Accelerating technological progress in experimental neuroscience is increasing the scale as well as specificity of both observational and perturbational approaches to study circuit physiology. While these techniques have also been used to study disease mechanisms, a wider adoption of these approaches in the field of experimental neurology would greatly facilitate our understanding of neurological dysfunctions and their potential treatments at cellular and circuit level. In this review, we will introduce classic and novel methods ranging from single-cell electrophysiological recordings to state-of-the-art calcium imaging and cell-type specific optogenetic or chemogenetic stimulation. We will focus on their application in rodent models of Parkinson’s disease while also presenting their use in the context of motor control and basal ganglia function. By highlighting the scope and limitations of each method, we will discuss how they can be used to study pathophysiological mechanisms at local and global circuit levels and how novel frameworks can help to bridge these scales. This pr1.2In the following sections, we will focus on the use of rodent animal models due to their wide adoption in the field and the availability of an extensive repertoire of genetic tools to investigate circuit (dys)function in PD. Neurotoxins, such as 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP), induce a selective degeneration of dopaminergic neurons in the SNc of rats and mice leading to motor impairment . They ar1.3Anatomical tracing methods have established an extensive network of brain regions involved in motor control, including and extending beyond the interconnected basal ganglia nuclei. For example, the striatal direct, indirect and hyperdirect pathways have been identified to serve distinct but complementary functional roles in movement . Novel a1.4DBS is a neurosurgical treatment using chronically implanted electrodes to deliver therapeutic electrical stimulation to specific brain regions, such as the subthalamic nucleus (STN), the internal globus pallidus (GPi) or the ventral intermediate nucleus (VIM) of the thalamus. It has been successful in treating motor symptoms of PD, dystonia and essential tremor with current efforts expanding the indications beyond movement disorders . Studies22.1A classic approach to study neuronal activity is based on the recording of electrical signals. When the recording electrode is in contact with the intracellular space, small amplitude subthreshold synaptic currents/potentials and suprathreshold action potentials (AP) can be measured, whereas extracellular recordings can only detect neuronal spikes . In the Ex vivo patch-clamp recording provides detailed information about electrical neuronal activity of single neurons. This technique allows direct recording of fluctuations in membrane potential or current at a high temporal resolution which enables for unambiguous detection of ion channel conductances, single APs and subthreshold excitatory or inhibitory postsynaptic potentials , the transition to burst spiking mode was dependent on inactivating sodium channels . As thisMulti-neuron patch-clamp recording reveals the synaptic properties of local microcircuits. To record and characterize functional monosynaptic connections in patch-clamp recordings, it is necessary to elicit action potentials in a presynaptic neuron and simultaneously record subthreshold responses in the postsynaptic neuron. This can be achieved by recording from multiple neurons within the same brain slice . StimulaCombining patch-clamp recordings with electrical or optogenetic stimulation can uncover pathway-specific synaptic properties and pathological plasticity changes. Another way to elicit presynaptic action potentials to study synaptic inputs is by electrical stimulation of afferent axonal tracts. This can simulate the impact of upstream regions even if they are not within the brain slice. Such approaches were combined with pharmacological interventions to identify the contribution of NMDA-receptor subunits and D5-receptors for synaptic transmission in STN neurons . Monitor2.2In vivo single cell recordings can identify cell-type specific spiking patterns that arise within an intact or pathologically altered circuit. Brain slices are useful to study local mechanisms in controlled and isolated systems, but in vivo recordings are necessary to understand the function of neuronal activity that arises from the complex interaction between local and long-range inputs across dynamical brain states. Sharp electrode and patch-clamp recordings can be used to detect sub-threshold activity while in vivo multi-neuron patch-clamp recordings are able to characterize synaptic transmission in an intact brain . Positioning the electrode juxtacellularly still allows the recorded neuron to be labelled with neurobiotin to pair precise post-mortem genetic, neurochemical or structural characterization with its in vivo electrophysiological properties. Such recordings have been used to characterize cell-type specific spiking activity in GPe  using extracellular microelectrodes can monitor the local population activity during movement and motor symptoms. Microwire electrodes in the extracellular space can detect large and fast voltage changes generated by nearby APs as spiking activity , Table 1 sorting . At the  sorting . These r sorting . Recordi sorting  and that sorting . However sorting . Chronic sorting . While sin vivo monosynaptic transmission  based fabrication process have led to the development of Neuropixels probes with a higher electrode density. The first version has one 10 mm shank with 960 recording sites that can be selected for 384 channels and the newer 2.0 version accommodates 5120 recording sites distributed over four shanks with 384 channels for each shank . This tein vivo at spiking resolution opens up new and exciting ways to study neuronal activity at local and global scales  can report changes in intracellular calcium concentration, termed calcium transients, by enhanced emission of fluorescent light  or carried in transgenic mouse lines, on the presence of specific enzymes. A popular system to be used is the Cre/ loxP system , allowin3.2miniscope) and allows the observation of calcium-related activity of up to hundreds of single cells in deep brain regions of freely moving animals  lenses with miniaturized, head-wearable microscopes  brain regions in freely moving mice , Table 1Overall, calcium imaging methodology yields large scale recording of neuronal activity within selected circuit elements such as cell types and their projections, as well as subcellular compartments such as dendritic spines and axonal boutons. While two-photon and micro-endoscopy approaches can reveal complex heterogeneity of individual cellular activity associated with specific functions or events, fiber photometry allows for understanding of average neuronal activity of a distinct cell type at its soma or synaptic level at a given time. This is especially useful for investigation of long-range projection pathways, such as those originating from SPNs. In contrast to electrophysiological techniques, calcium imaging potentially allows observation of a significantly larger number of identified neurons, so that neural ensemble dynamics related to motor behavior can be studied. In addition, further development of fluorescent sensors will not only enable researchers to improve calcium imaging, but also, for example, to measure release of neuromodulators, such as dopamine or serotonin . The dev44.1Complementing to the observational electrophysiological and calcium imaging recordings, optogenetics is a state-of-art method that allows targeted perturbation of identified cell groups within a specific (deep) brain region and their projections within a circuit by expression and activation of light-sensitive proteins –2. Sincein vivo calcium imaging techniques and inhibitory opsins  induced dyskinesia (LID), which is the main therapy-limiting adverse effect and very difficult to treat . As discFunctional dissection of afferent and efferent projections of the STN demonstrates their potential contributions to therapeutic DBS. Optogenetics is a powerful tool for studying the mechanisms by which DBS modulates neuronal activity in a cell-type and pathway-specific manner, thereby supporting future refinement of stimulation protocols and electrode placement . In lineOptogenetic dissection of the hyperdirect pathway and its long-range projections highlights its role in therapeutic DBS. Considering the network nature of motor function, and beyond a focus on local STN-DBS induced changes, several studies addressed the role of cortical inputs to the STN, the so-called hyperdirect pathway. An early study from Gradinaru et al. reported, comparing electrical stimulation and cell type-specific optogenetic perturbation of STN neurons in 6-OHDA mice, that neither activation nor inactivation of glutamatergic populations in the STN had a therapeutic effect on motor behavior . HoweverOn a microcircuit level, electric STN-stimulation increased firing rates of optogenetically identified somatostatin (SST) interneurons in the M1. Optogenetically activating those SST neurons reduced PD motor symptoms in 6-OHDA mice . These rIn the context of STN stimulation, optogenetics was used to understand the impact of stimulation frequency patterns. To support specific frequency protocols, the employed opsin needs to have suitable temporal kinetics . A direcModern circuit-centered neuroscience has uncovered brainstem circuits for locomotor control. Until recently, most studies on PD circuit mechanisms have focused on the BG network. However, the consequent refinement of optical and genetical methodology, such as miniaturisation of hardware and ever-increasing specificity and temporal precision of observational and perturbational tools, now enables systematic investigation of evolutionarily older subcortical brain regions instrumental for motor control. Specifically, brainstem circuits are crucial network elements for regulation of motor functions, such as locomotion and behavioral arrest . Importain vivo electrical recordings revealed a role of the lateral rostral medulla in forelimb reaching and handling (Besides behavioral arrest and freezing, brainstem circuits have long been implicated in the control of active locomotor patterns . More rehandling . These s4.2Another novel approach to manipulate neuronal activity of specific circuit elements utilizes genetically engineered ion channels or receptors exclusively activated by inert synthetic ligands which can be administered systemically . SimilarMotor symptoms in PD mouse models have been reverted by chemogenetics. Dopamine depletion in PD affects the normal activity of multiple basal ganglia nuclei. Therefore, chemogenetic manipulation of neuronal activity within specific neuron subpopulations can be used to counterbalance circuit dysfunction. For example, selective chemogenetic activation of dSPN was shown to mimic L-DOPA therapeutic effect in 6-OHDA-lesioned mice, whereas chemogenetic stimulation of iSPN reduced LID severity . SelectiBeyond chemogenetics, novel genetic tools allow inducible inactivation of endogenous proteins to perturb neuronal function and behaviour. In mice, inducing a knockout of D2-receptors during adulthood has been shown to cause PD-like phenotypes with typical pathological neural activity recorded using silicon probes . Patch-c5Technological advances in recent years have resulted in a vast array of promising neuroscientific tools that allow recording and perturbation of neural activity in unprecedented detail. This development now allows basic and clinical researchers to adequately address the complexity of the mammalian nervous system as a global network of interconnected microcircuits. By combining correlational and causative approaches, thereby functionally characterizing individual circuit elements and their input-output connectivity, truly mechanistic insights into the underpinnings of normal and pathologic neuronal conditions can be gained. In this review, we have introduced a range of advanced electrophysiological, optical, and genetic approaches –2 that ain vivo.Observational electrophysiological and optical methods provide different spatial and temporal resolutions. When deciding which method to use for a specific research question, it is vital to consider the spatial and temporal scale of the studied effect. Two-photon calcium imaging provides a very high spatial resolution as it can visualize neuronal activity in individual somata and even in subcellular compartments such as axons and dendrites. Furthermore, thousands of neurons can be recorded simultaneously depending on the regions scanned by the laser. Single-photon microendoscopy has a lower spatial resolution, but is suited to study deeper brain regions . HoweverCombining optogenetics with optical or electrophysiological large-scale recordings can help to bridge the macro- and microcircuit level. Previous technical constraints limited recordings to only a few hand-selected cells and theoretical frameworks focused on neurons as single computational units with properties such as firing rates or spiking variability . With thModern circuit neuroscience methods have a great translational value in advancing our understanding of PD as a circuitopathy and to develop novel DBS approaches. Recent studies that have manipulated precise circuit elements highlight the ability of optogenetic studies to uncover novel principles of basal ganglia computation  and addrKey questions remain as to how brainstem nuclei contribute to parkinsonian symptoms. Given that cortico-basal ganglia circuits mediate a large proportion of motor output through these areas, their output will inevitably be indirectly affected by pathological changes in those outputs. Ascending projections from the brainstem back to these input areas may then further contribute to network dysfunction. Importantly however, several areas of the brainstem may also be directly affected by neurodegeneration in PD, and this occurs before extensive dopamine depletion . Cell-tyin situ.However, a challenging aspect of perturbational circuit-centered approaches is the nature of the studied object itself - a network of connected circuit elements that interact to generate specific functions. It is therefore important to cautiously interpret effects due to manipulation of individual circuit elements . PerformFinally, the successful translation from mechanistic insights into PD circuit pathophysiology from rodent models towards human patients requires close collaboration between basic scientists and clinicians. This can be achieved within interdisciplinary research consortia, organised around a focus topic, and following unifying working hypotheses. Beyond bringing together researchers from different backgrounds, they also provide ideal environments for the training of a new generation of clinician and medical scientists, which are capable of integrating complex circuit neuroscience with theoretical knowledge of neurodegenerative disease and practical experience of the clinical routine.


Acute hypoxemic respiratory failure is one of the leading causes of intensive care unit admission and is associated with high mortality. Noninvasive oxygenation strategies such as high-flow nasal cannula, standard oxygen therapy, and noninvasive ventilation (delivered by either face mask or helmet interface) are widely available interventions applied in these patients. It remains unclear which of these interventions are more effective in decreasing rates of invasive mechanical ventilation and mortality. The primary objective of this network meta-analysis is to summarize the evidence and compare the effect of noninvasive oxygenation strategies on mortality and need for invasive mechanical ventilation in patients with acute hypoxemic respiratory failure.We will search key databases for randomized controlled trials assessing the effect of noninvasive oxygenation strategies in adult patients with acute hypoxemic respiratory failure. We will exclude studies in which the primary focus is either acute exacerbations of chronic obstructive pulmonary disease or cardiogenic pulmonary edema. The primary outcome will be all-cause mortality (longest available up to 90 days). The secondary outcomes will be receipt of invasive mechanical ventilation (longest available up to 30 days). We will assess the risk of bias for each of the outcomes using the Cochrane Risk of Bias Tool. Bayesian network meta-analyses will be conducted to obtain pooled estimates of head-to-head comparisons. We will report pairwise and network meta-analysis treatment effect estimates as risk ratios and 95% credible intervals. Subgroup analyses will be conducted examining key populations including immunocompromised hosts. Sensitivity analyses will be conducted by excluding those studies with high risk of bias and different etiologies of acute respiratory failure. We will assess certainty in effect estimates using GRADE methodology.This study will help to guide clinical decision-making when caring for adult patients with acute hypoxemic respiratory failure and improve our understanding of the limitations of the available literature assessing noninvasive oxygenation strategies in acute hypoxemic respiratory failure.CRD42019121755PROSPERO  Acute hypoxemic respiratory failure (AHRF) is among the leading causes of intensive care unit (ICU) admission in adult patients, , with ar2:FiO2 < 200 mmHg) HFNC decreased the need of invasive ventilation when compared with the other two modalities. Notably, the highest mortality was seen among the FM-NIV treatment arm. This study started to raise concerns for potential harmful effects associated with FM-NIV, which were further supported by two large observational studies [Previous studies have aimed to identify the optimal initial oxygenation strategy to avoid the receipt of invasive mechanical ventilation in adults with AHRF but have often produced conflicting results –14. Clas studies , 19. Pot studies . As a re studies . InteresThe use of a helmet interface to provide NIV (H-NIV) poses an attractive alternative to provide both oxygenation and ventilation in AHRF, potentially with better patient tolerability, patient-ventilator interaction, and the ability to provide ventilation for longer periods of time . The potDetermining the optimal strategy to decrease rates of endotracheal intubation in patients with AHRF is essential to minimize morbidity and mortality associated with the need for invasive mechanical ventilation. Although HFNC has become widely used, evidence is lacking regarding its effect as compared to standard oxygen, and there are no head to head comparisons between HFNC and H-NIV. The limitations of the current evidence are clearly reflected in clinical practice, where there exists high variability among clinicians in the use of these four noninvasive oxygenation strategies for AHRF . FurtherNetwork meta-analysis (NMA) is a strategy to summarize the evidence of clinical studies which include multiple treatments . By incoThe primary objective of this NMA is to assess the association between different noninvasive oxygenation strategies and all-cause mortality in adult patients with AHRF. The secondary objective is to assess the association between different noninvasive oxygenation strategies and the receipt of invasive mechanical ventilation in adult patients with AHRF.This protocol has been written in accordance with the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols 2015 statement (PRISMA-P) and its extension for NMA , 34. ThiWe will include any studies that enrolled adult patients (18 years of age or older) with AHRF, defined by the new onset of clinical signs , radiologic signs , and gas exchange alterations (different degrees of hypoxemia). We will consider studies including patients treated in the intensive care unit, intermediate care unit, medical wards, and emergency department. Studies primarily focused in the treatment of acute exacerbations of chronic obstructive pulmonary disease (AE-COPD) or congestive heart failure (CHF) will be excluded. Specifically, we will exclude such studies if 50% or more of the included population are patients with either AE-COPD or CHF, acknowledging that a lower threshold will exclude too many studies in the field. Our pre-planned sensitivity analysis will specifically address this issue. The rationale for exclusion is based in the already shown superiority of noninvasive ventilation for these conditions. Furthermore, we will also exclude studies including patients after extubation for major thoracic surgery.2 up to 1.0 and a flow rate that can be as high as 60 l/min. NIV provides positive pressure ventilation that can be delivered with different modalities: continuous positive airway pressure, bi-level positive airway pressure, or pressure support ventilation. The interfaces used can be either a face mask (FM-NIV) or a helmet (H-NIV). Given the potential differences in reported outcomes, these interfaces will be considered as two different interventions in the network. SOT (low-flow systems) comprise traditional nasal cannula, Venturi system masks, or non-rebreather masks. All of these modalities can provide oxygen at low flow (< 15 l/min) with varying levels of FIO2.All interventions considered in this review are noninvasive oxygenation strategies. HFNC oxygenation refers to oxygen delivered through a nasal cannula, at a FIOThe primary outcome of this study will be all-cause mortality, defined as the longest available in the first 90 days after randomization. The secondary outcome will be receipt of invasive mechanical ventilation (longest available at 30 days). Given that the criteria for endotracheal intubation might vary between studies, we will collect information on whether decisions to intubate were based on pre-specified criteria or not.We will include only randomized controlled trials focused on the treatment of acute hypoxemic respiratory failure if a combination of any of these interventions was assessed: HFNC, FM-NIV, H-NIV, and SOT.Ovid MEDLINE, (2) Ovid EMBASE, (3) PubMed (non-MEDLINE records only), (4) Ovid EBM Reviews-Cochrane Central Register of Controlled Trials, (5) EBSCO CINAHL Complete, (6) Web of Science, and (7) LILACS. We will also search ClinicalTrials.gov, WHO International Clinical Trials Registry Platform, and International Standard Randomized Controlled Trial Number Registry for all registered clinical trials and randomized controlled trials. The search strategy will be structured according to the Peer Reviewed Electronic Search Strategies (PRESS) 2015 Guidelines [Cochrane Handbook for Systematic Reviews of Interventions Version 5.1.0, Section 6.4.11 [Ovid MEDLINE, Ovid EMBASE, and PubMed. We will adapt a pre-tested search filter for randomized controlled trials from the Scottish Intercollegiate Guidelines Network [EBSCO CINAHL Complete and Web of Science. No limits will be applied to language, publication year, gender or race. We will manage all references and duplicates using EndNote X8 citation management software.The following seven electronic bibliographic databases will be searched using a comprehensive search strategy developed by an information specialist (MR): (1) idelines  (refer tn 6.4.11  to scree Network  for EBSCTwo reviewers (BLF and FA) will screen independently the titles and abstracts retrieved from the search strategy and the additional sources in order to identify those meeting the mentioned eligibility criteria. Subsequently, we will obtain full texts of the articles meeting these pre-specified criteria and review again in a second stage. Any disagreement between the reviewers will be discussed and referred to final decision by a third investigator .2:FIO2 ratio < 200)), details of the interventions , outcome data for each endpoint of interest.The two reviewers (BLF and FA) will perform data extraction independently in a pre-piloted data extraction form created in Excel (Microsoft®). Any differences will be resolved by consensus or discussion with a third author (LM). Abstracted data will include study characteristics , patients’ characteristics  between direct and indirect comparisons will be estimated using the node-splitting approach contrasting estimates from both direct and indirect evidence [We will summarize the included studies based on study and patient characteristics, outcome measures, and risk of bias. We will perform a series of pairwise Bayesian meta-analyses with a random-effects model, followed by a network meta-analysis using a Bayesian framework to derive head-to-head treatment effect estimates comparing all interventions. Analyses will be based on Markov chain Monte Carlo methods using prior distributions for event rates derived from previous literature, minimally informative treatment effect estimates, and informative prior distributions for heterogeneity estimates derived from external evidence . We willevidence , 40. We We will assess for the presence of publication bias by examining the comparison-adjusted funnel plot . We will2:FIO2 ratio < 200) vs less severe AHRF (PF ratio > 200), hypothesizing that HFNC is better than SOT for patients with severe AHRF, and (2) immunocompromised patients  vs non-immunocompromised patients, hypothesizing that NIV may be more effective in immunocompromised patients.Where information is available, we will assess for credible subgroup effects using a random-effects NMA meta-regression Bayesian model to determine whether the estimated treatment effects are affected by the following factors: (1) severe AHRF  Working Group . The netIn summary, this Bayesian network meta-analysis will provide a comprehensive summary of the direct and indirect evidence on the efficacy of different noninvasive oxygenation strategies to prevent mortality and the receipt of invasive mechanical ventilation in adult patients with acute hypoxemic respiratory failure. The current protocol was written in accordance with the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols 2015 statement (PRISMA-P) and its extension for NMA. This protocol is also registered in the International Prospective Register of Systematic Reviews (PROSPERO). The overall quality of the evidence will be assessed using the Grading of Recommendations Assessment Development and Evaluation (GRADE) approach.We expect this study will help with clinical decision-making when caring for adult patients with AHRF. Furthermore, the systematic approach will improve our understanding of the limitations of the available literature to inform the treatment of patients with AHRF. Finally, we believe that our results will help identify predictive enrichment strategies that may improve the design of future randomized controlled trials.Additional file 1. NMA-CRFRI.


Although a positive association has been established, it is unclear whether lower respiratory tract infections (LRTIs) with respiratory syncytial virus (RSV) cause chronic wheezing illnesses. If RSV-LRTI were causal, we would expect RSV-LRTI prevention to reduce the incidence of chronic wheezing illnesses in addition to reducing acute disease. We aimed to evaluate the strength of evidence for a causal effect of RSV-LRTI on subsequent chronic wheezing illness to inform public health expectations for RSV vaccines.b) quantify differences between exposure and comparator groups on the loge odds ratio (loge OR) scale.We did a systematic review and meta-analysis of observational studies evaluating the association between RSV-LRTI and subsequent wheezing illness (exposure studies) and studies evaluating the association between RSV immunoprophylaxis and subsequent wheezing illness (immunoprophylaxis studies). Exposure studies were included if the exposure group members had an LRTI with laboratory-confirmed RSV and if the exposure ascertainment period began before 2 years of age and ended before 5 years of age. We required a wash-out period of more than 30 days between the index RSV-LRTI and the outcome measurement to allow for resolution of the acute illness. Comparisons between RSV-LRTI and non-RSV-LRTI were not included. Immunoprophylaxis studies were included if they measured the association with subsequent wheezing illness relative to a control group, either in a randomised controlled trial (RCT) or an observational design. For the immunoprophylaxis drugs in question, we required evidence of efficacy in targeting RSV-LRTI from at least one RCT to ensure biological plausibility. All variations of wheezing illness were combined into a single outcome that refers broadly to asthma or any other respiratory illness with wheezing symptoms. Ovid MEDLINE and Embase databases were searched from inception up to Aug 28, 2018. We evaluated whether data from exposure studies could provide evidence against the most viable non-causal theory that RSV-LRTI is a marker of respiratory illness susceptibility rather than a causal factor. Additionally, we tested whether RSV immunoprophylaxis reduces the odds of subsequent wheezing illnesses. We used a random-effects modelling framework and, to accommodate studies providing multiple correlated estimates, robust variance estimation meta-regressions. Meta-regression coefficients  compared with those that did not , a significant difference . Infants who were not protected with RSV immunoprophylaxis tended to have higher odds of subsequent wheezing illness, as we would expect if RSV-LRTI were causal, but the effect was not significant . There was generally a high threat of confounding bias in the observational studies. Additionally, in both the observational studies and immunoprophylaxis RCTs, there was high risk of bias due to missing outcome data.From 14 235 records we identified 57 eligible articles that described 42 studies and provided 153 effect estimates. 35 studies estimated the direct effect of RSV-LRTI on wheezing illnesses (exposure studies) and eight evaluated the effect of RSV immunoprophylaxis (immunoprophylaxis studies). Exposure studies that adjusted for genetic influences yielded a smaller mean adjusted OR estimate (aOROur findings, limited to exposure and immunoprophylaxis studies, do not support basing policy decisions on an assumption that prevention of RSV-LRTI will reduce recurrent chronic wheezing illnesses.Bill & Melinda Gates Foundation. Lower respiratory tract infections (LRTIs) caused by the respiratory syncytial virus (RSV) contribute substantially to infant aged 0–1 years) morbidity and mortality, years moEvidence before this studyAlthough there is a well established positive association between respiratory syncytial virus lower respiratory tract infections (RSV-LRTIs) and subsequent wheezing illnesses, it is unclear whether RSV-LRTI is a causal factor, a marker of susceptibility to respiratory illness, or both. Before doing the literature search for this Article, we searched PubMed and Google Scholar for articles published from inception until May 12, 2018. The full list of search terms is available in the appendix (pp 28–37). We identified two relevant meta-analytic reviews: the first reported a positive mean association (odds ratio [OR] 3·84) between RSV hospitalisation and subsequent asthma or wheezing in 15 studies; and the second also reported a significant mean association (OR 4·03) between RSV-bronchiolitis and asthma. In terms of causal evidence, a population-based birth cohort study showed that asthma risk was temporally associated with birth timing in close proximity to winter viral bronchiolitis epidemics, consistent with a causal effect. In contrast, twin-registry studies suggested that RSV-LRTI is more likely a marker of genetic risk than a causal contributor to asthma. One of the two randomised controlled trials of RSV immunoprophylaxis that evaluated effects on wheezing illness showed a reduction in parent-reported wheezing outcomes in those aged between 1 and 6 years, but neither trial found evidence of reductions in medically-attended wheezing illness.Added value of this studyAlthough previous meta-analyses provided estimates of the association between RSV-LRTI and recurring wheezing illness, this Article was, to our knowledge, unique in that it had the explicit goal of appraising the strength of evidence for a causal effect. We could have increased confidence in a causal effect by providing evidence against the most plausible non-causal theory  and showing that RSV immunoprophylaxis protects against childhood wheezing illness. However, our findings did not support either of these conditions. These findings do not rule out a causal effect but suggest that the current evidence does not support basing policy decisions on an assumption that prevention of RSV-LRTI will reduce recurrent wheezing illnesses.Implications of all the available evidenceOur findings, in combination with previous RSV twin studies, are consistent with the hypothesis that shared genetic predisposition accounts for a substantial proportion of the association between RSV-LRTI and subsequent wheezing. Individual studies and meta-analyses that do not account for shared genetic risk could substantially overestimate the effect of RSV-LRTI on recurring wheezing illness. Additionally, we found insufficient evidence that RSV immunoprophylaxis prevents wheezing illness, which we would expect if RSV-LRTI were causal. Long-term follow-up data from ongoing trials are needed before assuming RSV-LRTI prevention might lead to reduction in recurrent wheeze or asthma. Additionally, more research is needed to assess the potential effects of RSV infection without LRTI and potential host–viral interactions.We present three possible models for the established association between RSV-LRTI and wheezing illness .10, 11 IIn this Article, we use meta-regression to consider both causal and non-causal models of the association between RSV-LRTI and subsequent wheezing illness and evaluate whether their unique implications  are consistent with empirical data.vs placebo) affects risk for wheezing illness only by limiting the severity of RSV-LRTI exposure and not through any other mechanisms.25Studies evaluating the association between RSV immunoprophylaxis and subsequent wheezing illness (immunoprophylaxis studies) can also help to appraise the strength of evidence for a causal effect . If RSV-We were commissioned by WHO to do a systematic review and meta-analysis to assess the evidence for a causal effect of RSV-LRTI on subsequent wheezing illness to inform public health expectations for RSV vaccines.PanelRespiratory syncytial virus (RSV)-associated lower respiratory tract infection (LRTI) exposure studiesEvaluate the direct association between RSV-LRTI and subsequent wheezing illness using an observational designViral surveillance studiesDo viral surveillance over a discrete exposure ascertainment period: those developing RSV-LRTI during this period form the exposure group and those with no LRTI form the comparison groupMedical event studiesCompare children (aged 0–5 years) with an RSV-LRTI medical event  during the exposure ascertainment period to those without an LRTI-related medical event (comparator group)RSV immunoprophylaxis studiesEvaluate the association between receipt of RSV immunoprophylaxis with established efficacy and subsequent wheezing illnessRandomised controlled trialRandomly assign infants (aged 0–1 years) to receive either RSV immunoprophylaxis or placeboObservational (non-randomised) studyCompare infants (aged 0–1 year) receiving RSV immunoprophylaxis in their normal clinical care to those not receiving immunoprophylaxis The protocol for this systematic review and meta-analysis provides complete methodological details (appendix pp 3–27). As there was no involvement of human participants or identifiable information, institutional review board approval was not required. We have described the two types of published peer-reviewed studies included in the analysis and all the inclusion criteria , table 1RSV-LRTI exposure studies were included if the exposure group members had an LRTI with laboratory-confirmed RSV. LRTI was considered present if children were diagnosed with relevant illnesses , had relevant clinical indications , or received treatment in hospital for RSV-related illnesses. As we were interested in early life RSV-LRTI, the exposure ascertainment period had to begin before 2 years of age and end before 5 years. These exposure studies could be further categorised by study design. Some exposure studies determined RSV-LRTI status by doing viral surveillance over a defined ascertainment period (surveillance studies), whereas others compared individuals with an RSV-LRTI medical event  to those without an LRTI medical event . We required a wash-out period of more than 30 daysWe included RSV immunoprophylaxis studies if they measured the association with subsequent wheezing illness relative to a control group, either in an RCT or an observational design. For the immunoprophylaxis drugs in question, we required evidence of efficacy in targeting RSV-LRTI from at least one RCTAn expert systematic review information specialist did a literature search of Ovid MEDLINE and Embase databases using the population, intervention or exposure, comparator, outcome, study design framework,Four study investigators  with doctoral degrees in health-care sciences completed record reviews and data abstraction. Literature search records were reviewed in two stages. Stage 1 eliminated records that were irrelevant based on the study abstracts alone. Two of these investigators evaluated each abstract independently, blinded to the others' ratings. Any article deemed potentially relevant by either reviewer was retained. In stage 2, two investigators independently and redundantly reviewed the full text of the articles that passed stage 1 to determine whether the full inclusion criteria were met.For each article, one of the four investigators abstracted relevant data and a different investigator did quality assurance reviews, resolving discrepancies by consensus. For estimates from RSV-LRTI exposure studies, we noted whether or not analyses controlled for genetic predisposition to wheezing illness and neonatal health proxies that might be markers of respiratory illness predisposition . As RSV often co-occurs with other respiratory infections that are also positively associated with subsequent wheezing illness,e odds ratio (logeOR). Whenever possible, for RSV-LRTI exposure studies and non-randomised immunoprophylaxis studies, we included estimates from analyses in which there was explicit effort to adjust for confounders, as these estimates were generally expected to be less biased than unadjusted estimates. Some studies reported other adjusted ratio-based effect estimates . Rather than excluding these estimates or replacing them with unadjusted ORs, we included them in our analyses as the best-available estimates. 17% of estimates (26 of 153) used non-OR ratio estimates as stand ins for OR estimates, which pulls the estimated mean OR systematically toward the null.We quantified differences between exposure and comparator groups using the logR version 3.5.0. RVE allows studies to contribute multiple correlated estimates while providing accurate point estimates and confidence intervals.b) are reported to quantify covariate effects on the logeOR scale.To accommodate studies providing multiple correlated estimates, we did robust variance estimation (RVE) meta-regressionsWe used the Newcastle-Ottawa ScaleThis study was commissioned by WHO through a grant from the Bill & Melinda Gates Foundation. A WHO RSV expert (DRF) contributed to the study conceptual isation, protocol and search strategy development, interpretation of results, and manuscript editing. AJD was supported by the National Institute of Health (NIH). The NIH had no role in the study design, data collection, analysis, interpretation of data, writing of the report, or in the decision to submit the paper for publication. The Gates Foundation had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.From 14 235 initial records, we identified 57 eligible articles, describing 42 studies, and providing 153 effect estimates . In addi+) indicated that children exposed to RSV-LRTI had a 3·39 times increase in odds of subsequent wheezing illness (95% CI 2·72–4·24). OR+ remained positive when limiting the analysis to effect estimates of RSV-LRTI on asthma outcomes measured at age 6 years and older .Among RSV-LRTI exposure studies, the unconditional mean OR . As shown in +) was considerably smaller when estimates accounted for genetic influences  relative to those that did not . The one studyb 0·63, 95% CI 0·05–1·20). However, when sequentially removing one study at a time  from the analysis, eight studiesIn our primary model, effect estimates for the association between RSV-LRTI and wheezing illness differed depending on whether estimates were adjusted for genetic influences (+ 1·21), indicating that those not receiving immunoprophylaxis (with presumably greater risk of RSV-LRTI) tended to have higher odds of subsequent wheezing illness, but the 95% CI (0·73–1·99) included the null . Owing to the small sample size (eight estimates), there was considerable uncertainty around the mean estimate among the two RCTs and no evidence of increased odds of wheezing illness among children who did not receive RSV immunoprophylaxis .In our primary model of RSV immunoprophylaxis studies, the mean effect estimate was positive  contributing to the model, our primary estimate of the effect of immunoprophylaxis on wheezing illness had a wide 95% CI (0·73–1·99), indicative of insufficient evidence for benefit or harm. Additional, preferably large, RCTs would improve precision.Future observational studies evaluating the association between RSV-LRTI and wheezing illness are unlikely to be helpful in resolving the question of causality unless they accurately account for genetic influences. Additional twin registry studies would be valuable, particularly if the data could be combined to improve precision. Although we have focused on genetic risk as a potential confounder, future studies should also evaluate potential gene and RSV interactions.Regarding RSV immunoprophylaxis studies, RCTs are likely to provide less biased estimates than observational studies.Ultimately, evidence of causality in epidemiological research is always uncertain.The primary limitation of this Article is that it did not address all the evidence relevant to assessing whether RSV-LRTI causes wheezing illness. Several important studies did not meet our inclusion criteria. Notably, in a study of monozygotic and dizygotic twins, Thomsen and colleaguesThis study had notable strengths. We used RVE meta-regression allowing for inclusion of multiple correlated estimates from the same study. Additionally, our search strategy was peer-reviewed by an expert information specialist. Finally, we provided all analytic code, allowing for replication and extension of our findings. Our assessment of findings from observational studies and immunoprophylaxis RCTs could neither discount a plausible non-causal model of the association between RSV-LRTI and subsequent wheezing illness, nor confirm that RSV immunoprophylaxis protects against wheezing illness. Consequently, this study does not provide compelling support for a causal effect of RSV-LRTI on subsequent wheezing illness. RSV-LRTI prevention will probably have a substantial public health effect by reducing complications associated with the acute infection;All analyses and R code are available at https://brunwasser.github.io/whorsv.github.io/.


Patients with vascular Ehlers-Danlos syndrome (EDS) occasionally suffer from arterial dissection. Eagle syndrome, which is caused by an elongated styloid process and also causes arterial dissection, is difficult to diagnose and could sometimes be overlooked. Little is known of the coexistence of these two diseases, and treatment strategy is not established. Here, we present a case of bilateral internal carotid artery (ICA) dissection due to Eagle syndrome in a patient with vascular EDS.A 30-year-old man was admitted to our hospital because of sudden onset of mild sensory disturbance in his left limbs. He had a history of Ehlers-Danlos syndrome (EDS) and also had left cervical internal carotid artery (ICA) dissection 3 years before. Diffusion-weighted imaging showed acute cerebral infarcts in the right hemisphere. Cervical computed tomography angiography (CTA) revealed the right ICA narrowing at the cervical portion in addition to the previous left cervical ICA dissection. Cervical magnetic resonance imaging (MRI) revealed double-lumen and intramural hematoma at the narrowing portion of the right cervical ICA, which indicates arterial dissection. CT also revealed bilateral elongated styloid processes which are close to each side of cervical ICA. We diagnosed him as bilateral ICA dissection due to bilateral Eagle syndrome. Considering vascular complications due to vascular EDS, we performed closer follow-up with transoral carotid ultrasonography (TOCU). In 4 months, his right ICA dissection gradually improved without stroke recurrence or deterioration of dissection.Since patients with vascular EDS easily develop arterial dissection, Eagle syndrome may be overlooked. Clinicians should consider Eagle syndrome in the case of vascular EDS with extracranial ICA dissection and close follow-up should be prioritized in cases of Eagle syndrome with vascular EDS. Patients with vascular Ehlers-Danlos syndrome (EDS) occasionally suffer from vascular events including arterial dissection. Because of the high incidence of vascular complications, surgical treatments that include catheter intervention is controversial. Eagle syndrome, which is caused by an elongated styloid process and also causes arterial dissection, is difficult to diagnose and could sometimes be overlooked. Here, we present a case of bilateral internal carotid artery (ICA) dissection due to Eagle syndrome in a patient with vascular EDS.A 30-year-old right-handed man was admitted to our hospital due to mild sensory disturbance in his left limbs. He had histories of sigmoid colon rupture and pneumothorax and underwent surgical therapies. Three years before, he was admitted with transient amaurosis due to left ICA dissection, and a gene mutation analysis revealed the presence of vascular EDS with a missense mutation of COL3A1 cDNA (c.1196 G > A). On current admission, he only had slight sensory disturbance and a National Institutes of Health Stroke Scale score of 1. There were no other neurological deficits including his cranial nerves. In brain magnetic resonance imaging (MRI), diffusion-weighted imaging showed multiple acute cerebral infarcts in the right middle cerebral artery territory (Fig. This is a case of bilateral ICA dissection caused by bilateral Eagle syndrome in a patient with vascular EDS. There are several reports on arterial dissection in vascular EDS and those on arterial dissection in vascular Eagle syndrome; however, this is the first known case of coexisting vascular EDS and Eagle syndrome.Vascular Eagle syndrome refers to a set of symptoms associated with an elongated styloid process, by which the cervical ICA is compressed, resulting in ICA dissection and cerebral infarction . AlthougTreatment strategy was controversial because there was no case of coexisting Eagle syndrome and vascular EDS. In cases of carotid artery dissection due to Eagle syndrome, either surgical resection of the elongated styloid process or carotid artery stenting are sometimes conducted . On the In conclusion, Eagle syndrome should be considered as an important causative condition of extracranial ICA dissection even in vascular EDS patients. Close follow-up should be prioritized in cases of Eagle syndrome with vascular EDS.


Bolaamphiphiles (BAs) are structurally segmented molecules with rich assembly characteristics and diverse physical properties. Interest in BAs as standalone active agents or as constituents of more complex therapeutic formulations has increased substantially in recent years. The preorganized amphiphilicity of BAs allows for a range of biological activities including applications that rely on multivalency. This review summarizes BA-related research in biomedically relevant areas. In particular, we review BA-related literature in four areas: gene delivery, antimicrobial materials, hydrogels, and prodrugs. We also discuss several distinguishing characteristics of BAs that impact their utility as biomedically relevant compounds. Synthetic bolaamphiphiles (BAs) are segmented amphiphilic molecules that take structural inspiration from archaeal membrane lipids  creates opportunities to combine self-assembly with multivalent ligand presentation. Here, the segmented amphiphilicty facilitates particle organization while the multivalent hydrophilic segments enhance biological function.Archaea microorganisms famously utilize ether-linked BA lipids to assemble and stabilize a range of membrane organizations but especially the monolayer type . As shown in In some situations membrane stabilization is desirable  but in other applications destabilization becomes the goal. The use of BAs as ion-conducting species was one of the earliest and most robust lines of research inside the synthetic amphiphile community , asymmetric BAs are attractive as they can encode a higher density of information vs. symmetric BAs. For example, Jain et al. , 2012 stAnother notable feature of segmented amphiphilicity is the organization of functional groups into dense clusters. Another cross-cutting theme that emerges from the literature is the ability of BAs to impact stability of nanostructures either alone or as a component in a more complex formulation. Archaea-like or archaea-inspired BAs are well-known in this regard. Their membrane spanning ether linked lipid backbone along with hydrophilic head groups stabilize the liposomal membrane  or co-formulated with helper lipids. In 2010, they reported the synthesis and complexation of DNA by asymmetrical BAs featuring ornithine and gluconic acid polar head groups  head groups  to delivery long RNAs  is a threat to global health s including several BA-type structures with central hydrophobic tethers ranging from 3 to 12 carbons s but it was also cytotoxic.In 2016 the Gokel group studied a collection of bis-THA]Cl2, which is structurally similar to dequalinium  block essential microbial transcription and therefore represent a powerful weapon against antimicrobial resistance  then the particular TFD. It was found that nanocomplexes formed in this way preferentially accumulated in bacteria.Gonzalez-Paredes et al.  took a sin vitro antisense treatment to inhibit Clostridium difficile using a bolasome-based approach  that were joined by a hydrophobic isoprene linker see  Caron e. These BHydrogels offer a unique pathway for the conversion of small molecules (low molecular weight gelators) into macromolecular structures. BAs are attractive building blocks for hydrogels as their segmented amphiphilicity promotes both hydrophobic and polar interactions  leading to a rich design space. The self-assembly proclivities of these materials are diverse and influenced by a portfolio of weak interactions including hydrogen bonding, π-π interactions, aromatic interactions, and hydrophobic effects.Romera et al.  show howPhotodynamic therapy is another potential solution to the onset of multi-drug resistant bacteria  to use BAs as a standalone nucleic acid complexing agent (bolaplex) or as just one ingredient of a multi-component lipid formulation? BA-based hydrogels also remain an exciting area of research where creative design can produce multifunctional materials with dynamic properties. Overall, innovative synthetic design will continue to drive discovery. The synthesis of new BA constructs with intermediate molecular weights, falling somewhere between traditional small molecule BAs and segmented polymers, is interesting and somewhat underdeveloped.JH, AM, CW, GV, and PI wrote the paper. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Yet the majority of current bone and skeletal muscle tissue engineering strategies are still premised on a direct contribution of stem cells as building blocks to tissue regeneration. Our review of the recent literature finds that researchers continue to focus on the quantification ofde novo bone/skeletal muscle tissue following treatment and few studies aim to address this mechanistic conundrum directly. The dichotomy of thought is reflected in the diversity of new advances ranging fromin situ three-dimensional bioprinting to a focus on exosomes and extracellular vesicles. However, recent findings elucidating the role of the immune system in tissue regeneration combined with novel imaging platform technologies will have a profound impact on our future understanding of how stem cells promote healing following biomaterial-mediated delivery to defect sites.Stem cell–based tissue engineering is poised to revolutionize the treatment of musculoskeletal injuries. However, in order to overcome scientific, practical, and regulatory obstacles and optimize therapeutic strategies, it is essential to better understand the mechanisms underlying the pro-regenerative effects of stem cells. There has been an attempted paradigm shift within the last decade to think of transplanted stem cells as “medicinal” therapies that orchestrate healing on the basis of their secretome and immunomodulatory profiles rather than acting as There are considerable scientific, practical, and regulatory hurdles that so far have restricted the translational use of stem cells for these applications. Underlying all of these hurdles is a dearth of understanding of the mechanism by which transplanted cells promote regeneration. This has imposed significant limitations on our ability to optimize key parameters such as stem cell purity, dosing, priming, delivery, or the design of scaffold biomaterials. In fact, the majority of clinical trials are not using ASCs/MSC for direct differentiation and contribution to tissue repair but rely on paracrine mechanisms. Yet few recent studies have focused on improving our understanding of the fate of transplanted cells. In spite of this, a convergence of stem cell–based tissue engineering with recent advances in imaging technologies and immunoengineering may open a window of opportunity for further exploration and the discovery of new insights.Bone and skeletal muscle both have high self-healing capacities that become overwhelmed when loss of large tissue volumes results in critical-sized bone defects or volumetric muscle loss (VML). Current pre-clinical tissue engineering strategies for musculoskeletal defects employ bone marrow–derived mesenchymal stem cells (MSCs), adipose-derived stromal/stem cells (ASCs), other tissue-derived stem cell populations, and induced pluripotent stem cells (iPSCs). However, although there are over 200 clinical trials involving ASCs and MSCs in the US  cultures of MSCs and in a nude rat model6. Implanted chondrogenically pre-cultured MSCs, but not non-differentiated MSCs, seeded on polycaprolactone (PCL) scaffolds exhibited mineralization, formation of trabecula-like structures, and chondrogenic and osteogenic gene expression profiles at 8 weeks. Interestingly, scaffolds seeded with chondrogenically pre-cultured MSCs were the only group with human DNA present at 8 weeks and with 45% human RNA content. The findings from these studies suggest that the direct contribution of transplanted cells to bone regeneration might depend heavily on the pre-implantation priming6.The current gold standard of treatment of the critical-sized bone defect is the cancellous autograft, often harvested from the iliac crest12. However, recent studies have called into question whether ASC-derived cells are contributing tode novo myofiber regeneration directly: Goreckaet al. injected autologous ASCs into the tibialis anterior (TA) of mice following a crush injury and found that while the cell transplantation resulted in increased fiber cross-sectional area and improved muscle contractility, ASC-derived cells did not differentiate into myofibers or fuse with endogenous muscle fibers13. Similarly, Gilbert-Honicket al. seeded ASCs onto electrospun fibrin hydrogels and transplanted this construct into a mouse model of VML injury14. Although there was an increase in fiber cross-sectional area, only limited expression of myogenic markers in the donor cells was observed. Alternative mechanisms for the therapeutic benefit of ASC transplantation have been posited. Secretion of paracrine factors by transplanted stem cells may improve regeneration by activating endogenous muscle stem cells, by reducing inflammation at the site of the injury, or by promoting angiogenesis17. In particular, a number of recent studies have documented anti-fibrotic effects of transplanted stem cells. Di Summaet al. found that differentiated ASCs incorporated into fibrin nerve conduits and transplanted into a rat nerve gap model reduced fibrotic tissue formation, enhancing axonal regeneration and remyelination18. Milosavljevicet al. injected MSCs or MSC-conditioned medium intravenously into mice and demonstrated that CCl4-induced liver fibrosis was attenuated19. They found that MSCs acted on fibrosis by means of decreasing levels of inflammatory T helper 17 (Th17) cells while increasing anti-inflammatory CD4+ interleukin 10–positive (IL-10+) T cells. These anti-fibrotic effects of ASCs and MSCs may play a role in their regenerative capabilities upon transplantation into injury models.A variety of stem cell types have been explored as a potential source of myogenic cell replacement therapy. ASCs, which provide a high rate of stem cell proliferation and can potentially be sourced directly from the patient, bypassing immune rejection, have been evaluated extensively in the treatment of musculoskeletal damage22. Raoet al. generated the first 3D contractile skeletal muscle constructs from human iPSCs23. After transplantation, the cells formed densely packed, aligned myofibers and retained functional responses. Wuet al. injected iPSC-derived myogenic progenitors into cardiotoxin-injured mouse TA and observed engraftment and contribution of the iPSC-derived cells to new myofiber formation24. However, long-term survival of iPSC-derived cells in the transplanted environment is a limitation, multiple groups have reported high levels of cell death upon transplantation23, and further work is necessary to characterize the therapeutic mechanism of transplanted iPSC-derived cells. An emergent alternative to the treatment of VML is the use of autologous minced muscle grafts, or 1 mm3 pieces of muscle, which have also been demonstrated to attenuate T lymphocyte and macrophage responses to severe muscle injury. These results may indicate a promising therapeutic role for cell aggregates and immunomodulatory therapies in the treatment of VML25.A number of recent studies have explored the use of human iPSCs as a source of myogenic cell transplantations. Multiple groups have demonstrated that iPSC-derived myogenic cells contribute directly to the formation of new myofibers in damaged tissuede novo bone/skeletal muscle tissue and the positive effects of stem cell delivery. However, most studies do not track implanted cellsin vivo or quantify their viability over a long time course. Multiple studies that have tracked transplanted cells have demonstrated that few cells survive more than 4 to 8 weeks following transplantation, suggesting that their pro-regenerative outcomes might be more correctly attributed to indirect mechanisms such as cytokine secretion, immunomodulation, and signaling to endogenous cells27. For example, a recent study in which ASCs were injected systemically or locally into a wound bed showed that systemically delivered ASCs became trapped in the lung and could not be detected 72 hours after systemic injection but that locally injected cells remained strongly detectable up to 7 days at the wound site, yet both groups exhibited enhanced wound healing28. Recently, some groups have investigated stem cell aggregation and its impact on metabolic and secretory profiles while other studies of exosomes and extracellular vesicles (EVs) have been performed in an effort to provide greater insights into the potency of transplanted stem cells.Tissue engineering studies continue to prioritize the “direct contribution” paradigm. They focus largely on the quantification of30. Yet the lack of standardization renders it impossible to accurately correlate the impact of aggregate sizes, methods of aggregation, and mode of implantation on the therapeutic outcomes. Recent investigations into bone tissue engineering have used periosteum-derived stem cells embedded in collagen type 1 hydrogel31, MSCs embedded in a platelet-rich plasma construct32, or arginine-glycine-aspartic acid (RGD) functionalized alginate33. The studies employed 250 cells per aggregate, randomly sized spontaneous aggregates, and 500 cells per aggregate, respectively. Aggregation increased osteogenic and chondrogenic markers and paracrine secretions. When compared with monodispersed cell-laden counterparts, aggregate-laden scaffolds showed increased bone formation32 but no beneficial impact on cell survival or construct vascularization when implanted subcutaneously31. However, when stem cell aggregates were used in conjunction with bone morphogenetic protein 2 (BMP2) stimulation, there was increased blood vessel formation, BMP2 production, presence of hypertrophic chondrocytes, and remodeling31, although there was no increase in bone volume or torsional strength of the resulting bone33. For skeletal muscle tissue engineering, recent studies have tested human umbilical cord–derived MSCs34 as aggregate sheets encasing porcine heart decellularized extracellular matrix and green fluorescent protein (GFP)-labeled murine MSCs35 (500 cells per aggregate) injected in phosphate-buffered saline, respectively. In both cases, the investigators observed increased recovery and peak isometric torque observed from aggregates compared with single cells35.Implanting stem cells as aggregates rather than monodispersed cells has been shown to enhance their viability, migration, and differentiation and modifies their secretion of cytokines, immunomodulatory factors, and EVs to improve therapeutic outcomes36 and skeletal muscle myogenesis37, but further investigations are necessary in order to elucidate the mechanisms in which these occur. For bone tissue engineering, recent studies of MSC-derived EVs or exosomes embedded in a hydrogel for treating critical-sized calvarial defect have demonstrated therapeutic benefit with significant increases in bone volume fraction, bone mineral density, and new bone area40. Furthermore, combining EVs with MSCs resulted in increased bone volume and bone volume fraction compared with either component delivered separately41. To regenerate skeletal muscle, recent studies have explored the use of ASC-derived EVs42 and MSC-derived exosomes15 injected at various time points at the site of injury or intravenously. These studies also measured various outcomes from an increase in cross-sectional area of newly formed fibers17 to increased regulation/expression of myogenic genes42, capillary density, myofiber diameter, number of centrally located nuclei, and decreases in fibrotic area15.EVs such as exosomes or microvesicles carry important cargo for cell communication and are influential in cell signaling. Exosomes and microvesicles are distinguished by their sizes and origins . They both contain lipids, nucleic acids, and protein cargo. Studies have shown that EVs can enhance cell differentiation and viability, which in turn may affect therapeutic outcomes, although the molecular underpinnings of this are not yet understood. It has recently been suggested that EVs are heavily involved in bone homeostasis43. Conversely, innate immune cells exert an effect on stem cells in musculoskeletal tissues, as demonstrated by the induction of calvarial osteoblast mineralization by macrophages in 2D culture, the induction of osteogenesis in MSCs grown in media from IL-4–stimulated macrophages44, and the link between macrophage activation and pro-osteogenic gene expression in MSCs in 3D culture43. A further understanding of the crosstalk between implanted stem cells and immune cells will help us to maximize the regenerative capacity of stem cells in musculoskeletal therapy.The immunomodulatory effects of MSCs have been studied for over two decades. MSCs regulate immune cell activity via direct cellular contact as well as cytokine and growth factor secretion. Recently, MSCs were shown to negatively regulate the activation and proliferation of T cells during injury and to enhance the immunosuppressive capacity of regulatory T cells in culture46 combined with light sheet microscopy46 and quantitative confocal microscopy47, have enabled the spatial mapping of endogenous stem cells in their native 3D environment within musculoskeletal tissues and the monitoring of stem cell location and viability after implantation. These advances in imaging have been applied to the visualization of the bone marrow cavity in whole mouse femurs45, the quantification of the abundance of cell populations previously underestimated by standard flow cytometry, and the definition of subpopulations on the basis of location and morphology47. Other advances in magnetic resonance imaging48, bioluminescence50, photoacoustic imaging51, ultrasound, and magnetic particle imaging52 may also be applied to thein vivo visualization of implanted stem cells.Recent imaging advances, including novel optical clearing techniquesFuture advances toward the clinical application of stem cells for musculoskeletal treatments will require that tissue engineering studies move beyond empirical readouts and employ more rigorous tools to identify the molecular mechanisms underlying regenerative outcomes. Specifically, there needs to be a greater emphasis on the development and use of novel imaging techniques  as well as on coupling these data with single-cell analytics. The combined application of these advanced molecular tools will enable further insight into the actual role that stem cells are playing and will facilitate better targeting and optimization of their use in promoting tissue regeneration.


The epoch of nanotechnology has authorized novel investigation strategies in the area of drug delivery. Liposomes are attractive biomimetic nanocarriers characterized by their biocompatibility, high loading capacity, and their ability to reduce encapsulated drug toxicity. Nevertheless, various limitations including physical instability, lack of site specificity, and low targeting abilities have impeded the use of solo liposomes. Metal nanocarriers are emerging moieties that can enhance the therapeutic activity of many drugs with improved release and targeted potential, yet numerous barriers, such as colloidal instability, cellular toxicity, and poor cellular uptake, restrain their applicability in vivo. The empire of nanohybrid systems has shelled to overcome these curbs and to combine the criteria of liposomes and metal nanocarriers for successful theranostic delivery. Metallic moieties can be embedded or functionalized on the liposomal systems. The current review sheds light on different liposomal-metal nanohybrid systems that were designed as cellular bearers for therapeutic agents, delivering them to their targeted terminus to combat one of the most widely recognized diseases, cancer. Cancer ranks as a primary cause of death and a vital barricade to elevate life probability worldwide . AccordiNanotechnology offers an adaptable platform of biocompatible and biodegradable systems capable of delivering conventional chemotherapeutic agents in vivo, improving their bioavailability and accumulation around tumor tissues, thus enhancing their release profile . NanomatNanoliposomes are spherical lipid-bilayer vesicles in the nanometer range; they are self-assembled, biodegradable, and biocompatible . This syThe therapeutic perspective of metal and metallic complexes dates back to ancient times . Metals To address the restrictions of liposomal and metal nanoparticles, a new generation of therapeutically active carriers known as liposomal-metallic hybrid nanoparticles has been established. This hybrid system is advantageous due to long circulation time, controlled drug release, targeting, and specificity . FurtherNanohybrid liposomal-metal systems are an opportunity in the advance of multipurpose drug-delivery platforms. Even though existing cancer nanomedicines enhanced tumor accumulation and reduced drug side effects, there are still many concerns that should be endeavored before they can be considered for clinical applications. The suitable screening of the hybrid system components is essential for a novel structure that avoids drug leakage, provides high encapsulation efficiency, and maintains favorable pharmacokinetics . MoreoveIn this review, an updated overview of the role of nanohybrid liposomal-metallic systems in cancer therapy, as well as, recent novelties and applications of the aforementioned system is provided. The paper sheds light on the different mechanisms of liposomal-metal systems, namely, targeted and non-targeted carriers. Nanohybrid liposomal-metal systems theranostic applications in cancer research are the core of the article.The term “targeted therapy” itself refers to a new generation of anticancer agents or mechanisms intended to impede a specific molecular target that is responsible for tumor growth or progression . To idenExogenous stimuli-responsive delivery systems precisely control the release of drugs by external factors to overcome interpatient variability that affects drug pharmacokinetics. Different external stimuli can be used such as light, temperature, and magnetic field .Light as an external stimulus for drug-delivery systems is dominant for many reasons, such as its noninvasive nature, temporal control, and high spatial resolution, in addition to its convenience and ease of use . Thus, nUltraviolet-visible light has been employed in drug delivery because of its ease of manipulation, versatility, and its ability to induce chemical modification on the therapeutic nanocarriers. Nevertheless, the photo regulated drug release is not always accomplished by UV light due to shallow tissue penetration with depths less than 1 mm and may cause cellular damage, which is why it is mostly replaced by NIR as mentioned later . Table 1Near-infrared light at a wavelength ranging between 650 to 950 nm is characterized by negligible phototoxicity and deep penetration into biological tissues (1–3.5 mm) . NIR ligGold nanoshells loaded with doxorubicin (Dox) in hollow silica core shells employing nanoliposomes as a template were prepared by Wu et al. . After d2 method for a photothermal triggered drug release and tumor therapy ) with the addition of hydrophilic organic dye, Texas red (TR) for dual fluorescent imaging. To inspect the targeting efficacy of the prepared system, Her2/neu antibodies were decorated on Lipo(TR)[MNP@m-SiO2(FITC)] surfaces. Incubation with normal 3T3 fibroblast and SKBR-3 breast cancer cells for 24 h showed no Lipo[MNP@m-SiO2]-induced cytotoxicity, even at high concentrations, ensuring its biocompatibility. The nanohybrid magnetic-liposomal system was not internalized by breast cancer cells even after 6 h, until the temperature was altered from 37 to 4 °C, proving that cellular internalization was a time- and temperature-dependent process. These results supported the use of Lipo[MNP@m-SiO2] for targeted drug delivery in Her2/neu positive breast cancer cases; nevertheless, further work is recommended to evaluate its in vivo behavior and to monitor its therapeutic outcomes [For additional benefits of hybrid magnetic nanoparticles and liposomes, particularly for targeting cancer cells, Jang et al. synthesized mesoporous silica core/shell magnetic nanoparticles encapsulated by liposomes  liposomal system with superparamagnetic iron oxide nanoparticles (SPIONs) and cilengitide for dual-imaging-guiding cancer therapy. The formulation revealed a complete encapsulation of SPIONs and QDs in liposomes as confirmed by TEM images and good stability in several media . The in 2O4/Au core/shell nanoparticles were found to be encouraging agents for combined chemo/phototherapy.Lastly, Rodrigues et al. designed a multifunctional nanohybrid liposomal-metal system containing manganese ferrite-gold core/shell nanoparticles in two forms; solid magneto-liposomes (SMLs), and aqueous magneto-liposomes (AMLs), which acted as nanovehicles for simultaneous chemotherapy to deliver the fluorescent thienopyridine derivative into the lipid membrane. In this investigation, the prepared nanohybrid systems showed diametric sizes below or around 150 nm and high encapsulation efficiencies (above 90%). Thienopyridine derivative-loaded AMLs (with the magneto-nanoparticles entrapped in liposomes) displayed a good interaction with model membranes by fusion improving its ability to transport the antitumor drug in the lipid membrane. SMLs, where the core/shell nanoparticles were covered by a lipid bilayer, showed their ability to be employed in photothermal applications as revealed by the normalized fluorescence intensity. Thus, the multifunctional nanohybrid liposomal systems containing MnFeMagnetic hyperthermia is dated back to 1957 when Gilchrist et al. heated the tumor zone selectively utilizing magnetic particles in the presence of an alternating magnetic field . The intInspired by this feature, Hardiansyah et al. prepared citric-acid-coated magnetic nanoparticles (CAMNP) by the coprecipitation method, encapsulated along with doxorubicin (Dox) in liposomes by thin film hydration method; Dox and CAMNP were loaded during the hydration process . HyperthThe authors further prepared drug-loaded PEGylated magnetic liposomes by encapsulation of the oil phase of MNP and curcumin in the PEG-modified liposomes, and it was tested on MCF-7 breast cancer cells . CurcumiIn another study performed by Farcas et al., thermosensitive betulinic acid-loaded magneto-liposomes were prepared by thin-film hydration method and assessed for their potentials in fighting human breast adenocarcinoma . The finContinuing with the magneto-liposomes platform, Cardoso et al. developed two systems: stealth (PEGylated) and non-PEGylated magneto-liposomes containing calcium-substituted magnesium ferrite nanoparticles as curcumin nanocarriers . The impThe developed nanohybrid liposomal systems are promising nanocarriers for curcumin in therapeutic applications, making it possible to choose nanohybrid systems with enhanced drug release (PEGylated systems) or nanohybrid systems with the triggered release by AMF (non-PEGylated systems).A novel formulation based on photoresponsive magnetic liposomes was synthesized to ablate cancer cells . The iroAnticancer drug doses that reach the diseased cells in a controllable manner have contributed to the development of nanocarriers with a controlled release in response to high glutathione levels of cancer cells, which is being four times higher than that of normal cells. By integrating a glutathione stimulus-responsive factor into the nanoparticulate systems, the release profiles of the drugs can be targeted and improved. Thus, pH/redox responsive nanocarriers have been suggested to be good stimulant drug-delivery systems due to their ability to accumulate and internalize cancer cells .® and PTX–PEG@GNP sodium carboxymethyl cellulose suspension, respectively. The presented values were evident by the successful release of PTX from the hybrid and ability of liposomes to protect and improve the stability of PTX thus, enhancing its circulation time. The prolonged circulation time was due to the distribution of PTX in organs, where the amount of drug distributed to the liver (at 12 h) and reached, 18.2- and 5.6-fold compared to Taxol® and conjugate suspension, respectively which ensure a passive targeting to the liver. The study lacked essential experiments that could give a deeper knowledge of this novel system. Primarily, MTT assay on cancerous cell lines and normal cell lines could evaluate the cytotoxicity and safety of the formulation, compared with a free Taxol® and conjugated system. Secondly, the cellular uptake and molecular assays would shed light on the ability of the developed system to uptake and the mechanism of death induced by the studied nanohybrid system.A hybrid system of PEG-modified paclitaxel (PTX) conjugated GNPs encapsulated liposomes was synthesized by Zhang et al. and referred to as (PTX-PEG400@GNP Lips) . LiposomIt is essential to develop strategies for anticancer drug delivery through the cell membrane and to enhance successive distribution to the biological target with high selectivity and efficacy. For this purpose, many biological ligands have been evaluated and investigated for enhancing the active targeting of the nanosystems. The ligand binds to a specific receptor on the outer surface of the target cancer cells and thus increases the cellular uptake of the active agents containing nanoparticles aiming at bioimaging or managing these cells . Compare®) with 385 ± 12.52, 99.7 ± 6.87, and 24.7 ± 2.4 ng/g, respectively, after 0.5 h of intravenous administration. The authors described such enhanced distribution of the targeted system by the augmented cellular uptake induced by liposomes, as well as the sustained drug release obtained, and the targeted delivery of transferrin that increased the blood–brain barrier (BBB) transport of DTX as an active delivery. The higher presence of transferrin receptors on BBB mediated much higher passage of liposomes via endocytosis. Targeted liposomes showed high fluorescent intensity compared to the control and non-targeted carrier, confirming the improved brain uptake and the liposomes ability to cross BBB mediated by the transferrin. These findings proved the potential application of transferrin receptor-targeted liposomes for brain tumor delivery of anticancer drugs and imaging molecules, for successful brain tumor diagnosis and treatment [Transferrin (Tf) is an iron-bounded glycoprotein that acts as an iron vehicle in the body. The presence of transferrin receptors expressed in certain cells allows targeting by transferrin-modified nanoparticles . Sonali reatment .® [Among targeting biological ligands, peptides have many unique features viz: feasible production cost, ease of preparation, and conjugation to the surface of nanoparticles at high density, and good stability . To targ® . This stThe role of peptide ligand in targeting cancer cells was investigated from different aspects one of which is the transdermal drug delivery system (TDDS). As known, TDDS has emerged as an auspicious alternative approach to other routes to deliver antineoplastic agents and to avert some of the oral delivery adverse effects . Patra eAptamers are small nucleic acid (RNA or DNA) consisting of numerous nucleotides. They are small, biodegradable, and have high immunogenicity, allowing them to be a good choice as active targeting ligands . Tumor-tFolate receptors (FA) are known to be overexpressed in macrophages and solid tumor cells making them attractive targets for numerous nanodelivery systems through receptor-mediated endocytosis . Folate-®). Docetaxel-loaded targeted liposomes revealed higher cytotoxicity with IC50 (0.23 ± 0.05), while that of the non-targeted and the commercial were 1.56 ± 0.19 and 9.54 ± 0.76, respectively. The multifunctional liposomes in the current study were effective as dual agents for targeted treatment and diagnosis of breast cancer; however, additional in vivo assessment is needed for better correlation and approval of theses in vitro results.In line with the previous study, another investigation was carried out by Muthu et al. that showed the importance of folic acid conjugated liposomes in imaging and targeting cancer cells. In this research, D-alpha-tocopheryl polyethylene glycol 1000 succinate mono-ester (TPGS)-coated folic-acid-conjugated liposomes for the co-delivery of docetaxel (DTX) and quantum dots (QDs) as a multifunctional theranostic tool for MCF-7 breast cancer cells was investigated. Confocal laser microscopy images showed higher red fluorescent intensity with the folic-acid-conjugated DTX-QDs liposomes than those non-targeted and which were more concentrated in the cytoplasm , confirmGreat effort has been applied for the development of controlled drug-delivery systems aiming to improve drug therapeutic efficacy and minimize their toxicities during cancer treatment. The drug release can be stimulated by light, pH, mechanical stimuli, temperature, or biological molecules . After d−1 than that of the nontargeting system with an IC50 value of 4.92 ± 0.57 μgmL−1. Moreover, it must be noted that no cell death was observed in cell lines treated with blank nanohybrid liposomal-metal conjugate system, thus ensuring safety of the nanohybrid liposomal-metal systems. Accordant with the in vitro observation, animal experiments revealed that synergistic application of dual therapy with the folic acid targeting liposomes could effectually inhibit the tumor growth in xenograft-bearing mice as shown in A unique nanocomplex design of gold nanorods and doxorubicin in a liposomal system conjugated with folic acid was investigated by Nguyen et al. to combine photothermal-chemotherapy and target doxorubicin (Dox) to tumor cells . The nanTumor-distribution of the non-targeted nanocargos is largely governed by the same principles as targeted ones. Thus, not only the targeted delivery systems are capable to transport drugs into the diseases sites, but also nanocargos with no aided molecules or external stimulants are able to effectively carry drugs into the damaged cells . TherefoIn order to enhance the cellular delivery of nanoliposomes without any physical or chemical interference, Karchemski et al. suggested their attachment to carbon nanotubes (CNTs) forming CNT liposomal conjugates (CLCs) . The CNTMetal-organic frameworks (MOFs) are hybrid unique structures, made of inorganic metal ions or clusters and organic units that represent another approach in passive delivery of nanosystems for multiple drug therapy. Illes et al. synthesized liposome-coated MOF nanoparticles composed of iron (III) and fumaric acid (MIL-88A nanoparticles), then coated with DOPC derived liposomes (Lip-MIL-88A) and efficiently loaded with irinotecan and floxuridine . Coated Gold nanoparticles (GNPs) were also applied for the non-targeted delivery of different agents. Epirubicin (EPI) is an epimer of doxorubicin with limited applications in cancer therapy due to multidrug resistance and side effects. To conquest these limitations, Kunjiappan et al. designed a passive delivery of EPI loaded in GNPs and encapsulated into the lipid bilayer of liposomes . The relA versatile nanovaccine composed of liposomes-coated gold nanocages (Lipo-AuNCs) modified with dendritic cells (DCs) was developed for the targeted codelivery of melanoma antigen peptide TRP2 and adjuvant monophosphoryl lipid A (MPLA) . The preNanosystems can be carried using linker DNA resulting in controllable and reversible changes in their physical properties which offer multifunctionality. This system has been tested for drug delivery, but since most of the systems utilized are nonporous, the drug surface attachment leads to limited loading capacity, and this limitation can be overcome by the functionalization of DNA on liposomes surface. The authors reported the reversible assembly of DNA functionalized gold loaded nanohybrid liposomal system. Contrary to other studies that employed the hyperthermia property of gold to promote leakage, the distance between gold and liposomes via DNA hinders heat transfer. AuNPs were functionalized with different thiol modified DNA and achieved stability. To test the stability of the nanohybrid liposomal system, calcein was loaded as a fluorescent agent which indicated that the rigid particle assembly did not increase the liposomal leakage. The DPPC liposomes showed a time-dependent leakage, which was attributed to UV-induced liposomal damage which was further supported by the use of ascorbate as a ROS scavenger. The AuNPs assembly on liposomes completely inhibited leakage of calcien, suggesting a protective effect. The authors also studied the distance-dependent heat transfer within the nanometer scale as the AuNPs can effectively absorb radiation energy. This study presented an approximately 8 nm distance between AuNPs and liposomes through rigid DNA, and this distance allowed protection of the system form leakage due to the prevention of heat transfer from AuNP to the liposomal surface . This stThe current review showed that liposomes apart from being used for the delivery of chemotherapeutic agents can also be considered as revolutionary aid for theranostic purposes. Liposomal delivery systems use has been widely accepted due to its ability to manipulate many drugs pharmacokinetics especially in cancer-targeted therapies. Additionally, metals represent an opportunity to be opportunistic to be exploited along with metal drug and metal nanoparticles as candidates for chemotherapeutic agents. Regardless of the different strategies used, and whether these nanohybrid systems were intended for targeted or active delivery, these prepared systems offered great prospects for the progress of combinational therapeutically modalities. The novel nanohybrid system builds a new pathway as an evolving system in drug delivery, yet the lack of deeply detailed in vivo performance, cellular toxicity, and metabolism, in addition to financial and quality considerations, are the main limitations for this current nanohybrid complex. Further studies will aid in the development of this system, thus facilitating their transition into clinical application.


Porites astreoides coral colonies on a shallow reef in St. John, U.S. Virgin Islands: near-coral , reef-depth (sampled 2 m above each colony) and surface seawater (sampled 1 m from the seawater surface). At all time points and locations, we quantified abundances of microbial cells, sequenced small subunit rRNA genes of bacterial and archaeal communities, and measured inorganic nutrient concentrations. Prochlorococcus and Synechococcus cells were consistently elevated at night compared to day and these abundances changed over time, corresponding with temperature, nitrite, and silicate concentrations. During the day, bacterial and archaeal alpha diversity was significantly higher in reef-depth and near-coral seawater compared to the surface seawater, signifying that the reef influences the diversity of the seawater microorganisms. At night, alpha diversity decreased across all samples, suggesting that photosynthesis may favor a more taxonomically diverse community. While Prochlorococcus exhibited consistent temporal rhythmicity, additional taxa were enriched in reef seawater at night compared to day or in reef-depth compared to surface seawater based on their normalized sequence counts. There were some significant differences in nutrient concentrations and cell abundances between reef-depth and near-coral seawater but no clear trends. This study demonstrates that temporal variation supersedes small-scale spatial variation in proximity to corals in reef seawater microbial communities. As coral reefs continue to change in benthic composition worldwide, monitoring microbial composition in response to temporal changes and environmental fluctuations will help discern normal variability from longer lasting changes attributed to anthropogenic stressors and global climate change.Reef organisms influence microorganisms within the surrounding seawater, yet the spatial and temporal dynamics of seawater microbial communities located in proximity to corals are rarely investigated. To better understand reef seawater microbial community dynamics over time and space, we collected small-volume seawater samples during the day and night over a 72 hour period from three locations that differed in spatial distance from 5  Microorganisms play fundamental roles in coral reef ecosystem nutrient cycling , 2. MicrMost of our knowledge of reef seawater microbial community composition and function is obtained from opportunistic field sampling of reef seawater collected across reefs at a single point in time. This sampling approach has contributed knowledge about coral reef seawater microbial dynamics along various environmental and anthropogenic gradients –16, but On coral reefs, there are significant temporal changes due to the availability of light (influencing primary production), influences of tides, and diel vertical migration and grazing by zooplankton . The avaAcropora formosa changed between day and night , but some of the trends we observed in this study have been observed in other marine microbial communities. Gammaproteobacteria exhibited higher activity at night [Differential enrichment tests of reef seawater bacterial and archaeal communities  between day and night revealed ASVs exhibiting diel enrichment. Excluding the consistent diel changes in at night  in the Mat night  and up-rat night . These tat night , 73.Prochlorococcus, SAR11 (clades 1a and 1b), the NS4 marine group, and “Candidatus Actinomarina,” taxa that are associated with oligotrophic environments and that are commonly detected in reef seawater.The observed richness of bacterial and archaeal ASVs was higher in seawater collected from reef-depth and coral ecosphere seawater relative to surface seawater during both day and night, demonstrating spatial heterogeneity in the microbial community over the water column and elevated microbial diversity at depth. Enhanced diversity closer to the reef may reflect a wider variety of microbial niches, increased nutrient availability, as well as less photoinhibition compared to surface seawater. Taxa enriched in reef-depth and coral ecosphere seawater compared to surface seawater bacterial and archaeal communities included Prochlorococcus ASVs were identified as Prochlorococcus marinus (MIT9131), a low-light adapted Prochlorococcus ecotype. We suspect that there are more Prochlorococcus ecotypes in reef seawater, but did not have the resolution to capture this diversity by comparing differences in the V4 region of the 16S rRNA gene because ecotypes can differ by <1% variation [reviewed by Prochlorococcus ecotypes, MIT9313 is usually found at the base of the euphotic zone and has the genetic capability of using and reducing nitrite as a source of nitrogen [Prochlorococcus could be advecting onto the reef from offshore currents, but if this were the case, we would expect Prochlorococcus to be evenly distributed across the shallow water column. We consistently detected this ecotype at 7 m depth as opposed to 100 m in the open ocean [reviewed within Prochlorococcus because of photoinhibition [nitrogen , 76. Prohibition .Prochlorococcus, heterotrophic bacteria within the SAR11 clade are abundant in oligotrophic marine environments and coral reef seawater [P. astreoides stimulate SAR11 growth rates and there is evidence that P. astreoides also grazes on these cells in mesocosm experiments [Like seawater , 79. In eriments . EnrichmCa. Actinomarina” are very small and their distribution tracks with abundances of Synechococcus [Ca. Actinomarina” have also been identified in coral reef seawater and coral mucus previously [NS4 marine group bacteria within the phylum Bacteroidetes are commonly detected in marine microbial communities –83 and ehococcus . “Ca. Aceviously . We haveP. astreoides does not influence coral ecosphere bacterial and archaeal communities to the same degree that other coral species do. For example, P. astreoides’ ecosphere bacterial and archaeal communities sampled in Cuba were more similar to reef-depth seawater microbial communities compared to ecosphere communities sampled surrounding other Caribbean coral species including P. astreoides, Orbicella faveolata, Montastrea cavernosa, Pseudodiploria strigosa, and Acropora cervicornis [P. astreoides could be site-specific and depend on local environmental conditions and/or on the health state of the coral colony, similar to observations that have been made about common coral-associated microorganisms [P. astreoides were mostly dominated by Endozoicomonas bacteria whereas Endozoicomonas were present but cryptic (average relative abundance of 0.11%) in coral ecosphere and reef-depth seawater. These findings demonstrate that interactions between corals and planktonic microorganisms may be nuanced and depend on factors that have been previously unexplored, necessitating more research in this area.Coral ecosphere and reef-depth seawater bacterial and archaeal communities had generally similar compositions and metrics of alpha diversity (richness and Shannon’s diversity index) even though distance  significantly influenced community similarity as revealed by the PERMANOVA test. Furthermore, no ASVs were significantly enriched in coral ecosphere compared to reef-depth seawater. These conflicting results potentially suggest that minor compositional differences do exist between ecosphere and reef-depth seawater, but that most of the compositional differences are driven by temporal changes. To elaborate, we expected to find taxonomic differences between these two seawater environments as well as enrichment of Gammaproteobacteria within the coral ecosphere seawater based on previous observations , 37, butrganisms . For insProchlorococcus and Synechococcus cell abundances at night and changes over time that corresponded with changes in temperature and increases in nitrite and silicate. We also identified diel patterns in bacterial and archaeal diversity, enrichment of different taxa by day and night, and specific taxa exhibiting rhythmic population fluctuations in reef-depth and coral ecosphere seawater. Bacterial and archaeal alpha diversity was higher in reef-depth seawater compared to surface seawater, suggesting that there may be enhanced microbial niches close to the reef, a hypothesis held widely, but rarely observed. Comparatively, temporal changes superseded spatial differences in terms of influence on the seawater microbial community, emphasizing the need for a more comprehensive understanding of how these communities change over short term  and longer term  time-scales. Coral ecosphere and reef-depth seawater bacterial and archaeal communities had generally similar compositions in this study, suggesting that colony or site-specific conditions may influence the outcome of coral-microbial interactions within the coral ecosphere. Overall, these findings demonstrate the small-scale population dynamics that take place over a diel cycle and the relative influence of temporal compared to spatial changes on microbial communities sampled across one reef.High-resolution sampling of surface, reef-depth, and coral ecosphere seawater microbial communities revealed several novel observations of seawater microbial dynamics on tropical coral reefs. Even though reef seawater bacterial and archaeal communities were highly similar over three days, there were consistent increases in At the scale of the reef, understanding variability in microbial composition as well as dominant forcings on these communities is essential for describing baseline temporal and spatial dynamics in productive, diverse, and sensitive coral reef ecosystems. As coral reefs continue to decline, these changes may not only impact coral reef health and the composition of microbial communities on reefs, but also the variability of microbial population fluctuations.S1 FigEach point represents a sample. Point shape corresponds to sampling distance from the coral and point color reflects the colony adjacent to where sampling was conducted. Gray shading indicates samples collected at night. Lower and upper edges of the boxplot correspond to the first and third quartiles, the whiskers extend to the largest or smallest value at 1.5 times the interquartile, and the black bar across the box represents the median.(DOCX)Click here for additional data file.S2 FigEach point represents a sample. Point shape corresponds to sampling distance from the coral and point color reflects the colony adjacent to where sampling was conducted. Gray shading indicates samples collected at night. Lower and upper edges of the boxplot correspond to the first and third quartiles, the whiskers extend to the largest or smallest value at 1.5 times the interquartile, and the black bar across the box represents the median.(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.S1 Appendix(XLSX)Click here for additional data file.