There were also significant associations with exhaustion markers FoxP3+, PD-1+, and PD-L1+ . This new qNPA technology is useful to quantify intratumoral immune cells on FFPE specimens through RNA gene expression in metastatic melanoma. As previous studies have shown on other solid human tumors, we also confirm that the expression level of a single gene may be used to represent a single IHC immune cell marker in melanoma.Immune cell infiltrates in melanoma have important prognostic value. Gene expression analysis may simultaneously quantify numbers and function of multiple immune cell subtypes in formalin-fixed paraffin-embedded (FFPE) tissues. Prior studies report single gene expression can represent individual immune cell subtypes, but this has not been shown in FFPE melanomas. We hypothesized that gene expression profiling of human melanomas using a new RNA expression technology in FFPE tissue would correlate with the same immune cells identified by immunohistochemistry (IHC). This retrospective study included melanoma specimens analyzed by IHC on tumor tissue microarray (TMA) cores and by gene expression profiling with EdgeSeq Immuno-Oncology Assay using qNPA technology on the corresponding tumors. Standardized gene expression levels were analyzed relative to enumerated cells by IHC using Spearman rank test to calculate r-values. Multivariate analysis was performed by Kruskal–Wallis test. 119 melanoma specimens had both IHC and gene expression information available. There were significant associations between the level of gene expression and its quantified IHC cell marker for CD45 A more traditional approach for defining immunologic features of the TME has been to quantify numbers of selected intratumoral immune cells within the TME which has also been found to predict responses to certain immunotherapies or overall prognosis from melanoma12 and other cancers14. Pathologists can report immune cell density on formalin-fixed paraffin-embedded (FFPE) tissue using hematoxylin and eosin (H&E) stains or immunohistochemistry (IHC), but these modalities are cumbersome and costly to integrate into routine clinical practice as quantitative assays. Furthermore, those traditional approaches limit the simultaneous analysis of multiple immune markers and can be tedious and time consuming, with automated techniques still years away from becoming fully integrated into routine clinical practice16. In research environments, flow cytometry can accurately define immune cell subsets but this requires viable single cell suspensions which are not routinely available and cannot be performed on FFPE specimens which are most typically available in clinical settings. Gene expression profiling measures mRNA levels in tissue, and traditional methods are most accurate when the tissue is preserved in RNAlater or flash frozen within about 15 min. This allows tissue samples collected in selected research studies available for gene analysis but not with FFPE tissues handled routinely in pathology laboratories. On the other hand, newer technologies have overcome some of these challenges to enable gene expression analysis on FFPE tissues; however, these remain fairly new techniques20.Gene expression profiling of cancer tissue offers a window into the immunobiology of the tumor microenvironment (TME) of human cancers. Several gene expression profiles have been reported to predict clinical responses to systemic immunotherapy21 and respective cell populations in purified in vitro assays23. One study assessed correlations between these same gene markers from tumor lysates with flow cytometry of peripheral blood mononuclear cell (PBMC) and also performed a limited analysis of associations between CD3+ and CD8+ cells by IHC in 19 ovarian FFPE cancer specimens24. To our knowledge, however, no previous study has assessed associations of gene expression in FFPE tissue with a wider range of lymphoid and myeloid markers and checkpoint blockade molecules in human solid tumors. We have addressed this in the present study for over 100 FFPE human melanomas.A few studies have validated these techniques by identifying genes specific to different immune cells and showing associations between expression levels of these genes27. The unique advantage proposed by the quantitative nuclease protection assay (qNPA) with Next Generation Sequence (NGS) technology is that it does not require nucleic acid extraction, cDNA synthesis, or gene amplification, thereby reducing the risk of RNA degradation and biases due to molecular artifacts, while also requiring less tissue31. The qNPA technology creates DNA to RNA hybridization, called the nuclease protection probe (NPP), which is then detected and sequenced using standard next-generation sequencing protocols after S1 nuclease is added to digest excess non-hybridized DNA probes and non-hybridized RNA31. This technology has previously been validated on human lymphoma and canine cancers30, but not on human solid cancers such as melanoma. In this present study, we assessed the validity of qNPA technology on human melanomas by comparing the levels of gene expression for single immune cell marker subtypes24 with these same immune cells identified by IHC performed on the same FFPE tumors. If valid, these gene expression profiles would reflect the densities of intratumoral immune cells measured by IHC and allow the simultaneous measurement of multiple immune cell markers within the TME even when limited amounts of FFPE tumor tissue are available.Gene sequencing techniques on FFPE tissue are generally performed through real-time quantitative PCR (RT-qPCR), yet RNA extracted from FFPE tissue can be degraded, can be crosslinked to other proteins, or can lack the poly(A) tail needed to generate cDNA, all potentially leading to inaccurate outputs and poor yields of transcript10. Briefly, areas of FFPE tumor tissue blocks from these surgical specimens were identified by a pathologist on H&E slides to identify central areas within tumor cell nests for TMA sampling. TMAs were constructed from 3 to 4 cores (1.0 mm diameter) through the tumor regions of each specimen from the selected FFPE blocks. No duplicate samples were represented from the same surgical specimen. Control tissues from liver, spleen, placenta, and kidney were included in each TMA block. TMA tissue sections were previously evaluated for immune cell infiltrates by immunohistochemistry (IHC) staining using standard protocols. Positive controls included lymph nodes (LN) and placenta for CD34 antibody. Negative control slides used PBS instead of primary antibody, with other conditions constant. Immune cells within each core were enumerated by a surgical pathologist10.Adult patients diagnosed with melanoma had previously been identified by the Anatomic Pathology Software System in the Department of Pathology, University of Virginia and archival tumor specimens from 1982 to 2007 were sampled to create a melanoma tissue microarray. All methods were performed in accordance with the appropriate guidelines and regulations from the University of Virginia IRB  with waiver of consent due to retrospective design of study. Melanoma specimens with ample tissue and appropriate clinical follow-up were selected for creation of a melanoma tissue microarray (TMA), as previously described+ immune cell infiltrates relative to CD34+ vasculature10. In each core, intratumoral immune cells were scored as 1 when immune cells (CD45+) were absent or sparse (no more than 50 immune cells per 1 mm diameter core); 2 when intratumoral immune cells were present but were limited to perivascular cuffing around intratumoral blood vessels; 3 when immune cells were diffusely present among tumor cells in different areas of the core. Mean scores were calculated for each tumor sample and three Immunotype groups were created to reflect these three patterns of infiltration: Immunotype A for mean scores < 1.75, Immunotype B for 1.75–2.4, and Immunotype C for ≥ 2.5. Additional details have been reported10. The TMA samples were also evaluated for the density of a range of immune cells including CD8 (CD8+ T cells), CD4 (CD4+ T cells), CD56 (NK cells), CD20 (B cells), CD138 (plasma cells), DC-LAMP (mature dendritic cells), CD163 (M2 macrophages), FoxP3 (regulatory T cells), as well as for cells expressing PD-1, PD-L1, and PD-L232.We previously reported that melanoma can be categorized into 3 Immunotype groups based on the extent and patterns of CD4510. These cores from each tumor sample were also evaluated for homogeneity. When the Immunotype scores were consistent across all three or four replicates, these samples have been considered homogeneous for immunotype. However, when they were not all consistent, these samples have been considered heterogeneous for immunotype.For each tumor specimen, mean values of the quantified immune cell populations were recorded among the triplicate or quadruplicate cores for each tumor sample, as previously described33.Among the FFPE tissue blocks that had been analyzed by IHC in the TMAs, 119 were also evaluated in the present study for expression of immune-related genes (IRB #17816). These included 112 metastases (94%) and 7 primary melanomas (6%). One five micrometer section of each FFPE tumor specimen was submitted to HTG Molecular . The HTG EdgeSeq Immuno-Oncology Assay was performed for gene expression analysis, using 558 probes with 15 housekeeper genes, 5 negative and 4 positive processor controls. For this assay, functional DNA Nuclease Protection Probes (NPPs) are flanked by universal wing sequences that are hybridized to the target RNAs, which can be both soluble and cross-linked in the biological matrix. Universal DNA wingmen probes are hybridized to the wings to prevent S1 nuclease digestion. S1 nuclease is added to digest excess non-hybridized DNA probes and non-hybridized RNA. This reaction then results in a stoichiometric quantity of fully intact NPPs:RNA hetero-duplexes of interest. The released and labeled DNA protection proves are concentrated, pooled, and sequenced using standard next-generation sequencing (NGS) protocols on the Illumina NextSeq™ platform. Gene expression data from the NGS instrument are processed and reported through the HTG EdgeSeq host system software. Data was standardized through a procedure that log-transformed Counts Per Million (CPM) and adjusted for total reads within a sample24 and for which gene primers were available in the HTG EdgeSeq Immuno-Oncology Assay. Several markers of immune cell exhaustion were also selected for study34.To evaluate concordance of cell enumeration by IHC with those identified by gene expression, genes chosen for study were those previously shown, through meta-analyses, to be expressed selectively on certain immune cell populationsp values < 0.05 were considered significant.Statistical comparisons among multiple subgroups were performed using the Kruskall-Wallis test. Correlation coefficients  were performed using the Spearman rank test. Statistical analysis was performed using Prism 8.0 . This research study was conducted retrospectively from data obtained for clinical purposes. IRB approved study  with waiver of consent due to retrospective design of study.10. Of the 183 tumor specimens previously analyzed by IHC, we included FFPE blocks that were still available and included sufficient tumor for gene expression analysis. A total of 119 tumor specimens had both IHC and gene expression data from the same FFPE block, and included both primary  and metastatic  tumors. No duplicate primary or metastatic tumor specimens were represented. To categorize the patterns of intratumoral immune cell populations among the 119 tumors, we used the Immunotype Score from a previous analysis that was based on immune cell density and distribution relative to the tumor and proximity to intratumoral vasculature10. The Immunotype Scores represent a continuous variable from 1.0 to 3.0. These scores were previously categorized into Immunotype Groups, where Immunotype A tumors  have very little to no immune infiltrates, Immunotype B tumors  have greater immune infiltrate but mostly limited to perivascular cuffing, and Immunotype C tumors  were heavily infiltrated. The intratumoral cell densities of immune cell subtypes including CD45+ immune cells, CD8+ T cells, CD4+ T cells, CD20+ B cells, CD138+ plasma cells, CD56+ natural killer (NK) cells, and CD163+ macrophages/monocytes among the three different Immunotype Groups are shown in Fig. + immune cells per mm2, while Immunotype B and C have significantly greater numbers , but the difference between Immunotype C versus A did not reach statistical significance  specimens were heterogeneous in the Immunotype patterns for immune infiltrate among the 3–4 TMA cores evaluated for the same tumor specimen. Despite this heterogeneity found on IHC analysis, the mean density of intratumoral CD45% specime24, and assessed whether expression of these genes was significantly associated with counts of those cells per mm2. Expression levels of these genes were most strongly and significantly correlated with the density of CD8+ T cells , CD45+ cells , CD20+ B cells , CD4+ T cells . Similar results were also found with exhaustion gene markers and their concordant IHC markers , FoxP3+ cells  and PD-1+ cells .Tumor microenvironments vary in their composition of T cell subsets, B cells, myeloid cells, and other subsets, and these may have prognostic relevance or value in predicting response to immune therapies. To investigate whether qNPA gene expression profiling of FFPE tumors may provide comparable data to those obtained with enumeration of the cell subsets by IHC, we selected single genes to identify individual immune cell subtypes as reported previously24. Among these three NK cell gene candidates , we did not find significant correlations with CD56+ NK cell density by IHC (data not shown). Thus, we chose to evaluate another NK-associated gene, FCG3A35, and identified a relatively weak, yet still significant, association with CD56+ NK cell density . We did not assess chemokines as possible gene cell markers candidates previously used in the literature due to the possible confounding effect of the pro-inflammatory TME .Three gene markers for NK cells were included in the current RNA assay, among 31 gene candidates previously used in the literature+, CD8+, CD20+, and PD-1+ and their respective gene expression levels, compared to the data from the full dataset , and of Immunotype A (n = 42) tumors. Those analyses did show that, among Immunotype B + C tumors, the correlation coefficients were increased between IHC counts for CD338. Tissue autolysis at room temperature, prior to fixation, can also affect mRNA levels, especially after 24 h38. Newer technologies overcome some of these limitations but warrant additional validation in human tissues. Also, a potential limitation of gene expression analysis for quantitation of immune cells in tumor tissue is that the expression levels of some marker genes  are not constant per cell, and thus may be modulated by immune activation or other features of the microenvironment so that their expression level may be an imperfect correlate of cell number. Finally, spatial variations in immune cell distribution can lead to differences in results based on sampling different areas of the tumor. In a prior study, quantitation of T cells, and of CD8+ T cells in particular by flow cytometry of PBMC samples, was compared to quantitation based on gene expression in FFPE tissue with Nanostring technology, with findings that the correlation r values were 0.657 (p = 0.002) and 0.481 (p = 0.037), respectively24. The present study has provided an opportunity to assess similar correlations with a different technology for a wider range of immune cells in FFPE solid tumor tissue.Gene expression analysis provides a valuable tool to assess immune cell populations in tumor microenvironments. Most human tissues available for analysis are FFPE; thus, new technologies for gene expression analysis in these tissues have opened the door to evaluate archived specimens and those collected as part of clinical care. However, several factors can modify those measures, and thus can impact their accuracy. Formalin fixation leads to cross-linking and degradation of mRNA and can have differential effects on different mRNAs10. In prior studies, we have evaluated how heterogeneity impacts the relevance of CD8+ T cell counts in small tumor samples. We have shown that a single core sample often misrepresents the CD8+ T cell density of the whole tumor, and that there can be significant differences in CD8+ T cell densities among pairs of metachronous tumors. However, a large biopsy of one tumor may be representative of multiple synchronous metastases40. Also by modeling different ways to sample a lung cancer, we found that different sampling strategies yield different densities of CD8+ infiltrate, but that the values most concordant with whole tumor counts were predicted by a random core sampling of the tumor or sampling of the tumor center41. These studies support our approach of using triplicate or quadruplicate 1 mm diameter cores from each tumor specimen as a reasonable representation of the tumor. Each tumor specimen was from a specific surgical resection. Multiple metastatic sites were not analyzed in this study, but 42% of tumors in our analysis had heterogenous tumor cores on IHC, yet data from these tumors were still very significantly associated with gene expression of immune cells as a measure of melanoma Immunotype.Assessing the qNPA technology, we found that expression of the PTPRC gene was significantly associated with the immune infiltration pattern (Immunotype score), though correlation coefficients were only 0.32 overall, and somewhat higher (r = 0.48) for the subset of tumors heterogeneous for the Immunotype by IHC. Tumor heterogeneity is a common features of cancers; so, sampling of selected tumor areas may not reflect the entire tumor. We have found that immune cell infiltrates in melanoma metastases strongly predict clinical outcome, when assessed with mean values derived from 3 to 4 core samples of central tumor areas+ T cells and CD3+ T cells, respectively. These are comparable to, or slightly better than, similar correlation coefficients for the Nanostring approach24. In addition, significant positive correlations were observed for PTPRC expression with CD45+ cells by IHC. This was also true for B cells and CD4+ T cells, as well. Significant correlation was also observed for CD56+ cells and FCGR3A expression but with lower correlation coefficient and wide variance across the population. There was a significant association for FoxP3+ cells, but the gene expression values were limited to a narrow range, making the findings less useful than the others for extrapolation to actual cell counts. Interestingly, expression of PD1+ and of PDL1+ were closely correlated with the counts of cells expressing them, by IHC. Thus, these data support the value of gene expression analysis by the qNPA method for evaluation of sets of tumors for all of these markers, but patient-level data appears to be more reliable for only a subset of these immune cell markers.The densities of various immune cell sub-populations by IHC, were also significantly associated with expression of the cell-associated gene, with correlation coefficients of 0.67 and 0.54 for CD82 (Immunotype B + C tumors) than among those without significant infiltrates within tumor nodules (Immunotype A tumors), which is likely explained in part by disproportionately high impact of peritumoral immune cells when tumor cell nests exclude immune cells, as is observed with Immunotype A tumors. Immune cell heterogeneity within tumors may contribute to decreased associations between gene expression analysis and IHC quantification40. In particular, collections of immune cells present not within the tumor itself but within tertiary lymphoid structures (TLS) on the peripheral margins of tumor43 may have been represented by the gene expression analyses, but not by IHC of tumor cores taken from the center of tumor nodules. Other possible factors that could impact association between gene expression and IHC analysis are differences in time course for preservation of each tumor specimen and the length of time each specimen was stored, especially if protein is more stable than mRNA. Considering these limitations, the strongly significant associations for multiple immune cell markers is more remarkable and supports use and further investigation with this qNPA technology.This study was limited in part by the fact that the IHC studies were performed on 3–4 cores (1 mm diameter) collected from tumor cell nests, rather than from peritumoral regions, whereas whole tissue sections were sent for the gene expression analysis. Thus, the associations of cell counts by IHC and corresponding gene expression may reflect some differences in the portion of the tumor that was sampled for each. Most associations were stronger among tumors with greater than 50 immune cells per mm+ cells by IHC. This may reflect low numbers of CD163+ cells in the tumors evaluated, or the need for other gene markers for CD163+ cells. Previous studies have shown correlations with 50 single gene markers as possible candidates to capture macrophage cell populations in solid tumors, and the current assay included only 5 of them24. We did not customize this assay for our analysis and so other gene markers such as CD163, MS4A4A, ARG1, and CD84 genes24 could also be assessed in future studies.Of all the immune cell subtypes investigated, we did not find a gene marker in this particular qNPA Immuno-Oncology Assay panel that was significantly associated with density of CD163Overall, the use of qNPA technology on human metastatic solid tumors such as melanoma is supported by the present analysis to identify FFPE tumor specimens as having high- or low- densities of varied immune cell infiltrates. As previous studies have shown on solid human tumors, we also confirm that in selected cases, the expression level of a single gene may be used to represent a single IHC immune cell marker in melanoma. Even when heterogenous tumor samples were found through analysis of 3–4 individual TMA cores, the intratumoral immune cell populations were still highly significantly associated with the level of single gene expression. Thus, quantification of multiple immune biomarkers simultaneously using qNPA technology may have significant value in future analysis of the TME, and possibly on peritumoral TLS, even with the use of small FFPE tumor specimens.


The mammalian brain can form associations between behaviorally relevant stimuli in an animal’s environment. While such learning is thought to primarily involve high-order association cortex, even primary sensory areas receive long-range connections carrying information that could contribute to high-level representations. Here, we imaged layer 1 apical dendrites in the barrel cortex of mice performing a whisker-based operant behavior. In addition to sensory-motor events, calcium signals in apical dendrites of layers 2/3 and 5 neurons and in layer 2/3 somata track the delivery of rewards, both choice related and randomly administered. Reward-related tuft-wide dendritic spikes emerge gradually with training and are task specific. Learning recruits cells whose intrinsic activity coincides with the time of reinforcement. Layer 4 largely lacked reward-related signals, suggesting a source other than the primary thalamus. Our results demonstrate that a sensory cortex can acquire a set of associations outside its immediate sensory modality and linked to salient behavioral events.  Previously, the only known triggers of apical dendritic spikes were “bottom-up”events, such as appropriate sensory stimuli or an animal’s location in space. Lacefield et al. show that reinforced associations are powerful triggers of apical dendrite activity and that reward can manipulate perceptions at their earliest stages of cortical processing. Apical dendrites are a common feature of pyramidal neurons throughout the mammalian neocortex, suggesting a general function in cortical computation. Pyramidal neurons in cortical layers 2/3 and 5 send apical dendrites to the surface of the cortex, where they arborize in layer 1. Layer 1 is composed almost entirely of these apical dendrites and axons from both local and distant sources. Being devoid of somata except for a sparse population of inhibitory cells, layer 1 has been largely inaccessible by electrophysiology during behavior. Consequently, the role of apical dendrites in cortical processing remains mysterious.In the whisker representation of rodent primary somatosensory cortex (“barrel cortex”), long-range axons from diverse areas, including motor cortex, secondary somatosensory cortex, and secondary somatosensory thalamus, synapse extensively onto these apical tufts , potentiThe only previously identified triggers of global apical tuft dendritic spikes are the same events that drive strong somatic spiking. Sensory stimuli are effective triggers of apical dendrites in primary sensory cortex, limb movement in the case of primary motor cortex, and location within an environment in the case of the hippocampus . Here, w+/NeuN+ cells), including their apical tuft dendrites in layer 1, without expression in the pyramidal cells of other layers (N-methyl-d-aspartate (NMDA) receptor-mediated spikes. These calcium events could additionally reflect backpropagating somatic action potentials.To examine the activity of apical dendrites within layer 1 during behavior, we first combined a transgenic mouse line expressing Cre recombinase specifically in layer 5 pyramidal neurons (Rbp4-Cre) with viral expression of the genetically encoded calcium indicator GCaMP6f . This ter layers . This por layers . Water-rr layers . Mice wer layers , we usedr layers . We obseWe first averaged fluorescence over the imaged region to assess overall population activity in apical tufts of layer 5 pyramidal neurons (44 sessions in 4 mice). Within a behavioral session, single-trial fluorescence showed a prominent short-latency peak  immediately following the presentation of the pole , which p−4, n = 33 sessions in 4 mice).Neurons in the primary visual cortex are sensitive to reward timing , and we To further examine the effects of reward on apical dendritic activity in the absence of active contacts, we randomly administered water rewards during a small percentage of the inter-trial intervals (ITIs). Unexpected random rewards during inter-trial intervals elicited a calcium influx of qualitatively similar timing and amplitude to the long-latency peak during trials . These dApical activity during rewards could result from motor inputs into layer 1 . Reward Before they were trained on pole detection, the mice were pretrained for 1 week to press a lever for a water reward while freely moving and then for 2 to 3 additional weeks while head fixed. Mice were imaged during the last 2 days of head-fixed pretraining and were therefore highly habituated at the time of imaging. Water rewards given to mice that were proficient at the lever-pressing task did not elicit dendritic calcium , in contSince average responses to trial and random rewards are similar in amplitude, they could represent the activity of a single population of reward-sensitive dendrites or, alternatively, activity in distinct populations. To assess the activity of individual apical dendrites activated at distinct times during the task, we segmented movies using a sparse non-negative matrix factorization method that forms an overlapping clustering of pixels according to their temporal covariance . Thus, wAs a population, individual dendritic arbors had peak activity times that tiled both pre-trial and trial epochs . A subsein vivo demonstrations that layer 2/3 apical dendrite activity strongly depolarizes the soma , which lack apical dendrites reaching layer 1, showed average activity only during the initial whisker contact period, at a time corresponding to the first peak in the layer 2/3 and layer 5 responses  and 4E. Activity in individual layer 2/3 apical dendrites and somata  was consUsing mouse transgenic lines to target calcium indicators to specific layers, we demonstrated that apical dendrites can incorporate non-modality-specific information into sensory representations. For mice experienced at an operant whisker-based task, an event involving no immediate whisker contact whatsoever (delivery of a reward water droplet) elicited pronounced long-latency dendritic spiking (and somatic output) in the barrel cortex. In retrospect, such a secondary peak is visible in previous imaging of layer 5 apical dendrites during a similar task . Our stoAny of the many areas synapsing on apical dendrites of barrel cortex neurons—for example, motor cortex, secondary somatosensory cortex, secondary somatosensory thalamus (POm) — could be a trigger for these apical dendritic events. Even the apical dendrites that were not locked to an obvious behavioral event  appeared to have a preferred time of activity within or around a trial , perhapsAlthough dopamine is often implicated in reinforcement effects on neural circuits, dopaminergic terminals are relatively sparse in primary sensory areas. Neurons in rodent primary visual cortex, however, have been found to be sensitive to reward timing during operant tasks, an effect that is mediated by a cholinergic-dependent mechanism . AcetylcIn the motor cortex, reward is able to potentiate the somatic discharges of weakly active cells when paired with the firing of the cell . SimilarOur method for segmenting individual dendrites based on spatiotemporal covariance  does notOur study suggests that salient behavioral events, such as rewards, can modify the occurrence of apical tuft spikes, presumably through the plasticity of long-range connections encoding context. This could afford a powerful generalized mechanism for encoding task-relevant information to any given cortical area, including associations with multiple sensory modalities and motor behaviors, as well as predictions about upcoming inputs.Detailed methods are provided in the online version of this paper and include the following:randybruno@columbia.edu).Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Randy M. Bruno , Cux2-Cre  (MMRC), www.openmaze.org. Prior to training on the tactile detection task, mice were trained to press a lever for a water reward for one week prior to headpost implantation and > 1 week while head-fixed. Lever-trained mice were then either injected with virus (below) or trained to a criterion of 70% correct responses on the tactile detection task before injection. Two weeks after injection, animals were implanted with a chronic cranial window and imaged during the detection task.Behavioral experiments were performed with the Arduino-based OpenMaze open-source behavioral system, whose designs are fully described at The tactile detection task requires that the mouse hold a lever down for > 1 s to initiate a trial, in which a stepper motor moved a small pole , which started from a position 3–4 cm below the animal. The stepper motor rotated the pole to ~2 mm anterior of the nose and ~10 mm lateral of the nose  or in the opposite direction even further away from the whiskers . Whiskers were not hit passively by the pole during stimulus movement, except in a minority (a few percent) of trials where mice whisked or held their whiskers in a protracted position during pole presentation. Typically, mice initiated whisking after they heard the motor begin to move at the start of the trial. During each trial, the mouse had to lift its paw from the lever within 3 s to indicate the presence of the pole, or keep the lever depressed if the pole was absent. Correct pole/Go trials (“Hits”) were rewarded with a small droplet of water (~8 μl) from a water port, whereas incorrect lever lifts during the catch/NoGo trials  were punished by an 8–10 s timeout before another trial could be initiated as well as a white noise sound. Imaging during the detection task was from animals that had achieved > 70% correct responses during a previous session. Animals performed 100–200 trials during a behavioral session, which typically lasted 20–30 minutes.Licks were detected with a capacitance-based touch sensor (Sparkfun). Whisking was monitored with a high-speed imaging camera (at 300 fps with a PhotonFocus CCD camera or at 187 fps with Sony PS3eye camera) and automatically measured offline using published software . For expThe overall reward rate, which varied across sessions and mice, was approximately 5–10 rewards/min for the pole task and 10–15 rewards/min for the lever task.13 cfu/mL). 100 nL of virus was injected at 1:2–8 dilution in ACSF using a pulled pipette (20–30 μm ID) at a depth appropriate for the cortical layer of interest , 1.6 mm posterior to bregma and 3.2 mm lateral of the midline. Two-photon imaging was performed ~2 weeks after viral infection. For cranial window implantation (a few days prior to imaging), animals were injected with dexamethasone 1 h prior to surgery, at which time they were anesthetized with isoflurane. A 3-mm hole was drilled in the skull overlying the barrel cortex, and the dura removed from the region of the craniotomy. A 3-mm glass coverslip was inserted into the craniotomy and cemented into place with Vetbond.Animals were anesthetized with isoflurane and implanted with a light-weight stainless steel headpost embedded in dental acrylic affixed to the mouse’s skull after application of a thin layer of Vetbond (3M). Mice recovered for one week before habituation to head fixation. For virus injections, mice were anesthetized with isoflurane and injected with adeno-associated virus (serotypes 1 or 9) encoding the fluorescent calcium indicator GCaMP6f in a Cre recombinase-specific manner. The human synapsin promoter  was used for Rbp4 and Cux2 lines, and the CAG promoter for the Nr5a1 line  using software custom-written in Labview .Two-photon imaging was performed using a Sutter movable objective microscope under the control of the ScanImage software package . Scanning was performed at 4 fps using a Chameleon Ultra II laser (Coherent), tuned to 940 nm, and focused through a 16×/0.8NA water immersion lens (Nikon). Emitted light was collected with an HQ535/50 filter (Chroma) and GaAsP photomultiplier tubes (Hamamatsu). Images were acquired at a resolution of 128 × 128 or 256 × 256 pixels. Apical tuft dendrites in layer 1 were imaged at depths of 40–80 μm from the pial surface, and Layer 2/3 and 4 somata were imaged at 200–300 μm and 400–600 μm, respectively.Movies were motion corrected using the SIMA image processing package . SpatialData and software are available upon request to the Lead Contact.12


This combined top-down/bottom-up approach can be used to produce composites with outstanding hydrophobicity properties, affording superhydrophobic materials that are capable of retaining water droplets on their surfaces, even when the material is inverted by 180°, with a wide range of potential applications in oil/water separation technology and for selective cell recognition in biological systems.The three-dimensional hierarchical morphology of surfaces greatly affects the wettability, absorption and microfabrication properties of their hybrid materials, however few scalable methods exist that controls simultaneously complex geometric shape and spatial scattered location and their physical properties tuned. Consequently, this report describes a synthetic strategy that enables the position of well-ordered biomorph nano-microstructures on hydrophobic surfaces to be precisely controlled. The hierarchical architecture can be accurately positioned on polydimethylsiloxane (PDMS) surfaces in an unprecedented level by leveraging a solid/liquid/gas triphase dynamic reaction diffusion system strategy. The effect of salt concentrations, pH, CO In particular, the well-ordered patterns and aligned hierarchical morphologies produced by synthetic inorganic materials  can impart unique anisotropies, large surface areas and enhanced charge transport properties to materials for applications in opto-electronic devices4, catalysis5, mechanics7, superwettable materials9 and molecular diagnostics10. However, the development of efficient self-assembly techniques that enable directed mesoscale complex construction using low-cost scalable protocols for the production of a wide range of materials is still urgently required.Self-assembly processes present in nature have inspired scientists to develop the artificial supramolecular self-assembly systems for the synthesis of complex and hierarchical structures that allow functional materials that exhibit useful physical/chemical/structural properties to be produced at scales ranging from nanometers to millimeters2 into alkaline solutions of sodium silicate (affording CO32− and H+ ions) that results in the coprecipitation of SrCO3 and SiO217. This simple approach involves mixing of cheap ingredients (Na2SiO3 and Sr/BaCl2) to create silicon composites with unprecedented complex morphologies  that are generated through randomly dispersed growth processes18. The key factor in these bioinspired mineralization systems is the presence of front reaction-diffusion processes that create the dynamic environment required for production of these precisely sculpted arbitrary architectures24. These processes serve to create an artificial microenvironment that creates exactly the right chemical and thermodynamic conditions required for spatial control of the microstructures that are produced25. Initial reports into generating silica biomorph systems in dilute silica sols or silica gels involved immersion of a substrate into a solution containing reactive silicon precursors which resulted in the random generation of microstructures with no positional control17. More recently, a bubble mediated substrate water/air interface strategy has been developed to induce crystallization at solid-liquid-gas triphasic contact lines that can be used to control the spatial arrangement and morphology of the self-assembled surfaces on polydimethylsiloxane (PDMS) surfaces26. This approach relies on the hydrophobic micropillar-based PDMS surfaces containing air gaps that enable reagents in solution to be effectively transported to their edges where they can then react to form defined microstructures. In these processes, the micropillars of the PDMS act as a solid support that simultaneously interacts with air (containing CO2 gas) and the solution (containing reactive silicon reagents) to produce a solid-liquid-gas triphasic reaction that deposits microstructures on the surface of the micropillars. The incorporation of soft lithography techniques into this hydrophobic micropillar fabrication process, can potentially control the location of the self-assembly growth process to afford selectively patterned materials. Three-dimensional microscopic computed tomography (3D-CT) technology has been used to determine the amount of air in contact with the liquid/solid phases, thus enabling the structure of the gas transfer networks present in these systems to be determined. These studies have shown that this gas transfer network is essential, because it sustains the integrity of the liquid phase and facilitates solution based gas diffusion-reaction processes that are required for self-assembly to occur at room temperature. Aebisher and co-workers27 have reported other types of reactive solid-liquid droplet-based systems, employing silicon phthalocyanine sensitizing particles to study the role of hydrophobic photosensitizers that generate oxygen gas in droplets for the functionalization of PDMS micropillars. Furthermore, Wang28 and Wu29 have reported diffusion studies on H2 and H2S at triphasic interfaces under nonequilibrium conditions that enabled gold and metal sulfide dish-like microstructural arrays to be grown on the top surfaces and edges of PDMS pillars. Therefore, the ability of well-organized hydrophobic micropillar substrates to act as structural templates to precisely locate gas-liquid-solid triphasic interfacial self-assembly reactions has been firmly established.Self-assembly processes that occur during biomineralization at ambient temperature afford highly functional materials with hierarchical architectures and stunning beauty that have prompted the development of rationally designed silicon-based materials. For example, silica-carbonate biomorphs comprised of encapsulated crystalline carbonate and amorphous silica have been synthesized by diffusing CO3–SiO2 microstructural arrays on hydrophobic micropillar substrates. These protocols generate stable CO2 concentrations at the reactive triphasic interface of these hydrophobic self-assembly processes, thus enabling the location and hierarchical structures of the microstructures formed on the pillars to be studied and controlled. This new triphasic interface approach represents a novel platform for investigating crystal growth mechanisms for the formation of carbonate-silica biomorphs and is likely to be of use for the production of silicon-based arrays of use in functional devices.In this study, we report how self-assembly processes generated through bottom up biomineralization and top-down lithography processes that occur at hydrophobic gas-liquid-solid interfaces can be used to generate multiscale patterned architectures on pillar templates. Optimization of reaction times, reagent concentrations, pillar geometries and temperatures of this triphasic self-assembly process has enabled reproducible tunable protocols to be developed that enable the scalable interfacial growth of uniform SrCO2 and Na2SiO3 (Sigma Aldrich) using the set-up shown in Table 2 from the atmosphere to diffuse into the system. The pH of the solution was measured using a Mettler Toledo FE20 FiveEasy pH meter and adjusted to between 10 to 13 by adding HCl or NaOH, respectively ; a FEI Nova NanoSEM scanning electron microscope (SEM) equipped with a SE2 detector and a four-quadrant electron backscatter detector; and a JEOL JEM-2100F TEM  equipped with a F-216 digital camera , a high angle annular dark field (HAADF) detector and an INCA X-SIGHT EDX system . Thin film X-ray diffraction (Shimadzu 6000 with Cu Kα radiation (λ = 1.5418 Å)) was used to analyze phase compositions of all coatings using a grazing angle of 1°. High resolution transmission electron microscope (HRTEM) imaging and energy dispersion X-Ray spectroscopy (EDS) were employed to further characterize the crystalline structure and chemical composition of the crystalline products.Determining Superhydrophobic Properties of Materials: A water droplet was placed onto the surface of the self-assembled composite materials using a syringe needle. Pictures of formed droplets were taken using an optical microscope from the side view and the resultant images analyzed using “Image J Software”, that calculated contact angles for materials with the different number of arms and gaps.2–Na2SiO3 solution (via air pockets) in a CO2 atmosphere at room temperature  and Na2SiO3 (8.2 mM) at pH of 11.8. The beaker was then fitted with a loose cover that allowed gas diffusion from the atmosphere to the surface of the pillars  revealed that the carbonate-silica biomorphs with microflower and micrograss morphologies were present on the surface of the PDMS pillars   Fig.  insert. 2 required for the reaction is less at this more exposed interface  is slower than that at the edges because the concentration of COace Fig. . The airent Fig. . Therefo2 and SrCO3 resulted in the liquid entering the gaps to disrupt the self-assembly process. Consequently, all self-assembly reaction was carried out using vertically aligned PDMS templates to avoid the air-liquid interface from being disrupted and ensure free CO2 exchange between the atmosphere and the triphasic reaction sites  in the solution, then the uncontrolled aggregation of SiOtes Fig.  S2.3 and SiO2 were present at the reactive triphasic sites in these systems. Initial nucleation events occur at the pillar top edges that provide high surface energies and tensions to establish effective triphasic interfaces for functionalization of the micropillar surface. After an hour, self-assembles stems were observed with a statistical average length of 5 μm, whose formation occurs because of continuous diffusion of CO2 gas through the ambient-connected gas network, which facilitates the solid crystallization process. Over the next five hours, the width of the stem remains constant at 2 μm and its length rising to 40–45 μm as the number of crystals present reached saturation level. Between seven and nine hours, the rate of the self-assembly reaction process decelerated significantly, which is probably due to the pH value at the interface being reduced and the surface wetting properties of the system change. A concomitant reduction in ion concentration levels results in a change in growth behavior to a radial mode that produces agglomerates with plate vase like shapes. To demonstrate that lower ion concentration was responsible for this change in structural morphology, we repeated pillar functionalization experiments using a solution containing half the concentration of the reactive silicon precursors, which resulted in the formation of vase shape assemblies. Notably, once the tops of pillars were uniformly coated by the hierarchical architecture of a definite size, then the wetting properties of the exposed surfaces became hydrophilic, with the pillar gaps still being exposed to CO2 gas. Interestingly, PDMS pillars whose surfaces were pretreated with oxygen plasma to produce hydrophilic surfaces produced microstructural assemblies wit rambling structures, similar to those produced in standard solution based self-assembly systems.The structural growth behaviors of root, stem and petal segments of interface-mediated silica biomorphs were investigated over time Fig. . Root seThe effect of different temperatures on the growth of silica biomorph architectures was also investigated, with increasing temperatures resulting in increased reaction rates due to air bubbles destroying the gas-liquid interface, which results in the liquid fully permeating into the pillar gaps to afford less morphologically defined microstructures. Conversely, when the temperature declines, the growth rate decreases, with no surface based self-assembly processes found to occur at 0 °C. As a result, it is recommended that a temperature range of between 25–45 °C Fig.  is used 3−/CO32− speciation equilibrium. This results in the concomitant release of protons that react near crystallites to dissolve silicate species, such as Si(OH)3O−. These localized protonation events raise the concentration of silicic acid, resulting in supersaturation of amorphous silica which precipitates out and polymerizes around the carbonate crystallites, thus preventing further growth at that site. Continuous oligomerization reactions remove protons from the precipitation site, which in turn affects local carbonate speciation by increasing the amount of CO32− ions present21. This causes an increase in supersaturation of barium carbonate, resulting in the nucleation sequence being repeated and further growth occurring. After an initial stage involving silica-induced fractal branching of a carbonate seed crystal, this dynamic interplay results in emerging nanocrystalline assemblies initially growing as cardioid shaped quasi-2D laminar sheets that are a few microns thick. These sheets can grow along the surfaces of vessel walls, or assemble on top of each other, curling, bending and twisting in the process to produce delicate helical or worm-like morphologies. Based on this knowledge, we investigated whether the morphologies of the silica biomorphs could be controlled by modifying salt concentration and pH levels used in these self-assembly reactions. Inspired by phase diagram theory, there were four factors affecting the chemical reaction  at a gas-liquid biphasic interface  at the front the oscillatory coprecipitation reaction. Three types of micro-environment interfaces where COace Fig. ; (2) at ace Fig. ; and (3)ace Fig. . CO2 dir3-SiO2 nanostructures with different morphologies to be generated depending on the reaction conditions employed 30. As for the mixed CBM-Wm state (lower/macro-Cassie–Baxter and upper/micro-Wenzel) with the water droplet pinned on the top of arms and an air layer is trapped by nano-microstructure arms  state in the middle makes the high CA hysteresis and petal effect (Fig. The surface of the template substrate in conventional processes does not influence on the nucleation and growth of crystal formation. However our methodology generates silica biomorphs at a triphasic interface that enables SrCOyed Fig. . Interesyed Fig. . The supyed Fig. . The capyed Fig. . Increasyed Fig.  resulted80° Fig. . As reporms Fig. . It is nrms Fig. . Togetheect Fig. .Figure 4The long-term stability of the superhydrophobicity of the obtained surface after humidity, outdoor exposure and UV for various time intervals was also investigated. The relative air humidity was controlled by using the saturated salt solution method according to EN ISO 483. The samples were put into a closed container with different kinds of saturated salt solutions by which the relative humidity in the container could be kept constant. Hydrophobicity transfer characteristics of microstructure arrays are shown in Fig. 2SO4 with pH = 1, NaOH with pH = 13 and neutral 3.5 wt.% NaCl solution for the superhydrophobic surface. From Fig. 2SO4 and NaCl solutions, suggesting a good stability. However, the CA is lower than 150° after 3 h of contact with NaOH solution and finally decreased to 124° after 24 h of contact with NaOH solution. This is due to the occurrence of the chemical reaction between stearic acid and NaOH. It was investigated the pH and surface wetting properties changing before and after the reaction. Through the rate of the self-assembly reaction process carry on, the pH value decelerated and the CA increased first then decreased in Fig. In order to analyze the chemical stability, the influence of pH of an aqueous solution and saline solution on the superhydrophobicity was studied for the as-prepared surface. A dry sample was placed inside a closed experimental cell, with water vapors saturated with respect to the test solutions. Figure Therefore, we propose that our triphasic approach is a potentially versatile approach to directly position hierarchical combinatorial architectures on to the surfaces of pillar shaped PDMS substrates, thus producing self-assembled superhydrophobic materials without the need for multiple operational processes and complicated instrumentation.2 concentration at the site of reaction. The artificial hierarchical structure shows remarkable super-hydrophobic property and petal effect. The super-hydrophobic property and tunable adhesion are related to the number of arms and the gap distance between each branch. SiO2-SrCO3 array exhibited ultrahigh durable superhydrophobic properties under UV irradiation, humidity and pH condition. It opens intriguing perspectives for designing artificial surfaces on the basis of complex hierarchical structure to form a super-hydrophobic surface. This one-step, mild fabrication method can be used to produce various different types of silica biomorph microstructure, thus providing a versatile method for fabricating materials with superhydrophobic surfaces that have potential applications for self-healing/self-regulation32, oil/water separation34 and cell culture/diagnostics/recognition36.A triphasic interface-mediated strategy has been used to attach hierarchical silica biomorph microstructural arrays to the surfaces of vertical hydrophobic micropillars PDMS substrates. The factors that affect solid/liquid/gas interface coprecipitation of the local chemical field have been evaluated to identify what parameters are responsible for controlling the self-assembly reaction at the growth front, which has enabled conditions to be identified that enable the morphology, size and direction of the resultant self-assembled structures to be controlled. Triphasic interface growth behavior was found to occur via liquid-gas and solid-liquid growth processes, with overall growth rates controlled by the ambient COSupplementary information.Supplementary information2.Supplementary information3.Supplementary information4.Supplementary information5.


The cardiovascular outcome trials (CVOTs) have shown that glucagon like peptide-1 receptor agonists (GLP1RAs) have varying degrees of cardiovascular (CV) safety in patients with type 2 diabetes mellitus (T2DM.) The lack of any head-to-head comparative trials among GLP1RAs urged the need for an indirect comparison of these agents. Therefore, this study was conducted to indirectly compare the CV safety and mortality effects among different GLP1RAs in patients with T2DM using network meta-analysis (NMA).Medline was searched to identify GLP1RA CVOTs to date. The outcomes of interest were CV death, myocardial infarction (IM), stroke, and death from any cause. An NMA with binomial likelihood logit link model was used for the binary outcomes. We conducted both fixed effects and random effects models for each outcome, and selected the best model based on the deviance information and the average posterior residual deviance. This NMA was reported in accordance with the preferred reporting items for systematic reviews and meta-analyses (PRISMA-NMA).A total of seven GLP1RA CVOTs were included having 56,004 patients. The NMA results showed that oral semaglutide was statistically better than exenatide , dulaglutide , albiglutide , lixisenatide  in reducing CV death events. No significant differences were detected between most of the treatments regarding reducing death from any cause, MI and stroke events. The ranking results showed that oral semaglutide had the highest probability to be ranked first (> 90%) in reducing CV death and death from any cause. Moreover, once weekly semaglutide had the highest probability to be ranked first in reducing MI and stroke events.The GLP1RAs have shown significant benefits in terms of CV safety. The indirect comparison and ranking probability results have shown that one weekly semaglutide and oral semaglutide seems to be the preferred option in patients with T2DM and established or at high risk of CVD. This result can aid health care providers, pharmacy and therapeutics committees in hospitals, and insurance companies when deciding which GLP1RA to start or add to their formulary. Diabetes mellitus is a major health condition that affects more than 382 million adults worldwide, and this number is expected to reach 592 million by 2035 . PatientGlucagon like peptide-1 receptor agonist (GLP1RA) based therapies have been in use since 2005 and GLP1RAs have been extensively studied for the treatment of type 2 diabetes mellitus (T2DM). Most of the GLP1RAs, including lixisenatide (short-acting) and dulaglutide (long-acting), are administered as a subcutaneous injection either once daily or weekly . RecentlIn 2008, the US FDA mandated that all novel antidiabetic drugs must be evaluated by manufacturers to assess their CV safety profile . Since tPubMed was searched from inception to November 2019 with the aim to identify GLP1RA CVOTs. Published randomized double blind placebo-controlled studies that have addressed the CV safety of GLP1RA as their primary outcomes were targeted. The outcomes of interest were major adverse cardiovascular events  and nonfatal stroke termed three-point MACE). In addition to MACE, death from any cause, stroke, nonfatal stroke, MI, nonfatal MI and hospitalizations for heart failure (HF) outcomes were included. The search terms used were “glucagon like peptide-1,” “GLP-1 receptor agonist,” “liraglutide,” “semaglutide,” “albiglutide,” “exenatide,” “stroke,” “nonfatal stroke,” “myocardial infarction,” “nonfatal myocardial infarction,” “heart failure,” “CV death,” and “CV mortality.” Results were further limited to randomized controlled trials and English language. Published data were extracted from the published studies by two authors (OMA and OAA) independently, and reviewed by a third one (OSA). The risk of bias was assessed using the Cochrane risk of bias assessment tool , and albiglutide compared to lixisenatide . Moreover, the weekly injections of semaglutide, albiglutide, liraglutide (daily) and dulaglutide showed a statistically greater reduction in MACE when compared to placebo. The ranking results showed that weekly administration of semaglutide had the highest probability of being ranked first at 52% followed by oral semaglutide at 26% and albiglutide was ranked third at 20%. The indirect comparison of GLP1RAs is presented in Fig. The indirect comparison showed that oral semaglutide significantly reduced CV mortality compared with exenatide , dulaglutide , albiglutide , and lixisenatide . Moreover, oral semaglutide had the highest probability of being ranked first (> 90%) in reducing CV mortality in the rankogram comparison.The indirect comparison showed that oral semaglutide significantly reduced death from any cause than all the GLP1RAs except liraglutide. Therefore, oral semaglutide had the highest probability of being ranked first at 96% in the rankogram comparison.No significant differences were detected regarding total MI between the treatments except when albiglutide was compared with exenatide and lixisenatide. Albiglutide was associated with a significant reduction in MI events compared to exenatide  and lixisenatide . The ranking results showed that weekly administration of semaglutide was ranked first at 51%, followed by albiglutide at approximately 40% in the rankogram comparison.Similar to the results in total MI outcome, albiglutide was associated with a significant reduction in nonfatal MI compared to exenatide  and lixisenatide . However, weekly administration of semaglutide had the highest probability of being ranked first at approximately 52%, followed by albiglutide at 42%.For total stroke events , no significant differences were identified between the treatments, except when the weekly administered semaglutide was compared to lixisenatide . Moreover, ranking probability showed that weekly administered semaglutide was ranked first at 59%, followed by oral semaglutide at 27%.Similar to the results in total stroke outcome, no significant difference was identified between the GLP1RAs, except when the weekly semaglutide was compared to lixisenatide . Furthermore, weekly semaglutide had the highest probability of being ranked first in reducing nonfatal stroke events at 58%, followed by oral semaglutide at 29%.The indirect comparison did not show any significant difference among the GLP1RAs and placebo in reducing HF related hospitalizations. The ranking results showed that oral semaglutide had the highest probability of being ranked first, followed by albiglutide.The results from meta-regression analyses of the effect of potential covariates did not suggest any association between the covariates and the relative effect of GLP1RAs. The 95% CrI of the interaction coefficients for the included covariates crossed the null value (0). These findings were presented in Additional file The study was conducted to indirectly compare the CV safety and mortality effects among different GLP1RAs in patients with T2DM using network meta-analysis (NMA), to identify the likely preferred agent with respect to CV safety. The study did not find any significant difference between GLP1RAs in reducing death from any cause, MI and stroke events. However, the ranking results showed that oral semaglutide had the highest probability to be ranked first (> 90%) in reducing CV death and death from any cause, while once weekly semaglutide had the highest probability to be ranked first in reducing MI and stroke events.Several meta-analyses were performed to compare the CV safety of GLP1RAs. Furthermore, NMA was also performed to explore the CV safety of GLP1RAs in comparison with other hypoglycemic drugs such as SGLT2is –26. FiveWhen examining the individual components of MACE, a significant reduction in CV mortality (12–13%) and stroke (16–17%) was consistently reported for GLP1RAs, with no heterogeneity, in the five aforementioned meta-analyses. However, the reduction in the MI events (8–9%) was not as robust as the results for the CV mortality or the stroke outcomes, as this was only significant in three of the five meta-analyses –19. MoreDeath from any cause was also reported to be significantly reduced by GLP1RAs. The reduction ranged between 10 and 12% in four of the five meta-analyses, with no significant heterogeneity reported –18, and For the HF related hospitalization, the use of SGLT2is was previously found to be associated with the most cardioprotective effect in patients with HF, or even at risk of having HF, compared to all other antidiabetic classes, including GLP1RA , 30. HowThe risk of CVD, CV mortality, and death from any cause varies among patients with T2DM based on age, HbA1c level, history of CVD, and the duration of diabetes –35. The In light of the recent results of CVOTs, the diabetes guidelines have changed their recommendations regarding T2DM treatment. This change was heavily influenced by the CV effect of drugs regardless of the glycemic effect. The most recent American Diabetes Association (ADA) guidelines of 2020 recommend the use of GLP1RAs as an add-on to metformin or as a first line therapy for selected patients who cannot tolerate metformin . MoreoveIn this paper, other safety issues were not addressed, such as retinopathy. However, recent meta-analysis did not show significant increase in retinopathy events with the use of GLP1RAs . There aThe GLP1RAs have shown significant benefits in terms of CV safety. While utilizing data from the CVOTs to date, the indirect comparison and ranking probability results have shown that one weekly semaglutide and oral semaglutide seems to be the preferred option in patients with T2DM and established or at high risk of CVD. This result can aid health care providers, pharmacies and therapeutics committees in hospitals, and insurance companies when deciding which GLP1RA to start or add to their formulary.Additional file 1: Table S1. PRISMA Network Meta-Analysis Checklist.Additional file 2: Figure S1. Flow diagram for study selection.Additional file 3: Figure S2. Network plot of all interventions in the analysis.Additional file 4: Table S2. Risk of bias assessment of glucagon like peptide-1 receptor agonist (GLP1RA) cardiovascular outcome trials (CVOTs).Additional file 5: Figure S3. Inconsistency plots for the outcomes from the fixed effect model.Additional file 6: Table S3. Results from the meta-regression analyses for the interaction coefficient, median (95% CrI).Additional file 7: Table S4. Summary of the results from the previous pairwise meta-analyses.


Organ congestion is a mediator of adverse outcomes in critically ill patients. Point-Of-Care ultrasound (POCUS) is widely available and could enable clinicians to detect signs of venous congestion at the bedside. The aim of this study was to develop several grading system prototypes using POCUS and to determine their respective ability to predict acute kidney injury (AKI) after cardiac surgery. This is a post-hoc analysis of a single-center prospective study in 145 patients undergoing cardiac surgery for which repeated daily measurements of hepatic, portal, intra-renal vein Doppler and inferior vena cava (IVC) ultrasound were performed during the first 72 h after surgery. Five prototypes of venous excess ultrasound (VExUS) grading system combining multiple ultrasound markers were developed.p = 0.001). The association remained significant after adjustment for baseline risk of AKI and vasopressor/inotropic support (HR: 2.82 CI 1.21–6.55 p = 0.02). Furthermore, this severe VExUS grade offered a useful positive likelihood ratio (+LR: 6.37 CI 2.19–18.50) when detected at ICU admission, which outperformed central venous pressure measurements.The association between each score and AKI was assessed using time-dependant Cox models as well as conventional performance measures of diagnostic testing. A total of 706 ultrasound assessments were analyzed. We found that defining severe venous congestion as the presence of severe flow abnormalities in multiple Doppler patterns with a dilated IVC (≥ 2 cm) showed the strongest association with the development of subsequent AKI compared with other combinations (HR: 3.69 CI 1.65–8.24 The combination of multiple POCUS markers may identify clinically significant venous congestion. Hemodynamic management in critically ill patients has traditionally focused on maintaining adequate cardiac output and arterial blood pressure by relying on fluid administration and vasopressor/inotropic support , 2. HoweAn exceedingly challenging aspect of hemodynamic evaluation is determining what represents clinically significant venous hypertension. Central venous pressure (CVP) measurements performed in the intensive care unit (ICU) remain invasive and are subject to important measurement errors even among experienced operators . FurtherPoint-Of-Care ultrasound (POCUS) enables the clinician to visualize the vascular anatomy and assess blood velocity using Doppler imaging. Within any venous vascular system, the additional volume associated with congestive factors will eventually reach the upper limits of the systemic venous capacitance, causing a rapid rise in venous pressures. Several markers of the high pressures associated with this congestive process have been proposed including the assessment of large veins  as well as detecting abnormal venous waveforms suggestive that the limit of the systemic venous compliance in the portal vein, hepatic veins and intra-renal veins . All of The primary objective of the exploratory study was to develop a prototypical Venous Excess Ultrasound (VExUS) grading system of the severity of venous congestion, and to validate its potential clinical value in predicting the occurrence of acute kidney injury (AKI) after cardiac surgery using existing data from a recent prospective cohort study. The secondary objective was to compare its clinical usefulness to CVP measurements.This is a post-hoc analysis of data collected during a prospective cohort study at a tertiary cardiac surgery center from August 2016 to July 2017 (NCT02831907) . Written2 calculated using the Modified Diet in Renal Disease formula [Non-critically ill patients 18 years and older undergoing cardiac surgery with the use of cardiopulmonary bypass were eligible to participate. Complete inclusion and exclusion criteria have been previously described . Notably formula ) or dialAll patients underwent repeated POCUS assessment the day before surgery, at ICU admission after surgery and daily from post-operative days 1 to 3. Each ultrasound assessment consisted of hepatic vein Doppler, portal vein Doppler, intra-renal venous Doppler and inferior vena cava (IVC) ultrasound. The complete method for hepatic, portal and renal Doppler assessment has been previously published . The assThe IVC diameter was measured in its intra-hepatic portion at 2 cm of the junction with the hepatic veins using a longitudinal view from a sub-xiphoid position . When thBased on an original concept (P.R.), a multidisciplinary team composed of intensivists , anesthesiologist (A.D.), emergency physicians  and nephrologist (W.B.S) developed five VExUS grading system prototypes based on the severity of venous ultrasonographic markers Fig. . For theDemographic, baseline information, cumulative fluid balance information, hemodynamic parameters at the time of assessment and outcomes during hospitalization were all collected prospectively during the study as previously published . The pret-test or Mann–Whitney U test, as appropriate, and comparison between two groups for categorical variables was done using Chi squared test. The prevalence of each VExUS grade was presented as descriptive data for each timepoint. The association between the VExUS grades and the risk of new‐onset of AKI was assessed using a Cox proportional hazards model with the VExUS grades considered as segmented time‐dependent covariates. After identifying the VExUS grade most associated with AKI in univariable analysis, multivariable Cox regression was performed. A first model was constructed by including the pre-operative risk of AKI as performed by Birnie et al. [Results are presented in number (%) for dichotomous variables and in mean ± standard deviation (SD) or median and interquartile range (IQR) for continuous variables, where appropriate. Comparisons between two groups for continuous variables were done using Student e et al.  as an a The sensitivity and specificity as well as the positive likelihood ratio (+LR) and the negative likelihood ratio (−LR) of the different VExUS grades assessed at ICU admission to predict AKI after cardiac surgery were presented. The same analysis was performed using different cut-offs of CVP , as well as individual ultrasound findings included in the VExUS grading systems. The CVP cut-off used was chosen based on current literature and expert opinion , 13, 35.p < 0.05) with the time of assessment. Consequently, we presented the association for each timepoint and for each VExUS grade. The difference between the different grades was assessed using one-way ANOVA or Kruskall–Wallis test, where appropriate depending on the distribution of data. In the presence of a significant result (p < 0.05), multiple post-hoc pairwise comparisons with Bonferroni correction were performed and significant results were presented. Statistical tests were performed in SPSS version 24 .The association between the VExUS grading system prototypes and commonly used markers of venous congestion  and VIS was first assessed using generalized estimating equation models using a robust estimator for the covariance matrix and an exchangeable structure for the working correlation matrix was used. This type of analysis accounts for the repeated measures’ design, implying that the sample was not independent. In addition to the VExUS grades, the time of assessment (4 timepoints: Day 0 to Day 3) was included as a factor in the analysis and the interaction between the studied variable and the time of ultrasound assessment was tested. We found multiple significant interactions (2) was present in 37 patients (25.5%). At the start of the surgery before cardiopulmonary bypass, trans-esophageal echocardiography revealed low LVEF in 37 patients (25.5%) and systolic right ventricular dysfunction in 18 patients (12.4%). After surgery, 49 patients (33.8%) developed AKI but no patients received renal replacement therapy.Complete data from all 145 participants included in the original prospective study were included in the analysis. Baseline characteristics of patients have been previously published  and a suThe complete distributions of all VExUS grades (0–3) according to candidate grading systems (A–E) during the peri-operative period are presented in Fig. p = 0.001). After adjustment for the baseline risk of AKI and pharmacologic support (VIS), severe congestion (Grade 3) defined by the VExUS C grading system remained associated with the subsequent development of AKI after surgery  (Table p = 0.036).In the post-operative period, the presence of severe congestion (Grade 3) was associated with the development of subsequent AKI for all VExUS grading systems Table . Severe While only considering the assessment performed at ICU admission after surgery, severe congestion Grade 3) defined by the VExUS C grading system had a high specificity (96% CI 89–99%) but low sensitivity 27% CI 15–41%) for the development of subsequent AKI resulting in a moderate +LR of 6.37 (CI 2.19–18.5) which surpassed the performance of other grading systems  and a higher CVP  without statistical interaction with the time of assessment. While an interaction was present for the analysis involving NT-pro-BNP, a significant difference in NT-pro-BNP measurements among grades was also present at each studied timepoint was as shown in Fig. Other congestion markers including cumulative fluid balance, NT-pro-BNP and CVP were associated with severe congestion (Grade 3) in all studied VExUS grading systems , offered the strongest association with the development of subsequent AKI compared with other combinations of ultrasonographic features. Examples of VExUS C grading are presented in Fig. While there is now a widespread agreement that systemic venous hypertension resulting from fluid overload and right ventricular failure is associated with multiorgan injury and adverse outcomes . POCUS mIn the present work, the low sensitivity to predict AKI is not surprising since venous congestion is unlikely to be a contributive factor in all cases. A multitude of other factors, venous congestion being only one of them, may trigger AKI in the peri-operative period . HoweverThe present work has several limitations. First, we performed a relatively small single-center study including only cardiac surgery patients which limits the generalizability of the findings. Most importantly, the formation of interstitial edema also depends on vascular permeability which may be quite variable depending of the underlying disease. Consequently, caution should be taken when interpreting these finding in other clinical contexts. Furthermore, while the relationship between portal Doppler patterns and other echocardiographic variables during cardiac surgery has been previously described by our group  and otheIn conclusion, we used existing data to propose a novel grading system for venous congestion, the Venous Excess UltraSound (VExUS) grading system based on the combination of multiple ultrasound findings. The presence of at least two severe alterations of hepatic vein, portal vein or intra-renal venous flow on pulse-wave Doppler ultrasound with an IVC of ≥ 2 cm of diameter at ICU admission after cardiac surgery indicates a high risk of post-operative AKI. Further studies should aim to validate this grading system in different clinical settings, confirm the optimal criteria for diagnostic performance and determine whether it could be used to personalize interventions to improve organ perfusion.Additional file 1. Quantifying systemic congestion with Point-Of-Care ultrasound: development of the Venous EXcess UltraSound (VExUS) grading system.


A 60-year-old woman with diabetes and hypertension presented with a decreased vision OD to 6/18 N10 due to cystoid macular edema (CME) following vitrectomy for removal of the dislocated posterior chamber intraocular lens (PCIOL) and scleral-fixated intraocular lens (SFIOL) implantation performed five months before. Spectral domain optical coherence tomography (SD-OCT) showed a central foveal thickness (CFT) of 484 µm. Intravitreal ranibizumab  was administered; however, the CFT increased to 533 µm after one month. She received intravitreal dexamethasone (DEX) implant , which resulted in best corrected visual acuity (BCVA) improvement to 6/12 N10 and CFT reduction to 404 µm within a month. Three months later, CFT increased again to 677 µm and she received a second DEX implant. Seven weeks following the injection, she presented with pain, corneal haze, and intraocular pressure (IOP) of 38 mmHg. A week later, IOP decreased to 16 mmHg on brimonidine tartrate 0.2% and timolol maleate 0.5% BD; however, the DEX implant was seen in the anterior chamber . Complete pupillary dilatation in this vitrectomized eye allowed the implant to migrate forward through the gap between the IOL edge and the pupil .There was no corneal edema and BCVA was maintained at 6/12 N10. Specular microscopy showed an endothelial count (EC) of 1144 cells/mm Anterior chamber migration of the implant is a potential, though uncommon complication, in eyes with a compromised lens capsule, prior to vitrectomy and/or iris defects.,3 Very few cases of migration in eyes with an SFIOL have been reported,,4,5 as has occurred in this case, reinforcing that the presence of an IOL alone does not prevent the implant migration when the posterior capsule is not intact. As a precaution to minimize recurrent migration, the pupil size was kept reduced enough to cover the edge of the optic.Intravitreal DEX implant is a safe and effective therapeutic option for post-surgical macular edema.,4 Early migration (within three weeks of injection) could result in higher incidence of corneal edema as higher rigidity of the implant in the first weeks could cause greater mechanical endothelial trauma. This would explain why our patient did not develop corneal edema and could avoid the additional procedure of immediate repositioning or removing the implant, which reduces the likelihood of permanent corneal edema.,3Corneal endothelial decompensation is the most serious complication resulting from the migration of implant into the anterior chamber.While vitrectomized eyes without an intact lens capsule are at risk for anterior migration of a DEX implant, this subset of patients may also benefit most from having a DEX implant administered for post-surgical macular edema. A regular post-injection follow-up can recognize and manage this rare, though potentially vision threatening, complication at the earliest. An individualized approach is recommended that takes into account the potential anterior segment complications as well as loss of drug effectiveness. A single episode of anterior chamber migration of the DEX implant should not be considered as a contraindication for further injections if warranted.Nil.There are no conflicts of interest.


To present long-term results of modified bleb-limiting conjunctivoplasty as a successful treatment for intractable bleb dysesthesia and to review the literature on the surgical management of dysesthetic bleb.  Consecutive case series and literature review. We present four cases of surgically reduced painful blebs. Our technique consisted of the following steps: (1) conjunctival, radial incision to the bare sclera in the desired limit of the bleb; (2) suturing with buried, interrupted sutures at the nearest edge of the filtering bleb; (3) lower limbal peritomy including unwanted area of the extended bleb; (4) dissection and removal of the underlying fibrous tissue when present; (5) conjunctival and resorbable sutures. In addition, a systematic literature review was performed. Only reports presenting outcomes of surgical treatment of bleb dysesthesia after filtering procedure were included in review. n = 123) with rates of success within 46–100%, favoring less aggressive approaches to reduce bleb size.  Four eyes were included consecutively in the study in a period of 4 years. On average, they developed circumferential bleb dysesthesia 9.3 ± 4.7 months after uneventful combined phacotrabeculectomy with Mitomycin C as primary procedure. Surgical reduction was decided after failure of lubricants in controlling ocular discomfort. Two cases showed a dense fibrous tissue beneath the conjunctiva that was excised to ensure filtration. In all cases, a rapid disappearance of symptoms with very good aesthetic and functional outcome was observed. After 12-month follow-up, patients remained asymptomatic and maintained intraocular pressure of 10.7 ± 1.2 mmHg without treatment. A systematic review of the literature obtained 15 eligible case series  is able to target the underlying etiology providing ocular discomfort relief while maintaining bleb function and may be considered as first-choice surgical treatment. In 1977, Cohen et al. proposed classifying dysfunctional filtering blebs into three basic categories: underfiltration, overfiltration, and excessive size –4 due toThere are many described methods for managing dysesthesia. Intensive surface lubrication is universally used. Other nonsurgical methods that stimulate scarring  can lead to IOP increase . PlannedThe objective of this study is to present the characteristics and outcomes of consecutive cases of bleb dysesthesia refractory to medical lubricant treatment that underwent bleb-limiting conjunctivoplasty surgery from 2015 to 2019 and, secondarily, to perform an extensive online literature search to identify other studies reporting on the surgical management of bleb dysesthesia.This case series is a prospective study of consecutive patients that developed refractory bleb dysesthesia after successful filtration surgery in a 4-year time period. This study was approved by the Ethical Committee and was conducted in accordance with the tenets of the Declaration of Helsinki. Written informed consent was obtained from all the participants.All the participants underwent a thorough ophthalmic evaluation before and 1 day, 1 month, 6 months, and 1 year after conjunctivoplasty: best-corrected visual acuity (BCVA) in Snellen  scale, standard automated perimetry with Swedish Interactive Threshold Algorithm (SITA) standard strategy, program 24–2 of the Humphrey Field Analyzer , Goldman applanation tonometry, slit-lamp biomicroscopy with IBAGS scale  AND [dysesthesia]. All terms were used as MeSH and as free terms. All articles were revised by 2 investigators independently . Considering the rarity of this condition, articles were included if they were prospective or retrospective case studies presenting outcomes of surgical treatment of circumferential dysesthetic blebs after filtering procedure. Additionally, data of surgical management and outcomes were extracted from the article body.Four consecutive patients with refractory bleb dysesthesia were included in this study during the 4-year time period. We present their baseline characteristics and postoperative outcomes of a modified bleb-limiting conjunctivoplasty procedure and an overview of previously described cases in the literature.The study group consisted of 4 eyes of 2 women and 2 men whose ages ranged from 69 to 81 years . All patients were from European descent and had primary open-angle glaucoma treated with combined phacoemulsification and trabeculectomy with Mitomycin C (MMC) as primary procedure see . During n = 3) or temporal (n = 1) displacement  between 43%  and 100%n = 13) obtaining very good results (100% success) but still with 3 cases needing additional hypotensive topical treatment [n = 2/4, 50%) with the idea to individualize treatment depending on the most predominant causing mechanism. In all of our four cases, discomfort relief and good IOP control were achieved and maintained after one year of follow-up without any additional glaucoma treatment or significant glaucomatous deterioration.There are several methods for limiting bleb size without removing conjunctival tissue. Bleb compression sutures have been used as an effective alternative , 7, 8, breatment . EncouraOur study has several limitations. First, due to the rarity of the condition, a small number of cases were included in the study, and this needs to be considered when interpreting the results. And second, our systematic review could not find enough reliable and prospective information to provide more robust data. To partly address these limitations, a thorough and comprehensive evaluation and summary of the available data in the literature was provided.In conclusion, bleb dysesthesia is an uncommon complication that very likely needs surgery due to failure of medical treatment. Aggressive surgical techniques removing bleb tissue may be less effective in terms of subsequent IOP control. A modified bleb-limiting conjunctivoplasty technique may be a treatment for bleb dysesthesia and allows deciding intraoperatively which mechanisms, avascular or fibrous, predominate, choosing then the most appropriate procedure of bleb reduction, with or without excision of subconjunctival fibrous tissue in each case. After 12-month follow-up, this technique provided dysesthesia relief with good IOP control in the four studied cases.