The emergence of multicellularity is strongly correlated with the expansion of tyrosine kinases, a conserved family of signaling enzymes that regulates pathways essential for cell-to-cell communication. Although tyrosine kinases have been classified from several model organisms, a molecular-level understanding of tyrosine kinase evolution across all holozoans is currently lacking. Using a hierarchical sequence constraint-based classification of diverse holozoan tyrosine kinases, we construct a new phylogenetic tree that identifies two ancient clades of cytoplasmic and receptor tyrosine kinases separated by the presence of an extended insert segment in the kinase domain connecting the D and E-helices. Present in nearly all receptor tyrosine kinases, this fast-evolving insertion imparts diverse functionalities, such as post-translational modification sites and regulatory interactions. Eph and EGFR receptor tyrosine kinases are two exceptions which lack this insert, each forming an independent lineage characterized by unique functional features. We also identify common constraints shared across multiple tyrosine kinase families which warrant the designation of three new subgroups: Src module (SrcM), insulin receptor kinase-like (IRKL), and fibroblast, platelet-derived, vascular, and growth factor receptors (FPVR). Subgroup-specific constraints reflect shared autoinhibitory interactions involved in kinase conformational regulation. Conservation analyses describe how diverse tyrosine kinase signaling functions arose through the addition of family-specific motifs upon subgroup-specific features and coevolving protein domains. We propose the oldest tyrosine kinases, IRKL, SrcM, and Csk, originated from unicellular premetazoans and were coopted for complex multicellular functions. The increased frequency of oncogenic variants in more recent tyrosine kinases suggests that lineage-specific functionalities are selectively altered in human cancers. Tyrosine kinases propagate cellular signals through the phosphorylation of tyrosine residues on protein substrates. Forming a monophyletic group within the larger protein kinase superfamily, tyrosine kinases diverged from serine–threonine kinases prior to the emergence of opisthokonts  , which aA classification of the protein kinome into evolutionarily and functionally related families (here on referred to as the KinBase classification) was achieved two decades ago following the sequencing and comparative genomic analyses of model organism genomes including human , mouse , sea urcIn addition to the uniquely evolved features across different tyrosine kinase families, similarities across some tyrosine kinase families have also been noted. For example, the recently termed “Src module,” which consists of a tyrosine kinase domain and N-terminal SH3 and SH2 domains, is found across the Src, Abl, Tec, and Csk families, and structural and solution studies have determined that a similar autoinhibitory configuration of the Src module is shared across members of the Src, Abl, and Tec families . BecauseHere, we determine a novel hierarchical, constraint-based classification of the tyrosine kinome that newly identifies three evolutionary subgroupings of tyrosine kinase families based on the selective conservation of sequence motifs in the kinase domain, which encode common autoinhibitory conformations. In addition, we illustrate an evolutionary timeline of how unique kinase functions have expanded on shared subgroup-specific features through duplication events, selection of family-specific motifs, and domain shuffling to give rise to the vast repertoire of tyrosine kinase signaling observed throughout metazoans. A closer examination of tyrosine kinase phylogeny in light of constraint-based tyrosine kinase subgroups reveals new insights into the evolutionary conservation or divergence of subgroups, as well as the unique signaling features that may have emerged from three separate monophyletic clades of receptor tyrosine kinases. In particular, we note the early emergence of two major clades of holozoan tyrosine kinases distinguished by the presence (or absence) of an insert between the αD and αE helices of the kinase domain, where tyrosine kinases containing the insert comprise the majority of metazoan receptor tyrosine kinases. Our classification of the tyrosine kinome and the approach used in this study set a new precedent for the classification and evolutionary study of protein kinases and other large protein families.To generate a comprehensive classification of the holozoan tyrosine kinome, we generated a multiple sequence alignment of 44,639 tyrosine kinase sequences spanning 586 species . We then used the Bayesian Partitioning with Pattern Selection (BPPS) algorithm to classify aligned sequences into hierarchical clusters based on the patterns of conservation and variation in aligned tyrosine kinase domain sequences A Neuwal, 2014. ENext, we reclassified 34,954 tyrosine kinase sequences from the UniProt reference proteomes database into the optimized hierarchy by quantifying the extent to which individual sequences match cluster-specific motifs . We define this reclassification as a constraint-based classification because this post-processing step eliminates spurious or divergent sequences from clusters that do not score over an optimal cut-off score due to their lack of cluster-specific patterns. The spurious sequences eliminated from each cluster are categorized within the unclassified family .The new constraint-based hierarchical classification of tyrosine kinases, which is broadly similar to the KinBase classification of the tyrosine kinome, reveals several novel subgroupings. In particular, the constraint-based classification defines three new subgroupings of tyrosine kinase families which account for nearly half of the tyrosine kinome: the Src module (SrcM) subgroup, the insulin receptor kinase-like (IRKL) subgroup, and the fibroblast, platelet-derived, and vascular growth factor receptors (FPVR) subgroup . The SrcBy examining the sequence constraints that define each of the three novel subgroups in light of existing crystal structures, we observe that subgroup-specific motifs are located in known regulatory regions of the kinase domain. For example, the SrcM subgroup conserves a highly distinguishing GxM motif in the β3-αC loop and a GxKF motif in the activation loop that both form important interactions associated with a common Src-like inactive conformation in the activation loop A Xu et . This SrIn order to infer when each of these tyrosine kinase subgroups and families emerged in evolution, we organized tyrosine kinase subgroups and families based on taxonomic conservation . TyrosinIn order to further explore the functional diversity of SrcM, IRKL, and FPVR tyrosine kinases, we surveyed the diversity of protein domains present across these subgroups and analyzed their conservation across holozoan taxa . As prevThe IRKL and FPVR subgroups encompass the majority of receptor tyrosine kinase families and, despite sharing common kinase domain mechanisms within these subgroups , have diHis260) in the αE helix which tyrosine kinases selectively lost upon diverging from the serine/threonine kinases . By integrating our constraint-based classification of tyrosine kinases with our phylogenetic tree A, we can kinases  online  group of serine/threonine kinases. Holozoan tyrosine kinases (Group A) also form a monophyletic clade that is distinct from a paraphyletic group of divergent tyrosine kinase sequences found in pre-opisthokonts (Group B), such as those found in the amoebozoans nhardtii  . This correlation between the presence of the longDE insert with the presence of transmembrane and extracellular domains, alongside evidence that the insert plays important roles in kinase activation and protein recruitment for downstream signaling, suggests that the longDE insert evolved as a means to facilitate downstream intracellular signaling upon the activation of receptor tyrosine kinases by extracellular signals. In addition, though the DE insert is difficult to align across families due to the lack of sequence conservation, the DE insert is alignable within families and often conserves sequence motifs including phosphorylatable tyrosine, serine, or threonine residues, suggesting that individual receptor tyrosine kinase families along the longDE clade have rapidly and frequently evolved the longDE insert in family-specific contexts, presumably to carry out family-specific downstream signaling functions. We also note that the longDE tyrosine kinases highly conserve a unique activation loop methionine, which is not observed in shortDE tyrosine kinases C; howeveInterestingly, the shortDE clade in the tyrosine kinase phylogeny, which predominantly consists of cytoplasmic tyrosine kinases, includes two monophyletic clades of receptor tyrosine kinases: the EGFR family of receptor tyrosine kinases and a separate monophyletic clade that includes the Eph and choanoflagellate-specific RTKC families of receptor tyrosine kinases. Thus, our phylogeny suggests at least three independent origins of highly expanded receptor tyrosine clades, with the majority of receptor tyrosine kinases emerging from the longDE clade. That these disparate branches along the tyrosine kinase phylogeny have convergently evolved to include transmembrane and extracellular domains highlights the importance of relaying extracellular signals into intracellular responses across various signaling niches. Although the longDE receptor tyrosine kinases are distinguished by extra functionalities imparted by the longDE insert, the Eph and EGFR families also exhibit unusual signaling functions so far unobserved in other longDE receptor tyrosine kinases. Eph receptor tyrosine kinases have a unique capacity for bidirectional signaling, where the binding of ephrin ligands, which are also membrane bound, can activate signaling both in the receptor-bearing cell, as well as in the ligand-bearing cell . FurtherThe classification of protein kinases into evolutionarily related families has provided the foundation for decades of comparative sequence-structure-function studies on protein kinases . Here, wWe constructed a new representative phylogenetic tree of the holozoan tyrosine kinome which revealed larger evolutionarily related clades of tyrosine kinases associated with additional defining features A. DividiThe expansion and diversification of the tyrosine kinome across the animal kingdom highlights its central role in metazoan biology. Although many previous studies have speculated on the role of tyrosine kinases in the evolution of multicellularity , our finGiven the important roles of tyrosine kinases in multicellular metazoan biology, it comes as no surprise that sequencing efforts have identified many disease-related variants across the tyrosine kinome . MultiplWe sampled alternative classification hierarchies ab initio, using the omcBPPS algorithm  which emPhe43) and ending at the αI helix (PKALys292). Kinase sequences which did not span from at least the β3-Lys to the DFG-Asp were deemed fragmentary and removed from the alignment. The UniProt sequence set contained 12,137 tyrosine kinase sequences. The nr sequence set was further purged at 98% sequence identity and contained 17,071 tyrosine kinase sequences. We performed hierarchical clustering on both sequence sets using omcBPPS. For both sequence sets, we optimized the “minnats” parameters (minnat = 1 and minnat = 5) which changed the minimum log-likelihood required to form a cluster. All runs were performed twice. To create a consensus of the hierarchical classification schemes found by multiple runs of omcBPPS, we used the mcBPPS algorithm. Clusters which were consistently identified throughout multiple runs were refined using the mcBPPS algorithm  and NCBI nonredundant (nr) proteins database  . Within lgorithm . We ran Using our consensus model, we developed quantitative means of evaluating how well any given sequence fit into each of the clusters defined by our model  which implements NumPy array-based sequence alignment manipulation. All sequence features  were represented as Boolean arrays as a function of the full sequence alignment. More complex queries pertaining to multiple sequence features (such as the presence of a given domain in a given taxon) were constructed by Boolean algebra. These Boolean arrays were applied as filters to our sequence alignment using NumPy indexing routines . SequencAfter we classified our representative set of tyrosine kinase into discrete clusters, we determined the taxonomic conservation of each cluster. We determined the source of each tyrosine kinase sequence using the organism identifier number (OX) provided in the FASTA header of UniProtKB sequences. OX numbers were traced back to their parent node identifiers using the nodes dump file provided in the NCBI taxdump database. All node identifiers were translated to their respective scientific names using the taxonomy names dump file. We determined the distribution of taxa across each cluster of tyrosine kinases and selected an optimal mix to depict diverse taxa ranging from unicellular pre-metazoans to more complex metazoans such as chordates  online. Intron/exon annotations were mapped using Scipio . Intron We produced protein domain annotations for each full-length tyrosine kinase sequence using the NCBI Conserved Domain Database  database of conserved protein domains  one randomly selected sequence from each cluster-taxon pair based on  online etellanii , and 4) tellanii , with Motellanii . Branch tellanii . Resultstellanii  online. The consensus tree with the highest bootstrap support values for the three major tyrosine subgroups was selected as the final tree. The optimal substitution model for our final topology was determined to be LG+R8 based on the Bayesian Information Criterion as determined by ModelFinder . We rootWe compared our tree to previously published phylogenies which also sampled diverse tyrosine kinase families . HoweverMolecular Biology and Evolution online.msab272_Supplementary_DataClick here for additional data file.


Although clinical guidelines recommend that people showing signs of cognitive decline engage in Advance Care Planning (ACP) while they still have decision-making capacity, too often this opportunity to clarify values and treatment preferences is missed among patients and their families. In reflection of the paucity of empirical data on factors influencing how this planning process begins for families experiencing cognitive decline, this study explored facilitators and barriers to ACP among adult children of parents showing signs of early- to mid-stage dementia, with a particular focus on relationship quality. Among this sample (N = 315), relationship quality positively and significantly predicted advance care planning engagement . Financial burden weakly and positively predicted ACP engagement , while both psychological burden  and financial burden  negatively and significantly predicted relationship quality. This study validates the use of the ACP Engagement Survey (ACPES) adapted for surrogates among adult children of people experiencing cognitive decline and contributes to a scarce literature on the impact of relationship quality on ACP engagement.


Current 3D cardiac organoid cultures have shown their utility in modelling key developmental hallmarks of heart organogenesis, but the complexity of the organ demands a more versatile model that can investigate more fundamental parameters, such as structure, organization and compartmentalization of a functioning heart. This review will cover the prominence of cardiac organoids in recent research, unpack current in vitro 3D models of the developing heart and look into the prospect of developing physiologically appropriate cardiac organoids with translational applicability. In addition, we discuss some of the limitations of existing cardiac organoid models in modelling embryonic development of the heart and manifestation of cardiac diseases.Medical research in the recent years has achieved significant progress due to the increasing prominence of organoid technology. Various developed tissue organoids bridge the limitations of conventional 2D cell culture and animal models by recapitulating  Congenital heart defects (CHD) affects approximately 0.4–5% of live births worldwide, making it the most common form of congenital disease . Despitein vivo (The advent of pluripotent stem cells (PSCs), of both embryonic stem cells (ESC) and induced pluripotent stem cell (iPSC) origins differentiated into various cell lineages, provides novel insights to embryonic development and regenerative biology . Human iin vivo . These fin vitro tissue cultures have been widely used in multiple research fields to model their in vivo organ counterparts. Self-organized from stem cell populations, these organoids have the potency to undergo multiple divisions and differentiate into various appropriate cell types to confer structural and cell composition resembling in vivo organs  o organs . Kidney,o organs . Cell-ceo organs . As suchin vitro model that can capture such a complex system, maintain, and mature the various cell types will be even harder to achieve. While cardiac organoid technology is still in its infancy, a number of research groups have developed methods to generate 3D cardiovascular tissue using either a pure CM starting population or a mixture of CMs, CFs and ECs and aggregating them into Engineered Heart Tissues (EHT) or on scaffolds , epicardial cells, endocardial cells, smooth muscle cells, cardiac fibroblasts (CF) and endothelial cells (EC) with compositions that can vary with age or due to myocardial damage and remodelling . PSC-dercaffolds  (Eschenhcaffolds . A summaEmbryoid bodies (EBs) are aggregate masses of PSCs cultured in suspension and are widely used for differentiation into various tissue lineages. CMs were first derived using EBs generated using murine stem cells . Since tTo overcome the stochastic nature of EB-CM models resulting in widely varied cell compositions and cell number, cardiac spheroids have been aggregated using specific cell composition and number. A number of research groups have explored aggregating tissues of varying compositions appropriate to study specific archetypes of cardiac developmental diseases or health. Hanging drop culture has been used to aggregate CMs, ECs and CFs in a 3:1:6 ratio for spheroids to measure the cardiotoxicity of doxorubicin, a widely used cancer drug . Anotherin vivo early heart and foregut endodermal anlagen of the developing embryo, with layered myocardium and endocardium, vascular network, and anterior-posterior foregut patterning (On the other hand, cardiac organoids are more strictly defined as 3D cardiac structures that were self-organized from either PSCs or multipotent progenitors. This direction is highly popular of late, with many recent works on utilizing either murine and human PSCs to generate cardiac organoids attempting to recapitulate cardiogenesis . Interestterning . By natuA popular method to overcome the maturation limitation of these spheroid and organoid models is long-term culture within bioreactors. Bioreactors supply biochemical signals by improving media circulation around the organoids to ensure higher mass transfer, physiological relevant gradient of nutrients and sufficient removal of waste products . In addiEngineered heart tissues are amongst the oldest and most utilized 3D tissue construct made up of aligned CMs with the ability to propagate contractions along a plane . While tEHTs have also been adapted to contain the appropriate cell composition for modelling specific diseases. ECs and/or CFs have been aggregated to study neovascularization and cardiac fibrosis . PurifieHowever, there are limitations to EHTs as a modelling platform. It requires a high initial cell count for aggregation, within a complex equipment setup that prevents high throughput screens . In addiEHT constructs of 3D CM tissues was popularized alongside several other hydrogel/polymer based matrixes for patterning . ConceptSimilarly, hydrogels and polymers have been utilized to create 3D scaffolds as a biomimetic approach to recapitulate human cardiac tissue . While EWhile cardiomimetic scaffolds have been developed to provide a geometrically and environmentally appropriate 3D architecture for CM seeding, simple folded scaffolds have also been developed to promote cardiac chamber formation . These hAlternatively, precise control of cellular composition and spatial distribution of individual cells can be achieved via 3D bioprinting. Hydrogels and decellularized extracellular matrix have been developed into bioinks that serve as a scaffold for individual cell types to be printed on . Of notein vitro heart models limit research in understanding these disorders. While much effort has been poured into generating the 3D CM models described in in vivo development.Congenital heart defects occurring during the first 8 weeks of embryonic development are detrimental to the growing embryo’s ever-increasing metabolic demands during embryogenesis. However, inaccuracies in the in vitro models were shown to recapitulate various aspects of cardiogenesis. Earlier work by There have been significant recent advances in the field of developmental cardiac organoids, which were intrinsically formed by taking advantage of the self-organizing and patterning capabilities of either murine or human hPSCs. These Consistently, in vivo heart (in vitro organ/tissue formation to mimic cardiogenesis from the early to mid-gestation stages, and thus does not require complex differentiation protocol. Therefore, the heart organoids represent a promising research tool to study developmental diseases for drug testing.Developmental organoids can also possess the capability to be used as a disease modelling tool, particularly for drug testing. Heart organoids were generated using mouse ESCs by utilizing FGF4 and extracellular matrix, emulating the developmental processes of the vo heart . These hAs mentioned, several organoid platforms have focused particularly on the disease modelling and drug screening purposes of the model . These mvia Wnt signaling modulation, and extensively detailed its capability in modelling cardiac development and diabetes-induced congenital heart disease. These hHOs were mainly comprised of myocardial tissue, with epicardial tissue organized near the exterior surface of the organoids. In addition, these hHOs recapitulate the functional and structural features of the developing fetal heart, exhibiting robust contraction and action potential waves reminiscent of the PQRST waves, and expressed well defined sarcomeres surrounded by gap junctions, mitochondria and t-tubules. As a proof of concept, pregestational diabetes impacted the cardiac development of these hHOs, resulting in irregular arrhythmic contraction, metabolic dysfunction and structural organization in accordance with the phenotype observed in vivo.Of note, Myocardial infarction and drug cardiotoxicity has been recently modelled in a cardiac microtissue organoid model as well . The uniin vivo. However, current cardiac organoid models fall short in developing important structural and morphological elements unique to the heart. Particularly, cardiac architecture, most notably being the heart chambers are absent, and thus related elements such as the septa and valves are also missing. As such, CHD affecting these missing elements in the in vivo developing heart cannot be accurately modelled.Collectively, developments in 3D cardiovascular disease models have demonstrated the ability of patient-derived cardiac organoids to recapitulate developmental processes of cardiogenesis, as shown by ultrastructural, gene expression, histochemical characteristics with resemblance to the developing heart in vivo (Cardiac progenitors within the embryonic heart specifies their subtype early in development, primarily during heart field specification . Howeverin vivo . Furtherin vitro, working in tandem with classical models for accurate phenotyping of disease and drug effects. However, the lack of functional cardiac chambers limits the utility of these organoids in the disease modelling space. The closest model in achieving self-organized chambers currently available is the hHO model by Lewis-Israeli et al., detailed previously in + endocardial cells (Lastly, the missing structural elements of current cardiac models results in the failure to model mechanical functional changes such as myocardial wall thickness, pressure loading and ejection fraction. Heart disease often present with pathological cardiac remodeling, causing hypertrophic growth, irreversible decompensation and thinning of the myocardium and dilatation of the failing heart . Classical cells . Howeveral cells . Such anDrawing from the approaches of the tissue organoid systems currently generated, intrinsically formed chambered cardiac organoids should theoretically be achievable. Judging by the rapid pace of advances in the space of cardiac organoid development in recent years, there is promise that such an organoid model will soon be developed and be scalable for high-throughput disease modelling. Lately, there has been increasing interest in utilizing microfluidic systems such as organ-on-a-chip platforms for use in preclinical analysis. These chip-based systems are the preferred platform for industry-level research—completely automated high throughput derived organ-models are screened within highly reproducible fabricated chips. Organ-chip systems have been adapted to allow for vascularization in a number of tissue types, recently reviewed by in vivo heart in place, future in vitro cardiac modelling will be able to better support downstream preclinical pharmaceutical research in discovering cures and preventing cardiotoxic drugs from slipping through.While 3D CM cultures currently exist and are routinely used for studying development, drug screening and disease modelling, they often portray an incomplete picture of associated effects on a human heart. The strong demand required to bridge the technology and knowledge gap will enable researchers to better study heart development and cardiovascular diseases for research and translational purposes. Hence, future direction for cardiac organoid research is currently focused on generating physiologically appropriate chambered cardiac organoids paired with an appropriate high-throughput modelling platform that can simultaneously assess multiple cardiac functional parameters. With more faithful models of the


Socioeconomic status (SES) is one of the most robust predictors of health. The source of SES-health associations is heavily debated; one approach is investigating neighborhood-level environmental characteristics. Challenges include selection effects and the possibility of reverse causation: people choose their neighborhoods. Longitudinal twin research can overcome these issues by assessing location choice over time as well as twin similarity; however, few existing twin studies have incorporated neighborhood-level data, and none of those focus on aging. Using longitudinal data from the Swedish Adoption/Twin Study of Aging, the current study examined the impact of location at various points in life. Location at birth and in 1993 were available for 972 participants. Birth years ranged from 1926 to 1948; mean age in 1993 was 54.55 (range = 35-67). Thirty-nine percent of the sample had moved to a different county between birth and midlife: individuals who moved had significantly higher parental SES and had achieved significantly higher education. Moreover, identical twin concordance for geographic mobility (77%) was significantly higher than fraternal twin concordance (65%), indicating a modest but significant genetic contribution. Geographic mobility did not impact identical twin similarity on a functional aging factor (corrected for age and education), but fraternal twins concordant for mobility were more similar than discordant twins, suggesting genetic contributions to mobility may also impact health. Ongoing retrieval of location information for twins born 1900-1925 and geocoding of location information available at 9 waves of data collection will allow for expanded investigation of the SES-health relationship at the neighborhood level.


METHODS/STUDY POPULATION: This study involves refractory epilepsy patients with implanted intracranial electrodes and moderate-to-severe traumatic brain injury (m/sTBI) survivors. In epileptic patients, we will identify connectivity of cortical regions via the ANT, which probes components of attention  and a CC task that probes implicit learning. We hypothesize that modulation of attention and learning can be seen at the network level. In TBI we will assess improvement following two attention rehabilitation paradigms behaviorally; and use our results from epileptic patients to guide measurement of treatment-related neuroplastic change via scalp electroencephalography. RESULTS/ANTICIPATED RESULTS: When the proposed objectives are complete, we expect to determine how the implicit learning rate in m/sTBI changes as a result of both direct attention and metacognitive-strategy training, and discern the neuroanatomical networks associated with attention and implicit learning based on connectivity results. We expect to identify intracranial regions and networks that exhibit modulatory effects associated with attention and implicit learning. Additionally, we anticipate that deficits in attention will be mitigated following training and hypothesize that implicit learning rate will improve in TBI patients as a result of both attentional rehabilitation paradigms. DISCUSSION/SIGNIFICANCE OF IMPACT: Characterizing intracranial activity in epilepsy patients will give electrophysiology data unattainable in TBI patients. This intracranial perspective will enable us to propose mechanisms of action that may result from our interventions and enable critique of current rehabilitation treatments.OBJECTIVES/GOALS: 1) Investigate the network level interactions of attention and learning during an attention network task (ANT) and an implicit learning contextual cueing (CC) task. 2) Assess the effect attention rehabilitation strategies have on


In the last decade, cold water immersion (CWI) has emerged as one of the most popular post-exercise recovery strategies utilized amongst athletes during training and competition. Following earlier research on the effects of CWI on the recovery of exercise performance and associated mechanisms, the recent focus has been on how CWI might influence adaptations to exercise. This line of enquiry stems from classical work demonstrating improved endurance and mitochondrial development in rodents exposed to repeated cold exposures. Moreover, there was strong rationale that CWI might enhance adaptations to exercise, given the discovery, and central role of peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) in both cold- and exercise-induced oxidative adaptations. Research on adaptations to post-exercise CWI have generally indicated a mode-dependant effect, where resistance training adaptations were diminished, whilst aerobic exercise performance seems unaffected but demonstrates premise for enhancement. However, the general suitability of CWI as a recovery modality has been the focus of considerable debate, primarily given the dampening effect on hypertrophy gains. In this mini-review, we highlight the key mechanisms surrounding CWI and endurance exercise adaptations, reiterating the potential for CWI to enhance endurance performance, with support from classical and contemporary works. This review also discusses the implications and insights (with regards to endurance and strength adaptations) gathered from recent studies examining the longer-term effects of CWI on training performance and recovery. Lastly, a periodized approach to recovery is proposed, where the use of CWI may be incorporated during competition or intensified training, whilst strategically avoiding periods following training focused on improving muscle strength or hypertrophy. Cold water immersion (CWI) is a strategy aimed at enhancing recovery from strenuous exercise, typically involving the submersion up to the waist or mid-torso for ~5–20 min in temperatures between ~8 and 15°C (Versey et al., Meta-analyses and experimental research in general show that CWI can beneficially influence the recovery of physical performance (Montgomery et al., In this mini-review, we have adopted an introspective approach in discussing the molecular mechanisms and rationale surrounding CWI and endurance exercise adaptations. Moreover, we review and discuss key studies which provide information on the applied scenarios where CWI can be utilized to promote physical recovery and adaptation whilst avoiding potential negative effects on hypertrophy and strength gains.Cold stimulus is a physiological stressor capable of triggering primary signals and downstream cascades implicated in exercise-induced improvements in muscle oxidative function (Ihsan et al., Seminal work by Spielgeman's group in the late 90's generated major breakthroughs in the mechanisms underpinning mitochondrial biogenesis (Puigserver et al., Whilst CWI was not conceived as a strategy specifically meant to supplement exercise adaptations, there was substantial interest in examining how recovery-based CWI might influence skeletal muscle adaptations to endurance exercise (Ihsan et al., Although such molecular responses would expectedly improve endurance performance in the longer term, Yamane et al.  reportedIn contrast to Yamane et al.  initial While these findings refute suggestions that CWI might counteract endurance adaptations, it nevertheless questions whether post-exercise CWI is an effective strategy to promote muscle adaptations resulting in improved exercise performance. Indeed, changes in acute signaling response (Ihsan et al., While some studies have shown that CWI can enhance physical recovery following resistance exercise (Vaile et al., 2 supply and utilization (Ihsan et al., Complimenting these mechanisms, we suggest that the attenuated increase in muscle mass observed following CWI and resistance training may be part of a macro-level mechanism protecting the oxidative profile of the muscle. This is supported by D'Souza et al.  demonstrWhile we rationalize that the dampened increase in muscle mass observed following CWI is a compensatory mechanism improving oxidative function, further research is needed to understand how this might influence athletic function and performance. For instance, it is currently unknown if CWI influences the regulation of muscle mass following aerobic exercise, and whether this hypothetical trade-off involving the attenuated increase in muscle mass and strength might be beneficial to endurance performance. On the other hand, co-assessment of muscle aerobic function within these resistance training studies (Frohlich et al., Athletes embark on a variety of training sessions such as cardiovascular conditioning, strength/resistance training, technical, and tactical work. In sports science practice, CWI is likely to be incorporated at various instances to promote recovery, particularly when recovery time between sessions is limited. Caution should be warranted against the regular use of CWI particularly following resistance exercise sessions.Recent work  examininTraining frequency reported within these applied studies surmounts to at least 10 sessions per week . PerhapsAnother key feature of these studies , and perCold water immersion is widely utilized by athletes during training and competition. Given that both a cold stimulus and exercise are independent stressors capable of enhancing muscle oxidative function, there remains substantial interest in examining how this modality might influence adaptations to exercise. Although post-exercise CWI up-regulates mitochondrial-related signaling, longer-term changes in protein content and result in vascular adaptations, these changes do not seem to translate to improved endurance performance. As such, further research is required to elucidate how endurance performance can be improved through its positive molecular signaling outcomes for CWI to be incorporated to enhance exercise-induced oxidative adaptations. It must be re-iterated that CWI does not impair aerobic training adaptations, and can be incorporated as a recovery modality following endurance training if needed. In contrast, regular CWI recovery incorporated into a resistance training program will dampen strength adaptations, and therefore the use of this modality following resistance exercise sessions should be discouraged. However, there is emerging data showing no impairments in strength gains in athletes incorporating regular use of CWI during intensified training periods; this either indicates that the recovery benefits conferred by CWI may outweigh its dampening effects on hypertrophy response, or the negative effects of CWI on strength may be circumvented by programing CWI following technical or aerobic conditioning sessions. In this regard, “recovery periodization” may be an important approach, where the use of CWI may be incorporated during competition or intensified training, whilst strategically avoided following training focused on improving muscle strength or hypertrophy.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The aerial portion of a plant, namely the leaf, is inhabited by pathogenic andnon-pathogenic microbes. The leaf’s physical and chemical properties, combined withfluctuating and often challenging environmental factors, create surfaces that require ahigh degree of adaptation for microbial colonization. As a consequence, specificinteractive processes have evolved to establish a plant leaf niche. Little is known aboutthe impact of the host immune system on phyllosphere colonization by non-pathogenicmicrobes. These organisms can trigger plant basal defenses and benefit the host by primingfor enhanced resistance to pathogens. In most disease resistance responses, microbialsignals are recognized by extra- or intracellular receptors. The interactions tend to bespecies specific and it is unclear how they shape leaf microbial communities. In naturalhabitats, microbe–microbe interactions are also important for shaping leaf communities. Toprotect resources, plant colonizers have developed direct antagonistic or hostmanipulation strategies to fight competitors. Phyllosphere-colonizing microbes respond toabiotic and biotic fluctuations and are therefore an important resource for adaptive andprotective traits. Understanding the complex regulatory host–microbe–microbe networks isneeded to transfer current knowledge to biotechnological applications such asplant-protective probiotics. Microbial colonization of above-ground parts of plants is a dynamic and interactiveprocess that requires a high degree of adaptation. Understanding complexhost–microbe–microbe interactions is key to new strategies for plant protection. This review examines how aerial parts of plants, particularly leaves, are colonized bymicrobes. The first section (‘Colonizing leaf surfaces’) dissects biotic and abiotic factorsthat shape leaf microbial communities and determine the quality and quantity ofcolonization. We discuss pre-formed barriers, such as the cuticle, that restrict plant hostcolonization, and environmental conditions that enhance selection pressures. As aconsequence of the extreme conditions on leaves, the properties and generation of biofilmsthrough quorum sensing (QS) are considered. Leaf colonization by microbes is not onlyimpacted by host and environmental factors but also by resident microbes, includingpathogens that can severely perturb microbial communities. The second section(‘Microbe–microbe–host interactions’) discusses effects of microbial communities on hostsusceptibility to pathogens and the impact of plant pathogens and endophytes on microbialhost colonization and community composition. A particular focus is on microbe–microbeinteractions that are often mediated via the plant host. Individual plant cells also havethe capacity to steer microbial activities by pre-formed or induced structures and compoundsthat influence microbial growth on the leaf surface. The third section (‘Role of the plantimmune system in shaping the phyllosphere microbiome’) considers the role of the plantimmune system on microbial host interactions with a focus on plant leaf colonization,leaf–microbe outputs, and microbial diversity.6–107 cells cm–2 of leaf area ,lettuce (Lactuca sativa), and neotropical forest and poplar treesconsists of four major bacterial phyla, namely Proteobacteria,Firmicutes, Bacteroidetes, andActinobacteria , and other treeleaves , and nitrogen fixation (nifH) gene markers,Methylobacterium and Sphingomonas species were highlyabundant in the plant leaf environment of three species, namely A.thaliana, Trifolium repens, and Glycine max fungal infection  andEscherichia coli O157:H7 (EcO157) colonize fresh leafy vegetables suchas lettuce (Lactuca sativa) via damaged leaf tissue and can causefood-borne disease outbreaks  whichregulates colonization as an important priming event in microbial community interactionswith the plant .ResearcPseudomonas syringae pv. syringae B728a strains onbean (Phaseolus vulgaris) leaves indicated that this hygroscopicbiosurfactant increases diffusion of water across a waxy leaf cuticle surface whichattracts moisture and nutrients to benefit the bacteria , ormation . SimilarN-acyl-l-homoserine lactone  and quinolones as QS molecules, whereas modified oligopeptides  are commonly used by Gram-positive bacteria for communication betweencells  enhancedsusceptibility of various Brassicaceae species to fungal mildew pathogens and Erwinia toletana(olive knot cooperator) stabilize the community and exchange QS signals, and thiscooperation results in a more aggressive disease on olive plants (Oleaeuropaea)  leaf-associated strains with QQ activity fordisruption of AHL-mediated QS, by using the biosensor reference strainChromobacterium violaceum CV026. These bacterial quorum quenchers canbe used as effective biocontrol agents against plant pathogens (Endophytes utilize QS to act against pathogenic microbes by expressing QS inhibitors(QSIs) to attenuate the activity of AIs, or quorum quenching (QQ) enzymes to disruptsignaling molecules. For example, AHL lactonase enzyme (a potent quorum quencher) presentin endophytic bacteria has been reported to inhibit the plant pathogens rotovora , 2001, Bngiensis , and Entasburiae . Ma et aInplanta experiments on Phaseolus lunatus have shown that, if apathogen was introduced to the plant on the same day or before inoculation with anendophyte, disease resistance was more strongly reduced than when the endophyte hadalready colonized the host . Similarly, different strains of the pathogenic fungusFusarium oxysporum can act as microbial antagonists against otherF. oxysporum strains  and intracellular receptors which recognize microbial or modified host molecularsignatures and retain plant health and secure plant propagation. Surface immune receptors are members of a diversefamily of ligand-binding proteins that sense microbial, environmental, developmental, andnutritional cues . The intThe activation of plant immune responses by mobilizing a network of defense and stresshormone pathways has been extensively characterized in binary plant–pathogen interactions. High-thIn this section, we consider evidence that abiotic and biotic stress responses modulatemicrobial consortia on leaves and discuss the consequences for plant fitness. It is becomingclear that microbial community structure throughout a plant host’s life cycle is dynamic andmodulated by the innate immune system, which itself is tuned to environmental changes.P. syringae bacteria and riceresistance to the rice blast fungus, Magnapothe oryzae (ethylene-insensitive2 (ein2) mutantdisplayed an altered bacterial leaf community compared with wild-type plants, suggestingthat ethylene signaling is important for modulating the leaf microbiota ] that is defective inPTI and the MIN7 vesicle trafficking pathway (affecting the aqueous apoplasticmicroenvironment) and a constitutively activated cell death1(cad1) mutant had altered endophytic bacterial leaf diversity  in protecting Arabidopsis against infection by afungal necrotrophic pathogen, B. cinerea (Actinobacteria(Strepotmycetes sp.) are able to activate plant biosynthesis ofsalicylic acid (SA) and promote leaf defense responses against fungal pathogens , which favor NLR expression in thephyllosphere , is often mediatedby intracellular NLR receptors which recognize certain pathogen-delivered virulencefactors (effectors) to induce immunity  reduce microbial diversity within leaf habitats and stabilizemicrobial communities among wild plants  in leaf tissues  and grapevine against necrotrophic (B.cinerea) fungi have been studied extensively in vitro (P. syringae pathovar tomato (Pst) to Arabidopsis rootsattracted Bacillus subtilis and led to IP upon Pstinfection  affecting microbial diversity  on crops such as potato against biotrophic(The plant phyllosphere is a highly competitive and challenging habitat for microbes tocolonize. Pre-formed barriers such as the hydrophobic cuticle, stomata, or cell wallstructures require specific adaptation for the microbes, and persistence of microbesstrongly depends on their ability to interact with others. Thus understanding microbiotaassembly and persistence in the plant phyllosphere requires investigating ecological factorsthat shape pre-formed plant structures and therefore directly act on host–microbe andindirectly microbe–microbe interactions. Microbe–microbe interactions in turn not onlyimpact microbial behavior but can impact host fitness by antagonizing plant pathogens.Pathogen invasion generally has a significant negative effect on host fitness caused bytissue damage, nutrient loss to invaders, and reallocation of resources to immuneactivation. Successful pathogenesis on the other hand is a complex process that requiresmultiple steps of host colonization and reproduction, and is generally the result oflong-term co-evolution (The downside is, however, that this pool is highly dynamic and probably requires a stableco-existence of different microbial species in one habitat in order to express beneficialtraits. Interconnected networks between organisms can be an important element in providing abuffer against perturbations since such links help to recruit microbes to fulfill specificfunctions in cases where another organism that, for example, provides importantantimicrobial compounds or enzymes within the network is lost.A main goal to develop strategies to protect the phyllosphere from pathogen invasion, suchas wheat from rusts, is to identify probiotics that can either stabilize the naturalcommunity or become stable on its own. One major effort to develop such probiotics istherefore understanding the multistep process of establishing a niche and defending thisniche. Only once we know how to combine traits for stability with our desired traits such asplant protection will we be able to develop products that can replace the majority of ourcurrent agrochemicals.


Geum peckii Pursh, Rosaceae) is a globally rare and endangered perennial plant found only at two coastal bogs within Digby County  and at several alpine sites in the White Mountains of New Hampshire (USA). In Canada, the G. peckii population has declined over the past forty years due in part to habitat degradation. We investigated the culturable foliar fungi present in G. peckii leaves at five locations with varying degrees of human impact within this plant species’ Canadian range. Fungal identifications were made using ITS rDNA barcoding of axenic fungal cultures isolated from leaf tissue. Differences in foliar fungal communities among sites were documented, with a predominance of Gnomoniaceae . Habitats with more human impact showed lower endophytic diversities (10–16 species) compared to the pristine habitat (27 species). Intriguingly, several fungi may represent previously unknown taxa. Our work represents a significant step towards understanding G. peckii’s mycobiome and provides relevant data to inform conservation of this rare and endangered plant.Eastern Mountain Avens ( Phialocephala scopiformis, produces rugulosin, which inhibits the feeding of the spruce budworm, Choristoneura fumiferana. Consequently, the Canadian forestry industry inoculates spruce seedlings with P. scopiformis to improve host health ..https://Geum peckii. We documented 51 ascomycete fungi from the classes Sordariomycetes, Leotiomycetes, Dothideomycetes, and Eurotiomycetes. Endophytic fungal species richness changed with habitat degradation status, suggesting that this may influence the fungal assemblage present within leaves. A core fungal assemblage was documented for the first time in this plant host, from multiple sites throughout its Canadian range. Additionally, we present new host records for Rosaceae and make valuable linkages between host plants in the habitats and shared endophytes within Geum peckii that warrant further ecological investigation. By approaching plant conservation from a mycological perspective, we provide the first mycobiome assessment of G. peckii, an understudied component of the habitat. This new knowledge aids in ongoing conservation and propagation of this endangered plant species.In summary, we present the first preliminary survey of foliar endophytic fungi from the rare and endangered Eastern Mountain Avens,


Objective determination of burn wound healing potential remains elusive and significantly impacts decision making for surgery, the extent of tissue excised intraoperatively and the use of donor site-sparing alternative tissue therapies. Indocyanine green angiography (ICGA) has promise as an adjunct to evaluate healing potential, but feasibility has limited adoption in clinical practice. Delayed fluorescence imaging of indocyanine green (ICG), in a method called second-window ICG (SWIG), is a new technique used intraoperatively to guide tumor resection via increased peritumoral endothelial permeability. The objective of this study is to examine ICGA and SWIG fluorescence in burns requiring excision and grafting, and to correlate SWIG fluorescence to microscopic localization of inflamed and necrotic tissue.ex-vivo to determine the presence of fluorescence in the tissue compared to that remaining within the wound bed. Excised tissue was processed for histologic analysis of cellular architecture, viability, inflammation and necrosis. Macroscopic ICGA and SWIG fluorescence images were compared to the associated microscopic tissue sections to determine the presence of inflammatory infiltrate, localization of non-viable tissue, and co-localization of ICG fluorescence.Deep partial thickness, indeterminate depth or full thickness burns were identified in adult patients scheduled for excision and grafting. 24 hours prior to surgery, baseline bright light and fluorescence images were obtained before the administration of up to 5 mg/kg ICG intravenously. ICGA was performed within 5 minutes of infusion initiation. On the day of surgery, bright light and SWIG fluorescence images were obtained before and after burn excision. The excised tissue was imaged ICGA imaging performed preoperatively demonstrated variable fluorescence throughout the burns without a clear cutoff value to delineate deep partial versus full thickness burns. SWIG imaging revealed a speckled fluorescence pattern prior to burn excision that became diffuse after excision suggesting a potential utility of SWIG to intraoperatively identify excision completion. ICGA and SWIG fluorescence demonstrated an inverse relationship, and SWIG fluorescence was associated with non-viable tissue.ICGA imaging alone was unreliable to delineate the need for surgical intervention. SWIG imaging of burn injuries may represent a valuable tool to guide the extent of excision intraoperatively and reduce unnecessary excision of viable tissue. Further studies are needed to understand SWIG fluorescence at the inflammation-necrosis border and how ICGA fluorescence along with SWIG can synergistically improve detection of healing potential in burn patients.


Biomolecules, titled “Oral Regenerative Medicine: Current and Future”, covers the dental field and focuses on craniofacial tissue regenerative therapy. Dental pulp and periodontal tissue-derived cells are well-known as major cell sources in the dental field. Dental pulp stem cells (DPSCs) are promising cell sources for biomineral tooth complexes [2+-ATPase (PMCA) in rat and human odontoblasts mediates dentin mineralization [2+ homeostasis in odontoblasts. They suggested that PMCA not only plays a critical role in physiological dentin formation but also in the pathological reactionary dentin formation induced by multiple external stimuli. It is also used in the application of high-pH dental materials on the dentin surface. Dental and oral tissues maintain homeostasis through potential reparative or regenerative processes. This native biological regulation ensures appropriate oral function and provides systemic health conditions through ideal digestion and intake of nutrition. Even if some tissues break down, general dental treatment or the administration of growth factors can help them to recover. However, a large disruption of oral tissues due to tooth loss, malignant diseases, and severe trauma can cause irreversible tissue loss in the oral cavity. To compensate for this, artificial materials, such as prostheses and dental implants, support the improvement in human quality of life. We need to consider both native biological elements and biomedical products. This Special Issue of omplexes . Pilbaueomplexes . The automplexes . In thisomplexes . Schmidtomplexes . The autlization . This stAmong oral tissues, the periodontium is also permanently exposed to mechanical forces resulting from chewing, mastication, or orthodontic force. Brockhaus reported on the preservation and remodeling processes within the periodontium through periodontal ligament fibroblasts during orthodontic tooth movement. Tooth movement by mechanical stress initially decelerates the periodontal ligament fibroblast cell cycle and proliferation. After adapting to environmental changes, cells can regain periodontium homeostasis, affecting their reorganization . DieterlThis Special Issue suggests the importance of understanding the relationship between both the cell source and effective regeneration. Physiological functional recovery is the most essential problem and the ultimate purpose of regenerative therapy. A deeper understanding of the pathological background and genetic basis and cellular signaling in local-systemic regulation is essential in dental tissue regeneration.


Following ischemic stroke, polymorphonuclear neutrophils (PMNs) are rapidly recruited to the ischemic brain tissue and exacerbate stroke injury by release of reactive oxygen species (ROS), proteases and proinflammatory cytokines. PMNs may aggravate post-ischemic microvascular injury by obstruction of brain capillaries, contributing to reperfusion deficits in the stroke recovery phase. Thus, experimental studies which specifically depleted PMNs by delivery of anti-Ly6G antibodies or inhibited PMN brain entry, e.g., by CXC chemokine receptor 2 (CXCR2) or very late antigen-4 (VLA-4) blockade in the acute stroke phase consistently reduced neurological deficits and infarct volume. Although elevated PMN responses in peripheral blood are similarly predictive for large infarcts and poor stroke outcome in human stroke patients, randomized controlled clinical studies targeting PMN brain infiltration did not improve stroke outcome or even worsened outcome due to serious complications. More recent studies showed that PMNs have decisive roles in post-ischemic angiogenesis and brain remodeling, most likely by promoting extracellular matrix degradation, thereby amplifying recovery processes in the ischemic brain. In this minireview, recent findings regarding the roles of PMNs in ischemic brain injury and post-ischemic brain remodeling are summarized. PMNs are first line immune invaders in the ischemic brain. PMN recruitment after experimental ischemic stroke is highly coordinated in a spatio-temporal way. PMNs accumulate within capillaries and venules of the ischemic brain territory within the first hour after ischemic stroke , followeEvidence in humans supports the idea that PMNs have detrimental consequences on stroke outcome. Hence, PMNs of ischemic stroke patients produce and release significant amounts of reactive oxygen species (ROS) and proteases, such as neutrophil elastase, as shown in peripheral blood within the first 6 hours post-stroke . Under ivia which PMNs in peripheral blood aggravate secondary ischemic brain damage are PMN stalls resulting in microvascular occlusions even under conditions of successful arterial reopening  within 6 hours of symptom onset aggravated neurological recovery assessed by the mRS score, increased stroke mortality and increased infection susceptibility . BesidesEx vivo, the treatment of blood clots obtained from ischemic stroke patients with DNAse-1 significantly increased tPA-induced thrombolysis in comparison to tPA alone  and Hertie-Stiftung .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.


Key challenges include poor graft survival, low donor neuron localization to the host retina, and inadequate dendritogenesis and synaptogenesis with afferent amacrine and bipolar cells. In this review, we summarize the current state of experimental RGC transplantation, and we propose a set of standard approaches to quantifying and reporting experimental outcomes in order to guide a collective effort to advance the field toward functional RGC replacement and optic nerve regeneration.As part of the central nervous system, mammalian retinal ganglion cells (RGCs) lack significant regenerative capacity. Glaucoma causes progressive and irreversible vision loss by damaging RGCs and their axons, which compose the optic nerve. To functionally restore vision, lost RGCs must be replaced. Despite tremendous advancements in experimental models of optic neuropathy that have elucidated pathways to induce  The retina is embryologically derived from central nervous system (CNS) neuroectodermal progenitors. The mammalian retina, therefore, like the rest of the mammalian CNS, lacks inherent regenerative capacity . RetinalRetinal diseases have been pioneering targets for therapeutic cell transplantation in medicine ,12, withendogenous RGCs following experimental injury [Molecular targets relevant to achieving critical milestones in functional RGC replacement, particularly the achievement of RGC survival and axon regeneration, have been elucidated by studying l injury . For exal injury ,20. Insul injury , overexpl injury , inductil injury , deletiol injury ,25,26, al injury  in endogl injury . RGC-extl injury ,29. Morel injury , establil injury .By comparison, in vivo transplantation of exogenous primary RGCs or stem cell derived RGCs remains a field in its infancy. Nonetheless, pioneering transplantation studies have recorded light-evoked electrophysiological responses from donor RGCs  and docuRGC transplantation strategies require generating lineage committed progenitors or terminally differentiated neurons in sufficiently large quantities. The source of donor cells for transplantation  into humBRN3b-dependent tdTomato and murine Thy1.2 have facilitated purification of RGCs and longitudinal cell tracking in transplantation studies [ESCs are derived from the inner cell mass of embryonic blastocysts , and the studies .Adult human somatic cells, after appropriate induction, can dedifferentiate and resemble ESCs in morphology, and have the capacity for multilineage differentiation and self-renewal . When auA limitation to cellular differentiation in two dimensional culture is a lack of multi-cellular organization, which is overcome by three dimensional cultures of self-organizing organoids . These “Taken together, characterization of robust RGC differentiation from multiple cell lineages and culture conditions have paved the way for stem cell-based replacement strategies as a promising approach to optic nerve regeneration. The tools now exist to study, in earnest, RGC transplantation in vivo. In order to maximize generalization of protocols and experimentation worldwide, standard descriptions of donor cell characteristics are critical when reporting RGC transplantation results. Such studies should explicitly describe the parental stem cell line or source, differentiation culture conditions, the stage of RGC or progenitor at harvest, RGC purification technique and yield, and the expression of a standard set of canonical RGC markers. Ideally, critical results should be replicated with multiple independent lines of donor RGCs. Critically, we do not know the developmental state for donor cells—whether fully mature RGCs, developing RGCs, or lineage-committed progenitors—that will yield the best engraftment results. Whether stem cells or organoids are used as a source of donor RGCs, the isolation process should maximize purity in order to limit aberrant tissue overgrowth or teratoma formation from undifferentiated cell populations. Furthermore, RGCs survive poorly in organoids during prolonged culture , presumaA critical prerequisite to studying interactions between donor RGCs and the host retina is robust survival of transplanted neurons. Unfortunately, this has been a formidable obstacle in most studies to date (see below). One of the difficulties in comparing survival rates across multiple methodologies stems from unstandardized methods of reporting survival, either as an absolute number, a percentage of transplanted cells, or by providing the density of surviving cells. The transplantation route plays a critical role in discerning donor cell survival. Administration to the basal (vitreous) side of the retina via intravitreal injection provides the most direct access to the inner retina, without the need for potentially disruptive transparenchymal migration . MoreoveIn nearly all cases, where reported, intraocular transplantation of RGCs has yielded low rates of neuronal survival, far below what would be required to restore visual function in advanced optic neuropathy . Develop2 [2 [Purified postnatal mouse RGCs transplanted into rat recipients survive at a typical rate of about 1% , althoug2 . However2 [2   pathway is particularly relevant to RGC survival and regeneration following injury . DownregBax subfamily of Bcl-2-related proteins is essential for JNK-dependent apoptosis [Dual leucine zipper kinase (DLK) and leucine zipper kinase (LZK) are other key sensors in the RGC injury response . Upon dapoptosis , and itspoptosis . Initiatpoptosis . Deletiopoptosis . In addipoptosis , indicatpoptosis . Based on extensive work identifying critical regulators of injury-related RGC death, there are myriad intracellular pathways that could be targeted pharmacologically or transgenically to promote donor RGC graft survival following transplantation in the eye. Although manipulating neuronal survival pathways may seem like an obvious and feasible method to attenuate undesirable death of transplanted cells, we must also acknowledge the importance of these cellular pathways in driving malignant transformation. For instance, deregulation of PI3K/Akt mTOR signaling contributes to the initiation, development, and acceleration of multiple types of cancer . A majorPromotion of donor RGC survival by modulation of neurotrophic factor delivery may be an alternative to or compliment to directly targeting cell-intrinsic death signaling cascades. Soluble neurotrophins, such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), and glial cell line-derived neurotrophic factor (GDNF), regulate neuron growth and survival . The binBDNF and NGF signal via tyrosine-receptor kinases (Trk) receptors to promote cell survival, or via neurotrophin receptor p75 (p75), which induces neuronal apoptosis. Indeed, the effects of NGF on RGC survival depend on the relative expression of these dueling receptors . SpecifiBDNF is developmentally critical to RGC survival. By binding to the TrkB receptor, BDNF triggers activation of the Ras/MEK/extracellular signal-regulated kinase (ERK) pathway . Under nCNTF belongs to the IL-6 family of cytokines and binds a heterotrimeric membrane receptor complex composed of CNTF receptor-α, gp130, and the leukemia inhibitory factor receptor (LIF-R) . CNTF prGiven their strong potential for neuroprotection, trophic factors delivered concomitantly with RGC transplantation may substantially improve graft survival. However, clinical translation is limited by inefficient delivery of these agents. Intravitreal protein injection has a short therapeutic duration due to rapid clearance. Slow release formulations are being explored and have yielded promising results . Given tNumerous aspects of the recipient ocular microenvironment into which donor RGCs are transplanted influence their survival, including innate and adaptive immune system reactivity; blood flow, oxygen, and nutrient supply; and reactive gliosis .Autologous cell transplantation is generally well accepted by the recipient, although some genetic manipulations can generate immunological responses . However+ T cells are activated through the binding of MHC class I and T cell receptors, particularly in the presence of non-matching major histocompatibility complex  [Whereas the vitreous cavity and the subretinal space are relatively immune privileged when compared to other sites, damage to the blood ocular barrier and local inflammatory cytokine production can be induced by both neurodegenerative disease and the transplantation procedure itself . Intraocen, HLA) ,125,126.en, HLA) ,127.Editing of individual HLA genes in iPSCs to establish “universal donor cells” has been proposed for evasion of both T cell activity caused by HLA mismatching and natural killer (NK) cell activity. Genetic manipulation, such as the transgenic expression of HLA-E or HLA-G on porcine endothelial cells inhibits NK cell cytotoxicity and adhesion . GeneticThe transplantation technique itself is critical to consider when evaluating factors that might affect transplanted RGC survival. To date, the majority of studies have injected cells directly into the vitreous cavity, where the supply of oxygen and nutrients is subject to passive diffusion from the uveal vasculature. During development, the lens and the developing inner retina are first vascularized by the hyaloid canal, composed of the hyaloid artery, vasa hyaloidea propria, and tunica vasculosa lenti, which lie within the vitreous cavity. However, at birth in humans and postnatally in mice, these vessels regress as retinal vessels are prioritized, leaving the vitreous cavity unperfused . In ordeIt is critical to recognize that RGC transplantation will only be necessary because a primary neurodegenerative process caused extensive death of endogenous RGCs. Glaucoma, optic neuritis, and ischemic optic neuropathy are the most common neurodegenerative diseases characterized by RGC loss, but each one has unique pathophysiologies ,133. OncInnate neuroinflammatory processes influence donor RGC survival following injury and are principally mediated by retinal macroglia (astrocytes and Müller glia) and microglia. Under normal conditions, astrocytes play a key role in maintaining homeostasis in the CNS, controlling angiogenesis, contributing to the extracellular matrix (ECM), and maintaining the blood retinal barrier. Upon disruption or injury, reactive glia undergo complex alterations in gene expression, morphology, and function. Reactive astrocytes are promoted by JAK/STAT signaling, which leads to the release of a variety of soluble mediators that activate inflammation within the CNS ,136. His2 (PGE2) synthesis, which contributes to neuronal death [2+ concentrations and induces CREB-mediated transcription of apoptotic proteins, resulting in cell death [Some of the effectors of neuroinflammatory toxicity have been elucidated. In response to CNS injury or neurodegenerative disease, multiple cell types produce cyclooxygenase-2 (COX-2), leading to prostaglandin Eal death . Anti-inal death . Glial aal death ,145. Nonll death . Thus, nDonor cells arriving at the retina from the vitreous cavity encounter the retinal basement membrane (the ILM), local astrocytic and Müller glial reactivity, and local inflammatory cells that impede spontaneous cell migration into the retina ,147. EndThe ability of transplanted RGCs to migrate spontaneously into the recipient retina is inconsistent across published studies, which may be related to differences in graft and host species, or to the methodologies of determining graft localization. Our previous ex vivo work has identified that the ILM is a major barrier to both human RGC somal and neurite engraftment; we have found that donor human RGC migration into the host mouse retinal parenchyma rarely occurs spontaneously . DisruptAvailable evidence supporting donor integration is typically derived from en face confocal micrographs illustrating close proximity between donor cells and host RGCs, or from histological sections, again relying on proximity to the endogenous RGCs. However, such demonstration typically lacks the spatial resolution to distinguish the anatomical layers at the vitreoretinal interface. Because the RGCL is in close proximity to the ILM, donor RGCs that are external to the retinal parenchyma (within the posterior vitreous cavity) but juxtaposed to the retina can easily be mistaken for having “integrated” into the retina, both in retinal flat mounts and in histological sections . TherefoA combination of analyses using histological sections and en face evaluation of retinal flatmounts will provide a compromise between complete topographic imaging and depth resolution;Flatmount microscopy should be performed with high resolution confocal or multiphoton microscopy using a high magnification objective in order to create shallow depth of field;High depth resolution is attained by minimizing slice interval distance and pinhole aperture diameter (if single photon microscopy is employed);Z-stack reconstructions should be assessed using an orthogonal slice viewer to compare the localization of the donor cell to those of nearby endogenous cells;A secondary marker that outlines the boundary of the retinal parenchyma should be included when possible, such as immunofluorescent laminin delineation of the ILM .Although retinal cross sections can clearly demonstrate the relationship between transplanted RGCs and endogenous RGCL, applying this method to completely survey entire eyes is unfeasible. To help meet this challenge, we propose the following considerations for microscopic inspection of donor RGC localization within the host retina : A combiScrutinizing donor RGC integration using this robust approach sets a benchmark for accurate assessment of donor cell localization. The reported outcomes from the localization analyses should include the absolute number and percentage of total surviving RGCs in each of the retinal layers  as well as outside of the retinal parenchyma. Somal localization is important because donor RGCs with cell bodies remaining outside of the host retina can rarely exhibit parenchymal neurite ingrowth, which may be distinguished from fully integrated donor cells.In our experience, we observe negligible spontaneous donor human RGC integration into the host RGCL when the ILM remains intact, whereas somal integration is greatly enhanced by enzymatic disruption of ILM , suggestWe have observed a propensity for transplanted RGCs to cluster when cultured on organotypic retinal explants with intact ILM . ProteolConsidering this challenge, studies of RGC transplantation should include quantitative characterizations of cellular dispersion and retinal coverage. We have employed spatial analytic tools to describe the topography of donor RGCs cultured on the retinal surface ex vivo , and theAfter overcoming barriers to localization of transplanted RGCs within the recipient retina, dendrite targeting of the IPL necessarily precedes synaptogenesis to achieve true functional integration into the visual neurocircuitry. Donor RGCs are unlikely to sprout neurites without specific external cues . One sucThe proper targeting of donor cell dendrites to the mature host IPL is an understudied aspect of RGC transplantation. Developmental studies reveal that RGC dendritic lamination in the IPL is guided by signaling through cell surface receptors, and dendrite localization is reinforced and stabilized by neuronal activity . RGCs utRetinal flatmounts are superior to histological section for characterizing complex dendritic arbors of RGCs within the IPL;Additional secondary tissue samples for histological sectioning can, however, be useful for correlation of sublaminar neurite localization with multiple immunohistochemical markers that define RGC subtype;It is of interest to characterize the topographical spacing among integrated RGCs. Reported metrics may include nearest neighbor and density recovery profiles of integrated donor RGCs;When possible, metrics describing dendritic architecture should be described for individual cells; if individual arbors are not resolvable from overlapping RGCs, then metrics should be normalized to the number of donor RGCs contained within a region of interest;Dendritic architecture and localization within the recipient retina should be assessed using high resolution microscopy; it may be necessary to tile images in order to capture entire dendritic arbors at high resolution;Integrated dendritic arbors can be convoluted and should be resolved and traced using 3D rendering software ;Useful metrics to describe dendritic arbors include total neurite length, number of neurite segments, neurite density, dendritic Sholl analysis , and neuIn order to robustly characterize the dendritic outgrowth of donor RGCs, we raise the following considerations:developing RGCs express both MAP2 and Tau until definitive specification of an axon [If neurite integration is to be evaluated, detailed characterization of neurite identity will be needed. In mature RGCs, dendrites and axons are defined by the expression of MAP2 and Tau, respectively . Neurite an axon . Therefo an axon . This fiThere are over 40 subtypes of RGCs, each having dendritic arbors precisely restricted to specific lamina within the IPL . Such heViral transsynaptic tracers make it possible to decipher complex circuit connectivity. Individual viruses have defined directionalities of travel and benefit from intracellular replication that propagates reporter signal generation . The mosThere are numerous paradigms available to restrict uncontrolled propagation of virus across multiple synapses, to specify neuronal subtypes for primary infection (starter cells), and to not only label but also genetically modify synaptically coupled partner neurons through payload packaging. Typically, viruses used for synaptic circuit tracing are modified and incapable of infectivity and/or replication without helper genes  that are supplied to starter neurons separately. Through the use of Cre-dependent helper gene expression in conjunction with Cre reporter lines, one could, for instance, restrict starter cells to retinal neuronal subtypes of interest and probe donor RGCs for evidence of viral transsynaptic spread following transplantation . A compaLarge scale visualization of neuronal activity can be achieved by imaging genetically encoded fluorescent indicator proteins. Various optical reporters have been devised in response to vesicle release, membrane voltage change , and calElectrophysiology and synaptic histology remain useful for characterizing specific examples of functional synapses. Whole cell patch clamping of donor RGCs facilitates recording of light-evoked electrophysiological responses in retinal flatmount preparations. So long as controls are included to exclude the possibility of recording from intrinsically photosensitive RGCs, electrophysiologic responsivity to light implies stimulus conduction from bipolar cells downstream of photoreceptor transduction. Characterization of specific responses to light permits classification of donor RGCs based on electrophysiological properties.Synapses in retinal neurons are unique in that they require rapid and sustained release of neurotransmitters in order to achieve graded synaptic output in response to a dynamic range of light signal intensity. Consequently, retinal synapses require a several fold higher number of vesicles than conventional synapses that transmit action potentials elsewhere . SynaptiThese techniques may prove useful in future transplantation studies examining factors that regulate synaptogenesis between donor RGCs and the mature recipient retinal neurons. Synthetic synaptic organizers have been developed that are capable of inducing functional excitatory synapses within the CNS . Their fOne major consideration that is required for all transplantation studies is the possibility of intercellular material exchange ,189,190,Using human stem cells in RGC transplantation requires balancing safety and efficacy against potential risks and ethical considerations. Ethics concerns surrounding ESC use has largely been avoided with iPSCs as an alternative stem cell source. However, colonies expanded from a single iPSC display more heterogenous gene expression compared to ESCs , elevatiRecent scientific advances have enabled RGC production from a wealth of human stem cell sources for both disease modeling and RGC replacement within the visual pathway. Developments in stem cell biology and regenerative neuroscience have brought the field of optic nerve regeneration from science fiction to scientific possibility. Nonetheless, tremendous challenges for translating this potential to actual vision restoration for human patients lie ahead. Improving transplant survival remains the primary short-term goal, as it sets the foundation for subsequent study of functional integration. Advanced differentiation protocols may provide reliable RGC subtype specification and expand the donor source repertoire. As imaging techniques continue to improve, we anticipate significant progress in the ability to assess migration, localization, synaptogenesis, and axonogenesis in vivo.It is not well established how closely transplanted human stem cell derived RGCs resemble developing or mature neurons. This distinction may be important because the molecular machinery governing growth and regeneration potentially involves separate sets of regulatory mechanisms . For exa


This study explored the role of tested contextual factors  in high Medicaid (under resourced) nursing homes performance. Four nursing homes in geographically diverse states were purposefully selected for site visits based on high and low performance  indicators. Eight nursing home administrators and directors of nursing, and twenty-one nursing staff  and providers of support services were interviewed. Data were analyzed using an inductive thematic approach with NVivo 12 Plus. Within and across case analysis was used to compare participants’ perspectives across nursing homes and across administrators and staff. Several themes provide insight into varied influences of contextual factors on these nursing homes’ performance: focus on quality care, team-based approach, community support and engagement, and staffing retention. Providing quality care to residents was strategic priority in all facilities, which was enhanced by an adopted team-based leadership approach, open-door policy and home-like atmosphere. Community reputation and availability of local training opportunities for CNAs affected nursing staffing which some facilities addressed using creative retention strategies. These research findings will facilitate interventions, such as leadership training and organizational development activities, aimed at improving the performance of low performing facilities in terms of lower costs and better quality.


The purpose of this study was to examine whether frailty status moderates the association between social participation and attitude towards using gerontechnology. The sample was Korean older adults without cognitive impairment  who completed an online survey. The attitude towards using gerontechnology was measured with two questions from the Senior Technology Acceptance Model , asking whether using technology is a good idea and whether they like the idea of using technology. Social participation was assessed by asking whether the participants engage in social or community activities on a scale of 1–10. Frailty status was determined based on the Korean Groningen Frailty Indicator (K-GFI). Covariates were age, gender, marital status, employment status, education level, and household income. Results from regression analyses showed significant interaction between frailty status and social participation on attitude towards using gerontechnology. Specifically, social participation was associated with positive attitude towards using gerontechnology among non-frail older adults. This association was not significant among frail older adults. Our findings suggest that the relationship between social participation and attitude towards using gerontechnology might differ by physical health status. Among older adults who are physically healthy and actively participate in social activities, the attitude towards using gerontechnology might be more positive due to greater exposure to new technology-related information. Future studies need to address alternative ways to enhance technology-friendliness among older adults with poor physical health.


Caenorhabditis elegans), fruit flies (Drosophila melanogaster), and zebrafish (Danio rerio): prominent model systems in which anatomical and genetic analyses have defined fundamental principles by which epidermal cells govern SSN development.Somatosensory neurons (SSNs) densely innervate our largest organ, the skin, and shape our experience of the world, mediating responses to sensory stimuli including touch, pressure, and temperature. Historically, epidermal contributions to somatosensation, including roles in shaping innervation patterns and responses to sensory stimuli, have been understudied. However, recent work demonstrates that epidermal signals dictate patterns of SSN skin innervation through a variety of mechanisms including targeting afferents to the epidermis, providing instructive cues for branching morphogenesis, growth control and structural stability of neurites, and facilitating neurite-neurite interactions. Here, we focus onstudies conducted in worms ( Why focus on these model systems? Our understanding of patterns and mechanisms of Somatosensory neuron (SSN) innervation in human skin is limited by several challenges. First, human skin exhibits remarkable diversity in its structure across anatomical locations, varying in thickness, permeability, and cellular composition. Single-cell RNA-seq (scRNA-seq) studies demonstrate the presence of multiple distinct subpopulations of fibroblasts, keratinocytes, and other dermal cells at various locations in mammalian skin  provide solutions to many of these problems. Chief among them, these organisms offer transparent skin and ex utero development that renders SSNs optically accessible, providing a direct window into SSN development in vivo. These systems also offer sophisticated genetic toolkits that facilitate manipulation of gene function with single-cell resolution, reagents to simultaneously and independently visualize skin cells and SSNs, and a repertoire of epidermal cells and SSNs whose developmental origins and peripheral morphologies are defined.The model systems discussed here  and motor neurons that traverse the body and receive instructive epidermal cues. The touch receptor neurons (TRNs), sensory neurons PVD and FLP, and motor neurons provide instructive examples of different modes of epidermal signaling that contribute to skin innervation patterns. First, the bipolar mechanosensory TRNs ALM and PLM  extend distinctive anterior and posterior processes, and their polarized outgrowth is controlled by epidermal cues. Epidermal cells ensheath axons of these neurons, providing insight into the developmental origin and function of this specialized epidermis-SSN interaction  and is surrounded on the basal surface by a basement membrane  has served as a powerful experimental system for analysis of SSN dendrite morphogenesis, cell spacing, and dendrite-epidermis interactions that shape innervation patterns. Unlike vertebrate dorsal root ganglion (DRG) neurons, cell bodies of Drosophila SSNs are located in the periphery, where sensory organ precursors delaminate from the ectoderm early during embryogenesis and give rise to neurons in a highly stereotyped spatiotemporal birth order  organs or chordotonal (cho) organs; as discussed below, studies of cho neuron development have revealed roles for epidermal cues in guiding SSN migration and orienting dendrite outgrowth  on the basis of larval dendrite arborization patterns , and muscle.Each larval segment contains >10 different epidermal cell types, transcriptionally specified on the basis of their position along the anterior-posterior (AP) axis within each parasegment  have distinct experimental advantages for the analysis of SSN/skin interactions. Zebrafish are amenable to forward and reverse genetic screens; reverse genetic manipulation is particularly attractive since the large, externally fertilized eggs are easy to inject with antisense morpholino oligonucleotides or CRISPR-Cas9 ribonucleoprotein complexes. The small size of the larvae and automated behavioral assays additionally make high-throughput chemical screens feasible  periderm layer is derived from the enveloping layer  organs contain bipolar mechanosensory neurons that extend a single unbranched dendrite, and a subset of these neurons (v’ch1 and lch5) migrate along the epidermis to their final position, rotating during migration to orient dendrite outgrowth  or frazzled (fra), which encodes an attractive DCC  family Netrin receptor, prevent v’ch1 migration and randomize the direction of dendrite outgrowth  in mouse skin, in which a local secreted factor orients neurite positioning  and Drl2, which signal through the Rho guanine nucleotide exchange factor (GEF) Trio and Rho1 to locally destabilize the actin cytoskeleton and prevent dendrite extension beyond the Wnt5 source  and Wnt (Wingless) ligands illustrate two additional principles of peripheral guidance by secreted cues. First, extrinsic guidance signals can work in concert with intrinsic mechanisms for neurite spacing to fine-tune peripheral arborization patterns. Self-avoidance signaling mediated by the Drosophila homophilic adhesion molecule Dscam1 promotes sister dendrite spacing in da neurons  controls the density of body wall innervation . Third, how are short- and long-range Ret signaling coordinated? Mav exerts short and long-range effects on C4da dendrite growth, yet Mav exhibits limited diffusion, so internalized Mav likely regulates growth throughout the arbor. In vertebrates, internalized Ret-GDNF complexes mediate long–range retrograde signaling from the periphery together with GFRα1–3  as well as increased epidermis-ECM adhesion that is thought to reduce the permissivity of the ECM to dendrite growth , NLP-29, triggers this degeneration , which promotes ECM adhesion , whereas non-peptidergic nociceptors project through the SS and innervate the stratum granulosum  likewise regulates dendrite positioning of Many types of cutaneous receptors form specialized terminal structures with epidermal components that contribute to somatosensation -enriched microdomains on epidermal membranes adjacent to sensory neurites  to the cortex of the epidermal membrane surrounding the invaginating neurite  and numerous septate junction proteins  family member, is required for sheath formation, and EPB41 proteins function as interaction hubs that organize specialized plasma membrane domains , controls this developmental restriction of C4da plasticity  and engulf dendrite debris generated via laser-induced damage . What are the cells that mediate engulfment and digestion of neurite debris in the skin? Surprisingly, neither macrophage-like hemocytes in Drosophila, many studies have focused on Draper (drpr), an engulfment receptor, as an important component in both epidermal and non-epidermal phagocytic clearance of axon debris  downstream of engulfment receptors  often rely on the same set of phagocytic machinery to recognize and engulf synapses, cell corpses, or debris . In DrosDrosophila requires the CD36 family member Croquemort  and in human skin samples from patients with atopic dermatitis (AD), epidermal fibers often penetrate through the tight junction barrier and avoid pruning by keratinocytes. These observations raise the interesting possibility that aspects of pathological itch in AD may be due to aberrant SSN pruning by epidermal cells. It is possible that similar mechanisms may be at play in other skin diseases, lending to their pathologies, but this requires more careful investigation.Questions remain about epidermal involvement in neurite pruning and debris removal in adult animals, as well as whether these features are conserved in mammalian systems. Intriguingly, one recent study in mice found that epidermal SSN fibers often reside directly beneath keratinocyte tight junctions that form below the outer, cornified layer  and time? C. elegans presents an appealing system to address these questions, given the morphological stereotypy and limited cellular diversity. Finally, how do different SSNs achieve type-specific innervation patterns in response to similar extracellular cues? Neuron type-specific expression of receptors for these cues has been a focus of recent study, but additional mechanisms likely contribute including cell- and context-dependent signal transduction, as well as spatial tuning of receptivity to signals within SSN arbors.Despite the recent progress, substantial questions remain to be answered about epidermal control of SSN innervation. One pressing question is the extent to which epidermal diversity contributes to innervation patterns. A necessary prerequisite to answering this question is a deeper sampling of epidermal cell types. Even within model systems, this question is understudied, hence leveraging positional information embedding in the CY, EP, JR, and JP wrote this manuscript. All authors contributed to the article and approved the submitted version.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.


Me & My Wishes is a novel systematic approach for long-term care residents living with dementia to record videos about their care preferences that can be shared with staff and families in care plan meetings. To understand how the videos were utilized in Goals of Care (GOC) conversations, we coded and analyzed transcripts of recorded care plan meetings at the time of sharing the video using a priori codes derived from GOC conversation elements. Coding discrepancies were resolved in team meetings; finalized codes were summarized to derive themes. Thirty-four care plan meeting conversations between residents (n=34), family members (n=29) and staff (n=35) were analyzed. Residents appreciated sharing personal histories and preferences via video, while staff members appreciated deeper understanding of residents’ care preferences. Two themes described care plan meeting conversations: Everyday Care - a checklist-style assessment of the resident’s daily care , activities engaged in and satisfaction with care; and Clarifying Care Goals - checking the resident’s treatment preference , explaining hospice, or confirming the resident’s contact person. Several elements of GOC were not discussed  and conversations lacked depth and comfort evidenced by apologetic language and abrupt transitions of topics rather than exploring alignment of goals with care preferences. Me & My Wishes videos are a mechanism for residents to voice preferences. Standardized guidance, which is lacking in long-term care, is needed to help care teams engage in meaningful conversations to ensure alignment of goals and treatment preferences.


Whether increased formal long-term care (LTC) reduces informal LTC use by serving as a substitute or has a complementary role that boosts both informal and formal LTC use has been an important issue for evaluating LTC policy effectiveness. We described trends in in-home LTC use among older adults and LTC availability in relation to changes in LTC policy in Japan. In addition, we examined whether these trends differ by living arrangements, gender, income, and disability levels. We used five waves of repeated cross-sectional data starting in 1999 to 2017. The use of both informal and formal LTC types combined increased until 2006 and then gradually decreased while remaining higher than in 1999. Although implementing the LTC program may have temporarily contributed to the complementary use of both LTC types, eligibility limitations brought about by LTC reform potentially reduced the effects of formal LTC’s complementary role.


Microbial communities are constantly challenged with environmental stressors, such as antimicrobials, pollutants, and global warming. How do they respond to these changes? Answering this question is crucial given that microbial communities perform essential functions for life on Earth. Our research aims to understand and predict communities’ responses to change by addressing the following questions. (i) How do eco-evolutionary feedbacks influence microbial community dynamics? (ii) How do multiple interacting species in a microbial community alter evolutionary processes? (iii) To what extent do microbial communities respond to change by ecological versus evolutionary processes? To answer these questions, we use microbial communities of reduced complexity coupled with experimental evolution, genome sequencing, and mathematical modeling. The overall expectation from this integrative research approach is to generate general concepts that extend beyond specific bacterial species and provide fundamental insights into the consequences of evolution on the functioning of whole microbial communities. Microbial communities perform pivotal functions, from the cycling of elements through Earth’s ecosystems to the digestion of complex foods that shape human health and disease. Many of these functions emerge from the interaction of multiple species working as a collective . For exaAchieving such understanding is extraordinarily difficult given that microbial communities are incredibly diverse in terms of numbers of different microbes, genes, and interactions. Progress on this problem requires both top-down studies that deal with the complexity of natural communities and bottom-up research that uses simpler experimental setups . My reseRapid evolution can impact short-term ecological dynamics, and these altered ecological dynamics can feedback to affect subsequent evolutionary change . Eco-evo4–Acinetobacter johnsonii and Pseudomonas putida, two bacterial species that were originally isolated from a polluted aquifer in Denmark   . Thus, bA. johnsonii and P. putida  in natural communities  and different genotypes (evolutionary responses). What is the relative contribution of ecological versus evolutionary processes to mediating responses to change? We previously discussed how adaptive evolution mediated by settings . What resettings , 20. Filsettings . This exOne important ecosystem function is plant litter decomposition, which is performed by microbial communities . I have recently joined a collaboration, The Loma Ridge Climate Change Experiment, aiming to understand how the plant litter microbiome responds to drought in Southern California , 23. We Using an integrative research approach based on evolution experiments of simple microbial consortia, whole-genome sequencing, and mathematical modeling, we have gained general insights into the mechanisms by which microbial communities respond to environmental change. We are currently working to understand how these responses affect community-level functions important for ecosystem functioning. The next exciting research directions will be (i) to study whether natural selection can act on community-level properties  and (ii)