his cheek and ankle were appeared Figure  which weAny occurrence of KS lesion has not been observed until now (21 months after excision of the last lesion) and the patient did not describe any relapses of PV or KS lesions.3KS is an angioproliferative neoplasm of low‐grade malignant potential.It is hypothesized that altered immunity weakens the immunological surveillance and leads to reactivation of a latent HHV‐8 infection in genetically susceptible patients.KS is classified into four types: classic, endemic, immunosuppression‐associated or iatrogenic and AIDS‐associated or epidemic.Association of pemphigus (mainly PV) and KS has been previously reported.Iatrogenic KS often has similar clinical features to the AIDS‐associated KS including fulminant course with widespread lesions, tendency to visceral involvementAs mentioned before, most of the cases of KS arises in immunocompromised patients have an aggressive and fulminant course, whereas the relatively fair course and good response to treatment in our case might be in favor of the fact that the appearance of KS was not secondary to immunosuppression. It is worthy to note that occurrence of the second and third KS lesions in our case were 1 month after tapering off the prednisolone.Altered immunity due to receiving immunosuppressive agents is not the only explanation for association of PV and KS. Although not proven yet, HHV‐8 has been introduced as a triggering factor of bullous disease, especially PVResults of the recent cohort study showed that half of pemphigus‐associated KS cases developed their KS lesions before the start of immunosuppressive therapy that is fairly in favor of the role of HHV‐8 infection as a triggering factor in patients with pemphigus.HHV‐8 detection in tissue samples of our patient could have shed some light on this matter of debate and suggested the pivotal role of HHV‐8 at least, in PV‐associated cases with a relatively fair course of disease, but regretfully, we could not test it which is a major limitation of this study.The fact that tuberculin test in our patient was positive without any clinical manifestation of tuberculosis, might be inferred as the relative strength of cell‐mediated immunity that resulted in a benign course of KS in our case.Treatment of KS depends on the clinical presentation, extent of involvement and associated comorbidities. In local diseases, radiation, photodynamic therapy, intralesional vinblastine, topical alitretinoin gel, cryotherapy, curettage and electrodesiccation are main therapeutic options. For extensive disease, a combination of surgery, chemotherapy and radiation is advocated. Some recent therapeutic agents such as imatinib and matrix metalloproteinase inhibitors are also being triedIn conclusion, we reported a case of KS with an unusual presentation to emphasize the fact that careful examination and clinical suspicion are the key factors for correct diagnosis. In fact, throat burning and odynophagia in PV patients might not be merely due to pemphigus erosions and should raise the suspicion of occurrence of other pathologic processes such as KS even in HIV‐negative patients.Furthermore, we highlighted the fact that not all of patients with pemphigus‐associated KS have fulminant course. Therefore, the crucial question to be resolved is how we can discriminate between steroid‐associated KS and disease‐associated KS in patients with pemphigus.Implementing some proceedings such as skin tuberculin test might be useful in answering this question and predicting the course of disease. Further studies are needed to shed more light on this topic.Kamran Balighi: Conceptualization ; supervision ; visualization . Maryam Ghiasi: Data curation ; methodology ; project administration . Zeinab Aryanian: Investigation ; writing – original draft . Zahra Nikyar: Investigation ; project administration ; writing – original draft . Parvaneh Hatami: Investigation ; visualization ; writing – review and editing .The authors declare that they have no conflict of interest regarding the publication of this article.The patient provided written informed contest to publication of this case report and accompanying images.