Centromeres are essential genetic elements that enable spindle microtubule attachment for chromosome segregation during mitosis and meiosis. While this function is preserved across species, centromeres display an array of dynamic features, including: (1) rapidly evolving DNA; (2) wide evolutionary diversity in size, shape and organization; (3) evidence of mutational processes to generate homogenized repetitive arrays that characterize centromeres in several species; (4) tolerance to changes in position, as in the case of neocentromeres; and (5) intrinsic fragility derived by sequence composition and secondary DNA structures. Centromere drive underlies rapid centromere DNA evolution due to the “selfish” pursuit to bias meiotic transmission and promote the propagation of stronger centromeres. Yet, the origins of other dynamic features of centromeres remain unclear. Here, we review our current understanding of centromere evolution and plasticity. We also detail the mutagenic processes proposed to shape the divergent genetic nature of centromeres. Changes to centromeres are not simply evolutionary relics, but ongoing shifts that on one side promote centromere flexibility, but on the other can undermine centromere integrity and function with potential pathological implications such as genome instability. Saccharomyces cerevisiae ..DrosophiArabidopsis thaliana [Oryza sativa [Zea mays [Arabidopsis centromere has a 180 bp monomer   H [21], yIndeed, primary constriction size appears invariant and with a constant scale of magnitude from yeast to human . Thus, dA rapid and heterogeneous evolution of centromere components across eukaryotes is in disagreement with its vital and conserved centromere function ,124. YetEven though this evidence elucidates the advantage of centromere evolutionary changes, deleterious effects must also be taken into consideration, including unbalanced segregation that could generate incompatible post-zygotic hybrids contributing to speciation ,134.Centromere rearrangements are protagonists in karyotypic divergence, as in the case of the horse and donkey. Changes in centromere repositioning created chromosomal structural variations that act like a “genetic barrier” between these two species due to the odd rate of meiotic chromosome recombination, which causes the gametogenic failure in mules .Mimulus aurantiacus displays many CenH3 duplication events under a divergent process in which paralogs differentiate with distinct sub-specialized functions , , 189], oAs an alternative, circular 3′ ssDNA (single stranded DNA) templates generated at the D-loop lead to the induction of rolling circle replication (RCR) E which oIndeed, Rice  assignedThe aforementioned processes cause amplification, expansion and large-scale remodeling of the genomic landscape at the centromere. However, they must also be intersected by localized mutagenesis, including that which triggers divergence between monomers. In the example of the human centromere, individual monomers of α-satellites share only 50–70% sequence identity between each other, while HOR blocks are nearly-identical. Thus, large-scale processes may be rarer and have operated on a wider timescale than small-scale changes and micro-mutations that may continue to shape and diverge centromeres. Notably, BIR seems sufficient to create mutations within the replicated sequences  andC. albicans and C. tropicalis, in which the loss of these inverted repeats provokes the formation of a new centromere. When the essential function of centromeric HIRs is missing, a CENP-A-rich zone influences the seeding of evolutionary new centromeres (ENCs) in order to reestablish the eroded centromere region [A supplementary mechanism to accomplish concomitant mutagenizing and homogenizing of the centromeric repeats is based on inter-chromosomal translocations guided by the organization and proximity of spatial repeats. A high percentage of translocation events has been demonstrated in centromeric homology inverted repeats (HIRs) of common progenitors of e region . The plae region ,147, mutThe DNA organization at human centromeres is a notable example of repeat amplification, homogenization and mutagenesis. One of the first studies on the evolution of human satellite DNA was advanced by Smith in 1976 , with thMore recent advances in methodologies and sequencing allowed the construction of centromere phylogenies to compare centromeres among different organisms, as well as between the same species. Intra- and inter-species analyses are a very helpful tool for the recognition of ancestral and new properties of centromere repeats, exposing evolutionary constrains and adaptive changes over different timescales . In factAlexandrov and colleagues advanced a very interesting model about the formation of HOR in Great Apes from an old ancestral monomer in lower primates . SupposeWhile evolutionary processes underlying centromere divergence remain unclear , a new aThe model’s structural frame is based on the spatial organization of three types of ~170 bp monomeric repeat units ,232 thatAmong these three monomeric repetitive units, intra-array competition exists. It is based on the capability of centromeric core repeats to extend and migrate towards the flanking heterochromatin region, contrasting it. Thus, this new and interesting model highlights the contrasting forces and high level of evolution caused by the amplification (BIR process), shirking (SSA process) and homogenization of HORs .Inside human HORs, the number of monomers ranges from two (as in chromosome 1 ) to 34 mWhile chromosomes can contain more than one centromere array with its own set of HORs , SullivaEven if these processes are valid and important attempts at placing repetitive pieces of a large puzzle together, experimental evidence is needed to validate their action within human centromeres.Changes in centromere DNA can reasonably induce chromosome segregation errors and result in chromosome instability (CIN) ,240. HowS. cerevisiae, where there is a cohesin enrichment [The pericentromere region is responsible for sister chromatid cohesion and has been found to contribute to centromere integrity . Except richment ,245, therichment . Heterocrichment . This isrichment , yet it richment . Hypo-acrichment , as wellrichment ,250,251,richment ,250,251 richment . DNA hyprichment . Collectrichment . Epigenerichment , maintenrichment . Notablyrichment .From recent evidence, a shift in CENP-A localization or its depletion led to a change in chromatin status that could interfere with local and long-range transcription processes . This waCentromeres hold multiple paradoxes, including rapidly evolving DNA and molecular players, tolerance to changes in position and size, evidence of profound mutational processes, intrinsic fragility dictated by repetitive DNA and possibly secondary structures, all while maintaining a fundamental and conserved function. Interestingly, centromere sequences reveal the preferential accumulation of tandem repeats and a conserved epigenetic identity as the driving force for maintaining centromere function in spite of their high mutational rate. Such molecular processes affect the clusterization of satellite DNA and its higher order assembly, but it is not exactly clear at which level they operate and especially what the mechanisms are that preserve centromere DNA stability and mitigate ongoing mutagenesis. Because of its genetic variability, the unifying definition of a centromere refers to its functionality in enabling chromosome segregation. Under evolutionary forces, each organism modulated this essential structure based on their evolutionarily benefits, mitigating drawbacks and tolerating adaptation. Thus, the changes in DNA content, size and positional shifts, as well as the three-dimensional arrangements of increasing complexity that can converge to sustain a loop of mutagenesis that feeds centromere evolution, are both an advantage or a hindrance depending on the timescale snapshot (within a few generations or selected over millions of years). Looking at centromere abnormalities widely found in cancer and other disorders, the precarious equilibrium between rapid changes and functional preservation in the quest for the sustained propagation of centromeres likely comes at a cost in conflict with DNA stability. We do not yet know the complete journey that makes a centromere so, but it is certainly an exciting and eventful one that we hope will soon fully emerge.


It is now apparent that platelet function is more diverse than originally thought, shifting the view of platelets from blood cells involved in hemostasis and wound healing to major contributors to numerous regulatory processes across different tissues. Given their intriguing ability to store, produce and release distinct subsets of bioactive molecules, including intercellular signaling molecules and neurotransmitters, platelets may play an important role in orchestrating healthy brain function. Conversely, a number of neurodegenerative conditions have recently been associated with platelet dysfunction, further highlighting the tissue-independent role of these cells. In this review we summarize the requirements for platelet-neural cell communication with a focus on neurodegenerative diseases, and discuss the therapeutic potential of healthy platelets and the proteins which they release to counteract these conditions. Platelets are small anucleate blood cells that have been gaining recognition as important mediators of several regulatory processes. Emerging research has identified novel functions that reach well beyond the traditional role of platelets in hemostasis and wound closure, revealing them to be crucial players during immune responses and tissue remodeling processes. We have recently summarized the evidence highlighting the capacity of platelets to contribute to brain homeostasis under physiological circumstances . WhereasUntil recently, platelets were primarily known for initiating coagulation following tissue injury and endothelial disruption. Although the platelet count in healthy humans ranges from 150,000 to 400,000 platelets per microliter of blood , only a Platelets can communicate with other cell types in multiple ways, with their flexibility and mechanistic diversity suggesting that they likely act as inter-tissue messengers, including between blood and brain cells. Although the secretion of bioactive molecules from α- and dense granules represents a likely route of intercellular communication, additional mechanisms via which platelets may support crosstalk between the brain and the systemic environment are possible. Platelets release extracellular vesicles containing active cytoplasm components such as exosomes and microparticles . Both rein vitro, and that mice which have been depleted of platelets fail to show the expected exercise-induced increase in neural precursor cell proliferation in vivo , glutamate, serotonin, epinephrine, dopamine, and histamine. This suggests that platelets can send and receive signals to and from the nervous system and may act as an important relay between the brain and peripheral organs. The monoamine neurotransmitter serotonin is stored in dense granules, and peripheral serotonin release-associated regulatory functions of platelets have been described , 22. Alte medium .Given these similar mechanisms of neurotransmitter uptake and metabolism, platelets have been suggested as a model system of glutamate and GABA transport in patients suffering from neurodegenerative conditions , 31. A mIt is becoming clear that neurodegenerative diseases do not solely involve cells and tissue of the central nervous system, but rather that systemic influences also play a fundamental role in the development and exacerbation of brain pathologies. As discussed above, platelets are of particular interest as important mediators of this two-way relationship. Several review papers have concluded that these blood cells can serve as potent systemic biomarkers of neurodegenerative diseases, mirroring the pathological phenotypes of neural cells –34. In tAD is a slowly developing progressive form of dementia that is accompanied by unpredictable behavior, lack of enthusiasm and memory loss. The neuropathological hallmarks of AD include neuronal and synaptic loss, neuroinflammation, the formation of intracellular neurofibrillary tangles and the deposition of amyloid-ß in brain tissue and cerebral vessels. Increasing evidence has linked platelet dysfunction to this disease, in particular in the context of amyloid-ß secretion from platelets.Although neural cells, including astrocytes and neurons, produce and secrete amyloid-ß , the pepRather than alterations in platelet count or size, changes in platelet activation appear to play a prominent role in AD, with increases in activation detected in the blood of AD patients, likely as a result of increased lipid peroxidation . SimilarA recent parabiosis study, in which the blood circulation of APPswe/PS1dE9 transgenic AD model mice was connected with that of their wildtype counterparts demonstrated that human amyloid-ß originating from the transgenic mice accumulated in the brains of their healthy littermates, forming amyloid-ß plaques and amyloid angiopathy following 12 months of parabiosis . MoreovePrior to amyloid-ß plaque formation, platelet inclusions in cerebral blood vessels are among the first symptoms to appear in the brains of APP_SweDI AD model mice . AnotherAn interesting alternative theory is that amyloid-ß release represents a defense mechanism against septic agents , 90. RecAlthough the studies described above focused on amyloid-ß, this peptide does not represent the only known link between platelets and AD, with other investigators examining the involvement of neurofibrillary tangles and impaired neurotransmitter homeostasis. These studies have been reviewed elsewhere .HD is a hereditary autosomal dominant neurodegenerative disorder caused by a CAG repeat expansion in exon 1 of the huntingtin gene, resulting in the production of a mutant huntingtin protein (mHtt). This protein accumulates in neurons, thereby leading to their eventual death and a progressive loss of motor and cognitive functions. Extensive research has shown that a number of cell subpopulations in the blood are altered in HD patients, with platelets having the highest levels of mHtt .2AR) signaling  have also been suggested to be critical contributors to HD pathology. Nitric oxide metabolism has been found to be dysregulated in platelets during the late stages of HD progression , and MAOA proposed model of HD pathogenesis is the “excitatory hypothesis,” based on the observation that excitatory amino acids and N-methyl-D-aspartate receptor agonists, including aspartate and glutamate, recapitulate the striatal neuron degeneration observed in HD . AlthougMitochondrial dysfunction has also been implicated in the pathogenesis of HD. A significant decrease in mitochondrial complex I activity per platelet was observed when patients were grouped according to disease severity; however, when normalized to mitochondrial DNA content, no differences were detected . In contPlatelets are also important in maintaining normal vascular integrity . RecentlPD is a degenerative disorder caused by the loss of dopaminergic neurons in the substantia nigra, thereby resulting in an impairment in motor and cognitive functions. Although the cause of sporadic PD, the most common form of the disease, is unknown, one major causal factor is mitochondrial dysfunction. This was first suggested by the finding that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a neurotoxin that selectively kills dopaminergic neurons, acts by inhibiting complex I of the electron transport chain . A plethSeveral studies have suggested that MAO also plays an important role in MPTP toxicity and the etiology of PD. Increased MAO-B activity has been observed in PD patients –111, potA number of other alterations in the platelets of PD patients have also been suggested as potentially useful biomarkers. These include a reduction in vesicular monoamine transporter 2 mRNA , an incrALS is a fatal neurodegenerative disorder that is characterized by progressive and selective loss of motor neurons in the brain and spinal cord. Patients suffer from progressive muscle weakness and paralysis of their voluntary muscles, ultimately leading to respiratory failure and death. There is accumulating evidence that in addition to affecting motor neurons, ALS also affects platelets.Almost all ALS cases (~97%) are characterized by pathology due to the TAR DNA-binding protein of 43 kDa (TDP-43) , 115. InMitochondrial abnormalities, particularly impairments of complex IV (cytochrome c-oxidase) activity, have been implicated in ALS, although the exact role of mitochondrial dysfunction remains unclear. In addition to mitochondrial dysfunction in motor neurons of ALS patients, mitochondrial changes have also been reported in muscle, liver and blood cells, suggesting systemic involvement –118. ComThrombospondin is a glycoprotein that is released from platelet α-granules following thrombin-induced platelet activation. Changes in blood thrombospondin levels have been detected in a number of pathological conditions, including a marked increase in thrombospondin deposition in the muscles of ALS patients , 121. ThMS is an inflammatory disease, where the immune system attacks the myelin sheaths that cover nerve axons in the spinal cord and brain. The resulting nerve damage leads to communication deficits between the brain and other tissues, and depending on the affected nerves provokes a range of symptoms, including impairments in vision, deficits in motor control of the arms and legs and neuropsychological symptoms such as depression and memory loss. To date, there is no known cure for MS, as the underlying cause is still unknown.A few studies targeting platelets and their involvement in MS and its mouse model, EAE, have shown that these conditions are associated with abnormalities in platelet function. One of these investigations found increased platelet activation in the blood of clinically stable relapsing-remitting MS patients who had not yet received treatment . This waRecent work investigating the brains of EAE-induced mice demonstrated that platelets were also present in the parenchyma of the hippocampus, including in the fimbria and in close proximity to neuronal cell bodies in the dentate gyrus and CA1 region . This phPrion proteins (PrPs) comprise a class of amyloid-forming proteins, with some isoforms being associated with a group of fatal neurodegenerative diseases termed transmissible spongiform encephalopathies. Once diagnosed, these conditions progress rapidly and are characterized by the chronic deterioration of physical and mental abilities, including profound memory impairments. The scrapie isoform of PrP is an abnormal, misfolded, protease-resistant isoform , 127 whic) is carried by blood cells, including platelets, in which PrPc is present on the membranes of α-granules (c can be released from activated platelets, mainly in the form of microparticles and exosomes (c under these circumstances is unknown, although it has been reported that the protein is unlikely to play a role in the aggregation or adhesive actions of activated platelets (Cellular PrP (PrPgranules , 129. Foexosomes . The funlatelets . The rellatelets . Other wlatelets . In a shlatelets . These dlatelets , 80, evelatelets –132.Impairments in platelet function are a common observation in neurodegenerative disorders; however, healthy platelets and their secreted factors also represent a possible approach for the development of therapeutic interventions for the treatment of neurodegenerative conditions. Among the primary applications are the use of platelet lysate and platelet-rich plasma, both of which are easy to obtain from immune-compatible healthy donors. The beneficial effects of platelet-rich plasma treatment are likely to be attributable to the abundant variety of growth factors that platelets carry in their granules. Neural and glial cells express surface receptors for a range of these growth factors, including vascular endothelial growth factor, epidermal growth factor, fibroblast growth factor-2, platelet-derived growth factor, brain-derived neurotrophic factor, platelet factor 4, transforming growth factor-ß, insulin-like growth factor-1, connective tissue growth factor and bone morphogenetic protein-2,−4, and−6, suggesting a fundamental role of platelets in tissue growth and regeneration, including in the brain –135. MorPlatelet-rich plasma can easily be prepared from whole blood using a slow centrifugation speed and physiological washing buffers that support platelet purification. This method achieves a nearly pure population of platelets [>99.99% purity ], and th® technology to isolate platelet-rich plasma from human blood have demonstrated that treatment with these preparations significantly reduces amyloid-β plaque density in the hippocampus and improves cognitive function in APP/PS1 AD model mice (Beneficial therapeutic effects of platelet-rich plasma treatment have been reported in numerous tissues, including during burn healing , 142, cadel mice . Anotherdel mice . The samdel mice . These dSimilar to platelet-rich plasma, platelet lysate can be easily obtained from whole blood samples. Platelets are first enriched by centrifugation steps, followed by freezing and thawing of the samples. An additional centrifugation step then separates the freeze/thaw-triggered secreted platelet factors, which constitutes the platelet lysate, from the remaining cell debris.in vivo applications (Given their essential role in wound healing and tissue repair, platelet lysates are being investigated as a therapy for a number of neurodegenerative diseases. Human platelet lysates have been investigated as a novel biotherapy for ALS and PD patients. In an NSC-34 cell-based model of ALS, human platelet lysates conferred a neuroprotective effect against staurosporine-induced apoptosis and menadione-induced oxidative stress, indicating that neuronal loss can be diminished by platelet factors in those conditions . In a Luications . Followiications . The intications , making Although we have not addressed stroke and other brain injuries in this review, human platelet lysate treatment has also been shown to produce positive outcomes in these conditions. Following stroke, human platelet lysate injections into the lateral ventricles of rats had neuroprotective effects . The plaIn addition to platelet-rich plasma and platelet lysate preparations, an interesting approach is emerging, whereby platelets are used as a physiological vehicle to deliver molecules to target regions that might otherwise be difficult to access. With their context-dependent and specific cell-cell communication capacity, platelets could serve as a selective and non-toxic drug delivery system in order to target specific cells and tissues. This approach has been extensively discussed previously, with a particular focus on the use of platelets to deliver chemotherapeutic agents to tumors . HoweverAs summarized in this review, data connecting platelets and the factors they secrete to neurodegeneration have accumulated over recent years. However, it remains unclear whether platelet malfunction initiates the pathophysiological events that occur in neurodegenerative conditions, or whether platelet dysfunction arises as a consequence of other unfavorable changes that occur at early stages of these disorders. More data regarding the origin of platelet dysfunction are therefore required. During the onset of neurodegenerative conditions, factors released from healthy platelets could also have a protective role, as suggested by recent studies of AD  and cancAlthough platelets and their released factors are gaining recognition for their potential therapeutic value in regenerative medicine, research is still in its infancy. Furthermore, the origin of platelets, the bone marrow, should not be overlooked, as a functional predisposition may also be inherited from their parent cells, the megakaryocytes. In conclusion, it remains highly interesting, but at the same time extremely challenging, to understand how platelets exert manifold actions across different tissues in physiological as well as pathological conditions. Their functional complexity clearly demands interdisciplinary approaches in order to develop novel therapeutic interventions which benefit from the multifaceted nature of platelets, including their capacity to facilitate crosstalk between the systemic environment and the brain.OL and TW wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This series of 13 articles  is presented by international leaders in adenovirus-based cancer therapy. Virotherapy strategies provide new options for treatment of various diseases, including cancer. Oncolytic adenovirus (OAd) is one of the most promising anti-cancer agents, and it has been employed for anti-tumoral potency via its strong oncolytic effect and intratumoral amplification. Additionally, virus-mediated cell lysis releases tumor antigens and induces local inflammation , which contributes significantly to the reversal of local immune suppression and development of antitumor immune responses (“cold” tumor into “hot” tumor). Thus, OAd-based therapy is becoming increasingly popular for the treatment of many different forms of cancer. This Special Issue features recent advances in adenovirus vector technologies, as well as combinations with other therapies, toward clinical application.Among many clinical trials with OAd-based therapies, Ad2 and Ad5 serotypes are most widely used and studied for oncolytic therapy. Although their biology is very well characterized and they can effectively transfer genes in vitro and in vivo, various limitations still exist. For instance, their infectivity is extremely low in many cancers poorly expressing the adenoviral primary receptor . The efficacy is also potentially hampered by neutralizing antibodies, hampering the use of conventional adenovirus-based vectors for systemic injection. To address these issues, several of the articles discussed that various strategies have been employed and demonstrated to be effective in achieving a more specific targeting of cancer cells: for example, de-targeting and specific re-targeting modifications in the host-interacting domains of the adenovirus protein, such as HVR regions of hexon and AB-loop or HI-loop regions of fibers . As a neWhile many preclinical studies of OAd-based therapy are promising, clinical results of OAd monotherapies show limited efficacy, so far. There is a growing body of evidence from recent preclinical and clinical studies indicating that the host immune response may provide a critical boost for the efficacy of oncolytic virotherapy. Indeed, Franco-Luzón et al. reported Celyvir  therapy caused increased infiltration and changes in the quality of immune cells per unit of tumor volume when compared to untreated mice. After this treatment, the tumor microenvironment showed a less protumoral and more inflammatory profile . Yousaf The collection also includes insightful reviews discussing the important features needed to be considered when expressing transgenes from oncolytic adenoviruses, also known as “arming OAds” . The insFrom the 13 high-quality papers and reviews, we observe the diversity of approaches and great potential of oncolytic adenovirus as a cancer therapy agent. The flexibility of DNA manipulation to drive de-targeting and re-targeting of adenoviruses, combined with conditional replication and targeted expression, allows for the combination of multiple useful tools into one. In order to enhance the therapeutic effect of OAds and improve the patient outcome, there is a shift in the OAd therapy field away from OAd monotherapy and toward combining OAd with additional synergistic strategies: radiotherapy, chemotherapies, immune checkpoint blockade, or the use of other cellular therapies including CAR-T cells. We hope that this Special Issue will provide new and significant inspiration for future research of adenovirus-based cancer therapy.


Chronic pain surrounding the temporomandibular joints and masticatory muscles is often the primary chief complaint of patients with temporomandibular disorders (TMD) seeking treatment. Yet, the neuro-pathophysiological basis underlying it remains to be clarified. Neuroimaging techniques have provided a deeper understanding of what happens to brain structure and function in TMD patients with chronic pain. Therefore, we performed a systematic review of magnetic resonance imaging (MRI) studies investigating structural and functional brain alterations in TMD patients to further unravel the neurobiological underpinnings of TMD-related pain. Online databases  were searched up to August 3, 2019, as complemented by a hand search in reference lists. A total of 622 papers were initially identified after duplicates removed and 25 studies met inclusion criteria for this review. Notably, the variations of MRI techniques used and study design among included studies preclude a meta-analysis and we discussed the findings qualitatively according to the specific neural system or network the brain regions were involved in. Brain changes were found in pathways responsible for abnormal pain perception, including the classic trigemino-thalamo-cortical system and the lateral and medial pain systems. Dysfunction and maladaptive changes were also identified in the default mode network, the top-down antinociceptive periaqueductal gray-raphe magnus pathway, as well as the motor system. TMD patients displayed altered brain activations in response to both innocuous and painful stimuli compared with healthy controls. Additionally, evidence indicates that splint therapy can alleviate TMD-related symptoms by inducing functional brain changes. In summary, MRI research provides important novel insights into the altered neural manifestations underlying chronic pain in TMD. Temporomandibular disorders (TMD) are a subgroup of craniofacial pain disorders involving pain and dysfunction of the temporomandibular joint (TMJ), masticatory muscles and associated musculoskeletal structures of the head and neck. It is the commonest cause of nondental pain in the orofacial region . PatientInitially, TMD-related chronic pain was considered to be caused primarily by peripheral factors, such as chronic inflammation of the TMJ, microtrauma of the masticatory muscles, and oromotor dysfunction. However, the correlation between pain severity and tissue pathology is often poor, and not all patients have clearly identifiable peripheral etiological factors –8. MoreoPrevious studies have thoroughly investigated changes in brain activity when experiencing clinical pain . PersistTherefore, this systematic review was conducted to provide an overview of neuroimaging MRI studies that shed light on neuro-pathophysiological basis underlying TMD-related pain by defining associated structural and functional brain alterations. Studies reporting brain changes after splint treatment that have potential implications for clarifying the therapeutic mechanism of TMD were also reviewed. Finally, while reviewing progress in this field, consideration was also given to describing the limitations of previous work and suggesting future directions for neuroimaging investigations of TMD.We followed the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelines for data collection . StudiesA customized 9-point checklist based on the Strengthening the Reporting of Observational Studies in Epidemiology statement  was usedThe present results of brain structural and functional differences between patients with TMD and controls were qualitatively described according to the specific brain system or network the brain regions were involved in. Altered brain activations in response to mechanical stimuli, the effects on the brain of splint treatment, as well as the association between brain alterations and other pain-related measurements were summarized and presented descriptively.Figure 1H MRS) to identify the metabolites in the brain of TMD patients; of these 2 studies [1H MRS.Of the 25 studies, 4 studies , 39, 41  studies , 59 focu studies  used botNotably, there was some overlap in the patient cohorts. Six studies from the University of Toronto recruited the same subjects  , 46, 54.Most studies include either only female subjects , 54, 59 Four studies –49, 57 rQuality scores for each study are reported in Additional file In MRI studies, patients with TMD displayed significantly lower FA , 42, higReports of volumetric changes in the trigeminal principal sensory nucleus (Vp) in TMD are inconsistent. Wilcox et al.  found deWilcox et al.  also useThe thalamus and S1 are chief projections from the trigeminal nerve system  and playAnatomical MRI studies of S1 changes in TMD have generated less consistent results, with different studies reporting decreased GMV , increasThe lateral and medial pain systems are involved in the nociceptive processing of pain and responsible for individual differences in its experience. The lateral pain system mainly carries information to the lateral thalamic nuclei which projects to S1, secondary somatosensory cortex, posterior insula and mid-cingulate cortex (MCC), etc., and is believed to encode pain intensity, laterality and somatotopy . The medThe insula and cingulate cortex are the most consistently activated forebrain regions when someone experiences pain. Younger et al. , studyinIn regard to MCC involved in the lateral pain system, Zhang et al.  reported1H MRS studies [The involvement of posterior insula in TMD-related chronic pain has also been noted. Two  studies , 59 inve studies , and its studies . MRS stu studies  found a The DMN is a group of functionally interconnected brain regions known to be active when people are mind wandering and not involved in any specific task, which becomes correspondingly deactivated during goal-oriented tasks . ActivitThe PAG-raphe magnus system is the best-studied pathway for descending pain modulation . Wilcox A number of higher-level brain areas have also proved to be engaged in pain control, including the cingulo-frontal regions, amygdala, and hypothalamus . IncreasThough this is less well-explored than the sensory-discriminative and affective-motivational components of pain, there is increasing evidence supporting involvement of the motor system in patients with TMD-related pain. Wessman-Fogel et al.  found thIn addition to M1 and SMA, the striatum has also been implicated in the motor response to pain in TMD. TMD patients with myofascial pain had increased GMV in the right putamen and right globus pallidus relative to controls . TMD patPrevious studies found that patients with TMD had abnormal vibrotactile sensibility on the face, with elevated detection threshold  and impaMultivariate analysis techniques have begun to be used to identify the central mechanisms underlying pain processing in TMD. Roy et al.  investigHarper et al.  exploredLickteig et al.  first usHe et al.  used rs-In this review, 25 original MRI studies were retrieved to investigate the neuro-pathophysiological manifestations of TMD-related pain. Notably, our findings provide evidence for both peripheral and central neural basis for pain in TMD , and shows anatomical alterations in other chronic pain conditions , 99. SinIt has been established that pain can induce attentional biases , and attNeuroticism is one of the ‘Big Five’ personality traits and individuals with high neuroticism are more likely to interpret common situations as threatening and minor frustrations as hopeless. It is a personality feature associated with increased levels of current and future anxiety and distress , 110. HiNeuroimaging studies in patients with TMD pain have reported structural and functional changes in the motor system, including M1, SMA, and striatum. Persistent pain can inhibit protective movement and impair motor performance due to maladaptive neuroplasticity in the motor cortex , 114. InUncontrollable stressors like persistent chronic pain can lead to learned helplessness, a maladaptive response featured by reduced motor escape behaviors and deficits in motivation and learning . The SMAAs the major component of basal ganglia, striatum receives input from cortical regions and thalamic nuclei, and sends output to other structures of basal ganglia, serving as a critical site where cognitive, motor and limbic signals from other brain regions overlap and are integrated , 118. FiCompared with healthy controls, TMD patients display perceptual amplification of pressure stimulation across a wide range of physical intensities, from gentle and innocuous to strongly painful . fMRI alAlthough TMD, like many chronic pain conditions, is accompanied by motor impairment, few MRI studies have directly investigated neural activity during a motor task in chronic pain. Roy et al.  first inTreatment for TMD has been focused on alleviating the main symptoms, especially chronic pain, and the palliative approaches which prevail over surgery have relatively satisfactory clinical outcomes , 122. SpThe recent fMRI studies by Lickteig et al. , 84 and The present review, and the field of TMD brain imaging research, have several limitations. The first important consideration is the difference in pain origins and TMD patients recruited in studies. Since TMD is an umbrella term encompassing a number of painful conditions involving masticatory muscles, TMJ and associated structures , the larAnother consideration is the gender of participants. The sex ratio of patients seeking medical advice has been reported as ranging from 3:1 (women: men) to as high as 9:1 , 130. AcA third factor that impacts neuroimaging studies of TMD is the potential impacts of analgesics, which may reduce some functional alterations and induce other functional changes that might impact the experience of persistent TMD-related pain. Studying untreated TMD patients at illness presentation, as in other disorders, would be a strategy to advance clarity of illness related alterations.Fourth, causal relationships are inherently difficult to establish in cross-sectional studies. Among the brain alterations in TMD patients, differentiating features that are the consequence of persistent pain or represent compensation efforts to reduce pain can not be confidently determined at this point, which requires further research. Moreover, findings from existing studies suggest the possibility that certain structural characteristics of the brain associated with levels of anxiety and distress and helplessness may either make some people more vulnerable to developing TMD or represent responses to TMJ-related pain with their own impact on brain anatomy and function.Last, given that TMD is a multifactorial disease, understanding the personal difference in central sensitization is critical for choosing proper clinical management. Therefore, the combination of neuroimaging techniques and machine learning algorithms such as SVM may serve to benefit future studies of the central mechanisms underlying TMD pain, and eventually facilitate clinical practice by providing new diagnostic strategies, as well as objective measures of therapeutic efficacy.There are structural and functional changes in the classic trigemino-thalamo-cortical system, including peripheral trigeminal nerve roots, brainstem (SpVc and Vp in particular), thalamus, and S1, which provides support for a peripheral origin of TMD. Specifically, the results of increased GMV in the thalamus were consistent, while whether there are alterations in S1 in TMD patients remains to be clarified.There are alterations in several cortical regions implicated in pain perception and pain modulation in TMD. Neurochemical alterations are identified in the posterior insula. The altered FCs among anterior insula, pgACC and MCC are correlated with pain intensity. The dysfunctional DMN in TMD patients is characterized by reduced FC in mPFC-PCC/PCu and mPFC-PAG. TMD related pain-attention interaction is mediated by reduced FC in aMCC-dlPFC and pgACC-amygdala. Structural changes in the PAG-raphe magnus pathway may impair the efficiency of the endogenous pain inhibitory system of TMD patients.Regional functional brain changes in M1 and SMA, as well as increased GMV and decreased FC in the striatum, indicate the compensatory changes or maladaptive neuroplasticity of the motor system in patients with TMD pain.TMD patients displayed different brain activations in the fronto-insulo-thalamo-parietal network under both innocuous and painful stimulus compared with healthy controls, reflecting the involvement of aberrant central pain processing in TMD. Multivariate analysis techniques like SVM may help distinguish the subtypes of TMD patients, i.e. identify whose pain has a more peripheral or a central etiology, which could be clinically useful for optimal treatment.Splint therapy can impact the neural function of brain regions in TMD patients, accompanied by improved clinical symptoms, suggesting a central mechanism underlying the therapeutic effects of splint therapyThis article systematically reviewed the existing literature investigating brain changes in TMD patients for revealing the pathogenic mechanisms or consequences of TMD-related pain. MRI neuroimaging techniques have provided a deeper understanding of what happens to brain structure and function in patients with TMD, suggesting both peripheral and central neural basis for the most common conditions of TMD, i.e. hyperexcitability to external stimuli and disrupted pain perception and pain modulation. This review can enhance our understanding of the pathophysiology underlying symptoms of TMD-related pain as targets for treatment development and planning. Primary novel observations include:Additional file 1: Table S1. The checklist of quality assessment for included studies in the present systematic review.Additional file 2: Table S2. Regions with significant group differences between patients with TMD and controls reported in functional MRI studies included in the present systematic review. Table S3. Regions with significant group differences between patients with TMD and controls reported in structural MRI studies included in the present systematic review.


Glioblastoma (GBM) is a particularly devastating tumor with a median survival of about 16 months. Recent research has revealed novel insights into the outstanding heterogeneity of this type of brain cancer. However, all GBM subtypes share the hallmark feature of aggressive invasion into the surrounding tissue. Invasive glioblastoma cells escape surgery and focal therapies and thus represent a major obstacle for curative therapy. This review aims to provide a comprehensive understanding of glioma invasion mechanisms with respect to tumor-cell-intrinsic properties as well as cues provided by the microenvironment. We discuss genetic programs that may influence the dissemination and plasticity of GBM cells as well as their different invasion patterns. We also review how tumor cells shape their microenvironment and how, vice versa, components of the extracellular matrix and factors from non-neoplastic cells influence tumor cell motility. We further discuss different research platforms for modeling invasion. Finally, we highlight the importance of accounting for the complex interplay between tumor cell invasion and treatment resistance in glioblastoma when considering new therapeutic approaches. Glioblastoma (GBM) is the most prevalent and malignant primary brain tumor of adults, with a mean incidence of 7 cases per 100,000 per year and a mean overall survival of about 16 months , with siOne of the clinical hallmarks of GBM is extensive infiltration of the tumor surrounding parenchyma ,8. GliomReinvasion of distant GBM cells to the primary tumor location and invasion to distant areas may lead to colonization of these sites, thus triggering local and distant bulky relapse. Recurring tumors share many early tumor mutations with their progeny , indicatSeveral patterns of invasion have been described ,17 whichThough the mechanisms leading to GBM invasion are increasingly understood in vitro and in animal models, results from the laboratory have not been translated into clinically relevant therapeutic approaches so far . Within GBM BTIC and differentiated cells may exploit conserved genetic programs that are fundamental during neuro- and gliogenesis to migrate through the brain parenchyma . These pMigration of both neural progenitor and glioma cells is guided by specific structures, such as white matter tracts  and blooClinical evidence suggests that some GBMs are more disseminated than others . GBMs shThis general heterogeneity is mirrored by BTIC that possess stem cell characteristics ,39 and aA number of transcription factors were identified as key regulators of transdifferentiation from an epithelial-like to a mesenchymal state  in a variety of different tumor types, including glioma. ,45,46. DEven though, in glioma, transition to a mesenchymal state emerges rather from a proneural than from an epithelial phenotype, similar factors contribute to this plasticity. Among the most prominent TFs involved in glioma EMT are ZEB1 ,50, TWISRecent publications clearly indicate that glioma subtypes are subject to high plasticity ,56. UponTo add even more complexity to the matter, it is not clear for many of the involved processes whether they are predominantly regulated at the genomic, transcriptional, proteomic or metabolomic level. Recently, single-cell profiling has become available for DNA , RNA 6262, the eA full understanding of the highly complex GBM ecosystem that drives infiltration may be masked by bulk analysis of the whole tumor. High resolution-analyses at the single cell level may better depict relevant molecular events . On the Reflecting the method of analysis used, it is not entirely clear at this time if single leading cells or the tumor bulk coordinate invasion; several distinct patterns of invasion have been described in GBM ,17 whichA large number of publications have addressed either single-cell or bulk high-throughput analysis . Based oGBM cells migrate in a highly coordinated manner . Tumor cGBM cells also attract cells such as microglia, astrocytes and endothelial cells that secrete proteases to enhance migration . By moviInvasive programs are coordinated by overexpression of transcription factors like STAT3 and C/EBPβ, which have been shown to be synergistic initiators and master regulators of mesenchymal transformation in BTICs . NF-κB mGBM invasion results from a continuous bidirectional interaction between tumor cells and their microenvironment . The micECM molecules provide structural support and act as a guiding scaffold or barrier ,119. TheWe and others have shown that the ECM itself, as well as cell–cell and cell–ECM connecting molecules and proteases, have an impact on glioma cell migration ,124,125.The composition of the ECM generates specific stem cell niches and facilitates migration and invasion in distinct ways ,131. The most prominent example of a niche within the diseased brain is the hypoxic niche. A hypoxic environment stimulates the expression of the hypoxia-inducible factors (HIFs) HIF1α and HIF2α that favor an invasive phenotype . An acidAnother example of a brain-intrinsic niche that may enhance invasion is the angiogenic niche. Endothelial cells interact closely with BTICs and secrete factors that maintain these cells in a stem-cell-like state  and enabAmongst the nonimmunogenic cell types that are prevalent in the brain, endothelial cells are important inducers of invasion, especially in the angiogenic niche. The activation of angiopoietin 1 and its crosstalk with Tie2 increases invasion .The most important immunogenic cell type able to promote invasion in GBM are microglia. GBM progenitor cells closely interact with microglia  and therIn vivo animal models allow the study of GMB invasion within the microenvironment of a living host. However, they are often complex, expensive, time consuming, impaired by a low reproducibility and associated with difficulty in dissecting the different components involved in the process. Therefore, a number of in vitro models have been developed in recent years . These mTwo-dimensional models are based on monolayers of GBM tumor cells on surfaces that are coated by, for example, ECM molecules. These models are simple to use but have important limitations. Cells in monolayers do not invade (but migrate) and are separated from their natural microenvironment, therefore behaving differently . TransweThree-dimensional models can be used to investigate intraparenchymal  and periTumor spheroids on the other hand are generated as organotypic multicellular spheroids that are obtained by culturing resected fragments of tumors . They reAs a three-dimensional ex vivo model, organotypic slice cultures (OBSC) have been developed to allow long-term imaging of cancer cell migration and invasion ,158,159 The combination of an orthotopic GBM model and time-lapse in vivo microscopy of human brain tumor initiating cells through a cranial window allows in-depth analysis of tumor cell infiltration to acquire a better insight of GBM tumor cells invasion dynamics within their environment . Mice caModels of tumor progression offer the possibility to predict the behavior of tumor invasiveness and, consequently, to define more accurate prognostic estimates and therapeutic strategies. In the last 20 years, several in silico models have been developed to elucidate the main mechanisms that induce glioma proliferation and invasion . AlthougThe advent of high-throughput data generation, largely coincident with the development of next-generation sequencing, has boosted the knowledge of many cancers—including gliomas—allowing computational modeling of large datasets to assess and predict tumor dynamics. For example, the Cancer Genome Atlas (TGCA) and Repository for Molecular BRAin Neoplasia DaTa (REMBRANDT) are two large-scale efforts containing genomics, transcriptomics, DNA methylomics and proteomics data as well as clinical data ,167. SucRecently, the Ivy Glioblastoma Atlas Project (Ivy-GAP) has generated a transcriptional atlas for known anatomical features of GBM . StartinTo understand infiltrating single cells on a deeper level, novel targeted approaches for single cell sequencing have been developed. For example, topographic single cell sequencing (TSCS) is a method that combines laser microdissection and cell catapulting  with sinTMSB10) can predict an invasive phenotype [Although DNA-seq and RNA-seq are useful to unravel cancer heterogeneity, clonal evolution and mechanisms of invasiveness in gliomas, they do not provide insight into cell-to-cell communications and interactions with the tumor microenvironment. Recently, spatial transcriptomics has therefore also interrogated cell-to-cell interactions in their natural context, revealing an unexplored landscape of heterogeneity in different types of cancers ,174,175.henotype . An applSpatial transcriptomics may also unravel expression patterns in the inflammatory environment of cancers. Recently, a dissection of prostate cancer tissue has shown that reactive stroma and inflammation are present at early stages and promote tumor growth by activation of oxidative stress and integrin-linked kinase (ILK) signaling . SimilarA bioinformatic integration of the mentioned scRNA-Seq and spatial transcriptomics data has been recently developed and applied to infer the tissue architecture in nonglioma tumors, for example in pancreatic ductal adenocarcinoma . Such inPOSTN and CXCL12 [MYC overexpression and inhibition of NF-KB inhibitor-alpha (NFKBIA) support an invasive imaging signature [PDGFRA, PTEN, TP53, 1RB and EGFR [In the clinical setting, high-quality MRI data are nowadays transformed into radiomics, a method of use for building predictive models for diagnosis, prognosis and therapeutic response ,183. Radd CXCL12 . In addiignature . Similarand EGFR . Furtherand EGFR .In summary, these multidimensional approaches may enable much better models and concepts for the clinical behavior of invasive GBM, allow to further fine-tune the landscape of an individual’s GBM and support the development of more precisely targeted and therefore personalized therapies.Molecular mechanisms of invasion and drug resistance overlap to a high extent and are influenced by cell-intrinsic and microenvironmental factors. An invasive tumor phenotype may therefore also increase resistance to antitumor systemic therapy and vice versa. Glioma cells stimulated to migrate showed downregulation of proliferation- and apoptosis-associated genes while genes involved in cell motility became overexpressed . SimilarIn general, members of pivotal signaling pathways such as p53, the Ras/MAPK, PI3K and mTOR pathways, growth factors, chemokines and integrins are hubs of mechanisms that jointly support tumor cell survival and resistance as well as migration and invasion. One prominent example is the tumor-suppressive transcription factor (TF) p53, which is altered in cancer, including glioma, via loss- or gain-of-function mutations. In both cases, apoptosis and cell cycle arrest are impaired via, e.g., downregulation of proapoptotic molecules like Bax and lack of p21 activation, respectively ,193. ConIn the future, it will be highly important to precisely define focal points that influence both invasion and resistance to therapy to select high priority candidates for a targeted drug intervention in patients with GBM. Malignant gliomas are, amongst other pathogenetic features, characterized by a unique ability to invade. From a clinical point of view, invasion renders gliomas microscopically nonresectable and presents a major obstacle for curative treatment. The immense interpatient and intratumoral heterogeneity of gliomas, as well as the pivotal role of the tumor microenvironment that makes a profound understanding of the involved mechanisms difficult, are increasingly appreciated in this context. Even so, research on the complex interplay between tumor cells of different subtypes and the surrounding brain parenchyma is still at its infancy. Current standard therapies are unable to target infiltrative tumor cells, and effective anti-invasive treatment strategies are not available so far. Therefore, there is urgent clinical need to investigate the mechanisms leading to invasion of glioma in more detail. Sophisticated sequencing approaches and advancing new in vitro and in vivo models will hopefully provide a better understanding of these interactions and soon present new targets for anti-invasive therapeutic strategies.


While inner ear disorders are common, our ability to intervene and recover their sensory function is limited. In vitro models of the inner ear, like the organoid system, could aid in identifying new regenerative drugs and gene therapies. Here, we provide a perspective on the status of in vitro inner ear models and guidance on how to improve their applicability in translational research. We highlight the generation of inner ear cell types from pluripotent stem cells as a particularly promising focus of research. Several exciting recent studies have shown how the developmental signaling cues of embryonic and fetal development can be mimicked to differentiate stem cells into “inner ear organoids” containing otic progenitor cells, hair cells, and neurons. However, current differentiation protocols and our knowledge of embryonic and fetal inner ear development in general, have a bias toward the sensory epithelia of the inner ear. We propose that a more holistic view is needed to better model the inner ear in vitro. Moving forward, attention should be made to the broader diversity of neuroglial and mesenchymal cell types of the inner ear, and how they interact in space or time during development. With improved control of epithelial, neuroglial, and mesenchymal cell fate specification, inner ear organoids would have the ability to truly recapitulate neurosensory function and dysfunction. We conclude by discussing how single-cell atlases of the developing inner ear and technical innovations will be critical tools to advance inner ear organoid platforms for future pre-clinical applications. Over 6% of people worldwide suffer from hearing loss  and likeDifferent approaches of modeling the human inner ear in vitro have been demonstrated using human pluripotent stem cells (hPSCs) –24, adulDuring early embryogenesis, pluripotent cells in the epiblast generate the ectoderm germ layer, which splits into the non-neural  ectoderm and neuroectoderm. The otic placode arises at the border region between the cranial non-neural ectoderm and neuroectoderm, known as the otic-epibranchial placode domain (OEPD) , 32. SevUpon embryonic otocyst formation, asymmetric gene expression leads to dorsoventral and anteroposterior patterning that sets up the coordinates for subsequent development of the semicircular canals, endolymphatic sac, vestibular organs, and cochlea . SignalsThus, the inner ear is patterned through the integration of a multitude of signaling pathways across space and time. These signals arise from within epithelia and from surrounding tissues, which enables the differentiation of otic progenitor cells to subsequent cochlear and vestibular fates. Our collective knowledge of these mechanisms has come from animal models and few, if any, mechanistic studies have been performed on human fetal inner ear tissues  plays an important role in functional development and is linked with noise-induced hearing loss . Recent Schwann cells are the myelinating cells of peripheral nerves, including the VIII cranial nerve in the inner ear. Unlike inner ear neurons, which arise from the otocyst, Schwann cells arise from the cranial neural crest. Remarkably, the inner ear organoid model appears to co-produce S100B + Schwann cells that closely associate with sensory neurons in the culture Fig.  21]. Th. Th21]. Organoids could also be used to investigate neural pathfinding and synapse formation of otic afferent neurons with central nervous system targets. A co-culture system of a developing inner ear organoid paired with developing hindbrain tissue, would potentially mimic elements of the otic ganglion-to-cochlear nucleus circuit . The typIn addition to the role of POM in endolymph ionic homeostasis, it plays an important role in otocyst patterning . ShortlyThe inner ear has a unique relationship to the rest of the body. In many ways it is isolated: largely immune privileged and with a vascular barrier system called the blood-labyrinth barrier (akin to the blood-brain barrier (BBB) in the central nervous system) . It is uMost recently, it has been shown that forms of genetic hearing loss are linked to autoinflammation in the inner ear . Like maAs we have discussed, major breakthroughs have been made in inner ear modeling using hPSCs, yet current models are limited in their applications for translational research. To induce greater cell diversity in inner ear organoid models, it is essential to identify key regulatory pathways that play a role in determination of vestibular versus cochlear, epithelial versus mesenchymal, and sensory versus non-sensory cell fate decisions. Manipulation of these pathways should be systematically incorporated into current differentiation protocols Fig. . More fu


Nosocomial infections (NIs) are hospital-acquired infections which pose a high healthcare burden worldwide. The impact of NIs is further aggravated by the global spread of antimicrobial resistance (AMR). Conventional treatment and disinfection agents are often insufficient to catch up with the increasing AMR and tolerance of the pathogenic bacteria. This has resulted in a need for alternative approaches and raised new interest in therapeutic bacteriophages (phages). In contrast to the limited clinical options available against AMR bacteria, the extreme abundance and biodiversity of phages in nature provides an opportunity to establish an ever-expanding phage library that collectively provides sustained broad-spectrum and poly microbial coverage. Given the specificity of phage-host interactions, phage susceptibility testing can serve as a rapid and cost-effective method for bacterial subtyping. The library can also provide a database for routine monitoring of nosocomial infections as a prelude to preparing ready-to-use phages for patient treatment and environmental sterilization. Despite the remaining obstacles for clinical application of phages, the establishment of phage libraries, pre-stocked phage vials prepared to good manufacturing practice (GMP) standards, and pre-optimized phage screening technology will facilitate efforts to make phages available as modern medicine. This may provide the breakthrough needed to demonstrate the great potential in nosocomial infection management. Nosocomial infections (NIs), also known as hospital-acquired infections (HAI), are infections that are newly acquired in a hospital or other healthcare facility. They arise from various sources including healthcare staff, contaminated equipment, bed linen, and air droplets. NIs represent the most frequent adverse events during care delivery and typically result in prolonged hospital stays. There can be massive additional costs for health systems and patients, and unnecessary deaths .Enterobacteriaceae (CRE), carbapenem-resistant Acinetobacter baumannii (CRAB), MDR Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-resistant Enterococcus (VRE). In the absence of effective treatment for bacterial infections that have AMR, modern medical care becomes inadequate and the infections persist in the body, increasing the risk of spread to others. Even worse, increasing global connectivity facilitates the rapid transport of infectious agents and their resistance genes through the world , which has become a major public health problem. In 2017, the World Health Organization (WHO) highlighted, as a global priority, a list of multidrug-resistant (MDR) bacterial pathogens  to help in prioritizing the research and development of new and effective antibiotic treatments . These hhe world .NIs pose a high healthcare burden in both developed and developing countries , this prde novo phage screening from nature. For this reason, both the quantity and quality of phage libraries need enhancement.A key precondition in phage application is an eligible phage library (phage bank) with broad-spectrum, poly antimicrobial properties, where many well-characterized phages are constantly collected and arranged according to the host species. Although an increasing number of phage libraries has been established recently, the overall scale is small and the information in most of the existing phage collections is incomplete  Yerusha. This stA first step to build a phage library is to prepare a library of pathogenic bacteria that provides the baits for phage hunting. Phages can be isolated from diverse environmental samples, however, not all phages are useful for therapeutic use. Criteria of eligible phage candidates have been extensively discussed and, although estimates of index values vary among groups, there’s a general consensus that the key features that must be characterized include: host species, complete genome sequence , host range testing, and high lytic activity . Some otE. faecium, S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa, and Enterobacter species) (Helicobacter Pylori and Clostridia difficile are temperate (https://phage.directory) are also conducive to pool a global-scale phage library and to connect demands for therapeutic phages.In contrast to the limited number of antibiotics available, the extreme abundance and biodiversity of phages in nature make it possible to build an ever-expanding phage library that collectively provides sustained broad-spectrum and poly microbial coverage against the most common nosocomial pathogens such as the ESKAPE group (species) . Howeverspecies) . For insemperate . Phage eRapid tracking of pathogen sources with appropriate subtyping tools and immediate initiation of appropriate antibacterial treatment are crucial in the management of nosocomial infections. However, conventional bacterial genotyping methods such as pulse-field gel electrophoresis (PFGE), multi-locus variable tandem repeat analysis (MLVA), multilocus-sequence typing (MLST), and whole-genome sequencing (WGS) are time-consuming, normally needing more than three workdays to yield results. In contrast, bacterial phenotypic technics such as antibiotic susceptibility test, immunological methods (serotyping), or phage-typing are limited in discriminatory power but advantageous to save time . For theA phage susceptibility test is a method followed by bacterial diagnosis, to simultaneously test the contents of a sub-library  of phage candidates against the bacteria isolated from a patient, thereby identifying phages for patient-specific precision therapy. Laboratory-developed phage susceptibility tests are widely used for therapeutic phage screening . AdditioNevertheless, on the basis of known epidemiological situations and pre-established phage collections, phage susceptibility testing may serve as a rapid and cost-effective method for bacterial subtyping to routinely monitor nosocomial infections. It may also be a prelude to preparing ready-to-use phages for patient treatment and environmental sterilization. Data from several preliminary studies have shown good concordance of profiles between phage-typing and other subtyping methods in distinguishing bacterial isolates . Howeverprêt-à-porter) or customized screened (sur-mesure), that is the question for therapeutic phage selection (sur-mesure). However, due to the high species/strain specificity of phages and the frequently evolving phage resistance in bacteria, it is difficult for a fixed phage cocktail to provide broad coverage on clinical strains. There have been some well-conducted clinical trials on the effectiveness of fixed phage cocktails but they have failed to obtain positive results  and the US Food and Drug Administration (FDA) placed phages under the medicinal  product regulation; this means that marketing a phage product requires proof of safety and efficacy, also of quality by manufacture under Good Manufacturing Practice (GMP) regulations  allowance .Good infection prevention practices such as hand hygiene, daily and terminal cleaning, and disinfection have been demonstrated to reduce the incidence of healthcare-associated infections . HoweverMeanwhile, there has been great interest in developing novel sterilizing methods, such as “self-disinfecting” surfaces, continuous room disinfection with diluted chemical sterilant, and other reagents . Among t7 PFU (plaque forming unit)/ml for a ∼ 27m3 room. A significant reduction in CRAB-associated NIs was observed when adding a single aerosolized phage treatment to the conventional chemical-based disinfection performed in ICU rooms at the patient discharge  of General Medicine wards, reporting a rapid and significant decrease in Staphylococcus spp. load on treated surfaces, up to 97% more than PCHS alone (Mycobacterium smegmatis in different multiplicity of infection (MOI) and aerosol generation time. The result showed that, MOI of 10 000 for 10 seconds significantly blocked recovery of M. smegmatis colony from the culture medium  for environmental applied phages should be addressed. It is certain that manufacture and QCS of phage disinfectants do not require as much as therapeutic phages , however, this strategy needs mass consumption of phage and thus is production-capacity dependent. Although phages should not replace broad-spectrum agents in terminal disinfection, they offer great potential for continuous target-specific applications where the target is drug-resistant and antimicrobial chemicals are either relatively ineffective or their use would result in unintended detrimental consequences.The use of phages is consistent with the need of nosocomial infection control to diminish both bacterial infection and transmission, as well as to relieve the overuse of antibiotics. However, little has been described regarding the systemic application of phages in infection management. Nevertheless, a similar concept has been successfully practiced in several other settings  , where aHow might phages be integrated into the clinical workflow at a healthcare establishment? First, a system of cooperation between different departments  is essential. Second, an established phage library is needed for screening lytic phages against a given pathogenic bacterium isolated from a patient or from a hospital surface. Third, ready-to-use phage vials need to be properly manufactured prior to application to humans (GMP-approved) or environments; qualified phage vials should be pre-stocked to accelerate phage delivery. In parallel to use in phage therapy, the same phage formula can be aerosol-applied to the room or even the entire ward of the treated patient to prevent nosocomial transmission. Fourth, the accumulated phage-typing database of many bacteria can serve to monitor nosocomial infection and as a prelude to preparing phage stocks for patient treatment and environmental sterilization. Fifth, both retrospective and prospective phage susceptibility tests can be united  to design fixed-composition phage cocktails with broad-spectrum, poly antimicrobial properties against the epidemic strains; the fixed-composition cocktails could then be applied in routine environmental sterilization or in treatment of serious infections such as septic shock or severe pneumonia that call for rapid initiation of treatment .The extreme abundance and biodiversity of phages in our surrounding environment and inside our body provides an opportunity to establish an ever-expanding phage library that collectively provides a sustainable broad-spectrum and poly microbial coverage. This library might play a major role in tackling the global AMR crisis. Whilst acknowledging the remaining obstacles for clinical phage application, we believe that by taking advantage of pre-established phage libraries, pre-stocked phage productions, and pre-optimized phage screening technology, phages can become available as modern medicine. This availability may constitute a breakthrough in both clinical treatment and environmental disinfection in the context of nosocomial infection management.NW and TZ reviewed the literature, summarized the tables, conceptualized the figure, wrote the manuscript, and acquired funding. Both 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.


Rejuvenation research is a rapidly expanding field aimed at restoring cell and tissue function of aged individuals. Pharmacological approaches include senolytic therapies which rejuvenate aged tissues via the removal and/or suppression of senescent cells . Biologiin-vitro as well as plasma EVs isolated from old mice reconstituted with young or old bone marrow. However, EV microRNAs were similar in both models, suggesting that circulating cells have a minor contribution to the microRNAs identified this study. Further investigation into potential cell sources revealed that induction of senescence in-vitro and in-vivo, using gamma irradiation, mimicked the changes observed in old mice such as increased levels of circulating EVs and increased expression of EV associated miR-146a, mIR-21, and let-7a. Interestingly, senolytic therapy using dasatinib + quercetin (D+Q) reduced the expression of these microRNAs in the plasma of old mice, supporting that senescent cells or the pathways targeted by these compounds contribute to increased expression in the circulation. Collectively, this data demonstrates that aging and cellular senescence leads to increased levels of circulating EVs, and that these EVs impair cellular responses to activation. Pharmacological targeting of senescent cells partially rejuvenated the microRNA profile and functional effects of old plasma EVs.Extracellular vesicles (EVs) are membrane bound vesicles which vary from nanometer to micrometer in size and carry a diverse set of factors  that reflect the secreting cell’s milieu . EVs areSenescent cells have emerged as key players in the aging process. Senescent cells secrete cytokines, growth factors, and proteases which alter neighboring cell function. This secretome is collectively referred to as the senescent associated secretory profile (SASP) and the adverse effects of senescent cells are largely attributed to this profile. More recent definitions of the SASP have been expanded to include EVs ,5. DurinCirculating factors that change with age can be divided into multiple categories but include factors enriched while young that decline with age, and aging-associated factors which are present at undetectable to low levels and increase . The lat


Extensive damage to skeletal muscle tissue due to volumetric muscle loss (VML) is beyond the inherent regenerative capacity of the body, and results in permanent functional debilitation. Current clinical treatments fail to fully restore native muscle function. Recently, cell-based therapies have emerged as a promising approach to promote skeletal muscle regeneration following injury and/or disease. Stem cell populations, such as muscle stem cells, mesenchymal stem cells and induced pluripotent stem cells (iPSCs), have shown a promising capacity for muscle differentiation. Support cells, such as endothelial cells, nerve cells or immune cells, play a pivotal role in providing paracrine signaling cues for myogenesis, along with modulating the processes of inflammation, angiogenesis and innervation. The efficacy of cell therapies relies on the provision of instructive microenvironmental cues and appropriate intercellular interactions. This review describes the recent developments of cell-based therapies for the treatment of VML, with a focus on preclinical testing and future trends in the field. Volumetric muscle loss (VML) is the traumatic, surgical or degenerative loss of a substantial portion of bulk skeletal muscle in a manner that overwhelms the endogenous repair capacity of the muscle and results in impaired scar tissue formation. VML is associated with chronic functional disability in many military and civilian populations following battlefield injuries, car accidents, tumor ablation or degenerative diseases ,2. VML lSkeletal muscle consists of bundles of oriented multi-nucleated muscle fibers (myofibers) that are surrounded by connective tissue, along with branches of blood vessels and peripheral nerves that supply blood flow and electrical signals to the muscle. Thus, a variety of cell types work in concert to form the skeletal muscle structure . FollowiA variety of cells reside within skeletal muscle, including muscle satellite cells, pericytes, vascular lineages, interstitial stem cells and fibro/adipogenic progenitors (FAPs). Among these cell types, satellite cells are primarily responsible for natural skeletal muscle repair and regeneration. Satellite cells are muscle stem cells (MuSCs) that reside beneath the basal lamina surrounding each myofiber. These cells are a heterogenous population, but commonly express paired box protein-7 (Pax7), a transcription factor that plays role in myogenesis and regulates the proliferation of MuSCs. In response to myofiber injury, quiescent satellite cells activate, proliferate, and give rise to myogenic progenitor cells (MPCs). MPCs differentiate into myoblasts and can fuse to form multinucleated myotubes, which mature into newly formed myofibers. Although MuSCs are considered promising cell sources for VML treatment, some of the limitations associated with satellite cells include their low abundance within muscle, challenges in isolating and purifying these cells, limited self-renewal and differentiation potential in vitro, and low engraftment post-transplantation ,7. The iGiven the limitations of MuSCs, mesenchymal stem cells (MSCs) are an alternative cell source. The intramuscular injection of MSCs has accelerated muscle repair in injured skeletal muscle in animal models . MSCs arInduced pluripotent stem cells (iPSCs) represent another attractive myogenic cell source for skeletal muscle regeneration, due to their unlimited self-renewal capacity and capacity to differentiate into myogenic cells. The iPSCs can be directly reprogrammed into skeletal muscle cells via the overexpression of myogenic transcription factors, such as Pax7 or MyoD, or by small molecules ,16. GeneIn addition to resident MuSCs, the normal functioning and regeneration of skeletal muscle tissue are supported by fibroblasts, endothelial cells, neural cells, fibro/adipogenic progenitor (FAP) cells and infiltrating immune cells. FAPs are muscle-resident progenitor cells of mesenchymal origin, expressing stem cells antigen-1 (Sca-1) and platelet-derived growth factor receptor beta (PDGFR-β) surface markers, which can differentiate into fibroblasts and adipocytes. Following muscle injury, FAPs proliferate and enhance myogenic differentiation by generating pro-differentiation signals for MPCs . The intMulti-cellular culture systems have been developed to study the intercellular and paracrine interactions that are necessary for muscle formation and function. For example, Ostrovidov et al. demonstrated that the co-culture of a PC12 neural cell line with a C2C12 myoblast cell line could induce C2C12 myoblast differentiation and myotube formation . Neural Approximately 90% of preclinical models of VML have been conducted in mice and rats . HoweverThe direct transplantation of MuSCs and MPCs into damaged tissue results in poor cell survival and limited engraftment due to a lack of sufficient cell support and the harsh injured tissue microenvironment . Scaffol+/Pax7+ in the cross section of engrafted muscle demonstrated that the transplanted MuSCs, labeled with LacZ, could restore satellite cells. In addition, LacZ+ donor-derived cells contributed to an average of 26% of the new vessels formed in engrafted regions . Ad. Ad43]. Besides muscle stem and progenitor cells, MSCs have been tested in rodent VML models ,46. ReceSimilarly, another study using adipose-derived MSCs (ASCs) in collagen hydrogels to treat rat VML demonstrated improved therapeutic outcomes. Laser Doppler perfusion imaging 2 weeks post-injury demonstrated that blood perfusion was doubled in groups with ASC-loaded hydrogels compared to cell devoid hydrogels. The upregulation of endothelial cell markers, such as α-SMA, in these groups also confirmed the angiogenic effect of ASCs. In groups with ASC-loaded hydrogels, anti-inflammatory cytokines such as IL4 and IL10 were increased, and pro-healing macrophage markers such as CD163, CD206 and Arg-1 were upregulated. In addition, myogenic markers, including myogenin and MyoD, were upregulated, and collagen deposition was decreased, which together indicated the immunomodulatory effect of ASCs in attenuating the inflammation and enhancing tissue regeneration . Using aIn addition to ASCs and BM-MSCs, amniotic MSCs demonstrated positive therapeutic effects in VML. Amniotic MSCs were induced using 5-azacytidine, a biochemical induction factor, and delivered to rat TA muscle with VML within a gelatin methacrylate (GelMA) hydrogel. Treatment with MSCs significantly increased neovascularization and functional tissue repair, compared to groups that received just GelMA scaffold without cells or blank groups 2 and 4 weeks post-implantation . TogetheAlthough research on the use of iPSC-derived myogenic studies is still underway, a recent study by Rao et al. evaluated the efficacy of human iPSCs as a source of myogenic cells for muscle regeneration in a mouse model. Human iPSCs within patterned polydimethylsiloxane (PDMS) expressed Pax7 protein and directly contributed to the formation of new myofibers, generating 3D vascularized and contractile muscle constructs . It is lMuSCs and MSCs are the two main cell sources that have been evaluated in preclinical cell therapies in VML rodent models, and have demonstrated the capacity for muscle regeneration. However, the absence of a standardized VML surgical model as well standardized functional measurements of muscle regeneration makes it difficult to compare the results of various studies. Since almost all the present preclinical studies have been performed in small rodent models, the scalability of cell-based therapies for large animal models is a big challenge. MuSCs lose their stemness during in vitro expansion, so the development of a robust in vitro expansion method for MuSCs without losing their potency is necessary. Additionally, more studies are needed in order to understand the molecular mechanisms behind the differentiation and self-renewal behavior of MuSCs. Both murine and human MuSCs have been used for studies in this area, but due to differences between human and murine MuSCs, in vitro culture systems are required to be optimized specifically for human MuSCs. MSCs show superiority in terms of abundance for large defects in humans, but these cells have a poor myogenic differentiation capacity. Human iPSC-derived MuSCs may currently be the only source of infinite autologous cells for scalable therapeutic use, but the tumorigenic potential of iPSCs remains a major concern for clinical translation, and no preclinical studies have evaluated the potential of these cells for VML as yet. For the translation of cell-based therapies into the clinic, developing a delivery vehicle to promote cell survival in in vivo implantation, and obtaining appropriate protocols for cell expansion and storing in vitro, is required. The success of cellular therapeutics in treating VML strongly depends not only on the therapeutic cells themselves, but also on microenvironmental cues presented to the cells. Exploiting tissue engineering strategies can improve cell-based therapies for muscle regeneration. However, more work is still needed in order to elucidate the optimized microenvironment for cells capable of regenerating functional muscles, vascularization and innervation. Poor innervation is a major obstacle to achieving the full-force recovery of new muscles, and although the in vitro co-culturing of neurons and muscle cells has been promising, preclinical studies of muscle–nerve constructs are limited. In addition, repairing myotendinous junctions in VML remains an unmet challenge. Further studies are needed that focus on establishing muscle–tendon junctions and the generating of new nerves with new muscles in VML.Although cell-based therapies to date have shown exciting potential, we believe that it is the combination of cellular therapeutics and tissue engineering approaches that will open up more possibilities for finding a clinical therapy for VML. An improved understanding of the molecular and cellular interactions of muscle regeneration will facilitate the engineering of an optimal microenvironment to guide therapeutic cells toward restoring muscle function after VML. Exploiting emerging technologies, such as on-chip disease models and Slide-Seq, will help in probing the role of different cells present at VML injury sites at the molecular and genomic levels ,55. Unde


Gene therapy is an innovative treatment for Primary Immune Deficiencies (PIDs) that uses autologous hematopoietic stem cell transplantation to deliver stem cells with added or edited versions of the missing or malfunctioning gene that causes the PID. Initial studies of gene therapy for PIDs in the 1990–2000's used integrating murine gamma-retroviral vectors. While these studies showed clinical efficacy in many cases, especially with the administration of marrow cytoreductive conditioning before cell re-infusion, these vectors caused genotoxicity and development of leukoproliferative disorders in several patients. More recent studies used lentiviral vectors in which the enhancer elements of the long terminal repeats self-inactivate during reverse transcription (“SIN” vectors). These SIN vectors have excellent safety profiles and have not been reported to cause any clinically significant genotoxicity. Gene therapy has successfully treated several PIDs including Adenosine Deaminase Severe Combined Immunodeficiency (SCID), X-linked SCID, Artemis SCID, Wiskott-Aldrich Syndrome, X-linked Chronic Granulomatous Disease and Leukocyte Adhesion Deficiency-I. In all, gene therapy for PIDs has progressed over the recent decades to be equal or better than allogeneic HSCT in terms of efficacy and safety. Further improvements in methods should lead to more consistent and reliable efficacy from gene therapy for a growing list of PIDs. Allogeneic hematopoietic stem cell transplantation (HSCT) has been a definitive therapy for the most severe PIDs over the past 4–5 decades. Replacement of the patient's hematopoietic stem cells (HSC) that carry the defective PID-causing gene with allogeneic HSC from a healthy donor can lead to production of genetically normal blood cells and restore immunity. However, immunological differences between the patient and the HSC donor may lead to significant complications, including rejection of the donor HSC graft or attack by the transferred donor's immune cells against the recipient – graft vs. host disease (GVHD). In addition to pre-transplant myelosuppressive agents that open up space in the HSC niche, potent immune suppressive agents are often administered to minimize risks of donor cell rejection. Post-transplant, immune suppression is often given to prevent development of GVHD. While allogeneic HSCT is a mainstay of treatment and has had progressively improved outcomes, the immunological complications remain a major source of morbidity and potential mortality.If PIDs can be treated by engrafting allogeneic HSC, it should be possible to treat PIDs by engrafting autologous HSC that have been gene corrected, either by gene addition using a viral vector (as will be discussed here) or by gene editing (not discussed here) to regain normal function . Upon reWhile gene therapy is conceptually attractive, it took more than a decade for any evidence of efficacy against a PID to be realized in clinical trials , 2. SincAbsence of adenosine deaminase (ADA) enzyme in a SCID patient was first deduced to be a cause of human SCID in 1972 by Eloise Giblett . SubsequAn early treatment for ADA SCID was frequent transfusions of red blood cells (RBC) that transferred ADA enzyme contained in the RBCs to catabolize adenosine nucleosides and allow survival of some lymphocytes . This leHSCT for ADA SCID was first reported by Parkman et al. , with thex vivo manipulation . The first clinical gene therapy for ADA SCID was performed at the National Institutes of Health in 1990, targeting a normal ADA gene into peripheral blood lymphocytes . There wpulation . While tGene therapy for ADA SCID was first made efficacious through studies performed in Milan, Italy . A gammaA gene therapy for ADA SCID has been approved by the European Medicines Agency (EMA), Strimvelis, which was derived from the work done at the Milan center. Unfortunately, one recipient of this gene therapy developed lymphoproliferation several years after treatment,A lentiviral vector has also been studied for gene therapy for ADA SCID in the U.S. and the U.K . Forty eWhile the gene therapies described above have led to high rates of correction of the life-threatening immune deficiency, the other complications in ADA SCID, such as hearing loss and neurodevelopmental deficits, may not be corrected by any of the therapeutic interventions that only correct hematopoietic cells. It is hoped that early identification of ADA SCID patients by newborn screening, the prompt implementation of ADA ERT, and the subsequent curative treatment by HSCT or gene therapy may reduce these complications, but this is not known at present.IL2RG gene . X-SCID is the most common form of SCID, accounting for approximately one-third of cases  to drive high level expression of IL2RG throughout the lymphoid lineages. The majority of patients (18/20) had appropriate T cell reconstitution after gene therapy. But six of the patients in the initial trials experienced serious adverse events, developing T cell acute lymphoblastic leukemia 2–14 years after undergoing gene therapy (LMO2) proto-oncogene, driving its over-expression, thought to be the leukemic initiating event.Initial gene therapy trials for X-SCID enrolled patients without an HLA-identical sibling donor, 10 patients in France and 10 patients in the United Kingdom –29. Pati therapy –32. Gammtrans-activate neighboring endogenous proto-oncogenes  cause autosomal recessive T-B-NK+ SCID (Art-SCID). Artemis is an endonuclease required to repair double stranded DNA breaks using non-homologous end joining. Artemis is used in the processes of V(D)J recombination that create a diverse repertoire of genes encoding B cell immunoglobulin receptors (and antibodies) and T cell receptors. Art-SCID patients are radiosensitive, with increased adverse effects after radiation or alkylating agents, due to their inability to repair double-stranded breaks. Like other forms of SCID, Art-SCID patients can be treated with HSCT, with patients receiving HSCT from matched sibling donors surviving at a rate of 85% and from haplo-identical donors at a survival rate of 65% . Thus far, the only reported side effect has been autoimmune hemolytic anemia of uncertain etiology occurring early after treatment in two infants and one previously treated patient; this problem resolved after T cell immunity recovered. New vectors and gene editing strategies for gene therapy of Art-SCID are also being assessed by other investigators.The first clinical trial to treat Art-SCID is currently enrolling patients, with 5 newly diagnosed patients and 3 patients previously treated with allogeneic HSCT reported in a meeting abstract . PatientWAS are found throughout the 12 exons of the gene, with defects in the C terminus associated with more severe disease symptoms  is an X-linked pan-hematopoietic defect that classically presents with bleeding , eczema, and infections, and frequently involves autoimmunity and lymphoid neoplasms. The Wiskott Aldrich Syndrome Protein (WASP) is expressed only in hematopoietic cells and is an important regulator of actin cytoskeleton. WASP dysfunction impairs normal leukocyte migration, phagocytosis, creation of immune synapse, and intracellular signaling. Abnormal WASP also creates abnormalities of megakaryopoiesis, with micro-platelets and thrombocytopenia. Autoimmunity  results from insufficient Treg functionality, defective apoptosis, and increased auto-reactive B cells . WAS patsymptoms . Pathoge protein .Patients with severe WAS  typically have a life expectancy of <15 years in the absence of HSCT due to hemorrhage, malignancy and severe infection . HSCT ofLMO and MECOM loci  accounts for approximately 65% of CGD. While use of prophylactic antibiotics and antifungals and aggressive treatment of infections that do occur can reduce morbidity in CGD, nevertheless patients offer suffer recurrent infections, inflammatory complications, hospitalizations and early mortality.Chronic Granulomatous Disease (CGD) was recognized as a distinct disorder in patients who had recurrent infections and cutaneous and visceral granulomas without detectable micro-organisms, but with apparently intact antibody production and delayed hypersensitivity (T cell-mediated) responses . CGD is Allogeneic HSCT can be curative for all forms of CGD and the development of reduced intensity conditioning protocols has led to relatively high rates of survival and correction of the immune deficiency . A recenHence gene therapy for CGD has been under study for almost three decades. Early gene therapy efforts for CGD in several trials were performed in the 1990's using gamma-retroviral vectors, but no cytoreductive conditioning . In gene2, respectively  and Cathepsin-G (CTSG)], to prevent trans-activation of proto-oncogenes at the stem and progenitor stages that are vulnerable to transformation, but express sufficient levels of the needed oxidase in mature myeloid cells  required for neutrophils to adhere to ICAM molecules on endothelium to leave the bloodstream and move to sites of infections , X-Linked Lymphoproliferative (XLP), Immune Dysregulation, Polyendocrinopathy, Enteropathy, X-Linked Syndrome (IPEX), X-linked agammaglobulinemia (XLA) . In theoRAG1 gene may be tempered by the need for simultaneous co-expression of endogenous RAG2, it is unknown if ectopic expression of the RAG1 recombinase may lead to toxicities.Of the more than 20 genes capable of causing human SCID when defective, gene therapy is currently in the clinic for three: ADA SCID, XSCID, and Artemis SCID. A clinical program for gene therapy for Rag1 SCID is underway by collaborative effort in the European Union, using a lentiviral vector with a constitutive retroviral long-terminal repeat enhancer/promoter to drive expression. Their early studies showed high level RAG1 expression is necessary to restore T cell production at sufficiently high levels to prevent autoimmune complications . While fIL7R gene may be drivers in pediatric T ALL  can all lead to higher levels of engraftment of gene-corrected HSC. The lessons learned from the use of retroviral vectors with strong enhancer elements that led to insertional oncogenesis and leukoproliferative complications has led to the development and application of safer SIN vectors that lack strong enhancer elements and have significantly reduced risks for causing transformations.Other elements of the gene therapy A major limitation of current approaches to both allogeneic HSCT and autologous gene therapy for PIDs is the use of cytoreductive chemotherapy agents to facilitate engraftment of the corrective HSC. These drugs have multiple acute and long-term complications, such as acute organ toxicity  and potential late adverse effects on growth, skeletal and dental development, neurodevelopment, and risks for secondary malignancies. Therefore, there is currently a great deal of effort to develop monoclonal antibody-mediated methods to achieve safe and effective cytoreduction, without the complications of chemotherapy. Antibodies to ckit, CD45, and other stem cell markers are in clinical trials for conditioning, either as unmodified antibody or as antibody drug conjugates with toxins that may increase the elimination of targeted HSC , 93. It As the field of gene therapy matures, the treatments are moving from early-phase research studies performed at one or a few academic medical centers to become licensed products that are produced commercially and may be administered at multiple institutions with experience in HSCT for PIDs. Ideally, this move to market gene therapies will make them widely available and not require patients and their families to relocate to the few sites that provide them under research trials.A major issue for commercially prepared gene therapies will be the costs for these treatments. Producing these complex gene and cell therapy products under the strict requirements mandated by FDA, EMA and other oversight entities comes with high costs, in terms of the multiple trained personnel, highly controlled facilities, aspects of a quality assurance program and regulatory requirements. Ultimately, gene therapy will compete with allogeneic HSCT as treatments for PIDs with efficacy, safety and cost all being determining factors of which approach gains acceptance.While both allogeneic HSCT and autologous gene therapy can alleviate all or most of the immunological and hematological complications of diseases, they often do not address non-hematological complications. For some disorders there may be cross correction of non-hematological tissues by protein made from hematological cells. In ADA SCID, expression of ADA enzyme in a fraction of cells can lower total body pools of adenine nucleotides and rescue survival of ADA-deficient lymphocyte. However, in most cases the non-hematological manifestations, such as the skin or other organs involved in Nemo Syndrome, may not be improved by successful engraftment of HSC with the normal gene. Correction of the gene defect in HSC of Art SCID patients does not correct their general somatic DNA repair defect, and conditioning carries risks for damage to other tissues.In all, gene therapy for PIDs has progressed over the recent decades to be equal to and even better in some cases than allogeneic HSCT. Further improvements in methods should lead to more consistent and reliable efficacy from gene therapy.LK and DK co-wrote the article. All authors contributed to the article and approved the submitted version.LK is the recipient of an A. P. Giannini Foundation Postdoctoral Research Fellowship. DK is a member of the Scientific Advisory Board for Orchard Therapeutics that markets Strimvelis and has licensed a lentiviral vector for gene therapy of ADA SCID from the University of California, Los Angeles for which he is an inventor. DK is also the principal investigator for clinical trials discussed in this article on gene therapy for XCGD  and LAD-I . He is the Chair of the Data Safety Monitoring Board for Leadiant Biosciences for Revcovi PEG-ADA ERT.