In nutritional ecology the intake target is the diet that maximises consumer fitness. A key hypothesis of nutritional ecology is that natural selection has acted upon the behavioural and physiological traits of consumers to result in them Selectively Consuming prey to match the Intake Target (SCIT). SCIT has been documented in some herbivores and omnivores, which experience strong heterogeneity in the nutritional quality of available foods. Although carnivores experience a prey community with a much more homogeneous nutrient composition, SCIT by carnivores has nevertheless been deemed highly likely by some researchers. Here we test for SCIT for micronutrients (amino acids) in two freshwater carnivores: the river blackfish and the two-spined blackfish. Although both blackfishes exhibited non-random consumption of prey from the environment, this resulted in non-random consumption of amino acids in only one species, the river blackfish. Non-random consumption of amino acids by river blackfish was not SCIT, but instead an artefact of habitat-specific foraging. We present hypotheses to explain why wild populations of freshwater carnivores may not exhibit SCIT for amino acids. Our work highlights the need for careful, critical tests of the hypotheses and assumptions of nutritional ecology and its application to wild populations. A key hypothesis of nutritional ecology is that consumers selectively consume foods to match the consumer’s ‘intake target’4 . In nutritional ecology, the ‘intake target’ is defined as the diet with the nutritional composition that conveys maximal fitness. Regulation of dietary intake increases fitness by reducing the costs of nutrient deficiency and excess. Deficiency occurs when demand for a nutrient exceeds supply. Deficiency can alter the processing of a nutrient so that it is allocated to critical maintenance functions, with little or none remaining for growth and/or reproduction. Such responses can occur when animals are confronted with diets deficient in only a single nutrient (Liebig’s Law of the minimum)5. Excessive consumption of particular nutrients can also reduce growth and reproductive performance due to the metabolic costs associated with processing and excreting superfluous nutrients, and negative feedback (termed ‘jamming’) on feeding control systems7. Jamming can occur when excess nutrients desensitize taste receptors8 or act as blood-borne suppressors of feeding9. This can prevent satiation of other nutrients. To mitigate these costs natural selection may have acted upon the sensory and behavioural traits of organisms to facilitate detection, tracking and selection of prey that match their intake target.Nutritional ecologists aim to understand the effects of dietary composition on the demography and persistence of populations4, but the generality of such findings remains limited for three reasons: (1) food selection to regulate nutrient consumption has mostly been demonstrated under laboratory conditions where the composition of diets is tightly controlled and contrasted, and consumers have more freedom to discriminate and select foods, when compared to natural settings; (2) most studies focus on macronutrients ; and (3) most studies concern herbivores and omnivores11.SCIT has been demonstrated in diverse taxa10. This contrasts with herbivores and omnivores, which are faced with greater nutritional challenges due to the nature of plant constituents, i.e., digestibility, caloric value, inhibiting factors14. As such, carnivores are largely assumed to optimise the size of prey and/or the rate of prey capture to maximise energy intake while foraging15; this is the fundament of optimal foraging theory. Optimal foraging theory is supported by ecological stoichiometry studies which suggest that carnivores are most likely to be C (energy) limited, as their diet is sufficient for their requirements of other nutrients and is more efficiently assimilated17. Contrasting with those stoichiometric studies, recent laboratory studies have indicated that carnivorous invertebrates, fish, and mammals may regulate the intake of macronutrients through selective consumption of whole prey or particular tissues, or selective extraction from pre-digested prey (i.e. some web-building spiders)22. In addition, carnivores in the wild have been observed to select prey non-randomly from the environment25, selectively consume specific tissues/organs of prey10, and achieve consistent pattens in macronutrient intake28. The latter has been seen in Australasian gannets (Morus serrator) for example, where males consistently consumed a diet that had higher protein-to-lipid ratios compared to their female counterparts, possibly in response to sex-specific nutrient requirements26. Although these studies have documented selection of nutrients from the environment, they have not demonstrated selection towards a nutrient intake target, and so it is unknown whether these cases of selective foraging represent selective foraging for a fitness advantage. Studies documenting selective foraging for macronutrients by carnivores raises the question: do carnivores selectively forage for micronutrients?Active nutrient regulation has historically been thought to be unnecessary in carnivores34. Recent experimental work on the nutritional ecology of blackfishes demonstrated that diets composed of an amino acid profile that is dissimilar to their intake target reduces performance (e.g. reduced protein synthesis and increased nitrogen wastage)35. As wild animal prey—including birds, mammals36, teleosts, cephalopods, crustaceans37, and insects42—vary in amino acid composition, this produces potential for carnivores to gain fitness by selecting the right balance of amino acids, to avoid the costs of under- and over-consumption.Individual amino acids can act as a structural unit, an energy source, and can play other functional roles. Diversity in the roles of different amino acids suggests that animals may have different requirements for specific amino acids. This is seen in laboratory studies of production animals (used in agriculture and aquaculture), where supplementation of individual amino acids promoted growth and in other instances, inhibited growthDicentrarchus labrax) selected foods with concentrations of taurine (a sulfonic acid derived from cysteine) that promotes growth43; rainbow trout (Oncorhynchus mykiss) discriminated between diets deficient in lysine within 3–17 days, but not those deficient in methionine44; and rainbow trout were also seen to increase their intake when restricted to diets deficient in particular suites of essential amino acids45. While these studies used artificial diets, they demonstrate the roles of olfaction and gustation46, neurological feedback, and/or associative learning48 in regulation of amino acid intake. Determining whether wild carnivores can accomplish these patterns of intake in complex natural habitats is far more difficult. A recent study provided evidence that common kestrels  select prey in the wild with high contents of sulfur amino acids41, but as has been the case for studies of macronutrient selection by carnivores, this study did not characterise the intake target. If we are to determine the extent to which the well-developed theoretical framework of nutritional ecology applies to carnivores, then tests of its hypotheses and assumptions are required using wild carnivore populations whose nutrient intake targets are defined.In experimental settings, carnivorous fishes use behaviours and sensory systems that may enable them to detect and select amino acids in their free or polymeric forms (bound in peptides or protein) . For example, sea bass (Gadopsis marmoratus) and the two-spined blackfish (G. bispinosus), exhibit SCIT for amino acids. We determined the prey and amino acid composition of blackfish diets and their surrounding environment. Our previous work has shown that the amino acid composition of blackfish bodies approximates the amino acid intake targets of these carnivores35. Our first hypothesis (H1) was that the intake targets of both carnivores would be different from the amino acid composition of the environment , the nutritional composition of the environment that is available to blackfish, (b) the composition of the diet that was consumed by blackfish, and (c) composition of the blackfish themselves—the intake target .2 quadrat. Samples from the water column were collected using drift nets , deployed overnight (approx. 1700–0800 h) to coincide with the nocturnal feeding of blackfish. The nets were positioned with the opening above the water surface to facilitate the capture of both floating and drifting prey. Triplicate measurements of water velocity were taken at four points within each drift net at deployment and collection. All samples were stored in ethanol, which does not alter the primary structure of proteins, hence amino acid composition50.To precisely characterize the relative abundance of prey and amino acids throughout our study reach, macroinvertebrates were randomly sampled from the benthic substrate , ‘edge habitat’ (steep banks of compacted sand and silt), and the water column , with fine tubing  connected, was filled with clean stream water. The tubing was inserted past the oesophagus and water was discharged into the stomach to trigger each fish to disgorge its stomach contents. Stomach contents were immediately preserved in ethanol. As blackfish are primarily nocturnal, all samples were taken between 0730 and 1200 h to minimise biased estimation of gut-composition caused by digestion. Sample collection was extended over four days, within an eight-day period, to ensure the estimate of diet was not biased by prey availability on a single night. All fish were released at the point of capture, except for 18 individuals (9 of each species) that were humanely killed, transported back to the laboratory on ice and stored at − 20 °C. These were then dissected to assess the efficiency of stomach flushing. Flushing accounted for 93% (± 1 SD) of all prey individuals. These 18 individuals were also used in subsequent amino acid analysis to characterise the intake target of the blackfishes.51.Macroinvertebrates from gut and environment samples were separated from debris, enumerated, and identified to genus (where possible) with the aid of stereo microscopic examination . Where environment samples contained copious debris or animals, a 100-cell Marchant sub-sampler was used to randomly select cells that were successively sorted until at least 250 animals were collected in total. A total of 135 gut samples (42 from river blackfish and 93 from two-spined blackfish) and 68 environment samples  were used to test for prey selection. Of these, ten samples from each habitat type and 20 gut-content samples from each fish species were individually analysed for amino acid composition. These sample sizes were adequate to precisely characterise prey and amino acid composition of the nutritional environment and the diet, as confirmed by the multivariate dissimilarity-based standard error . High-performance liquid chromatography (HPLC) and Empower software  were used to separate, identify, and quantify amino acids. See Dwyer et al.38 for further details.Individual gut, environment and whole fish body samples were used for amino acid analysis. Gut and environment samples were heated to 55 °C overnight to remove ethanol via evaporation. These samples, along with the fish body samples, were then freeze dried. Dried samples were weighed, reconstituted in Milli-Q water and homogenized. Triplicate aliquots of each sample were then freeze dried and hydrolysed with 6 N HCl containing 0.02% Phenol at 116 °C for 20 h. As acid hydrolysis causes the deamination of asparagine and glutamine, we use 39. These comparisons included (H1) the nutritional environment and the intake target (significant differences indicate nutrient mismatch), (H2) the nutritional environment and the gut contents (significant differences indicate selective consumption), and (H3) the gut and the body of each species (no significant difference indicates minimized metabolic regulation). These analyses were completed for each blackfish species separately. Further comparisons were made between the body composition of the two blackfish species, between the gut contents of the two blackfish species, and among the drift, edge, and benthic prey communities. Where analyses involved comparisons with the nutritional environment (H1 and H2), weighted LDA was used. This allowed the density of prey and amino acids available within the three habitat types  to be weighted by the surface area  or volume (water column) of each of the three habitat types within the study reach at the time of sampling. Pairwise comparisons of blackfish gut contents with each habitat individually illustrate the direction in which these results would change with increasing importance (weighting) of each habitat  was used to compare prey taxa or amino acid compositions of sample sets40. Prey and amino acid data from environment, gut, and body samples were transformed to proportional abundance, as is necessary given the disparate nature of the samples being compared . For the prey data, the noise-inflating effects of rare species were removed using McArdle’s algorithm53, leaving only those taxa that had a ≥ 95% probability of being detected given our sample size. This resulted in the retention of 62 of the 114 taxa found in the diet of the two-spined blackfish, and 41 of the 86 taxa for the river blackfish for analyses. Taxa which were removed in this process were found in fewer than 3 gut samples. For the amino acid data, extreme outliers for each amino acid (identified using Tukey's range test) were removed from the data set and replaced with the mean value within each sample set. A total of 35 outliers were identified ], methionine, and valine were in higher proportions in two-spined blackfish, whereas serine and threonine [correlated with alanine(–)] were higher in river blackfish.The amino acid composition of bodies of the blackfish species–their intake targets–differed significantly , threonine(–), tyrosine(–), Glx(+)] and Asx. In this analysis, lysine was also negatively correlated with phenylalanine(–) for river blackfish. Of these amino acids, lysine, Glx, and alanine were in higher proportions in the fish, and so may be limiting, whereas tyrosine, proline, threonine, and phenylalanine were in high proportions in the environment, and so may be available in excess of requirements. For both carnivores, lysine was the only essential amino acid in higher proportions in the body compared to the composition available in the amino acid landscape , Acruroperla (Plecoptera), Baetidae (Ephemeroptera), and Tanypodinae (Diptera) than found in the available prey community. The prey community instead was characterised by high proportions of Atelophlebia, Nousia and Centroptilum (Ephemeroptera), Sternopriscus larvae (Coleoptera), Ecnomus and Aphilorheithrus (Trichoptera), and Parakiefferiella and Polypedilum (Diptera). This was not a consequence of the blackfish foraging selectively in one of the three habitats  as all three prey communities differed from the gut contents of both species , Talitridae (Amphipoda), Orthocladiinae, Rheotanytarsus, Paramerina and Nilobezzia (Diptera), and Sternopriscus larvae (Coleoptera) in the gut contents of river blackfish. By contrast, the gut contents of two-spined blackfish contained higher numbers of Chironominae, Austrosimulium, Tanytarsus and Chironomidae pupae (Diptera), Sclerocyphon larvae and terrestrial adults (Coleoptera), Taschorema complex (Trichoptera), and Gripopterygidae (Plecoptera).A comparison of river and two-spined blackfish gut contents revealed significantly different prey compositions  but may reflect habitat-specific foraging. The gut contents of river blackfish were not significantly different from the drift , but were different from the edge  and benthic prey communities  , tyrosine(–), Asx(+) and Glx(+), and phenylalanine(–)] and threonine, which were higher in the intake target. By contrast, retention was lower for isoleucine [correlated with leucine(+) and cystine(–)] and proline, which were higher in the gut , Glx(+), phenylalanine(–), alanine(+), and threonine(–)], which were higher in the body. In contrast, proline [correlated with leucine(+) and isoleucine(+)], valine, methionine, and serine were higher in the gut . For two-spined blackfish separation of the gut and intake target was largely influenced by lysine [correlated with alaninegut Fig. .55? To our knowledge, our study is the first to test this hypothesis within the context of amino acids. Our data showed that there is a mismatch between the composition of amino acids in the environment and the intake target of both blackfish species. Consequently, random foraging by the carnivores studied here would result in amino acid diets significantly different from their intake targets. Although selection of prey species by two-spined blackfish was non-random, the amino acid composition of their diets was not significantly different from that of the environment. By contrast, non-random selection of prey species by river blackfish did equate to an amino acid diet that was significantly different from that expected under random foraging from the environment. However, our data showed that this non-random selection of amino acids by river blackfish was not in the direction of the intake target, but was instead likely due to habitat-specific foraging, whereby the amino acid composition of their diets resembled that of the prey available in the drift. We therefore found no evidence for SCIT in our study system.A key question at the frontier of nutritional ecology is: do carnivores exhibit SCIT56. Further, none of these studies obtained data on the micronutrient composition of different habitats that the carnivores forage within, nor did they have data on the micronutrient intake target of the carnivores. As such, these studies were not able to determine whether non-random selection of micronutrients by carnivores was due to selection towards the intake target; nor could they rule out non-random micronutrient diets due to habitat-specific foraging. By including detailed data on the amino acid composition of habitats and characterisation of the intake target35, the present study was able to better elucidate the likely cause of non-random consumption of amino acids—habitat-specific foraging by the carnivore , and not selection towards the intake target (both blackfishes).Few other studies have documented non-random selection of micronutrients from the environment by carnivoresThis study shows that the environment is mismatched to the consumers’ requirements, but that the consumers are not selecting amino acids towards their intake target. Although the fitness cost of this mismatch is unknown, the physiological literature (see H1 Nutrient mismatch) indicates that such a mismatch would result in some energetic costs to the fishes we studied. Thus, our results raise the question: why do wild blackfish not exhibit SCIT?57, such as macronutrients, or possibly other micronutrients like fatty acids. Under this hypothesis, natural selection may have optimised blackfish foraging behaviour towards achieving a different nutrient intake target, trading-off traits that lead to selective amino acid foraging. Supporting this, Jensen et al.58 found carnivorous beetles regulated their lipid and protein intake rather than the intake of micronutrients or other chemical components58. Few other studies have investigated the trade-offs between different intake targets; however, much laboratory evidence suggests that carnivores exhibit SCIT for macronutrients59.The fitness costs incurred by not achieving the amino acid intake target may be small relative to not achieving the intake target of different nutrient groups60. In the wild, carnivores encounter foraging costs, including chemical and physical prey defences, lost foraging time due to antipredator behaviour61, and high metabolic activity costs. These foraging costs must be significant, as natural selection has favoured many foraging strategies, including ambush predation, co-operative hunting, trapping and increased handling capacity, which reduce such losses64. While these foraging traits maximise net energy gain, they also intensify the opportunistic nature of predation and so may preclude selective foraging for amino acids. Two arguments supporting a strategy aimed at maximising energy intake over one that minimises nutrient imbalance are: (1) carnivores are adapted to over-ingest protein10 as they have a greater capacity to metabolise excess amino acids for cellular needs and energy demands; and (2) they have greater control over nitrogen excretion to avoid toxic accumulation65. For aquatic ammoneotelic animals (e.g. most bony fishes), in particular, catabolism of amino acids into ammonia requires little energy and the ensuing ammonia passively diffuses across the epithelia into the ambient water66.Alternatively, a foraging strategy aimed at minimising nutrient imbalances through SCIT—which, by extension, implies certain prey available to the carnivore are rejected—may be less fit than a strategy aimed at maximising energy intake and digestive regulation of any nutrient excesses post consumption. Under this hypothesis carnivore growth is more limited by energy intake, in accordance with ecological stoichiometry (energy represented by carbon) and optimal foraging theory (joules)67. However, the relative importance of amino acid composition compared to other nutrients (i.e. fatty acid composition) or energy content is still largely unknown. In addition, while one study has shown that increasing intake (high quantity) does not ameliorate dietary imbalances in amino acid composition 45, little research has investigated whether nutrient inadequacies that may occur in the wild can be rectified by increasing consumption to maximise energy gain. Without a full understanding of these fitness costs, it is difficult to understand what aspects of the nutritional landscape are guiding prey selection.In conclusion, this study has shown that selective consumption of prey by wild carnivores is unlikely to be driven by selective foraging for amino acids. If the amino acid composition of prey communities varies among habitats, then selective consumption of amino acids by carnivores may be an artefact of habitat-specific foraging. Further critical tests of the application of nutritional ecology to wild carnivore populations are required. To determine whether selection of amino acids from the environment is a trait that improves carnivore fitness—and hence is consistent with nutritional ecology theory—future studies will need to (a) characterise the nutrient intake target; and (b) characterise the nutrient composition of different foraging habitats. To resolve whether competing nutritional demands or energetic costs are constraining blackfish from regulating their intake of amino acids in the wild, further research in micronutrient ecology needs to be directed towards understanding the fitness advantages and disadvantages of selecting groups of micronutrients in relation to others. For instance, numerous aquaculture studies have shown that the amino acid profiles of foods influence the fitness of consumers, and consumers may also select foods balanced in amino acid composition over total protein levelsSupplementary Information.


Brain tissue contains the highest number of perivascular pericytes compared to other organs. Pericytes are known to regulate brain perfusion and to play an important role within the neurovascular unit (NVU). The high phenotypic and functional plasticity of pericytes make this cell type a prime candidate to aid physiological adaptations but also propose pericytes as important modulators in diverse pathologies in the brain. This review highlights known phenotypes of pericytes in the brain, discusses the diverse markers for brain pericytes, and reviews current in vitro and in vivo experimental models to study pericyte function. Our current knowledge of pericyte phenotypes as it relates to metastatic growth patterns in breast cancer brain metastasis is presented as an example for the crosstalk between pericytes, endothelial cells, and metastatic cells. Future challenges lie in establishing methods for real-time monitoring of pericyte crosstalk to understand causal events in the brain metastatic process. Breast cancer (BC) remains the leading cause of cancer death and disability-adjusted life years in women , and appBC is a heterogeneous disease that can be categorized based on multiple morphological and molecular properties ,11. One BCBM can occur in different CNS regions. Rostami et al. reported that 52.2% of BCBM were supratentorial and 24.1% infratentorial, and 14% of patients had metastases at both locations . MagnetiBrain metastatic colonization is a multistep process that starts at the primary tumor site and includes epithelial-to-mesenchymal transdifferentiation (EMT) to enable BC cells to detach from the primary BC tumor, penetrate extracellular matrices, and invade neighboring tissues. The invasion of blood and lymph vessels signals a major metastatic leap and provides a selective advantage for the evolution of a heterogeneous group of tumor cells that have developed strategies to survive as circulating tumor cells (CTC) in the blood and lymph and crucially have the ability to colonize distant organs . The estThe neurovascular unit (NVU) is an anatomically complex functional brain microenvironment that the brain metastatic BC cells encounter first when entering the brain vasculature . This NVThe NVU is the site of entry of BC cells for the establishment of BCBM in the brain parenchyma ,28. ActiThis review focuses on pericyte populations at the NVU, their molecular markers, and the roles of these brain pericytes during BC cell colonization of the brain.Pericytes were first identified in 1873 when Charles-Marie Benjamin Rouget described a population of contractile cells surrounding the endothelial cells (EC) of small blood vessels. Originally, these cells were called “Rouget cells”, but later Zimmermann termed these cells “pericytes” (PC), as this name best describes their location around capillary vessels . The vasPericytes regulate the BBB by reducing the expression of endothelial genes involved with transendothelial permeability ) and promote astrocyte end-feet polarization ,33,44. CPericytes also have critical roles in tumor vasculature. In fibrosarcoma and osteosarcoma mouse models, pericytes were shown to display aberrant PDGF signaling and, similar to mice lacking PDGF-B and its receptor, this coincides with increased blood vessel diameters and reduced endothelial cell junctional circumference in the tumor vasculature ,50. PeriImproper identification and frequent mix-ups with adjacent cell types, e.g., vascular smooth muscle cells (vSMCs) and juxtavascular microglia, have resulted in conflicting data being published . Hence, Several distinct types of pericytes have been described based upon the morphological appearance of their processes. This includes ensheathing, mesh, and thin-strand pericytes  26]. Th. Th26]. The expression of CD146, also referred to as melanoma cell adhesion molecule (MCAM) or cell surface glycoprotein MUC18, was shown to be primarily confined to vSMC. High αSMA expression in CD146+ cells in vitro suggests that CD146 identifies a subpopulation of vSMC, but a role of CD146 as a pericyte marker is questionable . These fAmong the most common and adverse scenarios associated with BBB disruption are intracranial metastatic lesions . The estDisruption of BBB integrity in BCBM arises from direct cell–cell interactions between BC cells and the NVU and the secretion of a broad range of cytokines/chemokines derived from tumor cells. In fact, CX3CL1 and CXCL13, also known as B cell-attracting chemokine (BCA-1), are notably elevated in patients with BCBM and associated with poor outcomes . CX3CL1 Vascular endothelial growth factor (VEGF) is implicated in the formation of BM and is frequently overexpressed and secreted by tumor cells in BM . PericytThe initial steps of BC cell brain colonization have been described: (1) arrest of BC CTCs in brain capillaries, (2) BC cell passage through the BBB, (3) extravasation of BC cells from capillaries that are surrounded by PDGFRβ+ pericytes, and (4) initial growth of extravasated BC cells in the perivascular niche ,82 Figu. Upon suThe intravascular arrest of BC cells at the NVU includes cell–cell interactions of metastatic BC with microvascular EC, and this is sufficient to activate astrocytes. Upon extravasation, the formation of micro-metastatic lesions coincides with the appearance of activated astrocytes that accumulate both around and inside of the metastatic foci and form direct contacts with tumor cells . The rol+ pericyte density and reduced CD31 staining, suggesting that pericytes take an active role in tumor microvessel formation [Despite the close proximity between pericytes, EC, and cancer cells during and after the trans-endothelial passage, the detailed role of brain pericytes in the process of BBB invasion and metastatic niche formation is largely unknown. BC dormancy in the brain perivascular niche was shown to be controlled by thrombospondin-1 secretion from well-differentiated EC . Sproutiormation . LineageCancer cells and other cellular components of the tumor microenvironment respond to depleted oxygen levels with increased activity of hypoxia-inducible factors (HIFs)  and the Imbalances in the cellular homeostasis may generate cell stress from aberrant protein folding, which leads to ER stress. This causes the activation of the unfolded protein response (UPR) as an adaptive cellular response aimed at restoring homeostasis and cell survival . ER streBM can be distinguished on the basis of their invasion patterns. Berghoff et al. proposed to categorize all metastatic brain lesions into three distinct types: well-demarcated, vascular co-option, and diffuse . AnotherMetastatic growth was accompanied by an increased pericytic expression of Serpin H1 (an enzyme involved in collagen biosynthesis) and αSMA, which coincided with elevated levels of collagen in the vessel walls . Hence, Several findings suggest a critical role of pericytes in the development of BM lesions by directly influencing key steps of tumor progression. In a syngeneic mouse 4T1 mammary carcinoma model of brain metastasis, pericytes were shown to be located adjacent to capillaries within BM lesions . In vitrPericytes have been predominantly investigated for their role in BBB functions. Several in vitro BBB models have demonstrated the role of pericytes in supporting BBB function ,140,141.Replicating the functions of pericytes for a functional BBB requires culture conditions that reflect the proper cellular and spatial organization of the NVU. Transwell inserts are most commonly used for in vitro models of the BBB  for studTranswell systems are limited by the large fluid-to-cell volume ratio and lack of flow, which is a critical parameter in blood vessels and a major contributor of shear stress in EC . MicroflMicrofluidic devices are promising BBB model systems that provide the in vivo microenvironment more closely and have the potential to be adapted for high-throughput clinical studies . Microfl(Pdgfrβ), notch receptor 3 (Notch3), and neural/glial antigen 2 (Ng2) depict pericytes in zebrafish. Transgenic lines are suitable for lineage tracing and monitoring pericyte plasticity in transgenic zebrafish, and the short developmental duration and the transparency of the zebrafish larvae allow for in vivo lineage tracing of mural cells during development [The high variability in the temporal and spatial occurrence of hematogenic brain metastasis imposes great challenges for in vivo tracing and monitoring of pericyte functions during the initiation and formation of brain metastases. Two-photon imaging through a chronic skull window has been used for in vivo pericyte imaging by monitoring calcium flux in anesthetized transgenic mice expressing genetically encoded calcium sensors in ensheathing pericytes . A multielopment  and makeelopment  suitableA better understanding of the interactions between BC and the NVU as well as the BCBM tumor microenvironment is critical in advancing effective treatments against fatal brain metastases. Newly emerging in vivo molecular imaging and tissue profiling technologies are expected to reveal detailed gene and/or protein expression patterns at a high contextual and spatiotemporal resolution. This will excel our understanding of the diversity and functional roles of pericyte populations in normal and diseased tissues, including the NVU and brain metastases. Putting these spatial tissue data to the test in sophisticated in vitro devices as well as genetic and tumor animal models will contribute major advances to our understanding of BCBM and be instrumental in the development of new and more efficacious treatment options for breast cancer and other cancer patients with brain metastasis.


Current treatment for chronic HBV infection is mainly based on nucleos(t)ide analogues, that in most cases need to be administered for a patient’s lifetime. There is therefore a pressing need to develop new therapeutic strategies to shorten antiviral treatments. A severe dysfunction of virus-specific T cell responses contributes to virus persistence; hence, immune-modulation to reconstitute an efficient host antiviral response is considered a potential approach for HBV cure. In this perspective, a detailed understanding of the different causes of T cell exhaustion is essential for the design of successful functional T cell correction strategies. Among many different mechanisms which are widely believed to play a role in T cell dysfunction, persistent T cell exposure to high antigen burden, in particular HBsAg, is expected to influence T cell differentiation and function. Definitive evidence of the possibility to improve anti-viral T cell functions by antigen decline is, however, still lacking. This review aims at recapitulating what we have learned so far on the complex T cell–viral antigen interplay in chronic HBV infection. CD8 T cells are pivotal elements of the antiviral immune response. Following recognition of antigen exposed on antigen-presenting cells (APC), CD8 T cell activation and differentiation can result either into a fully functional response with T cell memory generation, which is key to controlling infection, or to dysfunctional T cell responses of different severity, up to exhaustion and even physical T cell deletion if the pathogen is not successfully eliminated and antigen exposure persists overtime. T cell exhaustion during persistent viral infections is a well-documented phenomenon, which is characterized by the progressive and hierarchical loss of antiviral functions . Among mHepatitis B virus (HBV) infection targets the liver, which represents a unique immunological environment enriched with different antigen presenting cell populations which contribute to its tolerogenic properties . Thus, tAs many other viruses, HBV exploits the production of a large excess of viral antigens as a mechanism to evade the host immune response . Among HUnderstanding the effect of HBsAg over-expression on host immune responses can provide important information in the perspective of devising new therapeutic strategies for chronic hepatitis B and novel reliable biomarkers to monitor the effect of therapy and the evolution of infection. In this perspective, this manuscript will analyze studies aimed at improving our knowledge of the causal link between HBsAg expression and T cell dysfunction in chronic HBV infection, with the awareness that this represents just one among numerous factors playing a role in T cell exhaustion.HBV virions are composed of an icosahedral nucleocapsid, formed by core proteins (HBcAg) containing partially double-stranded DNA (rcDNA: relaxed circular DNA) and a viral polymerase. The nucleocapsid is surrounded by the envelope, consisting of the surface antigen (HBsAg). Six polyadenylated transcripts derive from the viral genome, including the pregenomic RNA (pgRNA), and seven proteins : HBeAg (The HBV envelope consists of three proteins: the large (L), middle (M) and small (S) proteins, originated by starting translation at different sites of the HBV genome. The small (or major) protein contains only the HBs antigen, which is referred to as the S domain; the middle protein contains an additional 55 AA sequence that represents the PreS2 antigen and finally the large protein, in addition to HBs and PreS2, is composed of an additional antigen sequence which is called PreS1 . BesidesThe precore/core gene codes for an amino acid sequence corresponding to the whole core protein plus 29 residues. The N-terminal 19 amino acids represent a signal peptide that drives the newly synthesized protein toward the secretory pathway. Following removal of the signal peptide and further proteolytic cleavages at the C-terminal end of the precore sequence, a 17 kDa protein is generated which is the predominant secreted moiety of the pre-core gene translation that is called e antigen ,14. Inte10 U/mL, that would preclude its application to patients with low viral replication. However, new and more sensitive tests are being developed [Recently, the simultaneous detection of serum HBcAg, HBeAg and p22 precore-derived proteins, all sharing an identical 149 amino acid sequence, together referred to as Hepatitis B core-related antigen (HBcrAg), has been proposed  Figure  as a poteveloped , which cAnimal models of infection have been used to elucidate the interdependence of antigen load and T cell exhaustion in persistent infections.The amount of antigen and the duration of antigen presentation were shown to negatively influence the T cell function, ultimately leading to impaired viral clearance in mice with chronic LCMV-CL13 infection . In thisThese studies, however, did not specifically address the main immunological issue related to hepatotropic infections, i.e., the antigen presentation to T and B cells within the peculiar tolerogenic environment represented by the infected liver . To elucper se would promote a defective differentiation program that became partially irreversible and gradually triggered an exhaustion profile [The notion that high levels of antigen expression can induce T cell dysfunction in CHB has recently been challenged, taking advantage of different mouse models, allowing the expression of variable levels of antigen within the liver, and the activation of different pathways of antigen presentation by hepatocytes, or by Kupffer cells (KC) and hepatic dendritic cells upon transduction with a LCMV-based vector. As expected, HBV-core expression by 100% hepatocytes led to CD8 T cell dysfunction, but the scenario did not change when the amount of hepatocellular antigen was dramatically reduced by more than 15-fold. Instead, antigen-transfected KC induced a correct T cell differentiation into effector cells, with a completely divergent transcriptional and chromatin-accessibility profile. Thus, hepatocyte T cell priming rather than high antigen levels  profile ,30. Thes profile .3–104 IU/mL), 1–2 log10 antigenemia decline induced by small interfering RNA compounds targeting all HBV transcripts (shHBV) did not result in improved CD8 T cell function. However, decline of HBV antigen expression by RNA interference, but not inhibition of HBV replication by NUC treatment, allowed a significantly stronger T cell response with control of infection upon a subsequent heterologous therapeutic vaccination (TherVacB) with protein priming followed by Modified Vaccinia Ankara (MVA) virus vector boosting [In line with these findings, in two different immune-competent mouse models of persistent HBV infection expressing clinically relevant HBsAg titers  targeting viral transcripts , nucleicDefinition of extent and duration of viral protein decline sufficient to allow the restoration of a functionally efficient immune response will be also very important, as well as the identification of reliable biomarkers to predict possible virus rebounds. Finally, a reasoned combination of more than one approach to target different mechanisms of immune dysfunction and tailoring therapy also by patient age will have to be considered in the objective of extending functional cure to a larger proportion of infected patients. In particular, the possibility of an earlier start of therapy will have to be considered in the light of the concept that younger patients with shorter duration of exposure to the virus could likely get the maximal benefit from a successful immune rejuvenation.


Mycobacterium abscessus (Mab) are rapidly growing mycobacteria that cause severe and persistent infections in both skin and lung tissues. Treatment regimens involve the extended usage of complex combinations of drugs, often leading to severe adverse side effects, particularly in immunocompromised patients. Current macrolide therapies are gradually proving to be less effective, largely due to emergence of antibiotic resistance; there is therefore an increasing need for the discovery of new antibacterials that are active against Mab. This review highlights recent research centred upon a number of potential therapeutic targets from Mab , and discusses the various approaches used to discover small molecule inhibitors, in the search for future antibiotics for the treatment of Mab infections. Mycobacterium absecessus (Mab).Recently reported inhibitors developed against targets from  Mab are rapidly growing NTM, that cause severe and slow to repair tissue damage (chiefly in the skin and lungs), and have strong clinical correlation with the progression of decline in lung function in a number of pulmonary disorders, including cystic fibrosis (CF).2 Current preferred CF treatment involves prolonged use of a complex cocktail of drugs3 including a macrolide antibiotic, such as azithromycin (1), in combination with amikacin (2) and a third antibiotic   and sensory disturbances (hearing and sight).3atment)] . DiseaseMab over recent years, alongside research into the discovery of small molecule inhibitors of these targets as potential new medicines. This review aims to collate recently reported advances within this area of research (within the last 6–8 years of scientific literature), by way of reference to five key protein targets that have been identified , and highlights the differing approaches utilized, as well as some of the more important factors that were taken into account, as part of these various studies.The urgent need for improved treatment opportunities for such difficult to treat conditions has led to an increase in research into the discovery of potential therapeutic targets from 1.1.Mab are commonly occurring contaminant bacilli of soil and water samples. They are acid-fast, and stain positive using the Gram stain procedure. As with all mycobacteria,5 the bacilli are coated with a thick and complex waxy outer membrane that is rich in mycolic acids (and mycolates), rendering them relatively impervious to many antibiotics. Mab are rapidly growing mycobacteria (RGM), in stark contrast to other more notorious mycobacteria that have caused major health issues around the globe for centuries; Mycobacterium leprae (Mlep) and Mycobacterium tuberculosis (Mtb) [the causative agents of leprosy and tuberculosis (TB) respectively] both have extremely long doubling times. Compared to both Mlep  and Mtb , Mab are a more recent discovery1 and were only formally recognised as a species6 as recently as 1992. Subsequent subdivision of the Mab species into three subspecies  occurred as late as 2013, the two major subspecies being abscessus and massiliense.7 Although numerous new Mab strains have since been recorded from clinical isolates , in general, knowledge of Mab remains relatively scant.1.2.8 including Mab, constitute a serious clinical issue, being associated with high morbidity and mortality.9 Limited precise mortality data for Mab exists, however a five-year follow-up of the effect of Mycobacterium avium (Mav) in non-immunocompromised (HIV-negative) patients receiving standardized treatment (in the absence of any macrolide antibiotics), noted a 36% mortality rate.9 Prognosis with macrolide therapy is also relatively poor. Five year mortality rates amongst HIV-negative adults, including many who had received macrolides as part of a more complicated cocktail of drugs, have been reported from studies in both Denmark10 and Japan11 as 40% and 24% respectively.Pulmonary infections caused by a number of closely related non-tuberculous mycobacteria (NTM),Mab show a high degree of resistance to many antibiotic drugs that might normally be prescribed for the treatment of bacterial infections. The waxy and relatively impermeable Mab cell walls act as a natural barrier to the entry of drug molecules into the organism, in a purely physical sense (via size exclusion phenomena based upon compound molecular volume) and also physicochemically (due to the highly hydrophobic nature of this heavily lipid laden barrier), so preventing the absorption of drugs that possess lower cLog P. Added to this, Mab also possess further resistance mechanisms, both intrinsic and inducible,12 against xenobiotics that show higher degrees of cell permeability, including several medicinally important macrolides.13 A number of varied factors play a role, including target-side modification, efflux and drug inactivation through metabolism.14 Target-side modification, involving either post-transcriptional ribosomal RNA methylation15 or gene mutation, results in the macrolide binding with much reduced affinity, thereby decreasing treatment effectiveness against the bacterium.Target-side modification is a key mechanism responsible for microbial resistance to macrolide therapies, that inhibit bacterial protein synthesis. When bound to their ribosomal target, macrolides prevent the peptidyltransferase from adding the growing peptide attached to tRNA onto the following encoded amino acid and inhibit ribosomal translocation.Mab infections. Of the five superfamilies of multidrug efflux pumps that are present in bacteria ,16,17 categorized based upon the mode of transport and energy coupling methods that they utilize, many are expressed in Mab ,18 contributing towards resistance to macrolides19 and other antibiotics. In comparison to other mycobacteria, the number of genes in Mab encoding for these proteins is high. As a representative example, the mycobacterial membrane protein large (MmpL) family of proteins20 are members of the RND superfamily of efflux pumps. Five such proteins have been noted in Mlep and fourteen in Mtb (H37Rv strain), whereas in Mab there are thirty one21 .Efflux of drug molecules constitutes a major issue in the treatment of via a number of mechanisms. Drug-modifying enzymes such as β-lactamases, esterases and hydrolases are common, as are other proteins that are able to reduce the effectiveness of xenobiotics by way of processes involving methylation, phosphorylation and specific oxygenation. In mycobacteria, some or all of these mechanisms may be prevalent for a given antibiotic, meaning that some classes of drugs are more at risk that others. The front line drugs are most at risk, and multidrug resistant strains of mycobacteria are becoming more and more prevalent with in the clinical setting. In Mab, a number of such processes are known, and have been recently highlighted.23Drug inactivation by way of metabolism may occur Mab, the highly complex cell structure, coupled with the various important resistance mechanisms outlined above, make the task of discovering new drugs a difficult and complex one. New antibiotics, when they do arise, may sadly have a much shorter timescale of clinical utility that initially expected, due to overuse, with the resulting excessive circulation within the environment leading to acquired resistance. This is a serious problem, and may be even more acute should the antibiotic have only a singular molecular target against which it acts, as only a slight mutation of the target gene or single site modification in the target may be needed to render it effectively useless.There is therefore an increasingly pressing need for the discovery and development of new antibiotics against a number of key bacterial pathogens. In the case of 1.3.Mab infections, generating a variety of lead chemical structures against a wide variety of molecular targets. These approaches can essentially be classified as being of one of two types: those involving initial screening and subsequent drug discovery against a known molecular target (target-based screening approaches), and those where compounds are initially screened against whole-cell mycobacteria and the exact molecular target of the resulting active hit compounds is subsequently identified (phenotypic screening approaches). Both approaches have been successful, and each has a particular set of problems to be overcome to enable compound progression.A number of differing approaches have been used in attempts to discover new therapeutic agents for use in combatting Target-based screening approaches are most common, and have proven to be particularly useful in cases where a new target has been discovered using molecular biology and structural biology techniques, when precise structural information regarding the target active site may exist. This is particularly true when the target has been found to be essential for bacterial viability; compounds that inhibit such a target are likely to prove effective when suitably developed into drugs. Such approaches are amenable to a variety of screening processes, either by screening more advanced chemical compounds (from screening libraries or corporate collections), or by using a screening library of much smaller compounds (fragment screening), a powerful technique when used in combination with protein X-ray crystallography, allowing for precise binding information to be used to aid subsequent compound progression. The downside to these approaches is that the inhibitory information gathered may be somewhat artefactual, as it is collected against the isolated target. Affinities of advanced compounds from such approaches are often much reduced when measured against whole-cell bacteria, , compounds having invariably been optimised specifically for binding affinity against the target, with less regard having been placed on them having the correct physiochemical properties for optimal cellular penetration.24 In most cases, the compounds screened in such approaches are larger and already more developed that in target driven methods , so as to ensure that the cellular potency observed is of the appropriate level. In many cases, the compounds screened in the search for inhibitors of Mab by such means are compounds that are already known to be effective against Mtb, because of the considerable sequence similarities between all mycobacterial species.25 The screening of Mtb active compounds tend to give relatively high hit rates,26 allowing for the relatively quick and easy identification of active inhibitors against other mycobacterial species, including Mab.In contrast, phenotypic screening approaches involves the screening of libraries of compounds against whole-cell bacteria to ascertain which are able to retard the growth and multiplication of the organism. Compounds that show reasonable potency  may then be further developed in a medicinal chemistry approach that involves progression of the compound to further improve the whole-cell affinity down to the appropriate level. In addition, these approaches require subsequent efforts to allow the identification of the precise molecular target (or targets) against which the compound acts. Possible approaches that allow for effective target validation in mycobacteria include the combination of RNA sequencing, chemoproteomics, morphological profiling and metabolomics.2.Mab. Protein targets that are also found in other important mycobacteria are thought to be particularly useful as potential therapeutic targets, as in theory it may prove possible to utilize novel antibiotics discovered by such means in multiple clinical applications. Key individual findings from discovery research might thus be translated between mycobacteria, potentially increasing the rate of compound development. The sheer wealth of literature data that exists from research efforts aimed at more notorious and better understood bacilli (such as Mlep and Mtb) offers a wealth of such opportunities.Multiple approaches might be considered in research efforts aimed at inhibition of In most cases, particular molecular targets that have been actively pursued have focussed upon those that are believed to be involved in a number of key infective processes, such as bacterial host invasion; as well as those targets that have been shown to be essential for bacterial virulence. In addition, those targets whose function is related to the maintenance of the complex cell superstructure of the bacillus, such as those involved in both the synthesis and transport of the key building blocks necessary for cell wall repair have been deemed worthy of investigation. Likewise, those targets that maintain the biochemical integrity of the mycobacterial cell environment, such as key efflux pumps, have also been seen as targets worthy of pursuit.Mab that have been the source of such discovery efforts within the last 6–8 years, and highlight key findings in this area, with a particular focus on the distinct classes of chemical structures that these efforts have generated.We emphasise these ideas below, by way of reference to five potential therapeutic targets from 2.1.27 catalysing the reactions that produce both mycolylarabinogalactan (mAG) and trehalose dimycolate (TDM) from trehalose monomycolate (TMM). These transesterification processes, producing the essential highly lipophilic components of the mycobacterial cell envelope, thus play a key role in maintaining the cell impermeability, as well as the infectiousness and viability of the bacterium. A very recent study has explored the binding of the Ag85 complex to fibronectin in multidrug-resistant strains of Mab, an important step in host invasion,28 thus making this interaction a key factor to be potentially exploited in the design of new drugs against Mab infections.28Antigen 85 (Ag85), a complex of surface proteins found in mycobacteria, is comprised of three isoform subunits . These structures, which are the most abundantly secreted of mycobacterial proteins, are essential in the maintenance of the complex mycobacterial membrane,Mtb (particularly so in the case of Ag85A).29 The degree of helical rigidity of both α5 and α9 appears to play a crucial role in both substrate specificity and catalytic activity, and may also be an important factor in intercalation of these proteins with the insoluble cell envelope. X-ray crystal structures of the three Mtb isoforms show key differences in the residues at the secondary site which binds trehalose.Ag85C is the most active of the three isoforms, the primary function of which is the specific mycolylation of TMM to TDM. Although all three isoforms show a high degree of similarity at the active site regions, they are less homologous at a second distal putative carbohydrate binding site. Molecular dynamics calculations have shown that mutations in the alpha helices located at the ligand entry site can dramatically alter the flexibility of Ag85C in comparison to similar mutations in both Ag85A and Ag85B from 30 when screened against mycobacteria from a number of sources, with eight derivatives showing good levels of potency both in vitro and in vivo.30,31 Using both biochemical and structural approaches,31 the target for these novel antibacterial compounds was shown to be Ag85C. Fluorescent assays showed a significant loss of fluorescent intensity when a TAMRA-FP fluorescent probe (known to bind to serine dependent proteins such as Ag85C) was incubated with Ag85C that had been pretreated with the cyclic phosphonate derived inhibitors, suggestive of protein phosphonation as a mode of action. This was confirmed by X-ray crystallography,31 with structural data highlighting the attachment via covalent linkage of the inhibitors to an important catalytic serine residue located at the active site, that is key to the acyltransferase activity of the proteins (a specific residue identified previously32 using mass spectrometric techniques).A series of twenty seven cyclophostin and cyclipostins analogues,Mab. Four compounds (10–13) were found to be effective to varying degrees (50: 370 nM). Activity-based protein profiling (ABPP) experiments in combination with protein mass spectrometry confirmed that these compounds active against serine or cysteine containing proteins in Mab,33 and were likely acting be affecting lipid metabolism and cell wall biosynthesis. As per previously, experiments involving the use of a TAMRA-FP fluorescent probe confirmed that Ag85C was a major target of these inhibitors in Mab, suggesting their mode of action to be similar to that in other mycobacteria previously studied (outlined above). Ag85C has not been confirmed to be the only target in Mab against which this important class of compounds acts, and this is promising news, as Mab inhibitors that act by way of multiple targets may prove more effective, and may help to reduce the possibility of rapid mutations occurring in the bacillus that allow the organism to acquire resistance to new antibiotics.Subsequently, the series was expanded to thirty eight members, and was rescreened against  degrees , with  (MIC50: 33 μM) impaired both intra- and extracellular growth of Mab, albeit to only moderate degrees. As outlined above, ABPP experiments were used in combination with mass spectrometric analysis and showed that the activity of the compound was derived from the inhibition of enzymes that utilize either a catalytic serine or cysteine residue. In Mab, these enzymes are mainly involved in processes that involve control either lipid metabolism and/or generate the constituent materials required for cell envelope synthesis.The oxadiazolone class of antibiotic derivatives (14a–j and 15a–d) , previouMab Ag85C in the presence of (15b) resulting in significantly reduced fluorescence of the TAMRA-FP fluorescent probe used (as detailed above). In addition, significant resistance was observed, resulting in an increased MIC value (by 2–3 fold), upon overexpression of the Ag85C gene.35 A possible mechanism of action was elucidated using mass spectrometry studies, the data from which suggested covalent attachment between (15b) and the key catalytic serine residue at the active site of Ag85, so resulting in functional inhibition. Such mechanistic understandings are key to allow the further elaboration of these compounds, in order to target Ag85C more effectively, and so greatly increase antibacterial activity.One such target was shown to be Ag85C, with fluorescence based experiments performed with purified 2.2.E. coli as a two-component ATP-dependent protease (ClpP) comprised of fourteen subunits forming a complex tetradecameric structure from two heptameric rings that form a hollow cylinder, inside of which lie fourteen proteolytic sites. ClpP is highly dependent upon a series of ATPase active chaperone subunits, such as ClpA, ClpX or ClpC, which form key associated complexes.36 The ClpP proteins are considered to be valid drug targets, and as ClpC1 is found in Gram-positive bacteria and cyanobacteria, it has been viewed as a promising target in mycobacteria.Caseinolytic protease proteins are a family of large homologous proteins that are ATP-dependent, first identified in Mtb in order to identify inhibitors of the ClpP1P2 unit. ClpP1P2 is able to degrade small peptides, so this peptidase activity was monitored using a fluorescence-based approach by conjugation of a fluorophore to the hydrolysed peptide and measuring the increase in fluorescence upon hydrolytic release. As these studies aimed to find compounds that bound to the cofactors of ClpP1P2, ATP-dependent assays were also developed. A kinetic assay, in which the degradation of a substrate (GFPssra) was measured by fluorescence, was developed, and validated as a possible HTS option. A significant drop-off in fluorescence would be expected in the presence of ATP.37 In order to identify inhibitors that reduce the hydrolysis of ATP by ClpC1, the release of the hydrolysis product (ADP) needed to be measured, using a coupled assay, where ATP is regenerated from ADP using a mixture of pyruvate kinase and lactate dehydrogenase enzymes (which require NADH), the oxidation from NADH to NAD+ can be observed by way of a drop in absorbance.37 With these suitable assay methods in place, representative members from a library of over 1.8 million compounds were screened via these methods (due a limit in the amount of available protein for screening), and from these, GSK18 (16) (High Throughput Screening methods (HTS) methods have been used against K18 (16)  was idenMtb.38 ClpC1 was identified as the target of inhibition via affinity chromatography [using the active derivative (18) covalently linked to sepharose beads] in combination with proteomic analysis and isothermal titration calorimetry. ClpC1 is essential for growth in Mtb, so extension of these studies against ClpC1 from other mycobacteria is clearly pertinent. Fluorescence studies show that in the presence of cyclomarin A (17) there is an enhanced rate of breakdown of the peptide hydrolysis, indicating that (17) increases the proteolytic activity at the ClpP sites.The natural product cyclomarin A (17)  has been38 thus a mode of action involving allosteric binding to the ClpC1 hexameric ring was suggested, since the presence of (17) results in increased proteolysis. A co-crystal structure of (17) bound to the N-terminal domain of ClpC1 has since been published,39 and is suggestive of the idea that upon binding, (17) significantly reduces flexibility in this key domain, so resulting in a reduction in the ability of ClpC1 to partially close, and thereby restrict access to, the ClpP protease tunnel, hence allowing greater access of various proteins to the key catalytic machinery contained within, thus resulting in the greater degrees of hydrolysis and degradation that are observed experimentally.The mechanism of action of these analogues against ClpC1 was not determined, but affinity chromatography in the presence of ATP suggests that they do not acting by way of competition with ATP binding;Mtb: 160 nM;], as are another class of cyclic peptides, the rufomycins  . Subsequently, (17), (19) and (20) were screened against Mab. Most notable was that two different mechanisms of action were observed for (19) and (20).40 Whereas (19) promoted ATPase activity and uncoupled ATPase activity from proteolytic activity (thereby stopping the enzyme from eliminating proteins), (20) significantly decreased the proteolytic capabilities of the complex, so keeping the ATPase activity relatively constant.Other cyclic peptides, such as ecumicin (ECU) (19), are also known to target ClpC1  and DNA gyrase B (GyrB), that convert relaxed, closed-circular DNA into negatively supercoiled DNA in order to pack DNA more efficiently in cells. GyrA bears the breakage-reunion active site which is coupled with ATP hydrolysis activity promoted by GyrB. DNA gyrase is the single type II topoisomerase expressed in Mtb,43 which presumably also carries out similar functions to canonical gyrase and topoisomerase IV. As a result of this apparent multifunctional role, this enzymic system has been touted as a key target in Mtb, and by extension, in other mycobacteria, including Mab.DNA gyrase is a type II DNA topoisomerase .45In 2008, a structure-guided approach was used to optimise novel benzimidazole ureas against the ATP binding site of DNA gyrase and topoisomerase IV enzymes in antibiotic resistant strains of key Gram-positive bacteria. The original screening hit, compound (21) , was dis46 the dual inhibition of both gyrase and topoisomerase IV activities being seen as beneficial, as a potential aid to help minimise the frequency of resistant mutations (a major problem with current antibiotics). Multiple clinical Mtb strains (both drug-sensitive and drug-resistant) were shown to be inhibited by (22), both in vitro and in vivo, with the phosphate based pro-drug (23) proving to be even more effective that (22). Twenty two clinical isolates of Mab were also treated with (22), and growth inhibition noted, yielding MIC values on a par with those shown by both clarithromycin and moxifloxacin (8). Mode of action of (22) in Mab isolates was confirmed via gene knockout studies, which showed that as expected, showed the effect of the compound to occur by inhibiting the gyrB gene.Compound (22) was subsequently screened against a number of mycobacteria,Mab, possible insight into the precise mode of action of such GyrB ATPase site inhibitors may be gleaned from previous work, in which a series of pyrroloamides47 were screened against Mtb. By isolating spontaneous resistant mutants and mapping the point mutations produced, a homology model for the GyrB site in Mtb was generated, and allowed for the deciphering of the structure–activity relationship (SAR) and binding interactions in mycobacterial GyrB. A co-crystal structure of (24) (M. smegmatis) showed the compound to bind to the enzyme via a water mediated hydrogen bonded interaction with Ser208, a key amino acid identified as the site of the important point mutation (Ser208Ala) that confers resistance.Although no crystallographic information is available of (22) bound to GyrB from  of (24)  with Gyr48 little or no clinical success has been achieved. Early work initially centred around infections that showed resistance to penicillins, later other resistant strains, such as methicillin-resistant Staphylococcus aureus (MRSA) came to the fore. Only relatively recently have efforts been directed towards mycobacterial related diseases. It is clear that for further progress to be made in this area, particularly against Mab, more work still needs to done to aid understanding of the precise binding modes of key compounds that are active against GyrB from Mab.Despite research efforts against GyrB (and topoisomerase IV) being such a heavily worked area,2.4.Mab, and are a major contributory factor in the poor performance of many antibiotics against mycobacterial infections;49 they are therefore seen as important targets for anti-mycobacterial research efforts, particularly so MmpL3, as it essential for the growth and viability of Mtb. MmpL3 is large (>100 kDa) and complex, consisting of a periplasmic pore domain and a 12-helix transmembrane domain. It is responsible for exporting the bulky hydrophobic substrates that are essential for the synthesis and maintenance of the cell envelope ;50 MmpL3 has also been reported to bind haem.51 In addition, inhibitors of MmpL3 have also been shown to act synergistically with a number of key antibiotics,52 further strengthening the case of MmpL3 as an important therapeutic target against Mab infections.Mycobacterial membrane protein large protein 3 (MmpL3) is a member of the MmpL family of proteins (members of the RND superfamily of efflux pumps of mycobacteria), that are responsible for exporting endogenous lipophilic molecules. Such efflux pumps are heavily expressed in 53 involving the synthesis and testing of 63 000 closely related analogues of ethambutol with subsequent screening of the most potent compounds against Mtb, led to the discovery of a series of adamantyl derived inhibitors, including the antitubercular drug candidate (25)54 (55 A recent phenotypic screening approach translating novel hits against Mtb into Mab highlighted the piperidinol derivative (26). Whole genome analysis of Mab strains resistant to (26) noted several mutations in the gene which encodes for MmpL3, suggesting this to be the target of inhibition.56 The study also mapped the mutations which conferred resistance to (26) to generate a 3D homology model with a potential binding pocket.A combinatorial solid-phase approache (25)54 , which w57 Likewise, compound (25) is also believed to disrupt proton relay.58 Benzimidazole derivative (27) also shows promising anti-mycobacterial activity. Originally discovered as an active in a screen against Mtb,59 similar activity was recently also noted in Mab,60 and shown to occur via inhibition of MmpL3.61 Mapping of resistance mutations (as described above) again suggested that (27) bound to the transmembrane region of the protein, at the neighbouring D/Y amino acid pairs, and so also presumably blocks the proton relay.It is thought that (26) targets specific residues (a pair of Asp and Tyr neighbours) that are essential for the proton relay pathway that provides energy for substrate transport; this key functional tyrosine residue is located in the transmembrane region of the protein and is believed to be conserved in all MmpL proteins.62 of MmpL3 from M. smegmatis, along with co-crystal structures with several inhibitors bound suggests an active site comprised of five distinct regions, four of which are highly lipophilic in nature, with only the central one being polar in nature. This observation undoubtedly explains the binding of structures such as (25–27), all of which have a central protonatable functionality, flanked by highly lipophilic groups. When any of the inhibitors bind, the pair of lipophilic sub-sites in the proton-translocating channel are occupied, further confirming that inhibitors of MmpL3 disrupt the proton motive force for substrate translocation. As the binding region in M. smegmatis shares all but four residues with the MmpL3 binding site in Mtb, these crystal structures can be seen as useful aids in the rational design of drugs against TB, as well as perhaps in research aimed at combatting Mab infections. Other very recent studies63 have shown that (28), and other analogous conformationally constrained spiro-derived analogues64 are active in Mtb, and that very simple indole-2-amides, such as (29), are highly active against Mab.65 Both (28) and (29) show good activity in the appropriate animal models , and both have been shown to work via inhibition of MmpL3,63,66 presumably by way of mechanisms akin to those shown by (25–27).A recent X-ray crystal structure66 having the ability to bind a wide variety of chemical structures, as has the fact that MmpL3 inhibitors are able to work synergistically with several key frontline antibiotics,52 , potentially allowing lower doses of these compounds to be administered, so reducing the possibility of the emergence of resistance. MmpL3 is clearly a key target in the area of Mab anti-infectives, and X-ray crystal structures of important compounds bound to MmpL3 from Mab will no doubt add considerable guiding influence in research in the near future.The relative promiscuity of MmpL3 as a target has been commented upon,2.5.Mab S-adenosyl-l-methionine (SAM) dependent methyl transferase family of proteins known as the SpoU-TrmD (SPOUT) RNA methyltransferase superfamily or Class IV methyltransferases.67 There are nine other SPOUT enzymes, that recognise different motifs and methylate different positions to TrmD.68 TrmD is dimeric, and adopts a trefoil knot motif in order to constrain SAM within the active site.68 Its function is to catalyse N1-methylation of tRNA Guanosine 37 (G37) in order to prevent frameshift errors during translation on the ribosome.70 For TrmD to function, G36 and G37 must be the substrate RNA sequence on the anticodon loop, so the enzyme can recognise the sequence.69 Were these frameshift errors not suppressed by TrmD, protein synthesis would be terminated prematurely, leading to defective cell membrane proteins being produced, leading to cell death.70TrmD belongs to the 71,72 involving a fragment based drug discovery (FBDD) strategy has been utilised against TrmD from Mab to discover a series of novel inhibitors  based assay, of which twenty seven showed some electron density in the active site when soaked into apo crystals of TrmD, the key interactions being between the ligand in the area in which the adenine base of SAM binds.71A structure driven approachhibitors . FBDD isKd: 260 μM) and (31) (Kd: 170 μM) was pursued, to generate (32) (Kd: 110 μM) which led to more potent analogues. X-ray crystallography was used heavily throughout, and the combination of multiple X-ray structures with detailed SAR data allowed relatively rapid compound progression, resulting in a series of analogues that included lead compound (33a and 33b), which showed low nanomolar affinity against the target.72 The series showed reasonable potency against whole-cell Mtb, but much less so against Mab, possible due to poorer penetration in Mab, or due to much increased levels of efflux. Subsequent studies in which focus was more heavily weighted towards factors affecting the overall compound molecular profile led to improved compounds (34a–d), with a more balanced profile between whole-cell Mab and Mtb (unpublished results).Overlap of multiple X-ray structures of the weakly binding fragment hits gave rise to a clearer understanding of factors that that affected binding. Binding affinities were determined using isothermal titration calorimetry (ITC), in order to rank (and prioritise) fragment hits. A fragment merging strategy involving hits (30)  described within the recent literature as showing activity against s review  is worthe.g. from Mtb to Mab), whereas intermediate sized compounds can mostly be traced back to the output from HTS campaigns.Compounds with a much smaller size tend to originate in target directed campaigns, such as that described above against TrmD. Much larger compounds, such as the cyclic polypeptides mostly derive from attempts aimed at translating known activities from one mycobacterium to another  than others. It has been noted recentlyt factor .Mab, and what constitutes a favourable molecular property in an active compound? Clearly those targets that are essential, that are necessary for the growth or invasive property of the mycobacterium must be seen as worth pursuing. In addition, those targets that are located within specific, more readily accessible regions of the bacterium must also be seen as important . Those targets for which inhibitors can work synergistically with approved antibiotics (such as MmpL3) may be key to reducing resistance, as lower doses of the clinical antibiotics may be used, resulting in a lack of general environmental exposure of these vital important medicines. Also important are those targets for which compounds might work via a number of mechanisms (a prime example being ClpC1), again reducing the likelihood of resistance. Compounds that hit multiple molecular targets might be seen as preferable to those that are target specific; simple mutations may cause resistance to new medicines if the compound is to target specific. Above all, compounds developed need to maintain a high degree of control over physicochemical properties, especially for those that target intracellular proteins.So what constitutes a valid target against Mab as is possible; surrogate systems, including heavily truncated protein constructs, as well as those from closely related bacteria (very often Mtb) are extremely valuable, but are non-ideal, and can only ever deliver an imperfect degree of understanding.Phenotypic screening approaches are key, and afford an immediate indication as to the effectiveness (or otherwise) of compounds against whole-cell bacteria, but offer no information related to target engagement. In sharp contrast, with target-based approaches, target engagement is assured, but with no indication as to the ultimate effectiveness of compounds against the bacterium, often resulting in research efforts which prove to be fruitless. Thus phenotypic and target-based approaches might be considered to deliver orthogonal research outputs. In target-based studies, structural characterisation, such as X-ray crystallography, is important, and delivers a wealth of specific information on the binding between ligand and target (such as TrmD), allowing for rapid compound development. It is important, however, that the crystallography is performed using a protein construct that is as close to the natural situation in One possible way in which the two seemingly orthogonal approaches of target-based and phenotypic screening might yield improved compounds is to combine the use of both at a very early stage in the overall screening cascade, so utilising high-throughput X-ray crystallographic screening in conjunction with whole-cell assay readout for all compounds that are to be screened. Under such circumstances, X-ray crystal structures would offer assurance of target engagement whilst whole-cell activity greatly reduces the risk of failure of more developed hits at a later stage in the development cascade, with only those screening compounds that deliver high-quality X-ray crystallographic co-crystal structures between protein and ligand and also show acceptable phenotypic activity in a whole-cell assay being chosen for further development, and all other compounds being rejected for further consideration.Over recent years, research into the discovery of new antibiotics that are effective against diseases caused by mycobacterial infection have burgeoned, aided by technologies that have allowed whole genome sequencing of individual organisms, allowing for the discovery of new molecular targets and for them to be explored. The continued emergence of multidrug resistant strains within the clinical setting has further increased the urgency of need for therapeutic advances in this area.Mtb, the causative agent of TB, that continues to seriously affect millions of individuals worldwide,76 more recently Mab has come to the fore, due to the increasing prevalence of this bacterium in conditions such as cystic fibrosis, where therapeutic options are very limited and the prognosis for patients is often very poor.Most research over the past few decades has naturally focused upon Mab antibiotic research, covering the scientific literature published with the last 6–8 years, concentrating on five protein targets that show promise, and highlighting the successes and pitfalls of the individual approaches that have been taken in each case to further the discovery of potential new medicines. Success rate in the field of antibiotic research remains poor, nevertheless it is hoped that such continued research efforts may eventually bear fruit, and lead to new clinical treatments to aid in the fight against the diseases caused by these difficult to combat mycobacteria.In this review we have attempted to highlight some recent efforts within the field of There are no conflicts to declare.


Arabidopsis pollen-pistil interactions, ligand-receptor kinase signaling is utilized to mediate various checkpoints to promote compatible interactions. In Arabidopsis, the later stages of pollen tube growth, ovular guidance and reception in the pistil have been intensively studied, and thus the receptor kinases and the respective ligands in these stages are quite well understood. However, the components of the earlier stages, responsible for recognizing compatible pollen grains and pollen tubes in the upper reproductive tract are less clear. Recently, predicted receptor kinases have been implicated in the initial stages of regulating pollen hydration and supporting pollen tube growth through the upper regions of the reproductive tract in the pistil. The discovery of these additional signaling proteins at the earlier stages of pollen-pistil interactions has further elucidated the mechanisms that Arabidopsis employs to support compatible pollen. Despite these advances, many questions remain regarding their specific functions. Here, we review the roles of the different receptor kinases, integrate their proposed functions into a model covering all stages of pollen-pistil interactions, and discuss what remains elusive with regard to their functions, respective binding partners and signaling pathways.Successful fertilization of a flowering plant requires tightly controlled cell-to-cell communication between the male pollen grain and the female pistil. Throughout  Arabidopsis, is the presence of dry stigmas which possess cellular signalling pathways for selective pollen hydration  on the surface stigmatic papillae and leads to the papillae releasing water for pollen hydration. Hydration of the desiccated pollen grains is a critical step as it is required to initiate pollen metabolism and germination of a pollen tube. In contrast, pistils with wet stigmas such as tomato (Solanum lycopersicum) have little control over pollen hydration, and RKs begin to regulate these interactions during pollen tube growth  genes, and an increasing number of these RLKs have been shown to encode true RKs with the associated ligand(s) identified; thus, serving as primary sensors in the perception of extracellular signals  and the Leucine-Rich Repeat (LRR) RLK subfamilies have proven to be critical regulators, and they are involved at every stage of this complex, multifaceted process  and FERONIA (FER), are proposed to establish an autocrine signalling cascade to maintain these ROS levels in Arabidopsis sigmatic papillae in the absence of pollen  . Recentlf pollen . A connef pollen  and pollf pollen . The extf pollen . In the f pollen  and thesf pollen . Pollinaydration .pcp-bγ mutant pollen hydration defect would also be rescued relative to wild-type pollen, but this was not observed suggesting that additional regulators are present in the stigma. Recently, Arabidopsis LRR-VIII-2 RLK subgroup members have been identified as positive regulators in the stigma to promote pollen hydration and may be the missing link. RECEPTOR-LIKE KINASE IN FLOWERS 1 (RKF1) and its paralogue, RKF-LIKE 1 (RKFL1) were identified through a yeast two-hybrid screen to interact with a stigma-specific pseudokinase, BRASSIKIN 1 , and the RKF1 gene cluster has not been previously ascribed any function. CRISPR-Cas9 generated deletion mutants of the RKF1 gene cluster (rkfΔ mutants) were found to display some phenotypic changes in pollen-stigma interactions , it exacerbated the phenotype significantly, resulting in further reductions in wild-type pollen tube growth through the mutant reproductive tract at 6 hours post-pollination. Ultimately, some of these pollen tubes failed to reach ovules, as denoted by decreased seed counts in fully developed mutant siliques  and BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1/SERK3) . While psiliques . Overallpskr1-3 pskr2-1 double mutant pistils, wild-type pollen tubes were observed to grown more slowly through the upper reproductive tract resulting in an increased number of pollen tubes failing to reach an ovule  1 and 2, and ANXUR1 and ANXUR2 (ANX1 and 2) are responsible for maintaining pollen tube integrity as the pollen tube grows through the reproductive tract . BUPS1/2Arabidopsis involves an extensive network of RLKs, RKs and their corresponding ligands. While a number of these players have been identified, there are still many remaining gaps, particularly at the early stages. Starting with the stigmatic papilla regulation of pollen hydration, further work is needed to understand how the negative gating mechanism of the ANJ-FER complex in the papillae connects with the positive regulatory role proposed for the RKF1 cluster. Future investigations should seek to identify ligands for RKF1 and RKFLs as well as potential binding partners in the papilla plasma membrane. Similarly, does the same unknown ligands and binding partners function with RKF1 and the RKFLs in the reproductive tract. As discussed above, SERK1 and SERK3/BAK1 typically form complexes with other LRR-RKs .We thank members of the Goring lab for critically reading this article.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.