Although individual cancer cells are generally considered the Darwinian units of selection in malignant populations, they frequently act as members of groups where fitness of the group cannot be reduced to the average fitness of individual group members. A growing body of studies reveals limitations of reductionist approaches to explaining biological and clinical observations. For example, induction of angiogenesis, inhibition of the immune system, and niche engineering through environmental acidification and/or remodeling of extracellular matrix cannot be achieved by single tumor cells and require collective actions of groups of cells. Success or failure of such group activities depends on the phenotypic makeup of the individual group members. Conversely, these group activities affect the fitness of individual members of the group, ultimately affecting the composition of the group. This phenomenon, where phenotypic makeup of individual group members impacts the fitness of both members and groups, has been captured in the term ‘group phenotypic composition’ (GPC). We provide examples where considerations of GPC could help in understanding the evolution and clinical progression of cancers and argue that use of the GPC framework can facilitate new insights into cancer biology and assist with the development of new therapeutic strategies. Primary cancers often display high levels of phenotypic and genetic intratumor heterogeneity (ITH). Whereas the mutational and epigenetic mechanisms that generate this heterogeneity are relatively well understood, mechanisms responsible for maintaining this heterogeneity in cancer cell populations, as well as the impact of ITH on clinically important properties of the disease, are less clear. Yet, high levels of ITH have been linked to more aggressive tumor behavior, resistance to therapies, and overall poor prognosis . Thus, aITH can be subdivided into micro-heterogeneity  as well as macro-heterogeneity , yielding a co-existence of either independent genetic subclonal lineages or metastable phenotypic subgroups . DespiteIn particular, stable co-existence of distinct subpopulations or subclones within tumor cell populations that follow Darwinian dynamics appears to be counterintuitive. Indeed, the maintenance of stable subpopulations with heritable phenotypes within the same tumor indicates the presence of multiple niches producing complex patterns of co-existence, although weak stabilizing selection and drift may also contribute to phenotypic variability. In turn, the co-existence of different subpopulations can profoundly influence global tumor behavior such that elimination of one subpopulation can induce tumor regression or necrotic collapse . This coIn nature, the function and stability of a group is dependent on the characteristics of the constituent individuals  and their interactions, which are always in an evolutionary state of flux. Tumors have already been described as composed of functional compartments with a division of labor among these compartments  and concBox 1.Intratumoral phenotypic clustering despite genetic and epigenetic diversity.Cancer cells can reversibly and stochastically transit between states that differ in their competence to contribute to tumor progression . These sInterestingly, when a subpopulation is purified for a given phenotypic state, a return toward equilibrium proportions is then observed over time , suggestInnovative methods such as single-cell mass cytometry allow the detection of phenotypic clusters among single tumoral cells despite extensive heterogeneity in genetic and epigenetic content. For instance, a consensus clustering approach allowed hierarchical clustering of 45 epithelial clusters in breast cancers based on normalized epithelial cell marker expression . RecentlDaphnia magna for which predation risk (by the three-spine stickleback Gasterosteus aculeatus) is higher on larger individuals  and confusion effects operate, some conflicts between individuals with different phenotypes might emerge: both large and small individuals benefit from joining groups composed of large individuals, but the presence of small individuals enhances the predation risk experienced by large ones  costs, leading to mixed groups in the wild. This example illustrates the kind of complex and reciprocal links that may exist between individuals’ phenotypes and the GPC’s properties. More generally, individual heterogeneity and resulting GPCs result in multiple hierarchical effects that affect collective behavior, from within-group positioning, group coherence, leadership, and collective decision making to group functioning, fission–fusion dynamics, and among group assortment  be limited to cells that share a set of driver (epi)mutations , type , amount and composition of extracellular matrix, type and numbers of non-malignant cells, proportion of drug-resistant cells, etc.GPC can be dynamic both in terms of number, types, and proportion of subgroups, and codependency levels between subgroups. For instance, the emergence of macroscopic tumors is dependent on the presence of a threshold number of tumor cells that produce angiogenic factors . These fTumor growth and progression are associated with changes in microenvironmental contexts in space and time . In turn, these contextual changes would be expected to impact fitness of subgroups of tumor cells. Thus, a GPC that is highly ‘optimal’  during the early stages of tumorigenesis  may not be optimal in a large tumor (which requires vasculature) ; step 4.Thus, tumoral GPC, and its relationships with the fitness of individual malignant cells in different ecological contexts, may represent a crucial, previously unexplored aspect that influences evolutionary dynamics within tumor cell populations. In this context, GPC will influence selection pressures experienced by individual tumor cells, which will influence individual cell-level outcomes. As the relative proportion and type of interactions among subgroups of tumor cells vary in response to changing microenvironments, tumoral GPCs will also change resulting in either new optimal tumoral GPCs or, potentially, catastrophic imbalances producing tumor extinction  see also . IndividBox 2.Examples of GPC in cancer: CTCs.CTC clusters are defined as groups of two or more CTCs that have stable cell–cell linkages and travel together through the bloodstream . Aceto aFor instance, the presence of clusters as well as their size are correlated with a poorer clinical outcome compared to the presence of single CTCs in cancer patients . InteresA recent study might explain the advantage of clustered CTCs. In response to detachment from the extracellular matrix, reactive oxygen species are produced. The cell clusters could be protected from this phenomenon by the production of Hif1α and, subsequently, by a metabolic switch to glycolysis as well as increased survival and metastatic capacity . This inEpigenetics also seems to play a key role since DNA methylation is clearly different between single CTCs and CTC clusters. CTC aggregation results in the hypomethylation of binding sites for stemness and proliferation regulators . Interestingly, this methylation profile is reversible by dissociating CTC clusters into single cells .CTC cluster dissemination might also have an impact on a tumor’s developmental dynamics in the metastatic setting. In fact, heterogeneous clusters might seed polyclonal metastases, suggesting that this modality of cancer spread may increase the likelihood that a tumor will colonize distant sites successfully and might eventually show increased resistance to anti-cancer therapies .CTCs can form heterotypic interactions with non-tumor cells such as immune cells and cancer-associated fibroblasts . Szczerb CTC clusters can consist of similar or different tumor cells . Clusters can also include non-tumor cells  that can affect the fitness of cancer cells . AccordiThe GPC concept can also be extended to transmissible cancers see also . Here, wBox 3.Examples of GPC in cancer: transmissible cancers.5 or 106 viable DFTD tumor cells developed tumors after 7 and 4 weeks, respectively, and X–DFTD tumors reinoculated into NOD/SCID mice formed visible tumors after 5 weeks  would benefit one or both lineages via resource allocation and niche partitioning.The first steps during the emergence of a transmissible cancer are likely to be similar to non-transmissible ones , with similar constraints on the GPC composition required for a tumor to grow and metastasize. However, to become transmissible, a cancer must also gain the ability to shed a very large number of cells that can both survive in the environment outside the host’s body , but als5–107 cells were required in transmission experiments [Once inside the body of a new host, most transmissible cancer cells fail to establish a new tumor (10eriments ). This h MHC class I, expression has been completely lost  might be able to prevent plastic adaptive changes in GPC and improve therapeutic outcomes. Phenotypic plasticity should not be considered strictly as a means to increase individual/cell-level fitness. In our framework, phenotypic plasticity is also a way for cancer cells to both rapidly change the tumoral GPC to maintain the combination of phenotypes required for progression or overcome the therapeutic suppression of a dominant aggressive phenotype. This means that optimal or suboptimal tumoral GPCs could be restored after such interventions simply as a consequence of these non-genetic events. To circumvent these phenomena, targeting phenotypic plasticity should be relevant, either alone or in combination with targeted therapies . This isPhenotypic plasticity is extremely relevant especially during the epithelial-to-mesenchymal transition (EMT). EMT generates mesenchymal-like cells and a variety of intermediate cell states between the epithelial and the mesenchymal state and changes the GPC toward tumor progression and metastasis . Thus, tMore globally, insights can be gained on the role of phenotypic plasticity in affecting tumor GPC using dynamical systems theory  where reFourth, while currently relevant knowledge is almost entirely lacking, it will be important to understand whether and how the tumoral GPC is reestablished in a secondary tumor, and the role the tumoral GPC plays in metastatic colonization and outgrowth. We can speculate that since metastases can be seeded by individual cells or small groups of cells , the oddFifth, in addition to targeting keystone subgroups, tackling specific interactions  may be of equal or higher therapeutic benefit due to altering tumoral GPC quality. For instance, disrupting clonal cooperation by targeting key factors was already suggested , as wellTumors can be seen as examples of social heterogeneous groupings. A proper mechanistic understanding of the impact of ITH and tumoral GPC on cancer progression requires bridging oncological sciences with evolutionary ecology. What are the potential advantages of GPC consideration over traditional, reductionistic approach of viewing tumors as collection of specific genetic clones or phenotypic subpopulations? A first obvious benefit of the GPC concept resides in the fact that it permits assessment of some of the functional aspects of cancer dynamics, which are impossible to understand by simply examining cellular collections only. In the same way that we cannot fully understand the biology of any living organisms when examined in isolation from its abiotic and biotic environment (including its conspecific peers), the biology of malignant cells must consider the social context  to which it is exposed. The GPC concept should also permit us to understand phenotypic convergences, when cells differ at the genetic or epigenetic level but accomplish more or less the same tasks see .On the other hand, the utility of considering tumor GPC is currently limited by the lack of criteria to define relevant groups of tumor cells. Strong interdisciplinary research that integrates observational, experimental, and theoretical studies will be crucial to develop methodology to define and categorize GPCs, identify functionally relevant subpopulations within heterogeneous tumors, identify critical interactions between these subpopulations, and identify the parameter values of GPC descriptors that are associated with an ‘optimum’ GPC at each cancer progression step. Such studies will ultimately allow us to understand the impact of GPCs (and the interactions between metastable components of the GPCs and their microenvironments) on the collective behavior of tumors and their evolutionary and clinical trajectories.In this paper, we did not consider the potential existence of metaphenotypes of structured groups of cells, that is functional clusters, within tumors. Such a situation would yield to different and more complex GPC-related dynamics on tumor progression. While whole tumors are (most often) not in competition within tissues, this can conversely be the case for functional clusters within tumors, suggesting that the GPC of tumors could display a ‘nested structure’. That is, the GPCs of functional clusters within the tumor are also influenced by the competition between groups, but these groups also contribute together to a whole tumoral GPC that is not in a competitive context with other tumors. In addition, if a group within the tumor has a better GPC than others, it can increase in prevalence, with tumor growth then seeding the formation of new GPCs. More research is needed at the moment to explore the extent to which tumor dynamics are influenced by the whole tumor GPC, by GPCs of functional groups that are within tumors, and/or by a mix of both. Also, we need to determine whether there are temporal patterns in these processes, exploring for instance the importance of these different dynamics throughout tumorigenesis. Still, the formation of ‘the right GPC’, appearing in the right condition at the right time, is heavily influenced by chance, stochasticity and spatial context, and thus remains challenging to investigate and act on. For instance, cells that would form a viable GPC for host-to-host transmission may exist but are not co-localizing or cannot be transmitted as a unit during shedding/infection. Similarly, some phenotypes may benefit from an advantage simply because they are close to angiogenic cells, while other GPC-formation candidates in other parts of the tumor will die due to nutrient scarcity. Other information is also needed to determine whether the propensity for developing the ‘right’ GPC see  during tOverall, examining tumor GPC and addressing the effects of GPC on individual tumor cell fitness (selective consequences) and the responses of individual cells to the selective consequences of GPC (evolutionary consequences) should improve our understanding of tumor biology and cancer progression. At the moment, it remains mostly an abstract concept because we need to develop novel tools to describe, understand, and monitor the tumoral GPC functional dynamics and its resilience to local disturbances. Also, criteria used to describe groups remain by definition subjective, limiting the possibility at the moment to provide a unique definition of GPC in cancers (see


The presence of spiral ganglion cells (SGCs) is widely accepted to be a prerequisite for successful speech perception with a cochlear implant (CI), because SGCs provide the only known conduit between the implant electrode and the central auditory system. By extension, it has been hypothesized that the number of SGCs might be an important factor in CI outcomes. An impressive body of work has been published on findings from the laborious process of collecting temporal bones from CI users and counting the number of SGCs to correlate those numbers with speech perception scores, but the findings thus far have been conflicting. We performed a meta-analysis of all published studies with the hope that combining existing data may help us reach a more definitive conclusion about the relationship between SGC count and speech perception scores in adults. Cochlear implants represent the most successful example of a neural prosthesis that replaces a human sense with an electronic device. They do so by stimulating the auditory nerve directly, bypassing the normal mechanism that transduces sound into neural activity . HoweverPhysiological and anatomical variables have also been suggested as possible factors underlying individual differences . In partObtaining experimental support for this hypothesis is complicated because there is no reliable, precise, and noninvasive way to count the number of SGCs in live human subjects. The most direct way to establish the correlation between SGCs and CI outcomes is therefore to measure those outcomes  while patients are alive, and then analyze their temporal bones post-mortem to obtain precise SGC counts. Dedicated groups of otohistopathologists have taken on this challenging question and have published data on SGC counts and speech perception scores in CI users. However, results have been conflicting, and the first two studies to compare SGC counts and speech performance in patients who had used multichannel CIs found an unexpected negative correlation between CI speech perception scores and SGC counts ,12, whicHere, we performed a meta-analysis of all published studies of the relationship between SGC count and speech perception scores in adult patients who received a multichannel cochlear implant, to help us reach more solid conclusions about an unconfirmed but widely accepted hypothesis within the cochlear implant field.We followed the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) checklist to guide our study . This stThe population of interest was post-lingually deaf subjects who received the intervention of cochlear implantation. Most subjects had either consonant-nucleus-consonant (CNC) or Northwestern University (NU-6) word scores, two monosyllabic word identification tests that have been shown to result in “remarkably similar” scores . In 5 suThe literature search was performed by a medical librarian for studies reporting spiral ganglion cell counts and speech perception scores in patients with cochlear implants. The search strategy used keywords and controlled vocabulary terms . Studies reporting (1) speech perception scores and (2) total spiral ganglion cell counts in human subjects using multichannel cochlear implants were included in this study. In addition to word recognition scores and total SGC counts, available patient demographic information, type of implant, depth of insertion and presumed etiology of deafness was collected.Linear regressions of speech perception scores as a function of number of spiral ganglion cells were performed using Prism 9  for the whole dataset. Then, the process was repeated for two subsets of the data: studies conducted at Massachusetts Eye and Ear Infirmary (MEEI) and studies conducted at other institutes. Significance was evaluated before and after Bonferroni corrections to account for the fact that three tests were run, i.e., the original data set and the two subsets. Lastly, Fisher’s exact test was used to compare the proportion of patients with speech scores above 50% correct among those below or above a certain threshold of spiral ganglion cell count. This was done for two threshold values: 2800 and 3000 spiral ganglion cells.In addition to the univariate analyses listed above, several multivariate analyses were conducted to see if a relation between speech perception scores and number of spiral ganglion cells was present when including other independent variables in the analysis. In total, six multivariate linear regressions were conducted with the corresponding independent variables being: number of spiral ganglion cells (#SGC) and age at implantation; #SGC and age at death; #SGC and duration of deafness; #SGC and duration of implantation; #SGC, age at implantation, and age at death; #SGC and age at implantation, age at death, and duration of deafness.A total of 13 studies were found to contain relevant data ,24,25,262 = 0.084, p = 0.030), but this became insignificant after Bonferroni correction or after removing the data points with shallow or unknown insertion lengths.p = 0.0463). However, the significant result disappeared (p = 0.476) when a threshold of 3000 rather than 2800 SGCs was used.Post-hoc examination of the whole data set showed that 32% (19 out of 60) of the subjects who had SGC counts above 2800 scored above 50% correct in the word identification test, while none of the nine subjects with SGC counts below 2800 did. Fisher’s exact test comparing these two proportions (32% and 0%) yielded a significant result  yielded no compelling evidence of a systematic relationship between SGC count and speech perception scores. Some post-hoc analyses did result in statistically significant findings, but these disappeared after correcting for multiple comparisons or making slight changes in analysis parameters. An interpretation of our results is that the hypothesized relationship between SGC count does not exist. However, this would be a surprising result because spiral ganglion cells are the only known neurons that are stimulated by set word identification scores to approach zero.One possible explanation for the absence of correlation in our analysis is that a small number of SGCs may be sufficient to deliver the information conveyed via electrical stimulation by a cochlear implant, and speech perception is only affected at very low SGC counts. This would be consistent with the observation that the nine patients with an SGC count below 2800 failed to score above 50% words correct. However, this observation is completely exploratory, based on a post-hoc analysis, and it is highly dependent on the specific SGC count threshold that is chosen for the analysis. This result illustrates the point that one should be wary of post-hoc analyses that were conducted after having seen the data, instead of being determined in advance and pre-registered. Nonetheless, and consistent with this explanation, Otte, Schuknecht and Kerr looked at 41 human temporal bones and established that speech discrimination (with acoustic hearing) required at least 10,000 SGCs ; Blamey,Other possible explanations for the lack of correlation between SGCs and speech perception scores have been discussed in the studies included in our meta-analysis. These include varied etiology of hearing loss within the study population, and widely ranging intervals between time of last audiology test and death. Cognitive function at the time of testing is also not known, and it could have an impact on speech perception scores. Other known factors such as duration of bilateral deafness and compliance with using the CI every day are not taken into account either.To control for confounding factors, Seyyedi et al. examined within-subject correlations in bilateral CI users by comparing inter-aural differences in SGC counts to inter-aural differences in word identification scores for six patients . A weak After examining all available published data, the present meta-analysis did not find a significant correlation between SGC counts and word recognition scores in CI users. This result is consistent with the possibility that a relatively small number of SGCs is sufficient to understand speech with a cochlear implant, and therefore the correlation in question does not exist for patients with SGC counts between 1000 and 25000 (the range found in this study). The absence of a correlation is also consistent with the possibility that any influence of SGC count on speech perception by CI users is obscured by other factors that are more strongly associated with speech perception outcomes.


Lower extremity arterial occlusive disease (AOD) results in significant morbidity and mortality for the population, with up to 10% of patients ultimately requiring amputation. An alternative method for non-surgical revascularization which is yet to be fully understood is the optimization of the body's own natural collateral arterial network in a process known as arteriogenesis. Under conditions of conductance vessel stenosis or occlusion resulting in increased flow, shear forces, and pressure gradients within collaterals, positive remodeling occurs to increase the diameter and capacity of these vessels. The creation of a distal arteriovenous fistula (AVF) will drive increased arteriogenesis as compared to collateral formation with the occlusion of a conductance vessel alone by further increasing flow through these arterioles, demonstrating the capacity for arteriogenesis to form larger, more efficient collaterals beyond what is spontaneously achieved after arterial occlusion. Arteries rely on an extracellular matrix (ECM) composed of elastic fibers and collagens that provide stability under hemodynamic stress, and ECM remodeling is necessary to allow for increased diameter and flow conductance in mature arterial structures. When positive remodeling occurs, digestion of lamella and the internal elastic lamina (IEL) by matrix metalloproteinases (MMPs) and other elastases results in the rearrangement and thinning of elastic structures and may be replaced with disordered elastin synthesis without recovery of elastic function. This results in transmission of wall strain to collagen and potential for aneurysmal degeneration along collateral networks, as is seen in the pancreaticoduodenal artery (PDA) after celiac occlusion and inferior mesenteric artery (IMA) with concurrent celiac and superior mesenteric artery (SMA) occlusions. Further understanding into the development of collaterals is required to both better understand aneurysmal degeneration and optimize collateral formation in AOD. The incidence of lower extremity arterial occlusive disease (AOD) has continued to increase over the past several decades resulting in significant morbidity and mortality for the population. Symptoms progress slowly after onset, however between 5 and 10 years after diagnosis 20–30% of patients will experience progressive symptoms requiring intervention with up to 10% requiring amputation  is primarily composed of elastic fibers and various collagens and is necessary to provide structural stability to the vessel under conditions of hemodynamic stress. Elastin stores energy and distributes stress along the vessel wall while collagen, which is primarily located in the adventitia, prevents over distension of the vessel and provides a resilient framework, though it is unable to store energy . In the Elastin fibers consist of a heavily crosslinked, dense elastin protein core surrounded by peripherally oriented proteins and proteoglycans. These fibers have an estimated half-life approaching the human lifespan and are created predominantly in neonatal and early postnatal life with very little new elastin made during adulthood , 29. TheElastic fibers are present throughout the arterial wall but are most prominently featured in the dense sheets separating rows of resident VSMCs known as lamellae. These lamellae are present within the tunica media. Between the tunica intima and media, a prominent lamina underpins the endothelium and is better known as the internal elastic lamina (IEL). These elastic lamellae contain fenestrations which vary in size and frequency depending on the arterial branch order, and allow for cellular communication, diffusion, and molecular transport –39. MoreIn small arteries Type IV collagen forms the basement membrane while type I collagen bundles are abundant in the adventitia. Collagen fibers have a similar wavy appearance in all vessels. This baseline variability in ECM structure is significant in that it may have clinical consequences regarding remodeling capacity of these vessels. Alternatively, collagen fibers have half-lives as low as 2 weeks under experimental conditions of hypertension, and likely must be continually synthesized and replaced , 43.As arterial occlusive disease progresses, flow patterns automatically adapt as blood flow follows the path of least resistance. This may result in large flow rate increases among inter-territorial connections. The endothelial cells are influenced by the resulting shear stress elevations imparted by this increased flow and may become activated , 44. ArtAssuming laminar conditions, the primary forces acting on the vascular wall are fluid shear stress (FSS) and circumferential wall stress (CWS). FSS is experienced as the frictional force of blood exerted against the vascular wall, specifically the endothelium, which is believed to act as the primary modulator of this input . The forIncreased volume flow through a vessel has been shown to induce outward remodeling and diameter growth –53. As lLoss of a conductance artery necessarily contributes to altered pathways for blood flow due to shifting in pressure gradients. Pre-existing collateral connections between territories separated by a conductance artery occlusion will be subjected to increased flow and shear stress. The endothelium within these collateral pathways detects the shear alterations through mechanotransduction cascades that are complex and incompletely understood. The endothelial glycocalyx has been shown to be important in endothelial mechanotransduction and its absence leads to diminished arteriogenesis , 57. NucNitric oxide has been implicated in arteriogenesis, but its role is complex. Endothelia respond to increased shear stress with increased endothelial nitric oxide synthase (eNOS) expression and subsequent production of NO, a potent vasodilator , 69. PurAlthough short-lived vasoactive signals may produce immediate vasoconstriction or vasodilation mediated by VSMC shortening or lengthening, cessation of the signal results in return to baseline vessel diameter. Sustained signal, however, produces additional adaptation of resident VSMCs through “length autoregulation,” reorienting cell-cell and cell-ECM adhesion and thus increasing or decreasing VSMC overlap . Such ad2 purinergic receptor  and intercellular adhesion molecule 1 (ICAM-1). Adhesion molecule expression recruits circulating leukocytes to the developing collateral artery by promoting adherence and transmigration into the developing vessel wall –81. Inhireceptor –86, and receptor .Shear stress induced endothelial activation also promotes the release of several cytokines including monocyte chemoattractant protein-1 (MCP-1), TNF-α, and granulocyte macrophage colony stimulating factor (GM-CSF) which importantly attract monocytes , 88, 89.Adhesion molecule expression by activated endothelium and local cytokine production recruits a broad population of inflammatory cells to participate in arteriogenesis. Neutrophils appear to be recruited first, and produce cytokines such as midkine, which has been suggested to mediate VEGF release , 92. We Additionally, administration of lipopolysaccharide (LPS) stimulates TNF-a and is also capable of increasing perivascular concentrations of monocytes/macrophages leading to collateral growth and development . Once acCollateral arterial growth occurs with the proliferation of resident tissue cells, which can expand the vessel mass nearly 25 times the original . ActivatAs VSMC populations migrate and expand, a neointima forms in the developing collateral artery, appearing as early as 3 days following occlusion . FormatiDiameter increase along with ECM remodeling requires the rearrangement of elastic tissue in the internal elastic lamina. Ultimately, elastolysis allows for vessel diameter enlargement, and ECM remodeling is necessary to allow for increased diameter and flow conductance in mature arterial structures . In the The damage to the elastic fibers during arteriogenesis must be limited in order to maintain fiber integrity and prevent loss of IEL continuity. Tissue inhibitor of metalloproteinases (TIMP1) and plasminogen activator inhibitor 1 (PAI-1) also play vital roles in collateral development by inhibiting MMP function and preventing excessive breakdown of ECM . It woulCross linking of elastin and collagen fibers is mediated via the enzyme lysyl oxidase (LOX) and is essential to maintaining the integrity of the ECM. LOX expression is increased in remodeling of FAL + AVF arterioles and we found that inhibiting LOX [using β-aminopropionitrile (BAPN)] resulted in rapid fragmentation and loss of continuity of the IEL from collateral arteries. This suggests the role of LOX in the repair and stabilization of proteolytically digested elastic fibers during arteriole remodeling. This suggests the proteolytic balance between breakdown and repair during remodeling, possibly related to cross linking of newly synthesized tropoelastin monomers in the later stages of arteriogenesis –34. NotaLoss of elastic laminar definition is also a consistent histologic feature of arterial aneurysm development. In some cases, the development of specific types of arterial aneurysms have been linked to flow-mediated remodeling of the IEL, sometimes occurring within collateral arterial networks. For instance, aneurysm degeneration has been observed in mesenteric arterial collaterals in response to isolated celiac occlusion [resulting in pancreaticoduodenal arterial aneurysms –123] or In the case of cerebral aneurysm pathogenesis, focally increased fluid shear stress provides the local impetus for IEL degradation and enlargement of fenestrations –134. It in situ thrombosis, or even arterial transection in trauma. Evidence of collateral artery formation may be found in all instances.The use of arterial ligation in animal models of arteriogenesis typically creates an acute ischemia and is an important limitation regarding extrapolating animal models to human disease which tends to develop chronically. Numerous studies in larger animals, however, have employed surgically placed ameroid constrictors as a method to simulate more gradual arterial occlusion. Human arterial occlusive disease is variable, with (perhaps most commonly) a slowly worsening stenosis in the case of chronic atherosclerosis, but also via sudden arterial occlusion with thromboembolism or Interventions are currently being directed toward improving arteriogenesis as well as angiogenesis and are under clinical investigation with the hope that these will lead to more effective and non-surgical therapies for AOD. Unfortunately, however, current knowledge of arteriogenesis is limited, and methods to enhance the inflammation and positive remodeling of collateral arteries through growth factor or cytokine supplementation are known to have opposing effects by exacerbating atherosclerosis . DevelopArteriogenesis is a complex mechanism for collateral arterial pathways to develop quickly into larger and higher capacity vessels capable of effectively perfusing tissues distal to a conductance vessel occlusion . IncreasConceptualization and design of the study were performed by RM and RK. Original draft written by RK. RM and EA wrote sections of the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by a grant from the Department of Veterans Affairs (IK-2BX003509) and in part by the Vascular Cures Foundation.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Voice modulatory cues such as variations in fundamental frequency, duration and pauses are key factors for structuring vocal signals in human speech and vocal communication in other tetrapods. Voice modulation physiology is highly similar in humans and other tetrapods due to shared ancestry and shared functional pressures for efficient communication. This has led to similarly structured vocalizations across humans and other tetrapods. Nonetheless, in their details, structural characteristics may vary across species and languages. Because data concerning voice modulation in non-human tetrapod vocal production and especially perception are relatively scarce compared to human vocal production and perception, this review focuses on voice modulatory cues used for speech segmentation across human languages, highlighting comparative data where available. Cues that are used similarly across many languages may help indicate which cues may result from physiological or basic cognitive constraints, and which cues may be employed more flexibly and are shaped by cultural evolution. This suggests promising candidates for future investigation of cues to structure in non-human tetrapod vocalizations.This article is part of the theme issue ‘Voice modulation: from origin and mechanism to social impact (Part I)’. These include both the vocal apparatus itself and the main voice modulatory cues involved in vocal production.Voice modulatory cues that are shared and have similar functions in human and non-human tetrapod vocalizations as well as cross-linguistically can be hypothesized to result from anatomical, physiological and cognitive mechanisms that are evolutionarily conserved –6. TheseBecause this is a very large research program, our review will cover only some specific aspects of voice modulation. In the first section, we compare different voice modulatory cues across human speech and tetrapod communication, including pauses, fundamental frequency and syllable/unit duration. We discuss similarities and differences in the physiological mechanisms underlying these cues, and then discuss how the effort of producing and perceiving them may be linked to functional pressures in the environment. In the second part of the review, we take a comparative approach across languages, comparing whether different voice modulatory cues used for speech segmentation are similar between or differ among various human languages. Especially regarding the many voice modulatory cues for which animal data remain scarce, comparisons between different human languages may provide valuable insights as to whether the physiological and cognitive mechanisms behind those cues are species-typical , or more flexible language-specific. Finally, our review will identify research gaps and suggest avenues for further work that may help more clearly reveal the underlying physiological and cognitive mechanisms underlying the realization of different voice modulatory cues.Overall, our comparison between voice modulatory cues in tetrapod vocalizations and across various human languages will show that biological evolution can constrain cultural evolution, and that many of the structures and cues widely used in human speech rely upon basic acoustic and cognitive mechanisms that humans share with other tetrapods.. 2source generates acoustic energy using an airflow from the lungs. This source is the larynx in most tetrapods and the syrinx in birds, and consists of vibrating tissue that creates sound by oscillating at a particular rate termed the fundamental frequency (f0 hereafter). This source signal is then filtered in the supralaryngeal vocal tract (upper respiratory tract) via multiple formant frequencies that act as a series of bandpass filters, attenuating or enhancing certain frequency ranges. The actual vocal output fuses these two components (source and filter), which are mostly independent, meaning that f0 can freely vary independent of formants and vice versa. This process, summarized as the source-filter-theory of vocal production, is shared by humans and most other tetrapods )Since deceleration before pauses occurs across various human movements  and fina. 9In multiple speech segmentation experiments, similar pitch modifications led to different segmentation patterns in speakers of different native languages ,78. For production, stressed syllables are characterized by a co-occurrence of higher pitch and longer duration, and interestingly, cross-linguistically, duration seems to be a more consistent marker of word stress than pitch , and can work together with such statistical information or in some cases override it. Thus, although such statistical cues are important , they obscure the importance of voice modulation as a key factor in structuring animal communication signals.Summarizing, our review of human speech modulation shows that How language- or species-specific and cross-linguistic and cross-species cues interact certainly warrants further research. In those cases where comparative information is available, it suggests that the cues used to indicate a structure in the speech signal are both present in vocalizations of other species  and also can be used in similar ways . Nonetheless, there is currently far too little comparative data to allow any clear conclusions about the degree to which human-typical cues to structure are also used by other species. More research in this area—what we might term ‘animal phonology’—is needed to evaluate whether there are broad phylogenetic generalizations to be made, as we have hypothesized here. A rich comparative analysis of these issues could be expected to shed light not just on the evolution of communication across vertebrates, but also about the phylogenetic origins of universals in human speech production and perception.


Retirement can be a challenging life transition for mental health. Higher levels of IQ in young adulthood have been shown to be advantageous for different outcomes later in life such as quality of life and well-being. However, it remains unclear whether possessing higher cognitive abilities in early life also favors individuals’ mental health when facing challenges related to the retirement transition. In this study, we therefore investigated the relationship between IQ in young adulthood and depressive symptoms over the retirement transition. We used data of six waves from the longitudinal population-based HEalth, Aging and Retirement in Sweden (HEARTS) study, as well as data on IQ in young adulthood from conscription. In a piecewise structural equation model, we modelled trajectories of depressive symptoms  before and after retirement and in relation to young adulthood IQ (n = 1722 men). Results indicated an average decrease in depressive symptoms over the retirement transition for this sample of men. Higher childhood IQ was associated with further reduction in post-retirement depressive symptoms while controlling for education, retirement age, and memory ability and cardiovascular health at baseline. Our findings support the conclusion that higher IQ in young adulthood may act as a protective factor for mental health in the retirement transition. Individuals with higher IQ in young adulthood may have acquired coping strategies throughout their life-course, which they can apply when handling challenges related to retiring.


Currently, greenhouses are widely applied for plant growth, and environmental parameters can also be controlled in the modern greenhouse to guarantee the maximum crop yield. In order to optimally control greenhouses’ environmental parameters, one indispensable requirement is to accurately predict crop yields based on given environmental parameter settings. In addition, crop yield forecasting in greenhouses plays an important role in greenhouse farming planning and management, which allows cultivators and farmers to utilize the yield prediction results to make knowledgeable management and financial decisions. It is thus important to accurately predict the crop yield in a greenhouse considering the benefits that can be brought by accurate greenhouse crop yield prediction. In this work, we have developed a new greenhouse crop yield prediction technique, by combining two state-of-the-arts networks for temporal sequence processing—temporal convolutional network (TCN) and recurrent neural network (RNN). Comprehensive evaluations of the proposed algorithm have been made on multiple datasets obtained from multiple real greenhouse sites for tomato growing. Based on a statistical analysis of the root mean square errors (RMSEs) between the predicted and actual crop yields, it is shown that the proposed approach achieves more accurate yield prediction performance than both traditional machine learning methods and other classical deep neural networks. Moreover, the experimental study also shows that the historical yield information is the most important factor for accurately predicting future crop yields. Compared with field growing, currently, greenhouse growing is preferred by many crop growers. Growing crops in the greenhouse can extend their growing season, protect crops against temperature and weather changes and thus provide a safe growing environment. Moreover, environmental parameters . ItIn this work, we propose a deep neural network-based greenhouse crop yield prediction method, by combining two state-of-the-art networks for temporal sequence processing: recurrent neural network (RNN) and temporal convolutional network (TCN). The proposed deep neural network is developed for predicting future crop yields in a greenhouse based on a sequence of historical greenhouse input parameters  as well as yield information. According to the experimental evaluations of multiple datasets collected from multiple greenhouses in different time periods, it is shown that the RNN+TCN-based deep learning approach achieves more accurate yield prediction results with smaller root mean square errors (RMSEs), compared with both classical machine learning and other popular deep learning-based counterparts.Although there is much research related to crop yield prediction for the farming field, a relatively small amount of works focus on greenhouse crop yield forecasting. Approaches that have been developed for greenhouse crop yield forecasting are divided into two main categories: the explanatory biophysical model-based approach and the data driven/machine learning model-based approach.Explanatory biophysical model-based approach: Based on a series of ordinary differential equations (ODEs) reflecting a dynamic process, the explanatory model describes the relationship between some environmental factors and crop growth or morphological development. Different biophysical models have been applied for crop growth modelling which can thus be used for yield forecasting, based on greenhouse environmental parameters.The Tomgro model is proposed by Jones et al. in , which mposed in , which cposed in . This yion Model , which ion Model  and Vanton Model , is applon Model , Tomsim on Model , Greenhoon Model  and theion Model . The expon Model ,12 descr(i)There are many intrinsic model parameters associated with a biophysical model and the performance of an explanatory model is highly sensitive to its model parameters (as shown in ). Moreov(ii)In addition, most biophysical models are also not universal and restricted to model the growth for a specific type of plant. For example, the Tomgro model  and TomsThe explanatory model is practical to reflect the actual growth process of crops, which is bio-physically meaningful and explainable. However, the aforementioned explanatory models suffer from the following two main limitations:Due to the limitations of the explanatory biophysical model-based approaches, in this work, we refer to another category of approach–machine learning model-based approach for greenhouse crop yield prediction. More details of the machine learning model-based approach are introduced as follows.Machine learning model-based approach: Data driven/machine learning technique-based approaches have also been applied for greenhouse crop yield forecasting in many studies, which treat the crop yield output as a very complex and nonlinear function of the greenhouse environmental variables and historical crop yield information. In particular, linear and polynomial regression models are used in  for strak (DANN)  was implks as in ,17,18,19(i)Features extracted from data for building the classical machine learning models may not be optimal and most representative, thus deteriorating the performance for yield prediction .(ii)The classical machine learning models cannot effectively handle data with either high volume or high complexity.The aforementioned works focus on using classical machine learning approaches for greenhouse crop yield prediction. Given a certain amount of training data, classical machine learning models  are constructed to predict greenhouse crop yields based on certain factors . However, these works suffer from limitations due to the adoptions of simple and ‘shallow’ classical machine learning models, for example:Deep learning is a very popular machine learning technique and it has been successfully applied in a variety of applications  . RecentlIn this work, a novel deep neural network (DNN)-based methodology is proposed, to predict the future crop yield based on historical yields and greenhouse environmental parameters  component used in this work, as proposed in , applieslization  can be pAn additional 1D convolution operation is performed within each residual block, to adjust the dimension of the residual block input to be the same as that of the dilated casual convolution layer output to add them together. The output results obtained from one residual block are fed as the input of the next block and the final output is obtained from the last residual block. The final output of the last residual block from the TCN is then flattened and fed into a fully connected (FC) layer to output the final yield prediction result.The output of the TCN part is flattened and fed into a vector, which is then fed into a fully connected layer for the final yield prediction, as shown in In a summary, the proposed work investigates the combination of two state-of-the-arts deep neural networks for temporal sequence processing: LSTM–RNN and TCN, for greenhouse crop yield prediction. Based on an input temporal sequence containing both historical yields and environmental parameters information during a certain period, firstly, an LSTM–RNN is applied for pre-processing the original input to extract representative feature sequences, which are then further processed by a sequential of residual blocks in the TCN to generate the final features used for the future yield prediction. Compared with other deep learning methodology for greenhouse crop yield prediction by solely exploiting the LSTM–RNN as in , in our The experimental studies of the proposed DNN based crop yield prediction approach are presented in this section.Three datasets are collected from a tomato-growing site in Newcastle, UK, which contain recorded environmental parameters and crop yield information in different greenhouses during different time periods. The details of these datasets are described in the following As an illustration, the daily recorded environmental parameters  is applied to generate data samples, which contain recorded environmental parameters and yield information during one week as well as the associated future crop yield after one week. These generated data samples are exploited to train/test a network, for predicting the crop yield after one week given the collected environmental and yield information during the last week. Generated data samples are split into training and testing datasets with a proportion of 70% versus 30%. The training dataset is applied to train the network, which is then tested against another testing dataset for evaluating the performance of the trained network. Adam’s method in  is appliFurthermore, we use the root mean square error , random forest (RF), support vector regression (SVR), decision tree (DT), gradient boosting regression (GBR), multi-layers artificial neural network (MLANN)) as well as other deep learning-based ones (single/multiple layer(s) LSTM–RNN , LSTM–RN(i)The proposed approach can be applied for accurate greenhouse crop yield prediction, based on both historical environmental and yield information.(ii)The proposed approach can achieve much more accurate prediction than other counterparts of both traditional machine learning and deep learning methods.In this work, we have proposed a new methodology for greenhouse crop yield prediction, by integrating two state-of-the-arts DNN network architectures used for temporal sequence processing: RNN and TCN. Given an input temporal sequence containing historical yield and environmental information, firstly, an LSTM—RNN is applied for pre-processing the original inputs to extract representative features, which are then further processed by a sequential of residual blocks of the TCN module. The features finally extracted from the TCN are then fed into a fully connected network for future crop yield prediction. Comprehensive evaluations through statistical analysis of obtained RMSEs for multiple datasets have shown that:Furthermore, it is also shown in the experimental study that the historical yield information is the most important factor for accurately predicting future crop yields.With respect to future work, to further validate the general effectiveness of the proposed model, we will evaluate it on more datasets collected from different growers on different sites. Moreover, we will also test the model’s performance on yield prediction for different types of popular greenhouse crops. More advanced network architecture will also be considered, for example, the LSTM encoder–decoder component as in  will be


Human pluripotent stem cells (hPSCs), embryonic or induced, can be differentiated into large quantities of specialized cells in order to study development and model disease. These hPSC-derived iBMECs display endothelial-like properties, such as tube formation and low-density lipoprotein uptake, high transendothelial electrical resistance (TEER), and barrier-like efflux transporter activities. Over time, the de novo generation of an organotypic endothelial cell from hPSCs has aroused controversies. This perspective article highlights the developments made in the field of hPSC derived brain endothelial cells as well as where experimental data are lacking, and what concerns have emerged since their initial description.Brain microvascular endothelial cells (BMECs) possess unique properties that are crucial for many functions of the blood-brain-barrier (BBB) including maintenance of brain homeostasis and regulation of interactions between the brain and immune system. The generation of a pure population of putative brain microvascular endothelial cells from human pluripotent stem cell sources (iBMECs) has been described to meet the need for reliable and reproducible brain endothelial cells  Specifically, it has been reported that hPSC-derived iBMECs display EC-like properties including tube formation, low density lipoprotein uptake, high transendothelial electrical resistance (TEER), and select barrier-like transporter activity. Over time, this de novo generation of an organotypic endothelial cell from hPSCs has aroused controversies. This perspective article highlights the developments made in the field of hPSC derived brain endothelial cells as well as where experimental data are lacking, and what concerns have emerged since their initial description.The latest efforts to develop drugs targeting neurodegeneration and neurological disorders have been met with disappointment in recent clinical trials. The relative ineffectiveness of those drugs has incited the scientific community to develop better pre-clinical models by improving human cell-based models to capture the complexity of the brain. While the discovery of induced pluripotent stem cells and subsequent generation of brain organoids has advanced innovative avenues, these brain organoids are still rudimentary, lacking primordial non-neuronal cell types of the central nervous system (CNS) like microglia and most importantly functional blood vessels. During embryonic development, endothelial cells (ECs) acquire unique organ-specific molecular and cellular specializations that are crucial for the formation of the blood-brain-barrier (BBB) and therefore the maintenance of brain homeostasis. Human pluripotent stem cells (hPSCs), embryonic or induced, have been used in an effort to generate large quantities of specialized cells for development studies and disease modeling. The use of hPSCs to generate a pure population of these specialized brain microvascular ECs (iBMECs) has been described to meet the need for a reliable and reproducible Brain endothelial cells plays an essential role in the development of a multicellular vascular structure separating the central nervous system (CNS) from the peripheral blood circulation . In mammInitial signs for CNS angiogenesis and induction of BBB traits are given by the neural microenvironment on embryonic day E10 in mice . EndotheThis primitive BBB further mature by recruiting pericytes to the developing blood vessels. This step is critical to ensure proper BBB formation and function . A recenThe major endothelial transport systems, ions channels and GPCRs are described in detail elsewhere . The unitrans-endothelial electrical resistance (TEER) in vivo and reduced caveolar-mediated transport, along with the presence of selective transporters. Due to their significant structural and functional overlap, most of the current understanding of endothelial cell TJs has been derived from examination of their epithelial counterparts   known as brain microvascular endothelial cells (BMEC) that possess intercellular tight junctions (TJs), lack fenestrations, and greatly limit transcytosis. BMECs, acting in conjunction with various neural cell types and non-cellular elements, form the BBB which regulates the dynamic transfer of select molecules into and out of the CNS . These p ECV304) . Howeverin vivo . The Claudin family is comprised of at least 24 member proteins which contain two extracellular loops responsible for homophilic interaction as well as establishing a link with claudins of contiguous endothelial cells. This homophilic interaction forms the primary seal of the TJ in vivo  with Clain vivo , it has in vivo . Likewisin vivo , others in vivo . The gen–/– mice . Of note–/– mice . Thus, tAnother barrier property of the BBB lies in their ability to restrict immune cell infiltration to the CNS as BMECs generally has a low expression of leucocyte adhesion molecules during homeostatic conditions . Immune in vivo study in mice showed that E-selectin deficiency exert a neuroprotective effect characterized by reduced inflammation and neuronal apoptosis    but offe2 in some studies , and barrier-like efflux transporter activities  and marker genes was observed in both RNA sequencing platforms, concerns over the validity of the previously established protein expression data becomes apparent. iBMECs used in this study were shown to be transcriptionally identical to those previously published which mean that positive protein expression data could be due to non-specific binding by monoclonal antibodies, especially in the case of proteins from large homologous families such as Claudins ; however, some of these statistically significant differences denoted changes in FPKM values of less than 1  stipulating an overall lack of function difference in gene expression. Interestingly, differences in FPKM values of greater than 20 occurs exclusively in genes more closely related to an epithelial lineage, reinforcing the notion of an epithelial phenotype in iBMECs. Differences in FPKM of some marker genes were also shown to be not significantly different between samples even though the Row Z-score demonstrates a large difference in expression. Moreover, the average FPKM of some of these marker genes across all samples was under 0.1 indicating that the genes are barely expressed and unlikely to undergo any translation  infection of the brain. While characterizing the response of iBMECs to inflammatory factors brought on by Nm infection, the group notes no difference in VCAM-1 or E-Selectin RNA expression which shows a lack of an EC specific response to inflammatory stimuli. The lack of a functional vascular endothelial identity in iBMECs is further reinforced in a study by ETV2, ERG, and FLI1). These reprogrammed cells (rECs) harbor an EC transcriptomic profile, retaining a PECAM1+CDH5+KDR+ EC immunophenotype during passaging and expansion. Purified rECs can respond to inflammatory stimuli  and permeabilizing agents  in a manner congruent with vascular ECs. rECs were also shown to be capable of forming tubes in vivo using an immunocompromised mouse model, whereas iBMECs derived from the same hPSCs could not. This strategy of transcription factor reprogramming establishes a vascular EC identity in cells that otherwise lacked any phenotypic and functional aspects of bona fide ECs. However, further work is required to generate a reliable brain specific EC and that this only represents a crucial step toward the generation of true brain ECs suitable for in vitro modeling of physiological and pharmaceutical studies of the BBB.Before a functional hPSC-derived vascular BBB model can be developed, stable hPSC-derived vascular ECs must be generated. In a recent study , our groin vitro. Some groups were able to show that neural cell conditioned media could increase barrier resistance and decrease permeability in BMEC monolayers (in vitro BBB model.In addition to the co-culture systems referenced above, numerous culture conditions have been demonstrated to improve BBB phenotypes nolayers . Others SOX18, TAL1, SOX7, and ETS1 can enhance certain properties in EC such as barrier function. Their work shows that hPSC-derived ECs transduced with these TFs have increased transmembrane electrical resistance and tight junction protein expression while also decreasing paracellular transport (in vitro BBB models.Moving on from culture conditions, intrinsic transcriptional regulation may also be critical for the establishment of a vascular BBB model. A separate ransport . Taken tin vitro models attempting to mimic the physiological complexity of the BBB in vivo. Many difficulties have arisen during the course of these efforts as such models must phenocopy the high TEER observed in vivo as well as the intricate cellular transport mechanisms that are hallmarks of the BBB. It has been demonstrated that BBB traits are not intrinsic to brain specific ECs, but rather the result of a dynamic interplay with their microenvironment including multiple cell types such as astrocytes and pericytes. Consequently, primary brain ECs lose their barrier properties, especially high TEER, when cultured in vitro. Many laboratories have attempted to resolve this issue by developing various in vitro BBB models using neural cell co-cultures consisting of ECs, pericytes, and astrocytes. These models also include pluripotent stem cell differentiation methods as well as brain organoids and ‘organ-on-a-chip’ approaches.Over the past decade many groups have aimed to advance the study of the BBB by developing in vitro models relies mainly on using TEER and expression of tight junction proteins as a determinant of barrier function. Using these measurements as a proxy for functional BBB-specific tight junctions presents some limitations since it can only measure the paracellular junctions. The BBB has many transcellular permeability functions that are imperative for its function which cannot be measured in this way. Additionally, high TEER and many of the junctional proteins used to validate brain EC identity have been demonstrated in other non-endothelial cell types such as epithelial cells. ECs have also been shown to possess a polarized morphology (in vitro vascular barrier models. Other problems may also have arisen from assigning a vascular EC identity to hPSC-derived cells based on the expression of a restricted set of brain EC markers. False positive results of EC identity can occur in these cases due to antibody cross-reactivity with proteins present in the cell sample that are not specific to ECs. As shown, iBMECs may demonstrate a high TEER in vitro and express certain non-vascular specific junctional genes, however, they lack many functional phenotypes intrinsic to ECs. By not responding to inflammatory stimuli in an EC specific manner, their use in many models would yield results misrepresenting the in vivo BBB (Validation of these rphology  similar vivo BBB .PECAM1, CDH5, CLDN5, and VWF while also expressing epithelial cell genes including EPCAM, FREM2, and CLDN4. Expression of genes such as E-Selectin, VCAM-1, P-Selectin were also shown to be completely unaffected by inflammatory stimuli further decreasing the possibility for these cells to be used as a functional model of the BBB in vitro. This leads to the possibility that the barrier function observed in iBMECs could in fact more closely resemble an epithelial cell barrier such as the choroid plexus or intestinal epithelial barrier. As these cells lack a canonical vascular EC phenotype, the use of current protocols to generate iBMECs as prototypical human BBB model could results in inaccurate physiological studies and screening for misguided druggable targets or treatments with potential ineffective clinical outcomes. Thus, the application of a rigorous and thorough characterization of stem cell-derived products using the latest available technologies such as single cell multi-omics and metabolomics should be necessary, rather than facultative, for the development of faithful disease models and safe cell-based therapies.Taken altogether, the data presented by recent studies  contradiThe original contributions generated for this study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Panthera leo) presented with two ulcerative lesions on the nasal planum. Histopathology of the lesions revealed epidermal keratinocyte dysplasia and neoplastic basal- and supra-basal epithelial cells with dyskeratosis and evidence of basement membrane breaching and dermal invasion, consistent with a diagnosis of SCC. There was also evidence of laminar fibrosis and inflammation of the subjacent dermis suggesting that the SCC most likely resulted from UV-induced neoplastic transformation of the epidermal squamous epithelium following actinic keratosis. The lion was treated with hypofractionated radiation therapy and remained in remission until his death . This is the first report of cutaneous SCC in a lion with evidence of actinic damage and resolution after radiation therapy.Cutaneous squamous cell carcinoma (SCC) is a slow growing but locally invasive neoplasm, most commonly caused by prolonged exposure to ultraviolet (UV) radiation. Whilst SCC accounts for 15% of skin tumours in domesticated cats, cutaneous SCC in non-domesticated felids (apart from captive snow leopards) appears to be uncommon, with only three reports in the literature to date. In this report, a captive African lion ( Squamous cell carcinoma (SCC) is a malignant neoplasm arising from squamous epithelium. Squamous cell carcinomas account for 15% of skin tumours in domestic cats, with most feline cutaneous SCCs occurring on the head, often involving the pinna, eyelid and nasal planum   [De Oliveira et al. Panthera tigris) who had SCC on the left rear limb underwent surgery to remove the mass; however, recurrence was documented 2 years later  had severe swelling and draining sinus tracts on the front left paw and the lesions were unresponsive to 7 months of treatment with various antibiotic regimes such that her deteriorating condition resulted in humane euthanasia. At postmortem, SCC was confirmed, presenting as discharging sinuses lined by neoplastic squamous cells  had a swelling and an abscess on the right hind paw and biopsy revealed a well-differentiated SCC. Attempts at surgical excision and cryosurgery proved unsuccessful and subsequent recurrence of the mass led to a mid-femoral amputation , a common presentation in large captive carnivores in South Africa  and Zoletil  to allow samples to be taken for further analysis. Blood was collected from the femoral vein for routine haematology and clinical biochemistry analysis. The blood sample revealed all parameters to be within normal ranges . Four puPanthera tigris), which succumbed to severe metastatic disease 4 months later . Three months later, his condition deteriorated such that euthanasia was required. Histopathological findings at autopsy suggested chronic renal failure, which was most likely age-related. There were no neoplastic lesions observed in any of the organs examined .Older domestic cats are at greater risk of developing SCC, with the average age at presentation being 10–12 years Hauck . FactorsAlthough a range of different treatments have been used in domestic cats with nasal planum SCC such as strontium (Sr90) plesiotherapy (Berlato et al. In conclusion, cutaneous SCC, although rarely reported in non-domesticated felids, should be considered as a differential diagnosis for a long-term, non-healing skin wound in these animals and can be successfully treated with radiation therapy. It is hoped that further investigations of African lions, both in wildlife reserves and zoological facilities, will add to a better knowledge and understanding of cutaneous tumours in this species.


Abundant empirical evidence suggests that visual perception and motor responses are involved in language comprehension (‘grounding’). However, when modeling the grounding of sentence comprehension on a word-by-word basis, linguistic representations and cognitive processes are rarely made fully explicit. This article reviews representational formalisms and associated  models with a view to accommodating incremental and compositional grounding effects. Are different representation formats equally suitable and what mechanisms and representations do models assume to accommodate grounding effects? I argue that we must minimally specify compositional semantic representations, a set of incremental processes/mechanisms, and an explicit link from the assumed processes to measured behavior. Different representational formats can be contrasted in psycholinguistic modeling by holding the set of processes/mechanisms constant; contrasting different processes/mechanisms is possible by holding representations constant. Such psycholinguistic modeling could be applied across a wide range of experimental investigations and complement computational modeling. Over the past three decades, many areas of cognitive science have taken up the idea that cognitive representations are ‘grounded’ or ‘embodied’ via perception and action. Among these are animal cognition , neuroscHowever, “a close look at the literature reveals that the debate about the nature of the processes involved in language comprehension is far from settled, and key questions remain unanswered” . It has Regarding compositionality, effects of grounding are at least partially sensitive to constituent order and its semantic interpretation. This sensitivity suggests that in accommodating effects of grounding, we must pay attention to representations beyond the lexical level and their incremental interaction. In the present article I focus on using symbolic representational formalisms see  to accomExtant approaches do not yet offer a detailed description of the incremental grounding processes and compositional representations implicated in recovering the interpretation of a sentence (see Appendix A1 for details). But such a description is warranted: Psycholinguistic evidence suggests that grounding effects implicate compositional representations, and that action-based representations interact fairly incrementally and compositionally with visual attention and comprehension. Below, I review two studies in support of this view.The student will stagger along the trail to the picnic basket, staggering implies slow motion. By contrast, the same sentence with a different verb like run implies fast motion. To the extent that verb-implied motion affects visual attention and comprehension, participants listening to these utterances should exhibit distinct gaze pattern on a related image. An image showed, for instance, a man, a trail, and a picknick basket. These objects functioned as parts of an event  with the expectation being that verb speed, if re-enacted via eye movements, might influence how comprehenders attend to the goal and the path. Participants in the experiment by Lindsay, Scheepers, and Kamide , participants looked earlier to the goal as they listened to the verb , the rotation mismatch effect was localized on opened and the ensuing adverb. Moreover, the meaning of the adverb mattered. When a postverbal adverb kept the focus on the action , congruence effects in reading times lasted into the adverb. But if the adverb shifted focus to the agent , the effect emerged only at the verb, not the ensuing adverb. In addition to showcasing the incrementality of language grounding, these results also emphasize its compositional nature. Taylor and Zwaan  emerged during spoken comprehension and during reading. The locus of the effects was temporally coordinated with – and dependent upon – sentence interpretation beyond lexical-referential processes see also . AccommoGiven the evidence in favor of incremental and compositional grounding, implicating the conceptual level and aspects of meaning that reach into motoric representations/processes, we can assess models of language (processing) against the following benchmarks:Compositionality: How/to what extent are language representations related to visual perception or action beyond the lexical level?What level(s) of linguistic representation are grounded?Are representations implicated in attention and manual responses (and quantifiable linking hypotheses) included?Incremental processes: How/to what extent are compositional grounding effects modeled incrementally?Is the timing of grounding effects made explicit?What mechanisms are assumed?Together these benchmarks help assess to what extent and how the available models ground language processing. Could they, in principle, accommodate the target findings by Lindsay et al. , and Taymoon; features like [+round] can help enrich the noun’s meaning, and propositions capture compositional relations like The rocket races to the moon ], Jackendoff . In Jacckendoff . And indckendoff  assumes ckendoff . The embckendoff ).One distinction in grounding between these cognitive linguistic approaches and other, minimalist grammar accounts is the level of linguistic representations that is linked to representations of visual perception and action . Knott 3run vs. stagger in relation to further event representations.Representations of an immediate visually-perceived environment are thus explicit in the cognitive and minimalist grammar accounts; an explicit model of attention is central in Knott’s minimalist account, and links to visual attention are assumed in cognitive linguistics.Both representations of the non-linguistic environment and compositionality have also been included in models relying on distributed representations: Zeros and ones are used in connectionist networks to code features such as whether a representation stands for a noun (vs. verb), two different word orders, or case marking. This coding permits a computational network to process input representations via some function, and to generate output activation values that can be mapped into symbolic linguistic representations. In the Coordinated Interplay Account Network (CIANet), Mayberry et al.  created 5Jilly is outside, a “1” is assigned if she is outside, while “0” marks not outside. Probabilities resulting from such individual instances can be rendered in a vector and capture the reader’s belief of how likely it is for Jilly (or any other object) to be outside , p. 9) in a micro world and a mini grammar, representing both as vectors. Compositionality in language and world knowledge is captured via compositionality of propositions. Much of the appeal of DSS lies in its quantifiable link between vector representations, propositional representations reflecting states and events in a micro world, and human attention.Representations of attention, and of an immediate visually-perceived environment are, by contrast, not explicit in distributed situation space DSS, . But in  outside . Venhuiz outside  exploit In brief, accounts that achieve linking via a compositional representation at the conceptual/semantic level, including event representations appear to more easily capture the representational grounding required to accommodate the target findings. Not all of the approaches feature an explicit model of attention, and they differ in how directly they map visual attention to syntactic and semantic interpretation. Of the approaches, most seem to model action execution in a third-person view , as well as  attention of the language user, but not his/her manual responses , or later, if this process is assumed to take some time. For accommodating reading-time data, Bryant . Incrementality of grounding language comprehension in relation to attention in a scene is captured by connectionist models , and of attentional guidance (ii) are included in most of the models: In the implemented FUSE model, Roy and Mukharjee , as well as in the CIA, together with co-indexing and reconciliation. Knott  in additIn summary, attention as a mechanism seems key in most accounts and helps in grounding language guided by the unfolding speech and semantic interpretation ; attention is even tightly linked to recursive syntactic structuring ; or it iThis article assessed selected representational formalisms and  models via two benchmarks: grounding compositional language representations (1) and the incrementality of grounding effects, as well as implicated mechanisms (2).Did some representational formalisms or models fare better than others when assessed against the benchmarks in section 2? All captured compositionality of representations in language. But none of the discussed approaches seemed to feature all of the representations required to accommodate the compositional semantic grounding suggested by the target findings. Most approaches lacked representations implicated in a comprehender’s manual response , and some did not feature a representation of the comprehender’s explicit (object-directed) attention . All except one  model seem to assume that grounding occurs via compositional representations at the conceptual, interpreted level of language. At that level, meaning distinctions of action speed and directionality of movement can be captured.These differences in grounding notwithstanding, interesting convergences emerged for linking hypotheses . Bryant  and VenhIncremental grounding of language was modeled by many accounts and functionally most of these included mechanisms of attention and of reconciling language-based representations with representations of situation experience or of an immediate scene. Differences emerged at the level of realization of the mechanisms: attention-mediated co-indexing and reconciliation/verification  versus sBelow I motivate and give an example of a first step towards progress: by making explicit how incrementally grounded compositional linguistic representations are constructed. One might argue that the added specification of linguistic representations cannot contribute in a meaningful way to the  modeling of grounding language. After all, computational models make assumptions about grounding explicit; specifying linguistic representations would on this view have little added benefit. One might also be concerned that adding linguistic representations as another layer would not be fruitful since comparing connectionist with symbolic/linguistic representations is not straightforward e.g., .Why specify grounded linguistic representations incrementally? The present paper takes the stance that the combined specification of different models  offers added value over specifying only one of these. Perhaps linguistic and non-linguistic representational formats are best viewed as complementary levels in linking cognition to behavior  to steps in a computational model. Figure 1 illustrates how the Coordinated Interplay account can be related to – and complemented by – computational modeling (pink font). Specifying linguistic representations and processes could lay the foundation for predicting grounding effects broad coverage , something that would constitute a substantial psycholinguistic contribution to the computational modeling of language grounding. Any skilled (psycho)linguist familiar with a relevant formalism could engage in this process, and if pursued this would lead to broad coverage hypotheses  of grounding effects in language processing .Figure 1 but others could be adopted instead), we could enrich it with linguistic representations from different grammar formalisms (or simplified linguistic representations derived from these), and compare how substituting one representation format with the other affects model predictions. Figure 1 illustrates that the Coordinated Interplay account could be combined with different kinds of symbolic representations . To the extent that replacing one representation format with another does not change the predictions, one could use them in free variation. By contrast, better fit of predicted to observed response for one (vs. another) representational format across a substantial range of studies would lead to adopting that format (or aspects of it). Another possibility for making progress would be to keep the linguistic representations fixed but to contrast different mechanisms within a processing framework .If we adopt a processing framework  specify the grounding of language incrementally via linguistic representations (including of the language user) as we formulate hypotheses and interpret experimental results, complementing computational modeling; (b) contrast the assumed mechanisms and representation formats explicitly .


Through collating observations from various studies and complementing these findings with one author’s study, a detailed overview of the benefits and drawbacks of asynchronous email interviewing is provided. Through this overview, it is evident there is great potential for asynchronous email interviews in the broad field of health, particularly for studies drawing on expertise from participants in academia or professional settings, those across varied geographical settings , and/or in circumstances when face-to-face interactions are not possible (e.g. COVID-19).Benefits of asynchronous email interviewing and additional considerations for researchers are discussed around: (i) access transcending geographic location and during restricted face-to-face communications; (ii) feasibility and cost; (iii) sampling and inclusion of diverse participants; (iv) facilitating snowball sampling and increased transparency; (v) data collection with working professionals; (vi) anonymity; (vii) verification of participants; (viii) data quality and enhanced data accuracy; and (ix) overcoming language barriers. Similarly, potential drawbacks of asynchronous email interviews are also discussed with suggested remedies, which centre around: (i) time; (ii) participant verification and confidentiality; (iii) technology and sampling concerns; (iv) data quality and availability; and (v) need for enhanced clarity and precision. As such, our narrative review focuses on collating an overview of the benefits and drawbacks of asynchronous email interviewing. We also reflected upon the unique insights elicited by one of our author’s prior experiences utilizing asynchronous email interviews. The author conducted a study canvasing public health practitioners’ perspectives on tobacco policy tools, using email to both recruit participants and conduct asynchronous email interviews . These iAsynchronous email interviews enable data collection from beyond the researcher’s physical location. This ability to transcend geography is particularly useful during the COVID-19 pandemic, as asynchronous email interviewing allows continued engagement with research and provides participants with time to collect and articulate their thoughts, while concurrently ensuring the safety of both participants and researchers.Asynchronous email interviews can reduce the time required to conduct a research study while still generating in-depth data . The porFor participants of lower of socioeconomic status, access to strong, unobstructed internet connections can at times be a limitation to participating in synchronous electronic interviews. However, asynchronous email interviewing mitigates issues of internet drop-off and poor connection, by providing participants with the ability to formulate and send responses in spite of connection challenges. Moreover, this method can help prevent the exclusion of participants from lower socioeconomic status due to technological limitations. Even if participants cannot afford an internet connection at home, they can either bring their electronic device to community locations or use public computers (e.g. public libraries).The internet’s global reach also allows researchers to transverse geographic regions when recruiting, resulting in potentially larger sample sizes  and incrSnowball sampling involves one participant referring another potential participant. Asynchronous email interviewing allows the first participant to easily forward detailed requests, and additional participants to freely partake in the study, given the lack of geographic, time, and accessibility barriers typically faced in face-to-face interviews.In the tobacco policy project, participants volunteered to forward the letter of information and consent form to other potential participants. While it is difficult to discern whether this may have also occurred using other qualitative methodologies, we speculate that having the information clearly presented with an option to easily forward at the touch of a button without having to disclose the potential participants’ names to the researcher, made it substantially easier to pass along this information.In the study of public health practitioners, these individuals had professional obligations which likely entails availability constraints. Use of asynchronous email interviews was particularly beneficial for engaging this population, as they were free to respond at a time that was most convenient to them. Moreover, the professionals in the study were contacted due to their role at a particular organization or institution. If participants wanted to ensure they were accurately representing their role or organization, the asynchronous email interview method allowed them to carefully curate and edit their typed responses before submission to the researcher. While this may result in less spontaneous answers that occur during face-to-face interactions, it poses an opportunity to garner information from professionals who may have otherwise not obliged to interview requests due to their institutional affiliations.Allowing individuals to express their responses regarding a sensitive topic in a written form can also take on a therapeutic role  and may In this study of public health practitioners, participants were emailed at their institutional email address, aiding with participant verification .Time-pressure can result in inconsistent responses, spelling errors, and miscommunication. Therefore, by not requiring an instantaneous response, researchers alleviate sentiments of anxiety and stress amongst participants who struggle with expressing their thoughts instantaneously . By giviUsing computer applications to translate participant responses can mitigate traditional language barriers posed by face-to-face interviewing and eliminate the need for human translators and potentially scheduling challenges. Future studies can remedy language barriers posed by asynchronous email interviews by generating letters of information, consent forms, and interview questions in languages that are used by the target participants.If a predetermined window for response submission is not established, participants may take months to reply , 7. MoreEmail interviews require researchers to exhibit a high degree of diligence and carefully vet each participant . ParticiResearchers need to be cautious when constructing emails to avoid forwarding confidential details  or mixinWhile technology has afforded novel interviewing methods, it simultaneously poses challenges. For instance, poor internet connectivity may discourage individuals from participating or delay their responses, and system crashes can result in the elimination of data . Many poFurthermore, research participants may be from higher socioeconomic strata, limiting study generalizability and resulting in sampling bias . SimilarWhile the feeling of being anonymous may yield benefits, as discussed above, it is also important to consider how a feeling of lack of anonymity may result in sampling bias. For example, participants who opt out due to feeling uncomfortable with the asynchronous electronic interview method will result in sampling bias and potentially skew results. It is therefore crucial for the researcher to take actions to mitigate any feelings of lack of anonymity and consider any implications on sampling bias while undertaking recruitment.Asynchronous email interviewing fails to account for non-verbal and paralinguistic cues . While tRespondents may also perceive the lack of face-to-face meetings as researcher laziness and reciprocate with decreased effort in their responses (e.g. brevity), possibly resulting in numerous time-consuming follow-ups . ResearcThe use of emoticons has been suggested as an alternative to visual cues and can convey warmth . While vIn the tobacco policy study, one respondent indicated that “there are concerns and implications due to the regressive nature of tobacco taxation increases” . It was The above findings yield considerations for future studies seeking to employ the underutilized method of asynchronous email interviews, particularly pertinent during COVID-19. We highly advise all researchers who are assessing the utility of this method to carefully consider this compilation of benefits and drawbacks and reflect on the appropriateness for the aim of their study and type of participants being sought. It is important to note that while asynchronous email interviews may be a preferred option for one study, this may not be the case for another study.Through the inherent nature of asynchronous email interviews being able to collate data from across the world, we hope this method will also aid in elevating the voices from the global south and those of women—which is sorely needed in global health leadership —whether While this article collates both benefits and potential drawbacks of asynchronous email interviewing through drawing on various studies and observations from one author’s study, this list is not exhaustive and new considerations may emerge through undertaking this method.