Nonalcoholic fatty liver disease (NAFLD) has become a major public health issue as its progression increases risks of multisystem morbidity and mortality. Recent evidence indicates a more complex relationship between hypertension and NAFLD than previously thought. In this study, a comprehensive literature search was used to gather information supporting the comorbidity phenomenon of hypertension and NAFLD. Then, systems biology approach was applied to identify the potential genes and mechanisms simultaneously associated with hypertension and NAFLD. With the help of protein-protein interaction network-based algorithm, we found that the distance between hypertension and NAFLD was much less than random ones. Sixty-four shared genes of hypertension and NAFLD modules were identified as core genes. Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analysis indicated that some inflammatory, metabolic and endocrine signals were related to the potential biological functions of core genes. More importantly, drugs used to treat cardiovascular diseases, hypertension, hyperlipidemia, inflammatory diseases and depression could be potential therapeutics against hypertension-NAFLD co-occurrence. After analyzing public OMICs data, ALDH1A1 was identified as a potential therapeutic target, without being affected by reverse causality. These findings give a clue for the potential mechanisms of comorbidity of hypertension and NAFLD and highlight the multiple target-therapeutic strategy of NAFLD for future clinical research.  Nonalcoholic fatty liver disease (NAFLD) has emerged as the most common chronic liver disease worldwide and is becoming a major public health issue in China. According to previous reports, the prevalence of NAFLD in the Chinese population has increased to more than twice the rate in Western countries within a decade . More imIt is well accepted that NAFLD is a complex and systemic disorder with variations in genetic background, metabolic characteristics, cultural and socioeconomic factors, dietary habits, life styles as well as environmental risks, which could all contribute to NAFLD progression . ImportaUnfortunately, shared genes and mechanisms between NAFLD and hypertension are still poorly understood. Recently, bioinformatics scientists started to use biological networks as powerful resources for understanding the mechanisms in human complex diseases, for biological components such as genes and proteins always act via molecular interaction networks to generate, enhance or alter the disease phenotype . These nIn this study, we hypothesized that NAFLD and hypertension shared common pathogenic mechanisms and similar underlying signaling pathways. To understand the relationship between hypertension and NAFLD, a literature search and system biology-based method were used.A literature search was conducted in PubMed to identify relevant articles published on 31 March 2020. The search terms hypertension, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, liver fibrosis and combinations of them were used to identify articles. The search work was carried out by two authors independently. Only articles in the English language were included.http://www.disgenet.org/)13 and CTD (http://ctdbase.org/) [http://bioinfogp.cnb.csic.es/tools/venny/index.html) was used for overlapping analysis. Search Tool for the Retrieval of Interacting Genes database website (STRING: http://string.embl.de/) was used to construct an interaction network of overlapped genes [The list of genes for each phenotype of target diseases  was generated by searching two newly updated comprehensive discovery platforms designed to address the relationship between genes and human diseases: Disgenet (se.org/)  databaseed genes .The human protein-protein interaction (PPI) network was constructed from Professor Barabasi’s team , which wd between hypertension genes (x) and NAFLD genes (y). The Zscore is obtained by comparing the observed closest distance to a reference distance distribution between a randomly selected group of genes of matching size and degree distribution as the seed disease genes and target disease genes in the above human interactome. According to the standard normal distribution, Zscore ≤ −1.645  was considered as significantly proximal from one disease to another disease [To quantitate whether there is any statistical influence of hypertension on NAFLD, an efficient way to capture network proximity between one disease to another disease is by the z-score, which relies on the closest distance measured by the average shortest path length  disease .Zscore=According to previous study, complex disease genes rarely resulted by the abnormality in a single gene, and the specific disease modules could be identified via PPI network-based systems biology analysis. In this study, the hypertension and NAFLD disease modules are built through the DIAMOnD algorithm , which ihttp://www.metascape.org) is used for automated meta-analysis to understand common and unique pathways in NAFLD, NAFLD-related phenotype and hypertension [Metascape (rtension . Gene Onrtension . Kyoto Ertension .DGIdb (version 4.0) is an open-source project that help users mine existing resources and generate assumptions about how genes are therapeutically targeted or prioritized for drug development . All theAnalysis of gene (mRNA) expression levels in the liver tissue was conducted by retrieving data from The Genotype-Tissue Expression (GTEx) Consortium (Version 7) . Global We searched the GEO database and obtained a genetic dataset (GSE19817). This dataset contained 67 samples from male mice with different levels of blood pressure . GEO2R, a useful online tool for identifying DEGs in the GEO platform, was used to identify DEGs between hypertensive liver tissues and normal blood pressure liver tissues. |log2FC| > 0.3 and adj. P-value < 0.05  were set as the cutoff standard.In this article, to understand the relationship between hypertension and NAFLD, we first searched the PubMed database to gather clinical studies that supported a strong relationship between hypertension and NAFLD. Then we applied a protein-protein interaction (PPI) network-based systems biology approach to evaluate the significance of the network distance between hypertension and NAFLD. Finally, we identified some commonly shared genes and biological mechanisms of NAFLD and hypertension disease modules, which indicate that multiple-target therapeutic strategy could be more appropriate for future NAFLD treatments.We identified 26 studies published in the last ten years reporting either that hypertension may contribute independently to the development of NAFLD or NAFLD was considered as a risk factor of hypertension. Fourteen studies showed that hypertension is a risk factor for NAFLD, ten studies identified NAFLD as a risk factor for hypertension, and two studies reported a bidirectional relationship (Supplementary Table 1). All these new lines of evidence indicated a potential reciprocal causation of NAFLD and hypertension, and implied the existence of a complex and intertwined biological basis linking blood pressure regulation to metabolic and histologic conditions in the liver.Although the clinical phenomenon of comorbidity of NAFLD and hypertension has been known for a long time, the genes potentially involved in both diseases or related pathophysiological processes were studied only in a few existing works. Through database searching, we obtained 1292 unduplicated genes associated with NAFLD, NAFLD-related phenotypes and hypertension. The number of genes used for further analyses were as follows: 71 genes associated with NAFLD, 212 genes with hypertension, 421 with inflammation and 1013 genes with fibrosis(Supplementary Table 2). We used a web-based tool to compare overlapping genes between diseases with related phenotypes. As is shown in To identify potential biological mechanisms related to NAFLD, NAFLD-associated phenotypes and hypertension, we first identified all statistically enriched terms categorized by biological processes, molecular functions and cellular components, and then calculated accumulative hypergeometric P-values and enrichment factors for further filtering, as mentioned in the methods part. The overrepresented biological processes molecular functions and cellular components are summarized in x) and NAFLD genes and average shortest path length between random control genes, Zscore of hypertension and NAFLD was −8.84 (p < 0.05).To understand the relationship of hypertension and NAFLD, we used a network-based proximity measure to quantify the interplay between hypertension and NAFLD . After calculating average shortest path length between hypertension genes , in which liver expression (RNA-seq expression) levels generated using data extracted from The GTEx Consortium  were shoWe focused on the expression levels of the ALDH1A1 genes in the GSE19817 datasets. The results showed that the expression level of ALDH1A1 was statistically significantly elevated in hypertensive liver tissues compared with their expression in normal blood pressure liver tissues .Traditionally speaking, NAFLD was considered as simple ‘hepatic manifestation’ of MetS for its strong association with other components of MetS. Recently, researchers started to think over the role of NAFLD in the future development of other MetS components and the role of MetS components in the development and progression of NAFLD, which may have closer mutual interrelationships and limit NAFLD therapeutic developments . Here wedifferent mechanistic pathways treating NASH will become analogous to the treatment of hypertension . For fibIn the present systems biology analysis, some common BPs and MFs related to confirmed gene clusters of hypertension, NAFLD and NAFLD phenotypes included lipid binding, antioxidant activity, hormone activity, circulatory system process, myeloid leukocyte activation and small molecule catabolic process, which are involved in cardiovascular system, metabolic system, immune system and endocrine system. Of note, experimental evidence confirmed that some immune effector cells, including natural killer cells, macrophages, T cells, and B cells, promoted the progression in patients with NAFLD to steatosis, cirrhosis and hepatocellular carcinoma . We idenTo evaluate the possibility of hypertension-NAFLD interaction, we calculated the network proximity between hypertension genes and NAFLD genes in a human PPI interactome and compared it with groups of random selected genes. These data showed hypertension genes were more adjacent to NAFLD genes than random genes in the PPI network, indicating a strong association between these two diseases. Preclinical evidence showed that, compared to a normal chow diet, moderate high-fat diet improved blood pressures of maternal mice and their offspring. Importantly, the probability of offspring hypertension was particularly increased, if other factors remain unaltered . AnotherThe present overlap of genes of hypertension and NAFLD modules were enriched by the KEGG database. Insulin resistance and AGE-RAGE signaling pathway in diabetic complications signals were all enriched, indicating a complicated relationship between NAFLD, hypertension and diabetes. A consensus of international experts proposed that metabolic (dysfunction) associated fatty liver disease (MAFLD) was a more appropriate term than NAFLD, given the common association between NAFLD and metabolic dysfunction including hypertension, diabetes and obesity . Renin-aBy analyzing transcriptomics and proteomics data, we identified aldehyde dehydrogenase RALDH1  gene as a mediator related to both liver physiology and hypertension-NAFLD interaction, which has also been identified as differently expressed gene of a high blood pressure vs normal blood pressure mice gene series. ALDH1A1 is an important enzyme associated with metabolic diseases, which catalyzes the second and irreversible step of retinaldehyde oxidation to vitamin A (retinoic acid). In liver, ALDH1A1 has been reported to be present in rat hepatocytes, hepatic stellate cells (HSC) and Kupffer cells . In hepaIn this study, an important limitation of our systems biology approach was the lack of experimental validation of the roles of the identified gene list on the cross-talk between NAFLD and hypertension. Since the biological mechanisms of NAFLD and hypertension comorbidity are poorly understood, evidence supported the associations of the above genes with NAFLD, and hypertension comorbidity is also limited. Experiments using a complex model of NAFLD and hypertension should be designed in the future to identify the role of these genes in molecular agents or genetic manipulation.In conclusion, clinical evidence indicated a bidirectional effect of hypertension and NAFLD. The common biological processes and shared genes identified by the present systems biology approach could explain multiple molecular bases among hypertension and NAFLD to some extent. Our results indicate that NAFLD has common disease pathogenic mechanisms with hypertension and could be considered as a systemic disease which requires multi-target therapeutic strategies rather than a single treatment.Click here for additional data file.


The biological component of the biosocial theory of emotion regulation stipulates that borderline personality disorder (BPD) arises from biological vulnerabilities to heightened emotional reactivity. Comprehensive reviews have consistently implicated abnormalities in the amygdala, anterior cingulate cortex, and hippocampus in the neurobiology of BPD. While Dialectical Behavior Therapy (DBT) is the leading evidence-based psychotherapy for the treatment of BPD, there remains a paucity of literature examining changes in the neurobiology of BPD following DBT treatment. Nine studies were identified that examined neurobiological changes in BPD after the completion of DBT. Results indicated that there was significant deactivation of amygdala activity as well as the anterior cingulate cortex in patients with BPD after DBT treatment. As well, several studies found after DBT treatment, BPD patients had a decreased activity in the inferior frontal gyrus in response to arousing stimuli and increased activity in response to inhibitory control. Future research on the neurobiological change after DBT treatment can help clarify biological mechanisms of change in BPD. Borderline Personality Disorder (BPD) is a debilitating mental illness characterized by emotion dysregulation, poor impulse control, identity disturbances, recurrent suicidal or self-harming behaviors, and unstable interpersonal relationships , 2. The Comprehensive reviews have consistently demonstrated the multifaceted neurobiology of BPD in adults , 4, 5 aschanges in neurobiology following DBT treatment of BPD. The purpose of this article will be to review the literature on how DBT can influence the neurobiology of BPD.Dialectical Behavior Therapy (DBT) is the leading evidence-based psychotherapy for BPD that emphasizes the role of emotion regulation and targets the acquisition of skills and techniques to encourage control over maladaptive behavioral patterns –15. HoweA review of the current literature was carried out by searching for articles in Pubmed/Medline, PsycINFO, and Google Scholar, along with articles obtained from the references of other articles. The reference sections of the review articles mentioned above i.e., , 4–7] we–7 we4–7)A total of nine studies were identified for the review. Seven studies used functional magnetic resonance imaging (fMRI) and two used functional near-infrared spectroscopy (fNIRS). Within the nine studies, four of the fMRI studies , 16–18 aSeveral studies focused on neural changes observed using fMRI resulting from 12-week DBT treatment programs for individuals with BPD. Schnell and Herpertz  examinedAnother study compared patients with BPD with gender-matched healthy controls (HC) with no Axis I or II disorders or family history of an Axis I disorder using a 12-month DBT program . During reappraisal condition) or (b) view the pictures attentively without trying to alter their affective reaction (maintain condition). Results indicated that the BPD patients in the DBT group experienced a stronger activity decline in the anterior insula and dorsal ACC post-treatment compared to healthy controls, while there was no significant difference between activation of the orbitofrontal cortex (OFC) and the dlPFC between groups. Furthermore, in DBT patients, the decline of anterior insula activity during reappraisal (but not passive viewing) of negative pictures was significantly correlated with a reduction in BPD symptom severity. Moreover, BPD patients experienced increased anterior cingulate connectivity to medial and superior frontal gyrus, superior temporal gyrus, and inferior parietal cortices after DBT. Finally, DBT responders demonstrated diminished activity post-treatment in the right amygdala, subgenual, perigenual and dorsal ACCs, the medial and left OFCs, and right dlPFC during reappraisal compared to DBT non-responders. DBT responders also showed increased connectivity from the dlPFC to the OFC, superior temporal gyrus, posterior insula, cingulate gyrus, and thalamus over time compared to DBT non-responders  or (b) memorize letters  before being confronted with negative or neutral pictures in a distraction task during the pre- and post-treatment fMRI scans. After the presentation of the pictures, participants had to indicate as fast as possible when they (a) saw the letter “o” or (b) whether the letter presented was in the memorized string of letters. The memorization-negative condition was used as a way to measure distraction as an effective emotion regulation strategy in reducing negative affect, particularly when stimuli are highly arousing  with no current or lifetime psychiatric disorder and no psychotropic medication . Particiarousing . Resultsarousing .The third study  examinedThe fourth study compared female patients with BPD undergoing 12 weeks of DBT with female patients with BPD who were receiving TAU . Particin-back task whereby participants had to indicate when the current letter matches the one from n steps earlier in the sequence.In addition to examining the impact of a full DBT program on various brain structures, research has also been conducted on the specific modules of DBT. One study compared the neural correlates of BPD patients in a 10-week DBT mindfulness (DBT-M) group with BPD patients in a DBT interpersonal effectiveness (DBT-IE) group . DBT is n-back task compared to the DBT-IE group. However, there was no post-treatment difference in default mode network activation or deactivation across both groups, a brain region associated with various cognitive processes such as introspection, envisioning the future, and self-reflective thought  , 20. TheAfter the 6 months of DBT, differences in activation during inhibitory control were also observed based on changes in affective, cognitive, impulsivity, and interpersonal domains . More spThe main findings of this review suggest neurobiological changes in brain function after the completion of DBT treatment among patients with BPD. Firstly, two studies found a significant deactivation of amygdala activity after the completion of DBT treatment particularly among DBT treatment responders , 21. TheSecondly, two studies found significant decreases in activation in the ACC after DBT treatment among patients with BPD , 21, whiincrease in activation in the anterior insula  found stronger frontal region activation (Finally, there have been a handful of studies examining the neural impact of psychodynamic therapy among individuals with BPD , 43. Onetivation . Similartivation –44.after DBT treatment among BPD patients, there has also been several lines of evidence on specific neural correlates prior to the commencement of DBT that may be associated with improved outcome following treatment (In addition to neural changes reatment , 20, 45.reatment . In contreatment . Additioreatment . A folloA second study found that amygdala and parahippocampus activation during a cognitive reappraisal task after viewing negative pictures, severity measures of BPD psychopathology, and gray matter volume of the amygdala provided best predictive power for identifying DBT treatment non-responders . Prior tMoreover, one study of BPD patients undertaking psychodynamic therapy found that post-treatment improvements in constraint were predicted by pre-treatment right dorsal anterior cingulate cortex hypoactivation, while pre-treatment left posterior-medial orbitofrontal cortex/ventral striatum hypoactivation predicted improvements in affective lability . One stuIt is clear that there is much to learn about both the unique impact of DBT as well as other forms of treatment on individuals with BPD, along with learning more about how baseline genetics and neural patterns of brain functioning may be helpful in predicting treatment success in BPD , 19. HowAI conceived the topic idea and wrote the manuscript. EBL helped supervise the project. All authors were involved in the editing of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Finally, we discuss the use of membrane vesicles as excellent tools for investigating membrane lateral organization and protein sorting, which is otherwise challenging but extremely crucial for normal cellular functioning. We present current limitations in this field and how the same could be addressed to propel a fundamental and technology-oriented future for extracellular membrane vesicles.Lipids are essential components of cell membranes and govern various membrane functions. Lipid organization within membrane plane dictates recruitment of specific proteins and lipids into distinct nanoclusters that initiate cellular signaling while modulating protein and lipid functions. In addition, one of the most versatile function of lipids is the formation of diverse lipid membrane vesicles for regulating various cellular processes including intracellular trafficking of molecular cargo. In this review, we focus on the various kinds of membrane vesicles in eukaryotes and bacteria, their biogenesis, and their multifaceted functional roles in cellular communication, host-pathogen interactions and biotechnological applications. We elaborate on how their distinct lipid composition of membrane vesicles compared to parent cells enables early and non-invasive diagnosis of cancer and  Lipids are fundamental components of the plasma membrane across various living forms and other cellular compartments such as nuclear membranes, Golgi, and endoplasmic reticulum. They are also the essential building blocks of cell-derived membrane vesicles. As part of these, lipids conform to diverse biological functions ranging from structural and signaling roles as well as by fine-tuning protein activity. Historically lipids have held a secondary place-next to proteins-in both basic and applied research attributed to the lack of techniques for their isolation, visualization, manipulation and quantitation. However, due to their undisputable involvement in a spectrum of diseases, there has been a surge in development of effective tools and methods for investigating lipids with unprecedented details.Much we know about lipids and some of their presumed biological functions have come from the study of synthetic membranes with specific lipid composition due to the inability to study lipids in native environment. This is now fast changing with advent of super resolution fluorescence techniques, label-free mass imaging and lipidomics. However, the most impactful has been the discovery of membrane vesicles released from various kinds of cells . These membrane vesicles are known by various names such as exosomes, apoptotic bodies, oncosomes derived from eukaryotes and outer membrane vesicles from bacterial species. These rather universal components have contributed significantly in enhancing our knowledge on the multi-level role of lipids in physiology, disease, and interestingly as theranostic modules, which were mostly only affiliated with proteins. The most commendable use of cell-derived vesicles in the form of giant plasma membrane vesicles (GPMVs), bacterial outer membrane vesicles (OMVs)-and lately giant endoplasmic reticulum vesicles (GERs)—has been to query the functional relevance for lipid phase separation or nanoclustering to form distinct membrane domains with selective recruitment of specific lipids and proteins that orchestrate various cellular processes, i.e., signaling . MoreoveAligning with the varied roles of lipids within membrane vesicles, here, we review the latest development in this field. We elaborate on the various kinds of membrane vesicles and their biogenesis, comment on their composition and subsequently associated physical properties and how that is being exploited for early diagnosis in various diseases. The recent therapeutic prospects of membrane vesicles as delivery vehicles are discussed. We provide the breath of successful vaccine campaigns that have emerged from bacterial membrane vesicles and their constituent lipids components. Finally, we detail how the structural and compositional heterogeneity of various kinds of membrane vesicles are enabling exquisite insights into the functional roles of lipids in dictating membrane and protein function in eukaryotic and bacterial cells.Membrane vesicles are nano- or micrometer-sized membrane-bound lipid vesicles released from cells for systemic delivery of various kinds of molecular cargoes such as nucleic acids, lipids sugars and proteins to recipient cells. Cellular transport was the major functional role associated with membrane vesicles of diverse origins, both species and organelle-centric, however, recent decade has furnished unprecedented insights into the involvement of lipid vesicles in various biological phenomena ranging for cellular communication, bacterial and/or viral pathogenesis, aging, cancer progression and tumor invasion. In addition to these, their untapped clinical potential as diagnostic, therapenutic and prognostic modules is currently gaining attention . For up MVs are diverse in origin and consequently known by various names. MVs of Eukaryotic origin including fungi and other parasites are microvesicles (100 nm–1 μm), exosomes (50–150 nm), oncosomes (1–10 nm), migrasomes (50–100 nm), apoptotic (0.05–3 μm) bodies and chemically induced giant plasma membrane vesicles (>1 μm) ; the latde novo microvesiculation has also been implicated, but largely rejected due to the fact that eukaryotic lipid membranes have active mechanisms to resist spontaneous curvature generation. Though, triggering of membrane curvature due to alterations in lipid and protein composition has not been ruled out. Particularly, uneven distribution of lipids between the two leaflets of lipid bilayer, modulated membrane biophysical properties such as rigidity and curvature leading to membrane budding. Moreover, specific membrane components responsible for microvesicles formation have been identified including cholesterol, Ras-related small GTPase, ARF6, and Rab proteins. Recently, a more generic model for de novo microvesicle formation involves protein crowding at the cell surface that generates pressure due to protein-protein and protein-lipid interactions driving membrane bending. Thus, enrichment of protein cargo at the site of microvesicle budding could be an efficient and simple mechanism to drive the formation of microvesicles.Microvesicles also known as micro particles are formed by outward budding from cell surface and share some properties with bacterial MVs. Though the exact details of the biogenesis of microvesicles is unknown, some studies highlight the connection between ESCRT pathway and early stages of microvesicle formation . FurtherExosomes are formed from the outward budding of the late endosomal membrane leading to their accumulation in the multivesicular bodies (MVB) and the subsequent fusion of the MVBs with plasma membrane to release exosomes into extracellular space . ESCRT pApoptotic bodies, like exosome are another type of MV specific to eukaryotes and are formed during apoptosis by outward budding from the cell surface ; as a reApart from biological relevance, eukaryotic MVs also serve as molecularly complete scaffolds specific to plasma membrane and are used to investigate with intricate details the structure and function of native plasma membrane. These, giant plasma membrane vesicles (GPMVs), are on-demand MVs released from cells following chemically induced blebbing , and mosE. coli  and outer-inner membrane vesicles , E. coli . Furthervesicles . O-IMVs vesicles . In fact species .E. coli, and P. aeruginosa use OMVs to transport virulence factors. OMVs containing virulence factors attach to the outer membrane leaflet of recipient cells and rapidly fuse allowing delivery of the cargo into the recipient periplasm. Apart from the widely studied gram-negative bacteria, gram-positive bacteria also produce OMVs enriched with surface associated virulence proteins that play a key role in the bacterial pathology. The vesicles also help in the transfer of proteins to other bacteria and eliminate intracellular competition. Acid-fast bacteria like Mycobacterium tuberculosis also release membrane vesicles that are packed with molecules that modulate the host immune response. Mtb OMVs usually contain virulent lipids such as lipoglycans and lipoarabinomannan, which inhibit phagosome maturation , lactosylceramide (LacCer) in PC3 secreted exosomes and moderate enrichment of SM in reticulocyte . Compariexosomes . A recenC and SM . FurtherC and SM . SimilarC and SM . IntriguC and SM . Sun et  and Cer . Exosomeer cells .et al. using MALDI-TOF-MS and TLC compared lipidome profile of urinary microvesicles and exosomes and revealed that mannosyl-di-PI ceramides were detected only in exosomes, whereas PI-ceramides were found in microvesicles  and reduction of CL in MVs from bacteria . MVs frobilayers . Comparebilayers . MVs froabundant . Furtheret al. for the first time revealed high abundance of Chol in exosomal membranes with subtle similarities with detergent resistance fraction indicting that exosomes may have raft like membrane properties, namely, high order and rigidity  by fusion of HSL containing MVs with different bacteria; albeit with varying propensities , which is another highly hydrophobic molecule, thus hampering its free aqueous diffusion. MVs are therefore used to traffic this molecule within a population (Cell to cell communication or quorum sensing (QS) in bacterial species is the ability of individual cells to communicate with one another by the use of extracellular signaling molecules . The mosensities . This seronments  fosterinpulation , and thepulation . Thus, Mpulation .Along similar lines, MVs in eukaryotic cells also regulate cellular communication Particularly; their involvement in tempering with paracrine and autocrine communication in cancer is well studied , FiguresP. aeruginosa use OMVs, to deliver an array of virulence factors such as ß-lactamase, alkaline phosphatase, hemolytic phospholipase C, and Cif, into the host airway epithelial cells via the fusion of OMVs with host cell lipid membrane. Particularly, host cell plasma membrane lipid rafts have been implicated as the most exploited site for fusion with bacterial OMVs carrying virulence cargo. N-WASP mediated actin pathway then traffics the vesicle cargo into the host cytoplasm , proteins and carbohydrates that are involved in virulence. These studies have collectively elucidated the functional involvement of MVs for release of virulence factors in close proximity of the host cell facilitating targeted delivery of pathogenic moieties not the target host cell. For example, proteomic analysis of the sectretome of Leshmania donovani revealed that 98% of the secreted proteins lack identifiable signal sequences required for secretion pathways, thus implicating non-classical MV (exosomes and microvesicles) mediated release during pathogenesis.Similarly, eukaryotic microbes also release virulence factors packed with their MVs. Microvesicles ranging from 10 to 350 nm have been observed with various eukaryotic microbes such as P. aeruginosa OMVs activate TLR4 signaling pathway and contributes to the protection against pseudomonal lung disease in murine models  have elucidated them to be enriched with GLUT1 and glycolytic enzyme PKM2. For instance, Wan et al. showed enriched PKM2, and GLUT1 levels in secreted exosomes from hepatic stellate cells promoting metastasis, while another study demonstrated increased PKM2 exosomal content to be associated with pre-metastatic niche . ImportaOne of the most important treatment methods for cancer that is administered either on its own or along with chemotherapy is radiotherapy. As with any treatment for cancer, one of the major obstacles is the recurrence and relapse of the tumor and its resistance to radiotherapy (radioresistance) . Owing tin vitro and in vivo models of nervous system cancer. The provided evidence that these exosomes contain upregulated oncogenic and downregulated tumor suppressive contents. There were downregulated levels of tumor suppressive miRNA like miR-365 and miR-516 which increased cancer cell proliferation and upregulated level of miR-889 that increased resistance, metastasis and decreased apoptosis. These collectively show that radiation exposure leads to the release of altered exosomes that contain signals which decrease tumor suppressive cargo and concomitantly increase oncogenic cargo to be taken up by the recipient cell leading to proliferation and enhanced ability to survive radiation treatment (Glioblastoma (GBM) based radiation therapy has been shown to possess limited efficacy due to the enrichment of cancer stem cells (CSCs). CSCs have the capacity to cause recurrent rumors and are resistant to therapies usually used against GBMs . In thisreatment .Similar to radioresistance, cancer cells also relay cargo and other components that are important for survival of neighboring cells in response to stress conditions brought about by chemotherapeutic drugs. For example, multiple myeloma (MM), a plasma cell malignancy has a range of immunomodulatory drugs, proteasome inhibitors and antibody drugs developed against it. MM cells are seen to acquire resistance on long term exposure to these drugs. MM derived exosomes have been shown to play a role in the transmission of this information from cell to cell and subsequently help in the cancer progression and drug resistance of surrounding cells . Exosomein vivo during infection, and from sera of patients suffering from bacterial infections further reinforce the conclusion that MVs represent an excellent source of antigens during infection capable for eliciting a bonafide immune response. This aptly lays the foundation of exploring MVs in vaccine campaigns, in addition to serving as potential biomarkers as discussed in subsequent sections.Recently the ability of MVs from various pathogens to act as antigen decoys modulating antibody response has been reported. MVs released by various pathogenic organisms are nonviable, however are enriched with antigens that are recognized by the host adaptive immune system . MoreoveNeisseria meningitides (Nm) , caused by des (Nm) . The fordes (Nm) . Bexseroimmunity . In anotresponse . Similarimmunity , and thonfection . Furthere models . PNAG sularctica . Engineen strain . Likewis vaccine . In cont vaccine . Additiorferon-γ .et al. elucidated a non-overlapping immunomodulatory response from MVs derived from three distinct Leishmania species that cause different clinical manifestations  and specificity (100%), however reinforced the application of MS based lipidomic platforms as a routine method for clinical practice. This was due to the fact that various lipid classes displayed very little differences, which would go undetected using other semi-quantitative approaches . Specifiet al. isolated EVs from bronchoalveolar lavage fluid of asthmatics and healthy controls, and reported SM 34:to be highly abundant in EVs from second-hand smoke exposure asthmatics compared to healthy controls  found in the serum exosomes of patients suffering from these, compared to healthy controls. Interestingly, some of the healthy patients (absence of active TB) showed evidence of latent disease dependent on the abundance of specific biomarker mycobacterial peptides belonging to  prodrug (Pt (lau)), human serum albumin (HSA), and lecithin (2Se3 nanodots and DOX co-embedded microparticles (Bi2Se3/DOX@MPs) were fabricated. Using ultraviolet light irradiation-induced budding of parent cells preloaded with Bi2Se3 nanodots and DOX, the drug-loaded microvesicles displayed dual-modal imaging capacity and excellent tumor suppression  derived MVs facilitate issue repair of perivascular stem cells while providing an ‘off-the-shelf' substitute for bone tissue regeneration . InteresMVs are advantageous due to their low immunogenicity and innate ability to interact with target cells owing to their biocompatibility which is an important pre-requisite for clinical applications. Other factors that propel MVs based clinical use are high stability in biological fluids, cell-cell communication, active targeting by modification/functionalization, suitability for multidrug delivery and a wide range ofdrug encapsulation methods. However, MV-based therapeutics have their own challenges  such as sourcing of an optimal donor cell type, preserving the structural integrity of the MV, large-scale production, storage and high purity  are somePlasma membrane is a key component of eukaryotic cells that orchestrates various membrane-associated cellular processes by selective recruitment and sorting of lipids and proteins into distinct spatially organized lipid domains. The most investigated lipid domains central to signal transduction and membrane trafficking pathways are the lipid rafts. Lipid rafts are conceived as dynamic, transient and heterogeneous membrane platforms enriched in cholesterol, glycosphingolipids such as SM and other saturated lipids in addition to raftophillic proteins. The functional implication of lipid rafts and other specific lipid domains has been demonstrated in regulating lipid-lipid and lipid protein interactions critical for signaling, trafficking, cell growth, migration, pathogen entry, and immune response. Moreover, structural and compositional fluctuations in plasma membrane domains govern membrane biophysical properties, such as order, fluidity, adhesion, elasticity, and lateral organization, control lipid/protein diffusion  lipid/prOne obvious solution to query native membrane structure and function is the reconstitution of membrane-related processes outside the cell using artificial model membrane systems of manually tunable compositions. Well–established systems include detergent resistant membrane fractions (DRMs) , solid sInterestingly, many of these limitations have been addressed by the development of cell-derived giant plasma membrane vesicles (GPMVs) that readily recapitulates the native integrity of biological membranes. GPMVs are produced by chemically induced blebbing of cell plasma membranes and offer genetic controllability of fine-tuning their lipid and protein compositions. One of the earliest mentioned method for membrane blebbing was reported for sarcoma cells using sulfhydryl group based chemicals . More remisc than the ones with the L16P mutation implying the stabilization effect of the protein on the ordered membrane domains , have been developed. This proof-of-concept study by Grimmer et al. revealed GERS as an attractive and novel tool to elucidate ER-regulated process. By demonstrating the compatibility of various biophysical techniques on the newly developed membrane mode along with ability to incorporate additional fluorescent fused proteins of interest into the ER by genetic means post GERs production, the authors revealed the potential application of these systems to investigate enzyme functions and lipid metabolism regulated by ER membrane; a field less explored. Notably, in parallel, another studies revealed the ability of ER membranes to exhibit temperature-dependent phase segregation, similar to that seen in plasma membrane  as well Finally, the study of lipid rafts have been extensively investigated in eukaryotic cells, however evidence indicating their presence in bacterial is reinforcing the rather ubiquitous nature of lipid domains in regulating cellular processes across various living forms . In thisGPMVs, thus have proven a versatile system for studying plasma membrane organization dictated processes; lipid phase segregation, distribution of lipids and membrane proteins into distinct domains, protein sorting, regulation of protein function, and membrane fusion. However, there are certain limitations that signal caution in analyzing data collected with GPMVs. These are the lack of perfect plasma membrane asymmetry , effect Secretion of membrane vesicles is now a well-accepted phenomenon across various life forms and plays crucial roles in normal cell functioning. They serve varied functional roles, such as in communication of signals between and within a cell by decorating their surface with specific lipid and protein components. Consequently, they have occupied a center stage in disease biology focused on 1) discovery of pathogenic mechanisms at the interface of host and pathogen, 2) to facilitate effective and non-invasive source of biomarkers , and 3)


Spinal cord injury (SCI) is a devastating condition of the central nervous system that strongly reduces the patient’s quality of life and has large financial costs for the healthcare system. Cell therapy has shown considerable therapeutic potential for SCI treatment in different animal models. Although many different cell types have been investigated with the goal of promoting repair and recovery from injury, stem cells appear to be the most promising. Here, we review the experimental approaches that have been carried out with pluripotent stem cells, a cell type that, due to its inherent plasticity, self-renewal, and differentiation potential, represents an attractive source for the development of new cell therapies for SCI. We will focus on several key observations that illustrate the potential of cell therapy for SCI, and we will attempt to draw some conclusions from the studies performed to date. Spinal cord injury (SCI) is a devastating multifactorial event that affects approximately 39 cases per 1 million individuals in North America, with enormous healthcare costs. Cervical lesions, which in most cases result in tetraplegia, are the most common type of lesion, representing 60% of all SCI [Severe SCI damages gray matter neurons and white matter axonal tracts that carry signals to and from the brain, and involves the cellular loss of nervous tissue, demyelination, the generation of an inhibitory environment and glial scar formation. SCI repair therefore requires axonal regeneration, remyelination, the replacement of lost cells , and a permissive environment to increase survival and functional integration with host cells ,3. Over Numerous SCI animal models have been used to test a variety of cell types, including both tissue-specific cells and stem cells. Among the tissue-specific cells, Schwann cells , peripheThe advantages of stem cells over tissue-specific cells are manifold, of which the most appealing is their capacity to self-renew and their potential to differentiate into multiple lineages. Accordingly, various stem cell populations have been tested to repair SCI, including mesenchymal stem cells, neural stem cells (NSC) and pluripotent stem cell (PSC)-derived cells.Given the immunomodulatory potential of MSC, they have been used to try to mitigate the negative effects of the pro-inflammatory environment found at the lesion site. The results of animal studies with MSC were promising, and several clinical trials have been launched. However, clinical studies have generally failed to achieve significant functional recovery and restore neural circuits, although some positive results have been reported (reviewed in ).NSC also secrete immunomodulatory and neurotrophic factors, and generate neurons and glial cells, opening the possibility of cell replacement; they therefore seem to have better abilities to improve motor function in SCI . NSC of Pluripotent stem cells (PSC) represent a promising alternative source of stem cells, mainly due to the fact that they have a higher self-renewal and differentiation capacity than more committed stem cell types, albeit with considerable challenges limiting their implementation. PSC are typically obtained from pre-implantation embryos  or by reprogramming somatic cells into ESC-like cells , and they have the intrinsic capacity for differentiation into every cell type in the whole organism . DiffereParadoxically, while most researchers agree that PSC-derived cells may offer a permanent therapeutic solution to SCI, after more than 40 years and over a decade since the descriptions of ESC and iPSC, respectively, the adoption of these cell types for the development of novel SCI treatment modalities has been slow. In this review, we focus our analysis on the progress that has been made using PSC-derived cells in an effort to alleviate SCI. Studies wherein PSC have not been differentiated to a more committed cell type and have been directly transplanted into animals and humans have not been considered in this review, as the transplantation of undifferentiated PSC usually leads to tumor formation , and thuDuring the last two decades, numerous studies have explored whether the use of ESC-derived cells has some benefit for SCI repair . Many stESC-derived neurospheres (NS) containing NSC have also been used for SCI cell therapies. To evaluate its efficacy for SCI repair, Kumagai and colleagues  comparedAnother strategy applied for SCI repair has been the use of ESC-derived cells with forced expression of specific factors. For example, Butenschön and colleagues reported the effect of mouse ESC–NSC overexpressing BDNF, isolated by magnetic and fluorescent-activated cell sorting, in subacute SCI. Recovery of motor function was observed only in animals transplanted with SSEA-1-/PSAN-CAM+ cells overexpressing BDNF, but not in control NSC . AnotherIn general, the results reported using mouse and human NSC differentiated from ESC could be considered positive . In factAssuming that all injuries in the spinal cord are created equally, some investigators have chosen to transplant cells that are further along the differentiation path than NSC, including oligodendrocytes, astrocytes, and a variety of neuronal-type cells. This assumption entails a significant speculation considering the multiple tracks involved and the different causes of trauma in SCI. The obvious advantages of this approach are the fact that the tumorigenic potential will probably decrease, and that it allows some degree of control over the final cell types that are grafted, albeit at the expense of having to speculate the type of cells each particular lesion needs. In this regard, the first study to analyze the therapeutic effect of more differentiated cells used human ESC-oligodendrocyte progenitor cells (OPC) in a rat model of spinal cord injury . After gHuman ESC–OPC have also been tested in a cervical rat model, rather than the more commonly used thoracic model . GraftedMouse ESC-derived glial progenitors positive for the nerve glial antigen 2 (NG2) marker and matrix metalloproteinase 9 have also been tested. These cells could penetrate the glial scar formed after subacute SCI. In addition, the axons of these cells grew over long distances (>10 mm) with a preference to traverse white matter rather than gray matter. These facts support the notion that the expression of chondroitin sulfate proteoglycan in the injury scar is an impediment to regeneration, and that NG2-positive ESC-derived glial progenitors can breach this barrier and promote axon growth .Allodynia and hyperalgesia are the two forms of spontaneous neuropathic pain that affect approximately 50% of SCI patients and persist over time, complicating the rehabilitation and decreasing the quality of life. Hwang and colleagues, in an effort to address this problem, transplanted ESC-derived spinal GABAergic neural precursors, 3 weeks post-injury. They observed a reduction in neuropathic pain in injured animals 2 weeks post-transplantation, and the effect persisted for up to 7 more weeks, although locomotor function did not improve . Fandel In this context, other groups have tested a combination of cells. For example, Niapour and colleagues transplanted a combination of Schawnn cells isolated from the sciatic nerve and human ESC–NSC, with the goal of improving the differentiation of NSC after transplantation, into a subacute SCI rat model at thoracic level . The preTo date, the studies performed with ESC-derived cells have shown, in general, that these cells can be efficacious in acute SCI models , but furiPSC are artificially induced pluripotent stem cells from adult somatic cells , overcomRomanyuk and co-workers also reported the beneficial effects of human iPSC–NSC in a rat model using balloon-induced acute SCI at the thoracic level . Cells wNevertheless, motor recovery is not always achieved in iPSC-derived cell transplantation studies for SCI repair. Many studies have shown that transplanted cells survived and even differentiated into the three lineages in the host tissue, but this does not always imply integration or locomotor improvement. For example, Pomeshchik and colleagues  performeIn another fashion, the use of regionally specific iPSC–NSC has also been attempted. Kajikawa and coworkers hypothesized that, given that there are subtypes of NSC regarding their regional identity, spinal cord-type NSC would be the most adequate to inject into SCI for regenerative purposes. They injected forebrain and spinal cord-type iPSC–NSC into a mouse acute model of SCI. Both types of NSC were grafted onto host tissue; however, only the spinal cord type showed connection with the corticospinal tract of the host and, moreover, resulted in recovery of motor function .The level at which the SCI is located is an important factor to take into account in terms of achieving recovery in motor function, as cervical lesions entail a much more severe impairment than thoracic ones. The chronicity is also a relevant factor that should be considered. The maturation of the glial scar surrounding the lesion around 2–3 weeks post-injury is well documented to become a barrier to axonal regrowth and tissue regeneration due to the inhibitory environment within the lesion ,65. ThusWith the aim of modeling the clinically relevant condition of chronic cervical contusion, Nutt and coworkers tested human caudalized iPSC–NSC in a rat cervical model of chronic SCI. They chose to use the Forelimb Reaching Test (FRT), a very stringent test that offers advantages over others, as it provides a more objective behavioral comparison between and within treatment groups . Human iIn a different approach, some investigators have opted to use scaffold structures as a cell delivery platform, and to expedite cell differentiation and tissue formation. As an example, Lu and colleagues  embeddedCombinatorial therapies have also been applied. Some studies have chosen to condition cells with small molecules to improve their performance in vivo. For example, Shiga and collaborators conditioned iPSC–NSC with enzymatically inactive tissue-type plasminogen activator (EI-tPA), prior to grafting into an acute rat model with severe thoracic SCI. EI-tPA interacts with cellular receptors to mediate changes in cell physiology potentially relevant to the challenges of stem cell therapy; it is neuro-protective to cortical neurons, and promotes neurite outgrowth in neurons and neuron-like cells by activating cell-signaling factors, such as c-Src and ERK1/2. Furthermore, it may regulate innate immunity by suppressing toll-like receptor responses. Notably, they found that the cells differentiated, acquired markers of motor neuron maturation, and extended βIII-tubulin-positive axons several spinal segments below the lesion. Furthermore, they observed a decrease in muscle atrophy, and animals had significantly improved motor function, without exacerbating pain . BonillaIn the last few decades, SCI in the elderly population has increased substantially due to life expectancy ,67. In tAn important issue at stake is the safety of iPSC-derived cells, which must be rigorously evaluated before they enter the clinic . SeveralRecently, Chow and collaborators generated canine iPSC–NSC and transplanted them into pet dogs with chronic thoracic SCI after a traumatic accident. Observation via MRI did not show any changes in the lesion size or the glial scar of the animals. No adverse effects were observed either, demonstrating the safety of administering iPSC-derived NSC in dogs with follow-up for 6–12 months, particularly with respect to tumor formation at the injection site. However, the animals did not show an improvement in motor function .Similarly to the progress made with human ESC, various groups have tried transplanting more differentiated human iPSC-derived cells. Hayashi and coworkers  used mouUsing an induced thoracic contusion in NOD-SCID mice, Kawabata and collaborators grafted human iPSC–OPC into the lesion epicenter (subacute phase). The authors found that the grafted human iPSC–OPC contributed to remyelination, promoted axonal growth, and contributed to synapse formation with host mouse neurons, leading to enhanced functional recovery after SCI. However, when comparing the results with a previous study using human iPSC–NSC, they found no therapeutic differences in hindlimb motor function . Recentl6 cells treated with γ-secretase inhibitor, to promote cell differentiation and decrease the risk of tumorigenesis. The efficacy of the treatment will be evaluated secondarily, assessing motor function following the International Standards for Neurological Classification of Spinal Cord Injury (ISNCSCI) in comparison with a historical control. Sensory function, spasticity, and quality of life will be assessed as well [In summary, iPSC-derived cells have been shown to be efficacious in different animal models, especially in acute thoracic models . Nonethe as well .During the last 20 years of research with PSC-derived cells for SCI treatment, different animal SCI models have been assayed, using different types of PSC-derived cells, cultured under different conditions, grafted at different doses and time windows, and under different immunosuppression regimes . These sTo develop effective cures for SCI, it is necessary to obtain safe and fully characterized cells tested in a robust and repetitive SCI animal model that delivers, in every animal, a precisely measured force at the spinal cord. For initial experiments, validated and reproducible models in rodents such as MASCIS, Infinite Horizon or the Ohio Contusion device may be good options, but for therapy authentication, a model in large animals should be considered. Nevertheless, since human SCI is such a complex situation, it is very difficult to find a specific model that incorporates all variables. Therefore, the chosen model would depend mainly on the goals of each specific study . The useFunctional recovery assessments based on subjective evaluations or operator experience could be a red flag in many animal studies, and must be considered. An automated system for gait quantitative assessment would provide some parameters that can help standardize the evaluation of locomotion and forelimb function. Functional improvements should be measured by quantitative and objective protocols, such as the “Catwalk” automated quantitative gait analysis, among others .The establishment of optimal protocols for immunosuppression is an important aspect commonly overlooked when designing allogeneic cell therapies. The classically used immunosuppressants  are associated with important side effects in the host, and can affect the differentiation, proliferation and survival of transplanted cells ,79,80,81The precise type of cell(s), doses, and administration regimes required for significant sensory and motor function recovery in humans remain enigmatic. Furthermore, there is still no consensus on the preferred source of PSC, ESC or iPSC. Since ESC are derived from embryos, the use of ESC raises ethical concerns, and strict ethical committees regulate their use, complicating the development of ESC-based cell therapies . AlthougAnimal experiments with both ESC- and iPSC-derived products have shown that tumorigenic potential is an important safety issue. The PSC should be properly differentiated to avoid the transplantation of undifferentiated PSC that, due to their intrinsic self-renewal and differentiation potential, could form tumors (teratomas) in the host tissue . The traIn general, PSC-derived cell therapies are recognized as one of the most promising therapeutic options for SCI repair, but there are still important challenges to overcome before they can be widely used in humans . One of


Ardenna creatopus) breeding colonies on Isla Mocha  and Isla Robinson Crusoe (Juan Fernández Archipelago), Chile . The most commonly detected species were pink-footed shearwaters and introduced rats (Rattus spp.) on Isla Mocha, and European rabbits (Oryctolagus cuniculus) and pink-footed shearwaters on Isla Robinson Crusoe. Introduced mammals observed, in order of greatest catch-per-unit-effort, were rats, cats (Felis catus), dogs (Canis lupus familiaris), and European hares (Lepus europaeus) on Isla Mocha and European rabbits, cats, cattle (Bos taurus), rats, dogs, mice (Mus musculus), and southern coati (Nasua nasua) on Isla Robinson Crusoe. Especially noteworthy results for pink-footed shearwater conservation were the presence of cats during all monitoring months in shearwater colonies on both islands, that catch-per-unit-effort of rabbits was greater than shearwaters on Isla Robinson Crusoe, and that rats were the most observed vertebrates after shearwaters on Isla Mocha. Pink-footed shearwaters were regularly present on the islands from October through May. Presence and relative catch-per-unit-effort of pink-footed shearwaters qualitatively matched the species’ known breeding phenology. The regular presence and temporal overlap with shearwaters of cats, rats, rabbits, and cattle within shearwater colonies, coupled with the irregular presence of dogs, coati, hares, and mice, indicated a serious conservation threat for pink-footed shearwaters and other native insular fauna and flora. Finally, our study provides a widely applicable model for analysis of multi-year trail camera data collected with unstandardized settings.Biodiversity conservation planning requires accurate, current information about species status and threats. Although introduced mammals are the greatest threat to seabirds globally, data on introduced species is lacking for many seabird breeding islands. To inform conservation planning, we used trail cameras to document the presence, relative abundance, and seasonal and diel attendance of introduced and native vertebrates within pink-footed shearwater ( Felis catus) and rats (Rattus spp.) on island breeding colonies are the greatest threat to seabirds [Biodiversity conservation planning requires accurate, current information about species status and threats , 2. Consseabirds . Consequseabirds . Despiteseabirds , as do 2seabirds . Speciesseabirds . Thus, tArdenna creatopus) are known to breed worldwide [We used trail cameras to investigate the assemblage, relative abundance, and seasonal and diel attendance of introduced and native vertebrates on Isla Mocha and Isla Robinson Crusoe (hereafter IRC), Chile, two of three islands where pink-footed shearwaters , black and Norway rats (Rattus rattus and R. norvegicus respectively), and house mice (Mus musculus) [Bos taurus), horses , and other livestock that are limited to a low elevation coastal plain. On IRC, which has been inhabited by humans only post-European contact, introduced species with self-sustaining wild populations reported are cats, dogs, southern coati (Nasua nasua), goats (Capra hircus), European rabbits (Oryctolagus cuniculus), black and Norway rats and house mice [E. asinus x E. caballus) that range in wildlands [Both Isla Mocha and IRC are inhabited by humans, which is a strong predictor of the presence of introduced species on islands . On Islausculus) , 21–23. usculus) , 24. It usculus) , but it use mice –28. IRC ildlands , 26. PriThe goal of our study was to better inform conservation planning for pink-footed Shearwaters and other native species on Isla Mocha and IRC. Although the assemblage of introduced mammals on Isla Mocha and IRC is documented in published literature, we sought to advance our knowledge of the nature and extent of the threat of introduced species on these islands in several ways. First, though there are published records of the introduced species assemblages on both islands, most records were secondary citations of a limited number of primary sources . There wAnimal care and use was reviewed and approved by the BioBío Region of Corporacion Nacional Forestal (CONAF), Chilean Departamento de Areas Silvestres Protegidas of Chile. Bring a purely observational study, further steps were not required to ameliorate suffering nor was review required by other agencies.To quantify the presence and seasonal and diel attendance of animals on Isla Mocha and IRC, we used motion-triggered trail cameras with built-in infrared illuminators for capturing night images. We set cameras to capture images (both photos and videos) 24 hours a day. Details of the methods and duration of the study differed by island.2, 38.383°S, 73.900°W) is 34.2 km offshore of the mainland of south-central Chile. A densely forested central mountain range on Isla Mocha rises to 390 m elevation and is protected as a Chilean National Reserve. Approximately 650 people live in decentralized homesteads on the coastal plain skirting the mountains. Pink-footed shearwaters nest on steep slopes in the mountain above approximately ≥200 m elevation [We deployed trail cameras at five pink-footed shearwater colonies on Isla Mocha during 2015–2020 . Isla Molevation , 13.We monitored colonies in forested areas at 200–350 m elevation and with relatively large numbers of shearwater nests (>100 burrows). Each monitored colony was approximately 1–2 hectares in size. Monitored colonies were spatially separated by >2 km, with one exception in which two colonies were 0.9 km apart. Habitat features and topography were qualitatively similar in the five colonies, in forested areas on or near sleep slopes near the tops of ridges. One colony was located along a hiking trail popular with tourists (Colony 1), one colony was located along a trail mainly used by local people (Colony 3), and three colonies were in places without trails and not typically visited by humans other than researchers . All colonies were ≤1km from, but 200–300 m above, the nearest human settlements.2, respectively  is located 660 km off the South American mainland. Ninety-six percent of IRC is protected as a Chilean National Park. A human community of approximately 1,000 residents lives in San Juan Bautista, the only town on IRC. The island’s topography, volcanic in origin, is rugged and steep. Human settlement of IRC began in 1591 with post-European contact colonization; there is no evidence of prehistoric human occupation [We deployed trail cameras at one pink-footed shearwater colony, Piedra Agujereada (hereafter PAG), on IRC during 2019–2020. IRC  southeast-facing slope ranging from 100–300 m elevation. The area has been deforested and vegetation is characterized by non-native grasses and low-growing forbs . PAG is 2 in 10 m radius circular plots around the camera location. Cameras were 115–143 cm off the ground and were focused perpendicular to the angle of the slope. Vegetation at all camera locations was composed mainly of grasses and forbs ≤30 cm in height [We deployed three trail cameras at PAG from February 2019 to February 2020 and a fourth from January 2019 to December 2019 . Most imn height . Camera Rattus spp., native rodents on Isla Mocha, categorized as Rodentia spp., and “unknown animal” on IRC for unidentifiable animals . In describing methods and results, we refer to all categories of identified animals as “species” for understandability. For each photo or video, we recorded the number of individuals of each species, the date, and time to the second. We collapsed the dataset by 1 hour intervals because image contents in multi-image bursts were auto-correlated and because individual animals could not be identified. For each species in each hour, we used the highest single-image count as the count for that hour.For both islands, we manually reviewed each photo and video and identified all vertebrates to species. Exceptions were introduced rats, categorized as To accurately measure survey effort, we inserted zeros into the dataset as follows: 1) if an hour contained only “empty” images, we counted that hour as a zero for all species; 2) if an hour did not have a given species in it, we counted that hour as a zero for that species; 3) if an hour period passed with no camera triggers, we counted that hour as a zero for all species. We assumed that a camera was functioning the entire date if it was triggered on that date. If a camera was not triggered on a given date, we assumed the camera was working if less than seven days elapsed between the last photo of the previous event and the first photo of the next event , defined as the maximum number of individuals of each species recorded per camera per hour  . We thenOne limitation of this study was that trail camera monitoring was unstandardized on Isla Mocha. The colonies monitored and general focus of cameras on shearwater burrowing areas were consistent from year to year on Isla Mocha, but camera settings, number of cameras, types of images collected, deployment length, and exact camera locations were not. Because of the unstandardized nature of the Isla Mocha dataset, we developed analytical methods (described previously) to retro-actively standardize the data as much as possible, while attempting not to over-apply the data to comparisons for which more standardized data would be necessary. Thus, we pooled results from the five monitored colonies on Isla Mocha and did not attempt to compare metrics among them because they were monitored with differing efforts and methods. We do discuss differences in the basic presence/absence of species among colonies on Isla Mocha.A related limitation of the study is that trail camera methods were standardized on IRC and therefore differed from methods on Isla Mocha, such that the camera settings, positioning, and study duration differed between islands. We could therefore not make direct comparisons of the relative abundance of species on the two islands, and results for each species should be interpreted as island-specific and comparable only to other species on that island. An exception is the relative seasonal and diel CPUE metric scaled to the proportion of the maximum CPUE on each island; this metric can be used to compare attendance of species across islands, with the caveat that the maximum CPUE differed on each island.Lepus europaeus; Scelorchilus rubecula mochae), Mocha austral thrush , black-faced ibis (Theristicus melanopis), black throated huet (Pteroptochos tarnii), Chilean pigeon (Patagioenas Araucana), and Patagonian Sierra-Finch  at PAG. Identified species included seven introduced mammals, one introduced bird, and five native birds , cats, cattle, and rats, respectively, were the next most detected species (Columba livia), house mice, and southern coati (Pterodroma externa), Juan Fernández kestrel , and short-eared owl  or were not observed frequently enough to assess seasonality . There wWe observed pink-footed shearwaters, cats, and rats at all of the five monitored breeding colonies on Isla Mocha. Hares and dogs were observed only in two colonies (Colonies 2 and 3), both located near trails. Pudú were observed at all colonies except Colony 1, which is bisected by a regularly used tourist hiking trail. Humans identified as “tourists” were observed only at Colony 1 and 3, both of which had trails nearby. We did not make further comparisons of relative abundance or attendance of animals in each colony on Isla Mocha because of lack of standardization of trail camera effort on that island.We documented multiple species of introduced mammalian predators and herbivores within pink-footed shearwater colonies on Isla Mocha and IRC. With the exception of the European hare on Isla Mocha, the presence of the mammalian species we documented was previously described for these islands. However, our results provide the first detailed information on presence and relative abundance of introduced species within pink-footed shearwater breeding colonies, and an update and contemporary baseline for the status of introduced species on Isla Mocha and IRC. Collectively, Isla Mocha and IRC hold ≥90% of the world breeding population of pink-footed shearwaters [Though our study was not designed to directly assess the threats of each species to pink-footed shearwaters, there is ample evidence that introduced cats, rats, and dogs depredate burrow-nesting seabirds on other islands –38, and The primary limitation of our study was that trail camera monitoring on Isla Mocha was not standardized. This limited our ability to draw conclusions about inter-annual trends and inter-island spatial differences in relative abundance of animals. We acknowledged this important limitation and attempted to nevertheless use this valuable long-term, multi-colony monitoring data-set from the largest pink-footed shearwater breeding colony to its full potential. However, our CPUE results for pink-footed shearwaters matched well with known breeding phenology of the species, suggesting that the metric was biologically accurate for shearwaters. Our methods provide a template for quantifying unstandardized trail camera datasets collected for conservation purposes by binning data collected with variable settings into standardized time intervals analyzed at the lowest-common-denominator resolution. Though our results must be interpreted cautiously, we show that useful information on presence and relative seasonal and diel patterns of animals can be drawn from unstandardized trail camera datasets and applied to conservation planning. We also caution that the metrics we derived from trail cameras are representative only of what the trail cameras could perceive. More conspicuous animals  were more probably more likely to trigger cameras than small and cryptic ones . Other factors that could influence the seasonal or species-specific probabilities of detecting animals were seasonal changes in vegetation  and diurnal vs. nocturnal behavior . We qualitatively discuss factors likely to affect detection probabilities of various species in the interpretation of results.The near-constant presence of cats in shearwater colonies on both islands  is a serious conservation concern for pink-footed shearwaters. Though it was previously known that cats existed on these islands, these are the first data indicating the scope of the threat of cats as a near-constant presence in pink-footed shearwater colonies. Cats are seabird predators worldwide , 43. PinRats, present on both islands, had a particularly high CPUE on Isla Mocha, and were the second most observed vertebrate there after shearwaters. Rats negatively impact seabirds worldwide , 46, butDogs, coati, and mice are other potential predators of pink-footed shearwaters that were infrequently recorded in our study. Dogs are documented seabird predators on other islands , 49. TraCoati have been observed extracting shearwaters from burrows and killing them at the Vaquería shearwater colony on IRC . However, coati were detected only six times at PAG. Of these, most were in months when shearwaters were not present. These results suggest minimal overlap of coati with shearwaters at PAG, though greater study is needed on the presence of coati at other shearwater colonies on IRC. We recorded house mice only on IRC, where they were infrequently observed. It was unclear whether mouse abundance was actually low or if trail cameras were not easily triggered by mice because of their small size. House mice can have devastating impacts on seabirds, but their impact is situation-specific and depends on factors such as what other introduced vertebrates are present and the availability of plant-based food resources –52. FurtWe detected various mammalian herbivores that may have negative impacts on pink-footed shearwaters. European rabbits were the most abundant species at PAG, with a CPUE more than seven times that of pink-footed shearwaters. CPUEs were affected by each species’ behavior and visibility to trail cameras, and it is possible that the diurnal attendance of rabbits was more likely to be captured than the sometimes brief nocturnal visits of shearwaters, possibly because cameras could capture animals at a greater distance during daylight. Vegetation appeared unlikely to have influenced rabbit or shearwater visibility to cameras, as rabbit CPUE was fairly consistent during all seasons except for a brief, unexplained increase in February-early April, and the relatively low shearwater CPUE from January through May preceded vegetation growth during the wet season (beginning in April to May). Despite potential differences in the ability of cameras to perceive rabbits and shearwaters, the magnitude of the difference in CPUE between the two species suggests than rabbits likely outnumber shearwaters at PAG. This, coupled with rabbits’ year-round presence at PAG, suggests that competition from rabbits for burrow space is a serious conservation concern for pink-footed shearwaters. In another study on the Juan Fernández Archipelago, presence of rabbits in shearwater colonies had a negative impact on pink-footed shearwater burrow occupancy, and burrow occupancy of pink-footed shearwaters increased on Isla Santa Clara, another island in the Juan Fernández Archipelago, after rabbit eradication . Burrow Cattle were regularly present at PAG from June through mid-January 2019. Cattle are also present in low-lying areas of Isla Mocha but were not observed in high-elevation shearwater colonies. Cattle on IRC are managed and are grazed in the area around PAG, where they have negatively impacted shearwaters through burrow trampling and vegetation modification . Trail cThe only mammals on Isla Mocha that were not present on IRC were the European hare, the pudú, and native rodents. Ours is the first study documenting the presence of European hares on Isla Mocha. The European hare is considered a serious pest with negative effects on native ecosystems in continental South America , 55. Haren masse on both islands in early October, and shearwater CPUE was greatest during the pre-laying and incubation periods, which was similar to patterns observed in short-tailed shearwaters (Ardenna tenuirostris) [Calonectris borealis) [Trail camera monitoring allowed us to assess the seasonal and diel attendance patterns of pink-footed shearwaters, which had previously been reported only generally . Seasonarostris)  and Coryorealis) . This coorealis) , 60. On orealis) . We did Puffinus yelkouan) showed peaks in colony attendance during the three hours after dusk and the two before dawn, consistent with the pink-footed shearwater attendance patterns we observed [The strictly nocturnal behavior of pink-footed shearwaters at the breeding colony was consistent with results from a previous GPS-tracking study , and wasobserved . We did observed –65, thouOctodon pacifica) and Isla Mocha ground frog (Eupsophus insularis), both of which are critically endangered [Sephanoides fernandensis) hummingbird on IRC [Camera trap data proved useful for broadly describing the assemblages and patterns of native and introduced vertebrates within the breeding colonies of pink-footed shearwaters. Our results also highlight broader conservation issues on Isla Mocha and IRC, with introduced species having the potential to impact endemic and endangered plant and animal species. For example, on Isla Mocha, cats and rats may threaten multiple endemic bird sub-species , as welldangered , 67. Likd on IRC . Cattle d on IRC . Our resS1 Abstract(PDF)Click here for additional data file.S1 TableFull citations are listed in the main text of the manuscript with the exception of the articles with full citations listed below the table.(PDF)Click here for additional data file.S2 TableIn that study, 295 points were randomly generated across Isla Mocha in areas considered suitable habitat for pink-footed shearwater breeding based on elevations and slopes. The points that had burrows present are shown.(PDF)Click here for additional data file.S1 FigPAG is the Piedra Agujereada colony on Isla Robinson Crusoe. Each “cuadrante” on Isla Mocha is a separate breeding colony.(TIF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Figs the proportion of the island-specific maximum CPUE for each day on each island. The largest point represents the highest CPUE for that species on that island. Red line indicates no data. Black line indicates effort without detections on that date. Species with no colored data points were not observed on that island.Proportion of island-specific maximum CPUE was calculated by dividing each species’ hourly CPUE by the maximum recorded hourly CPUE for that species on each island, and averaging the hourly results for each day. This unitless relative CPUE metric can be used to compare seasonal attendance across islands, but does not reflect actual magnitude or relative abundance of species because the maximum CPUEs were species- and island-specific. Circle size indicate(TIF)Click here for additional data file.S5 FigProportion of island-specific maximum CPUE was calculated by dividing each species’ hourly CPUE by the maximum recorded hourly CPUE for that species on each island, and averaging the results for each hour of the day. This unitless relative CPUE metric can be used to compare diel attendance across islands, but does not reflect actual magnitude or relative abundance of species because the maximum CPUEs were species- and island-specific. Bay size indicates the proportion of the island-specific maximum CPUE for each day on each island. The longest bar represents the highest CPUE for that species on that island.(TIF)Click here for additional data file.S1 Data(CSV)Click here for additional data file.S2 Data(CSV)Click here for additional data file.S3 Data(XLSX)Click here for additional data file.