extfieldsofstudy: Medicine Biology

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Discovery of Actinomycetes from Extreme Environments with Potential to Produce Novel Antibiotics

Introduction Antimicrobial-resistant pathogens pose serious challenges to healthcare institutions and health of the public. Thus, there is an urgent need for the discovery of new and effective antimicrobial agents. Microorganisms that exist in extreme environments such as those with high salinity or alkalinity, are known as extremophiles, and include various species of actinomycetes. The goal of this study is to discover novel antibiotics from extremophiles found in Kazakhstan that are effective against drug resistant pathogens. Methods Soil from extreme environments of Kazakhstan was collected, and pure cultures of actinomycetes were isolated and cultured in modified Bennett’s broth with either high concentrations of salt or high pH to mimic extreme environments. Extracts obtained from selected actinomycetes strains were used to test for antimicrobial activity against Staphylococcus aureus, Escherichia coli and Aspergillus niger. Results A total of 5936 strains of extremophile actinomycetes were isolated; from these, 2019 strains were further isolated into pure cultures. Of these, 415 actinomycetes strains that demonstrated antagonistic antibacterial activities were selected. These actinomycetes were further classified into groups and subgroups based on their responses to different culture conditions. Antimicrobial antagonism activity for some of the actinomycetes strains was dependent on culture conditions and development of aerial mycelia under extreme conditions. Conclusion We identified several interesting candidate extracts with putative antimicrobial activities against several strains of drug resistant pathogens. Our research of the actinomycetes’ ability to produce antibiotics in the near-natural conditions provides a great opportunity to assess their biodiversity and distribution in the Central Asian region and to develop new methodological approaches to the screening of new antimicrobial agents.

of scholarly attention and have been harnessed as valuable sources of natural compounds, such as antibiotics, enzymes, and vitamins. More than 90 percent of chemotherapeutic antibiotics have been isolated from actinomycetes [3][4][5] .
The large-scale screening of antagonistic actinomycetes under neutral conditions (with a pH of 7.0 and no addition of salt [NaCl, Na2CO3] to the growth media) has led to the discovery of virtually all modern medical antibiotics 1,2 . During screening, the strains of actinomycetes that did not show activity in neutral environments were likely disregarded. Microorganisms that produce antimicrobials under extreme growth conditions may have gone undetected while being screened under neutral growth conditions, preventing the discovery of potentially valuable novel compounds from these strains.
Advances in PCR technology have revealed that actinomycetes do not always exhibit antagonistic properties when cultivated in vitro despite the presence of genes that are involved in antibiotic synthesis in the genome of the strain. This suggested that dormant genes may be present but not expressed. Certain natural conditions may exist under which these genes are expressed, and identification of these conditions is important when screening for the new natural antibiotic substances. Modern screening programs should take into account the relevant characteristics of the actinomycetes and should also include alternative conditions for antibiotic isolation. To that end, we undertook a study utilizing an alternative method of screening to enhance the chances of discovering unique bacterial strains from the environment that are potentially producing novel antibiotics for therapeutic use.
This study focuses on actinomycetes strains isolated from the extreme habitats of Kazakhstan. We investigated the characteristics of actinomycetes in the three most commonly occurring natural habitats/conditions (which are neutral, saline, and alkaline). We believe that this approach, which is different than the conventional screening methodology, enhances the chance of identifying actinomycetes strains from extreme environments that harbor the potential to produce novel antimicrobial agents. Kazakhstan possesses unique natural resources that can be sources of promising and novel biologically-active substances with antibiotic properties. Soil and water resources of the Republic of Kazakhstan provide a great variety of natural habitats for extremophilic microorganisms (such as solonchaks, solonetzes, and saline takyrs). These habitats differ both in morphological features and salinization (chloride, sulfate, soda, and mixed). The water resources of Kazakhstan present a wide variety of habitats for extremophiles; this includes the Caspian Sea, the Aral Sea, Balkhash Lake, and numerous saline lakes in which water salinity can reach up to 335 g/l. The area of saline soils in Kazakhstan (including solonetzes, alkaline soils and combinations with other soils) is 111.55 million hectares (0.4307 million square miles), or 41% of the entire territory of the country (1.052 million square miles) 6 . Most of these areas are marked by natural mineralization due to the presence of marine sediments. Overall, research on extremophilic microorganisms in Kazakhstan has been limited. The goal of this publication is to describe the process of screening for antagonistic activity against drug-resistant pathogens and characterize actinomycetes strains which are the potential producers of novel antibiotics.

Sampling sites and collection of soils
Natural substrate samples (soils, muds, and rhizosphere) were collected from the extreme habitats of Northern (Kostanay region, Auliekol and Mendykara district) and Southern (Almaty region, Balkhash district) Kazakhstan during several field trips in summer 2010-2011 (Fig. 1A) are typically developed in poorly drained arid or semiarid areas vegetated mostly by halophytes 7 . Solonetz are soils with a high content of exchangeable sodium and/or magnesium ions 8 . Takyr is usually formed in a shallow, depressed area with a heavy clay soil which is submerged by water after seasonal rains; after the water evaporates, a dried crust with fissures forms on the surface 9 . The soil samples were collected at the depth of 10 cm (~4 inches). The samples were packed in sterile plastic containers, transported to the laboratory, and refrigerated at 4°C (39°F) until ready for analysis. Natural substrate samples (soils, muds, and rhizosphere) were collected from the extreme habitats of Northern and Southern Kazakhstan during several field trips in the summer of 2010-2011. Figure 1A. Map of field trips collecting soil samples We chose the most common factors in nature, which are sodium chloride and pH, and grew the same actinomycetes in different growth media to mimic the natural environment of extremophiles. The conditions these organisms were originally isolated from are neutral, saline, and alkaline environments.
Strains of actinomycetes from extreme environments of Kazakhstan were cultured in neutral and alternative (saline and alkaline) conditions and their antimicrobial and morphogenetic properties were studied. Saline and alkaline conditions were created by using inorganic salts (NaCl, Na2CO3) and рН modulation.
The plates prepared with variants of modified Bennett's agar were incubated at 28°C and examined for growth after 1-2 weeks of incubation. The colonies with different cultural-morphological characteristics were inoculated from the variants of modified Bennett's agar (variant #1 and variant #2) into slants with the same variants of the medium. The purity of isolated strains was confirmed by standard microbiological methodologies whereby strains were isolated in pure cultures from separately growing colonies obtained from plates seeded with soil samples. Each colony was further tested by screening on specific agar growth media as described.
Purified isolates were maintained on the variants of modified Bennett's agar slants at 4°C for further antagonism tests and morphogenesis investigation. The percent of the total number of strains isolated in the relevant region was determined.

In vitro antimicrobial assay
Actinomycetes isolates were tested for their antagonistic activity against the selected microorganisms by the disc diffusion agar method 10 .
The first stage of screening: The isolates were cultured on the variants of modified Bennett's agar (#1 or # 2) for 10 days at 28°C. Agar discs (7 mm) were cut by a cork borer and transferred to the surface of agar plates, previously inoculated with bacterial test organisms (hospital strain MRSA # 3316). The petri dishes were kept in a refrigerator for 3 hours before incubation to permit the diffusion of antimicrobial substances. The diameters of the inhibition zones were measured after incubation for 24 hours at 37°C.
The second stage of screening: The isolates with antagonistic properties, selected based on the results of the first screening stage, were cultured on three variants of modified Bennett's agar (#1-3) for 10 days at 28°C. Variant # 3 of modified Bennett's agar corresponded to a neutral habitat: glucose (0.2%), peptone (0.2%), yeast extract (0.1%), and agar (2.0%) at рН 7.2. Agar discs (7 mm) were cut off by a cork borer and transferred to the surface of agar plates, previously inoculated with the test organism (MRSA # 3316, Escherichia coli pMG223, and Aspergillus niger). The petri dishes were kept in a refrigerator for 3 hours before incubation to permit the diffusion of antimicrobial substances. The diameters of inhibition zones were measured after incubation for 24 hours at 37°C for bacteria and for 72 hours at 28°C for fungi. Each test was repeated three times and the activity was expressed as the mean diameter of the inhibition zones (mm).

Study of morphogenesis
The degree of aerial mycelium development in isolates of actinomycetes was studied visually on three variants of modified Bennett's agar (#1-3).

Bacterial and fungal pathogens
The following bacterial strains were used in this study as testers for the antimicrobial activity of the isolated actinomycetes strains: hospital strain MRSA # 3316, Escherichia coli (pMG223) and Aspergillus niger (wildtype isolate).

Results
In Northern Kazakhstan (Kustanai region), samples were collected from the soils of the steppe and forest zones, sor solonchaks (non-perennial salt lakes in the area of Aman-Karagai forest), solods, solonets (steppe and meadow), rhizospheres of plants from halophytic meadows, and muds from the salt lakes (Fig.  1B). In Southern Kazakhstan (Almaty region), samples were collected from the soils of the arid zone, typical meadows and sor solonchaks, salinized takyrs, takyr-like salinized soils, and rhizospheres of the arid zone plants (Fig 1C). We collected 36 samples of natural substrates from extreme ecosystems in Northern Kazakhstan and 50 samples from extreme ecosystems in Southern Kazakhstan.
We identified a link between the ability of actinomycetes to grow under conditions reflective of three habitats, the ability to antagonize in each of these conditions, and the ability to form aerial mycelia. In the course of our research, we focused on three main types of ecological niches: neutral habitats (рН 7.0), saline habitats (рН 7.0), and alkaline habitats (рН 9.0).
A total number of 5936 actinomycetes strains were isolated on the two variants of modified Bennett's  The correlation of the changes in growth, morphogenesis, and antagonism of 415 strains of extremophile actinomycetes was determined under three conditions, modeling the most common natural habitats: neutral, saline, and alkaline. The actinomycetes were further classified into groups, subgroups, and variants (Table 1)  Subgroups IIa showed growth in neutral and saline conditions, IIb showed growth in neutral and alkaline conditions, and IIс showed growth in saline and alkaline conditions.
In each group, we established the following subgroups. Three subgroups were identified in Group I: IА, IB, and IC (Fig. 2). The subgroups of I are as follows; In summary, the groups of actinomycetes differ from one another in their ability to grow in different habitats, which are neutral, saline and alkaline. We define subgroups by the ability to produce antibiotics, or they may lose this ability to produce antibiotics in these specific habitats (neutral, saline or alkaline). The magnitude of the inhibition zone was determined by us, but for this study, only the presence or total absence of antibiotic production was important for the classification of actinomycetes that we identified in this study. Since it was important to determine the conditions under which antibiotic production is possible for each of the producer organisms, the strains were classified into these specific groups, subgroups and variants. Table 2 summarizes the data on the quantitative content of actinomycetes-antagonists of different group I subgroups in conditions mirroring those of Southern and Northern Kazakhstan. The data on the actinomycetes of Subgroup II is not discussed here, as the group did not have a sufficient size to be represented accurately.
We observed differences in the occurrence of Group I antagonists in the natural substrates of the Southern and Northern Kazakhstan. The antagonism against gram-positive bacteria in both researched regions is characterized by the predominance of Subgroup IA (55.4 -72.3%); Subgroup IB was less common (21.0 -34.0%), while Subgroup IC had the smallest amount of antibacterial activity against gram-positive bacteria (6.7 -10.6%). The antagonism against gram-negative bacteria and mycelial fungi also varied by region. In Southern Kazakhstan, the Subgroup IA prevailed (41.2 -41.4%), while the Subgroups IB and IC occurred less commonly. In Northern Kazakhstan, the antagonism against gramnegative bacteria was characterized by the predominance of the Subgroups IA (41.7%) and IC (38.9%). The antagonism against mycelial fungi in the researched substrates of Northern Kazakhstan is characterized by the predominance of the Subgroup IB (40.7%), while in Southern Kazakhstan, Subgroup IA predominates (41.4%).
Our studies of the dependence of antagonism on the degree of development of the aerial mycelium of actinomycetes under extreme conditions show that actinomycetes can be represented by the two main variants, which we designated as F and Q (Table 3) whereby "F" represents the "Fighters" and "Q" represents the "Quitters" which we describe further below.
The variant Q is represented by the actinomycetes whose antagonism is associated with good growth and abundant development of the aerial mycelium. Reduction of sporulation leads to a decrease in the production of antibiotics, and absence of aerial mycelium to the loss of this ability.
The variant F is represented by the actinomycetes that exhibit antagonistic properties only in the conditions when the formation of aerial mycelium is inhibited, and its absence is associated with the maximum formation of antibiotics.
The actinomycetes from the extreme environments of Kazakhstan were analyzed based on  Fighters") -antagonism in the absence of aerial growth (i.e., when they are fighting to survive); 2 Q ("Quitters") -antagonism associated with good aerial growth (i.e., quit producing antibiotics when growth is poor)

Figure 2. The classification of actinomycetes from extreme ecosystems into groups and subgroups
Group IA -antagonism in three habitats (neutral, saline and alkaline) Subgroup IBа -antagonism in neutral and saline habitats Subgroup ICc -antagonism in alkaline habitat their ability to show antagonism against MRSA in saline or alkaline conditions. The isolates with antagonistic properties, selected based on the results of the first screening stage, were cultured on three variants of modified Bennett's agar (#1-3) for 10 days at 28°C. Variant #1 mimics a saline habitat, while variant # 2 mimics an alkaline habitat and variant # 3 mimic neutral habitats. Agar discs (7 mm) were cut by a cork borer and transferred to the surface of agar plates, previously inoculated with the test organism (MRSA # 3316, Escherichia coli pMG223, and Aspergillus niger). The diameter of inhibition zones was measured after incubation for 24 hours at 37°C. Each test was repeated three times and the activity was expressed as the mean of diameter of the inhibition zones (mm). The actinomycetes were classified into these groups; I (growth in all three habitats) and II (growth in two habitats). The actinomycetes group I, showing the ability to grow in all researched habitats, had a greater variability of antagonistic properties. These actinomycetes groups were further classified into subgroups based on their ability to antagonize in the saline, alkaline and neutral habitats. Group IAantagonism in three habitats (neutral, saline and alkaline). Subgroup IBа -antagonism in neutral and saline habitats. Subgroup IBb -antagonism in neutral and alkaline habitats. Subgroup IBс -antagonism in saline and alkaline habitats. Subgroup ICb -antagonism in saline habitat. Subgroup ICc -antagonism in alkaline habitat.

Discussion
We have identified 415 strains of actinomycetes that demonstrate varying degrees of antifungal and antibacterial activities in saline and alkaline habitats. Some of these strains may be producing previously unknown antibiotics.
Traditionally, researchers have studied the diversity of actinomycetes using neutral growth media. Similarly, isolation, research of their properties, and production of natural, biologically active substances have been associated with strains grown in neutral media and conditions. These neutral conditions served as the basis of the discovery of contemporary natural antibiotics. However, the prevalence of drug resistance to known antibiotic substances necessitates the expansion of the boundaries of screening and changes in screening methods in order to identify new and promising antimicrobials 11,12 . Thus, unusual natural substrates and extreme ecosystems have currently become the most popular targets of research as they are the most likely to yield new microorganisms with unique properties. Marine environments, as well as soils with high levels of salinity and alkalinity, are being actively studied as sources of new secondary metabolites 13 With their unique metabolic pathway formed in special environments, microorganisms from extreme environments produce many special bioactive substances, such as enzymes and antibiotics 14-17 . The molecules formed by extremophilic microorganisms have unique properties that offer ample opportunities for a variety of applications 18-22 .
There are several limitations in this study. When investigating antagonism under changing conditions, we did not conduct studies in an acidic environment. In addition, in nature there may be other growth conditions that are not represented by the three media that we used to grow actinomycetes in the laboratory, therefore we may have missed the detection of other potential antibiotics produced by actinomycetes. The screening does not take into account the possibility of re-discovery of known antibiotics from certain strains, therefore we plan to develop approaches to rule out this possibility in future studies. The strains studied were not identified at the level of genus and species; however, in future studies we plan to perform these studies using traditional microbiology methods as well as molecular (PCR) methodologies.
It is important to determine how actinomycetes interact with their changing environment and with other microorganisms in relation to their ability to consume nutrients and produce antibiotics. The main characteristics of interest of actinomycetes from extreme environments of Kazakhstan are their growth, ability to form aerial mycelia, and their ability to antagonize bacteria and fungi.
We have determined that the most widespread antibiotic-producing organisms were the actinomycetes belonging to group I; they grew in each of the selected habitats -neutral, saline, and alkaline. This important characteristic accounts for the wide distribution of the actinomycetes in soils and water. These actinomycetes also constituted the majority of those isolated in our research. Actinomycetes of group II were very rare. When tested for growth in three different conditions (neutral, saline or alkaline), all strains grew in at the least two or three conditions. We did not find any isolate that grew only in one condition. Their absence may be a result of the isolation method used. However, their absence also indicates that despite their isolation from extreme habitats, actinomycetes are highly adaptable to different environmental conditions, including the neutral media. Thus, screening from any extreme natural substrates, including the less explored marine sources, will enable the isolation of actinomycetes that belong to the subgroups IA, IBa, IBb, which grow and secrete antimicrobials in neutral conditions and, perhaps also, secrete already known antibiotic compounds. Our proposed classification allows researchers to concentrate their efforts on studying antibiotic-producing actinomycetes that belong to the specific groups IBс, ICb, ICс, IIAc, IIBb, and IIBс, which do not show antagonism under neutral conditions but do show antagonism under saline and/or alkaline conditions; these actinomycetes have the greatest potential for yielding new and unexplored antibiotic compounds.
Actinomycetes are an unusual group of grampositive bacteria that have differentiated mycelia, both substrate and aerial. Information on the relationship of morphogenesis and antibiotic formation in actinomycetes is often contradictory. Some studies have correlated initial stages of antibiotic production with inhibition of aerial mycelium formation, while others indicate that the formation of antibiotics is linked to its abundant formation 23-28 . We found two variants of actinomycetes whose antagonism is inversely related to their morphogenesis. In the actinomycetes of the variant Q "quitters" (which constituted 39.8% of total actinomycetes isolated), antagonism was correlated to good growth and formation of aerial mycelium. The reduction of sporulation resulted in a decrease of antibiotic production, and its absence resulted in the loss of this ability. Actinomycetes of the variant F "fighters" (60.2% of total actinomycetes) exhibited antagonistic properties only when growth was poor and the production of aerial mycelium was inhibited, and its absence was associated with the highest level of antagonism.
Accordingly, in each previously selected subgroup based on the type of antagonism exhibition, the actinomycetes may be represented by these variants. Actinomycetes of the variant Q are more attached to certain habitats that ensure their comfortable existence, while the actinomycetes of variant F are capable of freer movement in a variety of habitats and colonization of new environments. We can conclude that in any natural environment members of both groups can be present simultaneously and can engage in certain relationships that ensure the survival of the actinomycetes community as a whole.
The research of the actinomycetes' ability to produce antibiotics in near-natural conditions provides a great opportunity to assess their biodiversity and distribution in the various regions of the world and to develop new methodological approaches to the screening of new antibiotics. Those groups of actinomycetes that produce antibiotics only under extreme conditions, even if these are neutrophile strains of the variant F, may be the most promising for modern screening. Once their growth is inhibited, the neutrophiles of the variant F may produce previously unknown antibiotics in saline and alkaline habitats. We consider the following subgroups to hold the greatest potential for screening: IBс, ICb, ICс, IIAc, IIBb, and IIBс. In future studies, we hope to further characterize the nature of these unknown antibiotics and to test them against known fungal and bacterial pathogens, particularly those strains that may be resistant to antibiotics currently available in the market. Additionally, one of our long-term goals of this work is to identify known strains of actinomycetes that exhibit interesting characteristics to the species level (which would be relevant to this study particularly for strains that produce potentially novel antibiotics), and/or to potentially identify novel actinomycetes strains.
An organism's environment may vary greatly, and a neutral habitat may gradually become saline, alkaline or acidic. Accordingly, actinomycetes ability to survive is largely determined by their adaptability. The study of the behavior of actinomycetes, which use antibiotics for their survival in changing habitats may allow for the development of new approaches to the identification of pharmaceutically valuable drugs. This study has a significant global health implication. The search for new antibiotic-producing strains will require collaborations of many countries and will significantly enhance global health if new antibiotic agents become available. The actinomycetes classification we propose here is neither complete nor indisputable, and it can be changed and supplemented with new data and hypotheses that make it possible to significantly expand it.


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A new consensus for evaluating CDKL5/STK9‐dependent signalling mechanisms

Mutation or inactivation of CDKL5 kinase is associated with a human neurodevelopmental condition commonly referred to as CDKL5 deficiency disorder.§ Two recent phosphoproteomics studies identify the first physiological substrates of mammalian CDKL5 and evaluate functional consequences of their phosphorylation and its loss in cells lacking functional CDKL5, highlighting potential roles for this kinase in regulating neuronal microtubule dynamics.

A new consensus for evaluating CDKL5/ STK9-dependent signalling mechanisms

Patrick A Eyers
Mutation or inactivation of CDKL5 kinase is associated with a human neurodevelopmental condition commonly referred to as CDKL5 deficiency disorder. § Two recent phosphoproteomics studies identify the first physiological substrates of mammalian CDKL5 and evaluate functional consequences of their phosphorylation and its loss in cells lacking functional CDKL5, highlighting potential roles for this kinase in regulating neuronal microtubule dynamics. The EMBO Journal (2018) 37: e100848 See also: IM Muñoz et al (December 2018) and LL Baltussen et al (December 2018) P rotein phosphorylation plays a fundamental role in basic cell biology, and the protein kinase superfamily has become a goldmine for potential drug-intervention strategies due to kinase mutation or dysregulation in human diseases. However, it is often (conveniently) forgotten that mechanistic information is lacking for much of the human kinome, and this includes an absence of basic knowledge pertaining to regulatory mechanisms and substrate specificity (Wilson et al, 2018). A case-in-point are the cyclin-dependent kinase-like (CDKL) kinases, a small family of five poorly characterised and structurally distinct Ser/Thr protein kinases from the CGMC evolutionary branch of the kinome, which contains GSK3a, MAPK and CDK families (Canning et al, 2018). Mammalian CDKL5 (also known as STK9) is widely expressed in cells, where it is targeted to a variety of subcellular structures (Barbiero et al, 2017;Oi et al, 2017). Of particular interest, inactivation of the X-linked CDKL5 gene, or disease-associated mutations commonly found in the Nterminal CDKL5 catalytic domain, causes a neurodevelopmental disorder termed CDKL5 deficiency disorder (CDD; Tao et al, 2004;Weaving et al, 2004), which has some overlapping features with Rett syndrome (Scala et al, 2005) and West syndrome (Kalscheuer et al, 2003). § Writing in The EMBO Journal, two groups simultaneously reveal the first physiological substrates for CDKL5, including potential new biomarkers for reporting cellular CDKL5 activity. Using distinct, but highly complementary mass spectrometry (MS)based phosphoproteomics approaches, the laboratories of John Rouse and Matthias Trost (Muñ oz et al, 2018), and Sila Ultanir (Baltussen et al, 2018) evaluate functional consequences of CDKL5-catalysed substrate phosphorylation and its loss in cells lacking functional CDKL5. These findings will be critical for understanding microtubule dynamics and cilium-based signalling in CDD cell models, including neurons from patients with pathological CDKL5 mutations.
Both teams exploited MS to establish, in as unambiguous a way as is possible, the direct substrates of CDKL5 in cellular models. Although the approaches taken were different, they (happily) converge on a subset of substrates related both through shared subcellular function(s) and conserved consensus site(s) of CDKL5 phosphorylation. Muñ oz and colleagues initially employed a clean CRISPR knockout/in approach in human U2OS cells AE CDKL5, which was coupled to a quantitative (6-plex TMT) phosphoproteomic workflow (Muñoz et al, 2018).
This high-quality, internally controlled experimental strategy revealed nearly 200 sites of intracellular protein phosphorylation (including CDKL5 autophosphorylation) whose increase was statistically relevant when CDKL5 was added back to CDKL5 genome-edited cells. In a distinct, no-less impressive approach, Baltussen and colleagues developed CDKL5 chemical proteomics, employing purified catalytically active CDKL5-F89A/C152A, an analogue-sensitive (as) version selectively using the bulky ATP analogue benzyl-ATPcS, to thiophosphorylate potential substrates in a mouse brain lysate (Baltussen et al, 2018). After affinitybased purification of phosphorylated substrate-derived peptides, mass spectrometry identified multiple potential CDKL5 substrates, including murine microtubuleassociated targets MAP1S (two sites of phosphorylation), EB2 and ARHGEF2 (Baltussen et al, 2018) and, independently, human MAP1S, CEP131 and DLG5 (Muñoz et al, 2018). At a single stroke, these consolidating approaches reveal, for the very first time, new signalling functions of CDKL5 that are relevant to microtubule assembly, ciliabased signalling and perhaps polarity-based cellular networks ( Fig 1A).
In the case of the microtubule-binding protein MAP1S, a biological function for phosphorylation was confirmed in elegant murine follow-up experiments, exploiting phospho-specific MAP1S antibodies to demonstrate that phosphorylation on Ser812 (equivalent to human Ser900) was impaired in microtubule binding in vitro, consistent with a significant reduction in microtubule dynamics in living cortical neuron dendrites derived from CDKL5 knockout mice (Baltussen et al, 2018). In order to probe signalling with additional reagents, both studies made use of additional phosphospecific antibodies. For example, the phosphorylation of MAP1S at Ser812 and EB2 at Ser222 is both markedly reduced in CDKL5 knockout mice regardless of age, validating phosphoproteomics data and establishing CDKL5 activity markers in cells based around proteins that modulate microtubule trafficking and dynamics (Fig 1B). Perhaps the icing on the cake, however, is the demonstration that endogenous EB2 phosphorylation (as judged by a phospho-specific EB2 antibody able to recognise an identical phospho-epitope in human and mouse EB2) is severely reduced (by~80%) in neurons derived from CDD patient fibroblasts after reprogramming into iSPCs (Baltussen et al, 2018). Importantly, many of the antibodies validated in the two studies can now be employed, or re-engineered as higher-affinity antibodies, to serve as diagnostic tools for quantifying cellular activity of CDKL5, most obviously to evaluate CDKL5 biology and diagnose CDKL5 phenotypes, but also for therapeutic approaches being developed to normalise CDKL5 expression (see below).
Another exciting outcome from these studies is the finding that catalytically active CDKL5 phosphorylates Ser/Thr in a sharply defined, specific amino acid consensus sequence in its target substrates (Fig 1A), as has been documented for many other protein kinases. A harmonious minimal CDKL5 phosphorylation consensus emerging from both papers is the amino acid sequence Arg-Pro-X-Ser/Thr-Ala/Pro, in which the Ser or Thr residue (heavily biased 85:15% Ser:Thr) is phosphorylated, adjacent to a C-terminal residue (commonly Ala or Pro). These latter findings, confirmed biochemically with model peptide substrates, agree with phosphoproteomics data sets, which demonstrated CDKL5 autophosphorylation sites in Ser/Pro motifs alongside a dominant general Arg-X-X-Ser motif (Muñ oz et al, 2018). Although the CDKL5 consensus overlaps to some extent with known sites of phosphorylation for other "basophilic" kinases (e.g. Arg-X-X-Ser-Ala for several AGC kinases) and MAPK/CDKs (minimally Ser/Thr-Pro), the absolute conservation of a Pro at À2, and a specific requirement for Arg at À3, may well be diagnostic for CDKL5 (and perhaps other CDKL orthologues). Further studies employing active CDKL1/2/3/4 proteins (which, somewhat surprisingly, do not appear to phosphorylate any of the physiological CDKL5 substrates identified in this study) should open up these unstudied human kinases to careful scrutiny using similar chemical genetic or genome editing approaches.
The functional consequences of pathological CDKL5 mutations were previously uncertain, with evidence for inhibition and preservation of activity, alongside Tyr phosphorylation in the TEY activation segment (Bertani et al, 2006). By quantifying the reduction of CDKL5-dependent MAP1S and CEP131 phosphorylation in cells expressing specific CDD mutants, and by using a new MAP1S S900 peptide assay to assess immunoprecipitated CDKL5 activity, Muñoz and colleagues now unequivocally identify them as loss-of-function mutations that inactivate CDKL5.
Intriguingly, biochemical analysis demonstrates that CDKL5 also possesses an inherent ability to phosphorylate itself on a conserved regulatory residue (Tyr171) in the activation segment Thr-Glu-Tyr motif. This autophosphorylation event appears to be intimately associated with an active kinase conformation when evaluated in vitro, and might also be relevant for generating active CDKL5 in cells (Bertani et al, 2006). Interestingly, a Thr-X-Tyr motif is conserved in all five human CDKL protein kinases; its mutation to an Asp-X-Glu phosphomimetic version in CDKL1-5 prior to crystallographic studies (Canning et al, 2018) provides supporting evidence of the importance of this motif for driving structural dynamics. However, how (or even whether) CDKL5 phosphorylation is regulated at this motif in cells remains to be established, since the mechanism through which it is triggered is currently unknown. It is intriguing to speculate that CDKL5 is not only able to control its own activity, but that mutations in CDD patients might also lead to a loss in CDKL5 catalytic output through a lack of activationloop Tyr phosphorylation. The best-known "dual-specificity" protein kinases are the MAPKK/MEK family of MAPK activators, which catalyse dual phosphorylation of a Thr-X-Tyr motif (Thr-Glu-Tyr for MEK1/2) in the activation segment of cognate MAPK substrates. A concerted autoactivation mechanism in CDKL5 suggests that it possesses (biochemical) properties of a dual-specificity protein kinase, and opens up this mechanistic event in CDKL kinases for further analysis.
In the related MAPKs, dual phosphorylation of the Thr-X-Tyr motif massively increases catalytic activity, whereas Tyr15 phosphorylation is strongly associated with inhibition at a motif distinct from the activation segment in CDKs. It will therefore be interesting to evaluate whether CDKL5 autophosphorylation on Tyr (and/or Thr) also occurs on the other four CDKL proteins (where the human sequence is Thr-Asp-Tyr, rather than Thr-Glu-Tyr), or whether Tyr is also phosphorylated on cellular proteins distinct from CDKL5. Finally, CDKL5 Tyr autophosphorylation does not rule out the presence of distinct Tyr or dual-specificity kinases lying "upstream" of CDKL5; putative activators include the interacting dual-specificity tyrosine phosphorylation-regulated kinase DYRK1A (Oi et al, 2017), which was previously shown to contribute to CDKL5 subcellular targeting. A central impact from this work is the discovery of the very first physiological CDKL5 substrates and the emergence of a framework for the development of multiple approaches to study and measure (and eventually normalise) CDKL5 activity in systems relevant to CDD, such as the brain. Further detective work will be required to uncover the complete set of cellular CDKL5 substrates (many more being predicted in these phosphoproteomics studies) and to decipher regulatory networks associated with CDKL5 catalytic output that are central to physiological function and underlie CDKL5 loss-of-function in CDD. Unravelling the molecular mechanisms of CDKL5 activation and inactivation could also help link subset(s) of the CDKL5 network that contribute to disease phenotypes to potential therapeutics. In terms of targetable outputs, factors that control CDKL5 activation include pharmacological agents linked to activity of the NMDA receptor (Tramarin et al, 2018), whose inhibition by experimental neuronal depolarisation has a significant experimental effect on EB2 phosphorylation at pS222 (Baltussen et al, 2018). Several avenues might also be explored in related areas of cell biology, most notably any links between human CDKL5 and the ciliopathy-associated protein CENP131, which is associated with centriolar stability and the DNA damage response. Centriolar and cilium-based analysis also provide fascinating new potential models for functional CDKL5 analysis. For example, human CDKL5 has recently been demonstrated to localise to the cilium, and effects of patient-derived mutations have been modelled in C. elegans, where CeCDKL1 (most similar to human CDKL1/4) regulates cilium length, and Chlamydomonas, where CrCDKL5 is also required for controlling aspects of cilial dynamics (Canning et al, 2018).
Finally, very recent studies have begun to analyse small molecule kinase inhibitors, including the pre-clinical GSK3a inhibitor tideglusib, which exhibits some promise for restoring memory function in immature CDKL5 knockout mice, although the specific intracellular target that mediates these effects is unknown . Further work is now required to link the specific subset(s) of phosphorylated CDKL5 targets that contribute to disease phenotypes, and to understand the function of the long C-terminal region, which is much longer in CDKL5 than other CDKL proteins, and presumably plays a key role in its physiological function. Rational drug design using recently available CDKL5 structures and small molecule screening data (Canning et al, 2018) could provide new ways to activate mutated (inactive) CDKL5 mutants, alongside the development of genetic therapies, or even protein-replacement therapies .


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Skin Cell Heterogeneity in Development, Wound Healing, and Cancer

Skin architecture and function depend on diverse populations of epidermal cells and dermal fibroblasts. Reciprocal communication between the epidermis and dermis plays a key role in skin development, homeostasis and repair. While several stem cell populations have been identified in the epidermis with distinct locations and functions, it is now recognised that there is additional heterogeneity within the mesenchymal cells of the dermis. Here, we discuss recent insights into how these distinct cell populations are maintained and coordinated during development, homeostasis, and wound healing. We highlight the importance of the local environment, or niche, in cellular plasticity. We also discuss new mechanisms that have been identified as influencing wound repair and cancer progression.

Skin cellular behaviour and fate are regulated by interactions at the level of single cells, termed microniches, as well as on higher scales.
Individual niche components and transcription factors can be identified that significantly affect stem cell maintenance and behaviour.
Cell population-specific mechanisms regulate cellular plasticity and behaviour during wound repair and tumour development.
Epidermal stem cell plasticity is achieved by a loss of lineage specificity during wound healing, and may become permanent in skin cancer.

Glossary
Arrector pili muscle: smooth muscle attached to the bulge region of the hair follicle that is responsible for piloerection. Bulge: hair follicle region that marks the bottom of the permanent portion of the hair follicle and the insertion point of the arrector pili muscle. The bulge harbours the stem cells of the hair follicle that divide infrequently. Cell lineage: developmental history of a differentiated cell based on the cell from which it arosean analogy would be a family tree. As cells progress along a lineage they undergo transcriptional and epigenetic changes associated with differentiation. Cells that have a common differentiated state can still arise from different lineages. Cellular state: describes the physiological condition of a given cell defined by molecular profiles, such as transcriptome or proteome. Cell state is distinct from cell lineage and may be transient, such as proliferative or quiescent, or the point of transition from undifferentiated to differentiated. Dedifferentiation: describes the process whereby differentiated cells lose their specialised characteristics and may acquire stem cell potential. Dermal papilla: specialised subset of mesenchymal cells located at the base of each hair follicle. Dermal papilla cells communicate with the neighbouring epithelial cells and provide essential signals for hair follicle formation and regeneration. Dermal sheath: connective tissue sheath consisting of mesenchymal cells that wrap around the hair follicle and span from the bulge to the dermal papilla. Gene ontology: bioinformatic initiative for a unifying representation of gene and gene product attributes across all species. It classifies gene functions according to cellular components, molecular function and biological processes. Hair follicle placode: thickening of the embryonic epidermis that marks the initiation of hair follicle formation. Infundibulum: upper part of the hair follicle, above the entry point of the sebaceous duct, which extends to the interfollicular epidermis.

Stem Cell Populations in the Epidermis
Multiple epithelial stem cell (SC) populations have been shown to contribute to skin homeostasis. In mice a highly diverse pool of SCs has been identified in the HF[ 3 3 6 _ T D $ D I F F ] , ranging from the lower HF (Lgr5 + [ 3 3 5 _ T D $ D I F F ] and CD34 + /Krt15 + ) bulge to the upper HF (Gli1 + and Lgr6 + ) and junctional zone (Lrig1 + ) ( Figure 1A). In undamaged skin the different cells populate distinct and restricted areas in the HF, whereas upon tissue injury these cells have the intrinsic ability to give rise to all epidermal cells, including the interfollicular epidermis (IFE), which is located between HFs and comprises the largest pool of keratinocytes in the skin [5,6]. How these differential cellular behaviours are regulated at the molecular level is currently being investigated (Box 1).
During development, the epidermis is formed by a flat single layered epithelium known as the surface ectoderm. Local induction of Wnt signalling in the epidermis and subsequently in the dermis leads to the formation of HF placodes, which are characterised by expression of adult SC markers such as Sox9 [7]. While adult skin SC maintenance and differentiation depend on signals from local niches such as the HF bulge, during hair bud development, SC specification is achieved by asymmetric cell division, differential levels of Wnt signalling, and the response to sonic hedgehog (SHH) of basal and suprabasal cells [8]. During further maturation of the HF placode, the SC markers start to segregate into the distinct HF structures including the HF bulge isthmus and sebaceous gland (reviewed in [9]). When the HFs form they further provide niche structure and signals to induce the specification of Merkel cells, a specialised subpopulation of keratinocytes that become organised into touch domes around primary HFs. These innervated mechanoreceptors mediate light touch sensation [10].

Beside epithelial cells (keratinocytes and Merkel cells), the adult IFE harbours melanocytes and immune cells (Langerhans cells and gdT-cells).
Until recently the organisation of epithelial SCs within adult mouse IFE was a matter of some debate. Initial in vivo clonal analysis in mouse tail and ear epidermis indicated that a single cell population is responsible for epidermal homeostasis, and variation in clone size could be explained by stochastic (random) cell division of a homogeneous population of keratinocytes, referred as the neutral drift model [11][12][13] [14,15]. The size of the scale and interscale regions is controlled by epidermal Eda and Wnt/b-catenin signalling, and there is a corresponding patterning of melanocytes and papillary dermal fibroblasts [14]. Lgr6 + [ 3 3 7 _ T D $ D I F F ] cells contribute to the interscale but not to the scale IFE [5]. The scale and interscale IFE can also be distinguished by expression of Slc1a3 and Dlx1, respectively [16]. The two tail IFE SC compartments differ in their proliferative dynamics, gene-expression profiles and ability to repair the epidermis after injury [16,17].
Single cell transcriptomic analysis of mouse dorsal epidermis and cultured human epidermis has identified at least two distinct IFE SC transcriptional signatures, even though there appears to be a single terminal differentiation programme [18,19]. It is not known at present whether the cellular heterogeneity in the IFE reflects differential susceptibility to initiating keratinocyte differentiation. In addition the proliferative properties of cells in the IFE basal layer are influenced by the HF cycle.
Lineage tracing experiments have revealed that while cell clones associated with HF show a rapid increase in size during the HF growth phase, distant clones cycle more slowly, yet can be mobilised upon tissue injury [20]. Thus, while in mouse tail IFE, distinct SC populations are associated with unique differentiation programmes, SC heterogeneity in mouse back skin IFE underlies a single differentiation programme and could reflect different cellular states.
To gain further insights into the proliferative dynamics of epidermal cells with age[ 3 4 2 _ T D $ D I F F ] , in recent yearsclonal analysis has been applied to human epidermis by making use of sunlight induced feature of some human skin disorders such as psoriasis.
Pericyte: contractile mesenchymal cell that wraps around the endothelial cells associated with small blood vessels in the body. Reticular fibroblasts: subpopulation of dermal mesenchymal cells that are located in the central dermis and surrounded by thick collagen fibres. These cells form the first wave of dermal repair during wound healing. Sebaceous gland: exocrine gland associated with the hair follicle, which secretes sebum to lubricate the skin surface and hair. Transdifferentiation: describes the process of a cell changing its lineage identity.
Tumour stroma: microenvironment associated with a tumour comprising all nontransformed tissue components such as extracellular matrix proteins, fibroblasts, immune cells and endothelial cells. Key intrinsic factors include SC-specific regulation of metabolism, gene expression, and chromatin arrangement. For example, HFSCs produce more lactate than other epidermal cells, suggesting that differences in cellular glycolytic metabolism influence SC activity [77]. In the HF bulge the transcription factor Foxc1 is specifically induced in HFSC upon activation of the hair cycle, and regulates the expression of BMP and nuclear factor of activated T cells 1 to maintain HFSC quiescence during HF growth [78,79].
Within the niche interactions between neighbouring cells are important regulators of cell behaviour. Upon epithelial SC ablation neighbouring cells are able to replace niche SC even if they are committed progenitors [73]. In addition, loss of single ECM components can be sufficient to dramatically impact SC maintenance in the niche. DNA damage induced proteolysis of collagen 17a1 in the HF bulge with age leads to a cyclic elimination of HFSCs through epidermal terminal differentiation [80]. Maintaining or replenishing collagen 17a1 in the skin [ 3 2 8 _ T D $ D I F F ] inhibits the ageing phenotypes, suggesting a possible therapeutic application [80,81].
Extrinsic niche factors can act on a single cell level and are either produced by the SCs themselves [82] or provided by surrounding cells such as progenitor cells, fibroblasts, resident immune cells, or sensory neurons [39,40,[83][84][85]. Sensory neurons in contact with the HF create a perineural niche microenvironment in the bulge and isthmus. The  [83]. A subset of resident regulatory T cells located close to the HF bulge and expressing high levels of Jag1 promotes HFSC proliferation and differentiation through direct activation of Notch signalling in HFSCs [84]. Impaired crosstalk between epidermal and dendritic T cells severely affects wound repair in aged skin, highlighting the importance of different resident immune cell types in homeostasis as well as tissue repair [85].
Physical parameters such as tension, pressure, or temperature contribute to the cellular microenvironment, are sensed by distinct cell populations, and influence cellular fate. For example, in epidermal SCs a mechanosensory complex consisting of emergin, nonmyosin-IIA, and actin affects gene transcription and lineage commitment [86]. Extrinsic forceinduced relocation of emergin and a change in cellular G-actin levels leads to a switch in histone methylation and loss of heterochromatin anchoring to the nuclear lamina, thereby inhibiting global gene transcription. It has also been noted in single cells that there is a potential interplay between the actin cytoskeleton and epigenetic modifications [87].
It remains to be determined whether force-induced changes in chromatin structure are transient or if SCs functionally adapt and memorise the stress response[ 3 3 1 _ T D $ D I F F ] . It was recently shown that epithelial SCs exhibit a memory of previous inflammation upon tissue damage by maintaining chromosomal accessibility at key stress response genes[ 3 3 2 _ T D $ D I F F ] , promoting faster transcription [88]. This memory does not require the presence of resident immune cells but is critically dependent on Aim2 (absent in melanoma 2), which is an activator of the inflammasome upstream of caspase-1 and interleukin-1b. A side effect of this increased responsiveness to stressors in epithelial SCs might [ 3 3 3 _ T D $ D I F F ] be increased susceptibility to autoimmune and hyperproliferative disorders including cancer.
Epidermal b-catenin activation not only results in cell-intrinsic effects but also alters the behaviour of neighbouring cells [89]. Recent single cell gene expression profiling has shown on cell intrinsic b-catenin activation, gene expression heterogeneity is reduced in neighbouring cells and the effect is most dramatic for genes associated with protein synthesis [90]. The effect is dependent on cell-cell contact and the changes in gene expression are accompanied by a shift to a more proliferative SC state.
It is conceivable that many more specialized SC subpopulations, maintained and defined by a complex array of niche factors, will be discovered[ 3 3 4 _ T D $ D I F F ] ; indeed that all epidermal cells in contact with ECM have SC properties. mutations in cancer-associated genes, such as p53, as markers [21,22]. This has led to conflicting conclusions about the relative importance of positive selection and neutral drift in clonal evolution. Recently, by sequencing larger areas of skin than previously and focusing on skin from patients who had previously developed a skin tumour, it has been possible to establish that some human mutant clones are too large to be accounted for solely by neutral drift. Rather, secondary mutations arising at the edge of a mutant clone have a selective growth advantage [23].

Mesenchymal Cell Heterogeneity and Behaviour in Dermal Homeostasis
Beside its role as an ECM-rich scaffold, the dermis harbours highly diverse fibroblast, pericyte, and immune and endothelial cell populations that dynamically change with age and influence the properties and cellular behaviour of the overlying epidermis [2,4,24] ( Figure 1B). Although the dermal layers can be easily distinguished by collagen structure and cellular density, the cellular events generating and maintaining dermal architecture have not been explored in detail until recently. During mouse embryonic development, dermal fibroblasts arise from at least two spatially and functionally distinct cell lineages that differentiate into distinct subpopulations and contribute to the dermal layers [25,26]. Neonatal dermis fibroblasts of the papillary layer are characterised by active Wnt signalling and proliferation, whereas populations in the reticular layer show increased expression of ECM and immune cell associated genes [26][27][28]. Whether bone-marrow-derived mesenchymal stromal cells (MSCs) contribute to the resident fibroblasts of mouse dorsal skin under homeostatic conditions or following wounding is controversial (reviewed in [2]).
During development, gene expression in dermal fibroblasts is highly dynamic and there is a swift change in dermal fibroblast behaviour on the tissue scale during dermal maturation [26,27]. While fibroblasts are highly proliferative during embryonic development they rapidly stop proliferating in the postnatal dermal growth phase, which is characterised by extensive ECM deposition and remodelling. Clonal analysis reveals that individual fibroblasts within clones start to be segregated by increased ECM deposition, leading to a dramatic reduction in fibroblast density postnatally [27].
In addition there is an increase in the adipocyte layer with age [29]. The different dermal fibroblast lineages are spatially segregated in P2 mouse skin, whereas mixing of lineages occurs during subsequent dermal maturation. Recent single cell RNA sequencing studies of human dermal fibroblasts identified several transcriptionally distinct subpopulations[ 3 4 3 _ T D $ D I F F ] , some of which are spatially segregated whereas others are not [28,30]. Further studies are needed to clarify whether the transcriptionally heterogeneous fibroblast subpopulations in human dermis represent different cellular states or functionally distinct fibroblast lineages.
While the nature of the switch between fibroblast proliferation and ECM production is unknown, gene expression analysis of neonatal (proliferative) and aged (nondividing) dermal fibroblasts suggests that it is controlled by epigenetic changes at the chromatin level [27,31]. In vitro studies suggest that there is not a common nonproliferative state in fibroblasts but that it is rather an accumulation of different states that are actively maintained [32,33]. [ 3 4 4 _ T D $ D I F F ] Besides actively reinforcing the nondividing cell cycle state and repressing the transition into senescence or terminal differentiation, aged fibroblasts remain highly metabolically active [34]. They increase expression of ECM proteins such as collagen I and III, which is partly due to changes in expression of miRNAs such as miR-29 [35].

Epidermal-Dermal Interactions via Reciprocal Niche Signals
Reciprocal signalling between epidermis and dermis plays a key role in skin development, homeostasis, wound repair, and cancer. A prime example of tight temporal and spatial regulation involves the DP fibroblasts at the base of the HF and the overlying HF bulb (reviewed in [36,37]). The transcriptional repressor Blimp1 is a key target and mediator of this interaction [38]. Upon HF induction during development epidermal Wnt/b-catenin signalling induces Blimp1 expression in the DP via transforming growth factor (TGF)b signalling. In the DP, Blimp1 promotes Wnt/b-catenin signalling activity and HF growth.
When the DP matures during HF growth, heterogeneity of mesenchymal signals at the single cell level involving gradients of Wnt ligands and bone morphogenetic protein (BMP) inhibitors creates distinct microniches along the epithelial-mesenchymal interface in the DP [39]. By secreting distinct combinations of factors these microniches coordinate the hierarchy of self-renewal and differentiation states of epithelial cells in the HF matrix, enabling formation of the different HF layers.

The HF bulge SC niche harbours a distinct combination of ECM components[ 3 4 5 _ T D $ D I F F ] . Bulge cells have been shown to create microniches for APM cells by depositing specific ECM components
including nephronectin in the basement membrane [40]. Nephronectin is specifically recognised by a8b1-integrin positive mesenchymal cells of the APM, which is essential for proper anchorage and function. Interestingly, regenerated HFs in wounds lack APMs[ 3 4 6 _ T D $ D I F F ] , indicating that additional factors or specific mesenchymal subpopulations are required for APM formation [26,27].
In contrast to the HF, in the IFE the spatial and temporal coordination of epidermal-dermal interactions has not been defined at single cell resolution. However, in tail skin, leucine-rich repeats and immunoglobulin-like domains protein 1 (Lrig1) expression is selectively upregulated in dermal fibroblasts that underlie the interscale epidermal compartment, pointing to different dermal niches in the interscale and scale [14].
One of the key pathways of epidermal-dermal communication is via canonical Wnt signalling. Activation of Wnt/b-catenin in basal keratinocytes of adult mouse epidermis induces a rapid increase in fibroblast proliferation and stimulates ECM remodelling [31,41].
It seems likely that, similar to the epidermis, dermal subpopulations are maintained by a complex combination of extrinsic and intrinsic factors (Box 1). On the one hand, reciprocal transplant experiments involving fibroblasts of different developmental origins (oral cavity vs skin[ 3 4 7 _ T D $ D I F F ] ) have revealed that their differential ECM deposition and migratory behaviour is maintained and thus are cell intrinsic features [25]. Furthermore Sox2 + DP fibroblasts maintain their identity in cell culture [42]. On the other hand, different fibroblast subpopulations respond to distinct paracrine signals from the epidermis. Epidermal SHH signalling stimulates proliferation and ECM remodelling of the upper dermis, while TGFb-2 influences proliferation, ECM deposition and differentiation of the lower dermis [41]. In addition, secreted factors of Wnt/b-catenin activated keratinocytes promote adipocyte differentiation and expansion of the hypodermis [29] whereas PDGF expressed by preadipocytes stimulates HF SC activity [43].

Epidermal SC Behaviour during Wound Healing
Wound healing and tissue regeneration are coordinated processes that involve epidermal, dermal, [ 3 4 9 _ T D $ D I F F ] endothelial and immune cells, and can be divided into distinct phases (reviewed in [44,45]). Upon wounding, a blood clot forms and immune cells infiltrate the wound [ 3 5 0 _ T D $ D I F F ] site (inflammatory phase). The cells from the epidermis and dermis start proliferating and migrate into the wound bed to close the wound (proliferative phase). Then, dermal cells deposit and restructure the ECM in the wound bed (resolution phase). Of note, wound healing responses differ between species: for example, in mice wounds close mainly through tissue contraction as a result of the action of the panniculus carnosus muscle[ 3 5 1 _ T D $ D I F F ] , a structure that is absent in human skin (reviewed in [46]). In mouse skin, several epidermal cell populations contribute to the skin wound repair process, leading to the picture that not only cells close to the wound edge but also HF epithelial cells are recruited to the wound site (Figure 2A,B). How distinct cell populations from different niches coordinate on the tissue scale and move into the wound bed, ensuring rapid wound closure and tissue regeneration, has only recently been addressed.
In the past, two distinct mechanisms for epidermal cell movement into the wound bed have been proposed. One involves a smooth flow of epidermis following a homeostatic rule of unidirectional basal to suprabasal transfer of cells with front edge movement [ 3 5 2 _ T D $ D I F F ] that is achieved by basal cells migrating across the wound [47,48]. The second, referred to as leapfrogging, involves suprabasal cells sliding over the leading basal cells to become basal cells themselves [49,50]. Two recent studies, combining in vivo live cell imaging, lineage tracing, and transcriptomic analysis, have mapped the anatomy and spatiotemporal dynamics of the wound healing response [ 3 5 3 _ T D $ D I F F ] and identified two concentric zones of cellular activity and differential gene expression [51,52]. The cell population closest to the wound edge is characterised by rapid migration and differentiation. Further from the wound there is a zone with high epidermal proliferation and little migration along the basement membrane. In the first (migratory) zone both basal and suprabasal cells actively migrate with increasing speed towards the wound centre and upregulate genes involved in ECM remodelling and cell adhesion[ influences cell division orientation towards the wound bed centre, demonstrating a spatial interplay between proliferation and migration. Clonal lineage tracing reveals that most of the committed progenitor cells become highly proliferative and rapidly differentiate in the early wound healing phase, whereas basal (SC-enriched) cells become activated and increase the pool of SCs later. These studies suggest a model whereby the nonproliferating leading edge functions as a scaffold, preparing the wound bed for efficient repopulation towards the wound bed centre and protecting SCs during tissue repair. The clonal dynamics of different SC populations from the HF and IFE are similar, indicating that the behaviour balancing proliferation and differentiation within the wound healing zones is independent of the original cell. Furthermore, the leading edge cell gene signature is specified independently of cell division or inflammation. This indicates that both intrinsic and extrinsic factors establish the wound healing zones.
Epidermal wound healing [ 3 5 5 _ T D $ D I F F ] induces plasticity in differentiated sebaceous duct cells such that they dedifferentiate, proliferate, and contribute to long-term maintenance of the IFE [53]. These cells are located in the suprabasal layer of the sebaceous duct that connects the HF junctional zone with the sebaceous gland and are defined by high expression of the transcription factor GATA[ 3 5 6 _ T D $ D I F F ] 6, a key regulator of the sebaceous duct lineage during homeostasis [53]. Upon wounding, differentiated GATA6 lineage cells in the HF duct become mobilised, migrate to the wound site suprabasally as individual cells, reattach to the basement membrane, and start proliferating to regenerate the IFE in the wound bed [53]. Mitogen-activated protein kinase [ 3 5 7 _ T D $ D I F F ] signalling is a key pathway that regulates keratinocyte differentiation, while the signals regulating dedifferentiation remain unclear [53,72]. In vivo live imaging reveals that downward cell migration is more pronounced at the wound edge than distally. This is in contrast to the  [6,51,53]. GATA6 lineage cells not only contribute to wound repair but also populate the lower HF and sebaceous gland of HF close to the wound [53]. Similar cell displacement has been observed in regenerating tail IFE. Upon wounding, SC populations in the interscale and scale migrate into neighbouring territories and contribute to tissue repair outside their normal niche [16]. This observation indicates a loss of lineage restriction during tissue repair, which is controlled by the transcription factors KLF5 and Sox9 [54].

Mesenchymal Cell Dynamics during Wound Repair and Fibrosis
Our understanding of epidermal wound healing has rapidly advanced, while the contribution of different dermal cell populations has been less clear (Figure 2A,C). Fibroblasts in the vicinity of a wound are known to become rapidly activated, are referred to as myofibroblasts, and change their behaviour and transcriptional program. TGFb signalling is the key pathway for fibroblast activation [ 3 6 0 _ T D $ D I F F ] [26,55,58,61]. During the whole wound repair process there is an intensive and highly dynamic crosstalk with multiple immune cell populations, which influences mesenchymal cell behaviour (reviewed in [44,45]). We and others have shown that distinct fibroblast populations show differential recruitment and contribution to wound healing [25][26][27]. Fibroblasts residing in the reticular dermis mediate the first phase of wound repair and are the major source of myofibroblasts, whereas fibroblasts in the papillary dermis enter the wound at a later stage and are essential for HF regeneration. Moreover, while fibroblasts from the DP do not contribute to Fibroblast-specific inhibition of Wnt/b-catenin signalling in mice enhances HF regeneration by preventing the early expansion of lower dermal cells [27]. In contrast[ 3 6 4 _ T D $ D I F F ] , sustained Wnt/b-catenin signalling in dermal fibroblasts impairs regeneration, induces fibrotic lesions in adult skin, and inhibits adipocyte differentiation [ 3 6 5 _ T D $ D I F F ] [59,60]. Lineage tracing experiments reveal that this is due to a TGFb-mediated conversion of adipocytes to myofibroblasts [ 3 6 6 _ T D $ D I F F ] [58]. Conversely, during wound healing, BMP signalling from regenerating HFs induces myofibroblasts to convert to adipocytes in the wound bed [61]. The signals promoting myofibroblast conversion to other mesenchymal cell populations are currently unclear.

Epithelial Plasticity in Cancer
Similar to wound healing, different epidermal and dermal populations contribute to cancer in different ways (Figure 3). This is well illustrated in the case of oncogenic b-catenin signalling, where depending on the epidermal SC type in which stabilised b-catenin is expressed, different tumours are formed. For example, targeting the Lgr5 + population promotes formation of pilomatricomas (benign HF skin tumours), while Lrig1 + cells develop trichoadenomas (a rare benign follicular tumour with cornifying cysts) and the Lgr6 + population gives rise to dermatofibromas within the IFE [5]. Similarly, activation of Hedgehog signalling demonstrates that only basal Krt14 + cells in the IFE and HF infundibulum can initiate basal cell carcinoma (BCC) formation [15,62]. BCC initiation and progression are highly dependent on expression of the transcription factor Sox9 [63]. In contrast, squamous cell carcinoma (SCC) can originate from Distinct epithelial cell populations give rise to different tumour types. Oncogenic b-catenin signalling in different hair follicle stem cell populations of the bulge (CD34/ Krt15 + , pink; Gli1 + , green; Lgr5 + , violet, and Lgr6 + , dark blue) and isthmus junctional zone (Lirg1 + , orange) induce different types of benign hair follicle tumours (grey circle), while BCCs only arise from basal (Krt14 + ) interfollicular epidermis (white cell) and Lrig1 + cells of the hair follicle isthmus and infundibulum (orange cell) upon Hedgehog signalling activation (dark grey circle). In contrast, a hyperactive Kras mutation is able to induce SCC in all epidermal lineages (light grey circle). If different tumour types are associated with distinct mesenchymal subpopulations (green, papillary fibroblast; red, reticular fibroblast; yellow, preadipocyte; white, undefined fibroblast; brown, APM, dermal sheath, dermal papilla fibroblast, pericyte [ 3 2 1 _ T D $ D I F F ] and adipocyte) (dashed arrows) and if mesenchymal subpopulation specific signalling pathways are involved (?), is unclear. Abbreviations: APM, arrector pili muscle; BCC, basal cell carcinoma; HF, hair follicle; SCC, squamous cell carcinoma. more than one epidermal population, including HF bulge SCs, as overexpression of a hyperactive Kras mutant in different epidermal lineages induces tumours with comparable efficiency [64]. Consistent with this, it has been shown that SCC cells harbour a distinct open chromatin landscape combining active gene signatures from distinct SC lineages [54]. This state of lineage infidelity can be transiently observed in wounds, [ 3 6 8 _ T D $ D I F F ] but persists during malignant progression, promoting uncontrolled growth and heterogeneous tumour cell behaviour [54,65].
During tumour initiation, mutant epidermal cells have the ability to engage nontransformed (healthy) cells via paracrine signalling, such as Wnt ligand secretion, to induce aberrant growth of the whole tissue [66]. Intriguingly, the converse is also observed: a tumour protective, 'neighbourhood watch like' mechanism, contributes to neoplastic tumour suppression. Using an in vivo live imaging approach, it was observed that healthy epithelial cells routinely recognise, surround, and eliminate mutant cells to restore tissue homeostasis, revealing an innate cellular ability to prevent over proliferation and tumour initiation [67]. So far the molecular mechanism of cell recognition and elimination and whether it is restricted to specific cell types remains unclear. The tumour-promoting as well as protective effects upon oncogenic b-catenin signalling rely on Wnt ligand secretion, suggesting that distinct Wnt ligand combinations balance cellular plasticity and behaviour.

Mesenchymal Heterogeneity in the Tumour Microenvironment
The tumour microenvironment, including the tumour stroma, comprising all nontransformed tissue components associated with a tumour, can have both tumour-promoting and -inhibitory effects. Besides endothelial and immune cells a major component of the microenvironment are cancer-associated fibroblasts (CAFs), which play an important role in the evolution of solid tumours. Similar to myofibroblasts, CAFs seem to originate from different mesenchymal populations, ranging from normal fibroblasts and MSCs to transdifferentiated epithelial and endothelial cells. In contrast to normal fibroblasts, CAFs either reside within the tumour margin or infiltrate the tumour mass and show increased proliferation, migration, ECM deposition, and secretion of growth factors and other ECM modulators (reviewed in [68,69]). Functionally, while CAFs are highly heterogeneous [ 3 6 9 _ T D $ D I F F ] in terms of gene expression, they show enrichment of similar gene ontology classes such as cell adhesion, immune response [ 3 7 0 _ T D $ D I F F ] and ECM modulation, suggesting that different cell types under similar conditions perform similar tasks. Thus it has been proposed that CAFs represent a dynamic cellular state of fibroblast-like cells in the vicinity of the tumour rather than a specific cell lineage [69]. This state could be maintained by a combination of genetic mutations, epigenetic alterations, and persistent environmental effects.
Similar to wound healing it seems likely that distinct fibroblast populations give rise to CAFs. Indeed, one study identified that in adult mice CD26 + [ 3 6 7 _ T D $ D I F F ] fibroblasts are the main contributor to ECM deposition in a skin melanoma xenograft model [25]. Depletion of this fibroblast subpopulation significantly reduced tumour growth, revealing that targeting distinct fibroblast subpopulations impacts tumour development. Inhibition of CD26 activity reduces the growth of wound-induced epidermal tumours, although it must be noted that CD26 is expressed [ 3 7 1 _ T D $ D I F F ] by both epidermal and dermal cells and that during tumour progression there are dynamic changes in dermal CD26 expression [70].
To date there have been few studies of how different fibroblast lineages contribute to [ 3 7 2 _ T D $ D I F F ] tumour stroma formation. Intriguingly, the different tumours induced by stabilizing b-catenin in Lgr5 + , Lgr6 + , and Lrig1 + epithelial cells exhibit both similarities and differences in stromal composition. In all three cases there are local increases in fibroblast proliferation, ECM remodelling, and expression of CD[ 3 7 3 _ T D $ D I F F ] 44, a major hyaluronic acid surface receptor. However, dermal CD26 expression is upregulated in the stroma of Lgr6 but not Lgr5 or Lrig1 tumours. Conversely Lgr6 and Lrig1 tumours have associated stromal inflammation while Lgr5 tumours do not [5]. It will be of interest to discover whether or not these differences reflect differences in the lineages of fibroblasts associated with each type of tumour or whether fibroblasts respond to tumourspecific signals independent of lineage.

Concluding Remarks
In recent years our appreciation of epidermal and dermal cell heterogeneity has grown, together with a realisation that cells are capable of exhibiting plasticity and changing fate through dedifferentiation and transdifferentiation. It is now clear that the responses of the skin to the challenges of wounding or tumorigenesis reflect a combination of changes in cell intrinsic properties and responses to different microenvironments. Microniches and cell memory are exciting new concepts that warrant further investigation, together with unravelling the distinction between cell types and states, for example in tumour stroma (see Outstanding Questions).
As more single cell gene expression profiles become publically available and tools to make those datasets readily accessible to researchers with a biological background improve [19,71], we anticipate greater appreciation of the significance of cellular heterogeneity. The combination of experimental data with computational modelling [16,20,23,72,73] not only allows rigorous evaluation of data quality but also fosters hypothesis generation. Ultimately we anticipate major benefits in terms of understanding tissue scale behaviour, dynamics, and the interplay of distinct cell populations during

Outstanding Questions
How is the behaviour of cell populations in the epidermis and dermis coordinated during wound repair and tumour development on the tissue scale?
What defines and [ 3 7 6 _ T D $ D I F F ] distinguishes cellular state and cellular type in the skin?
What are cell type specific and universal mechanisms for establishing and maintaining cellular identity and plasticity in the skin?
What are the key changes that create a pro-oncogenic environment?
How do distinct epidermal and dermal cell populations contribute to tumour heterogeneity and progression?
What are the dynamics and organisation of microniches in the dermis and epidermis?


Antler manipulation procedures for use in social and behavioral studies of deer

Theories describing the role of antlers in reproductive biology have lacked substantive evaluation due to the inability to differentiate the effects of antler size from other allometrically related morphological traits. We developed an antler manipulation technique to facilitate comparisons of antler size while controlling for body size and age using white-tailed deer Odocoileus virginianus. We describe the process by which antlers can be manipulated to further investigate the functions of antlers. We believe this process is a viable option for manipulating antler size across Cervidae with appropriate modification to scale.

Theories describing the role of antlers in reproductive biology have lacked substantive evaluation due to the inability to differentiate the effects of antler size from other allometrically related morphological traits. We developed an antler manipulation technique to facilitate comparisons of antler size while controlling for body size and age using white-tailed deer Odocoileus virginianus. We describe the process by which antlers can be manipulated to further investigate the functions of antlers. We believe this process is a viable option for manipulating antler size across Cervidae with appropriate modification to scale.
There has been much speculation as to the ecological justification for antler growth in Cervidae (Clutton-Brock 1982, Goss 1983. Antlers should enhance male fitness because they are costly to produce (Zahavi 1977, Ullrey 1983. Additional theoretical explanations of why males grow these structures range from predator defense to weapons for competition (Clutton-Brock 1982, Goss 1983), however few of these theories have been empirically evaluated. Supported theories of antler function include a display of dominance to other males, a weapon for intraspecific competition, and an ornament used by females to assess male quality (Demarais and Strickland 2011).
Although the dominance displaying function of antlers has some support in red deer (Bubenik 1983, Lincoln 1972, 1994, these studies were limited by the inability to manipulate antler size while controlling for allometrically related traits. Significant evidence suggests males use antlers as a weapon in intraspecific bouts against other males for access to females and to establish dominance (Bubenik 1983, Marchinton and Hirth 1984, Barrette and Vandal 1990, Miller et al. 2003. However, fighting ability and strength related to body size have also been related to success in malemale competition and reproductive success (Townsend andBailey 1981, Jones et al. 2011). Thus, conflicting results have been reported when age and body size have been accounted for (Clutton-Brock 1982).
To effectively assess the theorized functions of antlers, we must be able to manipulate antler size while controlling for the allometrically related trait of body size as well as age.
Using an innovative antler-manipulation technique, we provided the first definitive support for female choice of larger antlered males (Morina et al. 2018). While a previous antler manipulation technique exists (Lincoln et al. 1970), the procedure was not described in enough detail to be reproducible. Here we describe our antler manipulation technique so that other researchers can use it for further investigations into the theories of antler functions.

Study area
We housed captive deer at the Mississippi State University Rusty Dawkins Memorial Deer Unit (MSU Deer Unit), Oktibbeha County, Mississippi, USA. The MSU Deer Unit is subdivided into five 0.4-0.8 ha housing pens and 6 0.05-0.07 ha holding pens. We provided water and two feeders with 20% crude protein deer pellets (Cargill Sportsman's Choice Record Rack) supplied ad libitum in each housing pen. Natural forages including white clover Trifolium repens and various grasses and forbs were also available within each pen.

Design and fabrication of antler coupling
A male-female sleeve and socket coupling device ( Fig. 1) was designed to allow for natural positioning and rigid coupling of a foreign antler to a male after naturally grown antlers had been removed to a consistent height above the base of the pedicle. Preliminary measurements of representative antler base diameters to be coupled ranged from 19.1 to 44.5 mm. Therefore, the maximum inner diameter of the couplings was determined to be 44.5 mm to allow them to slip over any antler within the representative range. The couplings were machined from 50.8 mm outside diameter × 38.1 mm inside diameter 6061-T6 aluminum round tubing. The standard aluminum material is corrosion resistant and offers a strong and lightweight product that minimizes additional weight to the animal. Additionally, the dimensions of the standard tubing were consistent with the size needed for the couplings thus requiring minimal modification and machining. The couplings were designed and fabricated at the J. Charles Lee, Agricultural and Biological Engineering Machine Shop at Mississippi State Univ., Mississippi State, MS by means of standard precision machining procedures with a manual metal lathe to a dimensional tolerance of ± 0.05 mm.
The base coupler serves as the male component of the coupling device (Fig. 2). It is designed to slip over and be affixed to the cut-off portion of the antler above the base of the pedicle and in line with the natural direction of the antler's main beam. The mass of the base coupler is 60.8 g. The upper antler coupling serves as the female sleeve component of the coupling device (Fig. 3). It is also designed to slip over and be affixed to the end of selected foreign antlers. Mounting holes drilled at strategic locations radially and axially through the sidewall of the upper coupling allow the couplings to be secured to the antler with threaded mechanical fasteners. The mass of the upper antler coupling is 63.93 g. Both the upper and base couplers are designed to be installed axially in line with the natural direction of the antler main beam. The two-piece design allows the couplings to be preinstalled separately on selected antlers and deer. The radial design allows couplings to interlock concentrically upon installation and be rotated until a natural alignment and spread between antler beams is achieved. Combined mass of the coupling devices installed on both antlers of the animal is 124.75 g.

Mounting of antler in upper antler coupling
Prior to beginning the antler installation process, we prepared the upper antler couplings by preinstalling antlers with traits related to our trials (Morina et al. 2018). To evaluate female preference for antler size Morina et al (2018) selected smaller and larger antlers relative to the natural range of variation present in their study area. We placed each selected antler inside an upper coupling and positioned it to look as natural as possible when fully installed. We then held the antler in place with no. 10-16 × ¾" hex drive washer head 410 stainless steel self-drilling screws at three evenly spaced locations around the coupler connection. We then filled the interstitial spaces between the antler base and the coupling using Technovit (Jorgensen Laboratories, Loveland, CO, USA), a strong acrylic binding adhesive material (Fig. 4).

Removal and preparation of antler
The antler installation procedure begins with the removal of the naturally grown antlers. For the easiest installation, we sedated the deer prior to installing the antlers and followed sedation procedures outlined in Morina et al. (2018). We used a reciprocating saw to remove antlers at a point 5 cm above the burr at the base of the pedicle. Some antlers are more oval than round while others have burrs projecting above the base burr. These conditions dictated that we use a rotary tool with a cutting blade to reduce the burrs or alter the shape to allow the base coupler to fit.

Installation of base coupling
We slid the base coupler down onto the antler base, leaving 2 cm between the bottom of the base coupler and the antler burr (Supplementary material Appendix 1, BaseCou-  pler.mp4). One must ensure the coupler is in line with the natural direction of the antler main beam. Then, we drilled a pilot hole 10 mm from the bottom of the base coupler through both walls of the coupler and the antler base using a 4.76 mm (3/16" imperial) drill bit of sufficient length (≥50 mm). We inserted a no. 10-24 × 3" 6-lobe drive truss head grade 18-8 stainless steel machine screw (Fastenal Company, Winona, MN, USA) into the pilot hole and secured it using a no. 10-24 grade 18-8 stainless steel nylon insert lock nut (Fastenal Company, Winona, MN, USA). We removed the excess length of the machine screw that extended past the lock nut and filed down the new end to prevent any possible skin irritation. We then drilled a second pilot hole perpendicular to and 10 mm above the first fastener location and repeated the same fastening procedure.
After the fasteners were in place, we filled the interstitial spaces between the antler base and the coupling using anchoring adhesive (AnchorFix-1, Sika Corporation, Lyndhurst, NJ, USA). We applied tape at the bottom of the base coupler to prevent leakage of the anchoring adhesive onto the animal tissue at the base of the antler during the drying period. We then repeated the entire procedure for the second antler base and coupler (Fig. 5).

Installation of upper antler coupling
After we secured the base couplers, we slid each of the upper antler couplings onto their respective base coupling (Supplementary material Appendix 1, Upper.Coupling. mp4). We rotated the antlers to achieve a natural alignment and spread between antler. We then secured the antlers using no. 10-16 × ½" hex drive washer head 410 stainless steel self-drilling screws (Fastenal Company, Winona, MN, USA) at three evenly spaced locations around the coupler connection (Fig. 5). To provide a more natural look to the antlers when presented to females in trials, we wrapped the antler couplings with four layers of beige Color Duck Tape Brand duct tape (ShurTech Brands, Avon, OH, USA) (Supplementary material Appendix 1, NaturalLook.mp4).

Results
During the breeding seasons of 2015-2016 and 2016-2017, we installed antlers on six male white-tailed deer. Antlers remained installed for 10-52 days. The group of manipulated male deer was comprised of two six-yearolds, two three-year-olds and two yearlings. The time needed to adapt to the new antler size differed between age classes. The six-year-old males behaved normally after antler installation and did not require any acclimation period. The three-year-old males carried their heads slightly lower than normal and required a 2-24-h acclimation period, after which they could carry larger antlers with a natural, consistent appearance of the head and neck. The yearling males initially hung their heads significantly lower than normal and had trouble stabilizing movement of the large size antlers. They required a 24-48-h acclimation period. Qualitatively, there was no detectable differences in the behavior of manipulated and non-manipulated males after the acclimation period.
The six-year-old males had manipulated antlers installed for 52 days and the three-year-old males had manipulated antlers installed for 34 days. The yearling males had manipulated antlers installed for 10 days, at which point the yearling male with the large antlers installed broke them off at the fastening location, where the antler integrity was weakened. Upper antler couplings were removed from each male at the end of a set trial period conducted for a separate study (Morina et al. 2018). Base couplings remained on the animals until they fell off during the natural antler shedding period.

Discussion
Using our innovative process for size manipulation of deer antlers, we have provided a technique that can be used to disentangle the influence of antlers from other allometrically related traits in social dominance, intrasexual competition and mate choice. For instance, we used this method to test the ornament function of antlers by manipulating antler size while controlling allometrically related traits, like body size, to isolate the influence of antler size on female mate choice (Morina et al. 2018). Thus, the use of our antler manipulation process can be used in future social and behavioral studies to test current theories for the functions of antlers.
Our method provides more control of antler size manipulation than some previous methods attempted since it allows for increasing antler size in addition to simply size reduction (McComb and Clutton-Brock 1994). Lincoln et al. (1970) artificially increased antler size in red deer Cervus elaphus, but lack of a thorough description of the manipulation process warranted publication of a full description of a similar technique that others could repeat. We believe our technique will be a viable option across all species of Cervidae, although slight modifications may be required for some species due to antler diameter. Both the size of the couplings and the attachment hardware may need to be increased for species with larger and heavier antlers.
In manipulating the antlers of deer, the diameter of the antler bases need to be considered. The yearling male we attached a large set of antlers to had them break off with a forceful collision into a fence. His antler base diameter was approximately 2 cm and was not strong enough to withstand the force exerted on the attachment point of the base antler coupler. Though we did not determine the threshold of antler size to pedicle diameter, we would recommend limiting installation of large antlers to males with appropriate pedicle diameters.
Manipulated antlers worn by older males withstood abuse related to breeding behavior of male deer. Males rubbed their antlers on many surfaces within our research facility including fences, feeders and trees. No antlers manipulated using this technique came loose or broke off. In fact, one of the males in our study broke an antler tine off of his installed antlers with no damage to the antler coupling device. However, we did not allow males to directly interact with manipulated antlers installed, so it is possible there may be some limitation for their use in competition events. Based on their durability during our trials, we believe they would hold up in male-male combat but we cannot say what the failure threshold would be. Since the devices are relatively small, it would be possible to increase the length or thickness of the coupler devices to increase strength as needed.