Regionalization of the vertebral column can help animals adapt to different kinds of locomotion, including arboreal locomotion. Although functional axial regionalization has been described in both chameleons and arboreal mammals, no morphological basis for this functional regionalization in chameleons has been proposed. However, recent studies have described regionalization in the presacral vertebral column of other extant squamates. To investigate possible morphological regionalization in the vertebral column of chameleons, we took morphometric measurements from the presacral vertebrae of 28 chameleon species representing all extant chameleon genera, both fully arboreal and ground-dwelling, and performed comparative analyses. Our results support chameleons exhibiting three or four presacral morphological regions that correspond closely to those in other sauropsids, but we detected evolutionary shifts in vertebral traits occurring in only arboreal chameleons. Specifically, the anterior dorsal region in arboreal chameleons has more vertically oriented zygapophyseal joints, predicting decreased mediolateral flexibility. This shift is functionally significant because stiffening of the anterior thoracic vertebral column has been proposed to help bridge gaps between supports in primates. Thus, specialization of existing morphological regions in the vertebral column of chameleons may have played an important role in the evolution of extreme arboreal locomotion, paralleling the adaptations of arboreal primates. For example, arboreal primates and some marsupials exhibit increased forelimb length and range of motion in protraction, scapular mobility, grasping appendages and modification of the vertebral column (e.g. ). MorphoChamaeleo calyptratus) [Chameleons present a good opportunity to study the relationship between axial regionalization and arboreality. Like primates, arboreal chameleons exhibit adaptations for locomotion on narrow, discontinuous substrates such as grasping appendages, decreased lateral undulation, increased forward reach, mobile pectoral girdles and forearm pronation –9. In adptratus) . Finallyptratus) ,15. TherTo explore the relationship between vertebral morphology, regionalization, phylogeny and arboreal locomotion in chameleons, we took morphometric measurements from presacral vertebrae. Previous studies using mechanical joint testing, correlation with locomotor behaviour, and engineering beam theory have shown relationships between vertebral morphometrics and function in vertebrates including crocodiles, marine mammals and primates ,12,16–19Based on these relationships between morphometrics and function, we predicted that the arboreal and ground-dwelling chameleons show particular morphometric patterns related to their locomotor behaviour. In arboreal chameleons, we predicted that the anterior trunk vertebrae would show morphometric characteristics associated with decreased mediolateral compliance and range of motion, such as more vertically oriented articular facets and mediolaterally broader vertebral bodies. The rationale is that restricted lateral undulation in the anterior dorsal region has been reported in several arboreal chameleon species ,20. By c. 2. 2.1www.Morphosource.org) (Agama agama) was included in the analysis as an outgroup because it is a member of the family Agamidae, the sister group to Chamaeleonidae. Scan data with high resolution and good contrast were selected.Micro-computed tomography (CT) scans of 28 chameleons  representing all extant chameleon genera were downloaded from Morphosource (rce.org) . A scan . 2.2www.thermofisherscientific.com). Morphometric measurements were taken digitally from meshes based on segmented micro-CT scans. Taking measurements digitally from micro-CT scans allowed us to take precise measurements from very small structures and to reconstruct slices through the joints and measure the angles of articular facets. First, the entire skeleton was globally segmented using the threshold tool , width (CW) and height (CH); neural spine height (NSH), transverse process width (TPW) and lamina width (LW)) were taken from the mesh surfaces (a) using the three-dimensional measure tool. Pre-zygapophyseal angles were measured using the three-dimensional angle tool from transversely oriented slices taken through the zygapophyseal facets using the Slice tool in Amira.Reconstructed scans were imported into Amira v. 2020.2  is found in more than one habitat, but we included it among the ground-dwelling taxa because it does not exhibit the extreme arboreal lifestyle of most ‘true’ chameleons [Chamaeleo calyptratus [To test whether vertebral morphologies were statistically different by vertebral region or habitat, we compared metrics taken from two representative vertebrae from each taxon. Taxa were coded as ground-dwelling or arboreal  based onet al. . One chaameleons . Vertebryptratus . Becauseyptratus  on theseet al. [To evaluate regionalization patterns within each species, we followed Jones et al.  to fit set al. ,27,28. T. 2.4et al. [For phylogenetically informed analyses, we used a time-calibrated phylogeny based on Tolley et al. . To deteet al.  in the ‘et al.  to fit O. 3a). All else being equal, fewer vertebrae would produce a less flexible vertebral column. For nearly all chameleons we sampled, a three- or four-region model including cervical, anterior dorsal and posterior dorsal regions was most strongly supported. No phylogenetic or functional pattern in regionalization was obvious (a). The most obvious morphometric differences among species are pre-zygapophyseal angle (PZA) and neural spine height (NSH). Arboreal chameleons show a peak in NSH at the cervical–dorsal transition, but no major differences among groups were evident in the rest of the presacral vertebral column . However, the anterior dorsal region in arboreal chameleons has significantly more dorsoventrally oriented zygapophyses , suggesting greater mediolateral stiffness and increased leverage for dorsal extension. The evolutionary shift toward specialization of the anterior dorsal vertebrae most likely occurred around the ancestral node of arboreal chameleons (b).Compared with their ground-dwelling relatives, the vertebral column of arboreal chameleons has a greater number of presacral vertebrae and displays more variation between anterior and posterior regions. The ground-dwelling chameleons tend to have fewer presacral vertebrae than the arboreal chameleons, but there is some overlap in vertebral count  a. All el obvious a. The moameleons b.Figure. 3.1Agama, roughly corresponding to the cervical (light blue), anterior dorsal (dark blue) and posterior dorsal (purple) regions found in other extant sauropsids [a). Most chameleons (26 of 28 species) had either three or four regions. Of the 12 chameleons with four regions, five species had a two-vertebra ‘anterior cervical’ region, which may reflect the difference between C3, which does not bear ribs, and C4–C5, which do. Of the remaining seven species with four regions, six had a two-vertebra ‘lumbar’ region, and one had a three-vertebra ‘cervical–dorsal transition’ region. The chameleon with five regions (Kinyongia carpenteri) had both anterior cervical and lumbar regions. One chameleon, Brookesia perarmata, had only two regions with a division near the midpoint of the dorsal region. Brookesia perarmata also has the fewest presacral vertebrae [a). Inclusion of PZA in the regionalization model through principal coordinates analysis resulted in more variation in both the number of regions and the location of divisions between regions . However, no clear phylogenetic or functional pattern emerged.Two to five morphological regions were optimally supported across sampled species, and no phylogenetic pattern was obvious. Three regions were detected in uropsids  . Ground-dwelling chameleons show greater variation in neural spine morphology, ranging from the narrow, posteriorly inclined spines of Rieppeleon to the roughened accessory bridges of bone in Brookesia . No difference in the relative dimensions of the vertebral bodies or laminae between ground-dwelling and arboreal chameleons was apparent. Differences in vertebral morphometrics between regions and taxa was investigated further using phylogenetically corrected ANOVA (reported below) and models of trait evolution for NSH and PZA (see Evolutionary models section). Centrum length was not modelled because it closely parallels number of vertebrae.Although vertebral regionalization in squamates is less distinct than it is in mammals, the anterior and posterior dorsal regions in arboreal chameleons differ in several ways from those of ground-dwelling chameleons and other limbed squamates. Contrary to previous descriptions , the zygl spines d–f. Grourookesia a,b. No dAgama and the ground-dwelling chameleons are oriented more mediolaterally than dorsoventrally, reflecting specialization for mediolateral mobility. Pre-zygapophyseal angle in Agama lacks clear regional trend and remains close to 100° throughout the trunk (b). In most of the ground-dwelling chameleons, the zygapophyses are even more horizontally oriented. By contrast, the PZA in arboreal chameleons in the anterior dorsal regions is generally less than 90°. When we directly compared PZA of representative anterior and posterior dorsal vertebrae , differences in PZA between regions were statistically significant , while the p-value associated with differences between arboreal and ground-dwelling groups is slightly above the 0.05 threshold for statistical significance. Mean PZA in the anterior dorsal vertebrae of arboreal chameleons (mean (M) = 85.1°, s.d. = 10.4°) was smaller than in the posterior dorsal vertebrae of arboreal chameleons . In ground-dwelling chameleons the difference in angles between anterior and posterior dorsal vertebra was not statistically significant: PZA of anterior dorsal vertebrae  was not significantly different from PZA of posterior dorsal vertebrae . After accounting for the effect of phylogenetic relatedness, PZA in the anterior dorsal region of arboreal chameleons was smaller than that of ground-dwelling chameleons but fell just short of statistical significance at the 0.05 level (p = 0.056). No other metric demonstrated statistically notable differences between arboreal and ground-dwelling chameleons .As in most squamates , zygapophe trunk b. In mose–h; electronic supplementary material, figure S1). Archaius, thought to have independently acquired an arboreal lifestyle [Trioceros species, neural spines are tall relative to centrum length throughout the trunk . The ground-dwelling chameleons and Agama lack a sharp peak at the cervical–dorsal transition, and NSH decreases slightly or shows no trend throughout the trunk. Palleon, the earliest-diverging genus of chameleons, is the only ground-dwelling taxon in which we observed a pronounced peak at the cervical–dorsal transition . In both ground-dwelling and arboreal chameleons, size-corrected NSH was slightly greater in the anterior dorsal vertebrae than the posterior dorsal ones, but the differences were not statistically significant . For ground-dwelling chameleons, the mean NSH was 2.1 mm (s.d. = 0.7 mm) in the anterior region versus 1.8 mm (s.d. = 0.7 mm) in the posterior region, and for arboreal chameleons mean NSH was 4.8 mm (s.d. = 2.5 mm) in the anterior region versus 4.0 mm (s.d. = 2.1 mm) in the posterior region. NSH  was not significantly different between arboreal and ground-dwelling taxa at either the anterior or posterior position after accounting for phylogenetic relatedness (0.61 < p < 0.68).In addition to more vertically oriented pre-zygapophyses, the arboreal chameleons share a pattern of increasing neural spine height (NSH) around the cervical–dorsal transition followed by a decrease throughout the dorsal region . In arboreal chameleons, centra were significantly longer in the posterior dorsal region than the anterior dorsal region . Proportionally longer centra are associated with regions of increased flexibility [p < 0.484; electronic supplementary material, table S1), and, because summed centrum length was used to correct for body size, this metric was closely tied to number of dorsal vertebrae. Cervical vertebrae in all the squamates we examined are distinguished from dorsal vertebrae by anteroposteriorly shorter centra and broader laminae, although the cervicodorsal transition is less pronounced in Agama than in the chameleons.Common to chameleonids and xibility , suggestb–h; electronic supplementary material, figure S1). In the dorsal region in Agama, the transverse processes are relatively broad (TPW exceeds centrum length), and they increase in lateral breadth throughout the trunk. In the chameleons, however, the transverse processes remain relatively narrow throughout the dorsal region.All the squamates we studied share a peak in transverse process width (TPW) in the cervical region and a sharp increase in the posterior dorsal region . However, arboreal chameleons have a greater number of dorsal vertebrae on average; the same amount of disparity between individual vertebrae could produce larger differences between regions.Finally, disparity along the presacral vertebral column (excluding the proatlas and axis) was not significantly different between arboreal chameleons and ground-dwelling chameleons , which have larger PZAs , and finally in B. perarmata, which has PZAs smaller than most other ground-dwelling taxa but larger than most arboreal ones . The second shift occurred at the node representing the most recent common ancestor of arboreal chameleons, excluding Archaius , corresponding to the regional increase in PZA noted in the morphometric analysis. The OU model fitted to NSH scaled to trunk length showed shifts in the ground-dwelling clade including Brookesia and Palleon and in the node leading to Trioceros and within that genus, which includes the only fine-branch arboreal specialists in our analysis. Both shifts tended toward longer neural spines relative to trunk length.Based on best supported models of stabilizing trait evolution, evolutionary shifts in PZA most likely occurred at the node leading to arboreal chameleons and in an early diverging lineage of ground-dwelling chameleons. The earliest shift occurred at the node leading to  Palleon b, node 1Archaius b, node 2. 4We hypothesized that morphological correlates of decreased mediolateral compliance and range of motion would be found in the anterior trunk vertebrae of arboreal chameleons, and that this distinction most likely emerged at the origin of true arboreal chameleons. Morphometric data from 28 chameleon species representing all extant genera support this prediction, showing that the anterior dorsal region of arboreal chameleons exhibits a characteristic pattern of decreased PZA, correlated with decreased mediolateral mobility. Further, analysis of individual vertebrae from two locations along the vertebral column showed that PZA from the anterior dorsal vertebrae in arboreal chameleons is significantly smaller than posterior dorsal vertebrae in the same group and also smaller than anterior dorsal vertebrae in ground-dwelling chameleons, often resulting in facet joints that are oriented more vertically than horizontally. That pre-zygapophyseal angles smaller than 90° are found only in arboreal chameleons and almost exclusively in the anterior part of the presacral vertebral column suggests that more vertical facet joints play a role in climbing, possibly supporting the pectoral girdle and forelimb during bridging or stabilizing it against mediolateral excursions. When we mapped PZA onto a chameleon phylogeny, an evolutionary shift in facet orientation was recovered at the node leading to a radiation of large-bodied arboreal chameleons. Arboreal chameleons also showed increased neural spine height around the cervical–dorsal transition, possibly reflecting differences in locomotion and/or feeding. Analysis of morphological regionalization showed no clear change in the number and distribution of regions. As such, the trait evolution in arboreal chameleons seems to result from increased morphological differences between existing regions rather than an increase in regionalization.. 4.1et al.'s [C. calyptratus moving on a perch found that this lower contribution to step progression results mainly from restriction of undulation to the posterior region of the spine. They measured lateral flexion between five segments of the dorsal vertebral column, each corresponding to roughly 20% of trunk length  and fou [et al.  demonstr. 4.2b). Ancestral reconstruction indicates that the earliest chameleons most likely were ground-dwelling and that arboreal habits were independently acquired in the Eocene radiation and in Archaius [ca 51–53 Ma) [Although chameleons all share morphological specializations for arboreal locomotion such as prehensile tails and digits, their transition to fully arboreal way of life appears to be a relatively recent innovation. The family Chamaeleonidae originated in the Late Cretaceous period (90 Ma), whereas the origin of the clade including most fully arboreal species is thought to have occurred during the Eocene epoch (47 Ma)  figure b. AncestArchaius . Therefo1–53 Ma) , as an aThe evolutionary shift we observed in PZA is especially suggestive of a change in function, because zygapophyseal orientation is considered to be one of the best osteological clues to axial flexibility and mechanics, as close contact between zygapophyses constrains the range and direction of movement of the intervertebral joint ,33. MoreBrookesia and Trioceros, respectively). The shift in Brookesia + Palleon may be related to the elaboration of the vertebral processes in some Brookesia species: in B. superciliaris and B. thieli, the anterior and posterior articular processes are connected by a bridge of bone, and there is an accessory arch between the left and right anterior articular processes that is connected to the spinous process [Trioceros, the extreme elongation the neural spines in the trunk could be related to locomotion on very narrow substrates  as several Trioceros species including T. jacksonii and T. quadricornis are considered fine-branch specialists [Bradypodion also includes fine-branch specialists —probably representing recent radiation into open habitats [While the proposed link between PZA and arboreal locomotion is relatively straightforward, patterns of variation in NSH could be explained by more than one evolutionary hypotheses. The regional increase in NSH observed at the cervical–thoracic junction in most arboreal taxa may increase the leverage of muscles that attach to the pectoral girdle , facilitating the girdle rotation that contributes substantially to step length in arboreal chameleons ,38. In a process . The funcialists . Howeverhabitats ,41—no evChameleons and primates have in parallel acquired morphological and behavioural adaptations that allow them to navigate substrates narrower than their body width including decreased axial flexion. For animals that use slow arboreal locomotion, possible benefits to restricting undulation in the anterior dorsal region include minimizing head movement while stalking prey , decreas. 4.3Agama and ground-dwelling chameleons may not be functionally different, while the same two regions in arboreal chameleons may differ widely in terms of range of motion, stiffness, muscle leverage and other functional parameters. The difference between PZA in anterior and posterior dorsal regions as part of an evolutionary shift in PZA in the Eocene radiation of arboreal chameleons may indicate a functional shift in the vertebral column related to their adoption of a fully arboreal way of life.The trait evolution in arboreal chameleons reflects larger morphological differences between existing regions rather than an increase in regionalization. This result supports the hypothesis that morphological regions represent potential for functional differentiation that is not always realized. That is, a presacral vertebral column with multiple modules may have more than one functionally specialized region, but not necessarily so; for example, as mentioned in the introduction, the tegu lizard has four presacral morphological regions but only two functional regions . The antIn summary, a marked evolutionary shift toward reorientation of the zygapophyseal joints of the anterior trunk region in the lineage leading to extant large-bodied fully arboreal chameleons seems to have stiffened the anterior part of the trunk against mediolateral movements. This finding parallels a similar shift in vertebral morphology and function thought to help arboreal primates bridge large gaps between branches. Combined with previous kinematic data showing restricted lateral undulation in the anterior trunk, these results support the hypothesis that specialization of existing morphological regions was part of the suite of adaptations that allowed extant ‘true’ chameleons to adopt an extreme arboreal lifestyle.


Ruminococcaceae family taxon, one Clostridiales vadinBB60 group family taxon, and one Christencenellaceae family taxon– were connected to several additional butyrate-producing bacteria suggesting that deficits in butyrate production may contribute to clinical symptoms. Therefore, by considering the community nature of the gut microbiome in a real world clinical sample, this study identified a gut microbial co-occurrence network that was significantly associated with clinical anxiety in a cohort of depressed individuals.Alterations in the gut microbiome have been linked to a variety of mental illnesses including anxiety and depression. This study utilized advanced bioinformatics tools that integrated both the compositional and community nature of gut microbiota to investigate how gut microbiota influence clinical symptoms in a sample of participants with depression. Gut microbiota of 179 participants with major depressive disorder (MDD) in the Texas Resilience Against Depression (T-RAD) study were analyzed by 16S rRNA gene sequencing of stool samples. Severity of anxiety, depression, and anhedonia symptoms were assessed with General Anxiety Disorder – 7 item scale, Patient Health 9-item Questionnaire, and Dimensional Anhedonia Rating Scale, respectively. Using weighted correlation network analysis, a data-driven approach, three co-occurrence networks of bacterial taxa were identified. One of these co-occurrence networks was significantly associated with clinical features including depression and anxiety. The hub taxa associated with this co-occurrence module –one  Harnessing the potential of the microbiome for precision medicine approaches in psychiatry is at the forefront of clinical neuroscience. Much of the attention in this area has focused on the trillions of resident microbes in the gastrointestinal tract—referred to as the gut microbiome. The gut microbiome has been implicated in the etiology and pathogenesis of a number of diseases and disorders including: inflammatory bowel disease, irritable bowel syndrome, celiac disease, asthma, obesity, cancer, neurodegenerative diseases, autoimmune diseases, as well as mental health disorders, including, anxiety, schizophrenia, and depression –10. AccuTo garner the potential of examining the microbiome in psychiatry, it is important to recognize that the gut microbiome is a diverse ecosystem in which microbial cross-feeding and competition for nutrient resources influence the stability, composition, and function of the gut microbial community. Most studies to date have considered associations between single bacterial taxa and clinical phenotype, which does not consider the dynamic and community nature of the gut microbiome. This overlooked feature may be a critical factor to understand how bacteria-host communication influences host physiology. To fill this gap, we took a novel approach using advanced bioinformatics tools that integrated both the compositional and community nature of gut microbiota. Specifically, weighted correlation network analysis (WCNA), a data-driven tool commonly used in genomic analyses, was employed to identify co-occurring networks of gut microbiota. WCNA examines the biological networks’ structure by defining clusters of highly correlated taxa, identifying highly connected hub taxa, and comparing the network topology generated by different adjacency matrices . More imThis report provides a data-driven analysis of 16S rRNA microbiome sequencing data to identify networks of co-occurring human gut microbial communities in a sample of patients with current or lifetime diagnosis of depression. Once identified, these networks were used to answer the following questions: (1) Are these co-occurring networks related to clinical symptoms ? (2) What are the hub taxa for the co-occurring networks and are these hub taxa clinically relevant? This work demonstrates a novel bioinformatic approach to generate a microbial signature using 16S rRNA sequencing data that can be applied in clinical psychiatric research.n = 179) with current or past diagnoses of major depressive disorder (MDD) who had clinical assessments and a stool sample at the same visit.Participants in this study were recruited as part of the Dallas 2 K (D2K) study, a component of Texas Resilience Against Depression Study (T-RAD) . D2K folThe Patient Health Questionnaire (PHQ-9), a 9-item self-report questionnaire, used to assess depression severity (range 0 to 27 with higher scores between 20 and 27 reflecting severe depression) . The GenStudy participants were given a stool sample kit at each in-person visit to collect the stool sample. Explicit instructions were given to the participants on how to collect the samples. Once collected (within seven days of an in-person visit), the biological specimens were either chilled or frozen (if the sample could be returned within 48 hours of collection) by the participant. Once returned to the Center for Depression Research and Clinical Care, the samples were frozen at −80 °C until sequencing. All stool samples were collected within 1 week of the participant’s clinical assessments.Bacterial DNA was extracted from stool samples using methods previously described with some modifications . 16S rRNtax_glom function in phyloseq in R [Dimension reduction of quality controlled 16 S rRNA microbiome taxa was achieved using an in-house retain-resolve agglomeration strategy  in the WGCNA package. A soft-threshold power (β) of 3 was selected using WGCNA guidelines to satisfy scale-free topology criteria. Minimum module size of 10, 15, or 25 were used to generate networks; each network was assessed for individual module stability. Merging similar modules was attempted, however merged module was equivalent to original module sets for each network. Module taxa abundance in each sample was summarized as the module eigentaxon, which is a single value calculated using the moduleEigengenes function in WCGNA. Participants with high abundance of a particular module will have a higher module eigentaxon value. Then, association between module eigentaxon and continuous clinical traits  was determined using Pearson’s correlation coefficient according to WGCNA guidelines. Taxa in the 95th percentile for module membership were designated as hub taxa. Module membership was calculated by correlating taxa with each module eigentaxon. A higher module membership indicates a stronger relationship between the taxon and module abundance across samples. Module membership is also a proxy measure of taxa network node connectivity.The microbiome co-occurrence network was generated from 300 retain-resolved taxa using weighted correlation network analysis (WCNA) using the WGCNA package in R . CovariaTaxa significance was defined here as the signed Pearson correlation between an individual taxa and clinical trait . Correlation significance was adjusted using Benjamin-Hochberg correction. Higher taxa significance indicates that taxa is more biologically significant to the clinical trait. Moreover, high correlation between taxa significance and clinical trait suggests the module is robustly associated with the clinical trait. Lower correlation suggests a few select taxa within the module are likely driving the module’s association with the trait, rather than the microbial community. Correlation between module membership and absolute taxa significance – defined here as the unsigned correlation between individual taxa and clinical trait – is also provided for consistency with WCGNA’s methods.WCNA module stability was assessed by applying similar principles used in clustering stability analysis previously described . The modrcorr function in the Hmisc R package [r > 0.35 was set to simplify Cytoscape visualization. Edges were sized according to the strength of the correlation between taxa, with the thinnest connections corresponding to correlation of r = 0.35. Taxa nodes were also sized and colored by their brown module membership. Finally, taxa in the Clostridiales order are represented by oval-shaped nodes, while taxa belonging to all other taxonomic orders are represented by hexagonal nodes. A simple network visualization was also generated with a weak correlation threshold of r > 0.2. Taxa that did not connect to the main network after hard thresholding were removed from the visualization.The brown module network was visualized as a hard-threshold correlation network using Cytoscape (version 3.9.0) . Network package . An arbiglm function [randomForest package in R [ALDEx and random forest methods, which are often used in microbiome analysis, were employed to complement the results of the WCNA analytical approach and generate consensus taxa were associated with age, anxiety, and depression. Association between individual taxa and each continuous clinical trait  was determined separately using generalized linear models via ALDEx2 function . Taxa meage in R . Taxa me≥ 10), and 37% of participants had moderate or higher severity of anxiety  at the time of sample collection and clinical assessment. See Table The analytical sample included 179 participants  with average age of 46.2 years (SD = 15.9). Briefly, 50% of participants had moderate or higher severity of depression . Furthermore, brown module signed taxa significance is unidirectional, as all significant correlations between brown module taxa and GAD-7 score were negative  with primary hub taxa sp1198 Ruminococcaceae UCG-010   before correction . Although these individual taxa did not survive multiple correction in our analyses, this list represents potential taxa that may be considered as potential microbiota biomarkers for anxiety symptoms. PHQ-9 did not have any brown module hub taxa that achieved consensus criteria. However, GAD7 and PHQ-9 shared several positively associated consensus taxa including positive associations with Faecalitalea at both genus  and ASV-level  and genus-level Romboutsia . Genus-level Christensenellacea was also negatively associated with both GAD7 and PHQ9 . No consensus taxa were identified for DARS.To identify taxa of interest for potential biomarkers of depression, anxiety, or anhedonia we examined the consensus of all three analytical methods—WCNA, ALDEx2 and random forest. The taxa that met the criteria for significance in WCNA and either ALDEx2 or random forest methods are listed in consensus tables Tables . Brown mIn this sample of adults with depression, using 16S rRNA sequencing and clinical data, three co-occurrence communities of bacterial taxa were identified. One of these communities was significantly associated with clinical features including depression, anxiety, and anhedonia. Further, the microbial composition of this co-occurrence module was enriched with butyrate-producing bacteria. Importantly, these analyses demonstrate that applying weighted network correlation analysis to understand the community nature of the gut microbiome reveals stable microbiota networks in depressed individuals. Moreover, consideration of the bacterial community structure generated clinically relevant associations that were more robust than those generated by standard microbiome tools. Visualization of the clinically associated network demonstrated that numerous connections exist between co-occurring taxa and employing an integrative analytical approach may provide a more accurate and robust view of the community of bacterial associated with clinical symptoms.Ruminococcaceae family. The primary hub taxa belonged to the Ruminoccocaceae family. In addition, several module members belong to the Ruminococcaceae family and were significantly associated with anxiety symptoms following multiple correction. Notably, reduced abundance of Ruminococcaceae taxa was associated with increased depressive symptoms in two large cohorts, the Rotterdam Study cohort & Amsterdam HELIUS cohort, with 1054 and 1539 depressed individuals, respectively [Ruminococcaceae family taxa are butyrate-producing bacteria in the human gut [While anxiety symptoms are co-morbid in both physical and mental illness, clinical research design does not adequately address the presence anxiety or other co-morbidities . As suchectively  Ruminocouman gut , 42. Butuman gut , 43. Butuman gut . Indeed,uman gut . Intestiuman gut . Butyratuman gut . Furtheruman gut . A dysreuman gut , 47, resuman gut , and a human gut , 50. OveChristencenellaceae family. The Christencenellaceae family of bacteria have been reported to be heritable as revealed by microbiome studies in twins, and have been linked to metabolic health and aging [Christencenellaceae abundance has been associated with body mass index (BMI), with reduced abundance of Christencenellaceae in obese individuals (BMI > 30) compared to individuals with a normal BMI [Christencenellaceae has also been reported in individuals with metabolic syndrome [The second hub taxa of the clinically relevant microbiota module was a member of the recently identified nd aging . Christermal BMI . Reducedsyndrome , 53. IncClostridiales Vadin BB60 family emerging. Recent studies indicate it may be a marker of gut-brain axis health. Depressed patients were reported to have lower Clostridiales Vadin BB60 family abundance compared to healthy controls [Clostridiales Vadin BB60 was a robust biomarker for microbiome-based Alzheimer’s treatment in mice [Clostridiales Vadin BB60 family in the gut lumen is unknown, however, these taxa have been linked to increased dopamine metabolites or serotonin precursors in the brainstem [Clostridiales Vadin BB60 family taxa belong to the Clostridiales order, and other Clostridiales species are associated with reduced gut lumen inflammation via Treg expansion [The functions of hub taxa controls . Additio in mice . The biorainstem  and poterainstem . Clostrixpansion . While nFaecalibacterium and Coproccus bacteria were associated with higher quality of life, and a reduction of Coproccus and Dialister spp were linked to depression, an observation that was validated in a second cohort [Faecalibacterium and Coprococcus [Faecalibacterium in depressed individuals and reduced abundance of members of the Rumminococcaceae family [Faecalibacterium prausnitzii is an abundant commensal in the healthy human gut, plays an important role in gut physiology [Escherichia-Shigella and Alistipes, taxa that are suggested to be associated with increased inflammation [The significant relationship between anxiety and a gut microbial community identified in these analyses translates earlier neuroscience studies linking microbiota to anxiety-related behavior –62 to cld cohort . These prococcus . Severale family , 65, 66.ysiology , 68, andammation , supportOverall, the analysis of 179 current or formally depressed individuals presented here demonstrates the benefit of utilizing microbiome analysis to better understand the clinical heterogeneity in depression. The results of this study should be considered preliminary as replication of our findings in independent samples is required to validate the clinically relevant module, to verify the clinical utility of identified hub taxa, and to determine the generalizability of the results to a broad population of depressed patients. There are some limitations to these analyses: participants were recruited from a limited geographical region ; the analysis was cross-sectional and did not consider factors that influence microbiota composition and function, such as diet and exercise; and the lack of a healthy control cohort limits inference of the results to normal microbe-host interactions.In summary, this study identified three co-occurrence bacterial modules using 16S microbiome data from a broad sample of depressed participants. One of these co-occurrence modules was significantly associated with clinical depression and anxiety. Based on our results we propose that reduced abundance of butyrate-producing taxa and increased abundance of inflammatory-related taxa may drive increased anxiety and depressive symptoms in depression. An important feature of this work was the innovative, data-driven approach that integrated both the compositional nature of the microbiome, as well as the community structure. These results are a critical step toward understanding the association between the microbiome and depression, and how a community-driven approach may facilitate effective precision medicine to improve clinical outcome.Supplemental TablesSupplemental Figures


RS1 gene, is a common genetically determined form of macular degeneration. This report describes an unusual case of angle-closure glaucoma (ACG) with XLRS and discusses the treatment.X-linked retinoschisis (XLRS), due to mutations in the RS1 gene was diagnosed as XLRS and ACG, presenting with cystic macular lesions, shallow anterior chamber depth (ACD), and angle-closure with uncontrolled intraocular pressure (IOP). Malignant glaucoma occurred following trabeculectomy combining phacoemulsification with intraocular lens (IOL) implantation and goniosynechialysis. Subsequent anterior vitrectomy and irido-zonulo-hyaloid-vitrectomy (IZHV) effectively lowered IOP and deepened ACD, but the cystic cavity became larger.A 39-year-old Chinese man with an X chromosome-recessive inherited c.489G > A variant in the There is a potential risk of malignant glaucoma in ACG patients with XLRS after filtering surgery. Although anterior vitrectomy can effectively resolve aqueous misdirection, the macular retinoschisis may get worse. Awareness of this risk may aid in surgical planning and postoperative management in these patients. X-linked retinoschisis (XLRS), which is characterized by macular retinoschisis with or without peripheral retinoschisis, is a hereditary retinopathy in males resulting in significant vision deterioration . Young aA 39-year-old man presented with progressive blurred vision and ocular pain in the left eye for the preceding 8 months, and the left eye had undergone laser peripheral iridectomy previously. His right eye underwent combined phacoemulsification, intraocular lens (IOL) implantation, goniosynechialysis, anterior vitrectomy, and irido-zonulo-hyaloid-vitrectomy (IZHV) to resolve malignant glaucoma after trabeculectomy 5 years ago. The patient denied a family history of eye diseases or a history of ocular trauma or systemic diseases.RS1 gene. Genetic testing was also detected in other family members, and the heterozygous for the same variant in the RS1 gene was confirmed in his mother, while no variant in the RS1 gene was detected in his father or his son  was 0.1 in the right eye (OD) and 0.4 in the left eye (OS), and the refractive state was hypermetropic . IOP measurements were 11 mmHg OD and 45 mmHg OS. The fundus of the right eye cannot be seen clearly because of posterior capsular opacity. Positive findings on slit-lamp examination of the left eye revealed mild corneal edema, clear lens and shallow central and peripheral anterior chamber. Fundus examination of the left eye was notable for a spoke-wheel pattern in the macular area and a pale optic disc Fig. D. The paCarteolol, brimonidine tartrate, brinzolamide eye drops and oral acetazolamide were used to lower the patient’ IOP. Given the severe visual field damage and the elevated IOP on maximum anti-glaucoma medications, the left eye underwent trabeculectomy combined with clear lens extraction, IOL implantation and goniosynechialysis. On postoperative day 1, the eye showed a shallow central and peripheral ACD with a raised IOP of 35 mmHg. Malignant glaucoma was diagnosed, and anterior vitrectomy combined with IZHV was performed to balance the pressure between the anterior and posterior chamber. Following surgery, his IOP decreased to 11 mmHg and the ACD deepened.After 2 weeks, the right eye underwent Nd: YAG laser posterior capsulotomy and bilateral OCT was performed. OCT showed that the schitic cavities in the left eye were worse than that before surgery Fig. E and lasRS1 gene, which codes for retinoschisin, a secreted protein containing a discoidin domain [RS1 gene mutation.XLRS is associated with mutations in the n domain . Here wen domain –4. Furthn domain  also fouAlthough similar cases have been reported previously, this case had some unusual clinical course in that the macular cystic cavity of retinoschisis fluctuated during the treatment of glaucoma. In general, the natural history of XLRS displays long-term stability in most eyes . In thisThis case is unique also in its reminder significance of the high incidence of malignant glaucoma after filtering surgery for ACG patients with XLRS. In this patient, aqueous misdirection developed in both eyes after trabeculectomy probably because of young age, short axial length, and rotation of the ciliary body. In addition, retinoschisin can regulate fluid balance by binding to the NaK ATPase . One canTopical carbonic anhydrase inhibitors are beneficial in cases of XLRS with ACG, reducing IOP levels and particularly causing significant resolution in foveal retinoschisis. Selvan et al.  reportedIn the case presented here, we considered that the lower IOP was needed for the patient to prevent optic nerve damage further. Thus, trabeculectomy combined with clear lens extraction was performed to maintain lower IOP instead of clear lens extraction alone in the first surgery. However, aqueous misdirection syndrome developed following surgery. On the other hand, due to a lack of sufficient understanding of ACG patients with XLRS at that time, we performed the anterior vitrectomy, a routine surgical option for malignant glaucoma, to resolve aqueous misdirection instead of TCP, resulting in worse macular retinoschisis. Therefore, this case warns us that we should carefully select the treatment methods for ACG patients with XLRS in future clinical work.In conclusion, XLRS may be one of the etiologies for young ACG patients, and young ACG patients should undergo a fundus examination to exclude undiagnosed inherited retinal dystrophies. The present case demonstrates that malignant glaucoma occurs frequently in ACG with XLRS. Although anterior vitrectomy can effectively treat malignant glaucoma, the macular retinoschisis may get worse. It is a challenge for ophthalmologists to manage young ACG with XLRS.


The vasculature is characterized by a thin cell layer that comprises the inner wall of all blood vessels, the continuous endothelium. Endothelial cells can also be found in the eye’s cornea. And even though cornea and vascular endothelial (VE) cells differ from each other in structure, they both function as barriers and express similar junctional proteins such as the adherens junction VE-cadherin and tight-junction member claudin-5. How these barriers are controlled to maintain the barrier and thereby its integrity is of major interest in the development of potential therapeutic targets. An important target of endothelial barrier remodeling is the actin cytoskeleton, which is centrally coordinated by Rho GTPases that are in turn regulated by Rho-regulatory proteins. In this review, we give a brief overview of how Rho-regulatory proteins themselves are spatiotemporally regulated during the process of endothelial barrier remodeling. Additionally, we propose a roadmap for the comprehensive dissection of the Rho GTPase signaling network in its entirety. •Rho-regulatory proteins orchestrate spatiotemporal Rho GTPase activity patterns.•Cell architecture and post-translational modifications guide Rho regulator dynamics.•Systems analyses enable the identification of individual Rho GTPase signaling units.•Computational modeling integrates Rho GTPase signaling components into a network. Current Opinion in Physiology 2023, 34:100676EndotheliumThis review comes from a themed issue on Jeremy Pearson and Paul C EvansEdited by Endothelium”For complete overview of the section, please refer to the article collection, “https://doi.org/10.1016/j.cophys.2023.1006762468–8673/© 2023 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).The endothelium lines the luminal side of blood vessels where it controls the passage of molecules and immune cells into tissues With the use of experimental tools such as mutant analyses and GTPase activity pulldown assays, the basic principles of Rho GTPase signaling have been well established. With respect to the endothelium, this led to the general understanding that the Rho GTPases Rac1 and Cdc42 make the endothelial barrier more restrictive through the formation of lamellipodia and filopodia, respectively, whereas RhoA increases barrier permeability by forming contraction-related stress fibers Dysregulation of Rho GTPase signaling has been linked to a variety of diseases that involve endothelial barrier dysfunction, among which are vascular pathology Actin makes up roughly 10% of the total protein in endothelial cells To safeguard a reliable endothelial barrier stability, the cortical actin ring cooperates closely with two types of cellular adhesions, namely cell–cell junctions and cell-matrix adhesion complexes  [20]. EnThe Rho family of GTPases in humans consists of 20 members that can be divided into eight subfamilies Rho GEFs activate Rho GTPases by exchanging GDP for GTP Fundamental to Rho regulator specificity lies the structural core of Rho GTPases, as this determines both the conformation of its binding sites and its subcellular localization. Rho GTPases contain a G domain, a short Rho insert region, and a C-terminal hypervariable region. The G domain is where Rho regulators interact to adjust the activity status of Rho GTPases.This domain is characterized by five conserved sequence motifs (G1–G5), of which G- domain motifs G2 and G3 resemble the switch-I and switch-II regions, respectively A general concept that is believed to underlie the spatiotemporal regulation of Rho GTPases relies on the reaction-diffusion system One feature that contributes to the intracellular dynamics of Rho regulators during endothelial barrier remodeling is the spatial distribution of cellular components, including lipid structures, FAs, and components of the actin cytoskeleton In addition to the distribution of cellular components, endothelial cell geometry affects the spatiotemporal dynamics of Rho regulators d. For exAlthough over 150 Rho regulators are involved in the process of endothelial barrier remodeling, these numbers alone do not account for the wide range of actions that 20 members of the Rho GTPase family carry out during this process. In addition to the classical concept of GTP–GDP cycling, other mechanisms contribute to the regulation of Rho regulators to accomplish this plethora of actions Beside post-translational modifications, alternative mechanisms that manipulate Rho regulator dynamics include modifications at the level of gene expression Over the last decades, molecular perturbation strategies have been used to dissect the spatiotemporal regulation of individual Rho GTPase signaling pathways One way to integrate the dynamics of individual Rho GTPase signaling components, as measured by quantitative readouts of molecular perturbation, into a complete network, is by making use of computational modeling . ComputaBoolean models are known for their low computational complexity and their ability to integrate many molecular components, which is beneficial for the simulation of crosstalk with other signaling pathways. For example, Boolean modeling created new insights into the molecular mechanisms involved in endothelial-to-mesenchymal transition (EndMT). Upon drastic changes in tissue requirements, endothelial cells may differentiate into mesenchymal cells. This process inhibits the expression of VE-cadherin and thus leads to loss of cellular junctions, a scenario that is associated with the onset of various diseases. For example, in severe cases of Fuch’s dystrophy, a degenerative disease of the corneal endothelium, EndMT contributes to the development of corneal blindness In 2020, Müller and colleagues acknowledged the need for a systems analysis to reveal how Rho regulators contextualize and spatiotemporally regulate Rho GTPase signaling Understanding how Rho GTPases orchestrate endothelial barrier remodeling is a long- standing challenge. A central question is how in this course Rho-regulatory proteins are spatiotemporally regulated. Here, we provide an overview of the features that govern Rho regulator dynamics over space and time, including endothelial cell component distribution and post-translational modifications. These features together establish the framework for a model in which Rho GEFs, GAPs, and GDIs contextualize and spatially orchestrate the diffusional flux of Rho GTPases. Moreover, we evaluated comprehensive analytical approaches that may cooperatively map Rho regulator dynamics in the process of Rho GTPase signaling during endothelial barrier remodeling.1) the identification of individual Rho GTPase signaling network components with the use of family-wide expression-based systems analyses 2) validation and further characterization of Rho GTPase signaling components through quantitative readouts of molecular perturbation experiments 3) integration of individual Rho GTPase signaling component dynamics into a complete cellular network with the use of computational modeling A major challenge ahead will be to resolve Rho GTPase signaling in its entirety. Taking into consideration the most recent insights in comprehensive analytical approaches, we propose a roadmap for the dissection of spatiotemporal Rho GTPase signaling networks, including  and multiscale modeling  will likely by inevitable to resolving the exact position of GTPase signaling networks in the development of endothelium-associated diseases.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.Jeffrey M.A. van der Krogt: Conceptualization, Methodology, Formal analysis, Writing – original draft. Ivanka J.E. van der Meulen: Conceptualization, Writing – review & editing, Supervision. Jaap D. van Buul: Conceptualization, Writing – review & editing, Supervision, Project administration.Nothing declared.


Polymer semiconductorsare fascinating materials that could enabledelivery of chemical fuels from water and sunlight, offering severalpotential advantages over their inorganic counterparts. These includeextensive synthetic tunability of optoelectronic and redox propertiesand unique opportunities to tailor catalytic sites via chemical controlover the nanoenvironment. Added to this is proven functionality ofpolymer semiconductors in solar cells, low-cost processability, andpotential for large-area scalability. Herein we discuss recent progresson soft photoelectrochemical systems and define three critical knowledgegaps that must be closed for these materials to reach their full potential.We must (1) understand the influence of electrolyte penetration onphotoinduced charge separation, transport, and recombination, (2)learn to exploit the swollen polymer/electrolyte interphase to driveselective fuel formation, and (3) establish co-design criteria forsoft materials that sustain function in the face of harsh chemicalchallenges. Achieving these formidable goals would enable tailorablesystems for driving photoelectrochemical fuel production at scale. One emerging strategy is to use highlyscalable, printable, soft materials as semiconductor photoelectrodesfor photoelectrochemical (PEC) devices, which offer opportunitiesto further enhance the tailorability of local environments that drivemultielectron, multiproton redox reactions. Herein, we present a perspectiveon the basic energy sciences of polymer-based semiconductors and theway that these “soft” semiconductors may enable tunableand durable devices for solar fuel generation. Simultaneously, weaddress how basic energy science research can and should advance societalaspirations of a just energy future  used in conventional battery descriptions. The unique characteristicsof this “interphase” of a polymer-based photoelectrodeare illustrated in nanoenvironment. Chargessubsequently migrate to catalytically active sites through chemicalpotential gradients. The large molar absorptivity across the solarspectrum of organic chromophores enables active layers on the orderof hundreds of nanometers thick, which suggests the interphase canmake up the entire system while encompassing a range of local nanoenvironments.Ions and solvent from the electrolyte can insert into the polymerlayer and the resulting swelling and mixed electrical-ion conductionmay promote or impede (photo)electrochemical charge transfer processes.Critically, molecular-based systems do not exhibit charge-trappingsurface states due to dangling bonds but rather are comprised of narrowerdistributions of molecular-orbital-based electronic states with energiesthat can be easily tuned synthetically by altering the molecular buildingblocks of the polymer. The many components of a soft PEC system—e.g.,donor and acceptor polymers, catalysts, and electrolyte—willultimately function as a whole, and must be co-designed, rather than individually examined in sequence. The temporal evolutionof the interphase and local nanoenvironments under PEC operation requirethat each component be treated as part of the whole, ultimately enablingdesign based on the realized properties of polymers under operationfor more stable and durable systems.A defining characteristic of soft semiconductorsin the contextof photoelectrochemistry is that the electrolyte can—at highvolumetric electrochemical doping—penetrate deeply into thepolymer semiconductor, changing its structure and properties profoundlyrelative to the “dry” (as-deposited) material. We adoptthe terminology of 11 While some issues highlighted here are also relevant to the emergingfield of covalent organic frameworks (COFs) for PEC applications,we focus here on π-conjugated semiconducting polymers, and referreaders to related work focused on COFs.15While many of these unique characteristicsare factors understoodby comparison to other organic semiconductor-based technologies, PECapplications are under-explored and many questions remain. Major knowledgegaps are highlighted in 2 generation:photovoltaic-driven electrolysis (PV-EC) and photoelectrochemistry(PEC). In PV-EC ,30 with a substantial recentfocus on metal oxides.55 However, challenges of stability and scalability remain.30 Devices based on III–V semiconductorshave the highest PEC efficiencies42 but requireintensive tailoring of compositions, structures, contacts, and protectivelayers, all of which increase cost at scale.30 Additionally, water splitting reactions are most efficient at highor low pH, where many hard semiconductors are unstable;57 many of those which are stable are less-developed, resulting ina trade-off between efficiency and durability.58 Finally, electrocatalyst integration is often necessaryto increase PEC reaction rates. Although the field is targeting earth-abundantcatalysts60 and/or selective molecular catalysts,62 long-term stable integration of any catalyst on a hard semiconductorwith good charge transfer is a persistent problem.65An understanding of theunique challenges in soft photoelectrochemicalsystems emerges from the historical trajectory of PEC research. Abrief timeline of developments in “hard” inorganic semiconductorPEC is presented in 20 Perhaps most importantly, organic semiconductors offer synthetictunability in their optical and redox properties in ways which areinaccessible to hard materials. Foundational work for organic PECcomes from studies of doping of conductive polymers44 and the photophysics of diode behaviors which necessitate energeticoffsets in ionization potentials and electron affinities to drivecharge transfer.45 In organic photovoltaics(OPV), the early planar structures were replaced by nanometer-scaleblended heterojunctions (BHJs) of donors and acceptors which requireexquisite control of microstructure to simultaneously optimize chargetransfer and charge transport phenomena. The chemical nature of thesesoft materials has allowed for intricate spectroscopic interrogationsof excitonic and polaronic species, which have supported improvementsin OPV69 and commercialized organic light-emittingdiode (OLED) technologies.The fieldof organic electronics developed in parallel  conversions of 10–13%, while the highestreported organic half-cell photocathode STH is ∼3.78%.and edge 3a,b. Elensertion 3e. This lly fast 3f.7876 and thus alters charge mobility,79 but are rarely considered in the design or analysisof polymer PEC systems. For instance, these fundamental changes must influence the capacitance at the polymer|electrolyteinterphase, but are seldom,80 if ever,incorporated into the circuit models used to measure the voltage-dependentcapacitance. Electrostatic interactions with outer-sphere redox molecules,such as methyl viologen, can be harnessed to enable molecule releasefrom a surface after the redox reaction has occurred,81 but can equally hinder such release. If redox reactionsoccur at more extreme potentials than electrochemical doping and dopantsare easy to remove, then dopant loss can out-compete charge transferto a redox molecule.75 All of these factorsare further complicated by electrode cycling and the duration of operation,and illustrate the broad scope of scientific development needed forsoft PEC systems.Unlike hard semiconductor systems, where surfacestates and boundcatalysts may modify charge transfer and redox rates but the bulkof the material remains unaffected, charge transport and transferin soft semiconductors are highly dependent on the local micro- ornanoenvironment, which includes effective interchain wave functionoverlap that differs in crystalline and amorphous regions85 Demonstrations include both photoanodes86 and photocathodes,88 with complementary applications to photoelectrochemical processesof biological relevance such as artificial retina90 and the functions of living cells.92 Despite thissubstantial record in the literature, remarkably little is known aboutwhat photophysical changes occur when soft polymer semiconductorsare exposed to polar electrolyte environments and how these propertiescouple to changes in nanostructure, charge transport, and charge transferto determine material- or device-level functionality. Recent focushas targeted buried junction devices, where the photoactive organiclayer is not in direct contact with the electrolyte.96 Rather, a charge-selective layer is interfaced between the semiconductorand the electrolyte to limit recombination, protect the organic semiconductorlayer, and/or reduce the need to tailor the frontier orbitals of theorganic semiconductor to the electrochemical reaction.87 However, such layers can quickly fail83 (as they do even in hard PEC systems58) and the electrolyte will contact the organicsemiconductor. Thus, understanding the chemical and physical consequencesof the polymer-electrolyte interactions is critical. Moreover, theburied-junction approach precludes exploitation of the molecular tunabiltyinherent to soft materials to control energy levels and integrationwith catalysts, shifting the problem to the challenge of catalystson metal oxide supports.59 We postulatethat the distributed interphase created when a conjugated polymerbecomes electrochemically doped and swollen with electrolyte is likelyto afford new scientific and technological possibilities not availablethrough the buried junction approach.We now turn our attention to recent demonstrationsof soft semiconductorsin PEC applications. Many recent examples replace the top contactof OPV devices with an electrolyte/redox couple and consider half-cellperformance.31 there are emerging guidelinesto design robust and durable interfaces. Conjugated polymers are potentiallyadvantageous to withstand acidic and alkaline electrolyte media ifdurability is properly understood; their chemically tunable structurescould enable chemical and structural resiliency to external stressesfrom the harsh electrochemical environment.99 Developmentsin other soft, polymer-based technology platforms  provide insight into the effects of doping from acidic or alkalineelectrolyte ingress,101 electrochemical bias,102 light (particularly UV),103 and oxygen104 on conjugatedpolymers, although very few studies exist that explicitly considermultistress pathways such as those that soft PEC devices will experienceduring operation.Durability is increasinglybeing considered as a factor in therecent work on both hard and soft PEC systems, and as our understandingof changes to charge transport and transfer improves, new systemsmust be designed with retention of function in mind. Although PECcells based on conjugated polymers to date have operational lifetimesfar from that needed for practical lifespans of solar fuel generation(>10 years),–2 chemicallydegrading BHJ photocathodes performing Eu3+ reduction.83 However, other work suggests negligible physicalchanges to BHJ polymers after device operation, using optical andRaman spectroscopy, possibly indicating a different mechanism of degradationor that degradation is limited to the interphase.106 Another factor is the possibility of small molecule acceptors, whichenable higher performance OPVs than polymer acceptors, but which aremore soluble, possibly causing chemical and microstructural instabilitiesthat can reduce durability.108 For example, removalof small molecule acceptors improves the underwater stability of OPVdevices.107 Other work shows that a ternaryBHJ with both fullerene and nonfullerene acceptor molecules (PC71BM and ITIC) can enhance durability andresult in very high PEC performance, albeit in a device protectedfrom electrolyte ingress by a carbon plate.96 Another issue is electrolyte swelling-driven delamination of BHJlayers from the underlying electrode during PEC operation. Porousoxide electron transport layers110 under BHJlayers have been demonstrated to enhance electrode adhesion for photoanodes.Side-chain engineering–such as changes from alkyl to polarside chains in random copolymers—may hinder delamination duringelectrochemical cycles. While this is not an exhaustive list of factorsto be considered in soft PEC, it illuminates the need for fundamentalexploration to improve understanding of such systems in operation,which should then enable the development of design rules for improvedintegration of different polymer types, etc. We emphasize herein thatadvanced technique development, afforded by the number of in situspectroscopic signatures for soft materials, will be critical to understandingthe transformation of polymer semiconductors under these types ofstresses.111Several stress factors impacting durabilityhave recently beenexplored experimentally, often with conflicting results which callfor further investigation. For instance, charge accumulation in thepolymer film rather than successful transfer across the interphasemay degrade PEC performance, as shown for the accumulation of photogeneratedelectrons over the order of 100 nC cm84 after substantial hydrogen evolution was demonstratedusing colloidal nanoparticles decorated with Pt catalyst.112 Like many soft PEC devices, these complex nanostructuresinclude an internal heterojunction that splits neutral excited statesinto free electrons and holes. These nanoparticle systems demonstratethat polymer semiconductors decorated with catalysts can perform inner-sphereredox reactions such as HER without a protective oxide, but fundamentalquestions remain. Charge transfer mechanisms are difficult to understandon soft polymer systems because the fundamental behavior of the polymeritself is not understood before it is further complicated, e.g., withthe formation of heterojunctions or the incorporation of catalysts,which makes improvement difficult.113 Asin OPV, the formation of polymer heterojunctions alters overall morphology,114 which will be further modified with interphase formation, and thereis little direct insight into the photophysical consequences of thesechanges. Subsequent reports116 agree that a largefraction of the charge carrier population in these soft nanoparticleshave a remarkably long, millisecond scale lifetime, but that the overallyield, or possibly the mobility, of free charges within the particlesis substantially reduced relative to bulk blends of the same materials.116 Whether the long-lived but apparently trapped,charges in these particles are the species that actually participatein HER remains to be seen.Closelyrelated to soft PEC, the area of organic colloidal nanoparticlephotocatalysts suspended in water has recently received attention118 Linear polyfluorene-based copolymers were synthesized with varyingamounts of a Sulphone-bearing unit, and the Sulphone content correlatedstrongly with HER rate and quantum yield, independent of residualPd catalyst loading.119 The differencecould not be explained by shifts in excited state lifetime or theenergetic driving force for HER, implicating a specific chemical roleof Sulphone groups in mediating the reaction. This behavior was explainedthrough a combination of spectroscopic investigations and quantumchemical modeling of the interface.117 Thehydrophilicity of the Sulphone groups attracts sufficient water tosubstantially modify the local dielectric constant at nanometer lengthscales  and subsequently the energeticsof electron transfer, allowing faster oxidation of the sacrificialreductant (triethyl amine) molecules. Moreover, the Sulphone groupsappeared to also mediate electron transfer to the cocatalyst Pd particles,though the mechanism was not explained.117 The opportunity to modulate the local dielectric constant is anelegantly simple statement that may form an important design criterionfor specifically designing reactive sites within conjugated polymer-electrolyteinterphases for PEC: a local dielectric constant in these systemscan be engineered through chemical modifications of the polymer todrive specific reactions at specified locations.Another opportunity to tailor polymer-electrolyteinteractionsand nanoenvironments relevant to soft PEC is illustrated by the recentlyexplained correlation between photocatalytic activity and Sulphonefunctional substituents on the polymer backbone.at least the donor or the acceptor is certainly crucialto the generation of free charge. Fortunately, order can increasewith modest degrees of electrochemical doping in conjugated polymers,as the nanoenvironment is altered by solvent and ion ingress. Althoughthese factors are not yet well-understood, each provides an opportunityto discretely control (photo)electrochemical charge transfer. Controlof charge-transfer reactivity for polymer PEC may be achievable ifthe local environment of a polymer semiconductor/electrolyte interfacecan be tuned from the molecular- to mesoscale to promote selectiveredox pathways. The ability to achieve this will require (1) the developmentof models describing electric fields and the motion of charges inthe polymer/electrolyte interphase; (2) investigation of mechanismsof outer-sphere charge transfer to elucidate rate-limiting factorswithin the polymer; and (3) application of these structure–propertyrelationships to the mechanisms of inner-sphere redox reactions atpolymer|electrolyte interphases.In summary, we have detailed manyof the advances and new observationsin the area of soft photoelectrochemical systems with a focus on chargetransfer, charge transport, and durability, as shown in


The biological influence of physicochemical parameters of “targeted” nanoparticles on their delivery to cancer tumors remains poorly understood. A comparative analysis of nanoparticle distributions in tumors following systemic delivery across several models can provide valuable insights.Methods: Bionized nanoferrite nanoparticles (iron oxide core coated with starch), either conjugated with a targeted anti-HER2 antibody (BH), or unconjugated (BP), were intravenously injected into athymic nude or NOD-scid gamma (NSG) female mice bearing one of five human breast cancer tumor xenografts growing in a mammary fat pad. Tumors were harvested 24 hours after nanoparticle injection, fixed, mounted, and stained. We performed detailed histopathology analysis by comparing spatial distributions of nanoparticles (Prussian blue) with various stromal cells  and the target antigen-expressing (HER2) tumor cells.Results: Only BH nanoparticles were retained in tumors and generally concentrated in the tumor periphery, with nanoparticle content diminishing towards the tumor interior. Nanoparticle distribution correlated strongly with specific stromal cells within each tumor type, which varied among tumor types and between mouse strains. Weak or no correlation between nanoparticle distribution and HER2 positive cells, or CD31 cells was observed.Conclusion: Antibody-labeled nanoparticles were retained across all tumors, irrespective of presence of the “target” antigen. Though presence of antibody on nanoparticles correlated with retention, non-cancerous host stromal cells were responsible for their retention in the tumor microenvironment. This study highlights gaps in our understanding of the complex biological interplay between disease and host immune biology, and the need to account for the influence of underlying aberrant tumor biology as factors determining nanoparticle fate in vivo. Engineered nanoparticles display immense potential for diagnosis and therapy of various diseases including cancer Enhanced permeability and retention (EPR) arising from aberrant and leaky vasculature in the tumor remains the accepted mechanism for nanoparticle access and retention in the tumor microenvironment. “Passive” retention is thought to be dominated by nanoparticle size slowing reentry into blood For the past several decades, preclinical studies in cancer biology and drug development have relied heavily on immunocompromised mouse models. These models have utility in cancer drug development for human disease because they provide a permissive environment for tumor grafts to grow from human cancer cells, enabling efficient drug screening, and providing useful insights into gene- or pathway-specific therapy strategies. Small-molecule anti-cancer drugs development has been enhanced by use of these models because they provide a complete, living system for testing. More recently, a growing recognition that the host immune system plays a pivotal role in both disease progression and response to therapy has motivated calls for careful evaluation of such strategies, particularly for nanoparticle-based formulations .®, achieved approval on its reduced toxicity profile compared to free drug rather than on its ability to raise intratumor levels of chemotherapy or its superior efficacy Nanoparticles, by virtue of their size and other physiochemical properties, interact strongly with the host immune system, altering their fate in vivo and adding to the biological complexities of host and disease. Hence, there is increasing acknowledgement that interpretation of results obtained from pre-clinical studies relying exclusively on immunocompromised models is complex, as these models fail to recapitulate clinical realities. The original optimism fueled by early preclinical successes were not recapitulated in clinical trials, motivating the recent reevaluation of development strategies in the context of a perceived disconnect between expectations and realities of clinical translation A more recent survey of data from mouse models, suggests that on average, the amount of nanoparticle retained in the tumor is about 0.7% of the injected dose Taken together, these and other studies point towards unanticipated complexities in our understanding of nanoparticle pharmacodynamics, with a growing realization that we must implement a nuanced and careful approach to our use and interpretation of data obtained from animal models, in order to improve our understanding of the relevance of these results to clinical translation. Though immune deficient mouse models may fail to represent faithfully all complexities encountered in the clinical setting, they remain a superior class of living models to study effects of diverse tumor and host biology on nanoparticle delivery ®  protein) conjugated nanoparticles In our previous study, we observed that the immune status of the host, rather than antigen-antibody interactions with cancer cell membranes dominated retention of antibody-conjugated nanoparticles in tumor models. Flow cytometry analysis of nanoparticle-associated cells revealed less interaction between HER2 positive tumor cells and Herceptin®-conjugated iron oxide nanoparticles in tumors obtained from the five (human-mouse xenograft) breast cancer models used previously. The spatial distribution of the nanoparticles was then compared to that of blood vessels present in the tumor identified by endothelial cell marker CD31, proinflammatory and dendritic cells using CD11c, tumor cells with HER2 staining, macrophages identified using F4/80, fibroblasts using alpha smooth muscle actin (SMA) and collagen by Masson's Trichrome staining. We found that the nanoparticles predominantly accumulated on the outer quartile of the tumors, irrespective of tumor type (HER2 status) or mouse background. Additionally, the distribution pattern correlated better with intratumor stromal cell distributions than with HER2 expressing tumor cells across all tumor models. In each tumor type however, nanoparticle distributions correlated better with one or more type of stromal cells that differed among the models.Here, we revisit those initial findings with a detailed histopathology quantitation of the spatial distribution of HerceptinAll tissues analyzed in this study were collected as a part of our previously published work th mammary gland on both sides of either athymic nude (BALB/c) or NOD-SCID gamma (NSG) mice. When the tumor volume reached approximately 150mm3 measured with calipers, mice were randomly assigned to intravenously receive phosphate buffered saline (PBS), Bionized nano ferrite plain (BP), or Herceptin®-conjugated BNF (BH) nanoparticles (5mg Fe/mouse). After 24hrs, all mice were euthanized and tumors were collected. One-half of each tumor was fixed in 10% formalin and embedded in paraffin and sectioned for histological staining. Prussian blue staining was conducted on all slides to evaluate the distribution of iron nanoparticles. Immunohistochemistry analyses for endothelial cell marker CD31, target tumor antigen HER2, macrophage marker F4/80, fibroblast marker alpha- smooth muscle actin (a-SMA), dendritic and other proinflammatory cell marker CD11c, were performed on tumors from PBS treated mice. Masson's trichrome and Prussian blue staining were done on BH treated tumor slides through Johns Hopkins University reference histology core. All stained slides were then scanned with an Aperio ScanScope At or CS system  at 40× magnification. Tissue that were folded or damaged after staining were excluded from the analysis. Total number of tumors analyzed from each group for each stain are given in Briefly, five different breast cancer cell lines of varying human epidermal growth factor receptor 2 (HER2) protein  were orthotopically injected into the 4Detailed staining procedures for CD31 and HER2 have been described previously Briefly, following dewaxing and rehydration on board, slides were soaked in Target Retrieval Solution  for 48 minutes at 96 ºC for antigen retrieval. Primary antibody, anti‐ F4/80  was applied at 36ºC for 60 minutes, followed by rabbit anti-rat linker antibody  at 36ºC for 32 minutes. Linker antibodies were detected using an anti-rabbit HQ detection system  followed by Chromomap DAB IHC detection kit , counterstaining with Mayer's hematoxylin, dehydration and mounting.Immunostaining of CD11c and α-SMA were performed as follows. After deparaffinization and rehydration with successive soaking in 100%, 95%, and 70% ethanol/water, all slides were washed in deionized water followed by dipping in 0.1% Tween and citrate buffer. Antigen retrieval was performed for CD11c slides in citrate buffer by heating in a steamer for 45 minutes. No antigen retrieval was needed for α-SMA staining. After cooling slides were washed with PBS-T and blocked with peroxidase blocking solution  for CD11c staining and 30 min blocking with 0.5% BSA for α-SMA staining. Primary antibodies ; α-SMA  were then added and samples incubated at room temperature for 45 minutes. Following a wash in TBS-T, Poly-HRP anti-Rabbit IgG (Power Vision poly HRP anti rabbit (PV6119), Leica Biosystems, Deer Park, IL) secondary antibody was added and slides were incubated at room temperature for 30 min. Following a thorough wash, DAB  was used as chromogen and slides were counterstained with Mayer's hematoxylin  before dehydration and mounting.FIJI/ImageJ  software was used to visualize and characterize nanoparticle distribution patterns in Prussian blue and immunohistochemistry (IHC) stained tumor cross-sections.Digitized images of stained tumors were imported into ImageJ using the BioFormats plugin. Images were then transformed into RGB color space, color thresholded to select for blue pixels (representative of the nanoparticles), and then binarized. Color threshold parameters were optimized as follows:Prussian blue: Hue range: 123-196; Saturation range: 0-255; Brightness range: 0-222Imported stained tumor images were then processed to set threshold values to select for IHC stained pixels. The color threshold parameters for IHC stained tumors were as follows:IHC: Hue range: 0-25; Saturation range: 0-255; Brightness range: 0-150In particular, HER2 stained tumors possessed non-negligible and uniformly distributed background staining relative to other IHC stains. To address, additional processing was performed using the Color Deconvolution tool, which unmixes an RGB image produced by subtractive mixing into separate channels corresponding to one to three determined colors. For HER2 stained tumors, the H DAB vector was used, after which a threshold value of 110 was used to reduce background.Finally, imported stained tumor images were processed to select threshold values to select for positive pixels following the Masson's Trichrome staining procedure. The color threshold parameters for Masson's Trichrome stained tumors were as follows:Masson's trichrome: Hue range: 123-235; Saturation range: 0-255; Brightness range: 0-180The tumor boundary was defined by a user-drawn region of interest (ROI), and then partitioned into four bands, or quartiles, of equal area. This process was carried out for all slides using ImageJ in the following manner: First, an ROI was drawn to outline the tumor boundary (with a 50 μm extension beyond the outer edge of the stained tumor to ensure that the outermost edges of the tumor were included). This outline (the primary ROI) was duplicated and reduced in size to enclose a smaller segment that comprised exactly 75% of the total tumor area. The area between these two lines/ROIs formed the first quartile, which comprised the outer 25% of the tumor area. Next, a second ROI was duplicated and reduced to form a third ROI that enclosed 50% of the total area. The area between the second and third ROIs formed the second quartile . This process was repeated iteratively until a total of four quartiles, each encompassing 25% of the total tumor area, was defined.The “Enlarge” tool in ImageJ, which enables objects to be rescaled, was used to generate the nested ROIs. The “XOR” tool  was then used to calculate the area of each quartile by comparing pairs of adjacent ROIs to measure the region between. For example, when the first and second ROIs were selected, the XOR tool finds the overlapping area between them and then takes the inverse, which represents the area of the first quartile.The pixel density - the number of positive stained pixels divided by the number of all pixels within a tumor region - was calculated for each ROI using the ImageJ “Analyze Particles” tool. After evaluating pixel densities in each quartile, differences in nanoparticle distribution in tumors among tumor models were represented graphically. Necrotic regions, artifacts, and any suspicious areas of background staining within the tumor were removed from the initial user drawn ROI and subsequent analysis. Positive pixel density within each quartile was normalized by total positive pixel count (100%) within the respective tumor to compare positive pixel density over each of the four quartiles, allowing for comparisons in positive pixel distribution among nanoparticle and cell type distribution.2) was used to quantify the correlation of normalized pixel positivity of each quartile to total positive pixel count between different stains. R2 ranges between 0 and 1 where a higher value indicates a stronger correlation. In order to account for the unbalanced sample sizes encountered among the different groups representing individual stains, and to quantify the variability when estimating R2, a resampling-based approach was used. Specifically, within each quartile of each cell type, staining results were resampled with replacement to have a sample size of 100 and the corresponding sample median within each quartile was recorded. R-square of these sample medians between nanoparticles (PB) and each cell type (represented by stain-group) was calculated. By repeating the bootstrap resampling for 1,000 times, we recorded the 2.5th, 50th, and 97.5th percentile of the resulting 1,000 R2 and these were reported. Given the study nature and small sample size, we consider the correlation is strong if both 2.5th of R2 was greater than 0.8, and 50th percentile of R2 was greater than 0.9.All quartile datasets were analyzed using nonparametric Mann-Whitney test using Graphpad Prism software. Coefficient of determination  nanoparticle distributions correlated poorly with markers of vascular density or target antigen expression, we sought to test additional hypotheses that other cell phenotypes were responsible for the observed nanoparticle accumulation. Phagocytic (innate immune) cells take up nanoparticles, particularly circulating nanoparticles by macrophages circulating in blood and those residing in liver, spleen, intestines, and other tissues. Further, myeloid derived innate immune cells exhibiting a proinflammatory phenotype display enhanced tendency to uptake BH nanoparticles in vitro From comparisons of slides stained with CD11c that stains dendritic cells and some proinflammatory cells in the tumor microenvironment, we observed significant differences in immune cell distributions among the quartiles of all tumor types Figure . In addiAnalysis of the quartile distribution of macrophages in all tumors revealed that significant differences among quartile distributions was observed only in HCC1954 tumors Figure .MCF7/HER2 and BT474 tumors grown in nude mice and HCC1954 tumors grown in NSG mice showed significant differences in their distribution of fibroblasts among all quartiles Figure . When weMDA-MB-231 and BT474 tumors grown in nude mice and HCC1954 in NSG mice showed significant differences in positivity for collagen among their quartiles Figure . Except 2 calculated. Depending on the R2 value, the best-correlated cell type with that of nanoparticle distribution of each xenograft is shown in Figure From the observed results, we hypothesized that certain stromal cell types, depending on the tumor model and mouse background, were key players in determining nanoparticle retention in the tumor microenvironment. To better standardize and compare the gathered data, we normalized the pixel positivity of each quartile to the total positive pixel count in each tumor for all stains. Table Overall, four out of five tumor types showed a higher correlation with macrophage and collagen distribution in tumors collected from nude mice. When we evaluated tumors grown in NSG mice, collagen content and endothelial cells correlated best with nanoparticle distribution in the corresponding slides from MCF7/neo and HCC1954 xenografts Figure . In addiDespite advances in understanding the properties of nanostructured materials, achieving consistent, targeted delivery of an engineered nanoparticle to solid tumors for theranostic applications remains unaccomplished. Current designs of nanoparticle agents rely heavily on tuning the physicochemical properties of the material to a passive diffusion-dominated mechanism, rather than the biological characteristics of the host or disease. Both the biology of the host and disease play a crucial role in the fate of systemically administered nanoparticles. We can expect that once in the bloodstream, nanoparticles interact with blood proteins. Thus, their uptake and accumulation in various organs we can hypothesize is dictated, in part, by individual cell interactions with the protein corona Regardless of surface modification, the liver and spleen sequester most nanoparticles as part of their function in vitro, is negligible in vivoTo understand better the challenges posed by host and tumor biology, we considered that a further analysis of interactions between nanoparticles and host cells/tissue at a microscopic level was needed. Here, we evaluated the interaction between a plain (no antibody) and an antibody-coated nanoparticle and the tumor microenvironment among several models (representing antigen positive and negative tumor biology) across two different immune strains of mouse by conducting a comparative spatial analysis of tissue histology sections. We measured no appreciable retention of unlabeled nanoparticles in the tumor microenvironment providing little evidence supporting a passive diffusion-dominated retention mechanism. In an earlier study, we documented that the antigen-antibody interaction mediated uptake of antibody-conjugated nanoparticles to the tumor cells, while is significant This suggested to us that a complex interplay of biological and physical factors exist to influence uptake and retention of nanoparticles in the complex environment within tumors. We also report that among HER2-overexpressing tumor models  grown in two different immune strains of mouse, significantly different patterns of nanoparticle retention were observed, despite the similarities of blood vessel density measured among them. These data directly challenge the hypothesis that vascularity and access to target-expressing cancer cells is a primary factor that determines targeted-nanoparticle retention in a tumor. In other words, nanoparticles circulating in blood gain access to tumors as well as to all other tissues, but whether they remain in a specific (target) tissue depends on host or other potentially independent biological factors comprising the local tissue environment. Evaluation of different cell types in the tumor microenvironment revealed that in each tumor type, whether grown in nude or NSG mice, dominant cell types residing in the tumor dominated retention. Further analysis illustrated that in tumors grown in nude mice, it was the distribution of resident macrophages and collagen content that correlated predominantly with nanoparticle distributions within each tumor quartile. On the other hand, very few correlations between nanoparticle distribution and cell type were identified in tumors grown in NSG mice. Little correlation between nanoparticle localization and HER2 positive regions were documented in both strains and among three different human HER2-positive tumor grafts displaying varying amounts of HER2 expression. These findings highlight the need for understanding the complexity of the biological system for targeted delivery of nanoparticles. We visualized and analyzed collagen with Masson's trichrome staining. This is the most common method used in both research and clinical settings to visualize tissue collagen content and distribution, but it provides little information on the structural differences and types of collagen fibers in each tumor type in different strains of mice. Such structural differences in collagen fibers may influence nanoparticle retention in tumor microenvironment, and may merit further investigation. To analyze those differences, other high resolution and/or phase contrast imaging modalities such as those employing second harmonic imaging, coherent anti-Stokes Raman scattering (CARS) microscopy, etc. may prove beneficial Increased accumulation of nanoparticles in the tumor periphery has been documented with other i.e., liposomal nanoparticle types, which is usually attributed to increased vascular density within the tumor The phenotype displayed by phagocytic cells when they encounter nanoparticles can significantly alter the quantity of nanoparticle they will ingest. Pro-inflammatory (M1) macrophages or induced neutrophils ingest significantly higher amounts of both antibody-conjugated and unconjugated nanoparticles than their uninduced counterparts ® nanoparticles retained in the tumor, than did that of the HER2-overexpressing tumor cells. Moreover, when the mouse strain changed, the cell type that predominantly correlated with the nanoparticle distribution in the tumor also changed. We note that the models chosen represent human cancer tissue grafts grown on mouse hosts. Thus, tumor stroma, blood, and all other tissues are murine, whereas the cancer cells (HER2 positive or negative) within the tumor are human. While the biological complexities of these models for studies of cancer nanomedicine cannot be underestimated, the benefits to compare among a specific number of variables in living systems can provide important insights. Despite the obvious limitations, use of human tissue xenografts grown in permissive animal hosts, e.g. immune compromised mice provide an important, albeit limited, tool to ascertain potential effectiveness of research pharmaceuticals against human diseases. From another perspective, syngeneic models  yield important information regarding interactions of the pharmaceutical agent with intact, but disease-affected host immune system in the context of the disease model. In such cases, the effectiveness of the agent to treat human disease may not translate directly, but results obtained from such a model (e.g. murine tumor grown in matched strain of mouse) might give us more insight that is relevant to host immune- and stroma-specific influences on agent pharmacodynamics. Further, results obtained from such models require additional caution for interpretation when the studies involve clinically relevant human-specific “immunogenic” agents, such as a monoclonal antibody as used in this study. We anticipate further insights with our ongoing efforts with analysis of mouse tissues following treatment with Herceptin®-conjugated iron oxide nanoparticles using tumor grafts obtained from human HER2 overexpressing genetically modified mice.Our analysis suggests that the spatial distributions of many stromal cell types making up the tumor microenvironment correlated better with the observed distribution of trastuzumab-labeled BNF3 and the influence of tumor volume on outcomes is undetermined. As reported by Sykes et al., 2015, varied tumor pathophysiology with tumor size can influence nanoparticle uptake Another limitation of the study is the limited number of stromal cell lineages we considered at only one time point, and only over a small portion of the tumor. In this study, sizes of tumors were ~ 150 mmDespite the limitations, we conclude that host immune-biology, disease, and non-cancer cell composition comprising the tumor microenvironment exert powerful influences to dictate patterns of nanoparticle retention and distribution within solid tumors. A simple, passive and diffusion-dominated mechanism of nanoparticle retention in solid tumors though supported by data, inadequately explains complexities observed. This knowledge is vital for future development of cancer nanomedicine, and may motivate new approaches to query cancer biology with nanotechnology. Additional in-depth computational analyses of the interplay between nanoparticle distribution, host biology, and immune status will enhance our understanding of biomolecular patterns that drive the behavior of nanoparticles within a specific tumor microenvironment. The ability to characterize and predict nanoparticle localization is a crucial first step towards development of theranostic materials.Supplementary figures.Click here for additional data file.