Bone marrow mesenchymal stem cells (BMSCs) refer to a heterogeneous population of cells with the capacity for self-renewal. BMSCs have multi-directional differentiation potential and can differentiate into chondrocytes, osteoblasts, and adipocytes under specific microenvironment or mechanical regulation. The activities of BMSCs are closely related to bone quality. Previous studies have shown that BMSCs and their lineage-differentiated progeny , and osteocytes are mechanosensitive in bone. Thus, a goal of this review is to discuss how these ubiquious signals arising from mechanical stimulation are perceived by BMSCs and then how the cells respond to them. Studies in recent years reported a significant effect of locomotion on the migration, proliferation and differentiation of BMSCs, thus, contributing to our bone mass. This regulation is realized by the various intersecting signaling pathways including RhoA/Rock, IFG, BMP and Wnt signalling. The mechanoresponse of BMSCs also provides guidance for maintaining bone health by taking appropriate exercises. This review will summarize the regulatory effects of locomotion/mechanical loading on BMSCs activities. Besides, a number of signalling pathways govern MSC fate towards osteogenic or adipocytic differentiation will be discussed. The understanding of mechanoresponse of BMSCs makes the foundation for translational medicine. Bone is the largest loading-bearing organ in our body, which provides mechanical integrity for locomotion and protection. During the whole life span, bone tissue undergoes constant bone damage and remodelling to contribute to bone homeostasis , 2. BoneRecent studies have greatly expanded the knowledge of mechanoresponse of mesenchymal stem cells and their roles on the maintenance of bone homeostasis . PreviouThe differentiation of MSC towards an adipogenic or osteogenic lineage relies on various of signalling pathways and transcription factors. The most well-known key factors for adipogenic or osteogenic cell fate decision are peroxisome proliferator-activated receptor γ (PPARγ), the master regulator for adipogenesis, and runt related transcription factor 2 (Runx2), the main regulator for osteogenesis , 12. TheConsidering the ubiquity of BMSCs, there is no such a classical mechanoenvironment that describes the accommodation of BMSCs. Current studies majorly focused on the mechanical environment of bone marrow, as it houses the largest percentage of total BMSC population . To narrA study regarding to extensive necrosis of the bone firstly caught public attention on the effects of intramedullary pressure (IMP) . NumerouOn the other hand, the response of BMSCs to their mechanical environment also depends on fluid shear stress from bone matrix. However, the rheology properties of bone marrow make the in vitro simulation of BMSC mechanical environment very difficult, which leaves us with the question concerning what forms of mechanical cues BMSCs experience in local environment . So far,2, which is far higher than the threshold to activate BMSCs in vitro [Another factor related to BMSC mechanoresponse is the stiffness of extracellular matrix (ECM). “Stiffness” is termed as a metric of the rigidity which is sensed by cells via application of cell‑generated forces . Much stin vitro . HoweverThe complexity of BMSC mechanosensation comes from the mechanism of how mechanical cues are transmitted and terminally transferred into molecular signals which result in various cellular activities. To answer these questions, several studies have investigated the cell-ECM and cell–cell adhesion as the major structures for mechanical sensation . IntegriMoreover, in recent years, it has been found that Piezo1, a mechanosensitive ion channel protein, can transfer mechanical signals through deformation to the pore nucleus structure of its protein, thus opening channels and mediating ion into cells, regulating the transcription of downstream genes. At the mechanistic level, in response to mechanical loads, Piezo1 in osteoblastic cells controls the YAP-dependent expression of type II and IX collagens. In turn, these collagen isoforms regulate osteoclast differentiation .Taken together, the findings of integrin, actin bundles and their correlation to the downstream molecules like YAP and TAZ unlocked the previously unanswered questions about ECM-to-BMSC mechanotransmission.Primary cilia is a microtubule-based antenna-like sensory organelles critical for mechanical transduction. transducing extracellular mechanical and chemical signaling . Motor cIn the process of fracture healing, primary cilium act as sensory organelles that mediate several signalling pathways . MicrotuRecent studies have implicated primary cilia have been cited as a potential important mechanosensor in bone cells. Primary cilium are responsible for sensing the mechanical load in bone cells, and bone cells have the function of regulating the expression of primary cilium . PeriostThe bone marrow mechanoenvironment is easily changed by physical loading and activities, but it does not mean easy breakage of a balanced bone cell activity. In fact, the loading of whole bone  induced Mechanical stimulus induced morphological changes and migration of BMSCs occur prior to proliferation and differentiation . A confo2 could trigger 126% increase of proliferation [2 [During the past decades, a number of studies have revealed a close relationship between fluid shear stress and BMSCs activities, especially proliferation and osteogenic differentiation , 86. Notferation . Consideation [2  which station [2 –91, it iThe differentiation of BMSCs, on the other hand, requires more strict conditions. It occurs only when the appropriate strain magnitudes, loading frequency and duration are together satisfied . To achiConsidering the effects of mechanical loading on BMSC activities, the regulation of exercise in bone mass is believed to be intensity and duration-dependent Fig. . A recenThe Rho/Rho-associated protein kinase (Rho/ROCK) signalling is important in MSC mechanobiology owing the ability for lineage switch in MSCs . The iniThe IGF signalling is mainly comprised of IGF-1, IGF-2 and their type I, type II receptors . In the Bone morphogenetic proteins (BMPs) are known as another group of mechanically sensitive cytokines upstream to locomotion induced BMSCs differentiation , 118. WiWnt signalling is generally subdivided into canonical Wnt and non-canonical pathways characterized by its dependency on β-catenin , 127. CaActivation of Wnt signalling is a common response to mechanical loading . ClinicaOn the other hand, oscillatory fluid flow could also activate non-canonical Wnt signalling . The actAlthough both of canonical and non-canonical Wnt signalling activated by mechanical stimulation exhibit ability for bone formation, the activation of diverse Wnt pathways may not collaboratively enhances osteoblastic differentiation as crosstalk in between Wnt signalling could antagonise the effects on BMSCs differentiation . TherefoThe effects of locomotion are always the focus in bone-related research. It is believed that there is a close relationship between locomotion and osteogenic lineage commitment. Although many in vitro studies have suggested that BMSCs could sense the fluid flow shear stress and initiate osteogenic differentiation afterwards, how to develop a proper physical activity by understanding the mechanical characteristics of BMSCs remains a mystery. So far, it has been suggested that regular physical activities could enhance proliferation and osteogenic differentiation of BMSCs in vivo  and signThe characteristics of BMSCs provides a guidance of appropriate exercise mode for strengthening our bone. Both clinical trails  and animOn the other hand, low-impact or non-load-bearing exercises are not osteogenic. For example, over 1% bone mineral density (BMD) loss at the hip and lumbar spine was detected in female cyclists in a 12-month-period study . SimilarIn addition to the form of exercises, duration is another factor to strengthen skeleton. Since BMSCs require a period for the completion of differentiation, short-term exercises may not enough for osteogenic commitment. Therefore, long-term exercises are considered to be effective on bone health. For example, it has been reported that a 7-month-periode high-impact training, followed by 7 months of normal activities resulted in a 4% increase of femoral head BMC and area . SimilarThe mechanical characteristics of BMSCs also provides possibility for translational medicine. BMSC-based therapy in bone defects healing has been investigated in several previous studies –154. HowIn addition to bone tissues, combination of mechanical loading and BMSCs have been applied to other tissues. Considering their chondrogenic potential, BMSCs were recently applied to cartilage regeneration . To maxiAlthough there is insufficient evidence concerning the efficacy of BMSC mechanoresponse-based treatment in clinical trials, current progress in this area sheds light on a novel approach for tissue regeneration.In summary, recent studies have established a strong relationship between locomotion and bone health based on the mechanoresponse of BMSCs. So far, it is well confirmed that constant loading with appropriate intensity could promote the proliferation and osteogenic differentiation of BMSCs , 86. Acc


The ability to prioritize people living with HIV (PLWH) by risk of future transmissions could aid public health officials in optimizing epidemiological intervention. While methods exist to perform such prioritization based on molecular data, their effectiveness and accuracy are poorly understood, and it is unclear how one can directly compare the accuracy of different methods. We introduce SEPIA , a novel simulation-based framework for determining the effectiveness of prioritization algorithms. SEPIA expands upon prior related work by defining novel metrics of effectiveness with which to compare prioritization techniques, as well as by creating a simulation-based tool with which to perform such effectiveness comparisons. Under several metrics of effectiveness that we propose, we compare two existing prioritization approaches: one phylogenetic (ProACT) and one distance-based (growth of HIV-TRACE transmission clusters).Using all proposed metrics, ProACT consistently slightly outperformed the transmission cluster growth approach. However, both methods consistently performed just marginally better than random, suggesting that there is significant room for improvement in prioritization tools.We hope that, by providing ways to quantify the effectiveness of prioritization methods in simulation, SEPIA will aid researchers in developing novel risk prioritization tools for PLWH. Molecular data gathered on human immunodeficiency virus (HIV) is useful for understanding the systems of epidemic spread of HIV. Such understanding can better allow us to intervene and treat high-risk groups of individuals. Methods of epidemic intervention include treatments such as antiretroviral therapy (ART) and awareness programs . Adherenpol and gag regions). This information is often used to determine groups of individuals with high risk of future transmission, which can help public health officials better allocate limited resources [In many parts of the world, when testing and treating PLWH, it has become standard practice to record various metadata on the patients, including viral genomic sequences ? Thus, it is unclear how to quantitatively assess the performance of different prioritization methods.The following questions naturally arise: how well does a given prioritization method perform, and which method is superior in specific contexts? With real-world data, the ground truth of who transmitted to whom is typically unavailable or error-prone. Further, even To address this open problem, we introduce SEPIA , a novel simulation-based framework for measuring the effectiveness of prioritization algorithms. Previously, in Moshiri et al. (2021) , ProACT Metric 1: Direct Transmissions This metric aims to quantify the direct impact of each individual u on the spread of the virus within a population by counting the total number of individuals to whom u directly transmitted.Metric 2: Transmission Rate This metric aims to quantify the rate of transmission of each individual u, giving higher values to those who transmitted most frequently and most recently. We produce a step function representing the number of transmissions from individual u over time, and we measure the slope of a regression line inferred from the step graph : Platform independentProgramming language: PythonOther requirements: SciPyLicense: GNU GPL v3.0Any restrictions to use by non-academics: Contact authorshttps://github.com/Niema-Lab/SEPIA-paper-finalManuscript data:


Leviviricetes class. 11% of viral contigs were found to be most closely related to members of the Ourmiavirus genus, suggesting that members of this clade of plant viruses may be far more widely distributed and diverse than previously thought. These results contrast with soil DNA viromes which are typically dominated by bacteriophages. RNA viral communities, therefore, have the potential to exert influence on inter-kingdom interactions across terrestrial biomes.The distribution and diversity of RNA viruses in soil ecosystems are largely unknown, despite their significant impact on public health, ecosystem functions, and food security. Here, we characterise soil RNA viral communities along an altitudinal productivity gradient of peat, managed grassland and coastal soils. We identified 3462 viral contigs in RNA viromes from purified virus-like-particles in five soil-types and assessed their spatial distribution, phylogenetic diversity and potential host ranges. Soil types exhibited minimal similarity in viral community composition, but with >10-fold more viral contigs shared between managed grassland soils when compared with peat or coastal soils. Phylogenetic analyses predicted soil RNA viral communities are formed from viruses of bacteria, plants, fungi, vertebrates and invertebrates, with only 12% of viral contigs belonging to the bacteria-infecting  Viruses are the most common and diverse biological entities on Earth  and can Riboviria and possess a universally conserved RNA-dependent RNA polymerase (RdRP) gene , 19Negarathogens , howevern plants .in-situ soil RNA viral community of soil ecosystems. We detected 3462 viral contigs across five sample sites, and observed site-specific variation in viral contig relative abundance. The viral contigs we detected are predicted to be from viruses of a range of hosts, including fungi, bacteria, vertebrates, invertebrates and plants. Therefore, RNA viruses have the potential to influence the grassland soil ecosystem at multiple trophic levels. From a technical standpoint, further development of both wet-lab and bioinformatics techniques is needed to further improve the detection and study of soil RNA viruses. Many RNA viruses have segmented and multipartite genomes, complicating the recovery of full RNA viral genomes from meta-transcriptomics and metaviromics datasets. This study found comparatively fewer putative mycoviruses compared to a previous study [Using an altitudinal primary productivity gradient as a source of soils with contrasting ecological properties for RNA virome analysis, this study is the first to apply a direct viromics approach to examine the us study  examininus study , the lacCaudoviricetes, the balance in RNA viromes is heavily skewed towards eukaryotic viruses [Whilst environmental DNA viromes are typically dominated by viruses of the prokaryote-infecting class  viruses . There a viruses , 64. Ref viruses  and so HThe impact that soilborne RNA viruses have on their host organisms has only just started to be explored, and future work is needed to establish the many influences they may have on global terrestrial ecosystems. Grassland soil bacterial communities show clear responses to the effects of climate change that are mediated by plant–soil–microbial interactions  and viruSupplementary Information


Blindness from age-related macular degeneration (AMD) is an escalating problem, yet AMD pathogenesis is incompletely understood and treatments are limited. The intestinal microbiota is highly influential in ocular and extraocular diseases with inflammatory components, such as AMD. This article reviews data supporting the role of the intestinal microbiota in AMD pathogenesis. Multiple groups have found an intestinal dysbiosis in advanced AMD. There is growing evidence that environmental factors associated with AMD progression potentially work through the intestinal microbiota. A high-fat diet in apo-E-/- mice exacerbated wet and dry AMD features, presumably through changes in the intestinal microbiome, though other independent mechanisms related to lipid metabolism are also likely at play. AREDS supplementation reversed some adverse intestinal microbial changes in AMD patients. Part of the mechanism of intestinal microbial effects on retinal disease progression is via microbiota-induced microglial activation. The microbiota are at the intersection of genetics and AMD. Higher genetic risk was associated with lower intestinal bacterial diversity in AMD. Microbiota-induced metabolite production and gene expression occur in pathways important in AMD pathogenesis. These studies suggest a crucial link between the intestinal microbiota and AMD pathogenesis, thus providing a novel potential therapeutic target. Thus, the need for large longitudinal studies in patients and germ-free or gnotobiotic animal models has never been more pressing. Age-related macular degeneration (AMD) is a leading cause of irreversible blindness in the developed world, affecting 11 million people in the U.S. and 170 million people world-wide . Due to Inflammatory mechanisms of AMD have been described through a series of genetic, immunologic, and other mechanistic studies. These inflammatory mechanisms include aberrations in the complement cascade, inflammasome activation, inflammatory cytokine upregulation, and microglial activation. The complement system is part of the innate immune system that normally aids in the clearance of microbes and injured cells from the body, but when dysregulated can result in immune-mediated diseases. Genetic studies have shown that variants in the complement cascade genes were highly associated with AMD , while oThe commensal microbiota, or microorganisms that normally inhabit the human body, potentially provide a missing link between the genetic and environmental factors that contribute to AMD pathogenesis. The vast majority of the human commensal microbiota is composed of bacteria located in the gastrointestinal tract, which are now known to be integrally involved in host biological processes such as control of the immune system, breakdown of lipids found in the diet, and the formation and metabolism of bile acids. Metabolites and other components produced by or expressed on the commensal bacteria have wide-spanning functions, including modifying host leukocyte trafficking within the adaptive immune compartment and activating crucial innate pathways within the gut, such as complement and the NLRP3 inflammasome . BecauseEnterobacteriaceae species in the early gut microbiome of high-risk premature infants, who later developed severe retinopathy of prematurity needing treatment compared to high-risk infants that did not develop any retinopathy of prematurity [The pathogenesis of AMD may be similar to that of other age- and lifestyle-associated inflammatory diseases. A mechanism has been proposed to link atherosclerosis to the activity of bacteria in the human gut. Phosphatidylcholine obtained through the diet is metabolized by gut bacteria to form trimethylamine oxide (TMAO), and serum levels of TMAO have been linked to atherosclerosis and cardiovascular risk . There imaturity . While tThe authors hypothesize that the composition and function of gut bacteria may also modulate immune response in AMD. Because of these functions and what we currently understand about AMD development, intestinal microbiota are probable candidates for the interaction between environmental and inherited influences associated with AMD. This review highlights pre-clinical and clinical data that support the potential impact of alterations in the intestinal microbiota on AMD development or progression.Anaerotruncus, Oscillibacter, Ruminococcus torques, and Eubacterium ventrosium. In a different clinical case-control study , Lin and colleagues discovered an intestinal dysbiosis resulting in enhanced abundance of Prevotella, Holdemanella, Desulfovibrio, and other bacteria, but reduced abundance in Oscillospira, Blautia, and Dorea, in AMD subjects compared to control subjects [Prevotella in rheumatoid arthritis and Desulfovibrio in IBD) have been associated with other non-infectious systemic inflammatory conditions [Studies showing that an intestinal dysbiosis occurs in advanced AMD have revealed specific bacteria that are differentially abundant in healthy older adults compared to those with advanced AMD. In a study of 12 new exudative AMD patients compared with 11 control subjects, Zinkernagel and colleagues demonstrated differences in the intestinal microbiota . The stusubjects ,35,36. Snditions ,37. Dietary factors, oral supplements, and other environmental risk factors such as smoking may promote AMD through their effects on the intestinal microbiota. While analysis of the role of nutritional factors is notoriously difficult, an oral supplement containing the carotenoids lutein and zeaxanthin, or beta-carotene , minerals, and antioxidants was found by the Age-Related Eye Disease Studies (AREDS and AREDS2) to reduce the risk of AMD progression (in up to 35% of patients) and remains the only known therapy for primary prevention of advanced disease . How AREFirmicutes and Verrucomicrobia phyla and decreased Bacteroides, similar to Andreissen’s study. In this study, they describe high fat diet-induced dysbiosis concomitantly influences purine pathway in the gut- microbiota and in the retina in the same animals using multiomics studies [Firmicutes to Bacteroides ratio [Other dietary factors may also affect AMD pathogenesis and progression via interactions with the intestinal microbiota. Andriessen and colleagues investigated a murine model of choroidal neovascularization in which a high fat diet resulted in increased weight gain, alterations in the composition of the microbiome, increased intestinal permeability, and promotion of CNV growth compared to mice on a regular diet. Weight gain persisted after administration of oral antibiotics, but CNV growth slowed, suggesting that gut microbial alterations and not simply obesity were responsible for angiogenesis promotion . Skondra studies . Interes studies .Mice defes ratio , similares ratio  and Skones ratio . Furtheres ratio . Rowan and colleagues demonstrated that mice given a high-sugar diet developed an altered intestinal microbiota along with AMD-like characteristics, such as loss of photoreceptors, retinal pigment epithelial atrophy, and sub-RPE deposits, as well as buildup of lipofuscin. On the other hand, a low sugar diet did not impact the retina nor the intestinal microbiota in this manner . This saLachnospiraceae [Interestingly, omega-3 fatty acids, at the doses used in the AREDS2 study, were not found to prevent progression to advanced AMD by oral supplementation. However, they were shown in epidemiologic studies to be beneficial in AMD, particularly when consumed as omega-3 fatty acid-rich fish ,38. One piraceae . Butyratpiraceae ,48. Finally, the composition of the microbiota can be heavily influenced by exposure to oxidants from smoking and other environmental sources. Such exposure may result in a pro-inflammatory repertoire of commensal bacteria , a dysbiCFH) and CFI genes, both inhibitors of complement activation, were strongly associated with AMD. A gene variant in the ARMS2 gene, which may also be involved in the complement cascade, but is not well understood, is also highly associated with AMD. LIPC, APOE (lipid pathway genes), metalloproteinase (TIMP3), and other inflammatory pathway gene variants were associated with AMD. Both CFH and ARMS2 proteins are thought to be important in the alternative pathway of the complement cascade, and their deficiency may be responsible for reduced clearance of cellular debris and its accumulation beneath the RPE and along Bruch’s membrane in the form of drusen deposits [The most robust genome-wide association study (GWAS) in AMD completed up to the present time exhibited 52 genetic variants (34 loci) significantly associated with AMD . Gene vadeposits . These 5deposits ,51. Furtdeposits ,51. ARMS2 and CFH risk alleles and lowered alpha diversity. Additionally, we have found that the ARMS2 risk allele was associated with significantly higher rates of IgA-bound bacteria in the gut, signifying the immunological relevance of these gut bacterial changes [Negativicutes was found to be a potential biomarker for nvAMD and also appeared to positively correlate with an AMD-associated CFH risk allele [A dysbiosis of the gut or other part of the human body, resulting in inappropriate chronic over-activation of the complement cascade, could be one possible host environmental factor contributing to the pathogenesis of AMD. Lin and colleagues have found that genetic risk scores  are inversely correlated with alpha diversity of the gut bacteria in advanced AMD patients, and there is a particularly strong correlation between  changes . In a rek allele . A synergistic effect of genetic predisposition, high-fat diet, and aging has been revealed by the work of Skondra and colleagues using AMD animal models. A high-fat diet accelerated and exacerbated both wet- and dry-AMD features in genetically predisposed apolipoprotein-E deficient mice more robustly compared to wild-type mice . MicrobiUsing high-throughput RNA sequencing in GF mice, Skondra and colleagues has also revealed key aspects of retinal gene regulation important in AMD pathogenesis that are modulated by the intestinal microbiota. Their study revealed that the top modified pathways include vascular endothelial growth factor (VEGF) receptor signaling, mitochondrial biogenesis, oxidative stress, autophagy, longevity, and AMP-activated protein kinase (AMPK) signaling . In the L.paracasei KW3110, a lactic acid-producing bacteria that can activate macrophages but paradoxically suppresses inflammation, leads to an altered Firmicutes/Bacteroidetes ratio [L.paracasei resulted in a reversal of the deleterious Firmicutes/Bacteroidetes ratio and decreased both age-related inflammation and ganglion cell loss in the retina [Dysregulated immune activation via recruitment of microglia and macrophages into the subretinal and choroidal areas, mast cell activation, and RPE immune activation all likely play a role in AMD pathogenesis or progression ; howeveres ratio . Severales ratio ,44. Feede retina . Several groups have found that an intestinal dysbiosis occurs in AMD patients, and that certain dietary changes can cause AMD-like phenotypes due to changes in the microbiota. Centralized repositories analyzing bacterial sequencing and patient metadata, as well as a multi-centered effort (similar to the GWAS effort), are required to establish a clearer pattern of distinct microbial profiles that might be used as biomarkers for disease development and severity. Additional work proving the implication that AREDS and dietary changes may be beneficial in preventing the progression of AMD through their action on the intestinal microbiota, and will require experimentation in both animal models and longitudinal data collection in patients. Future studies using specialized GF and gnotobiotic animal models will be essential to delineate the interaction of the gut microbiome with other complex host factors in AMD pathogenesis as well as progression. The association between microbial changes and the genetic risk alleles of the complement pathway genes associated with AMD provide clues to what may be promoting non-genetic risk allele AMD patients to have aberrantly elevated systemic complement activation. As pointed out by Handa et al. , the lin


The use of airway clearance strategies as supplementary treatment in respiratory disease has been best investigated in patients with cystic fibrosis (CF) and non-cystic fibrosis bronchiectasis (NCFBE), conditions which are traditionally characterized by excessive mucus stasis and mucociliary dysfunction. A variety of airway clearance therapies both pharmacological and non-pharmacological have been shown to ameliorate disease progression in this population and have hence been assimilated into routine respiratory care. This self-propagating cycle of mucus retention and airway damage leading to chronic inflammation and infections can also be applied to patients with respiratory failure requiring mechanical ventilation. Furthermore, excessive trachea-bronchial secretions have been associated with extubation failure presenting an opportunity for intervention. Evidence for the use of adjunctive mucoactive agents and other therapies to facilitate secretion clearance in these patients are not well defined, and this subgroup still remains largely underrepresented in clinical trials. In this review, we discuss the role of mucus clearance techniques with a proven benefit in patients with CF and NCFBE, and their potential role in patients requiring mechanical ventilation while highlighting the need for standardization and adoption of mucus clearance strategies in these patient populations. Clearance of foreign particles and bacteria from the lungs is an important factor in host immunity, facilitated by the optimal functioning of the mucociliary system in conjunction with the cough reflex. The airway epithelium comprised of ciliated and secretory cells lines most of the conducting airways . The secThere are two primary etiologies for a defective mucociliary clearance: dysfunctional cilia and dehydration of the airways. In primary ciliary dyskinesia (PCD) dysfunctional cilia fail to propel the mucus upward to the pharynx, while in CF dehydrated airways cause mucus to thicken with inability to be propelled up the airway to the pharynx . These gPseudomonas aeruginosa (PsA), Haemophilus influenzae, and Staphylococcus aureus  vs. indirect effects resulting from comorbid critical illness  . Anesthelearance . High Filearance . Endotralearance .Recurrent pulmonary infections may act as a predisposition among these patients to further exacerbate mucociliary dysfunction . Cough iA theoretical framework of deficiencies in optimal airway clearance leading to the self-propagating cycle of chronic infections and inflammation is described in Chronic inflammation and infection commonly lead to mucus derangements in patients undergoing mechanical ventilation  . TherapiP = 0.026). Additionally, the volume of 24-h sputum in the N-acetylcysteine group was significantly reduced (P = 0.002) (Nebulized n-acetylcysteine (NAC) functions as a mucolytic by severing disulfide bonds that link mucin oligomers . In vitr= 0.002) . Other n= 0.002) . Data su= 0.002) .Dornase alfa is a highly purified solution of recombinant human deoxyribonuclease-I (rhDNase), an enzyme which selectively cleaves DNA polymers . DornaseHypertonic saline has been used as both a mucolytic and an expectorant, and has shown to decrease bacterial biofilm formation . In patiFew studies have explored a combination therapy of dornase alfa and HTS. The study from 2012 discussed above randomized patients to 7% HTS, dornase alfa, or normal saline. It found no statistically significant difference between HTS and normal saline in chest x-ray scores or oxygenation . A retro2= 89%) was appreciated  across 13 randomized control trials have shown no effect on the duration of mechanical ventilation or hospital stay, but the analysis demonstrated a small effect on reducing ICU length of stay in the mucoactive agent groups , have also been explored in mechanically ventilated patients. Mechanical insufflation-exsufflation works on the principle that following deep inspiration with positive pressure, air in the lungs is expelled out immediately and rapidly by a forced negative pressure exsufflation. This resultant high pressure differential between the mouth and the alveoli creates a gradient to assist in the clearance of secretions .One single-center RCT of 180 adult ICU patients examined the use of MI-E in early mechanically ventilated patients which demonstrated improved secretion clearance (measured by weight of aspirated secretions) and lung compliance .High-frequency chest wall oscillation (HFCWO) involves oscillatory external chest wall compression causing expiratory flows that exceed inspiratory flow enhancing expectoration of mucus. In a pre-clinical canine model tracheal mucus transport was increased by 340% with HFCWO . This haIntrapulmonary percussive ventilation (IPV) is a newer modality in airway clearance. It generates “high flow mini-bursts of air along with a bronchodilator to the lungs at a rate of 300-400 times per minute”. This produces a higher pressure amplitude than unassisted modalities  . IPV is Taken together, this data suggests equipoise regarding the use of chest physiotherapy and other cough augmentation strategies, but there is insufficient data to conclude that any single modality is superior. These data are summarized in Macrolide antibiotics are associated with a host of anti-inflammatory and immuno-modulatory effects achieved through multi-factorial mechanisms. One of the more commonly described pathways involves blockade of IL-8, leading to decreased airway neutrophil recruitment and neutrophil elastase elaboration . MacroliInhaled antibiotics represent a safe mechanism to deliver high doses of targeted antibiotics to the lungs. Benefits in outpatients with targetable infections suggests the potential for a role in improving outcomes in mechanically ventilated patients. A meta-analysis of 5 RCTs examining the use of prophylactic aerosolized antibiotics  in mechanically ventilated patients found a reduced rate of nosocomial pneumonia in patients randomized to receive aerosolized antibiotics . Interes2, pulse oximetry, and PaO2:FiO2), ventilation (PaCO2), ventilator physiology (peak and plateau airway pressures). Patient centered outcomes would include duration of mechanical ventilation, hospital length of stay, incidence of reintubation, occurrence of nosocomial pneumonia, and mortality. Determining appropriate clinical outcomes represents a challenge in this population given the significant heterogeneity of the patient population, poorly defined outcomes (including VAP), and difficulties identifying the best surrogate endpoints. However, since sputum characteristics, duration of mechanical ventilation, and reduction of VAP episodes seem most rigorously studied, we propose that these be a focus in developing therapies in the future from registry/observational studies to determine their role in surrogacy. Until further research is conducted, however, definitive conclusions from existing comparator studies will remain hampered in this patient population.The data presented herein demonstrates a plethora of chosen outcomes for both acute and chronic mechanical ventilation. Endpoints summarized in Mucus clearance therapies form an essential part in the armamentarium available for the treatment of respiratory diseases associated with reduced or abnormal mucus clearance. Adoption of mucus clearance strategies in this critically ill population aims to break the self-propagating cycle of chronic inflammation/infections, and prevent extubation failures as a result of excessive tracheobronchial secretions. While both pharmacologic and non-pharmacologic modalities of airway clearance have been studied and are the standard of care in patients with CF and NCFBE, these interventions are still being explored as adjunctive therapeutic options in those requiring mechanical ventilation. Furthermore, trials conducted to date lack sufficient power, equipoise, and sufficient standardized endpoints to draw strong conclusions about therapy options. We postulate that it is beneficial to use mucoactive therapies in combination with cough augmentation strategies, however more studies with robust validation and standardized endpoints are needed to complement the existing expert recommendations.RG, KV, and GS contributed equally to composition and editing of the manuscript. 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Current cranial repair techniques combine the use of autologous bone grafts and biomaterials. In addition to their association with harvesting morbidity, autografts are often limited by insufficient quantity of bone stock. Biomaterials lead to better outcomes, but their effectiveness is often compromised by the unpredictable lack of integration and structural failure. Bone tissue engineering offers the promising alternative of generating constructs composed of instructive biomaterials including cells or cell-secreted products, which could enhance the outcome of reconstructive treatments. This review focuses on cell-based approaches with potential to regenerate calvarial bone defects, including human studies and preclinical research. Further, we discuss strategies to deliver extracellular matrix, conditioned media and extracellular vesicles derived from cell cultures. Recent advances in 3D printing and bioprinting techniques that appear to be promising for cranial reconstruction are also discussed. Finally, we review cell-based gene therapy approaches, covering both unregulated and regulated gene switches that can create spatiotemporal patterns of transgenic therapeutic molecules. In summary, this review provides an overview of the current developments in cell-based strategies with potential to enhance the surgical armamentarium for regenerating cranial vault defects. The craniofacial region is an anatomically complex set of bone, cartilage, blood vessels, soft tissues and nerves. Compared with the endochondral ossification of the major part of human skeleton, craniofacial bones, derived from the cranial neural crest and paraxial mesoderm , displayBone autograft remains the safest and most effective procedure for cranioplasties in most of the cases. Given that autografts suffer from same drawbacks, the scientific community has long been searching for biomaterials to replace the lost bone. These materials are designed to mimic both the composition and structure of bone, although no single biomaterial has yet been discovered that possesses the biological properties of autologous bone. By implanting osteogenic cells on supportive biomaterials, bone tissue engineering offers an attractive alternative that enhances natural bone regeneration . This arA search of the literature was conducted until 15 October 2021. The eligibility criteria were as follows: (a) original articles reporting preclinical and clinical studies; (b) systematic and narrative reviews. Letters to editors, commentaries and conferences abstracts were excluded. PubMed and Web of Science online databases were searched using a combination of the terms: “cranioplasty”, “calvaria”, “skull”, “cranial”, “biomaterial”, “implant”, “scaffold”, “cell therapy”, “bone tissue engineering”, “3D printing”, “bioprinting”, “extracellular vesicles”, “extracellular matrix”, “conditioned media”, “gene therapy”, “transgene” and “growth factor”. A screening of the full texts of the retrieved articles was performed next to select relevant original articles and reviews addressing calvarial bone repair. Retrieved studies that were not directly related to calvaria bones were excluded. Finally, 196 studies published between 1985 and 2021 were considered by the authors as relevant for the topic and included in this review.A critical size cranial defect is that lacking enough bone tissue to heal spontaneously. The size of a defect to be considered critical depends on various factors that affect bone healing, such as the location of the lesion, patient age and the condition of the surrounding bone and soft tissues in terms of infection, previous radiotherapy or diseases affecting bone metabolism, such as diabetes or osteoporosis. Currently, autologous bone transplantation is the gold standard of cranial reconstruction surgery. Autologous bone grafts do not cause host rejection and they integrate well with the cranial cavity, resulting in a lower risk of fracture . AutologIn the case of decompressive craniectomies, in which the cranial defect is reconstructed only after resolution of the cerebral edema, subcutaneous or extracorporeal preservation of the patient’s bone allows further reimplantation ,14. A clThe use of cadaveric cranial grafts in cranioplasty was associated with complications secondary to infection and bone resorption. Therefore, the use of allografts in contemporary cranial reconstruction remains relatively rare ,19. HoweAs mentioned earlier, autologous bone transplantation is the gold standard of craniofacial skeleton reconstruction surgery. Although this method is suitable for small- to medium-sized defects, problems associated with morbidity of the donor site and tissue availability hamper its use in reconstructive treatments of large defects. In addition, the risk of bone resorption with an autologous implant remains high in children and adolescents when compared with adults ,22,23. TThe ideal biomaterial for cranioplasty should be biocompatible; radiolucent; non-toxic; with low complication rates; easy to use in the operating room; suitable for bending and molding, thus allowing the preparation of patient-specific implants; with excellent cosmetic results and low cost. The only material that might meet all these specifications is autologous bone, whereas the ideal biomaterial that fulfills all these requirements remains to be discovered . BiomateTitanium is biocompatible, chemically stable and suitable for the manufacture of patient-specific implants. Compared with other biomaterials used for cranioplasty, reconstruction using titanium provides for immediate and durable skull protection . TitaniuSynthetic polymer-based bone substitutes, a heterogeneous group of materials, include PMMA, polylactic acid (PLA), poly(ε-caprolactone) (PCL), polyethylene (PE) and PEEK. PMMA is an acrylic polymer that is formed by mixing a liquid methyl methacrylate monomer and a powder containing methyl methacrylate-styrene co-polymer or pre-polymerized PMMA. The powder contains an initiator, di-benzoyl peroxide, and N,N-dimethyl-p-toluidine is added to activate polymerization at room temperature. When the components are mixed, the liquid monomer polymerizes around the co-polymer or pre-polymerized PMMA powder particles to form hardened PMMA. Hydroquinone is also added to prevent premature polymerization due to light or high temperature exposure. Polymerization of PMMA occurs as an exothermic reaction, generating a paste that the surgeon can easily model and that slowly cools down and hardens into a solid within a few minutes. The result is a stable and strong material with compression and stress resistance higher than those shown by HA. In addition to being moldable, PMMA is cheap and radiolucent. Given that PMMA is brittle, it is often used in combination with titanium. Titanium meshes are often used to shield the PMMA from venous pulsation during the curing period as well as to prevent thermal injury to the underlying brain tissue. In general, PMMA is a safe biomaterial, with complication rates of 22.7%, similar to other materials used in cranioplasties . Lastly,Due to their similarity to the mineral phase of bone, bioceramics, and among them, calcium orthophosphates such as HA, have been extensively used to prepare implants for cranioplasty . HA obtaPLA and PCL are biodegradable polyesters that currently have a role in pediatric skull surgery as resorbable fixation plates that are replaced by bone during its natural degradation. They are highly biocompatible, with a slow degradation rate that preserves their mechanical features . Their uThe most popular plastic cranioplasty materials are PE and PEEK. Porous PE implants can be custom-tailored to meet patient specifications. The main disadvantage of these implants relates to their limited ability to support osteoconduction , being uCurrent cranial repair techniques combine the use of autologous bone grafts and biomaterials. In addition to being associated with harvesting morbidity, autografts are often limited by insufficient quantity of bone stock, a problem especially relevant in pediatric patients undergoing reconstruction of large skull defects. Reported complication rates in large autologous cranioplasty series are as high as 40%, with a mean reoperation rate of 25% ,52. Alth2. The amount of autologous cancellous bone available from the iliac crest to reconstruct the calvarial bone was very limited. To enhance the regeneration process, autologous stem cells derived from fat were applied in the defect, along with the autologous bone graft. Simultaneously with bone harvesting from the iliac crest, gluteal fat was obtained and processed during the ongoing surgical procedure to isolate autologous adipose-derived stem cells. The milled autologous bone was applied in the defect along with the stem cells which were kept in place using fibrin glue, obtained also from the patient who was subjected to plasmapheresis 2 days prior to surgery. Mechanical fixation was achieved using macroporous sheets based on PLA that also acted as barriers. The procedure was safe, with no signs of neurological deficits. Three months after cranioplasty, computed tomography showed marked ossification in the defect areas. Although it cannot be determined whether the effect was due to the conventional bone grafting or to the combination with autologous adipose-derived stem cell transplantation, the results encouraged subsequent studies on cranial reconstruction using stem cells.The clinical applications of stem cells in bone reconstructive surgery have been limited thus far to small case series. The first case report, published in 2004 , relatedThesleff et al.  publisheDue to impaired proliferation rates and senescence issues, it might be difficult to obtain large quantities of autologous stem cells from older patients ,58. A clPreclinical in vivo data continue to be the most relevant evidence of the potential translation of any experimental cell-based strategy. During the last 2 decades, a great variety of cells combined with carriers or scaffolds have been tested in animal models of calvaria bone healing ,61,62. MAlthough a few studies have explored the regenerative potential of terminally differentiated cells, e.g., osteoblasts ,64, mostOsteogenic and vasculogenic cells have been simultaneously co-transplanted into critical size cranial defects to stimulate de novo formation of blood vessels within the engineered bone . To thisCompared with adult stem cells, embryonic stem cells (ESCs) are much more powerful tools for regenerative medicine applications. Whereas adult stem cells show limited proliferation capacity, especially when obtained from elderly individuals or from patients affected by systemic conditions, ESCs exhibit unlimited proliferation and self-renewal ability, providing a continuous supply of stem cells that can differentiate into a multitude of cell types from each of three germ layers with far superior efficiency than adult stem cells. Liu et al.  induced Like ESCs, induced pluripotent cells (iPSCs) are also capable of differentiating to a wide range of cell types. However, iPSCs surpass the ethical constraints for human ESC use because they can be obtained by reprogramming somatic cells. Moreover, given that they can be obtained from the patient’s own cells, they avoid the concern over immunogenic sequelae raised by allogenic stem cells while enabling the design of patient-specific cell therapies. Also like ESCs, iPSCs are prone to form tumors, a risk that can be circumvented prior to implantation by their induction into progenitor cells, such as MSCs, or fully differentiated cells ,92,93,94Engraftment and survival rates of transplanted MSCs are typically very low, remaining active only for a short time. Delivery of products secreted by these cells often results in regenerative outcomes similar to those observed after cell implantation. These observations have recently led to the shift of a paradigm centered on the progenitor function of MSCs to another based on the paracrine control exerted on host cells, which offers the possibility of bypassing the use of living cells for bone tissue engineering applications . The useThe extracellular matrix behaves as a natural reservoir of a multitude of bioactive factors that are embedded in its structure. Chi et al.  preparedThe conditioned media from cultured MSCs has also been assayed in rodent cranial defects. Katagiri et al.  soaked cExtracellular vesicles, including exosomes and microvesicles, are lipid bilayer-delimited particles released from the cells that actively mediate intercellular communication. Among other biomolecules, they contain functional proteins, mRNAs, microRNAs and lipids, and they are very attractive candidates for cell-free applications in tissue engineering. Diomede et al. ,105 isolIn recent decades, extensive research on tailored biomaterials that provide a suitable matrix for cell delivery that supports regeneration of the native, vascularized cranial bone structure has been conducted. To control the rate of bone regeneration, the chemical composition, architecture and mechanical properties of many biomaterials have been manipulated to improve their in vivo behavior ,113,114.The formulation of bioinks with proper rheological properties is one of the main challenges in the development of functional constructs. Hydrogels are among the most commonly used base biomaterials for bioinks because their high water content facilitates cell entrapment . Early bLaser-assisted bioprinting (LAB) is based on the laser-induced forward-transfer effect, in which a laser pulse interacts with a sacrificial layer onto which the bioink film is placed, generating a cavitation-like bubble in the film. The expansion of the bubble propels the bioink to the substrate. The method allows droplet deposition of a fluidic phase at picoliter-level resolution . LAB tecTo address the limitations of the size and stability of constructs generated using extrusion and LAB technologies, a system called integrated tissue-organ printer was developed . It emplCell-based regional gene therapy, also known as ex vivo gene therapy, involves the implantation of genetically-modified cells to control the local production of a therapeutic transgene, and it has shown promise as a tissue engineering approach for bone regeneration. Prior to implantation of modified cells, this technology requires the collection of target cells from autologous or allogeneic sources, followed by a period of cell culture, expansion and gene transfer. Although this gene delivery method is rather cumbersome, the possibility of selecting well-characterized cell populations that can be further genetically modified with high efficiency is a major advantage over in vivo strategies. Moreover, ex vivo approaches in which target cells are transduced with viral vectors avoid the immunological risks associated with direct virus administration, and therefore are considered relatively safe. Numerous preclinical studies in animal models have demonstrated the ability of ex vivo regional gene therapy to safely and effectively heal bone defects, including critical size cranial defects ,126. DesTransgenes encoding potent osteogenic BMP are the most extensively tested in ex vivo approaches for cranial bone regeneration. BMPs are a group of related proteins that, based on primary amino acid sequence homology, belong to the transformation growth factor-β superfamily . Among tThe tetrameric cell penetrating peptide Tat has been combined with adenovirus encoding human BMP-2 as a strategy to increase transduction of human bone marrow-derived MSCs. It resulted in far greater new bone formation in rat critical size defects than in those that received cells transduced only with adenovirus . Improve2. Near-complete, full-thickness cranium defect repair was detected after cranioplasty using BMP-2-transduced cells, whereas the defects that received unmodified cells lacked consistent bone formation at the center of the defects. The stiffness of the engineered bone was indistinguishable from that of normal bone. A subsequent study from the same group [Particularly impressive were the results obtained by Chang et al.  with bonme group , in whicme group ,138.Differentiated cells have also been explored in preclinical gene therapies to enhance cranial bone regeneration through production of exogenous BMP-2. Lee et al.  transducBMP-7 is a member of the BMP family with strong osteoinductive activity. In 2001, the recombinant version of human BMP-7 received a limited US Food and Drug Administration approval for the treatment of recalcitrant tibial non-unions. A large, prospective, randomized-controlled, multicenter clinical trial did not show that BMP-7 treatment is truly non-inferior to iliac crest autograft and, consequently, there have been no products on the market based on this protein since 2009. However, research on ex vivo regional gene therapy to overexpress BMP-7 in cranial defects has yielded interesting data. Rat dermal fibroblasts that were transduced with an adenoviral vector carrying a BMP-7 gene led to complete repair of rat cranial defects . In addiBMP-4 is a major regulator of the development of axial and craniofacial structures of the skeleton. The potential of a human BMP4 transgene to regenerate skull defects using an ex vivo gene therapy approach was tested in retrovirally transduced rat bone marrow stromal cells that were included in a gelatin matrix and implanted in rat calvaria defects. The defects completely filled with new bone, while limited bone formation occurred in defects that received non-transduced cells . Rat musThe therapeutic potential of BMP-9 for promoting cranial bone growth has been tested in immortalized mouse calvarial mesenchymal progenitor cells that were transduced with an adenovirus carrying a human BMP-9 gene and then implanted in athymic mice . As suppNoggin is a secreted, high-affinity BMP antagonist protein, which functions by binding directly to several BMPs, including BMP-2, BMP-4 and BMP-7, thereby blocking their interaction with specific cell surface receptors . Levi etIn addition to BMP proteins and antagonists, secretion of other growth factors and ligands has been manipulated by means of gene transfer to enhance cranial bone regeneration. To overexpress VEGF, one of the most potent inducers of angiogenesis, human adipose-derived MSCs were transfected with a plasmid encoding the human VEGF gene . Bone heDiffusion of secreted transgenic proteins from cell constructs implanted in the cranial lesion can result in uncontrolled osteogenic and/or angiogenic responses. As an alternative, regional gene therapy involves the use of bone-specific transcription factors to drive differentiation of stem cells toward the osteoblastic lineage. Osteogenesis depends upon the activity of at least 2 transcription factors, Runx2 and osterix. Runx2 is an osteoblast-specific transcriptional activator that controls osteoblast development and maturation from MSCs. Mouse bone marrow-derived cells transduced with an adenovirus encoding Runx2 were adsorbed in gelatin sponges and implanted in craniotomy defects performed in syngenic mice . DefectsRat bone marrow-derived MSCs transduced with a lentivirus encoding HIF-1α increased bone volume and mineral density as well as blood vessel number and area after implantation in rat cranial defects compared with defects that were implanted with cells transduced with a lentivirus carrying a reporter gene ,162,163.Osteogenesis can be dramatically enhanced using regional gene therapy to express specific combinations of interacting regenerative molecules. For example, more effective healing was observed in cranial defects of mice implanted with a gelatin scaffold containing mouse fibroblasts transduced with adenovirus carrying BMP-2 and BMP-7 genes than in defects that received cells transduced with adenovirus carrying a BMP-2 or a BMP-7 gene . To studThe potential of proteins that participate in the regulation of osteoblast differentiation, other than secreted growth factors and lineage-specific transcription factors, has been explored in ex vivo gene therapies for cranial repair. Tested proteins include α5 integrin, which activates signaling pathways of human bone marrow-derived MSCs, resulting in increased Runx2 expression that promotes osteogenic differentiation ; ubiquitIn addition to specific signaling pathways and transcription factors, osteogenesis is tightly controlled by microRNAs (miRNAs), small non-coding RNAs that behave as key post-transcriptional regulators of gene expression through induction of either translational repression or cleavage of targeted mRNAs. Some studies have recently engineered MSCs to overexpress miRNAs that modulate their differentiation toward the osteoblastic lineage. miR-135 negatively regulates the levels of the homeobox Hoxa2, a protein that restricts bone mineralization in the calvaria during craniofacial development. Xie et al.  overexprAll the gene expression systems described thus far employ constitutively active promoters, which are unregulated and can produce excessive amounts of transgenic products, leading to adverse effects like those caused by delivery of recombinant proteins. Ideally, the expression of the therapeutic transgenes should be temporally coupled with the progression of healing to provide for the best control of the cranial regeneration process. However, unregulated promoters cannot limit transgene expression to the optimal therapeutic window. Currently available pharmacologically activated gene expression systems can regulate the level and duration of transgene expression, thus increasing the safety and efficacy of gene therapy.Temporal regulation can be achieved through the use of systems that function as on/off gene switches. They generally comprise a chimeric transcription factor that is activated or inactivated by a small-molecule ligand and a promoter linked to the transgene to be regulated, which is activated or silenced by the transcription factor. The Tet-On system employs transactivators based on a mutant of the tetracycline repressor protein that in the presence of tetracycline or derivatives such as doxycycline bind to a responsive promoter. Peng et al.  transducDimerizer-activated gene expression systems use a synthetic transactivator in which DNA binding and activation domains are distributed in 2 different non-interacting proteins, each of which has affinity to a dimerizer molecule , but not to the other protein. Only after a dimerizer capable of joining both molecules is added can the separate components form a functional transcription factor. Koh et al.  exploredDuring bone development and healing, cellular gene expression is tightly controlled in space ,186. CreRecent advances in nanotechnology have provided for noninvasive means of heating tissues. Near infrared (NIR) light from the 650–900 nm range is minimally absorbed by skin and underlying tissues, which leads to deep tissue penetration and minimal tissue damage. Various nanostructures and probes can absorb energy from irradiating NIR light and convert it into heat to increase the temperature of the surrounding environment . PhotothBone tissue engineering offers the promising alternative of generating constructs, composed of biomaterials including cells and/or growth factors that can enhance the outcome of reconstructive treatments. Thus far, the clinical applications of stem cells in cranial bone reconstructive surgery have been limited to small case series with varying outcomes. Although data collected from these studies have shown the safety of cell-based therapy, the great variability of indications, defect sites and sizes, as well as stem cell sources and biomaterials makes it difficult to draw solid conclusions on stem cell efficacy in cranial tissue engineering. In fact, the poor understanding of the therapeutic mechanism of action of the implanted cells, as well as the lack of technologies to control their fate determination, are the main obstacles for the clinical translation of cell-based therapies. Development of methods for identifying, isolating and expanding large quantities of specific populations of functional cells will result in more efficacious therapeutic products. Nonetheless, stem cell-based therapies have already emerged for the treatment of cranial defects, and it is expected that future clinical trials will succeed in showing their efficacy.Preclinical data obtained using animal models are, however, much more encouraging. Many cell types, in combination with biomaterials, some designed to stimulate cellular responses, have proven their ability to accelerate bone healing after implantation in defects created in various animal models. Data obtained using extracellular matrix, conditioned media and exosomes strongly support the hypothesis that cranial bone regeneration can be achieved through mobilization of endogenous cells using “cell-based but cell-free” approaches. Nevertheless, prior to manufacturing therapeutic products with standardized benefits, more fundamental knowledge on these released systems is needed to identify those key components that play a specific role in the bone healing cascade. Lastly, many studies have convincingly shown that bone regeneration of cranial defects could be enhanced by genetic manipulation of cells from various sources. The overexpression of genes encoding secreted growth factors and ligands, transcription factors and signaling molecules, alone and in combination, have been tested in cell-based approaches. More recently, the potential of miRNA overexpression has started to be explored. Given that viral vectors with improved safety profiles and non-viral vectors with improved gene transfer efficiencies are being developed, bone tissue engineering employing ex vivo regional gene therapy could become a clinical reality within the next few years. Moreover, recently developed transcriptional strategies could provide for tight spatial and temporal control of transgenic expression. Cell-based therapies will undoubtedly benefit from bioprinting techniques. With further development, bioprinting could produce clinically useful computer-assisted, patient-specific constructs that incorporate controlled patterns of multiple cell types, transgenic or not, to recapitulate the native structure and function of vascularized bone tissue. Particularly exciting are the data obtained by means of in vivo bioprinting, which pave the way to the intraoperatively application of this technology.Aging, systemic diseases, radiation and infection are factors that critically affect healing of critical sized bone defects. While most of the preclinical studies have been conducted with healthy young animals, research on the influence of those conditions in cranial repair induced by cell therapies is scarce. In most cases, materials harboring cells are implanted immediately after the critical defect is generated, an experimental approach that does not mimic the real clinical situations, e.g., cranioplasties resulting from decompressive craniectomies, which are only performed after resolution of the cerebral edema. The comprehensive consideration of these factors might accelerate the regulatory approval and clinical translation of cell-based products for cranial repair.Given the complexity of cell bioprocessing, manufacturing of cell-based therapies involves a high level of structural and economic resources. Research advancements on the field of cranial bone regeneration will be fruitless if the overall cell production pipelines are not cost-effective. In this regard, substantial efforts have been recently made by implementing automated process steps that reduce the cell production costs. Despite the challenges, cell-based technologies show unprecedented levels of therapeutic potential and are expected to radically change the landscape of cranial bone reconstruction in the near future.


DNA double strand breaks (DSBs) are among some of the most deleterious forms of DNA damage. Left unrepaired, they are detrimental to genome stability, leading to high risk of cancer. Two major mechanisms are responsible for the repair of DSBs, homologous recombination (HR) and nonhomologous end joining (NHEJ). The complex nature of both pathways, involving a myriad of protein factors functioning in a highly coordinated manner at distinct stages of repair, lend themselves to detailed mechanistic studies using the latest single-molecule techniques. In avoiding ensemble averaging effects inherent to traditional biochemical or genetic methods, single-molecule studies have painted an increasingly detailed picture for every step of the DSB repair processes. Maintenance of genome stability is paramount to the survival of all living organisms. Both extrinsic environmental factors, as well as intrinsic, routine cellular processes such as transcription and replication can lead to DNA damage and contribute to genome instability. Understanding DNA damage and genome maintenance is a crucial aspect of cancer research, as they are involved in carcinogenesis and cancer therapies .5 spontaneous DNA lesions that a cell experiences per day, double strand breaks (DSBs) pose a unique challenge to repair in that the physical continuity of the DNA molecule is disrupted  and canonical nonhomologous end joining (NHEJ). Together with pathways of alternative end joining  and single-strand annealing (SSA), these four mechanisms are tasked to minimize undesired loss of genetic information in the process of restoring the physical continuity of DNA. Canonical and alternative end joining repair pathways directly join and ligate the two broken ends of a DSB after minimal end processing . As thesOver the past two and half decades, single-molecule microscopy and spectroscopy have made significant contributions to characterizations of systems previously considered intractable, thanks in no small part to technological innovations in fields from physics to nanotechnology and protein engineering. In this review, we begin with a brief description of single-molecule techniques frequently used in the studies of protein-DNA interactions. The sections that follow will be dedicated to homologous recombination and nonhomologous end joining, where we first provide an overview of each of these pathways in mammalian cells. We highlight and discuss in detail the findings from single-molecule studies that contributed to mechanistic understanding of steps involved in each repair mechanism. While the focus is on eukaryotic DSB repair, insights from pioneering studies of bacterial repair proteins will also be presented when appropriate.in vitro single-molecule imaging studies of protein-DNA interactions must overcome is that as flexible polymers, DNA molecules, especially those significantly longer than their persistence length, collapse into random coils in the absence of external forces on the ends. Under most circumstances, unambiguous characterization of protein-DNA transactions is only possible when imaging is unencumbered by the presence of multiple DNA segments in close vicinity. To that end, several experimental approaches have been developed to maintain extended conformation of DNA molecules by exerting forces on their ends. In the sections below, we briefly describe these implementations.A crucial hurdle that all Broadly speaking, there are two strategies for extending single DNA molecules to a desired end-to-end distance: mechanical force extension, typically through the use of optical or magnetic tweezers, and hydrodynamic force extension.Optical Tweezers (OT) is an implementation of optical manipulation that controls and measures motion of trapped microscopic dielectric particle(s) using optical/electromagnetic forces . Beyond Similar to optical tweezers, magnetic tweezers utilize paramagnetic microspheres that are held in magnetic fields generated by external magnets . TypicalIn comparison to mechanical force extension, hydrodynamic flow represents a more straightforward, albeit less precise, method to unravel DNA molecules. Flow stretching readily complements optical tweezers where only one end of the DNA is attached to a bead held in an optical trap, as such combination was initially used to study DNA conformational dynamics and polymer physics models . When coA variation of surface-tethered single-molecule imaging technique named DNA curtains has been developed to minimize potential of surface interference and maximize parallelization . The plavia epi-fluorescence, total internal reflection, or confocal illumination. Each illumination scheme has its own advantages and disadvantages. For example, while total internal reflection reduces background noise significantly compared to epi-fluorescence, it is also restricted to imaging within ∼200 nm of the surface, due to the depth reachable by evanescence waves produced by total internal reflection at that surface , and a myriad of increasingly bright and photostable small-molecule fluorescent probes such as Alexa Fluor, ATTO, Janelia Fluor dyes . Fluores surface   experiments shed light on interactions that occur at much smaller distance scale . smFRET flection . Althougflection .Usually considered the error-free repair pathway for DSBs, HR can be divided into four distinct stages: end resection, formation of presynaptic filament, homology search, and repair synthesis . In mammEscherichia coli RecBCD is a helicase and nuclease complex that plays a critical role in repair of DSBs in bacteria by homologous recombination, whose function in promoting recombination is regulated by the Chi  sequence in DNA . At its core, the homology search process is highly similar to target search by other ubiquitous sequence specific DNA-binding proteins. A theoretical solution to the target search problem has been known for four decades as facilitated diffusion . UnderstMore detailed understanding of minimum sequence homology requirements and kinetics of sampling were uncovered in a pair of papers using single-stranded DNA curtains . In thesin vitro and homology search in vivo, its exact mechanism of action remained unknown  intermediate stages and stable (∼minute) synaptic complexes. Notably, these results support the two distinct stages of synapsis observed by Graham et al. using smFRET. The subcomplex containing Ku and DNA-PKcs and stabilized by PAXX (∼2 s) appears consistent with the long range synapsis, while the full complex further stabilized by XRCC4-LIG4 and XLF (∼66 s) would correspond to the short range synapsis  is identified as an interactor of XRCC4 and regulator of ligation . X-ray cMany DNA ends at DSB sites, regardless of their origins, require end processing before repair. The arsenal of NHEJ end-processing enzymes include nucleases, polymerases, kinases, phosphatases, and phosphodiesterases . The effSingle-molecule techniques have advanced and matured by leaps and bounds, thanks to technological improvements in equipment and reagents such as cameras and fluorescent dyes. The field has also expanded and benefited from commercialization of single-molecule instruments. An underlying technical challenge in single-molecule work has always been to achieve higher spatial and temporal resolutions. And this drive has steadfastly pushed technical innovations in the field. As single-molecule studies are typically built with a bottom-up approach, the field is constantly striving for increased levels of complexity in biological systems under examination. For mechanistic studies of homologous recombination, challenges remain, including but not limited to, in addressing functions of RAD51 mediator proteins and incorporating other accessory proteins in reconstituting the process from filament assembly to strand invasion, among others. Studying repair in general within the physiologically relevant context of chromatin has also proven challenging. Since multiple repair pathways exist and are available to cells for DSB repair, pathway choice is an overarching subject that bridges studies of individual repair mechanisms. Though initial work exists, the molecular mechanism for how competing repair mechanisms cooperate at the single-molecule level has largely been elusive. Biochemical reconstitutions of repair using purified recombinant proteins provide a clear, pre-defined set of parameters and have been the preferred system for single-molecule studies. However, functional cell extracts that already contain the proteins of interest may be the key to the pursuit of higher degrees of reaction complexity.in vivo evidence and require careful reconciliation. It is worth repeating that gaps exist among these approaches, such that a comprehensive picture is best constructed when all evidence is considered together. Indeed, differences exist even between comparable single-molecule studies using the same techniques, resulting in apparently incompatible interpretation of the mechanism. Building on existing imaging platforms that focus on studies of particular stages of the process, further developments of single-molecule imaging in vivo, complemented by biochemical and in vitro studies, will undoubtedly help uncover deeper understanding of DSB repair.The unprecedented level of detail in mechanistic insights from single-molecule experiments may at times be seemingly at odds with existing biochemical or


The emergence of additive manufacturing, otherwise known as 3D printing, was predated by significant advances in the understanding and controlled engineering of hydrogels.Hydrogels swell or contract dramatically as a function of their water content. They may be formulated to respond based on environmental factors, such as temperature, pH, mechanical or electrical field stimulation, ion or solvent concentrations or light stimulation ,2,3. BasAdditive manufacturing refers to a layer-by-layer creation of parts from a digital file. Additive manufacturing brings the ability to create complex, three dimensional geometries with hydrogels and to vary material composition throughout fabricated parts. Additive manufacturing of hydrogels typically involves liquid precursors, which are cross linked, layer by layer. This makes it easy to incorporate multiple materials of varied types during fabrication. Additive manufacturing process categories that are commonly used with hydrogel materials include vat photopolymerization by stereolithography (SL) or digital light processing (DLP) and material extrusion by direct ink writing .The potential benefits of hydrogels and the unique capabilities of additive manufacturing made additive manufacturing of hydrogels an early, advanced application area for additive manufacturing. Hydrogels received only two minor mentions in an early (1993) review of tissue engineering—for use in corneal tissue generation and as microcapsules for cell transplant . By 1998Comprehensive reviews of materials, processes and biomedical applications ,17,18,19Focused review of tissue engineering applications and strategies for biofabrication of artificial organs and organ-on-a-chip technology ,21,22;Soft robotics, smart materials and smart structures, including shape memory and self-healing materials, and complex, origami-inspired structures ,24,25,26Hydrogel materials: hydrogels with functionalized polymers; hydrogel/reactive filler/embedded cell bioinks; bioinks with nano-biomaterials for tissue engineering; and biomaterials ,28,29,30Bioresorbable electronics ;Multi-material additive manufacturing, with soft robotics and biomedical applications .The community has made substantial progress in the additive manufacture of hydrogels over the past two decades. There are a number of excellent reviews of this progress, including on these topics:Below are select recent advances toward compelling hydrogel applications, developed at least in part through additive manufacturing processes. Despite these advances, many of the potential applications will not be a long-term fit for additive manufacturing. Drawing on experience from other additive manufacturing application areas, the author proposes criteria for use cases that will be a good fit for additive manufacturing.as a research tool. The pace of discovery in this field is accelerating. Mechanical performance improvements include a wider range of control of the stress–strain relationships through hydrogel network composition and the creation of hydrogel materials that break the prior links between mechanical strength and parameters such as fatigue performance and recovery response time. There have been significant advances in the hybrid fabrication of hydrogel–polymer composite structures, as well as in the performance and reliability of hydrogel-based sensors and actuators.There are exciting advances promising the incorporation of hydrogels into functional designs in areas such as soft robotics and organ-on-a-chip technology. Many of these advances have been enabled by additive manufacturing processes Highly stretchable hydrogel structures may be covalently bonded with ultraviolet-cured polymers by a digital light processing (DLP) vat photopolymerization additive manufacturing process . This prAdditive manufacturing will certainly continue to put the latest improvements in hydrogel mechanical properties to full use. Dual-hydrogel networks significantly improve toughness and can be leveraged to create self-healing hydrogel structures and hydrogel structures engineered to strengthen on repeated stress . ResearcResearchers developed an autonomous, light-powered and oscillation-propelled hydrogel robot suitable for additive manufacturing, along with a model for the robot’s performance to tune oscillation frequency and locomotive force at varied robot sizes . AnotherVascularization of tissue grown on scaffolds is a key challenge. Researchers recently demonstrated the potential feasibility of DLP-based vat photopolymerization additive manufacturing of hydrogels with interconnected microchannels for use as synthetic skin grafts . The sciThe ability to rapidly fabricate complex geometries makes additive manufacturing a great tool for rapid iteration in design and research. Where might additive manufacturing excel for production part fabrication? The most compelling use case is the additive manufacturing of hydrogel parts with composition gradients. In many cases, production processes through traditional methods can be developed to produce parts that are designed with the help of additive manufacturing . This hoTypically, after a design is proven with rapid prototyping tools, it will be transferred to manufacturing using traditional processes for cost-effective production. This is the case as well for hydrogel applications. Traditional methods of hydrogel manufacture include laminating sheets of hydrogel or casting hydrogel in place ,48. HydrInjection-molded parts with laminated films are cost-effective manufacturing processes for lab-on-a-chip designs using traditional polymer materials . SimilarHydrogel scaffolds for tissue engineering and organ-on-a-chip applications are arguably more advanced than the use of hydrogels for soft robotics. These biomedical applications make good test cases for where additive manufacturing best contributes beyond rapid prototyping. Sacrificial molding is a common approach to incorporating hydrogels with complex internal geometries into organ-on-a-chip devices ,48. The Traditional manufacturing methods using multi-step fabrication thus offers a significant cost advantage over the additive manufacturing of hydrogels in most situations. Based on the compliant nature of hydrogels, it is hard to argue for mass customization of most hydrogel-centric design applications. Other than use cases that suggest a need for ‘manufacture on demand’ of hydrogel devices using additive manufacturing, the primary remaining long-term advantage that additive manufacturing holds over traditional processes is for designs that rely on the gradient composition manufacture to achieve highly localized performance. This would include designs for hydrogels, such as those already highlighted, to enable localized actuation, sensing and drug delivery ,39,40,41Until applications are developed that use the localized performance achieved through gradient composition manufacture, we should expect that the primary role for additive manufacturing of hydrogels will continue to be for rapid prototyping of research test beds and design candidates.


Erythropoietin (EPO), a glycoprotein produced and released by kidney interstitial fibroblasts, is well known since 1905 to stimulate erythropoiesis in the bone marrow . EPO wasThe aim of this research topic was to foster an interdisciplinary exchange of the advances in knowledge concerning EPO, its analogues and derivatives, receptor isoforms and target cells in the nervous system . We wereDey et al. review the effects of EPO on non-hematopoietic tissues and the mechanisms underlying metabolic regulation. Although they mainly focused upon the adipose tissue response to obesity-inducing diets, numerous issues raised concern the role of brain in this regulation. In fact, while EPO treatment may reduce fat mass and body weight by improving glucose utilization, within the brain the hypothalamus, a master regulator of appetite and energy expenditure, plays a key role. The Authors discuss a critical role for leptin, a Type 1 cytokine that belongs to the same superfamily of EPO. Interestingly, both EPOr and leptin receptors are localized in the same proopiomelanocortin producing neurons, which could suggest considerable functional overlap of the two cytokines. The Authors further discuss the sexual dimorphism of the regulation of metabolism by brain EPO and support the view that EPO/EPOr may have substantial benefits on human health by finely regulating the metabolism.Ottolenghi et al. review the potential use of EPO to reduce hypoxemic neurological damage in neonates with congenital heart defects. By downregulating the oxygen delivery to neurons, this devastating pathology challenges brain development though disruption of the equilibrium between redox imbalance and antioxidant defense, thereby causing irreversible brain damage and impairing brain development. Although representing a clinical therapy to counteract fetal hypoxia, maternal hyperoxygenation is not without risks. Thus, studies are under way to evaluate the potential role of rhEPO as neuroprotective factor based not only on its antioxidant properties, but also on the promotion of oligodendrocytes differentiation/maturation and stimulation of myelin production. Concerns remain as to route of EPO administration because the poor permeability across the blood-brain barrier requires high doses that may lead to excessive erythropoiesis.Arias-Reyes et al.. The Authors examine in rodents the dose-dependent impact of EPO in the carotid body (CB) response to hypoxia or hypercapnia, based on a controversial data previously obtained, which showed that high EPO doses failed to stimulate central nervous system discharges as compared to the opposite by lower doses. The Authors found that EPO produces a dual effect on CB chemoreception: at low doses (<0.5 IU/ml) EPO stimulates the hypoxic response to hypoxia, while at higher doses (>1 IU/ml) EPO inhibits central nervous system activity via increased nitric oxide (NO) production by type I CB cells. Besides representing an important caution when treating hypoxemic patients, these findings are particularly relevant because they suggest a critical interaction between EPO and the activity of NO synthases, especially the endothelial one, which may be particularly relevant in populations residing at high altitude and therefore under continuous erythropoietic stimulation.The critical issue of the EPO doses to be used clinically without causing adverse erythropoietic effects is the main objective of the original study reported by Newton et al. critically review the role of EPO and EPO derivatives in cognition. Besides providing beneficial effects in several psychiatric disorders , EPO appears as the only drug capable of delaying cortex thinning in schizophrenia. The mechanisms underlying these benefits encompass changes in gene expression, improvement of neurogenesis, synaptic plasticity, oligodendrogenesis, and anti-inflammatory activity. Remarkably, an EPO non-erythropoietic analogue, carbamoylated EPO, apparently elicits the same favourable effects elicited by EPO, but without increasing erythropoiesis. The Authors describe a stereochemical model that compares the erythropoietic vs. non-erythropoietic receptor signalling whereby the JAK2/STAT signalling is engaged by EPO only, but the PI3K-Akt and ERK signalling are engaged by both.Finally, • Further dissection of the roles of EPO in normal and pathologic neurobiology and neurodevelopment.• Dissecting the biological activities and molecular mechanisms underlying the EPO neuroprotective effects.• Dissecting EPO’s effect on cell metabolism as therapeutic target.• Identifying EPO analogues that act as potential therapeutic treatments in CNS.• Assigning roles for specific cell-type EPO receptor isoforms.• Elucidating which acute pathologies  or chronic neurodegenerative diseases  may be targetable for rhEPO -based therapies.• Determining effects of EPO/analogues on cognitive function/depression/schizophrenia.• Identifying specific stimuli, e.g., hypoxia, hyperoxia, brain hypoxia and ischemia, inflammation, iron dysfunction, oxidative/metabolic stress, which may have potential positive or negative interference with EPO-based mechanisms.• Introducing novel multidisciplinary experimental models aimed at covering the gap between basic mechanisms and clinical outcomes.• Assessing potential clinical advances .The articles published in this Special Issue aimed at assessing the potential role of EPO and its analogues in driving neuroprotection in acute injury and neurodegenerative diseases. Remarkably, these contributions are based on basic or clinical data in the perspective of multidisciplinary efforts “from bench to bed—and return”. Clearly, to get a full and exhaustive picture of the role of EPO in neurological disorders, much work is still required to address the following topics:


Oropharyngeal cancers related to the human papillomavirus are a growing segment of head and neck cancers throughout the world. These cancers are biologically and demographically unique with patients presenting at younger ages and with more curable disease. This combination of factors heightens the importance of normal tissue sparing because patients will live a long time with treatment sequelae. Proton therapy has demonstrated benefits in reducing normal tissue exposure, which may lead to less toxicity, a higher quality of life, less immunologic suppression, and lower cost. Research investigating deintensified radiation volumes and doses are also underway. These deintensification studies synergize well with the beam characteristics of proton beam therapy and can decrease that already reduced normal tissue exposure enabled by proton therapy. Future studies should refine patient selection to best allow for volume and dose reduction paired with proton therapy. As of 2020, oropharyngeal squamous cell carcinomas (OPSCCs) constitute most new head and neck cancer diagnoses within the United States. The annual number of OPSCC cases is projected to rise from a baseline of 20,214 cases in 2016 to an estimated 30,629 cases by 2029, a 51.5% increase in less than 2 decades . S. S24]. SThe concept of contralateral neck sparing with the use of photon therapy has been advocated in the past couple of decades for the management of well-lateralized, early stage oropharyngeal carcinoma, resulting in favorable oncologic outcomes, lower toxicity rates, and improved long-term function in well-selected patients . FurtherWith more-accurate diagnostic imaging modalities and improvements in radiation-delivery techniques, such as IMPT, the idea of further deintensification therapy for these patients has become more tangible. In the past few years, several institutions have reported their experiences with the management of locally advanced head and neck tumors and the use of PBT –29. The Manzar et al  reportedAnother mechanism to further deintensify therapy and improve the therapeutic ratio is sparing different lymph node levels from RT. The University of Chicago developed a phase II trial of dose and volume de-escalation for the treatment of HPV-OPSCC  32]. S. S32]. SThe physical properties of a proton beam, combined with volume reduction strategies, allow for substantial reductions in healthy-tissue radiation exposure. Nevertheless, patterns of failure data for oropharynx cancer often require treatment volumes that unavoidably cover at least a portion of healthy structures. For example, coverage of retropharyngeal lymph nodes invariably involves at least the lateral pharyngeal constrictor muscles. Attempts to avoid these at-risk areas, such as the medial parotid gland, for example, can be linked with increased risks of regional nodal recurrence. These overlapping areas between at-risk volumes and healthy tissue structures comprise unavoidable areas of toxicity with radiation therapy, particularly in serial structures. It is in these settings that proton therapy synergizes with radiation dose-reduction efforts underway with HPV-OPSCC. This section will review how the clinical benefit from radiation dose-reduction strategies can be maximized with proton therapy.The most straight-forward strategy for radiation dose deintensification is to have the radiation dose reduction as the sole alteration in the treatment strategy. For example, a phase II study by Chera et al  investigGiven the larger volume of disease, radiation doses for definitive treatment are necessarily greater than radiation doses required for subclinical or microscopic disease. Because long-term healthy-tissue toxicity is often linked with cumulative radiation dose, deintensification strategies using multimodality therapy to debulk disease burden have also been investigated. Induction chemotherapy provides one means for simultaneously debulking a tumor when chemoselecting patients for radiation deescalation. For example, a phase II trial by Chen et al  followedProton therapy offers a number of deintensification strategies both off and on a clinical trial. The inherent dose characteristics of PBT translate into healthy-tissue sparing for oropharyngeal cancer treatment, even with standard treatment volumes and doses. Furthermore, the reduced low-dose spillover may translate into hematologic and immunologic benefits, which should be explored more prospectively. These dose characteristics may result in cost savings because of reduced side effects and better toxicity management in the long term.Off of a clinical protocol, proton therapy can leverage prospective data concerning volume reduction. In the induction setting, generating the high-dose target volume based on the postinduction primary and nodal disease appears safe and reasonable. Similarly, postsurgical volume modifications can be optimized using proton therapy. Avoidance of the primary site for early stage tumors and avoidance of the dissected, N0 contralateral neck have both shown utility in prospective studies. Although deintensification efforts for HPV-related oropharynx cancer continue to mature, these efforts are also still investigational. At this time, we recommend enrolling patients in clinical trials similar to those listed in the


Implemented through five health plans, Ohio’s MyCare demonstration began in 2014 and was designed to coordinate primary, acute care, behavioral health and long-term services in the major urban areas of the state. Individuals who are dually eligible for both Medicaid and Medicare and who reside in specified geographic regions must enroll into a managed MyCare plan. MyCare beneficiaries are assigned to two primary categories: community well and those needing long-term services and supports (LTSS). Individuals receiving the integrated MyCare intervention were expected to have lower acute care hospitalizations, lower long-term nursing home use, better longevity and lower overall health and long-term care costs. Using a propensity score matching design, the evaluation compared MyCare enrollees to comparison group members in non-MyCare counties of the state, using Medicaid and Medicare claims data. The 120,000 MyCare program participants represented about half of the dual eligible individuals in the state.


Air pollution is linked to mortality and morbidity. Since humans spend nearly all their time indoors, improving indoor air quality (IAQ) is a compelling approach to mitigate air pollutant exposure. To assess interventions, relying on clinical outcomes may require prolonged follow-up, which hinders feasibility. Thus, identifying biomarkers that respond to changes in IAQ may be useful to assess the effectiveness of interventions.We conducted a narrative review by searching several databases to identify studies published over the last decade that measured the response of blood, urine, and/or salivary biomarkers to variations  of changes in indoor air pollutant exposure.Numerous studies reported on associations between IAQ exposures and biomarkers with heterogeneity across study designs and methods. This review summarizes the responses of 113 biomarkers described in 30 articles. The biomarkers which most frequently responded to variations in indoor air pollutant exposures were high sensitivity C-reactive protein (hsCRP), von Willebrand Factor (vWF), 8-hydroxy-2′-deoxyguanosine (8-OHdG), and 1-hydroxypyrene (1-OHP).This review will guide the selection of biomarkers for translational studies evaluating the impact of indoor air pollutants on human health. Since individuals spend about 90% of their time indoors, indoor air quality (IAQ) is a key driver of the effect of air quality on human health Malondial5 , reactive gases ], a wide range of VOCs and semi-volatile organic compounds (SVOCs), and particulate matter (PM), ranging from about 1 nm to 10 µm in diameter. Some compounds, such as polycyclic aromatic hydrocarbons (PAHs), perfluorinated compounds (PFCs), polychlorinated biphenyl (PCBs), and polybrominated diphenyl ethers (PBDE), are found in both the gas and particulate phases depending on partitioning behavior and emission source.Indoor air exposures are a mixture of ambient air pollution brought indoors via ventilation and infiltration and indoor generated pollution emitted from combustion , building materials and furnishings, and human behaviors such as smoking, cooking, and cleaning products –61. CommPoor air quality is associated with adverse clinical outcomes, which however take a long time to accrue and are thus challenging to use in translational research studies. Hence, the ability to rely on biomarkers as surrogate endpoints is critical to the conduct of observational studies as well as interventions. A previous review suggested that common mechanisms included inflammation and oxidative stress . HoweverThe present review extends prior knowledge by summarizing available data on the associations between biomarkers and IAQ. The mechanistic pathways associated with variations in IAQ include inflammation, coagulation, and oxidative stress. These pathways are known to be associated with chronic diseases, including cardiovascular diseases, respiratory diseases, and cancers supporting the biological plausibility of these associations.Some limitations of the reviewed studies should be mentioned. Most studies were cross-sectional and almost half of the studies measured biomarkers at only one time point during the course of the study. Methods varied considerably across studies and hence direct comparison was challenging. Randomized intervention studies measuring paired groups of individuals are recommended for future IAQ biomarker studies to reduce confounding variables and improve quality research. Additionally, power was mentioned in only 3 of the 30 reviewed papers, therefore precluding its systematic assessment. Six biomarkers were measured in more than one type of specimen , however, methods of measurements were not compared across specimen type. Thus, it is unclear if one specimen is more useful in measuring a particular biomarker than the other.Our review has a number of important strengths. We conducted a comprehensive literature review using a rigorous methodology. Our review provides the most current review of the literature over the last decade and useful guidance for the selection of biomarkers in translational studies of IAQ.Herein, we summarize the current evidence on the biomarkers which most frequently responded to variations in IAQ. The biomarkers which exhibit the most consistent association with IAQ were high sensitivity CRP, vWF, 8-OHdG, and 1-hydroxypyrene (1-OHP. This summary provides a guide to select the biomarkers for translational studies evaluating the impact of indoor air pollutants on human health.