The lack of effective treatments for most neurological diseases has prompted the search for novel therapeutic options. Interestingly, neuroinflammation is emerging as a common feature to target in most CNS pathologies. Recent studies suggest that targeted delivery of small molecules to reduce neuroinflammation can be beneficial. However, suboptimal drug delivery to the CNS is a major barrier to modulate inflammation because neurotherapeutic compounds are currently being delivered systemically without spatial or temporal control. Emerging nanomaterial technologies are providing promising and superior tools to effectively access neuropathological tissue in a controlled manner. Here we highlight recent advances in nanomaterial technologies for drug delivery to the CNS. We propose that state-of-the-art nanoparticle drug delivery platforms can significantly impact local CNS bioavailability of pharmacological compounds and treat neurological diseases. Neurological disorders disrupt the normal function of the brain and/or spinal cord and are a major cause of death and disability worldwide. Dysfunction in the central nervous system (CNS) leads to variable levels of impairments in speech, memory, sensorimotor, and autonomic functions, which can considerably diminish the patients’ quality of life. The underlying causes of neurological diseases are often complex and diverse, and can stem from degeneration, trauma, infection, tumors, vascular dysfunction, structural defects, or autoimmune conditions . Recent The term neuroinflammation has been used to describe different cellular and molecular pathological phenomena that can encompass activation of CNS-resident cells, CNS infiltration by circulating leukocytes, and production of inflammatory mediators in the CNS environment . MicroglSpinal cord injury (SCI) involves a complex time-dependent course of cellular and molecular events that have been elucidated in the last several decades. An SCI induces vascular damage and ischemia that lead to cell necrosis and apoptosis, followed by acute and chronic neuroinflammation, glial activation, scar formation, and demyelination and neurodegeneration . These eNanotechnology advances have recently yielded countless innovative biomaterials that can be custom designed and serve as drug delivery systems to effectively target biological tissues with cell selectivity. The majority of nanoparticle-based therapeutics has focused on targeting tumor cells and provide enhanced antitumor efficacy. Many of these technologies are now undergoing clinical evaluation, and some have been approved clinically to treat a variety of cancer diseases . The vasThe ideal delivery of a pharmacological anti-inflammatory agent to the CNS entails non-invasive administration of a stable compound at a therapeutic concentration followed by rapid targeting of dysfunctional pathways, and reversal of the disease to a healthy state without induction of off-target effects. However, in the case of drug delivery to the CNS we encounter several roadblocks that complicate effective targeting. First, most non-invasive routes of delivery are ineffective in providing access to the CNS, due to the distinctive properties of the neurovascular unit that restricts most pharmacological compounds from entering the CNS . In addiThe innate immune response by CNS-resident cells is initially driven by sensing the presence of extracellular or intracellular pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) by pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) and nuclear oligomerization domain-like receptors (NLRs). Some examples of DAMPs include aggregated proteins characteristic of neurodegenerative diseases, such as β-amyloid, α-synuclein, and microtubule-associated protein-tau; and molecules released from damaged cells, such as ATP, heat shock proteins, oxidized lipids, and chromatin . SensingA recent focus of research has been the investigation of common neuroinflammation mediators across different neurological conditions. The therapeutic potential of targeting DAMPs, PRRs, and cytokine signaling pathways is being explored in various preclinical animal models. Pharmacological DAMP inhibition has shown therapeutic potential in multiple neurological disorders. High mobility group box 1 protein (HMGB1), for example, is a key protein that initiates neuroinflammation in traumatic, infectious, and neurodegenerative conditions. Preclinical studies have examined HMGB1 modulation by administering small molecule inhibitor compounds or anti-HMGB1 monoclonal antibodies with encouraging results in a wide number of experimental models . TLRs arCurrent delivery of therapeutic compounds to the CNS have minimal control over targeting specificity and duration of treatment. Systemic high dosages and multiple administration paradigms are often needed to achieve therapeutic concentrations that induce the desired effects in target tissues. Consequently, systemic toxicity and undesired side effects frequently accompany such dosage regimen. Alternative strategies that enhance CNS penetration and targeting include artificial disruption of the blood-CNS barriers (B-CNS-B) and use of direct administration routes. Transient disruption of the B-CNS-B can be achieved by action of chemical or physical agents. Mannitol infusion, for instance, increases cerebral blood flow and vascular permeability, but can also induce toxicity, and provides minimal control over treatment duration and targeting. Alternatively, the use of microbubble-assisted focused ultrasound can induce a more localized and controlled action on the B-CNS-B permeability . Safety Nanotechnology is defined as the control or restructuring of matter at the atomic and molecular levels, within the range of nano to submicron dimensions . It involves the manufacturing of nanomaterials, their applications, and integration into physical, chemical, and biological systems . AdvanceLiposomes are self-assembled lipid bilayer vesicles that can incorporate hydrophilic drugs in the inner aqueous phase, and lipophilic drugs within the phospholipid bilayer. Liposomes are easy to formulate, biocompatible and biodegradable nanoparticles that can be administered via different routes  . Micellein vitro. Another recent study proposes the use of theranostic nanoparticles for long-term tracking of transplanted cells coupled with antioxidant treatment following stroke  from oxidative stress. Moreover, the imaging properties of these nanoparticles allow MSC tracking to guide intracerebral implantation and evaluate transplanted cell homing. More importantly, the theranostic nanoparticles also promoted angiogenesis, neurogenesis and functional recovery after ischemic stroke in mice.As technology advances, sophisticated multifunctional systems are gradually emerging and supplanting the pioneering single purpose first generation nanoparticles. These multifunctional nanoparticles can combine multiple properties in one single system, which has led to the development of theranostic tools. Nanotheranostic tools can incorporate simultaneously diagnostic entities, usually an imaging agent, and therapeutic disease-modifying drugs. For CNS therapeutic applications, nanoparticles that can be visualized using magnetic resonance imaging (MRI), single-photon-emission computed tomography (SPECT), or positron emission tomography (PET) are advantageous. g stroke . MesoporDue to the complexity of CNS pathobiology, combinatorial approaches are likely to be needed in achieving therapeutic efficacy. Multifunctional nanoparticles are amenable to functionalization with addition of surface-grafted biocompatible polymers, cell targeting agents, stimuli-responsive components, energy conversion moieties, or catalytic activities . Additioclinicaltrial.gov NCT03500627). These are first generation nanomaterials that rely on passive targeting to reach the CNS and accumulate in the pathological site. Understanding how nanoparticle intrinsic properties influence biodistribution and function is an important step in the design of more complex multifunctional nanosystems.The first preclinical feasibility study evaluating intracerebral injection of liposomes was published in the late 1970s and concluded that safety depends on liposome composition and dose. This work investigated administration of different liposome formulations and doses in rodent models, and opened the possibility of using liposomes as carriers of cell-modifying compounds into diseased CNS tissues . Since tSeveral studies have explored how physicochemical properties influence nanoparticle penetration and retention in the CNS. There is no golden rule that applies to all nanomaterials, and comparative biodistribution studies are an essential prelude to preclinical studies. Nonetheless, intrinsic nanoparticle properties such as size, shape, and chemical composition influence behavior in biological systems. Furthermore, extrinsic parameters such as surface charge, purity, and colloidal stability are also important determinants of performance. An example is the case of nanoparticles that tend to aggregate when exposed to certain media. Nanoparticle aggregates behave as large particles, with different properties from individual nanoparticles and this can lead to immunogenic effects . The sizIn CNS pathologies, different cell types respond distinctively to the same environmental alterations. Thus, achieving high targeting efficiency to specific cell populations can significantly enhance treatments. For instance, during neuroinflammation neurons undergo apoptosis in response to glutamate excitotoxicity. Neuron-targeted dendrimer nanoparticles carrying neuroprotective drugs have shown efficacy in attenuating neurological deficits in a brain injury model , supportDeeper understanding of neuroinflammatory pathways and CNS cellular biology have led to the development of methods to modify nanoparticle surface with ligands that increase cell specificity and CNS penetration. These include modifications with integrin-binding peptides , antibodActive cell targeting can be achieved through nanoparticle modifications that exploit ligand-receptor interaction mechanisms. In the initial stages of neuroinflammation, endothelial cells become activated and play a critical role in immune cell trafficking to the site of inflammation . Upon cyex vivo. These monocytes were then infused back into a mouse and were detected in areas of neuroinflammation demonstrating the potential to exogenously deliver nanoparticle-carrying monocytes to target inflammation areas in the brain  and production of cell membrane-derived nanoparticles (such as cell-membrane nanovesicles) are emerging methods that provide new diagnostic and therapeutic modalities for different types of diseases . Membranembranes . Becauseembranes .Using membrane-coating technologies to produce nanoparticles that mimic the targeting capabilities of immune cells is also being explored. Leukocyte-membrane coating of nanoparticles adds naturally occurring receptor-ligand interactions present in leukocyte membranes, and permits easy passage to inflamed tissues . Dong etIn summary, cell-coated nanoparticles offer an additional approach to avoid immune detection, increase sustained circulation, and targeted drug release. Different types of membranes can be leveraged for a range of distinct targeted drug delivery applications without significant biological modifications, which is clinically relevant and can accelerate FDA-approval. The recent advancements in the development of cell-coated nanoparticles capable of sustained drug-release in response to the microenvironment is steadily progressing toward more sophisticated therapeutic strategies and can lead to unique advantages for CNS drug delivery. There is a growing number of patents related to cell membrane coating technologies, which reflects the potential for clinical use . AlthougAs naturally occurring nanoparticles in biological systems, exosomes are also gathering interest as therapeutic drug delivery agents. Exosomes are lipid nanovesicles involved in intercellular communication during physiological and pathogenic processes through the transfer of small molecules, such as RNA. After release from their cells of origin, exosomes can be internalized by other cells thereby modulating their function . Their sAnother recent focus of research has been the development of nanoparticles that exert therapeutic effects in response to physiological variations or externally applied stimuli. These materials are commonly designated as stimuli-responsive materials, or smart materials, and mimic the responsiveness of living organisms. Smart nanoparticles possess the unique ability to alter their structure in response to slight environmental changes and revert to their original state after the stimulus disappears. Physiological stimuli, such as temperature, pH, redox, oxygen, or enzymes can act as triggers for drug release by causing disruption of covalent bonds between drugs or other molecules and the nanoparticle, or by destabilizing the nanoparticle structure that becomes more permeable for drug diffusion or unveils functional ligands. Stimuli-responsive nanoparticles can thus react not only by releasing therapeutic compounds, but also by exposing and activating surface ligands, such as cell penetrating sequences, targeting ligands, or other functionalities that can alter pharmacokinetics or biodistribution. In addition to the more commonly known pH-sensitive and thermosensitive nanoparticles, many technological advances have allowed materials to be designed to also respond to externally applied stimuli including light, ultrasound, electrical or magnetic fields. pH is a particularly useful stimulus that can be used to control intracellular drug release in response to a changing pH environment from the extracellular medium to endosomes and lysosomes. At the tissue level, it can be used to release drugs in inflammation sites that have typically lower pH levels.The detailed portrayal and timeline of inflammatory events in neurological diseases can allow the establishment and optimization of precisely timed therapeutic interventions. Administering stimuli-responsive anti-inflammatory nanoparticles can allow precise interventions by only targeting and releasing therapeutic compounds upon detection of microenvironment alterations. In addition to cellular and molecular events, inflammation is also characterized by local microenvironment alterations such as decreased pH, high oxidative stress and accumulation of ROS, and overexpression of matrix-remodeling enzymes . pH-respin vivo, and the anti-tumor activity was increased in mice receiving pH-sensitive liposomes. In addition to triggered drug release in acidic neuroinflammatory environments, pH alterations can also be used as a trigger for intracellular drug release.pH is the most widely studied endogenous stimulus for control of drug release where an acid sensitive spacer between a nanoparticle and cargo is placed . AlthougIn vitro studies in models of blood-brain-barrier and inflammatory environments indicated enhanced transcytosis and resveratrol release in response to ROS. These stimuli-responsive nanomaterials can bring important advantages in targeted drug delivery, but they remain largely unexplored in neuroinflammation. Formulating nanocarriers that release drugs only at the target site in response to predetermined signals of disease can have a great impact in the design of effective therapies for neuroinflammation.Since upregulation of ROS is another feature of neuroinflammation, bioengineered ROS-responsive nanoparticles can be useful in a targeted site-specific drug delivery approach. The responsiveness to intracellular oxidative conditions is achieved by incorporating ROS-labile groups in the nanoparticle, for example boronic ester, proline, or thioketal . A recenin vivo are invaluable in providing safety profiles and promise translatability. Nanoparticles come across as ideal and resourceful tools to provide real time diagnostics and tailored treatment of neuroinflammation and other CNS illnesses, promising to have a significant impact in global health. Continued collaborative efforts between materials scientists, engineers, chemists, neuroscientists, and clinicians can finally identify safe and effective therapies that target neuropathological mechanisms and ultimately mitigate the burden of devastating neurological diseases.Recent advances in nanoparticle production technologies are creating promising theranostic tools to provide targeted and controlled drug delivery at sites of neuroinflammation. The emergence of biomimetic, cell targeted, and stimuli-responsive multifunctional nanoparticles are among the most encouraging strategies to treat neuroinflammation. Nanotechnology-based approaches remain underexplored for CNS applications, and an expected increase in preclinical studies can accelerate the establishment of nanoparticle-based treatments for CNS pathologies. How can we ensure that this anticipated surge in publications and patents will properly translate into successful clinical applications? Careful consideration of the design, large-scale production ability and standardized characterization of nanoparticles seem essential preludes to ensure adequate conversion into therapeutic products. Systematic approaches to assess the impact of nanoparticle properties on molecular interactions are also critical. Importantly, thorough investigation of toxicity, degradation byproducts and clearance routes of nanoparticles SC, NA, and JL conceived the concept and idea of the present review and selected the topics to be discussed. SC did literature searches, read the references, and wrote the first outline of the review. NA and JL reviewed and edited the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Borrelia burgdorferi sensu lato complex, is the most common vector-borne disease in the Northern hemisphere. Spirochetes are transmitted by Ixodes ticks and maintained in diverse vertebrate animal hosts. Following tick bite, spirochetes initially establish a localized infection in the skin. However, they may also disseminate hematogenously to several distal sites, including heart, joints, or the CNS. Because they need to survive in diverse microenvironments, from tick vector to mammalian hosts, spirochetes have developed multiple strategies to combat the numerous host defense mechanisms. One of these strategies includes the production of a number of complement-regulator acquiring surface proteins (CRASPs) which encompass CspA, CspZ, and OspE paralogs to blunt the complement pathway. These proteins are capable of preventing complement activation on the spirochete surface by binding to complement regulator Factor H. The genes encoding these CRASPs differ in their expression patterns during the tick-to-host infection cycle, implying that these proteins may exhibit different functions during infection. This review summarizes the recent published reports which investigated the roles that each of these molecules plays in conferring tick-borne transmission and dissemination in vertebrate hosts. These findings offer novel mechanistic insights into LD pathobiology and may facilitate the identification of new targets for preventive strategies against Lyme borreliosis.Lyme disease (LD), which is caused by genospecies of the  Borrelia burgdorferi sensu lato complex  burgdorferi sensu stricto (hereafter referred as B. burgdorferi), B. afzelii, B. garinii, B. spielmanii, B. bavariensis, and B. mayonii, all of which are transmitted by Ixodes ticks and maintained in diverse reservoir hosts   is the most common vector-borne disease in the northern hemisphere  which are deposited on host cells/tissues to avoid non-specific damage by the complement cascade  which is essential for LD spirochetes survival in the infection cycle , CspA binds only moderately to C7 and C9 (KD > 5 μM). These results raise questions regarding the physiological relevance of CspA-mediated C7- and C9-binding activity  in infection cycle. CspZ was shown not essential for spirochetes acquisition from mammalian hosts to ticks , the difference of these variants may convey the observed strain-to-strain variation in binding activity to human FH  some of these variable regions are located in the binding site/interface with human FH. These results support the possibility that these variable regions of CspZ mediate the different levels of FH-binding activity and spirochete survival in the infection cycle  to other outer surface proteins on cp32, such as OspF  driven by the endogenous promoters, remains susceptible to complement-mediated killing in human serum  encoding erpP and erpA under the control by the endogenous promoters which remain susceptible to human serum. However, when those genes are expressed ectopically in a serum-sensitive B. burgdorferi strain using a strong and constitutive promoter, these spirochetes inactivate complement and survive when incubated with human serum  and in the bloodstream of vertebrate animals. A key evasion mechanism is to circumvent the complement components by producing complement- or CRP-binding proteins, including CRASPs, which facilitate complement inactivation. These CRASPs have been shown to confer spirochete transmission from ticks to hosts and promote infection and dissemination in vertebrate hosts. However, the concurrent production of CRASPs increases the complexity in delineating the contribution of these proteins individually in each of the stages within the infection cycle. Elucidating such mechanisms will provide new insights into how spirochetes survive in two distinct environments, ticks, and vertebrate hosts. Such information will provide foundation for the development of preventions through targeting CRASPs to block these infection mechanisms, which will ultimately reduce LD burdens in humans.Y-PL, AF, TN, and PK wrote the manuscript. TN and Y-PL prepared the figures.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.


With the development of novel targeted therapies, including exon skipping/inclusion and gene replacement therapy, the field of neuromuscular diseases has drastically changed in the last several years. Until 2016, there had been no FDA-approved drugs to treat Duchenne muscular dystrophy (DMD), the most common muscular dystrophy. However, several new personalized therapies, including antisense oligonucleotides eteplirsen for DMD exon 51 skipping and golodirsen and viltolarsen for DMD exon 53 skipping, have been approved in the last 4 years. We are witnessing the start of a therapeutic revolution in neuromuscular diseases. However, the studies also made clear that these therapies are still far from a cure. Personalized genetic medicine for neuromuscular diseases faces several key challenges, including the difficulty of obtaining appropriate cell and animal models and limited its applicability. This Special Issue “Molecular Diagnosis and Novel Therapies for Neuromuscular/Musculoskeletal Diseases” highlights key areas of research progress that improve our understanding and the therapeutic outcomes of neuromuscular diseases in the personalized medicine era. Neuromuscular diseases include a large number of different medical conditions that affect the peripheral nervous system and muscle ,2. Many A key challenge in genetic diseases, however, is the difficulty of obtaining cell and animal models that faithfully recapitulate the disease phenotype . In addiAnother key area in the personalized medicine era is an accurate and cost-effective genetic diagnosis . In thisThere are several approaches to mutation-specific personalized genetic therapy for DMD. These approaches aim to restore dystrophin expression using different techniques, including stop-codon read-through, antisense oligonucleotide-mediated exon skipping, and genome editing. In this Special Issue, the former two approaches are discussed in detail by Shimizu-Motohashi et al. . Genome-Although significant progress has been made in DMD and spinal muscular atrophy (SMA) therapeutics, patients with most neuromuscular diseases, such as amyotrophic lateral sclerosis (ALS), still have no effective targeted treatment option available . Since mIn conclusion, we welcome a new era of personalized genetic medicine as we move forward enthusiastically towards the next generation of therapeutic technologies. We hope this collection of articles can provide readers with a useful introduction to molecular diagnosis and novel therapies for neuromuscular diseases in the personalized medicine era.


Antimicrobial susceptibility testing (AST) of bacteria isolated in blood cultures is critical for optimal management of patients with sepsis. This review describes new and emerging phenotypic and genotypic AST methods and summarizes the evidence that implementation of these methods can impact clinical outcomes of patients with bloodstream infections. Sepsis, defined as an infection with dysregulated host response leading to life-threatening organ dysfunction, occurred in nearly 49 million incident cases and accounted for 19% of all deaths worldwide in 2017 , 4. In tCandida in blood cultures, but not susceptibility testing.Sepsis is frequently caused by BSIs. In a retrospective analysis of nearly 3 million adult encounters across 409 US hospitals, a positive blood culture was found in 17% of patients with sepsis . Knowledin vitro), use of culture media that are a poor mimic of the physiological environment of the body, and use of an inoculum size that is infrequently observed in clinical specimens   . BMD is CFU)/ml] –30. DespCFU)/ml]  with decCFU)/ml] . In geneCFU)/ml] . In contCFU)/ml] –35, and CFU)/ml] , 36–40.Historically, off-label use of positive blood culture broth as the inoculum for disk diffusion or automated AST methods  was widely performed in clinical laboratories to expedite time to results for AST from positive blood cultures . This wa® , which is both US Food and Drug Administration (FDA) cleared and Conformite-Europeenne in vitro Diagnostic (CE-IVD) approved, and the Alfred 60AST , which is CE-IVD. The PhenoTest BC performs rapid identification (ID) (1.5 h) and AST (~7 h) from positive blood cultures by performing fluorescence in situ hybridization and time-lapse imaging of bacteria under dark-field microscopy, respectively    methods  Sweden) , Fastino Sweden) , and Rev Sweden) , 38.mecA in a blood culture that also harbors Staphylococcus aureus predicts methicillin resistance, but additional testing is required to confirm susceptibility to other antimicrobial agents, such as vancomycin, daptomycin, or linezolid. Furthermore, it is rare for complete correlation between genotype and phenotype to be observed—key examples are the presence/absence of mecA or vanA/B, which predicts resistance/susceptibility to oxacillin in staphylococci or vancomycin in enterococci, respectively. Outside these two examples, antimicrobial resistance is almost always multifactorial and ever-evolving, making such determinations challenging, even with whole-genome sequencing . Genetic tests cannot assign a detected resistance gene to a specific bacterium in a polymicrobial specimen, which may result in overcalling resistance. For example, mecA detection in a specimen harboring both S. aureus and Staphylococcus epidermidis could lead to incorrect assigning of this gene to the pathogen, and not the contaminant. Alternatively, rapid detection of a resistance gene in a culture containing multiple Enterobacterales species with differing susceptibilities may reduce undercalling resistance. Furthermore, new mutations and resistance mechanisms are continually evolving, which may limit the ability of certain genetic tests to predict resistance, particularly if a mutation occurs in primer complementary regions  . Only a  regions  or involThe impact of rapid blood culture diagnostic methods on clinical and economic outcomes has been mixed. The majority of outcomes studies to date are single-center, observational studies that evaluate the impact of rapid organism identification with or without AST. Some of these studies have demonstrated decreased time to appropriate antibiotics, lower mortality, shorter durations of hospital and intensive care unit (ICU) stay, and reduced costs, although these are not consistent findings. These studies have been previously summarized elsewhere and have been incorporated in meta-analyses –69. LimiCandida as well as select resistance markers  using multiplex PCR. In this study, participants with positive blood cultures were randomized in the clinical laboratory to have blood culture characterization with either conventional testing methods including matrix-assisted laser desorption–ionization time of flight (MALDI-TOF), the BCID test, or the BCID test plus antimicrobial stewardship review. Participants in both BCID arms had faster time to antibiotic escalation, less treatment of contaminants, and less use of broad-spectrum antibiotics than had those receiving conventional testing. In addition, participants who received BCID paired with antimicrobial stewardship had more rapid antibiotic de-escalation than those who received BCID without stewardship. However, no differences were observed between the arms in mortality, length of hospital stay, infection with multidrug-resistant organisms or Clostridium difficile, or cost of care (blaKPC) from Gram-negative species, which was exceedingly rare at the time and did not provide rapid phenotypic susceptibility information for a full panel of antibiotics  to evaluate outcomes of rapid blood culture AST methods and are summarized in  of care . Notablyibiotics .The recently completed RAPIDS GN trial evaluated the impact of a rapid phenotypic AST method for Gram-negative bacilli bloodstream infection and addresses some of the limitations of the BCID trial . This muTaken together, these studies demonstrate that a variety of rapid AST methods can shorten time to optimal therapy and improve antibiotic stewardship for patients with bloodstream infections. However, most RCTs do not demonstrate that rapid AST methods result in significant reductions in mortality, hospital length of stay, or adverse events, perhaps because larger studies are required to detect differences in these rare events. It is also notable that with the exception of the study by Kim et al., most of these trials were conducted in areas with low rates of antimicrobial-resistant bacteria, and it is possible that more clinical benefit would be observed in areas with higher rates of resistant infections , 20. AddIn conclusion, technological advances that enable rapid AST from organisms growing in blood cultures have great potential to improve the care and outcomes for patients with sepsis. Numerous platforms are currently available or in development, all of which can provide rapid genotypic or phenotypic detection of resistance. However, the success of these technologies requires demonstration of clinical impact. Unlike trials that evaluate a direct action, RCTs conducted for rapid diagnostics are one step removed from the patient, requiring the physician to act on the result. This task has been shown to be best supported when ASPs are active participants in RCTs, showing significant reduction in time to therapy optimization through the use of rapid AST devices. However, the impact of more rapid therapeutic intervention remains largely theoretical. Shorter time to optimal antibiotic therapy should lead to reduced length of hospitalization and mortality, but studies conducted to date were not sufficiently powered to measure these endpoints. More subtle endpoints , alternative trial designs, and inclusion of patient preferences in endpoint determinations may all provide further insight into the value of these tests. Also worth noting is the fact that while significant improvement in hospitalization stays or mortality has not been demonstrated through rapid antimicrobial de-escalation, the opposite is also true—i.e., interventions conducted ~1–2 days earlier in the course of sepsis do no harm. These data provide a valuable foundation to aid improved stewardship of antibiotics.Staphylococcus aureus (anti-MRSA) therapy . Ongoing investment in infectious disease diagnostics and development of rapid AST technologies will be important for continued improvements in sepsis outcomes.Another area of much-needed future research is implementation science. While most large academic hospitals have adopted rapid AST methods for blood cultures, their use is not universal . SimilarRB and RH reviewed the literature, drafted, and revised the manuscript. All authors agree to be accountable for the content of the work.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.


Externally bonded fiber-reinforced polymer composites have been in use in civil infrastructure for decades, but their long-term performance is still difficult to predict due to many knowledge gaps in the understanding of degradation mechanisms. This paper summarizes critical durability issues associated with the application of fiber-reinforced polymer (FRP) composites for rehabilitation of concrete structures. A variety of factors that affect the longevity of FRP composites are discussed: installation, quality control, material selection, and environmental conditions. Critical review of design approaches currently used in various international design guidelines is presented to identify potential opportunities for refinement of design guidance with respect to durability. Interdisciplinary approaches that combine materials science and structural engineering are recognized as having potential to develop composites with improved durability. Externally bonded (EB) fiber-reinforced polymer (FRP) composites are one of the most economical technologies showing promise to recover deteriorated concrete structures as well as improve the resilience of critical infrastructure across the world. EB FRP can be used to upgrade columns, beams, and walls in a variety of concrete structures ranging from residential and commercial buildings to critical infrastructure  . FRP comEven though composites have been in use in civil engineering for close to 30 years ,3, EB FRParadoxically, exceptional durability of composites is often cited as one of the main reasons for the use of FRP over other materials in aerospace, automotive, and marine industries. Although composites have been successfully used in these industries without experiencing any significant durability problems, it should be noted that these “parent” composite systems quite significantly differ from those used in infrastructure applications nowadays. Quality of constituent materials, processing and curing conditions, and environmental loadings are often significantly different. For instance, to drive down the cost and constrained by the application-specific requirements, civil engineers resort to the wet-layup application process (over resin transfer molding and autoclave molding) and resins such as ambient-cured epoxies (over high-temperature cured epoxies) ,6. TheseFurthermore, materials used in the aerospace industry have to pass conservative specification and qualification testing requirements to yield extraordinarily high levels of safety  ,8. The qAcross the world, there is a rising need to implement EB FRP in harsh environmental conditions in concrete structures to prolong their service life and ensure hazard resilience. Some example applications of EB FRP in harsh environments include concrete bridges and dams, water-treatment facilities, and nuclear reactors, where EB FRP composites must endure combined effects of multiple environmental stressors and structural loadings while maintaining sufficiently long service life. Civil engineers designing such structural upgrading systems are often faced with a lack of guidance regarding the proper treatment of durability concerns. The intent of this paper is to provide an overview of the most common deterioration mechanisms in the EB FRP systems and a critical assessment of the existing design guidance worldwide for EB FRP materials used in harsh environmental conditions. The reader shall be aware that, even though care is taken to provide the most up to date information, the research in this area is still ongoing while new materials, durability design guidelines, and test methods are under development.Installation of EB FRP is a relatively simple process, but even relatively small deviations from the prescribed procedures can cause defects in the bonded systems that can lead to poor performance of the EB FRP in the long term. Usually, the most critical component of the system governing its effectiveness is bond quality  between EBFRP applications require strong adhesive bonding between EB FRP and the concrete substrate for effective stress transfer between the adherents; examples of such applications include flexural  and sheaWhether the EB FRP application is bond-critical or contact-critical will affect the importance of proper concrete surface preparation. Even though substrate preparation and quality of adhesive bonding are more important in bond-critical applications, recent evidence shows that defects at the EB FRP/concrete interface can significantly deteriorate the strength of EB FRP-confined columns . As bondThe EB FRP installation process starts with concrete surface preparation a. DependFollowing concrete surface preparation, the epoxy primer is applied to the concrete surface b. The prBefore application to a primed concrete surface, dry fiber fabric is impregnated with a resin either by hand  or via resin impregnation machine. Impregnated EB FRP fabric can be applied directly to the primed surface c, or it After installation, EB FRP is allowed to cure according to the manufacturer’s specification. Given that the resin cure is dependent on the ambient temperature, the proper cure may require several days. In certain situations, it is prudent to apply an appropriate coating to cured EB FRP surface for aesthetic purposes and as protection from ultra-violet (UV) exposure and other environmental factors. While the ability of the protective coating to preserve the durability of EB FRP is often assumed, experimental evidence proving their effectiveness is rather scarce.Following installation and initial cure, the current state of practice in the U.S. is to perform a visual inspection for any signs of debonding or incomplete resin cure, which is followed by a pull-off test per ASTM D7522 . Common Conducting pull-off tests is expensive and time-consuming while also not being entirely non-destructive. Therefore, there is a need to develop rapid non-destructive test methods to facilitate evaluation of the adhesive bond. In addition, concerns have been raised about stress concentrations introduced by the tested locations . AccordiIn general, the lack of standardized techniques and procedures hinders development of inspection protocols and ability to quantify quality of the FRP installation. Possibility of using thermography , acoustiEB FRP is a complex multilayer system consisting of three constituent materials and their corresponding interfaces. As schematically shown in During their service, EB FRP systems are exposed to a variety of environmental conditions, many acting simultaneously. High humidity, elevated temperature, freeze-thaw cycles, UV radiation, and de-icing agents can affect the performance of an EB FRP strengthening system ,37,38,39fib addresses this issue indirectly—through reducing stresses in the internal steel reinforcement [Loading conditions  is another important factor affecting long-term performance of FRP, since presence of load accelerates its degradation and shortens the service-life of the repaired structures . Both faorcement . Level oorcement ,58. FRP orcement ,60.Commonly utilized resins and adhesives in infrastructure applications are thermosetting polymers such as polyesters, vinyl esters, and epoxies. Although polyester and vinyl ester resins can be formulated to exhibit good mechanical properties, they can display excessive shrinkage during curing and are often susceptible to accelerated deterioration under moisture . AdditioModern wet-layup EB FRP systems almost exclusively utilize ambient-cured epoxy as a composite matrix as well as an adhesive between EB FRP and concrete. When properly formulated, ambient-cured epoxy exhibits superior mechanical properties and better chemical resistance than other types of resins. Epoxy resins can have a range of viscosities and can cure under ambient conditions with minimal shrinkage. The adhesive also exhibits good wetting ability and adhesion to a variety of engineering materials, including concrete. Tg). Besides the monomer and hardener, epoxy adhesives often contain additives that can modify adhesive’s properties. Common types of additives in epoxy are accelerators (or curing promoters), coupling agents, antioxidants, and toughening agents.Epoxy is a two-component adhesive consisting of two precursors: (1) an epoxide group-containing polymer (or monomer) and (2) a curing agent or hardener. The backbone of the resin is usually a Bisphenol A diglycidyl ether (DGEBA) molecule which hosts epoxide groups a. EpoxidTg remains relatively low during the service life of EB FRP  [Tg. This can lead to loss of resin’s mechanical properties, ultimately compromising the integrity of EB FRP/concrete adhesive bonding and stress transfer between the fibers and matrix.Durability properties of epoxy adhesives are not only affected by their service environment but also by the processing and curing conditions characteristic for in situ wet-layup applications ,63,64. Sd 75 °C) . Given td 75 °C)  or higheTg (as shown in Tg) and post-cure (improving crosslinking density and increasing Tg) are two competing mechanisms [Tg in the same resin. Further complicating the complexity of the problem, the effects of plasticization can be partially or fully recoverable [Besides affecting epoxy’s mechanical and thermal properties, the incomplete cure may also render epoxy more vulnerable to certain deterioration mechanisms. Unreacted polar sites can “attract” water molecules into the cross-linked epoxy network that results in an onset of plasticization, which leads to a reduction in elastic modulus (by up to 50%), reduction in strength, and significant depression of chanisms . As can overable ,71,72.In addition to their susceptibility to degradation under moisture, epoxy resins were also found to be sensitive to UV exposure, which leads to oxidation of the ether and nitrogen groups ,74. OxidComparison of stress-strain behavior for different types of FRP materials and common grades of steel used in construction is shown in 2) chains due to their reaction with hydroxide ions (OH−) as well as hydrolysis of the glass network by OH− [Glass fibers are economical and thus the most attractive for civil engineering applications. Their durability properties were thoroughly researched due to the desire in the construction industry to replace corrosion-susceptible mild steel reinforcement with non-corrosive glass fiber reinforced polymer (GFRP) bars . Typicalk by OH− . It is ak by OH− ,94. In tCorrosion-resistant (ECR-Glass) and alkali-resistant (AR-Glass) grades of glass fibers can alleviate some of the observed durability issues. While ECR- and AR-Glass offer better durability characteristics than E- and S-Glass, these fibers are still characterized by a low modulus of elasticity (in comparison to carbon) which limits their applicability in situations where EB FRP is used to address the serviceability  concerns. When compared to CFRP, GFRP composites are more susceptible to environmental degradation especially when immersed in solutions and they are not adequate for application in aggressive environment . When itWhen EB FRP is employed in harsh environmental conditions in infrastructure, carbon fibers are utilized almost exclusively. Carbon fibers are inert to all environments typically experienced by civil infrastructure. They also offer superior mechanical properties and high resistance to creep-rupture. Carbon fibers possess better resistance to chemical attack than glass and aramid fiber. However, degradation of the outer layer of the fiber, which involves ion exchange reaction between the fiber and metal ions in the acid, results in the degradation of the interphase .Tg is exceeded). Exposure to freeze-thaw cycling can result in reduced mechanical properties as a result of thermal incompatibility of constituent materials, as explained above [Many studies that were performed to assess the longevity of wet-layup carbon fiber reinforced polymer (CFRP) composites confirmed their excellent durability properties ,96. Thesed above ,99. Effeed above ,100,101.Though EB CFRP composites show excellent durability under accelerated conditioning, the same cannot be said for the EB FRP-concrete adhesively bonded joints. Accelerated conditioning studies on EB FRP bonded to concrete have revealed varying levels of bond deterioration depending on the conditioning environment, conditioning time, and stress state (tensile vs. shear stress). Multiple researchers evaluated the durability of bond between EB FRP and concrete under moisture ,102, dryTg of epoxy) loosens the mechanical interlock which, combined with hydrogen bond breakage due to interactions by water molecules, leads to loss of adhesion and reduction in bond strength after prolonged exposure to moisture [The bond between epoxy and concrete is formed through chemical bonding and mechanical interlock. The primary chemical interaction between the adherents was found to be hydrogen bonding ,111, whimoisture ,66,113. moisture .Tg of epoxy. As previously discussed, plasticization leads to a reduction in Tg, which increases the possibility of ambient temperature exceeding the Tg. Because of this, many design guidelines specify a minimum Tg for a resin. For instance, ACI 440.2R requires the Tg to be at least 15 °C higher than the maximum design temperature, while AASHTO FRPS-1 is a bit more conservative and requires the Tg to be at least 22 °C higher than the maximum design temperature. Blackburn et al. [Tg following hygrothermal conditioning for six typically used epoxy adhesives in FRP composites. The experimental data was used to compute maximum and minimum allowed service temperatures per ACI 440.2R and AASHTO FRPS-1 based on the maximum and minimum measured Tg in the accelerated conditioning experiments, respectively  with epoxy-functional silane coupling agents ,126,130.United States: American Concrete Institute (ACI) 440.2R-08: “Guide for the Design and Construction of Externally Bonded FRP Systems for Strengthening Concrete Structures”; American Association of State and Highway Transportation Officials (AASHTO) FRPS-1, : “Guide Canada: Intelligent Sensing for Innovative Structures (ISIS)  Design MJapan: Japan Society of Civil Engineers (JSCE) . “RecommUnited Kingdom: United Kingdom Concrete Society Technical Report 55 (TR55) . “DesignItaly: Italian National Research Council Technical Document 200 (CNR-DT200) . “Guide To date, there are no prescriptive design codes that specify the requirement for the design of EB FRP in concrete structures which is thought to be one of the one of the barriers to a more widespread adoption of EB FRP systems. There are, however, various design guidelines that were developed in different countries. Some of the notable documents providing design guidance are the following:Given that the focus of this paper is mainly on the environmental durability of EB FRP in bond-critical applications, pertinent information regarding environmental reduction factors and maximum usable strain in EB FRP for flexure design is summarized in ISIS and TR55, while not providing explicit environmental reduction factors, differentiate between the performance of different types of EB FRP based on the fiber material and manufacturing procedure by implementing the material safety factors. It is believed that these factors account for variations in durability between different types of EB FRP. In that regard, ISIS is a bit more explicit in that it provides a different set of material safety factors for buildings and bridges. The maximum usable strain in EB FRP in flexural design is limited to 0.006 and 0.008 in ISIS and TR55, respectively.Tg) determined per ASTM E1640, and characteristic strain determined per ASTM D3039 [AASHTO FRPS-1 guide specification does not propose explicit environmental reduction factors. However, it is specified that EB FRP composite shall retain at least 85% of glass transition temperature (TM D3039  followinMechanical anchorage of EB FRP can increase the effective usable strain in the composites in bond-critical applications ,139. EffEB FRP/concrete bond durability is not explicitly addressed by the existing design guidelines even though it may be the governing factor in long-term durability of EB FRP systems . There hA particularly popular test is a notched beam three-point bending test . The tesInformed by the research findings, ACI 440.9R also specifies a standard accelerated conditioning protocol for notched beams with EB FRP consisting of 3000-hour conditioning by water immersion at 50 ± 3 °C. The durability of the bond is quantified via bond strength retention (As the existing infrastructure is aging worldwide, there is an immense need to develop and implement novel strengthening methods to prolong the service life thereof. EB FRP composites represent one of the most economical solutions. However, the long-term durability of EB FRP in harsh environments may limit the efficacy of these systems. As it was demonstrated in this article, there are multiple competing degradation mechanisms in the epoxy resins, fibers, fiber–epoxy interface, and epoxy–concrete interface that can affect the performance of EB FRP. Recent research on nanomodified resins demonstrates potential to develop improved materials capable of withstanding harsh environmental and loading conditions.While design guidelines consider the durability of EB FRP an important factor, there is quite a lot of variability in how the durability concerns are addressed. Some design guidelines provide explicit environmental reduction factors, while in others either material’s qualification criteria are proposed, or durability is accounted for through material safety factors. Probably the most inconsistent between the guidelines is the maximum usable strain in FRP for flexural EB FRP design—some guidelines propose specific strain limits while others offer debonding strain equations that compute maximum usable strain in EB FRP based on the substrate and composite properties.The complexity of the involved deterioration mechanisms limits our ability to mechanistically model the EB FRP deterioration under realistic environmental exposures and develop accurate service life prediction models from the short-term accelerated conditioning test data. This is one of the top research priorities. Progress in EB FRP service life estimation will lead to refined durability design guidelines that will allow for economical yet safe EB FRP strengthening.Interestingly, even though the durability of the bond between EB FRP and concrete is a critical factor, explicit treatment of EB FRP/concrete bond durability is not suggested by any of the available design guidelines. It is, thus, imperative that these concerns be addressed either through qualification testing requirements, bond durability design factors, or both.Advancements in materials for EB FRP applications are necessary to meet the performance requirements imposed on EB FRP strengthening systems in harsh environments. Future progress in composites for construction likely lies at an intersection between classical structural engineering and materials science. With the emerging materials and technologies, an interdisciplinary approach to addressing the problems in the next-generation infrastructure will be paramount.Based on the review of relevant research, the authors offer the following insights:


Breakthroughs in anti-tumor immunity have led to unprecedented advances in immunotherapy, yet it is now clear that the tumor microenvironment (TME) restrains immunity. T cells must substantially increase nutrient uptake to mount a proper immune response and failure to obtain sufficient nutrients or engage the appropriate metabolic pathways can alter or prevent effector T cell differentiation and function. The TME, however, can be metabolically hostile due to insufficient vascular exchange and cancer cell metabolism that leads to hypoxia, depletion of nutrients, and accumulation of waste products. Further, inhibitory receptors present in the TME can inhibit T cell metabolism and alter T cell signaling both directly and through release of extracellular vesicles such as exosomes. This review will discuss the metabolic changes that drive T cells into different stages of their development and how the TME imposes barriers to the metabolism and activity of tumor infiltrating lymphocytes. Hanahan and Weinberg’s seminal paper ‘The Hallmarks of Cancer’ was revised in 2011 to include deregulating cellular energetics and evasion of immune destruction . Tumors Expanded understanding of the basic biology of T cell activation has enabled immunotherapy to combat cancer, and T cell metabolism now offers the opportunity to optimize and improve these therapeutic strategies. Two of the primary immunotherapies are immune checkpoint blockade (ICB) and adoptive cell transfer (ACT). ICB is based on the use of antibodies to neutralize inhibitory immune receptors such as CTLA-4 or PD-1 to reinvigorate T cells . In contThere is a growing appreciation that distinct metabolic programs drive different developmental stages of a T cell throughout its lifespan . After l+ T cell metabolism in vivo versus in vitro by tracing glucose metabolism with 13C-glucose IV infusions in a listeria monocytogenes infection mouse model  in cancer cells catalyzes oxidative catabolism of tryptophan, thus dampening antitumor immune responses by depleting this essential amino acid and producing kyneurenine, which generates immunosuppressive regulatory T cells (Tregs) . These Tfunction . Tumor-afunction . In contfunction . One posfunction . In addifunction . Overall2, and changes in physical characteristics of the TME such as pH, there are other discrete transferable factors that may also influence immune cell metabolism and function. Extracellular vesicles (EVs) encompass a diverse set of membrane vesicles secreted by most, if not all, cell types -T cells or other cells through in vitro manipulation prior to cell transfer may overcome barriers of unintended direct effects on the tumor cells. The potential side effects of metabolic modulatory drugs have also not been fully explored and must be considered as normal tissues may be affected. Nevertheless, metabolic inhibitors have had fewer toxicities than expected, possibly due to the lower metabolic activity and high degree of metabolic flexibility of most tissues. Key remaining questions include: Can we translate findings from experimental models to humans? How do we keep T cells alive long enough to form memory cells to reject future cancer cells? How do we balance activating anti-tumor T cells with autoimmune side effects or inadvertently enhancing cancer cell growth? Understanding the T cell metabolic program and how it underlies function and dysfunction represents a promising venue that can be exploited to improve immunotherapy efficacy.The authors declare no direct conflict of interest with the contents of this manuscript. JCR has hold stock equity in Sitryx and within the past two years has received unrelated research support, travel, and honorarium from Incyte, Sitryx, Caribou, Kadmon, Calithera, Tempest, Merck, and Pfizer. Within the past two years, WKR has received unrelated clinical research support Bristol-Meyers Squib, Merck, Pfizer, Peloton, Calithera, and Incyte. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.


Malignant tumor is a largely harmful disease worldwide. The cure rate of malignant tumors increases with the continuous discovery of anti-tumor drugs and the optimisation of chemotherapy options. However, drug resistance of tumor cells remains a massive obstacle in the treatment of anti-tumor drugs. The heterogeneity of malignant tumors makes studying it further difficult for us. In recent years, using single-cell sequencing technology to study and analyse circulating tumor cells can avoid the interference of tumor heterogeneity and provide a new perspective for us to understand tumor drug resistance. Malignant tumor is a common disease whose incidence increases yearly. It has become a considerable threat to human health. In recent years, the five-year survival rate of patients has substantially improved with the widespread use of anti-tumor drugs. However, the vast majority of tumors have drug resistance, which is a large barrier to treatment . HeterogScientists currently believe that two mechanisms lead to drug resistance in tumors: inherent drug resistance and acquired drug resistance Fig. . InherenImportant information such as mutation status, epigenetic status and related protein expression levels of tumor cells may be expressed only in few cells or even in a single cell . HeterogAs mentioned earlier, the heterogeneity of tumor cells, especially the transcriptome information, including time and space limitation, is likely to change constantly. Hence, studying the heterogeneity of tumor cells dynamically can better explore the problem of tumor drug resistance . The maiIn recent years, scientists studied CTC through single-cell sequencing technology and discovered new mechanisms of drug resistance of tumor cells. Miyamoto et al.  isolatedAnaplastic lymphoma kinase (ALK) gene, first discovered in a subtype of anaplastic large cell lymphoma, is responsible for encoding a receptor tyrosine kinase (RTK) called ALK. The common pathogenic mutation in the ALK gene is gene rearrangement, which can lead to tumors. ALK gene fusion often occurs in a subset of patients with non-small-cell lung cancer (NSCLC); thus, ALK inhibitors are useful in the treatment of such patients. However, such patients will inevitably become resistant to ALK inhibitors, leading to treatment failure. Pailler et al.  studied According to Miyamoto’s sequencing results, Schissler  studied Most breast cancers express estrogen receptor (ER); thus, targeted therapy has become the first choice for the treatment of ER-positive breast cancer . SelectiResearchers  found thMany studies fully demonstrated that using single-cell sequencing technology to analyse CTC can better study the mechanism of tumor drug resistance.Scientists currently believe that drug resistance of tumor cells is a complex biological behaviour with continuous evolution and dynamic change in tumor development. In this process, multiple factors affect the drug resistance of tumor cells with dynamic changes over time. The use of single-cell sequencing to monitor the abnormal signal pathway of CTC can predict drug resistance and realise individualisation and precise treatment.Malignant tumor cells involve a series of genomic changes, including copy number variations (CNVs), single nucleotide variations (SNVs) and insertions/deletions (indels). In lung cancer, the timely detection of mutations that lead to drug resistance is essential for selecting the appropriate treatment for patients. Lung cancer with EGFR mutation is initially well treated with tyrosine kinase inhibitor , but druFor small-cell lung cancer, no convincing treatment plan other than chemotherapy currently exists. However, its drug resistance also leads to the failure of many treatments, and several patients even develop drug resistance after the first use of chemotherapy drugs. Clinically, patients with small-cell lung cancer can be divided into a chemotherapy-resistant group and a chemotherapy-sensitive group according to the response within 3 months after initial chemotherapy. Tumor cell adhesion is poor in small-cell lung cancer. The expression characteristics of CTC may be higher than those of other cancer types. Su et al.  performeSingle-cell genome sequencing can provide genetic heterogeneity and cell lineage information, whereas single-cell transcriptome sequencing can more dynamically represent the sum of all RNAs produced by a species or a specific cell under a functional state, which can better define the current table of cells type. Cheng et al.  used hydAs mentioned previously, tumor heterogeneity is an important theoretical basis for drug resistance. Tumor heterogeneity is not only affected by tumor cells themselves but also closely related to infiltrating immune cells , endotheThe single-cell sequencing of CTC is a burgeoning technology, but it is not yet fully mature and has several flaws. For example, fewer nucleotide feedstocks may be more likely to cause biological noise than conventional sequencing. The latest linear amplification via transposon insertion method  can addrIn addition, the bioinformatics and computational methods of single-cell sequencing remain unable to match the corresponding data fully, and the new single-cell sequencing data make the existing analysis tools appear impractical. At present, single-cell sequencing technology has also produced unprecedented data types. For example, two new sequencing algorithms , 70, whiAlthough the single-cell sequencing of CTC remains immature, this cross-era technology has its natural advantages , and we The single-cell sequencing of CTC contributes to the study of genetic heterogeneity and drug resistance of tumor cells. The single-cell sequencing of CTC certainly provides more powerful tools to understand many of the remaining mysteries of cancer cell resistance.


Cytomegalovirus retinitis (CMVR) is a severe, vision-threatening disease that primarily affects immunosuppressed patients. CMVR is the most common ocular opportunistic infection in human immunodeficiency virus (HIV) infected patients and is the leading cause of blindness in this group; however, the incidence of CMVR in HIV patients has dramatically decreased with antiretroviral therapy. Other causes of immunosuppression, including organ transplantation, hematologic malignancies, and iatrogenic immunosuppression, can also lead to the development of CMVR. Herein, we describe the pathogenesis of CMVR and compare clinical features, epidemiology, and risk factors in HIV and non-HIV infected individuals with CMVR.  Cytomegalovirus (CMV) is a member of the Herpesviridae family ,2. CMV pIn this narrative review, the pathogenesis, ocular manifestations, treatment and complications of CMVR in HIV and non-HIV infected individuals are presented. There is an estimated global CMV seroprevalence of 83% in the general population . CMV infAlthough CMVR in humans has been extensively studied through clinical studies, the lack of an animal model due to the species specificity of CMV hindered in vivo research; especially those that mimic HIV induced CMVR, for a long period of time . The useThrough sub-retinally injected MCMV in MAIDS mice, one could not only observe the progression of CMVR disease but could analyze for the presence and absence thereof for various cytokines, immune cell infiltration, immune-activation and antigen presentation, and mode of cell death during active and latent infection. One of the most important findings from these studies includes the upregulation of suppressor of cytokine signaling (SOCS) proteins SOCS1 and SOCS3 ,17. SOCSIn the general adult population, the transmission of CMV through bodily fluids is uncommon given the fact that the number of virus-shedding seropositive individuals is very low . Like maIn immunocompetent individuals, CMV remains asymptomatic but has the potential to reactivate when the immune system is compromised . In immuWhile only 1% of newborns in the United States are infected with CMV, up to 5%–10% of these patients demonstrate signs and symptoms at birth and 1%–2% suffer from severe complications that may lead to death ,27,28. TCMV is the largest Herpesviridae virus with double-stranded deoxyribonucleic acid (DNA) of 235 kbp . The virIt is known that CMV establishes latency in blood and bone marrow of infected individuals, although the virus may not be isolated from host blood . Viral rActive CMV in the eye primarily infects vascular endothelial cells followed by retinal pigment epithelial cells in the retina causing viral cytopathic effect and subsequent retinal necrosis, as characterized in CMVR . The lar3 or by the presence of an AIDS-defining illness, regardless of CD4 cell levels [HIV causes progressive immune system failure once contracted. It is characterized by a single strand enveloped ribonucleic acid virus that, via reverse transcriptase, is converted to double stranded DNA and ultimately incorporates itself into the host genome, propagates and leads to T-cell depletion ,47. AIDSl levels ,46,47. Al levels ,46,47.CMVR is the most common ocular opportunistic infection and the leading cause of blindness associated with AIDS worldwide ,50,51,52In addition to a low CD4+ cell count, individuals with low CD8+ cell counts are also at high risk for CMVR development and independently, CD8+ cells are predictive for CMVR development . SpecifiHIV-related retinopathy is an ocular risk factor for CMVR development. HIV retinopathy manifestations include cotton-wool spots (CWS), intraretinal hemorrhages, microaneurysms and exudates. Like CMVR, HIV retinopathy is also associated with lower CD4+ cell count and higher plasma HIV-RNA levels but is a non-infectious microangiopathy . CWS in Non-HIV-related CMVR is less common than HIV-associated CMVR. CMVR in non-HIV cases have been observed in patients undergoing immunosuppressive therapies for hematologic malignancies, autoimmune disease, bone marrow and solid organ transplantation, and after local steroid administration . The preThe most common non-HIV form of immunosuppression associated with CMVR occurs in the setting of organ transplantation ,60,61,62CMVR has been associated with hematologic malignancies due to the immunocompromised status caused by the pathogenesis of the disease itself. In a report of three patients with chronic lymphocytic leukemia who developed CMVR, two patients had decreased CD4 counts . LeukemiCMVR following periocular or intraocular steroid injection has been documented in immunocompetent individuals. CMVR following sub-Tenon triamcinolone injection, intravitreal dexamethasone implant, and fluocinolone acetonide implant in immunocompetent individuals on no other immunosuppression have been reported ,74,75,76Case reports of CMVR developing following systemic immunosuppression with corticosteroids and steroid-sparing agents have also been reported ,78,79,80It is now known that genetic factors play a role in the susceptibility for CMV infection, CMVR progression and HIV mortality. Host genetic factors such as specific haplotypes of interleukin 10 receptor (IL-10R1), chemokine receptor 5 (CCR5), and stromal derived factor 1 (SDF-1) have been evaluated ,84.The CMV genome contains a human IL10R1 homologue that CMV uses to evade the human immune system. Sezgin et al. found that an amino acid changing variation in the cytoplasmic domain of IL-10R1 is protective against CMVR; however, the haplotype carrying an amino acid changing variation in the extracellular domain increases susceptibility to CMVR. There were variations in ethnicity, and this was observed in European-Americans but not African-American patients .CCR5 is a receptor involved in HIV cellular invasion. In another study, Sezgin et al. again found variations based on ethnicity. It was observed that European-American CMVR patients with the CCR5+.P1+ promoter haplotype had increased mortality compared to those without this haplotype; however, this haplotype did not have a significant effect on CMVR progression or retinal detachment occurrence in this population. African-American patients with the CCR5+.P1+ promoter haplotype had increased risk for CMVR progression and demonstrated a trend towards greater mortality risk . IncreasAlthough a number of candidate gene studies have been performed to determine other genetic polymorphisms that may increase susceptibility to CMV infection, most of the studies were limited by small sample size and have not yet been supported by genome wide association studies. CMV is the most common intrauterine viral infection affecting between 0.5–2% of all live births . While CThe differences observed in CMVR in children compared to adults may be related to the fact that children have primary CMV infection rather than reactivation of latent infection. Children′s immune systems are also immature therefore the systemic response to CMV may be more severe .In more than half of infected patients, CMVR is asymptomatic and is revealed during routine ophthalmic examination ,89. WhenThere are three main clinical variants of CMVR . The firCMVR is more commonly seen in HIV patients, likely due to the degree of immune suppression required for manifestation and there are few studies that directly compare HIV and non-HIV CMVR patients ,91. WhenIt has also been observed that vitreous inflammation, arteritis, and vascular occlusion outside of the areas of active retinitis can be more severe in non-HIV patients and the degree may vary depending on the level of immune suppression in non-HIV immunosuppressed patients ,54,91,93While CMV predominantly affects the posterior segment, additional clinical findings in immune-deficient patients with CMVR include diffuse, stellate corneal endothelial deposits that can be detected on slit-lamp examination. In a post-mortem evaluation in these patients, it was found that these deposits are composed of macrophages and fibrin and are devoid of lymphocytes . The preFollowing institution of antiretroviral therapy, increased immunocompetency may cause an inflammatory response to CMV antigens. Immune reconstitution inflammatory syndrome (IRIS) is a phenomenon where increased inflammation is observed in HIV patients who are on ART during the initial weeks of treatment. This exaggerated reactivation of the immune system can lead to worsening reaction to CMV ,51,98. IToxoplasma gondii [The diagnosis of CMVR is primarily clinical and based on the observation of characteristic retinitis on dilated funduscopic examination by a trained ophthalmologist. Polymerase chain reaction (PCR) analysis of aqueous or vitreous ocular fluid can further confirm the diagnosis and may be helpful in cases with atypical clinical presentations. It can help differentiate CMVR from retinitis caused by other members of the Herpesvirus family, such as varicella zoster or herpes simplex virus, or from other pathogens such as a gondii ,103.Isolation of CMV from blood leukocytes can provide insight into active shedding of the virus; however, this test is not sensitive and may require multiple weeks to become positive. Patients who are immunosuppressed may not develop detectable titer levels ,103. SerAnother study suggested ophthalmic screening in HIV patients with CMV present in urinalysis or “CMVuria”; CMVuria as a single finding, however, does not justify antiviral prophylaxis against CMVR . CMV antIn both HIV and non-HIV patients, high dose induction antiviral therapy is initiated when active CMVR is diagnosed. Induction therapy is typically administered for 14 to 21 days but the final duration is determined by the clinical response to therapy. Induction is followed by continuous maintenance therapy until CD4 count increase in HIV patients is observed, when ART is therapeutic, and/or when CMVR shows no progression . In HIV CMVR management involves intravenous (IV), oral, and intravitreal injections (IVI) of antiviral medications. The location of the CMVR lesions largely dictates the treatment algorithm. For patients with immediate sight-threatening lesions, intravitreal injections in conjunction with systemic therapy are currently recommended. For patients without immediately sight-threatening lesions, systemic therapy alone with close observation is reasonable. The main virostatic drugs employed today include valganciclovir , ganciclovir , foscarnet , and Cidofovir . Acyclovir is not used in the treatment of CMV as this drug specifically requires phosphorylation by viruses to become active, a mechanism which CMV is not capable of as it does not encode for virus-specific thymidine kinase . CompariGanciclovir was the first antiviral agent approved for the treatment of CMV . The priIV ganciclovir or foscarnet are effective options in individuals who are unable to tolerate oral therapy . FoscarnCidofovir is a monophosphate nucleotide analogue. In the body, cidofovir becomes phosphorylated by intracellular kinases and competitively inhibits the addition of deoxycytidine triphosphate into viral DNA via DNA polymerase . Due to IVI of virostatic medications can be considered for short-term management or as adjunctive therapy but sole intravitreal therapy is not commonly recommended except in circumstances where systemic therapy is contraindicated . Local mIn recalcitrant cases or relapsed infection, combination therapy is recommended. It has been observed that patients treated with both ganciclovir and foscarnet had a longer life expectancy than those given either drug alone. No additional toxicity was observed with combination of foscarnet and ganciclovir in patients with severe CMV disease ,113.Letermovir is an antiviral drug that, as of 2017, has been approved for prophylaxis in CMV-seropositive allogeneic hematopoietic-cell transplantation recipients. Letermovir inhibits the CMV–terminase complex and in the phase 3 randomized, double-blind, placebo-controlled, superiority trial, letermovir prophylaxis resulted in a significantly lower risk of clinically significant CMV infection compared to placebo . ValgancThere are a number of patents for new drugs evaluating gene regulation and molecular biology. Medications targeting ribonucleic acid (RNA) and blocking translation of RNA into proteins required for replication of CMV are being evaluated as are other therapies targeting various aspects necessary for CMV survival and replication .The epidemiologic features, incidence and prevalence of CMVR in HIV patients have changed greatly with the advent of anti-retroviral therapy ,56,108. Resistance to antiviral therapy for CMV can occur in patients receiving prolonged therapy with ganciclovir and valganciclovir. The duration of medication use, ongoing viral replication and degree of host defense influence drug resistance . In AIDSCMV virological factors influence drug resistance development. CMV latency with later reactivation allows for prolonged replication that can yield many CMV variants resulting in resistant strains . Drug rePatients with a detectable CMV viral load in the blood or ocular fluid can undergo genotype testing for drug resistant CMV via PCR. Real-time PCR assays can identify drug resistance mutations in UL97 by analyzing changes in melting curves, following amplification . The advNew advancements in treating ganciclovir-resistant CMV are developing, including leflunomide and T-lymphocyte infusion. Leflunomide, an immunosuppressive therapy for rheumatoid arthritis, has been used as an off-label treatment for drug resistant CMV ,124,125.T-lymphocyte infusion has also been successfully used in resistant cases. In a case report of CMV viremia in a patient with acute lymphoblastic leukemia, status post allogeneic stem cell transplant and on immunosuppressive therapy for graft-versus-host disease, the patient was originally treated with ganciclovir. Due to a confirmed CMV UL54 mutation resistant to foscarnet, cidofovir, and ganciclovir, the patient was switched to leflunomide . The patPrior to the development of ART, CMVR in an HIV patient required life-long anti-CMV therapy . Many reIn non-HIV and HIV patients on anti-CMV drug treatment, resolution of the retinal opacity with resultant atrophy is considered a reliable sign of CMV inactivity ,53. One CMVR may cause debilitating, typically permanent vision loss, which is largely preventable by routine monitoring in immunosuppressed patients and ART initiation in HIV patients. Independent risk factors may aid in predicting the final visual outcome of CMVR and include extent of retina infected with CMV, better presenting visual acuity and absence of macular involvement. Poor prognostic factors include the increased size and activity of lesions, lack of ART in HIV patients during infection onset and lack of ART-associated immune reconstitution ,130.The overall visual prognosis and clinical features of CMVR have not been found to differ between HIV and non-HIV patients ,6. DespiCMV retinitis is one of the most serious ocular complications in immune-suppressed individuals and can lead to irreversible vision loss. CMVR can be prevented with ART optimization early in the course of HIV disease; however, with the increased life expectancy of HIV patients, CMVR is still a problem today. With every increasing indication for immune suppression, CMVR is also presenting more frequently in non-HIV immune suppressed individuals. It is exceedingly rare to observe CMVR in healthy patients unless it is secondary to local ocular steroid treatment ,74,75,76There are many overlapping characteristics of CMVR in HIV patients and non-HIV immune suppressed patients. The retinal manifestations are similar; however, non-HIV immune suppressed patients tend to have a more robust inflammatory response in the other segments of the eye . Treatme


Mitochondrion is a pivotal intracellular organelle that plays crucial roles in regulation of energy production, oxidative stress, calcium homeostasis, and apoptosis. Kidney stone disease (nephrolithiasis/urolithiasis), particularly calcium oxalate , has been shown to be associated with oxidative stress and tissue inflammation/injury. Recent evidence has demonstrated the involvement of mitochondrial dysfunction in CaOx crystal retention and aggregation as well as Randall’s plaque formation, all of which are the essential mechanisms for kidney stone formation. This review highlights the important roles of mitochondria in renal cell functions and provides the data obtained from previous investigations of mitochondria related to kidney stone disease. In addition, mechanisms for the involvement of mitochondrial dysfunction in the pathophysiology of kidney stone disease are summarized. Finally, future perspectives on the novel approach to prevent kidney stone formation by mitochondrial preservation are discussed. Tricarboxylic acid (TCA) cycle (Kreb’s cycle or citric acid cycle) and electron transport chain (ETC) are the two major metabolic processes that play pivotal roles in this energy production mechanism  is circular double-stranded DNA that is not protected by histones . Generalechanism . Additioechanism . Mitochoechanism , obesityechanism , aging , from acetate in the form or acetyl coenzyme A (acetyl CoA), which is converted from pyruvate via pyruvate dehydrogenase after glycolysis of one molecule of glucose (2 molecules compared to glycolysis (2 are transferred to NADH dehydrogenase (complex I) and succinate dehydrogenase (complex II), respectively, of ETC in the MIM. The electrons were then shuttled to ubiquinol-cytochrome c reductase (complex III) by ubiquinone  and transferred to cytochrome c oxidase  by cytochrome c  oduction . The TCA glucose . In addiycolysis . Electrochrome c . During chrome c . The req2−) and hydrogen peroxide (H2O2). These ROS are important for cellular signaling pathways as well as cell cycles  emission . Electrol cycles , apoptosl cycles , proteinl cycles . Under pl cycles . For exaoduction . Subsequto water . During to water . As a reto water .In oxidative stress condition, the nuclear factor erythroid 2-related factor 2 (NRF2) can trigger transcription factors of genes encoding antioxidant enzymes to cope with the ROS overproduction . This unvia mitochondria) and extrinsic (via death receptors) pathways  and ATP, resulting in recruitment of procaspase-9 to form an apoptosome, in which procaspase-9 is cleaved to caspase-9. The caspase-9 then converts procaspase-3 to effector caspase-3, resulting in the completion of cellular apoptosis . For thein vitro studies , which is a component of mitochondrial permeability transition pore (mPTP)  . CypD ple (mPTP) . Additioe (mPTP) . On the e (mPTP) . Vice vey stones .Our previous mitochondrial proteome study has highlighted the response of renal tubular epithelial cells of the distal nephron segment to CaOx crystals . Using tMitochondria have also been investigated in Randall’s plaque model of kidney stone pathogenesis. Mitochondrial dysfunction has been found to be related not only to renal tubular injury but also to the impaired immune response and inflammation by decreasing monocytes’ mitochondrial functions in the CaOx stone formers, leading to the decline of crystal elimination that further enhances tissue inflammation . ProgresOxalate and calcium can alter mitochondrial activities, leading to changes in metabolic status that may induce loss or alterations of mitochondrial functions on energy production, ROS regulation, and intracellular calcium homeostasis, all of which affect mitochondrial biogenesis . The dynMitochondrial dysfunction can increase retention of CaOx crystals inside renal tubular lumens of the distal nephron segment. These stagnant crystals subsequently form the stone nidus, which is the central part of the stone generated from crystal aggregates that finally become the macroscopic stone. The loss or defect of mitochondrial energy production can enhance metabolic process in TCA cycle and ETC, resulting in the increase of ROS production and reduction of antioxidant enzymes . ConsequMitochondrial dysfunction causes the release of cytochrome c and induces apoptotic cell death, associated with cell shrinkage, blebbing of plasma membranes, organelle condensation, and fragmentation. Thereafter, apoptotic bodies, cell debris, and fragmented subcellular organelles are released into tubular lumen and then bind with oxalate and CaOx crystals as shown by several studies . Also, fMitochondrial dysfunction can initiate interstitial stone formation . In oxidative stress condition, mitochondrial damage can lead to disruption of mitochondrial membrane integrity followed by the release of ROS, mtDNA, ATP, and fragmented mitochondria to the cytoplasm. These components can then induce secretion of the proinflammatory cytokines and trigger the inflammatory cascades . The proIn summary, mitochondria may be considered as the central intracellular organelles that play pivotal roles in kidney stone pathophysiology. Alterations in their main functions, including energy production and regulation of oxidative stress and intracellular calcium homeostasis, are associated with kidney tissue injury and inflammatory response, leading to CaOx crystal nucleation, growth, aggregation, and deposition that are the key processes for kidney stone formation. Furthermore, mitochondrial fragmented products and proteins can bind directly with CaOx crystals and thus play roles in the stone nidus formation. In addition to the intratubular crystal deposition and nidus formation, mitochondrial dysfunction is also associated with Randall’s plaque formation by enhancing tissue inflammation and interstitial deposition of calcium phosphate (hydroxyapatite).It should be noted that mitochondrial dysfunction alone is not sufficient to induce kidney stone formation, of which mechanisms are multifactorial. For example, mtDNA mutations or defective nuclear-encoded mitochondrial proteins have been extensively studied in several mitochondrial disorders, e.g., Alzheimer’s disease, epilepsy, mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS syndrome), etc. . Neverthvia alterations of mitochondrial membrane activities and death signaling release, respectively. To recover the mitochondrial functions and to reduce oxidative stress, inhibition of CypD activation using cyclosporin A (Based on the aforementioned pathogenic mechanisms, recovering the mitochondrial functions by antioxidants may be the effective approach for prevention of new or recurrent kidney stone formation. Although antioxidant agents have been recommended to preserve mitochondrial activities and to antagonize ROS overproduction in various diseases, their efficacy and adverse events are still ambiguous and need to be further elucidated. Additionally, previous evidence has demonstrated that CypD  and Bcl-sporin A  and N-mesporin A  has beensporin A . This prsporin A . TherefoAlthough the roles for mitochondrial dysfunction related to oxidative stress and CaOx crystal deposition have been well documented, the roles for excessive calcium that is also common in the stone formers  may be oSC and VT drafted the manuscript, read and approved the final manuscript, and are responsible for all aspects of the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.