Intravital imaging is providing new insights into the dynamics of tumor progression in native tissues and has started to reveal the layers of complexity found in cancer. Recent advances in intravital imaging have allowed us to look deeper into cancer behavior and to dissect the interactions between tumor cells and the ancillary host niche that promote cancer development. In this review, we provide an insight into the latest advances in cancer biology achieved by intravital imaging, focusing on recently discovered mechanisms by which tumor cells manipulate normal tissue to facilitate disease progression. In these models, intravital imaging has been used to dissect the molecular events governing cancer progression and revealed unprecedented information on the behavior of cells in their native environment, both within primary tumors and at distant metastatic sites. In combination with traditional and static approaches used in cancer research, intravital imaging has significantly expanded our understanding of the complexity of cancer biology, as it allows us to directly image the spatiotemporal dynamics of cancer progression in live settings and at the whole-organ, cellular, subcellular, and molecular levels. Here, we outline the latest discoveries facilitated by intravital imaging of tumors, which could not otherwise be achievedin vitro, and discuss how intravital imaging techniques used in other disease contexts could be repurposed for cancer research.Within tumors, intricate crosstalk between cancer cells and the surrounding microenvironment supports cancer initiation and progression. Importantly, these interactions not only shape the development of the primary tumor but also are required at secondary sites to develop a microenvironment permissive to metastatic growth. This appreciation of the dynamic cancer-stroma interactions in tumor progression has led to a transition from traditionalin vitro for studying microvasculature systems2, several intravital imaging tools, such as fluorescent proteins or nanoparticles, have been employed to understand the interactions between cancer cells and blood vessels in live settings, an aspect that cannot be recapitulatedin vitro6. For example, fluorescently labeled lectins, such asLens culinaris agglutinin, have been developed to selectively bind to endothelial cells4. Intravenous co-injection of agglutinin and fluorescently labeled cancer cells was recently used with intravital imaging of the embryonic chicken chorioallantoic membrane (CAM). This is a surrogate assay for the study of cancer cell extravasation and metastatic colonization and has recently provided insight into the time course and molecular mechanisms involved in cancer cell extravasation4. Here, the authors demonstrated that intravascular cancer cells initially move along the luminal endothelium in an amoeboid manner and subsequently start to extend protrusions in-between endothelial cells into the extravascular stroma. Cancer cells then gradually push through endothelial cell-cell junctions, in some cases even displacing endothelial cells4. Interestingly, invadopodia marker proteins such as cortactin or MT1-MMP were shown to be localized at the protrusions of extravasating cancer cells, and knockdown of these markers impaired cell extravasation and the formation of metastatic colonies within the CAM4. In line with this, tail-vein injection of invadopodia-deficient cells into mice resulted in a decreased number of lung metastases, showing that invadopodia are a key feature for transendothelial cancer cell migration4. Similarly, providing intravascular cancer cells in the CAM assay with hyaluronic acid, which is a component of the extracellular matrix (ECM) overexpressed in many tumor types9, led to increased cell extravasation3. Using this probe therefore demonstrated that the ECM not only represents a physical barrier against chemotherapy delivery10 but can also be co-opted by cancer cells for secondary site colonization.Cancer cells use and co-opt the surrounding stroma to promote their expansion and dissemination. Tumor-associated blood vessels serve a dual function during cancer development: they provide tumor tissue with essential oxygen and nutrients but can also act as carriers for circulating cancer cells. While various models have been developedin vivo cancer research to investigate vascular integrity in live tumor tissues12. For example, intravital time-course imaging of QDs in the MMTV-PyMT mouse model of breast cancer allowed Ormandy and colleagues to demonstrate that cancer cells at the primary site can use the surrounding blood vasculature during the progression of invasive and metastatic cancer13. Here, ELF5 induction resulted in QD leakage and accumulation within the interstitial space, in line with the formation of hemorrhagic tumors, and increased the number of lung metastases (13). Furthermore, we previously used QDs to map the vascularization of pancreatic subcutaneous xenograft tumors and showedin vivo that Src activity and drug-mediated Src inactivation in cancer cells correlate with their position relative to intratumoral blood vessels11. New organic QDs with improved biocompatibility, stability, and two-photon absorption cross-section and fluorescently tagged viral nanoparticles have recently been developed for real-time intravital mapping of the blood vasculaturein vivo15. Fluorescently labeled red blood cells and mice with fluorescently labeled endothelial cells were also engineered to mark the blood vasculaturein vivo18, and together with QDs and dextran, these approaches are likely to improve our understanding of how the tumor-associated vasculature supports cancer progression.Fluorescent dextrans and quantum dot (QD) nanoparticles are also commonly used to visualize blood vessels. More recently, they have been employed duringet al. recently used intravital imaging of melanoma cells injected into the mammary fat pad with a lymphatic tracer dye to visualize cancer cell migration towards tumor-draining lymph nodes19. The authors demonstrated that lymphatic sinuses of the lymph nodes, but not peripheral lymphatics, secrete CCL8, which activates CCR1 on the cancer cell compartment to permit entry into the lymph node19. Inhibition of CCR1 did not impair primary tumor cells from entering the lymphatic system but blocked their exit into the lymph node and arrested the circulating cells within nearby lymphatic vessels. This indicates that lymphatic cancer cell dissemination not only occurs through passive transport but also is supported by paracrine chemotactic signaling19. Recently, a transgenic reporter of lymphatic vessels was generated by crossing LSL-tdTomato mice with Prox1-Cre-ERT2 mice to induce fluorescent reporter expression in lymphatic vessels and map cell trafficking within lymphatic networks20. While the authors used this model to track dendritic cells entering tdTomato-expressing lymphatic vessels upon acute inflammationin vivo, this technology could be repurposed for future use in cancer research to watch circulating tumor cells disseminating through the lymphatic system.The lymphatic system presents another route of cancer cell dissemination. For example, Das24. Here, dual intravital imaging of leaky blood vessels and immune cells has been used to identify novel mechanisms of drug action. In particular, the antitumor activity of bisphosphonates, which are commonly used in skeletal diseases, such as osteoporosis25, has been explored in a 4T1 mammary tumor mouse model26. Here, it was demonstrated that fluorescently labeled bisphosphonates enter the primary tumor site via the leaky vasculature and bind to microcalcifications within the tissue. Bisphosphonates were then incorporated by tumor-associated macrophages (TAMs), and the authors suggest that this might impair the function of TAMs26, which have been shown to potentiate tumor progression27. Thus, intravital imaging allowed the authors to delineate an unknown mechanism of action underlying the partial benefits of bisphosphonate treatment in breast cancer patients. In line with this, TAMs have also been identified in the tumor microenvironment of metastasis (TMEM), a hotspot of vascular permeability characterized by the physical interaction among tumor cells, TAMs, and endothelial cells28. Intravital imaging of blood vasculature using QDs and fluorescent dextran demonstrated that Tie2hi-expressing TAMs induce transient vascular leakage via vascular endothelial growth factor A (VEGFA) signaling, thereby facilitating tumor cell intravasation. Interestingly, intravital imaging also showed that transendothelial migration preferentially occurred in regions containing a TMEM, and specifically targeting this site could slow down the spread of cancer cells28. Intravital imaging has also been used recently to compare the motility of two immune effector cell types, natural killer (NK) cells and cytotoxic T-lymphocytes (CTLs)29. Here, the authors showed that while NK cells form short and fast contacts with tumor cells independent of calcium signaling, CTLs form long-lasting contacts with tumor cells that require calcium influx into CTLs. Interestingly, both cell types were shown to require calcium influx for efficient killing29, thus intravital imaging has provided new insights into the cell-type-specific interactions between tumor cells and the immune system which are needed for specific and efficient killing of tumor cells to occur and which cannot be mimicked faithfullyin vitro.During cancer progression, tumor cells interact with and manipulate the immune system to facilitate their growth and escape from cell deathet al. characterized a potential pathway through which tumor-derived antigen is locally processed and presented to generate a systemic immune response30. Injection of B16.F10 melanoma cells, transfected with tdTomato as a surrogate tumor-derived antigen, into the mouse footpad led to a robust immune response after 30 days measured by high titers of anti-B16.F10-tdTomato IgG30. Imaging of tumor-draining lymph nodes showed that tdTomato was localized in macrophages in the lymphatics and also in follicular dendritic cells (FDCs). Using mouse models of macrophage, FDC, or B-cell deficiency, the authors suggest that tdTomato is taken up by macrophages and subsequently localizes with FDCs, which are then scanned by B-cells, generating a systemic immune response30. Similarly, intravital imaging of mCherry-expressing mammary tumors was used to characterize antigen presentation at the tumor site31. Here, the authors showed that a subset of myeloid cells function as tumor dendritic cells by taking up and presenting tumor-derived antigen to CTLs. Interestingly, it was shown that CTLs engage with the antigen-presenting cells and subsequently become arrested in this engagement, potentially inhibiting cytolytic effects and tumor rejection. The authors suggest that tumor immunotherapy may help release the CTL blockade following their initial attraction and clustering with antigen-presenting cells at the tumor site31. Similarly, Boissonaset al. showed by intravital imaging that infiltrating T-lymphocytes can become trapped with tumor dendritic cells following chemotherapy, suggesting that tumor dendritic cells can function as a sink that retains the T-cell-driven anti-tumor immune response32. These findings enhance our understanding of the immune response to cancer development both at primary and metastatic sites, thus intravital imaging may help pave the way to the development of new presentation-derived therapeutics. For example, intravital imaging in the MMTV-PyMT mouse model of breast cancer enabled the identification of a type of macrophage-dendritic cell (M-DC)33. Here, the authors used fluorescent dextran to label M-DCs and revealed that the depletion of M-DCs decreases tumor growth and metastasis and could therefore present a promising future target33. Similarly, recent intravital imaging of CTL intratumoral migrationin vivo revealed that treatment with anti-CD137 mAb prolongs the interaction between CTLs and cancer cells, thereby improving the antitumor CTL activity34. This is in parallel with recent improvements in anti-PD1 immunotherapy to educate the immune system to recognize and kill melanoma cells35 and may represent a promising approach for the improvement of immune-based therapeutics in multiple cancer types.On a similar note, using intravital imaging, Moalli36, wherein a bicistronic cassette of Cre and tdTomato was engineered to be expressed under the neutrophil-specific locus Ly6G. Using this tool, the authors tracked neutrophil migration and studied neutrophil-specific gene functionsin vivo. Similarly, a transgenic mouse with ubiquitous expression of Kikume, a green fluorescent protein (GFP) that can be irreversibly photo-converted to red via violet light excitation, was used to characterize dendritic cell37 and neutrophil migration38in vivo (38). For example, upon bacterial infection, neutrophils were recruited to the mouse ear and were photo-converted. Further intravital imaging allowed the authors to track the photo-converted cells and to identify and map new patterns of neutrophil migration and homing into the lymph nodes38. Another example is the MacGreen transgenic reporter mouse in which c-fms-driven GFP expression is targeted to macrophages, trophoblasts, and granulocytes39. Using this reporter, Paiet al. could track circulating leukocytes during cerebral malaria pathogenesis and identified plasmodium-specific CD8+ T lymphocytes as important regulators of this disease40. Crossing these reporter animal models with mouse models of cancer may provide much-needed insights into how cancer cells manipulate and interact with the host immune system to promote tumor progression. For example, intravital imaging may allow us to identify the migration routes of immune cells during cancer progression and treatment or to characterize tumor immune subtypes42 in a dynamic manner to better inform on immunotherapies in cancer, a feature of cancer progression that cannot be modeled or assessed accuratelyin vitro.Several fluorescent reporter systems to specifically label and track immune cells within native tissues have been developed recently. One such example is the Catchup mouse model43. Here, the authors show that the stiffened ECM in turn activated adhesion-mediated signaling pathways, such as focal adhesion kinase (FAK), to promote cancer cell survival and so decrease anti-Braf treatment efficacy43. Interestingly, impairing cellular adhesion tension through FAK inhibition reduced the safe haven provided by the ECM and resulted in a significantly enhanced response to chemotherapy43.Fibroblasts are also frequently recruited to tumor sites, and intravital imaging has recently been used to show that stromal fibroblasts can regulate overall tumor response to chemotherapy. For example, intravital imaging of a xenograft model of melanoma through an optical window demonstrated paradoxical activation of cancer-associated fibroblasts (CAFs) at the primary site upon treatment with Braf inhibitors, thereby leading to increased stiffening of the ECM compartmentet al. usedin vivo whole-body bioluminescence imaging to investigate the mechanisms of resistance to anti-insulin-like growth factor (anti-IGF) treatment with cixutumumab44, which recently showed limited efficacy in clinical trials46. Here, the authors demonstrated that upon treatment with cixutumumab, cancer cells underwent a protective reprogramming characterized by increased production of IGF, which in turn led to the recruitment and activation of host fibroblasts to the primary site through IGF-2R-dependent paracrine signaling. Activated fibroblasts were then shown to secrete pro-angiogenic factors and to potentiate metastasis and disease relapse44. Their findings also correlated with changes in the tumor microenvironment of patients undergoing anti-IGF-based therapies44, suggesting that preventing fibroblast activation can impair extrinsic mechanisms of chemoresistance and in turn improve cixutumumab efficacy.Similarly, Lee47. In a recent study, Lawsonet al. developed an approach for longitudinal intravital imaging of myeloma cells that had lodged within dense bone tissue48. Using a dye-dilution system, the authors were able to label and track slowly proliferating, dormant cells over time and showed that these cells preferentially lodge in direct contact with the endosteal bone surface and interact with host bone cells to establish a protective niche for persistence of metastasis (see48). Importantly, this study demonstrated that cell dormancy is a reversible state, which can be turned “off” and “on” by signals emanating from host osteoblasts and osteoclasts. The authors suggest that using bone-active drugs to either prevent the reactivation of dormant cells or take cells out from dormancy before treating them with chemotherapy could improve outcome and reduce disease relapse. Their findings present new opportunities for the treatment of metastatic cancers with a bone tropism such as breast, prostate, or kidney cancer48.Intravital imaging has also been used to implicate stromal fibroblasts in cancer cell dormancy, an important cause of disease relapse, which remains poorly understoodin situ imaging of secondary organ colonization has been used to reveal how host fibroblasts are implicated in the preparation of the metastatic niche49. In an orthotopic model of breast cancer, dual monitoring of metastatic initiating breast cancer cells and lung fibroblasts recently revealed that reciprocal interactions between the two cell types are required for successful establishment of lung metastases. Here, the authors demonstrate that a biphasic intercellular crosstalk progressively modifies both compartments. Briefly, activation of a fibrotic program in the fibroblast population led to the initiation of a mesenchymal-to-epithelial transition within the cancer cell compartment, promoting the onset of metastasis49. Taken together, these studies suggest that preventing fibroblast activation and reprogramming driven by tumor cells during cancer progression may help improve sensitivity to chemotherapy and impair cancer dissemination. Intravital imaging of secondary sites prone to metastasis is required to directly visualize the stages of metastatic colonization; however, long-term and deep imaging of metastatic organs remains technically challenging. Optical imaging windows now enable longitudinal and deep imaging of events occurring at both primary sites and secondary organs such as the liver, spleen, kidney, and pancreas51. For instance, monitoring the early events promoting the formation of the liver metastatic niche was achieved using optical imaging windows52. Interestingly, this study demonstrated that single extravasated cells are initially highly motile and proliferative and form pre-micrometastases within the liver before merging into micrometastases with reduced migration. Importantly, impairing early tumor cell migration significantly reduced the metastatic burden52. Similarly, the formation of the liver metastatic microenvironment and response to chemotherapy has been studied using longitudinal scanning multiphoton microscopy in an intrasplenic model of colorectal cancer50 and informed on the response of metastatic cells to chemotherapy.Tumor cells have also been shown to co-opt fibroblasts to promote cancer spread, and recent55. Additionally, the probability of imaging an invasive or metastatic event can be considered very low; however, optogenetic intravital imaging can be used to specifically activate or inhibit signaling pathways and subsequently trigger such an event.One limitation to longitudinal intravital imaging is the movement of live tissue, such as heart or lung, which can perturb high-resolution intravital imaging, and current research focuses on motion compensation systems to correct for tissue movement58. For instance, live imaging of a chicken embryo model of fibrosarcoma indicated that exosomes promote cancer cell-ECM adhesion assembly and are required for persistent cell movement59. In addition, elegantin situ studies demonstrated that exosomes secreted from primary cancer cells localize at secondary metastatic sites, fuse with host cells, and can “educate” secondary organs through the activation of fibrotic and inflammatory programs57. In line with this study, direct intravital imaging of exosome secretion and uptake by different cell populations61 has recently been achieved. Here, Zomeret al. developed a Cre-loxP-based system whereby cells that take up Cre(+) EVs exhibit a change in color (see60). With this approach, the authors demonstrated that EVs are locally and systemically transferred from aggressive cancer cells to less malignant cancer cells within the same mouse to coordinate a whole-body systemic metastatic programin vivo and to enhance the overall metastatic ability of the tumor61. Recent findings also demonstrate that mitochondrial DNA (mtDNA) can be transferred from host stromal cells to cancer cells. Here, the authors genetically manipulated melanoma and breast cancer cells to lack mtDNA and showedin situ that they can acquire host mtDNA to restore mitochondrial functions such as bioenergetic dynamics and respiration. In addition, incorporation of host mtDNA by cancer cells led to increased tumor growth and metastatic spread, demonstrating that tumor cells can take up material from the host to drive cancer aggressiveness63. Interestingly, Osswaldet al. usedin vivo multiphoton scanning of astrocytomas over a year to study a novel mechanism of intercellular connection64, wherein tumor cells in astrocytomas use ultra-long protrusions (>500 μm in length) to form a network of multi-cellular communication. The authors show that this network supports tumor cell invasion and proliferation and protects cancer cells against radiotherapies64. Future developments in intravital imaging to directly visualize mechanisms of cell-cell interaction may help us understand how cancer cells and host tissues interact and together drive cancer aggressiveness, and in the future may help us to uncouple this support mechanism.Reciprocal interactions between cancer and stromal cells require an exchange of information, for example, in the form of physical cell-cell interaction or secretion of protein ligands or extracellular vesicles (EVs). Direct visualization of signal transmission between cancer cells and the host environment has long been technically challenging; however, the recent development of new intravital imaging tools has helped elucidate the mechanisms which enable intercellular exchange of information within both primary and metastatic sites. Recent studies showed that cancer cells can manipulate host stromal cells through the local and systemic transfer of exosomes and other EVs72. Multiphoton imaging technologies, such as second harmonic generation (SHG) imaging73, have been used to study the modulation of ECM characteristics during cancer progression and have recently identified new regulators of biomechanical tissue properties . For instance, the actin cytoskeleton and a number of its regulatory proteins, such as the family of Rho GTPases, are known to govern the remodeling of the ECM77, and live SHG imaging has provided new insights into the tight regulation of Rho GTPasesin vivo. In collaboration with Samuel and colleagues, we helped identify the intracellular signaling protein 14-3-3 ζ as a novel negative regulator of Rho-kinase-driven ECM stiffening in wound healing78. Here, live monitoring of stromal fibroblasts coupled with SHG imaging of collagen fibers in a 3Din vitro environment was used to show that loss of 14-3-3 ζ in dermal fibroblasts impairs their ability to remodel collagen and to stiffen the ECM, and such effects were also seen inex vivo animal samples78. Interestingly, this study also showed in squamous cell carcinoma that 14-3-3 ζ is downregulated while collagen deposition is increased, suggesting that ROCK signaling is no longer restrained and controlledin vivo in this disease79. These findings suggest new therapeutic options for re-purposing 14-3-3 ζ-based treatments to facilitate uniformed and controlled wound healing78, which in future can also be used in the context of cancer to re-establish normal tissue homeostasis of solid tumors81.Over the last decade, intensive work has shown that ECM abundance and features, such as stiffness, topography, and porosity, contribute to tumor cell proliferation, invasion, and chemoresistance83. For example, hypoxia was shown to inhibit prolyl hydroxylase domain protein 2 (PHD2) in CAFsin vitro andin vivo, leading to a reversion of CAF activation and a decrease in their ability to remodel and stiffen the ECM82. In addition, proteins secreted from hypoxic tumors were demonstrated to prepare and activate the ECM of future metastatic sites. One such example is the enzyme lysyl oxidase (LOX), which is upregulated in bone tropic breast cancer cells83.In vivo, injection of LOX resulted in the formation of osteolytic lesions and in turn provided circulating breast cancer cells with a focal pre-metastatic niche for bone colonization, whereas LOX targeting with a blocking antibody partially reverted this phenotype and decreased tumor burden, as visualized by whole-bodyin vivo imaging83. Intravital SHG imaging has also been used to directly monitor changes of the ECM structure and content during cancer progression in live settings. For example, Walshet al. performed intravital SHG imaging of breast cancer xenografts to study the effects of trastuzumab treatment on the ECM84. Interestingly, their study demonstrated that trastuzumab induces changes in collagen density and alignment and thereby identified a non-cellular response to drug treatment84. In addition, longitudinal SHG imaging of orthotopic breast xenografts was performed at the tumor margin to identify the key components of the tumor microenvironment that modulates cell invasion85. Here, the authors used dual imaging of single cells and collagen fibers and showed that slow-locomotion breast cancer cells form invadopodia to remodel and stiffen the collagen matrix while they move through the surrounding tissue. Further use of intravital SHG imaging will provide important insights into the role of the ECM and its manipulation during tumor progression.The hypoxic tumor environment was also recently identified as a novel regulator of the ECM ultrastructure and stiffness at both primary and metastatic sites86. In line with this study, biophysical analysis of collagen remodeling was conducted in human breast cancer biopsies (>20 samples) and revealed a progressive increase of collagen reorganization and orientation as invasive cancer lesions develop87. Similarly, in a cohort of matched human breast tissues with areas of high and low mammographic density from the same patient (>15 samples), we analyzed ECM organization by gray-level co-occurrence matrix (GLCM), an image texture-based approach used to quantify the organization of collagen fiber networks88. In this study, Britt and colleagues demonstrated that increased deposition and crosslinking of collagen fibers correlates with a high risk of developing breast cancer88. Taken together, these studies along with our knowledge from intravital animal models indicate that upon acquisition of human biopsies, characterization of ECM abundance and crosslinking using medium-throughput SHG imaging can potentially be used as a biomarker to predict patient prognosis and tumor stage. Moreover, the development of new probes to directly image distinct components of the ECM such as hyaluronan89, elastin90, or fibronectin91 is likely to facilitate our understanding of the role of the ECM in cancer development and may help us improve therapies targeting specific components of the ECM.Rapidly developing intravital technologies and tools have provided unprecedented insights into cancer complexity and have also opened up promising avenues for pre-clinical cancer research. For example, SHG imaging has recently been translated to study the properties of the ECM in biopsy samples of cancer patients. For instance, in the context of pancreatic cancer, we recently used SHG imaging to analyze the properties of the ECM in a human pancreatic tissue microarray (>80 samples) and detected a positive correlation between fibrillar collagen abundance, tumor stage, lymph node spread, and vascular invasion, suggesting that fine-tuned targeting of collagen crosslinking may be a valid approach in this diseasein vitro. Here, the authors showed that upon treatment with MEKi, Erk and PI3K pathways can maintain mTORC1 activity and in turn promote cell growth, thereby leading to resistance to treatment92. Furthermore, fluorescently tagged drugs were used to decipher mechanisms of drug resistance. For instance, a fluorescent analog of eribulin was designed to monitorin vivo drug pharmacokinetics. Here, the authors engineered a portion of tumor cells to express multidrug-resistance 1 (MDR1)-mApple fusion protein, and dual imaging of eribulin and MDR1-mApple demonstrated that resistance to eribulin was directly correlated with vascular architecture and MDR1-mediated drug efflux. This study also demonstrated that inhibition of MDR1 reversed the multidrug-resistant phenotype and improved eribulin efficacy93 (93).Fluorescence-based biosensors are emerging as a reliable tool to dissect mechanisms of drug target activity and chemoresistance. In a large panel of MEK inhibitor (MEKi)-resistant cell lines, live imaging of extracellular signal-regulated kinase (Erk) and S6K fluorescent biosensors was used to interrogate the molecular bases of resistance to MEKi in Kras-mutant and Braf-mutant cancer cell lines99 (96). Crossing these biosensor mice with animal models of cancer allows us to dissect the spatiotemporal mechanisms of the molecular events driving cancer in native tissues. For example, Kumagaiet al. recently crossed their Erk biosensor mouse with a mouse model of mammary tumor formation and described a stable heterogeneous pattern of Erk activity100. Interestingly, the authors showed that cells with low Erk activity were more successful in forming tumor spheresin vitro and expressed high markers of mammary cancer stem cells compared to cells with high Erk activity100. These findings indicate that tumor heterogeneity in Erk activity may be beneficial to establish cancer stem cells with low Erk activity for self-renewal, whereas high Erk activity supports rapid growth and expansion100. Similarly, Erk pulsing and propagation dynamics emanating from the skin and hair follicle have also recently been demonstratedin vivo101 and have significant implications for future research in melanoma102.Taking these approaches further, for the assessment of whole-tissue or whole-body protein activity, transgenic mice have been generated with ubiquitous or Cre-inducible expression of reporters of the activity of Erk, PKA, Rac1, cAMP, or cell cycle progressionin vivo to monitor cell-cell junction dynamics (103). Here, we used fluorescence recovery after photobleach (FRAP) and fluorescence loss in photobleach (FLIP) imaging along with kymograph analysis to quantify E-cadherin mobility of healthy and diseased tissue. Using this approach, we could correlate high E-cadherin mobility in the cell membrane with a decrease in junction strength and integrity and an increase in cell invasiveness103. Crossing this mouse with a genetically engineered model of pancreatic cancer allowed us to recapitulate the full spectrum of pancreatic cancer progression and to dissect the genetic stages of early cancer dissolution. This mouse was also used as a tool for drug discovery by testing new anti-invasive agents, such as dasatinib, in pancreatic cancer, where we demonstrated that dasatinib is able to strengthen cell-cell junction integrity in line with decreased invasiveness in this setting103. This is in line with current clinical assessment in patients and its known effectiveness in the KPC pancreatic cancer mouse model104.Another recent example of biosensor mouse imaging in cancer is our E-cadherin-GFP mouse, which enabled us to perform photobleaching experiments106. This requires the use of fluorescent proteins that can be excited with near-infrared wavelengths, such as iRFP or iRFP variants, which have been recently described108. Furthermore, computational models, such as adaptive optical correction, have been used to overcome light scattering and to increase imaging depth109. Another imaging technique, called photoacoustic tomography (PAT), makes use of the phenomenon that photon absorption forms pressure waves that can be detected. Although PAT increases imaging depth, it strongly reduces image resolution, which can be partially overcome by the use of near-infrared fluorescent proteins111. Furthermore, current proceedings to reduce the distal optics and mechanical components of intravital microscopes114 should move the field towards intravital endoscopy of cancer biology115.Mouse models with ubiquitous expression of molecular reporters can now be used to monitor events in a large range of organs and diseases; however, due to light scattering within tissues, the imaging depth with current two-photon lasers is limited to several-hundred μm, limiting intravital imaging of large organs and tumors. Current multiphoton setups include three-photon lasers in combination with optical parametric oscillators (OPOs) that increase the excitation wavelength, as red and near-infrared light can penetrate deeper into tissue and reduces tissue scattering116, is emerging as another promising tool to interrogate cancer dynamics. Optogenetics allows researchers to manipulate gene expression or the activity of cellular signaling pathways and has recently enabled photocontrol of proteins with subcellular resolution. These approaches have been used to modify protein conformation117, stimulate DNA binding118, control enzymatic119 or receptor tyrosine kinase activity120, induce protein localization121, manipulate protein-protein interactions122, and study signaling pathway events124. For instance, optogenetics was used to demonstrate that localized Rac1 activity is sufficient to produce precisely regulated cell protrusion events to control directed cell motilityin vivo128. Most importantly, optogenetics may enable us to modulate cancer-related signaling pathways in a defined spatiotemporal manner and allow us to follow the fate of de-regulated cells in theirin situ environment. In summary, intravital imaging has demonstrated its capacity to allow us to explore the ancillary mechanisms supporting cancer progression and provide novel routes for the development of future therapeutics to target and impair these support mechanisms.Lastly, optogenetics, which rely on the inherent properties of light to observe and accurately control complex biochemical and signaling eventsCAF, cancer-associated fibroblast; CAM, chorioallantoic membrane; CTL, cytotoxic T-lymphocyte; ECM, extracellular matrix; ERK, extracellular signal-regulated kinase; EV, extracellular vesicle; FAK, focal adhesion kinase; FDC, follicular dendritic cell; FLIP, fluorescence loss in photobleaching; FRAP, fluorescence recovery after photobleaching; GLCM: gray-level co-occurrence matrix; IGF, insulin-like growth factor; LOX, lysyl oxidase; M-DC, macrophage-dendritic cell; MDR1, multi-drug resistance 1; MEKi, MEK inhibitor; mtDNA, mitochondrial DNA; NK cell, natural killer cell; OPO, optical parametric oscillator; PAT, photoacoustic tomography; PHD2, prolyl hydroxylase domain protein 2; QD, quantum dot; SHG, second harmonic generation; TAM, tumor-associated macrophage; TMEM, tumor microenvironment of metastasis; VEGFA, vascular endothelial growth factor A.


Ranunculus auricomus using single-nucleotide polymorphisms confirmed neutral allelic sequence divergence within a short time frame, but rejected a hypothesis of a genome-wide accumulation of mutations in asexuals compared to sexuals, except for a few genes related to reproductive development. We discuss a general model that the observed incidence of facultative sexuality in plants may unmask deleterious mutations with partial dominance and expose them efficiently to purging selection. A little bit of sex may help to avoid genomic decay and extinction.Genome evolution in asexual organisms is theoretically expected to be shaped by various factors: first, hybrid origin, and polyploidy confer a genomic constitution of highly heterozygous genotypes with multiple copies of genes; second, asexuality confers a lack of recombination and variation in populations, which reduces the efficiency of selection against deleterious mutations; hence, the accumulation of mutations and a gradual increase in mutational load (Muller’s ratchet) would lead to rapid extinction of asexual lineages; third, allelic sequence divergence is expected to result in rapid divergence of lineages (Meselson effect). Recent transcriptome studies on the asexual polyploid complex  Ranunculus.Currently, the understanding of evolution patterns of genomes on different phylogenetic groups is a hot topic in evolutionary biology. The arrival of next generation sequencing (NGS) technologies and the generation of huge amounts of genomic data is allowing researchers to dig in the past and better resolve organisms’ natural history as well as evolutionary enigmas. One of such enigmas is the predominance of sex in nature . One of Sexuality is a crucial factor molding the genomic features of eukaryotes. In plants, the formation of a new individual through sexuality involves an alternation between the sporophytic (2n) and the gametophytic (n) generations via meiosis and gamete fusion, the two mechanisms that create new genetic combinations. Additionally, outcrossing further potentiates genetic variation in populations. Thus, with few exceptions, every single sexual organism has a distinctive genotype that differentiates it from parents and siblings. Therefore, meiosis is the main source of genetic recombination and mixis, and segregates genetic factors in the offspring creating genetic variation. By doing so, meiosis and sexuality allows natural selection purging a lineage from harmful mutations. Because plant meiosis produces spores (mega- and microspores) and these spores develop into female and male gametophytes, in which considerable percentages of genes are being expressed , selectiIn contrast, by circumventing or suppressing meiosis and syngamy, asexual organisms skip the alternation of generation cycle and hence elude the ploidy-phase change step. In angiosperms, asexually-derived individuals can be formed either as consequence of vegetative propagation, or of asexual seed formation (apomixis), a trait that is taxonomically widespread in plants . While tA central fact for genome evolution, however, is that apomixis in angiosperms is rarely obligate. Apomictic plants produce asexual and sexual progeny within the same offspring generation, i.e., from different ovules and seeds in the same mother plant, and therefore asexuality is facultative. Consequently, a proportion of the offspring represents recombinants, but frequencies of sexuality vary a lot among genera, species and different modes of apomixis e.g., , 2014a. Sexuality has the effect that deleterious mutations appear in various genotypic configurations in the offspring. Thus, harmful mutations expressed under different states  will negatively affect the genotype’s fitness and natural selection will remove such genotypes and purge the lineage from an increase in the mutational load. Evolutionary benefits of purging a lineage from mutation accumulation have long been seen as a major advantage of sexuality . ApomictApomixis in plants is nearly exclusive associated polyploids, and often it is a result of hybridity e.g., . PolyploAllelic sequence divergence, the so-called Meselson effect, is another consequence of long-term asexuality . AsexualRanunculus auricomus complex, a system of diploid sexual species and hexaploid apomictic hybrids, in fact showed Meselson-like sequence divergence effects, but data did not support the idea of mutation accumulation  were found in the apomictic-sexual comparison and thus indicated a significant enrichment of genes associated to reproductive shifts during ovule development when compared to those outliers in sexual (or apomictic) genomes. Whether these mutations have positive or negative effects, needs further investigations. However, most mutations that are under selection do have strongly deleterious effects  to have accumulated significant mutations and hence dN/dS ratios are still similar to those in sexual putative parentals.A recent transcriptome study of the mulation . Analysemulation . A numbemulation , and hents e.g., . Since wts e.g., —they wouRanunculus hybrids show varying proportions of sexually formed seed in all genotypes, with a grand mean of 29.1%  deleterious mutations lead to a larger decrease of fitness because of negative interactions of these genes, then even truncating selection can act and rapidly eliminate this genotype . Hence, turnover . Consequturnover  and/or hturnover . Hence, turnover ,b suggesRanunculus auricomus, quantitative expression of apomixis is dosage-dependent on the apospory factor (A), which is a dominant Mendelian factor with variable penetrance in the sporophyte, but with lethal effects in haploid or homozygous states (The evolutionary consequences of facultative apomixis in plants have so far received little attention. Mendelian genetic studies on control mechanism of apomixis in angiosperms suggest that the apomixis-controlling genomic regions occur -in general- in a heterozygous state . In Ranus states . Consequs states . The lons states  suggest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


There is evidence to suggest that microvascular disease, particularly diabetic retinopathy, plays a role in the pathogenesis of HF. However, whether changes in retinal vessel calibre predicts HF is unclear. The purpose of this study was to examine the association of retinal microvascular structure with prevalent heart failure (HF).The Australian Heart Eye Study (AHES) is a cross-sectional study that surveyed 1680 participants who presented to a tertiary referral hospital for the evaluation of potential coronary artery disease by coronary angiography. Retinal vessel calibre was graded using retinal photography and participants’ self-reported echocardiography-confirmed HF was obtained via an extensive medical questionnaire.There were 107 participants (8.1%) with prevalent self-reported HF. Persons with wider retinal arteriolar calibre (comparing highest versus lowest tertile or reference) were more likely to have prevalent HF  when adjusted for age and sex. After further adjustment for body mass index, hypertension, diabetes, smoking status, triglycerides and estimated glomerular filtration rate, this association remained significant . After further stratification, this association remained significant among participants with diabetes  but not in those without diabetes . The strength of this association was not dependent on the length of history of diabetes, or retinopathy status. There was no significant association between retinal venular calibre and prevalence of HF.Wider retinal arteriolar diameter was significantly and independently associated with prevalent HF in participants of a cross-sectional study. This association was significant stronger among participants with diabetes compared to without diabetes. No association was found between retinal venule calibre with prevalent HF. Chronic heart failure (HF) is one of the major causes of mortality and morbidity in the Western world, occurring in more than 5 million individuals and responsible for more than 1 million hospital studies. and hypeSeveral studies have supported the integral role of microvascular disease in the pathogenesis of HF in the general population. The Atherosclerosis Risk In Communities (ARIC) study demonstrated an independent association between retinopathy and congestive HF, even aftPotential links between retinal vessel calibre and HF would have important clinical implications, as individuals with early changes in the retinal microvasculature could be screened or monitored for development of HF. Retinal vessel calibre changes can be measured directly and appeals as a non-invasive test and potential marker for underlying cardiac disease. We therefore aimed to assess the independent association between retinal arteriolar and venule calibre with prevalent HF, in a cross-sectional study–Australian Heart Eye Study (AHES).Ethics approval was obtained from Western Sydney Local Health Network Human Research Ethics Committee. Written informed consent was obtained from all participating patients via a patient information sheet explaining the AHES objectives, the role of the research participants, risks, costs, potential benefits and other relevant information, as well as the signed consent form. The Western Sydney Local Health Network Human Research Ethics Committee approved the informed consent procedure.The AHES is a cross-sectional study of 1680 participants who presented to Westmead Hospital , between June 2009 and January 2012 to evaluate potential CAD by coronary angiography. All eligible patients presenting for assessment of suspected CAD were included in this study. All patients approached for the present study gave consent for invasive coronary angiography. Relative contraindications for invasive coronary angiography included pre-existing renal impairment and contrast allergy. However, these were not absolute and coronary angiography was performed if the risk benefit ratio was favourable. Most patients were recruited following their angiogram. As these patients are hospitalised, and investigations are performed prior to angiography on a routine basis, information collected by medical personnel was also included in the study.Examinations and measurements included a detailed medical history questionnaire, visual acuity testing, biochemical, angiographic, peripheral arteriolar wave form analysis, pulse wave analysis, ankle-brachial pressure index, peripheral blood pressure measurements, invasively measured blood pressure measurements, transthoracic echocardiography, electrocardiography, blood count analysis, genetic testing and retinal photography data. If there was incomplete information of retinal vessel calibre, and patient status of diabetic retinopathy or age-related macular degeneration (AMD), these patients were excluded. Ethics approval was obtained from Western Sydney Local Health Network Human Research Ethics Committee.A 252-item questionnaire was designed and used to obtain patient medical history, cardiovascular and familial risk factors. The questionnaire forms were collected and entered by two study personnel with single entry. The forms included current cardiac rhythm status, previous/recent history of angina, previous myocardial infarction, previous angiography, previous intervention , previous stroke, transient ischemic attack (TIA), hypertension, hypercholesterolemia, diabetes mellitus, and current management of these chronic conditions, current medications, smoking status and alcohol consumption.A comprehensive questionnaire was used by study personnel to obtain a detailed medical history, including year of heart failure diagnosis, classification of heart failure (NYHA Class I-IV), and treatment for heart failure including fluid restriction (liters per day), biventricular pacemakers, and medications. HF prevalence was defined as a positive response to both questions "When was it diagnosed?" and “Was diagnosis confirmed with transthoracic echocardiography or transoesophageal echocardiography”. Other information collected included past cardiovascular events, cardiovascular risk factors, other medical conditions, drug and alcohol history, and history of past angiography and/or interventions (coronary artery stent or coronary artery bypass graft).Retinal photography was used to assess retinal vessel calibre. Participants had dilated, digital photographs taken of the optic disc and macula of both eyes using a pre-calibrated Canon 60° fundus camera  with an attached digital camera . Retinal vessel calibre measurements for the right eye of each participant were used. Left eye measurements were used if right-eye photographs were un-gradable. One grader, masked to participant identity and characteristics, measured retinal vessel caliber using a computer-assisted program  with high reproducibility, this has been previously described, 10. TheTM OMRON digital automatic blood pressure monitor in the supine position . Invasive blood pressure measurements were performed using a fluid filled catheter in the central aorta and measured with a Mac-Lab hemodynamic system . Biochemical data, including renal function (creatinine), eGFR, full blood counts, cardiac enzyme , fasting blood sugar, HbA1c level, fasting cholesterol levels, and thyroid function tests were collected from the participant’s medical record. Diabetes status was determined as per self-reported diabetes history and current use of diabetic medications. Patients with unknown diabetes status were excluded from analysis.Diastolic and systolic blood pressures and heart rate were measured, with a single reading, from the right arm with an IntellisenseAll analyses were performed using SAS statistical software . Statistical significance was defined as P-value < 0.05.Multivariable analyses using logistic regression models adjusted for cardiovascular risk factors including age, sex, BMI, hypertension, diabetes mellitus, smoker, eGFR, were used to estimate odds ratios (OR) and 95% confidence intervals (CI), in order to determine whether associations exist between retinal vessel caliber  and prevalent HF. Adjustment factors were choosen based on confounders which significantly modified the effect of retinal vessel caliber in age-sex adjusted models in relation to prevalent HF, or significantly predicted the outcome variable of prevalent HF. In analysis of the potential association between retinal vessel caliber and prevalent HF stratified by diabetes status, the same cardiovascular risk factor covariates (except for diabetes) was adjusted for in multivariable analyses.If a significant association was found between retinal microvasculature and prevalent HF, subgroup analysis was performed to see if the association held in (1) diabetic versus non-diabetic patients, and (2) when patients with cardiovascular disease  and CKD (eGFR < 60) were excluded from analysis.Participants with prevalent HF were more likely to be older, hypertensive, have higher BMI, diabetes and lower eGFR . PatientAfter initial adjustments for age and sex , particiStratified analyses showed that the association between wider retinal arteriolar diameter (highest versus lowest tertile) and a greater likelihood of prevalent HF was more marked among those with diabetes  than in those without diabetes (p = 0.0006) . The strIn the present cross-sectional study AHES, we report an association between wider arteriolar retinal diameter and prevalent heart failure among participants with diabetes. Patients with wider arteriolar retinal calibre in the highest tertile were 4.5 times more likely to have HF compared to participants with narrower retinal arteriolar diameters in the lowest tertile, even after controlling for conventional cardiovascular risk factors. The association was stronger in those with diabetes, who had over 10-fold higher odds of having HF. No significant association was found between retinal venular calibre and prevalent HF in the AHES cohort.Retinal microvascular signs have inconsistently been shown in the literature to be associated with systemic cardiovascular disease –16. ChanFew studies have explored the role of retinal microvascular disease in other cardiovascular diseases such as congestive HR. One of the few studies to explore this relationship is the Atherosclerosis Risk in Communities (ARIC) study, a prospective study of four US communities. The 7-year cumulative incidence of HF was 5.4%, and retinopathy was found to be independently associated with a 3-fold increased risk of HF. While otThe mechanisms underlying the relationship between retinal microvascular structural changes and prevalent HF are not well established. However, the biological rationale for an association between retinal arteriolar widening and HF is plausible. It is reasonable to expect that there is a continuum of microvascular structural adaptations which represent overall microvascular damage or inflammation in an individual, progressing form early retinal arteriolar widening, to retinopathy and finally HF. Even as early as 1939, it was recognised that the relationship between retinal vessel changes and cardiovascular risk and mortality was dose-dependent. The “conTo test the continuum hypothesis, we stratified our analysis according to length of diabetes history and retinopathy status, with stronger associations expected in participants with longer history of diabetes and with diabetic retinopathy. No significant association was demonstrated, which may be due to the small number of patients with prevalent heart failure and retinopathy in our cohort. Consequently, the statistical power may have been inadequate to detect any actual differences between these subgroups.Current guidelines indicate routine screening for retinopathy in patients with diabetes. Multiple studies have also emphasised the importance of using retinal visualization for risk stratification in coronary artery disease, to enable more effective primary prevention strategies. However, few international guidelines have emphasised the role of screening for early retinal vessel calibre changes in this particular population subset. Our results suggest that patients with diabetes with evidence of significant narrowing of retinal arteriolar calibre could also benefit from cardiovascular examinations during follow-up. This could potentially help identify subclinical HF, 29, thuStrengths of the present study include the use of a relatively large population-based sample with high participation rates, as well as objective measurements of visual acuity and retinal calibre assessment using fundus photography. Retinal grading systems used were standardized and comparable to the grading systems employed in other prominent population-based studies in this field including the Beaver Dam Eye Study and the AIC. Other strengths including adjustment for major confounders and traditional cardiovascular risk factors, as well as stratification into diabetes versus no diabetes subgroups.The present study results are also constrained by several limitations. First, the echocardiography confirmed HF was obtained by self-report from participants, and the possibility of selection bias or underestimation of prevalent HF cannot be discounted. The AHES population is based on participants who underwent angiography for potential coronary artery disease at a tertiary hospital center. As such, the population was not randomly selected, and consisted of a higher proportion of hyperlipidaemic, diabetic and hypertensive male population. As such, caution should be taken when interpreting the present results, especially when considering other population subsets such as younger, healthy females. Hypertensive status wa also defined based on medical questionnaire responses or a single blood pressure test, which has limited validity and may undermine confidence in the results. A small number of patients were found to have HF, which resulted in larger confidence intervals when the relationship was stratified according to diabetes status. Furthermore, given that all patients were selected for coronary angiography, patients with positive coronary disease would theoretically have smaller arteriolar calibre. The cut-off values for retinal vessel calibre tertiles in this study were slightly lower compared to prior studies in non-angiography populations, thus poGiven the cross-sectional design of the AHES, no incident HF data was available for analysis. Thus, a definitive judgement regarding the risk of developing HF and its relationship with retinal arteriolar calibre cannot be concluded. Another unaccounted limitation is that HF patients may be on high doses of vasodilator medications, which may have affected the presented trends. Future prospective population-based studies should be designed to assess whether this association holds after long-term follow-up. Current trends observed in this study should be confirmed by future larger prospective, adequately powered population studies. Although multivariable adjusted models were used to account for traditional cardiovascular risk factors, the effects of the differential use of anti-hypertensive agents and hypoglycaemic agents may confound the presented results.In summary, we report a significant association between wider retinal arteriolar vessels and prevalent HF in individuals who presented to angiography for testing for potential coronary artery disease, independent of traditional cardiovascular risk factors. This association was stronger in participants with diabetes compared to without diabetes. These findings add further support to the potential role of the retinal microvasculature and disease in aetiology of HF in diabetes. Patients with diabetes with adverse retinal microvascular signs, particularly retinal arteriolar calibre dilation, may warrant more careful cardiovascular evaluation and follow-up.


Critically ill survivors may have functional impairments even five years after hospital discharge. To date there are four systematic reviews suggesting a beneficial impact for mobilisation in mechanically ventilated and intensive care unit (ICU) patients, however there is limited information about the influence of timing, frequency and duration of sessions. Earlier mobilisation during ICU stay may lead to greater benefits. This study aims to determine the effect of early rehabilitation for functional status in ICU/high-dependency unit (HDU) patients.Systematic review and meta-analysis. MEDLINE, EMBASE, CINALH, PEDro, Cochrane Library, AMED, ISI web of science, Scielo, LILACS and several clinical trial registries were searched for randomised and non-randomised clinical trials of rehabilitation compared to usual care in adult patients admitted to an ICU/HDU. Results were screened by two independent reviewers. Primary outcome was functional status. Secondary outcomes were walking ability, muscle strength, quality of life, and healthcare utilisation. Data extraction and methodological quality assessment using the PEDro scale was performed by primary reviewer and checked by two other reviewers. The authors of relevant studies were contacted to obtain missing data.5733 records were screened. Seven articles were included in the narrative synthesis and six in the meta-analysis. Early rehabilitation had no significant effect on functional status, muscle strength, quality of life, or healthcare utilisation. However, early rehabilitation led to significantly more patients walking without assistance at hospital discharge . There was a non-significant effect favouring intervention for walking distance and incidence of ICU-acquired weakness.Early rehabilitation during ICU stay was not associated with improvements in functional status, muscle strength, quality of life or healthcare utilisation outcomes, although it seems to improve walking ability compared to usual care. Results from ongoing studies may provide more data on the potential benefits of early rehabilitation in critically ill patients. Intensive care units (ICUs) and multi-disciplinary team management have evolved improving the survival of critically ill patients –3, but tICU-acquired weakness (ICUAW) is one detrimental effect of critical illness on physical function. This term refers to a wide variety of disorders characterised by acute onset of neuromuscular impairment for which there is no other plausible cause than the critical illness, typically associated with multiorgan failure . One criIn the ICU context, the term ‘mobilisation’ is used to refer to physical activity of sufficient intensity to produce physiological benefits, namely enhanced circulation, central and peripheral perfusion, ventilation, muscle metabolism and alertness. Mobilisation can also prevent deep vein thrombosis and venous stasis ,3. CommoSome data suggest that exercise/physical therapy started early may improve physical function in ICU survivors  and prevA systematic review of the literature was performed to determine the effect of early rehabilitation/ mobilisation on the functional status in patients admitted to the ICU or HDU. The protocol for this systematic review was registered at PROSPERO (CRD42013004535). This manuscript adheres to the PRISMA statement recommendations see .st, 2014. Grey literature was identified using OpenSIGLE.com. The search strategy included free text words  and controlled vocabulary adapted for every database , Clinicaltrials.gov, WHO International Clinical Trials Registry Platform (ICTRP), IFPMA Clinical Trials Portal, Current Controlled Trials, Scielo and LILACS from inception to April 1base see . Also, tEligible studies were randomised or controlled clinical trials comparing rehabilitation to usual care in ICU/HDU patients. Adult patients had to be admitted to ICU/HDU for at least 48 hours and be followed for outcomes until ICU discharge.We excluded studies that: compared passive therapies  to usual care; started rehabilitation after ICU/HDU discharge; evaluated interventions in the same patient ; enrolled more than 20% of patients under 18 years; or had patients admitted to an ICU/HDU due to neurological conditions , or trauma that could limit rehabilitation .Usual care: encompassed passive range of motion, position change every two hours while the patient was unconscious and once they regained consciousness, it aimed to increase the level of activity, first in an active-assisted way with exercise in bed, progressing to sitting on the edge of bed, then transition from sitting to standing to finally reach ambulation (assisted or independent) ) for change from baseline was not available (or median [interquartile range]) provided, authors were contacted in order to obtain these data. If the data were not obtained, they were imputed using the method described in the Cochrane Handbook  which reogeneity .A number of predefined subgroup analyses were planned: comparisons between patients with and without ICUAW, early (≤ 7 days) versus delayed (>7 days) start of rehabilitation since ICU admission, and low (≤150 min/week) versus high (>150 min/week) volume of rehabilitation. However, we were unable to perform pooled analyses due to limited data. As a result, the information on these subgroups is presented in the narrative synthesis.In order to explore if the methodological quality was a cause of observed heterogeneity, a sensitivity analysis was carried out. High methodological quality was defined as fulfilling four quality criteria: randomisation assignment, concealed allocation, blinding of outcome assessor and intention-to-treat analysis. An article was considered of low methodological quality if it did not fulfil one or more of the high quality criteria. PEDro scale scores were calculated, but not used as measurement of methodological quality, because higher scores do not always imply better quality and the four criteria before mentioned have been related to overestimation of intervention effect size .The search in all databases included in this systematic review yielded 5733 articles. One hundred and ninety two abstracts were screened and 65 full texts were read. Thirty-nine articles and 26 clinical trial registry entries were reviewed in full . CharactDetails about methodological quality assessment using the PEDro scale can be found in In total, 774 individuals participated in the clinical trials included in this review . Of thosA detailed description of the design, eligibility criteria, intervention and control groups for each trial included is reported in Two studies could be regarded as delayed (≥7 days from ICU admission) start ,31 and tIt was possible to calculate the volume (mean or median duration*frequency) of rehabilitation and/or usual care for six trials –33,35,36Three studies measured functional status ,35,36. OFive studies reported walking ability –32,35,36Although two studies showed no difference in the timed-up-and-go test at hospital discharge and three months follow-up ,36, DeneFour studies –32,34 meTwo studies reported outcomes measuring peripheral muscle strength ,32 Tabl. AlthougThree trials reported ICUAW as an outcome measure ,33,35 T. There wTwo studies reported quality of life as measured by the SF-36 Physical Functioning Subscale ,35 Tabl. Only onTwo trials reported the effect of early rehabilitation on the duration of mechanical ventilation ,33. SchwSix trials reported information regarding length of stay in ICU –34,36 T. None foNone of the included studies reported data on this outcome.This systematic review and meta-analysis included seven studies (774 patients). Due to substantial heterogeneity of patients, interventions, and outcome timing/measures among studies of early rehabilitation in the ICU/HDU that were included, it was not possible to pool results for the primary outcome  in this review. The included studies reported conflicting results on the effect of early rehabilitation on functional status at ICU discharge. However, early rehabilitation was associated with an increased probability of walking without assistance at hospital discharge. In addition, early rehabilitation was associated with improved distance walked at hospital discharge and a reduced risk of ICU-acquired weakness. Finally, no significant effects were found on the secondary outcomes of muscle strength, quality of life, duration of mechanical ventilation, and length of stay in the ICU or hospital.All the included studies showed an improvement in functional status from baseline to next evaluation, but overall, there was no positive effect. Only Schweickert et al (2009) reported significant differences in the Barthel index compared to usual care, but patients in the usual care group of this study spent a longer time with limited mobility compared to other studies included in this analysis ,36. ThisAccording to our results, there was a potential improvement in distance walked at hospital discharge, but not at ICU discharge. This potential benefit was mainly driven by the results of Nava  and BurtPoor peripheral muscle strength has been associated with decreased walking distance in the 6-minute walking test , longer Health related quality of life is a broad concept referring to how individuals rate their own existence and has been deemed as an important outcome for ICU survivors . In thisDuration of mechanical ventilation, length of stay in ICU and in hospital are health care utilisation outcomes. Previous systematic reviews have found positive effects of early mobilisation in the ICU using these measures ,17,44, hIt must be noted that the effect sizes reported are affected by the individual studies’ biases. Lack of appropriate concealment of randomisation schedule and blinding may lead to overestimation of results . BlindinDue to the strict inclusion and exclusion criteria defined for this systematic review, only one study examining the effect of electrical muscle stimulation during ICU stay was included, which had modest results. Nonetheless, two recent systematic reviews have been published, which suggest neuromuscular electrical stimulation could decrease muscle wasting in critically ill patients with lower acuity ,47. StudThe results reported in this systematic review highlight the necessity of defining the most appropriate outcome measurements for critically ill patients in order to capture and monitor their health care needs through early rehabilitation. Hospital discharge and ambulatory follow-up visits at three, six and 12 months are recommended time points to measure the effect of the intervention in order to determine whether improvements are due to phenomena such as regression to the mean and natural recovery of the impairment, or due to early rehabilitation during ICU stay.We only included studies in which usual care was defined, in line with European Society of Intensive Care task force recommendations . HoweverOne possible reason for variation of usual care used in different ICUs is the barriers and facilitators therapists find to carry out early rehabilitation. Leditschke et al  performeBased on the data collected, a structured exercise program might be enough to improve outcomes in critically ill patients. The addition of daily interruption of sedation seems to increase the effect of early mobilisation. It is advisable to generate a consistent intervention program that includes safety criteria to start and interrupt sessions. When a cycloergometer or electrical muscle stimulation is available, they could be included as long as they do not put the patient at risk of an adverse event.The studies included in this systematic review covered a wide variety of conditions and the descriptions of interventions were scant. In addition, definitions of usual care and early rehabilitation varied across the different studies, which implied more demanding sessions in some cases .Considering the number and variety of ongoing studies, an update of this review in at least one year is advisable. In particular, those clinical trials testing electrical muscle stimulation (NCT0070909124 and ISRCTN35179428), the addition of cycloergometry to routine physiotherapy (DRKS00004347) and rehabilitation in populations with specific diagnosis such as sepsis (ACTRN 12610000808044) and COPD (NCT00628992) should provide useful results. Also, an individual patient data meta-analysis would yield valuable information in relation to characteristics of patients with ICUAW and their influence on functional status.Early rehabilitation during ICU stay has no effect on functional status, although it improves patients’ walking ability measured by proportion of individuals walking without assistance at hospital discharge. There is a tendency towards improved outcomes assessed by incidence of ICUAW and 6-minute walking test in those patients who received early mobilisation/ rehabilitation. Due to lack of information, it was not possible to accomplish our primary aim: to determine the impact of time from ICU admission to first mobilisation session and the dose of physical therapy on functional status.Establishment of a standardised rehabilitation program during ICU stay would provide more benefit in those settings where currently there is no early intervention aiming to improve functional recovery. The available options for implementation are: physical therapy/occupational therapy and interruption of sedation, cycloergometer, physical and cognitive therapy, electrical muscle stimulation and programs of progressive exercise.Consensus is necessary about which outcomes to assess, which instruments to use and which time points are relevant in order to improve the evidence in favour of early rehabilitation which would impact the production of evidence-based recommendations in future clinical practice guidelines.Results from ongoing studies in this topic would give useful information to determine the real effect of different bundles of interventions.S1 PRISMA Checklist(DOCX)Click here for additional data file.S1 Supporting Information(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


A growing body of evidence implies psychological disturbances in amyotrophic lateral sclerosis (ALS). Specifically, executive dysfunctions occur in up to 50% of ALS patients. The recently shown presence of cytoplasmic aggregates (TDP-43) in ALS patients and in patients with behavioral variants of frontotemporal dementia suggests that these two disease entities form the extremes of a spectrum. The present study aimed at investigating behavioral and electrophysiological indices of conflict processing in patients with ALS. A non-verbal variant of the flanker task demanded two-choice responses to target stimuli that were surrounded by flanker stimuli which either primed the correct response or the alternative response (the latter case representing the conflict situation). Behavioral performance, event-related potentials (ERP), and lateralized readiness potentials (LRP) were analyzed in 21 ALS patients and 20 controls. In addition, relations between these measures and executive dysfunctions were examined. ALS patients performed the flanker task normally, indicating preserved conflict processing. In similar vein, ERP and LRP indices of conflict processing did not differ between groups. However, ALS patients showed enhanced posterior negative ERP waveform deflections, possibly indicating increased modulation of visual processing by frontoparietal networks in ALS. We also found that the presence of executive dysfunctions was associated with more error-prone behavior and enhanced LRP amplitudes in ALS patients, pointing to a prefrontal pathogenesis of executive dysfunctions and to a potential link between prefrontal and motor cortical functional dysregulation in ALS, respectively. Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by combined degeneration of upper and lower motor neurons. The resulting progressive paralysis involving loss of bulbar and limb muscle function eventually causes death due to respiratory failure within an average of 3 years after symptom onset  is usually understood as an indicator of conflict processing. In addition, it has been observed that RTs and ERs are not only influenced by the congruency of target and flanker stimuli on any trial, but they are also contextually modulated by the congruency sequence, i.e., by the interaction between the congruency on the current trial and the congruency on the previous trial. Specifically, RT and ER benefits are typically found when a congruent trial was preceded by another congruent trial  rather than by an incongruent trial (iC), and when an incongruent trial was preceded by another incongruent trial (iI) rather than by a congruent trial (cI). In the flanker task, this congruency sequence effect  separately for response repetition and response alternation trials. Inspection of Figure cC and iI trials enable repetition priming (via exact stimulus repetitions) in response repetition, but not in response alternation trials. The specificity of the CSE for response repetition trials suggests that priming plays an important role in contextual modulation of conflict processing as assessed by the flanker task  at posterior electrode sites. The SN is a negative ERP waveform deflection that has its onset at around 160 ms post-stimulus and persists for approximately 200 ms and is thought to indicate prioritized processing of attended stimulus features  than on incongruent trials preceded by incongruent trials  obtained on the flanker task to examine conflict processing (CE) and its contextual modulation (CSE) in ALS patients. Several hypotheses about potentially distinct cognitive impairments are conceivable. Some of them are not mutually exclusive; nevertheless, they allow to study the dynamic interplay between selective attention, conflict processing and motor preparation. First, ALS patients may suffer from disturbed attentional modulation of visual processing . Additionally, daytime sleepiness was assessed using the Epworth Sleepiness Scale . Twenty-one age-, gender-, and education-matched healthy  controls (HC) were examined . The HC group did not differ from ALS patients with regard to age, t(40) = −0.43, p = 0.672, or education years, t(40) = 0.53, p = 0.600. Control participants were either spouses or friends of the participants, or volunteers who were compensated for their participation with payment (30 €). One control participant had to be excluded from the analyses regarding the flanker task due to poor task comprehension. All participants were right-handed and had normal or corrected-to-normal vision and intact hearing. The study was reviewed and approved by the local ethics committee . All participants gave written informed consent in accordance with the Declaration of Helsinki.A cohort of 21 patients with ALS .With regard to posterior negativity amplitudes  = 3.54, p = 0.039, Post-hoc tests revealed that LRP-r amplitudes were enhanced in ALSef− (−4.37 μV) in comparison to ALSef+ patients (−2.61 μV), p = 0.040, while the remaining group comparisons revealed statistically non-significant results .With regard to LRP-r amplitudes  in ALS patients, indicating that better performance on the executive part of the ECAS was associated with more accurate performance. Further, LRP amplitudes  correlated with the M-WCST EFC in ALS patients, but not in HC . It is worthy of note that these two correlation coefficients differed between ALS patients and HC .Since the sample size of the ALS subgroup comparisons was rather small, correlation analyses were performed in order to substantiate the reliability of the effects. Table We examined 21 ALS patients and 20 age-, gender-, and education-matched control participants with a battery of neuropsychological tests and a non-verbal version of the flanker task. ALS patients showed normal performance in their response times (RT), suggesting that conflict processing and its contextual modulation are unaffected by the disease. This conclusion that can be drawn from the behavioral data was further corroborated by the ERP and LRP data in that no evidence for altered neural indices of conflict processing and its contextual modulation could be discerned. More specifically, neither N2 amplitudes nor s-LRP onset latencies were altered in ALS patients compared to HC. However, we made three more subtle, nonetheless potentially important, observations. First, ALS patients showed enhanced posterior negativity amplitudes at occipital electrodes, and this ERP enhancement was somewhat more pronounced in those patients who showed clinical evidence for some degree of executive dysfunctions (EDF) as assessed by the M-WCST EFC. Second, subgroup analyses and correlation analyses converged in the finding that the same subgroup of ALS patients showed enhanced LRP amplitudes in comparison to ALS patients without clinical signs of EDF. The LRP amplitude data suggest that the presence of EDF in ALS might be associated with functional alterations in motor regions of the cerebral cortex. Third, the presence of EDF in ALS as assessed by the ECAS EFS was associated with more error-prone behavior (ER) on the flanker task, putatively mediated through functional alterations in prefrontal regions of the cerebral cortex  for ALS-related alterations with regard to the well-documented (see Section Introduction) contextual modulation of conflict processing. Of course, null findings should be treated with caution; however, they stand in contrast to earlier findings from our group that we obtained from PD patients  are related to enhanced LRP amplitudes. The specificity of the relationship between EDF and enhanced LRP amplitudes suggests that the LRP amplitude enhancement in ALS patients with EDF might occur as a corollary of the dysregulation in prefrontal control networks that in turn may be related to additional mechanisms recruited for maintaining motor performance.The ECAS (Abrahams et al., Replication in independent, larger samples of ALS patients is warranted, in particular to ensure the validity of the subgroup comparisons. Future studies should also consider the multidimensional nature of executive functions (Miyake et al., This article demonstrates the utility of ERP and LRP measures that provide the means for decomposing psychological disturbances associated with ALS into constituent parts Figure . Here, wAccumulating evidence about the heterogeneous nature of ALS raised the awareness about its presumably multifaceted etiology and phenomenology. Recently, this has led to the hypothesis of ALS being a general term for a variety of related yet distinct disorders, rather than a clearly defined disease (Goldstein and Abrahams, Conceived the study: RD, SP, BK. Designed the experiment: CS, SF, SP, BK. Performed the experiment: CS, SF, MV, SA. Analyzed the data: CS, SF, MV, FL, SA, BK. Wrote the manuscript: CS, SF, MV, FL, RD, SP, BK. All authors provided critical revisions to the manuscript and approved the final version of the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Above all, the regenerative capacity of zebrafish presents a particularly exciting opportunity to discover new therapies for cardiac injury, including scar formation following myocardial infarction. This Review will summarize the current state of the field and describe future directions to advance our understanding of human cardiovascular disease.Over the past decade, the zebrafish has become an increasingly popular animal model for the study of human cardiovascular disease. Because zebrafish embryos are transparent and their genetic manipulation is straightforward, the zebrafish has been used to recapitulate a number of cardiovascular disease processes ranging from congenital heart defects to arrhythmia to cardiomyopathy. The use of fluorescent reporters has been essential to identify two discrete phases of cardiomyocyte differentiation necessary for normal cardiac development in the zebrafish. These phases are analogous to the differentiation of the two progenitor heart cell populations in mammals, termed the first and second heart fields. The small size of zebrafish embryos has enabled high-throughput chemical screening to identify small-molecule suppressors of fundamental pathways in vasculogenesis, such as the BMP axis, as well as of common vascular defects, such as aortic coarctation. The optical clarity of zebrafish has facilitated studies of valvulogenesis as well as detailed electrophysiological mapping to characterize the early cardiac conduction system. One unique aspect of zebrafish larvae is their ability to oxygenate through diffusion alone, permitting the study of mutations that cause severe cardiomyopathy phenotypes such as  Moreover, sophisticated videomicroscopic techniques have been developed for quantification of ventricular function and for heart rhythm analysis. Zebrafish are particularly well-suited to genetic manipulation to simulate human cardiac disease, and of the disease-related genes listed in the Online Mendelian Inheritance in Man (OMIM) database, 82% have at least one zebrafish ortholog  promoter (islet1 (isl1) and fgf8, genes implicated in mammalian heart development, demonstrated that Isl1 is required for cardiomyocyte differentiation at the venous pole, whereas FGF8 signaling is necessary for cardiomyocyte addition at the arterial pole. Furthermore, this second phase of differentiation is dependent on the expression of mef2cb, analogous to the second heart field marker mef2c seen in the mouse  block  block . Effortsin vivo, offering a complementary approach to in vitro endothelial cell sprouting experiments. For instance, a transgenic fli1:eGFP zebrafish model, in which the entire vasculature is green, has been used to investigate the role of extracellular bone morphogenetic protein (BMP) modulators in angiogenesis , and was characterized primarily by cell migration rather than cell proliferation . Subsequ process . These tdicer mutant lacking mature miRNAs, endocardial cushion formation was excessive, prompting the observation that miRNA-23 is necessary to restrict the number of cells that differentiate into endocardial cushion cells  regulation of valve formation. In a zebrafish on cells . Similaron cells . The genelopment . Thus, ztcf2. In addition to the previously identified regulators of AV conduction (notch and neuregulin) . In a transgenic zebrafish line expressing a cardiac-specific fluorescent calcium indicator (cmlc2:gCaMP), four distinct CCS developmental stages were identified . Beginniregulin) , severalKCNH2 gene that results in prolonged cardiac repolarization and life-threatening arrhythmias. As with the human disease, the zebrafish KCNH2 mutant breakdance exhibits prolonged ventricular action-potential duration, spontaneous early afterdepolarizations and 2:1 AV block , which is easily observed at the embryonic stage (Similarly, zebrafish have been used to recapitulate type 2 long QT syndrome, which is caused by a genetic defect in the ic stage . In a chic stage . Throughic stage . Zebrafiic stage .silent heart, was initially identified through a forward genetic screen and subsequently attributed to disruption of the gene TNNT2, encoding the cardiac contractile protein troponin T (silent heart mutation also demonstrate a reduction in α-tropomyosin and cardiac troponins C and I, resulting in severe sarcomere defects and cardiomyocyte disarray. These ultrastructural changes parallel those seen in human TNNT2 mutations, which lead to familial forms of hypertrophic and dilated cardiomyopathy. Similarly, the pickwickm171 mutation in zebrafish causes a weakly contractile heart and pericardial edema (ttn, encoding the protein Titin, which is necessary for proper sarcomere assembly in cardiomyocytes (ttn truncating mutations are an important cause of human idiopathic dilated cardiomyopathy, responsible for 25% of familial idiopathic cases and 18% of sporadic cases (Several cardiomyopathy models have been developed in the zebrafish. Unlike other vertebrate models, zebrafish larvae have the unique capacity to oxygenate through diffusion alone for the first 7 dpf. This allows for the study of mutations leading to severe cardiovascular compromise, including those that result in a non-contractile heart. One such mutation, oponin T . Zebrafial edema . This mumyocytes . It has ic cases .Similarly, a zebrafish model has been created to simulate amyloid light-chain amyloidosis, a plasma cell disorder that causes rapidly progressive cardiomyopathy . Direct Finally, treatment with cardiotoxic chemotherapies causes cardiomyopathy in zebrafish, analogous to human heart failure. Anthracyclines such as doxorubicin form a class of chemotherapeutic agents used to treat a number of common malignancies, including breast cancer, lung cancer, leukemia and lymphoma. They are extremely effective and form the backbone of many cancer treatment regimens used today. However, doxorubicin exposure causes dose-dependent cardiotoxicity, which can lead to clinically significant cardiomyopathy and congestive heart failure . Our grolox system with a GFP reporter to track cardiomyocytes, this process was shown to occur predominantly through limited dedifferentiation of proliferating cardiomyocytes, rather than through stem or progenitor cells (gata4, providing a molecular marker of regeneration (Given the limited treatment options for advanced heart failure, there is considerable interest in identifying ways to regenerate cardiomyocytes to restore myocardial function . In contor cells . In thisneration . In addineration . Importaneration . These sBecause of its small size and ease of breeding, the zebrafish has become an invaluable model for the study of human cardiovascular disease. There remain countless opportunities for high-throughput chemical screening to both elucidate biology and identify small-molecule modulators of disease-associated pathways. In addition to furthering our knowledge of cardiovascular pathology, the unique ability of zebrafish cardiomyocytes to regenerate offers a particularly exciting opportunity to develop much-needed therapies for advanced heart failure. Given the ease of genome editing, particularly using new mutagenesis techniques such as CRISPR/Cas , future


Analytically distinct estimators have been proposed for the calculation of population-based estimates derived from respondent-driven sampling (RDS), yet there have been few comparisons of the inferences from these estimators using empirical data. We compared estimates produced by unweighted analysis used to calculate sample proportions and by three available estimators that are used to calculate population proportions, RDS-I, RDS-II (Volz-Heckathorn), and Gile’s RDS-SS. Data were derived from a cross-sectional, RDS study of men who have sex with men (MSM) conducted from October 2010 to April 2013 in Moscow, Russia . Analyses investigated the influence of key parameters: recruitment depth, homophily, and network size on sample and population estimates. Variability in results produced by the estimators and recruitment depth were statistically compared using the coefficient of variation (CV). Sample proportions had the least variability across different recruitment depths, compared to the RDS estimators. Population estimates tended to differ at lower recruitment depth but were approximately equal after reaching sampling equilibrium, highlighting the importance of sampling to greater recruitment depth. All estimators incorporate inverse probability weighting using self-reported network size, explaining the similarities in across population estimates and the difference of these estimates relative to sample proportions. Current biases and limitations associated with RDS estimators are discussed. Respondent-driven sampling (RDS) has emerged as a common method in HIV research to recruit hidden populations including men who have sex with men (MSM), sex workers, and people who inject drugs., 2 TheseRDS recruitment begins with ‘seeds’—well-networked individuals from the target population who are purposively selected and represent a range of characteristics. Seeds participate in study activities and are asked to recruit a limited number of peers to participate. Recruited peers who participate, in turn, become recruiters and are requested to invite a new selection of peers to participate. This process continues through successive waves of recruitment until the target sample size is reached. The reliThe second benefit of RDS is the ability to generate population estimates based on the underlying network from which the sample was derived. Estimation methods build on Markov chain theory and assume that after several waves of recruitment the total sample is no longer influenced by the initial sample (seeds) and begins to represent the underlying population. InherentSeveral estimators have been proposed to produce theoretically unbiased population-based estimates for RDS data. One of the earlier estimators, RDS-I, developed by Salganik and Heckathorn  incorporated the use of referral patterns, network size, and the number of cross-relation ties between subgroups of interest., 9 A staDespite on-going improvements in these estimators, debates continue on how accurately these estimators capture true population values and to-date there is no consensus on which estimator is optimal. Among hiIn practice, reporting of RDS data has been inconsistent. Often, sample proportions are not reported, nor are recruitment depth or total sample size, limiting critical review or reanalysis of data. There isThe analysis presented here is derived from a study of HIV among MSM in Moscow, Russia where RDS was used as a means to recruit hidden populations of MSM into an epidemiologic study and engage MSM in safe HIV testing opportunities. This analysis aimed to demonstrate the performance of the RDS estimators in population inference of HIV prevalence and behavioral characteristics as they relate to natural variations of key parameters within the empirical dataset.Data were collected as part of a cross-sectional study to characterize the HIV epidemic among gay, bisexual, and other MSM from October 2010 and April 2013. Details of the sample, study site, recruitment, and measurement methods have been described elsewhere. To proteRecruitment began with 14 purposively selected seeds. These individuals were recruited in a staggered fashion over the course of the study and were given four study coupons with which to recruit peer MSM. Eligible and participating peers were in turn provided with three study coupons for peer recruitment and this process was repeated until the target sample size of 1,370 was reached.Participation in the study included a sociobehavioral survey and biological assessment of HIV and syphilis. All participants were asked personal network size questions used for RDS weighting ., 4Survey measures included demographic characteristics; sexual identity and relationships; substance use; and depression symptoms. Key variables for this analysis were selected to capture demographic, sexual identity and behavior, and infectious disease domains. We present the results pertaining to four variables: self-reported country of birth, sexual identity, consistent condom use during anal intercourse, and HIV infection. These variables were selected to represent high or low homophily, differences in average network size among variable subgroups, and included both self-reported characteristics and clinically determined outcomes. Country of birth was defined as being born in or outside of Russia. Sexual identity included three categories: homosexual, bisexual, or heterosexual/other. Condom use consistency during anal sex (last 6 months) was a binary self-reported measure: inconsistent  and consistent condom . HIV infection status was determined via OraQuick Rapid HIV 1/2 test  and either confirmatory testing at the local reference laboratory  or self-reported HIV infection without confirmatory testing. Participants were offered HIV testing, but were allowed to decline testing and still participate in the survey, resulting in a greater response rate for the other variables included in the analysis.Sample proportions without sampling weights were calculated for each variable. Population (RDS- proportions weighted) were separately calculated using the RDS-I, RDS-II, and RDS-SS estimators. Seeds were excluded from all analyses. RDS-I and RDS-II (Volz-Heckathorn) population proportions were calculated using weights derived from individual self-reported network size. Unlike RDS-I, RDS-II does not rely on the use of recruitment chains for estimation, though all statistical packages still require the underlying recruitment structure for estimation., 12, 14 Analysis of sample proportions, RDS-I, and RDS-II estimates were implemented in Stata 13  using the RDS statistical implementation package developed by Schonlau and Liebau. Non-paraTo assess the role of RDS parameters on sample and population estimates, homophily, recruitment depth, and average network size were estimated for each subgroup of the variables of interest. These parameters were calculated using the Stata implementation package. CalculatRDS estimators were compared using both graphical and statistical comparison methods. To analyze the role of recruitment depth (numbers of waves recruited), sample and population prevalence estimates were calculated for each variable of interest and recalculated when data were restricted to depths ≤5, ≤10, ≤20, and ≤31 waves, the last being the maximum recruitment of the sample. Estimates were plotted graphically with 95%CIs to compare the sample and population estimates of key subgroups of interest for each variable across specified recruitment depths. Graphs are displayed with Y-axis spanning a difference of 25% (i.e. they do not begin at 0%) for the purposes of displaying relatively small differences in estimates. The coefficient of variation was used to statistically compare the variability of estimates produced by the different RDS estimators.[Six of 14 seeds successfully propagated five or more waves of recruitment. One seed that was initiated early in the study recruited 74% of the sample. A total of 3,997 coupons were distributed, yielding 31 waves of recruitment with 1,429 recruits and an overall coupon return rate of 0.36. Of these recruits, 1,362 were eligible for the study . All convergence estimates reported to reach sampling equilibrium by a depth of 3 to 4 waves for each variable. For most variables, except inconsistent condom use, sample and population proportion estimates appeared to stabilize after 5–10 waves of recruitment Figs .SP = 0.043, compared to higher variability among the other RDS estimators  compared to when recruitment depth was equal to or less than 20 and 31 waves . There is still no consensus, however, for how and whether RDS estimates should be incorporated into regression analyses to estimate magnitudes of associations within RDS samples., 12 With