Intracellular delivery is a critical step in biological discoveries and has been widely utilized in biomedical research. A variety of molecular tools have been developed for cell-based gene therapies, including FDA approved CAR-T immunotherapy, iPSC, cell reprogramming and gene editing. Despite the inspiring results of these applications, intracellular delivery of foreign molecules including nucleic acids and proteins remains challenging. Efficient yet non-invasive delivery of biomolecules in a high-throughput manner has thus long fascinates the scientific community. As one of the most popular non-viral technologies for cell transfection, electroporation has gone through enormous development with the assist of nanotechnology and microfabrication. Emergence of miniatured electroporation system brought up many merits over the weakness of traditional electroporation system, including precise dose control and high cell viability. These new generation of electroporation systems are of considerable importance to expand the biological applications of intracellular delivery, bypassing the potential safety issue of viral vectors. In this review, we will go over the recent progresses in the electroporation-based intracellular delivery and several potential applications of cutting-edge research on the miniatured electroporation, including gene therapy, cellular reprogramming and intracellular probe. Cells are the basic building blocks of all living organisms, while molecules, as the currency of information in cellular exchange, are responsible for the cellular structure and orienting cells toward specific function. Many diseases such as immunodeficiencies , cancersIn the process of intracellular delivery, cargos and approaches for transportation are two key elements. Cargos for delivery are highly variable in size, sources, organization and physicochemical properties. Such diversity of cargo could make great impact on the delivery strategies and intracellular applications. Based on the diverse properties and applications of cargos, current techniques for intracellular delivery can be mainly categorized into two types: carrier-mediated delivery and membrane-penetrating delivery. As illustrated in When comes to the carrier-mediated delivery, biological and chemical strategies are principally covered, which include extracellular vehicles (EVs), viral vectors and organized nanoparticles. Viral vectors  containing packaged gene materials of interest inside can effectively target host cells and release the genetic molecules consequently. While viral transduction can achieve a high efficiency, it can inherently disrupt the immune system and thus consistently raise safety issues in vivo. For the extracellular vesicles, especially exosomes, enthusiastic attention have been concentrated on this biological field because of their important roles in cell-to-cell communication . These vMembrane-disruption modalities are primarily physical methods, which can be classified as direct penetration modalities and permeabilization. Direct penetration employs a solid conduit or vehicle to concurrently penetrate the membrane and introduce cargo. Prevalent samples here are mechanical cell membrane penetration (micro- and nano-injection)  and geneAmong several choices in permeabilization-based disruption delivery, electroporation has already been an established technique in medical field, but many of its biotechnological applications, especially intracellular delivery did not start to emerge until 1980s . Due to This review will mostly focus on the electroporation-based intracellular delivery, especially the combination with novel technologies, extended miniatured electroporation and its potential applications in clinic. We will first cover the brief history and mechanisms of electroporation. These include an overview of development of electroporation and principles of traditional electroporation system, as these characteristics are inextricably linked to the deficiencies and challenges involved in intracellular delivery. We then survey some of the most promising technical improvement in electroporation, especially in miniaturized electroporation. Technical advances will be compared with traditional electroporation system and potential clinical applications will be introduced. The interest of people in applying electricity in human and animal bodies could date back to 1700s, and it was found that electric field caused damage to the tissues presumably due to the irreversible electroporation . SystemaCell membrane is known to be basically composed of a lipid bilayer with thickness about 5 nm, which functions as a barrier to the cellular components from extracellular environment. As an electrical insulator exhibiting excellent dielectric property in normal physiological conditions, it maintains the electric potential (~0.07 V) across the membrane due to significant difference of ion concentration between cytosol and the fluid in extracellular microenvironment. Theoretically, electroporation is an effective strategy to form pores in cell membrane by the application of a potential difference across that membrane. When the potential difference reaches a specific magnitude of voltage, the probability of electroporation taking place on cell membrane drastically increases. In actual, the electric breakdown of cell membrane happens when the transmembrane potential across the lipid bilayer bout 1 V , becauset is the time, and τ is the time constant of the cell membrane “capacitor” (characteristic charging time ~1 μs). Therefore, in the steady-state condition, A variety of factors have been studied to model the transmembrane potential This equation is feasible for the estimation of le cells ,41,42. IElectroporation has been widely applied to deliver a diverse range of cargo molecules and materials of interest into the intracellular space. Conventional electroporation technique for the intracellular is done in cuvette-style parallel plate setups, where the cell suspension and molecules to-be-delivered are mixed together in the conducting buffer solution between two electrodes connected to a generator of high electric voltage, and thus it is called bulk electroporation (BEP). In such a BEP setup , an apprOn the strength of permeabilization triggered by electric filed, bulk electroporation has been used to deliver a diverse range of cargo molecules and materials of interest to the intracellular space. These cargos range from small molecules to proteins and macromolecules, including dyes, impermeable drugs, molecular beacons, proteins, siRNA, DNA plasmid and nanoparticle. As expected, intracellular delivery mediated by bulk electroporation is influenced by not merely the pore diameter, but also the cargo dimensions as well as the charge of the cargo molecule. For small molecules, diffusion across concentration gradient throughout the duration of a pore’s lifetime is the main route . If the Taken together, BEP in cell suspension is mainly determined by the pore diameter and the cargo properties. During BEP, transient electrical pulses are applied to create intense electric field across cell membrane and induce the permeabilization of cell membranes. Meanwhile, the biomolecules are simultaneously delivered inside (or extracted from) the cells. For a portion of cells, because they are inevitably exposed to an extremely high electric field, the membrane disruption become irreversible which gives rise to the failure of membrane repair or prolonged apoptotic responses. Apart from damage to the cell, delivery mechanisms of BEP are diffusion dominated, and for large transfection agents such as nucleic acids, entry into the cytosol is affected through an electrophoretic aggregation and further attachment onto the outside of the cell membrane followed by an endocytosis-like process, which to some extent renders BEP low efficiency in intracellular delivery. Nevertheless, it is worth noting that precise dose control has not been demonstrated using BEP.The early generation of electroporation system, especially BEP, has been widely applied in intracellular delivery because of its technical simplicity, fast delivery and almost no limitation on cell type and size. However, the cuvette bulk style with cargo suspension has its problems. As mentioned above, sharp reduction in cell mortality caused by large uncontrollable membrane disruption greatly impairs the efficiency of intracellular delivery. Moreover, this cuvette style electroporation is difficult to gain command of the precise dose of cargo delivered into target cells, which to some extent hampers the future clinical application of intracellular delivery. With the development of nanotechnology and microfluidic fabrication, electroporation is moving toward the concept of miniaturization, which largely solves the challenges in conventional electroporation. Below we will discuss the innovations that have been developed in the field of miniaturized electroporation, including micro-/nanoelectroporation and microfluidic-based electroporation.Recent years have witnessed a rapid progress on the development of microscale platforms for achieving electroporation at single cell level. The original MEP raised by Kurosawa’s group  aimed toInspired by the deficiencies in early works on micro-scale electroporation, Lee’s group further introduced the concept of nanochannel electroporation . As shownopillar , nanostrnopillar  and nanonopillar . As for As discussed in previous section, novel miniatured electroporation devices are basically micro/nanoscale lab-on-a-chip platforms, which largely relies on nanotechnology, microfluidic and microfabrication techniques. The fabrication of these micro/nanoscale electroporation devices often involves the standard semiconductor cleanroom-based wafer process steps, i.e., lithography, etching, deposition, etc. In many cases, the small features created in silicon master were replicated or transferred to polymer-based elastomeric materials, most notably polydimethylsiloxane (PDMS). Taking the fabrication of high-throughput 3D NEP chip for instance , the nanSince the electric field in electroporation, which accelerates the charged transfection agents and prorates the cell membrane, could diminish exponentially outside nanochannels, it is critical to achieve close contact between the to-be-transfected individual cells and the corresponding nanochannels. A variety of cell manipulation techniques were integrated with miniaturized electroporation system to achieve accurate cell trapping against nanochannel. Optical tweezer (OT) is a precision instrument in which a focused laser beam can manipulate the dielectric microscopic particle by a piconewton-level force. Owing to the conservation of momentum, the refraction of light path across the particle which essentially is equivalent to the change of the momentum of photons, will in return provide a force on the particle. OT has been broadly used in biophysics research for force-extension measurement of biomolecules. As a particle manipulation tool, OT has been proven to be successfully implemented with the first-generation NEP device A and it 2). In such DEP-NEP platform system, an alternating current (AC) field was used for DEP force generation which was uncoupled with AC-induced electrophoresis in NEP process. In another work of Lee and his colleagues [To meet the cell number requirement for clinical applications, the high-throughput electroporation system requires parallel cell manipulation technique. Dielectrophoresis (DEP)-assisted cell loading method was utilized to integrate with miniaturized electroporation platform. Lee’s group  has demolleagues , magnetilleagues , howeverGene therapy is a simple yet revolutionary concept that treating a disease by modifying the problematic gene. Despite its potential of radically curing otherwise highly lethal disease at gene level, intracellular gene delivery strategy is one of the biggest hurdles. Miniaturized electroporation is a competitive physical gene delivery candidate, with the benefits of instantaneous delivery process, well-defined delivery dosage, high transfection efficiency, minimal cell damage and being viral-free, circumventing the inherited safety concerns posted by viral vectors . Below iGenetic engineering of immune cells  is prerequisite for adoptive immunotherapy which harnesses the patient’s own immune system to combat diseases ,64. As sHowever, current technical challenge of such applications lies in the limited number of “trained” immune cells due to low yielding of plasmids transfection using conventional non-viral methods such as traditional BEP and nanocarriers ,67,68,69Specific knockdown of oncogene or proto-oncogene messenger RNA (mRNA) by small non-coding RNA, e.g., small interfering RNA (siRNA) and microRNA (miRNA) has been proven an effective and secure RNAi therapeutics by a set of clinical trials including the first treatment of an RNAi therapeutic targeting VEGF and KSP in liver cancer patient . DeliverManipulating the genome of the living cells by efficiently adding, deleting, or changing the DNA sequence has long fascinated biology community. In recent decades, the concept of “gene editing” has become a research “hot spot”, fueled by the breakthrough in the development of novel editing tools such as engineered nucleases. The CRISPR —Cas9 (CRISPR-associate protein) technology is the most widely-used platform available for gene editing because it has enormous potential to boost the development of molecular therapeutics against a variety of diseases . Electroporation, as a common membrane-disrupted intracellular cargo molecule delivery method, has been reported to be utilized for delivering the CRISPR/Cas9 gene editing materials and thus play a profound role in gene editing ,76,77,78Cell reprogramming can be performed on either readily available somatic cells (such as skin cells) or induced Pluripotent Stem Cells (iPSCs) . Since tLiving cells are complex dynamic systems that constantly interact with both intracellular and extracellular environment. Development and progression of diseases such as cancer always involve disseminating cells undergoing a variety of physically, physiologically, or pharmacologically induced events ,83 that Electroporation can deliver various range of molecular cargos into a wide variety of cell types with the precise control of pulse parameters. In this review, recent advance in electroporation-based intracellular drug delivery technology is presented with potential applications in clinic. Miniaturized electroporation, as a promising non-viral transfection method, starts to play an ever-increasingly important role in a variety of fundamental scientific research and translational clinical trials. Spatial confinement in micro-/nanoscale electroporation provides numerous benefits over conventional bulk electroporation, including rapid cargo uptake, precision dosage control and minimum cell disturbance.Although the new generation of electroporation platforms exhibited unprecedented transfection capabilities, a large number of challenges remains to be solved. Current miniaturized electroporation still cannot transfect large enough cell population comparable to the conventional delivery methods. In other side, the transfected cells require accurate targeting to reach the disease sites in vivo for nucleic acid therapy with minimal side effects. Special expertise in micro-fabrication and instrumental operation is indispensable for the following applications. It also cannot be ignored that micro/nanoscale of the miniaturized electroporation system leads to high demand of robust fabrication protocols, which is a great challenge to biomanufacture. In view of these problems, such novel strategies have not yet been demonstrated to supersede the basic cuvette-style electroporation in clinical use.The challenges of current electroporation systems notwithstanding, for many applications the merits outweigh the weaknesses. Instrumental progress in electroporation, together with newly-developed molecular tools, will certainly boost biomedical research from biomanufacture and clinical trials of cancer immunotherapy to ex vivo cell-based gene therapy and regenerative medicine.


Zika virus (ZIKV) infection was historically considered a disease with mild symptoms and no major consequences to human health. However, several long-term, late onset, and chronic neurological complications, both in congenitally-exposed babies and in adult patients, have been reported after ZIKV infection, especially after the 2015 epidemics in the American continent. The development or severity of these conditions cannot be fully predicted, but it is possible that genetic, epigenetic, and environmental factors may contribute to determine ZIKV infection outcomes. This reinforces the importance that individuals exposed to ZIKV are submitted to long-term clinical surveillance and highlights the urgent need for the development of therapeutic approaches to reduce or eliminate the neurological burden of infection. Here, we review the epidemiology of ZIKV-associated neurological complications and the role of factors that may influence disease outcome. Moreover, we discuss experimental and clinical evidence of drugs that have shown promising results in vitro or in vitro against viral replication and and/or ZIKV-induced neurotoxicity. Zika virus (ZIKV) has been known since 1947 . FollowiA spectrum of neuropathological conditions has been reported in newborn babies exposed to ZIKV in uterus, being microcephaly the most severe outcome. Infants who develop ZIKV-induced microcephaly show recurrent seizures , and sevExtensive research has focused on unraveling the mechanisms of congenital microcephaly induced by ZIKV ,7,8. HowNeurological complications following ZIKV infection are not limited to cases of congenital infection . MountinAnother matter of concern involves the ability of ZIKV to persist in certain organs and tissues, since the late consequences of viral persistence remain unknown. ZIKV usually reaches detectable levels in the serum of patients within 3–10 days after onset of symptoms, shortly returning to undetectable levels . Even afLittle is known about what factors can contribute to the development of late neurological damage associated to ZIKV infection, and what are the precise mechanisms involved. It is likely that several genetic and environmental factors interplay to increase one’s susceptibility to the development of neurological outcomes . A comprZIKV strains isolated from different geographical regions are derived from two main lineages: African and Southeast Asian . Even thMutations in ZIKV genome may also promote host immune system evasion and enhance infectivity of mosquito hosts ,38, whicVariations on the hosts’ genetic background are determinant to an appropriate immune response, and may thus influence the occurrence of ZIKV-associated neurological complications. Experiments in animal models support this notion, since infection is associated to either mild or severe signs of disease, depending on the genetic characteristics of the host. For example, effective systemic infection in mice requires a limited interferon-mediated immune response, which is obtained either by knocking out specific components of interferon (IFN) pathway ,46,47 orIn line with this, individual responses determined by either genetic or epigenetic backgrounds have been shown to modulate susceptibility of cells in the developing brain to ZIKV. In a recent study, neural stem cells (NSCs) derived from three independent donors were infected in vitro with ZIKV (Mexico isolate). The virus was shown to replicate and induce decreased cell proliferation in all NSCs strains. However, NSCs from one of these donors were resistant to ZIKV-induced decrease in neuronal differentiation. RNA expression patterns changed accordingly: ZIKV-sensitive strains showed pronounced reduction in pathways related to neurogenesis and higher expression in pathways responsible for innate immune response . In anotConcerning the development of CZS, it can also be speculated that a pregnant woman’s individual response to infection influences the efficiency with which ZIKV crosses the placental barrier. Trophoblasts, placental barrier cells, are permissive to ZIKV only during a narrow time-frame, likely in the first trimester of gestation ,68,69,70Despite factors specific to host and viral genomes, several other factors can have contributed to the unforeseen scale of ZIKV neurological damage seen in recent years. An interesting study in immunodeficient mice has suggested that sexual transmission of ZIKV favors congenital infection and the development of malformations of fetuses, suggesting that the route of infection may also play a role in the severity of congenital ZIKV syndrome .As recently reviewed by Barbeito-Andrés and colleagues , environSimilar to other mosquito-borne diseases, clinical management of patients with ZIKV infection is largely based on alleviating symptoms of the acute phase of infection. The American Center for Disease Control and Prevention (CDC) and the World Health Organization recommend generic measures such as resting, drinking fluids, and using over-the-counter medication to alleviate pain and fever ,78. HoweEarly reports using in silico and in vitro approaches suggested the potential of sofosbuvir (SFB), an antiviral prodrug clinically used against hepatitis C virus (HCV), to inhibit ZIKV replication ,101. LatAnother RNA polymerase inhibitor that has been proposed as ZIKV therapeutic is favipiravir . Early reports indicated its ability to inhibit seven different ZIKV strains in preliminary tests in vitro ,89. ConfRetallack and colleagues used a drug repurposing strategy to identify the macrolide antibiotic azithromycin (AZT) as a potential anti-ZIKV drug. AZT was found to be the most promising drug, out of 2177 clinically-approved compounds, in preventing infection of the glial cell lineage U87 by rescuing cell viability . In anot−/− mice) infected with the Brazilian ZIKV strain: daily oral treatment with 30 mg/kg of ME from days 3 to 6 post-infection prevented ZIKV-induced increase in intraocular pressure, ameliorated brain histopathological lesions and led to decreased microgliosis [Excessive glutamate release from synaptic terminals causes an overactivation of the nearby neuronal network leading to neuronal damage and death. Such phenomenon, named excitotoxicity, is a common feature of several neurological diseases . Consideogliosis . ME is aogliosis . FurtherConsidering the extensive pro-inflammatory profile of brain tissue during and after ZIKV infection, anti-inflammatory therapy might be of help to alleviate further detrimental consequences. Our group has recently evaluated whether the TNF-α neutralizing antibody infliximab (IFX) (20 μg/day) is able to prevent long-term consequences of ZIKV infection in neonatal  Swiss mice . IFX tre50 concentrations against ZIKV replication in culture. These findings open new avenues for drug design and in silico optimization. However, because viruses do not present cyclin-dependent kinases, it is possible that the effect of such drugs rely on the regulation of the host cell differentiation machinery. Therefore, such compounds might not be indicated for pregnant women as fetus development might be affected [Xu and colleagues were one of the first groups to make a large drug repurposing screen for ZIKV, obtaining exciting results . One of affected .Emricasan is a pan-caspase inhibitor which was shown to increase viability of human astrocytes and hNPCs infected with several different ZIKV strains . This stThe screening performed by Xu and colleagues also identified the anti-helmintic drug niclosamide as a powerful antiviral drug against ZIKV, being able to mitigate infection in a human glioblastoma cell line (SNB-19) and in human astrocytes . In another study, two doses of niclosamide  were tested in a chick embryo model of ZIKV-induced microcephaly. Niclosamide was able to partially rescue ZIKV-induced reductions in cranial size and significantly improved brain inflammatory profile . NiclosaChloroquine (CQ) is a classically used antimalarial drug. Its antiviral activity against dengue virus and other flaviviruses has already been demonstrated ,129 and 50 within the micromolar range, indicating that AQ is also an interesting scaffold molecule for drug development . AQ has been used for treatment of malaria in pediatric settings, but serious adverse effects were shown after its long-term use for prophylaxis [Another interesting antimalarial quinine with anti-ZIKV activity is amodiaquine (AQ). A high-content screening study identified its ability to prevent ZIKV infection and its toxicity in human pluripotent stem cell-derived neural progenitors . Moreovephylaxis . TherefoAdenosine analogs share the same mechanism of action which consists of impairing the function of viral RNA polymerase of several viruses such as measles and rabies . One speAnother drug with similar mechanism of action is BCX4430 , which showed strong antiviral activity against yellow fever and several others viruses in previous studies, and was recently tested against ZIKV . It was Ribavirin (RIB) is a nucleoside analogue used for the treatment of hepatitis C. In an early report, RIB was able to inhibit replication of several different ZIKV strains in vitro . Such reZ2 is a synthetic peptide derived from the stem region of ZIKV envelope protein which showed potent anti-ZIKV activity. Z2 was able to inhibit infection and diminish cytotoxic effects of ZIKV on Vero and BHK21 cells. In pregnant ICR mice, Z2 was able to penetrate the placental barrier and cause no visible birth defects or perinatal damage in doses up to 120 mg/kg. It also blocked vertical transmission of ZIKV in this same mouse model at 10 mg/kg i.v. In both A129 and AG6 mouse models, Z2-treated mice showed decreased ZIKV-induced mortality and smaller viremia . Given tThe unprecedented high number of neurological symptoms associated to ZIKV infection after its landing in the Americas in 2015 has left severe economic and social consequences that cannot be fully estimated. The observation that ZIKV infection leads to late neurological burden both in congenitally-exposed infants and in adult patients has stimulated the international scientific community enormously. Researchers of different fields worldwide have gathered efforts in order to identify the mechanisms underlying ZIKV neurotoxicity and to search for drug candidates that can halt infection and neurological complications. In this sense, drug repurposing becomes an attractive alternative because several steps of drug approval by regulatory agencies can be surpassed. Drugs of different classes and mechanisms of action, including RNA polymerase inhibitors and several antimalarial quinines have been tested against ZIKV replication and its neurological damage. However, despite the promising in vitro and in vitro results, none of them has reached clinical trials for ZIKV infection yet. In silico drug design should also be employed to generate analogs with improved efficacy and pharmacokinetics.In addition, further efforts should focus on investigating whether genetic and/or environmental factors contribute to neurological damage associated to ZIKV. Finding answers to this question will undoubtedly help defining public health strategies to prevent the occurrence of new epidemics with such devastating effects. In conclusion, the little available knowledge of factors that might contribute to ZIKV-associated neurologic complications and the lack of effective therapies approved to treat the acute and late stages of infection reinforce the importance of long-term clinical surveillance and follow up of individuals exposed to ZIKV.


OBJECTIVES/SPECIFIC AIMS: Microarray technology has produced large volumes of gene expression data profiling differences in gene expression in a vast array of conditions, much of which is publicly available. Methods to query these data for similarities in patterns of gene regulation are limited to comparisons between preannotated groups. In response, we developed openSESAME to find experiments where a set of genes is similarly coregulated without regard to experimental design. An important application of openSESAME is drug repositioning: if a pattern associated with disease is reversed by a given drug, the drug might target disease-related processes. METHODS/STUDY POPULATION: Experiments from the Gene Expression Omnibus (GEO) were normalized, signature-association (SA) scores computed for each sample, experiments assigned enrichment scores, and ANOVAs used to assign significance to experimental variables automatically extracted from GEO. SA scores were also generated for hundreds of publicly available signatures, and pairwise correlations used to create a relevance network. RESULTS/ANTICIPATED RESULTS: Using signatures of estrogen and p63, we recovered relevant experimental variables, and with the network approach, we recovered previously reported associations between disease states and/or drug treatments. DISCUSSION/SIGNIFICANCE OF IMPACT: openSESAME has the potential to illuminate “dark data” and discover novel relationships between drugs and diseases on the basis of common patterns of differential gene expression.


Brain metastases are common to the natural history of many advanced malignancies. Historically, whole brain radiation therapy (WBRT) has played a key role in the management of brain metastases, especially for patients with multiple lesions. However, prospective trials have demonstrated consistent neurocognitive toxicities after WBRT, and various pharmacologic and anatomic strategies designed to mitigate these toxicities have been studied in recent years. Memantine, an NMDA receptor antagonist, taken during and after WBRT improved cognitive preservation in a randomized trial over placebo. Deliberate reductions in radiation dose to the hippocampus, via hippocampal-avoidance (HA)-WBRT, resulted in improved cognition over historic controls in a phase II trial, and follow-up randomized trials are now ongoing to evaluate cognitive outcomes with HA vs. conventional brain radiation techniques. Nevertheless, some of the most promising strategies currently available to reduce the cognitive effects of brain radiation may be found in efforts to avoid or delay WBRT administration altogether. Stereotactic radiosurgery (SRS), involving focused, high-dose radiation to central nervous system (CNS) lesions with maximal sparing of normal brain parenchyma, has become the standard for limited brain metastases  in the wake of multiple randomized trials demonstrating equivalent survival and improved cognition with SRS alone compared to SRS plus WBRT. Today, there is growing evidence to support SRS alone for multiple (≥4) brain metastases, with comparable survival to SRS alone in patients with fewer lesions. In patients with small-cell lung cancer, the routine use of prophylactic cranial irradiation (PCI) for extensive-stage disease has been also been challenged following the results of a randomized trial supporting an alternative strategy of MRI brain surveillance and early salvage radiation for the development of brain metastases. Moreover, new systemic agents are demonstrating increasing CNS penetration and activity, with the potential to offer greater control of widespread and microscopic brain disease that was previously only achievable with WBRT. In this review, we endeavor to put these clinical data on cognition and brain metastases into historical context and to survey the evolving landscape of strategies to improve future outcomes. Paradigms for the management of brain metastases are evolving, with increasing treatment options and a greater focus on cognitive preservation. In an effort to mitigate the neurocognitive effects of whole brain radiation (WBRT) and prophylactic cranial irradiation (PCI), both anatomic and pharmacologic strategies have been studied in recent years, including hippocampal-avoidance radiation and the concomitant use of the drug memantine for neuroprotection , 2. In aWBRT has been the historic standard for the management of brain metastases and, prior to the more widespread availability of SRS, WBRT often represented the only means for treating unresected brain metastases in cases ranging from diffuse to solitary CNS lesions. As access to SRS technology increased a number of trials began comparing strategies of SRS alone to SRS plus WBRT for limited (1–3 or 4) brain metastases –25. The p < 0.001). Importantly, cognitive deterioration was also assessed at 12 months in long-term survivors, and the difference in cognitive decline persisted  (p < 0.0001), as well as an increase in 6-month cognitive deterioration among patients that received WBRT . Consistent with the aforementioned studies, there was no difference in OS   objective declines in neurocognitive function, (2) improved CNS disease control rates, but (3) no benefit in terms of OS –25. The  = 0.04) . A subseTogether the randomized trials above have detailed consistent improvements in CNS control rates with WBRT that do not translate into OS benefits, but are associated with objective declines in cognitive performance. Notably, an unplanned subgroup analysis of the Japanese trial by Aoyama et al. suggested that WBRT might improve OS in a subgroup of patients of patients with favorable prognoses; however, separate secondary analyses from both the NCCTG and EORTC trials have since refuted this finding –29. MoreOne of the largest analyses of the cognitive impact of PCI was reported by Gondi et al. who performed a pooled analysis of the RTOG 0212 and 0214 trials . The RTOThe consistent neurocognitive effects of WBRT and PCI are also accompanied by a variety of characteristic anatomic and pathophysiologic correlates. Moderate doses of radiation to the entire brain common to WBRT and PCI have been associated with cortical thinning, demyelination, attenuated capillary density, damage to the vascular endothelium, disruption of the blood-brain barrier, oxidative and pro-inflammatory stress, and impairment of neurogenesis , 37–42. p = 0.059), memantine did delay time to cognitive decline and reduced the rate of decline in memory, executive function, and processing speed  receptor, which has important roles in learning and memory. In the setting of vascular dementia, ischemia is associated with excessive NMDA receptor activation and excitotoxicity, and inhibition of the NMDA receptor with memantine represents a neuroprotective strategy , 43–45. ng speed . As a reng speed .Ginkgo biloba, in 34 patients receiving partial or whole brain radiation and reported improved neurocognitive function assessments over time  randomized 198 patients to placebo or donepezil, a reversible acetylcholine esterase inhibitor. Although donepezil did not improve cognitive composite scores (the primary endpoint), donepezil did result in modest improvements in memory . Donepezver time .Overall, the improved cognitive preservation with pharmacotherapy in the randomized RTOG 0614 represents a unique success in the radiation oncology literature, demonstrating proof of principle that radiation-induced cognitive decline can be attenuated with pharmacotherapy. It is also important to acknowledge, however, that the rates of cognitive decline after WBRT in the RTOG 0614 study remained suboptimal (cognitive preservation at 24 weeks was 31% with memantine vs. 20% with placebo), and further research into novel neuroprotective agents is warranted.A separate strategy to potentially mitigate neurocognitive toxicity in patients undergoing WBRT and PCI involves a reduction in radiation exposure to the hippocampus using conformal intensity-modulated radiation therapy (IMRT). It has been proposed that injury to the neural stem-cell compartment of the hippocampal dentate gyrus may represent an important pathophysiologic mechanism of radiation-induced cognitive decline , 49, 50.While WBRT remains an appropriate treatment for contemporary patients with diffuse brain metastases, there is a wealth of randomized evidence indicating that avoiding WBRT in favor of SRS for suitable candidates can offer superior cognitive preservation and equivalent OS –25. SRS The strongest current evidence in support of SRS for multiple metastases comes from a Japanese single-arm, multi-institutional prospective study of SRS alone in 1,194 patients with 1–10 brain metastases reported by Yamamoto et al.  This stude facto WBRT from a dosimetric standpoint  . The medandpoint .These data, along with a variety of other institutional reports –55, provWhile the role of SRS has been expanded for increasing numbers of brain metastases, the accepted indications for WBRT have also begun to shrink for patients with more limited prognoses. The QUARTZ trial enrolled a population of 538 poor-prognosis NSCLC patients  with brain metastases who were not considered candidates for SRS and randomized them to WBRT or best supportive care. This trial found no significant difference in quality-adjusted life years (primary endpoint) or OS, suggesting that omission of WBRT may be a reasonable recommendation in this population .Historically, for patients with metastatic disease, the CNS and extra-CNS have largely been viewed as distinct compartments, at least in terms of anticipated response rates to systemic therapy. This division has primarily been attributed to the blood brain barrier, which can reduce conventional chemotherapy concentrations in the CSF to levels much lower than the peripheral blood, making the CNS a potential pharmacologic sanctuary for disease progression. As result, strategies for spatially cooperative combined-modality therapy emerged, with systemic therapy being used conceptually for extra-CNS control and radiation for the treatment of the brain. These historic lines of distinction, however, are now beginning to blur as emerging molecularly-targeted and immunotherapy agents have begun demonstrating encouraging CNS response and control rates in prospective trials –20. BeloIn ALK gene-rearranged lung cancer, a pooled analysis of two single arm phase 2 studies of alectinib, with a median follow-up of 12.4 months, demonstrated objective CNS response rates of 64% in patients with measurable CNS disease, and a median duration of response of 10.8 months . SimilarIn BRAF-mutated melanoma, a phase II study of dabrafenib and trametinib for patients with brain metastases demonstrated intracranial response rates of 44–59% in cohorts stratified by BRAF mutation type, prior CNS therapy, symptoms, and performance status. Importantly, however, the durability of response appeared to be suboptimal, with median durations of CNS response of only 4.5–8.3 months across the cohorts .In evaluation of single-agent vs. combination immunotherapy, a randomized phase 2 study of patients with melanoma brain metastases reported objective intracranial responses with a median of 17 months follow-up in 16 of 35 (46%) patients treated with ipilumumab/nivolumab and 5 of 25 (20%) treated with nivolumab alone . A separThe emerging data on systemic agents with enhanced CNS activity are encouraging and have generated appropriate optimism regarding the expanding arsenal for the treatment and prevention of brain metastases. It is important to acknowledge, however, that there is limited prospective data comparing CNS-penetrant agents to strategies incorporating CNS radiotherapy. One recent trial compared icotinib alone, a first generation EGFR-TKI with modest CNS activity, to radiation and chemotherapy for brain metastases and found improved CNS control outcomes with icotinib . This trN = 34) and NSCLC (N = 211) patients with brain metastases treated with SRS alone and found comparable rates of OS, CNS control, and neurologic mortality in SCLC and NSCLC patients (N = 200) vs. WBRT for brain metastases, and observed favorable survival outcomes with SRS overall and on propensity-score matched analyses  brain metastases identified earlier in the context of MRI surveillance may be salvaged without negatively impacting survival and (2) that a meaningful subset of patients with SCLC who do not develop brain metastases can be spared the neurocognitive sequela of PCI altogether. Moreover, the design of the Japanese trial also points to the need for new studies in LS-SCLC comparing PCI to MRI surveillance strategies, as the trials included in the 1999 meta-analysis of LS-SCLC were all in the pre-MRI era and the majority of patients did not undergo brain CT staging or surveillance in those studies , 68. In The management of brain metastases remains a complex and highly-individualized discipline in oncology. As prognoses continue to improve for patients with brain metastases, efforts to minimize the cognitive sequelae of therapy will only become increasingly important. Numerous clinical trials have characterized the deleterious effects of moderate doses of radiation to the entire brain common to WBRT and PCI, challenging investigators to develop new strategies to attenuate, avoid, or delay the neurocognitive effects of these therapies. Pharmacotherapy and anatomic avoidance strategies are actively being investigated, as is the expansion of SRS candidacy to patients with increasing burdens of CNS disease. It is also clear that management of brain metastases will become increasingly multidisciplinary in the context of emerging systemic agents with enhanced CNS activity. A new generation of combined-modality trials involving local and systemic therapies will be needed to evaluate the optimal strategies for durable CNS disease control, neurocognitive function, and survival in the rapidly evolving landscape of therapies for metastatic disease.TR and CR: Conception and design, writing, and final approval of 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.


Human pluripotent stem cells (hPSCs) have extensive applications in fundamental biology, regenerative medicine, disease modelling, and drug discovery/toxicology. Whilst large numbers of cardiomyocytes can be generated from hPSCs, extensive characterization has revealed that they have immature cardiac properties. This has raised potential concerns over their usefulness for many applications and has led to the pursuit of driving maturation of hPSC-cardiomyocytes. Currently, the best approach for driving maturity is the use of tissue engineering to generate highly functional three-dimensional heart tissue. Although we have made significant progress in this area, we have still not generated heart tissue that fully recapitulates all the properties of an adult heart. Deciphering the processes driving cardiomyocyte maturation will be instrumental in uncovering the mechanisms that govern optimal heart function and identifying new therapeutic targets for heart disease. The maintenance and expansion of embryonic stem cells in a pluripotent stateHuman cardiomyocytes have many distinct features compared to rodent cardiomyocytes, which have been used as the model system of choice for decades . To gainin vivo are also observed in CM when mouse pluripotent stem cell and hPSC-CM are compared.Many physiological properties such as the size, contraction rate, cardiac output, and metabolism differ between human and mouse hearts.Decades of research in developmental biology have been essential for the generation of hPSC-CM. This research provided the developmental blueprint to differentiate hPSCs into cardiomyocytes. Originally, non-directed spontaneous differentiation approaches were used . However, these protocols are inconsistent and generate very low percentages of cardiomyocytes. Over the past 15 years, directed differentiation protocols have greatly advanced and have now improved the efficiency of cardiac differentiation to yield greater than 95%. Directed differentiation protocols enable robust and reproducible differentiation and follow known developmental stimuli such as endoderm co-culture,in vivo counterparts. This makes them a very useful and predictive model for many applications including studying hypertrophy, electrophysiology, drug toxicity and discovery, and fundamental biology .hPSC-CM maturation is not defined by a single property nor is it driven by a single stimulus. There are a wide array of properties which need to be assessed, covering many aspects of cardiac biology and function . These rin vivo organ-like structures.in vivo behave very differently from their in vitro counterparts on stiff, flat, un-patterned 2D substrates.2), a wider array of properties resembling an adult heart have only been shown thus far within 3D engineered heart tissue (EHT) formats.As in vivo-like cardiac organization,EHT has been used for decades to create highly functional heart muscle. EHT was originally derived from isolated chicken or neonatal rat heart cells and cultured inside collagen I/Matrigel hydrogels.in vitro. This may be an important step for maturation, as tension through cardiomyocyte-substrate binding in stiff 2D environments imposes different biological effects and may actually keep the cardiomyocytes in an immature stateIn addition to cellular organization, the EHT approach enables precise mechanical loading of the sarcomeres. In the native heart, most of the mechanical tension is actually imposed on the sarcomeric giant protein TITIN at physiological strains.A variety of different loading regimes have been utilized in the EHT format. Auxotonic mechanical loading, rather than static stretching or phasic stretching, is the most physiological means of EHT mechanical loading and results in enhanced functionality.in vitro. The application of auxotonic mechanical loading and pacingOverall, the EHT approach has many advantages in creating the most accurate model of heart tissue Advanced maturation of hPSC derived-EHT has been achieved by optimizing multi-cellularity and mechanical loading and pacing [In addition to the aforementioned stimuli, metabolism has also been recently identified as a major driver of maturation . In the in vivo. In the early postnatal window, there is a shift in many cardiac parameters and recapitulating some of these in hPSC-CM has been shown to result in maturation  ,The heart is the most metabolically active organ and consumes roughly 20 times its weight in adenosine triphosphate (ATP) daily.The holy-grail of cardiac bioengineering is the production of fully mature adult hPSC-CM, which is also a pursuit for many other organ and cell types.


Heparanase is a β-D-endoglucuronidase that cleaves heparan sulfate, a complex glycosaminoglycan found ubiquitously throughout mammalian cells and tissues. Heparanase has been strongly associated with important pathological processes including inflammatory disease and tumor metastasis, through its ability to promote various cellular functions such as cell migration, invasion, adhesion, and cytokine release. A number of cell types express heparanase including leukocytes, cells of the vasculature as well as tumor cells. However, the relative contribution of heparanase from these different cell sources to these processes is poorly defined. It is now well-established that the immune system plays a critical role in shaping tumor progression. Intriguingly, leukocyte-derived heparanase has been shown to either assist or impede tumor progression, depending on the setting. This review covers our current knowledge of heparanase in immune regulation of tumor progression, as well as the potential applications and implications of exploiting or inhibiting heparanase in cancer therapy. Heparanase is the only mammalian enzyme that directly cleaves heparan sulfate side chains of heparan sulfate proteoglycans (HSPGs), key components of the extracellular matrix and basement membrane. The cleavage of heparan sulfate by heparanase regulates a number of fundamental cellular processes including cell migration , 2, cytoThe first documentation of heparanase expression in leukocytes was in T lymphocytes where the production of an endoglycosidase was observed in assisting their migration and penetration of the basement membrane and blood vessel entry . SubsequTumor associated macrophages (TAMs) are often found within primary tumors and pre-metastatic sites, and their presence frequently contributes to tumor progression , 53. In During inflammation and inflammation-associated tumorigenesis, the source of heparanase is often the epithelium . This wain vitro, but when implanted into mice, exhibited increased heparanase activity   . Howeveror cells  -T cell therapy utilizes engineered recombinant receptors expressed on T cells containing an antigen-recognition domain of a monoclonal antibody and a T cell-activating domain . These CHeparanase overexpression has been documented across the majority of tumor types, including solid tumors –77 and h+ T cells were identified in the bone marrow of a sample of breast cancer patients, and were functionally reactive to heparanase-overexpressing tumor cells  generated CTLs that specifically targeted human tumor cell lines presenting heparanase on either HLA-A*0201 (The heparanase peptide has also been tested as a TAA in a prophylactic vaccine. Priming mice A-A*0201  or HLA-AA-A*0201  (Figure A-A*0201 . InjectiA-A*0201 .These data suggest that heparanase could be a robust tumor antigen, as when targeted, shows both reduction and protection against tumor growth in animal and human systems. However, targeting heparanase via a vaccine approach will rely on tumors maintaining heparanase expression to allow T cell recognition of heparanase-positive tumor cells. Regardless, the selective pressure from these vaccines on tumor cells to downregulate heparanase expression would still be advantageous in blocking tumor progression.The delivery of gene therapy as a cancer treatment requires specific targeting to tumor cells. A promising approach to target therapies toward tumors is through viral particles via attachment to T cells . An impoThe robust expression of heparanase across multiple cancer types and cell types makes it a useful target to manipulate and utilize its anti-cancer properties. All of these therapies described will rely on maintained heparanase expression for efficacy. As we will describe, heparanase inhibitors currently used to reduce tumor burden may not always have favorable outcomes on tumor progression, especially for patients undergoing the therapies described above.Our understanding of the relationship between heparanase and leukocytes during tumor progression remains limited. Similarly, much is still unknown about how heparanase inhibitors affect the anti-tumor immune response, despite their current use in clinical trials against a range of cancers –94. A nuPreclinical animal models show PG545 can reduce tumor and metastatic burden in several tumor models, including breast, prostate , liver , lung 2, colon , ovarianPseudomonas aeruginosa infection (Other clinically relevant heparanase inhibitors such as Roneparstat and ODSH also display immunomodulatory effects. Roneparstat, in development for the treatment of multiple myeloma, has been observed to effect macrophage polarization by inhibiting the expression of M1 related genes in LPS-stimulated U937 macrophages . In a monfection  and protnfection , 114, a nfection .More work needs to be done to define and understand the effects of heparanase inhibitors on cells of the immune system. Heparanase inhibitors have been used as anti-inflammatory agents, and have been shown to impair lymphocyte trafficking , 117 andThis review describes how leukocyte-heparanase can be a double-edged sword in tumor progression; it can enhance tumor immune surveillance and tumor cell clearance, but also promote tumor survival and growth. We also discuss the potential of using heparanase in leukocyte therapies against tumors, and the effects of heparanase inhibitors on tumor progression and immunity.We are just beginning to understand the influence of heparanase on a pro/anti-tumor immune response, and there are still many questions to answer. How do the pro/anti-tumorigenic effects of heparanase differ across different cancer types? Does the tumorigenic effect of heparanase change during cancer progression? And how does the expression or role of heparanase change during treatment regimens? Answering these questions may help guide the appropriate use of heparanase inhibitors, and the use of heparanase-assisted therapies for the treatment of cancer.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Pseudomonas aeruginosa, a ubiquitous Gram-negative bacterium, is harmless to healthy individuals, and yet a formidable opportunistic pathogen in compromised hosts. When pathogenic, P. aeruginosa causes invasive and highly lethal disease in certain compromised hosts. In others, such as individuals with the genetic disease cystic fibrosis, this pathogen causes chronic lung infections which persist for decades. During chronic lung infections, P. aeruginosa adapts to the host environment by evolving toward a state of reduced bacterial invasiveness that favors bacterial persistence without causing overwhelming host injury. Host responses to chronic P. aeruginosa infections are complex and dynamic, ranging from vigorous activation of innate immune responses that are ineffective at eradicating the infecting bacteria, to relative host tolerance and dampened activation of host immunity. This review will examine how P. aeruginosa subverts host defenses and modulates immune and inflammatory responses during chronic infection. This dynamic interplay between host and pathogen is a major determinant in the pathogenesis of chronic P. aeruginosa lung infections.Bacteria that readily adapt to different natural environments, can also exploit this versatility upon infection of the host to persist.  Pseudomonas aeruginosa (PA) is a ubiquitous Gram-negative bacterium commonly encountered in the environment and readily cleared by host defenses. However, PA is also a formidable opportunistic pathogen that can cause invasive and fulminant infections, such as acute pneumonia or bloodstream infections, in immune compromised hosts. Remarkably, the same pathogen also causes chronic infections that persist for months to decades, such as the chronic lung infection in individuals with the genetic disease cystic fibrosis (CF). Chronic PA infections thus result from a dynamic and complex interplay between pathogen and host, where bacteria persist without causing overwhelming host injury, and where host defenses fail to eradicate the pathogen.Bacterial pathogens are most commonly studied for their ability to invade and injure the host, causing acute and invasive infections. In contrast, chronic infections present a distinct paradigm in infection pathogenesis which may challenge conventional notions of bacterial virulence and host defenses. To healthy individuals, PA has a large genome (>6 Mb) that encodes many regulatory genes involved in sensing environmental signals, controlling expression of virulence factors, metabolism and resistance mechanisms. PA thus readily adapts to a wide range of environments and can exploit this versatility to enhance its long-term survival and persistence in the host. Importantly, host-pathogen interactions evolve over time and anatomical space, with the balance fluctuating between host recognition and vigorous activation of defense mechanisms, and immune evasion and tolerance by the host.Chronic PA lung infections in individuals with CF persist for decades and provide a unique opportunity to examine how a bacterial pathogen can adapt to its host, modulate host responses and shift between different infection phenotypes. It is widely recognized that CF disease is associated with several intrinsic host defects, including impaired mucociliary clearance, and immune and inflammatory dysregulation. The implications of these host defects to the development of CF lung disease are beyond the scope of this review but may be found in excellent other ones –3. In thPA possesses a single polar flagellum composed of polymerized flagellin, its major structural protein, and attached to a transmembrane motor complex. The flagellar-host interaction plays a major role in defining the immune and inflammatory outcomes of PA infection, as the flagellar complex interacts with immune and non-immune cells through its structural components and as well as motility function.The flagellar-host interactions have been extensively characterized at the cellular and molecular level. Flagellin is best known as a pathogen-associated molecular pattern that binds to the extracellular Toll like receptor TLR5  and intrrpoN and fleQ which lead to downregulation of flagellar expression and motility  is a complex needle-like secretion machinery found in gram-negative bacteria that allows the translocation of bacterial effectors directly into the cytoplasm of host cells, causing cytotoxicity, or subversion of host defenses . The T3SBeyond its role in cytotoxicity, the T3SS activates innate immune responses through secretion of IL-1ß . The T3SChronic infections appear to select against T3SS-expressing PA. Although many CF patients carry antibodies against T3SS effector proteins , suggestPA produces several secreted proteases, which include LasB , LasA, AprA, and protease IV. Secreted PA proteases interact with a wide range of host molecules, leading to diverse outcomes, from degradation of structural components to modulation of inflammatory responses. The PA proteases are most studied for their ability to cause direct tissue damage, and they are primarily known as virulence factors involved in the pathogenesis of acute infections. LasB, a broad specificity metallo-protease, degrades elastin , disruptIn vitro studies have shown that PA proteases potently degrades secreted mediators such as cytokines , chemokines   –66, hostES/CCL5) , 68, antES/CCL5) , and memES/CCL5) , 71. LasES/CCL5) . LasB meES/CCL5) , 74. ProES/CCL5) . FinallyES/CCL5)  and inflES/CCL5) . InteresES/CCL5) .in vitro, in vivo in murine models of chronic PA lung infections and in CF patients  chronically colonized with PA , 78. In patients . The impPA produces three extracellular polysaccharides (or exopolysaccharides), namely alginate, Psl, and Pel. They provide many protective properties and confer surface and self-adherence. They are constituents of the biofilm matrix, are involved in surface colonization and promote host immune evasion. A detailed review of these EPS and their distinct functions can be found elsewhere .2O2  is a major component of the outer membrane of Gram negative bacteria. LPS is composed of three components: the lipid A and core oligosaccharides that form the outer leaflet of the bacterial outer membrane, and the O-antigen polysaccharide which interacts with the extracellular environment. LPS is recognized by the Toll like receptor 4 and myeloid differentiation factor 2 complex (TLR4-MD2). The O-antigen consists of highly variable and immunogenic oligosaccharide repeats which elicit a strong humoral response .During chronic infection, the LPS undergoes important adaptive changes at the level of its synthesis and structure, leading to modification of the lipid A structure and loss of O antigen which likely promote immune evasion. Lipid A acylation patterns or addition of positively charged components, renders the outer membrane more resistant to host antimicrobial peptides –99, moduDuring the process of chronic infection, PA adapts to the host environment and undergoes changes which promote bacterial survival and evasion of host defenses. Certain adaptive processes occur at the phenotypic and regulatory level, while others occur through genetic mutations and evolution. We will review here the key regulatory and genetic adaptive processes that PA undergoes during chronic PA infection.in vivo host responses to PA biofilms is limited by the lack of animal infection models that mimic human biofilm infections. Our insights are thus primarily drawn from in vitro studies that examine the response of various cell types to biofilm bacteria. Biofilm formation and its role in disease pathogenesis have been the subject of recent reviews  -related gene , 115. Chon state . Conversfections  or exacefections .Cyclic di-GMP is an intracellular bacterial secondary messenger that regulates multiple bacterial behaviors, most notably those involved in biofilm formation. The cellular level of c-di-GMP are modulated in response to environmental and intracellular signals, and affect expression of genes involved in flagellar and type IV pilus mediated motility, exopolysaccharide production and surface adhesion . GeneticThe RetS/GacS and sensor pathway, cyclic di-GMP signaling and other global regulators  allow PA to coordinately regulate numerous factors that define distinct bacterial infection strategies, namely acute and invasive disease, or chronic and localized disease. It is plausible that the ability of PA to phenotypically switch between acute and chronic virulence modes contributes to the complex disease phenotype it causes: the natural history of chronic PA lung infections is characterized by slowly progressive tissue pathology, but is also interrupted by periods of acute and more fulminant disease termed acute exacerbations. It is possible to speculate that exacerbation episodes may be caused in part by a phenotypic switch to acute virulence.The bacterial genetic adaptation to host environments is a common theme during chronic infection. For PA, this has been best documented in chronic CF lung infection, and we suggest several excellent recent reviews , 119 forDuring its long residence in the CF lung, PA populations show both genetic diversification as well as convergent evolution. On one hand, PA undergoes significant genetic and phenotypic diversification during chronic CF infection, a process likely attributable to the divergent evolution of clonally related PA inhabiting different regions and micro-environments of the lung . On the Alternative or adjuvant therapies that minimize direct bacterial damage to the host, that enhance protective host responses or subvert pathological ones, can improve infection outcomes . Such thAnti-virulence therapies target bacterial virulence without disrupting bacterial growth or viability. Although numerous PA targets  and inhibitor molecules have been studied, very few have progressed past pre-clinical studies . Anti-viAntibacterial antibodies can neutralize bacterial virulence factors, induce complement mediated lysis and enhance opsonophagocytic uptake and killing . AdvanceConsidering the intractable nature of chronic PA infection, an important strategy is also to prevent infection through approaches such as vaccine, antibody, enzyme or antibiotic-based treatments. Although several anti-PA vaccine targeting antigens such as LPS O-antigen, alginate, outer membrane or flagellar proteins showed promise in pre-clinical trials, their clinical efficacy in reducing the risk of chronic PA infection in susceptible individuals (such as CF patients) has been overall disappointing to date , 135.in vivo models of chronic PA infection and tools to simultaneously probe host and pathogen over time is critical in order to gain a more integrated understanding of chronic infections.Chronic PA infection illustrates a paradigm of chronic bacterial infections where pathogens dampen host defenses, adapt and evolve within the host to persist. Understanding the pathogenesis of chronic PA infection thus requires an intricate assessment of bacteria, host responses, and their interactions over time. Host-PA interactions are exceptionally complex in chronic infections, as they involve numerous host cell types and bacterial factors. These interactions are further complicated by the common co-existence of other pathogens or polymicrobial communities that interact with both host and PA, and by the potential changes in the host due to factors such as aging or environmental exposures. While decades of research have provided us with vast mechanistic data on host-PA interactions, integrating these mechanistic insights into a whole system understanding of chronic infection and translating this knowledge into effective treatments remain a major challenge. The development of better All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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. The handling Editor declared a shared affiliation, though no other collaboration, with all of the authors KK, EF, and DN.


Genome-editing, a recent technological advancement in the field of life sciences, is one of the great examples of techniques used to explore the understanding of the biological phenomenon. Besides having different site-directed nucleases for genome editing over a decade ago, the CRISPR/Cas  based genome editing approach has become a choice of technique due to its simplicity, ease of access, cost, and flexibility. In the present review, several CRISPR/Cas based approaches have been discussed, considering recent advances and challenges to implicate those in the crop improvement programs. Successful examples where CRISPR/Cas approach has been used to improve the biotic and abiotic stress tolerance, and traits related to yield and plant architecture have been discussed. The review highlights the challenges to implement the genome editing in polyploid crop plants like wheat, canola, and sugarcane. Challenges for plants difficult to transform and germline-specific gene expression have been discussed. We have also discussed the notable progress with multi-target editing approaches based on polycistronic tRNA processing, Csy4 endoribonuclease, intron processing, and Drosha ribonuclease. Potential to edit multiple targets simultaneously makes it possible to take up more challenging tasks required to engineer desired crop plants. Similarly, advances like precision gene editing, promoter bashing, and methylome-editing will also be discussed. The present review also provides a catalog of available computational tools and servers facilitating designing of guide-RNA targets, construct designs, and data analysis. The information provided here will be useful for the efficient exploration of technological advances in genome editing field for the crop improvement programs. Cells have several inherent mechanisms for the repair of double-strand DNA breaks (DSBs) ,2. TheseFokI monomer and constructed to create a spacer of 5–6 bp, enabling the FokI to be functional on dimerization and create DSBs. . F. F129]. ALS and FTIP1e generated double mutants resulting in resistance to two herbicides [Although initially applied in animal systems aspiring to reduce the off-target editing, this system has also been effectively used in plant system—including wheat, maize, rice, cotton, potato, and tomato ,136,137—rbicides  and can rbicides . Even thrbicides ,139.The indels produced by wild type Cas9 are random and do not ensure knocking out of a gene, rather some indels may also enhance the function of the protein to be targeted. The BE3 systems have also been used for CRISPR-STOP and iSTOP for base editing mediated introduction of early non-sense codons ,141. CRIPds gene [AtPAP1 (Production of anthocyanin pigment 1) by up to 7-fold, and could also reverse gene silencing due to methylation in AtFIS2 (Fertilization-independent seed 2) gene in Arabidopsis changing the AtFIS2 expression levels by up to 400-fold [The CRISPR/Cas system can be used to manipulate the expression levels of a gene by using catalytically inactive dCas9 fused to a transcriptional regulator. Earlier experiments done with animal cells implied that fusion of dCas9 with an activator, like VP64, could activate the genes but 3-4 gRNAs targeting a single promoter is required to activate a gene efficiently ,143,144.Pds gene . Lowder 400-fold . One of 400-fold . These rArabidopsis, where a modified SunTag approach has been used along-with the previously characterized catalytic domain of Nicotiana tabacum DOMAINS REARRANGED METHYLTRANSFERASE (DRM) to act as a methylation effector, for the manipulation FWA and SUPERMAN genes. This work is also an insightful example of promoter bashing (using dCas9-VP64 fusion) and site-specific base editing [DNA methylation is a very important epigenetic modification that controls many biological processes and gene regulation during development. Post-translational methylation of histone proteins and modification of the DNA by methylation of cytosine residues are the most important approaches for epigenetic gene regulation in organisms. DNA methylation is the preferred mode for targeted regulation of gene expression as it is more stable and can result in long-term effects. DNA methylation maps with single-nucleotide resolution have been developed for animal cells and specific tissues ,149. The editing . HoweverThe off-target activity of Cas9 protein is a major concern as Cas protein can edit any DNA sequence having about five mismatches with its sgRNA ,154, howSelection of appropriate target sites is an imperative aspect for reducing the problem of off-target effects of Cas9 and for ensuring that the target site selected is in the coding region of the gene. Various bioinformatics tools are now available, which help in selecting appropriate target sites and designing the sgRNA accordingly, which virtually eliminate the need for applying the above said measures to reduce the off-target effects. Various other measures can also be kept in check, such as the GC content of the target site and restriction sites within the selected target. Some of these tools are given in Although CRISPR/Cas is an unparalleled and pioneering technology, still there are some bottlenecks for its effective application in the area of crop improvement and translational research. One of those bottlenecks is efficacious delivery of transformation vectors into the correct host cells and further successful regeneration of plants. Plant transformation progresses through two consecutive steps: Transient transformation and stable transformation. Stable transformation being responsible for the production of edited plants having heritable mutations, of which the integrated transgene of nuclease can be segregated away to produce transgene-free plants.Agrobacterium-mediated and biolistic transformation methods remain the most applied transformation methods, but they are incompetent for many crops with the main challenges being: (1) low incidence of stably transformed plants, (2) long tissue culture periods, (3) tissue damage due to biolistic transformation, (4) limitation of Agrobacterium-mediated transformation to very few genotypes in a species, (5) Agrobacterium induced browning and necrosis of tissues, (6) induction of somatic mutations, (7) difficulties in transforming monocot species using Agrobacterium, and (8) low quantities of DNA delivered via Agrobacterium-mediated transformation which are insufficient for efficient HDR. Also, most of the time, cells easily regenerated cannot be transformed or vice versa and meticulous optimization of tissue and cell culture media with appropriate growth regulators are required [required . TherefoAgrobacterium species with a broader host range should also be developed. Many bacterial species other than Agrobacterium, capable of transforming diverse plant species have also been reported. These include Sinorhizobium meliloti, Mesorhizobium loti, Rhizobium sp. NGR234 and other Rhizobium species, and Ensifer adhaerens [Sinorhizobium meliloti has been shown to be capable of infecting both dicots and monocots [Agrobacterium-derived DNA transfer machinery, but a Rhizobium species has been identified recently which is capable of transferring DNA with its protein apparatus [Tobacco rattle virus (TRV) based vectors used for virus-induced gene silencing (VIGS) have also been developed and demonstrated to work efficiently for the delivery of gRNA and Cas9 in Nicotiana benthamiana germline cells, bypassing the requirement for plant transformation, and can infect multiple plant species. TRV based system is also crucial because the viral RNA genome does not integrate into the plant DNA genome and thus is a desirable factor to generate transgene-free plants [Arabidopsis thaliana, it has been successfully applied to check transgene expression [Triticum aestivum) for the production of male-sterile lines by targeting Ms1  [Some advancements in strategies used for plant transformation have already been made, and much work is simultaneously going on. One such strategy is to increase the frequency of HDR because efficient HDR requires high titers of nuclease and repair template to be delivered into the plant cell. Viral vectors are being developed to deliver large numbers of nucleases and repair templates aiming at increasing the frequency of HDR . Agrobacdhaerens ,182,183,monocots . Most ofpparatus . Tobaccoe plants . Recentle plants . Many ote plants , agar-tre plants , mesopore plants ,191, care plants , and flue plants —have beee plants . The usepression . Howeverpression  applied ty gene) . Using aty gene) . DiscoveCRISPR/Cas mediated genome-edited plants can be classified as non-genetically modified. However, the most frequently used methods for the delivery of constructs also lead to the insertion of transgene/s in the plant genome, therefore resulting in transgenics and a need to segregate the inserted genes in the following generation. However, this process is more troublesome if transgenes are inserted at multiple sites into the genome. Additionally, RNP mediated genome editing is desirable for clonally propagated plants. Hence, more novel technologies to altogether bypass the transgenic regulations are required. One such method is the production of Cas9 RNPs, which are composed of pre-integrated Cas9 nuclease and sgRNAs, and are equally efficient to generate genetic alterations as DNA-based transformation methods. In case of plants, RNP complex delivery is inhibited due to the presence of cell walls and thus is incorporated into naked plant protoplasts. RNPs have been successfully used in wheat , PetuniaArabidopsis, but the efficiency of transmission of mutations to subsequent generations was much higher in rice than in Arabidopsis. The higher efficiency of mutation inheritance was evident from the fact that in experiments evaluating knocking out effects of CRISPR/Cas involving Pds, the albino phenotype was transmitted to subsequent primary transgenic generation in rice [Arabidopsis took several generations to obtain the homozygous mutants, with 1-bp deletions and chimeric mutations predominating and most of the mutations being in somatic cells [0 plants then one might have to wait till T2 or even further generations. Still, such promoters can be used to increase the gene editing frequency in recalcitrant species and offer vast potential for technological improvement.A lot of early experiments using CRISPR/Cas equally involved rice and  in rice . Howeveric cells . This phCRISPR/Cas based genome editing emerged as a game-changer in recent years due to its enormous potential to make desired modifications in the genome and also for versatile diagnostic purposes. The technological advances are being implemented not only for generating knockouts but also knock-ins, and for activation as well as repression of gene expression. Because of its utility in a practical system, many advancements have already been achieved in a very short period after its discovery, that includes DNA free genome editing systems (RNPs), multiple Cas9 variants, many multi-gene targeting approaches, precise base editing, and measures to increase the frequency of HDR. Advancements in the CRISPR/Cas related bioinformatics tools have also led to the elevated use of this powerful genome editing technique. However, the challenge of providing the public with sufficient knowledge regarding CRISPR/Cas mediated method of modifying crops cannot be discounted, as this is essential to take laboratory research to the masses, to provide agriculture with a sustainable future. In a case, it is essential to have laws that clearly mark a line between the gene-edited plants having foreign DNA and gene-edited plants without any foreign DNA, rendering the latter subject to be bypassed for any regulations, making the utilization of such plants very trouble-free. As compared to the commercial release of transgene-free genome-edited plants having a novel mutation, genome-edited plants with mutation precisely the same which exist with natural variation will be more relaxed, more particularly in countries where policies are not in the favor. In this regard, the precise genome editing approaches look promising. The gap between lab to the field is continuously widening because of the regulatory policies which are not yet well defined. Since gene editing may lead to genome modifications which occur in nature and the results of genome modifications implemented by the ‘traditional’ methods and genome editing cannot be distinguished in some cases, and, therefore, special regulation of the technology is unreasonable. Another opinion states that the legislator should concentrate on the specific characteristics of the final product rather than on the production method to identify the possible risk in any case. At the same time, one must not forget about the obligation for proper assessment and management of risks, regulation of the gene drive issue, and essential precautions for the use of this technology in the field of reproduction and human genetics. Apart from these issues, significant efforts are required to improve the transformation techniques which is not yet optimized for several crop species and highlighted as a bottleneck to explore recent advancement efficiently. The CRISPR/Cas based genome editing is still limited to sophisticated molecular biology lab which mostly focuses on the fundamental biological question. In contrast, crop breeders still need to make significant efforts to implement technological advances in crop improvement programs. Pay-per-use basis, public as well as private facilities providing service for construct development, transformation, and evaluation of genome-edited plants, will be game-changer to take the advancements from lab to field by facilitating crop breeder with most efficient genome editing tools. The information provided here will be helpful to understand the recent technological advances, knowledge gaps, problems with technological adaptations which are required for the efficient utilization of genome editing tools for crop improvement.


Background: Two classes of amine oxidases are found in mammals: those with a flavin adenine dinucleotide as a cofactor, such as monoamine oxidases (MAO) and lysine-specific demethylases (LSD), and those with copper as a cofactor, including copper-containing amine oxidases (AOC) and lysyl oxidases (LOX). All are expressed in adipose tissue, including a semicarbazide-sensitive amine oxidase/vascular adhesion protein-1 (SSAO/VAP-1) strongly present on the adipocyte surface. Methods: Previously, irreversible MAO inhibitors have been reported to limit food intake and/or fat extension in rodents; however, their use for the treatment of depressed patients has not revealed a clear anti-obesity action. Semicarbazide and other molecules inhibiting SSAO/VAP-1 also reduce adiposity in obese rodents. Results: Recently, a LOX inhibitor and a subtype-selective MAO inhibitor have been shown to limit fattening in high-fat diet-fed rats. Phenelzine, which inhibits MAO and AOC, limits adipogenesis in cultured preadipocytes and impairs lipogenesis in mature adipocytes. When tested in rats or mice, phenelzine reduces food intake and/or fat accumulation without cardiac adverse effects. Novel amine oxidase inhibitors have been recently characterized in a quest for promising anti-inflammatory or anti-cancer approaches; however, their capacity to mitigate obesity has not been studied so far. Conclusions: The present review of the diverse effects of amine oxidase inhibitors impairing adipocyte differentiation or limiting excessive fat accumulation indicates that further studies are needed to reveal their potential anti-obesity properties. The wide family of sympathomimetic agents has been a continuous source of anti-obesity drugs for decades, essentially because it encompasses at least two types of molecules that interfere with energy balance: those able to limit energy intake (exhibiting anorectic properties); and those that increase energy expenditure (exhibiting thermogenic properties). Furthermore, several of these drugs even share both activities. Numerous sympathomimetic agents have been studied so far for their anti-obesity potential ; however, for the purpose of this review, only the inhibitors involved in the prevention of the inactivation of aminergic neuromodulators will be considered. Thus, we will focus on the compounds that inhibit amine oxidase activities and therefore contribute to impair the “turn off” of the signalling neurotransmitters norepinephrine, dopamine and serotonin. However, before reviewing this subclass of pharmacological agents that could be useful to treat obesity, it is worth mentioning that the concept of “burning fat” should be revised. Any novel anti-obesity strategy has to limit fat deposition without obliterating adipose tissue totally. On one hand, numerous mouse models with genetic ablation of white adipose tissue (WAT) have unanimously indicated that life without adipose depots is extremely unhealthy and hazardous. On the other hand, intense lipid mobilisation is more likely to lead to lipotoxicity, ectopic fat deposition and insulin resistance rather than to massive energy dissipation and safe or permanent body mass loss. Therefore, nowadays, an adequate anti-obesity treatment or prevention should limit lipid accumulation in a moderate but sustained way rather than promising an explosive fat utilisation.Bearing this in mind, it is easier to understand how the powerful anorectic effects of amphetamines and monoamine oxidase inhibitors (MAOIs) observed in animal models for over half a century ,2 did noThus, this review presents a series of independent observations showing that an inhibition of amine oxidases can be concomitant with a reduction of fat accumulation, at least in laboratory rodents. Then, although far from recommending immediately the treatment of obese patients with amine oxidase inhibitors, we will discuss the current evidence that gives interest to novel anti-obesity strategies based on the inhibition of members of the amine oxidase family. To this aim, it is necessary to briefly describe the classification and biological functions of amine oxidases, including a presentation of their substrates and inhibitors, before reviewing the current knowledge and establishing hypotheses linking amine oxidase inhibition and energy balance control.Monoamine oxidases are enzymes capable of removing an amine group from a substrate  in the presence of oxygen, and producing the corresponding ketone or aldehyde, ammonia and hydrogen peroxide. MAO proteins exist in two major forms, MAO-A and MAO-B, encoded by two genes located on the X chromosome. Both contain flavin adenine dinucleotide (FAD) as a covalently bound cofactor and share about 70% of their peptide sequence. These flavoenzymes are key regulators for normal brain function, since they contribute to the catabolism of amine neurotransmitters  are homologous to MAOs in their structure and inhibition properties. These flavoenzymes are involved in gene expression regulation by removing methyl groups of histones in a reaction requiring their FAD cofactor. Since LSDs are present in chromatin and participate in epigenetic reprogramming by turning off the action of histone methyltransferases, they are currently the targets of various anti-cancer drugs under development . A growiHowever, many peripheral tissues and organs also express other amine oxidases that do not use FAD as a cofactor to perform oxidative deamination: the copper-containing amine oxidases (CAO or AOC). One of them is currently known as vascular adhesion protein-1 (VAP-1), which was initially called semicarbazide-sensitive amine oxidase (SSAO) since it was inhibited by semicarbazide but not by classical MAOIs. The SSAO/VAP-1 cupro-enzyme exhibits multiple functions. In addition to its capacity to contribute to the rolling and adhesion of circulating leucocytes to endothelial cells at the sites of inflammation , it is pThe lysyl-oxidase LOX and four LOX-like proteins are also copper-containing amine oxidases that oxidize lysine and hydroxylysine residues of insoluble substrates, such as collagens and elastin, and other soluble substrates as well . As mentThe use of LOX inhibitors derived from β-aminopropionitrile (β-APN) is recognised to limit metastasis, and therefore could restrict obesity-associated cancers, such as those affecting colon or liver, among others.A list of amine oxidase inhibitors for the different forms of MAOs, AOCs, APAO, SMO and LOX–LOXLs is included in A screening of thermogenic drugs as potential anti-obesity agents performed by Dulloo and Miller revealed that, among more than 30 sympathetic stimulants, MAOIs were endowed with a remarkable capacity to increase energy expenditure and reduce fat without loss of body protein in various mouse and rat models of obesity . In partThe MAO-B inhibitor selegiline was very recently reported by Nagy and co-workers to reduce adiposity induced by a high-fat, high-sucrose diet in male rats without influencing body weight . SimilarHowever, these observations are not the first indicating that an inhibition of amine oxidases other than MAO could be effective in limiting fattening in treated animals. To the best of our knowledge, the first observations indicating that repeated administration of an SSAO inhibitor limits weight gain were serendipitously made by Yu and co-workers in 2004 . This grInterestingly, other drugs initially studied for their beneficial actions on vascular complications have been shown to produce SSAO inhibition and alter fat content in treated animals. Besides its multiple cardiovascular effects, hydralazine was shown to inhibit SSAO ,36, as wSince all these molecules sharing SSAO inhibitory properties exhibited some anti-obesity effects, it was of utmost interest to check whether semicarbazide, the reference agent for inhibiting copper-containing oxidases, was unambiguously reported to reduce body weight and/or adiposity. Indeed, it was independently observed that semicarbazide reduces body weight gain in Sprague Dawley and in Brown Norway rats in response to repeated intraperitoneal administration at 900 µmol/kg body weight/day for 6–8 weeks ,24,38. W2-C=O-NH-NH2) belonging to the chemical family of hydrazines, and is thus a carbonyl reagent that binds to the SSAO active site, but which can react with many other targets. As a consequence of its use in industrial organic chemistry, it is now banned as food contaminant (suspected genotoxic) and considered as a marine pollutant capable of disturbing endocrine and reproductive systems in fishes [Semicarbazide is a small urea derivative , many factors exerting an inhibitory effect on lipid accumulation in differentiating preadipocytes have been qualified as promising anti-obesity agents deserving to be further tested in vivo. Conversely, several anti-obesity drugs that were developed in view of their in vivo capacity to reduce appetite, such as sibutramine or rimonabant, were reported later on to be effective in such in vitro models since they also directly activate lipolysis or inhibit lipogenesis in adipocytes . It wasLastly, a very recent report by Yang and co-workers indicates a decreased glucose utilisation and increased lipid trafficking in preadipocytes when adipogenesis-dependent SSAO emergence is defective, therefore confirming that SSAO is a regulator of energy utilisation processes in adipocytes . MoreoveIs has been recognized that it is more convenient to reduce body weight gain by promoting lipid mobilisation and utilisation during the onset of obesity rather than treating massively hypertrophied WAT. Hence, the most important readouts in future clinical studies have to be the determination of the percentage of body fat and the anatomical location of the fat stores rather than the body weight itself because any efficient anti-obesity agent should be able to prevent/reduce excessive lipid storage in the visceral fat without impairing lean mass homeostasis. Obviously, a limitation of ectopic fat deposition and a reduction of the visceral/subcutaneous adipose tissue mass ratio should be demonstrated before affirming that an inhibition of amine oxidase(s) may be protective against obesity and its complications.In humans, the development of an anti-obesity treatment based on the use of hydrazine derivatives will be probably limited by their potential toxicity, except for the abovementioned case of phenelzine. However, studies carried out in depressed patients also show that another MAOI, tranylcypromine, is not associated with weight gain or with a hepatotoxicity risk since this inhibitor is not a hydrazine derivative ,4. As trOn the other hand, most of the MAOIs or SSAO/VAP-1 inhibitors listed in this review also interact with other multiple targets. It cannot be ruled out that additional mechanisms other than the blockade of oxidative deamination are involved in their observed promising anti-obesity effects . It should be of utmost importance to determine whether there is a substantial spontaneous activity of these amine oxidases in obese individuals, since in the absence of a pathologically elevated activity, no inhibitor can induce substantial change in the function of its target(s) and downstream events. In fact, elevated SSAO expression/activity has been reported in WAT animal models of genetic  or diet-Finally, it is worth mentioning that obesity is accompanied by low-grade inflammation, and is characterized by an increased number of immune cells around the adipocytes. SSAO/VAP-1 inhibitors contribute to reduce inflammation in many experimental models ,46 and mIn conclusion, there are converging observations suggesting that MAOIs, known to provide clinical benefits in the treatment of mental diseases, also interact with metabolism regulation in cultured adipocytes and animal models of obesity. In addition, small molecules that inhibit SSAO/VAP-1, and that have recently been reported to be useful for the treatment of many inflammatory diseases, are also capable of influencing adipocytes, which highly express this cupro-enzyme. Although there is not yet a confirmed clinical demonstration of the usefulness of amine oxidase inhibitors for treating obesity and related complications, testing of such inhibitors  could bring about promising novel pharmacological treatments for obese subjects.


Positive-sense single-stranded plant RNA viruses are obligate intracellular parasites that infect many agriculturally important crops. Most known plant RNA viruses are characterized by small genomes encoding a limited number of multifunctional viral proteins. Viral pathogens are considered to be absolutely dependent on their hosts, and viruses must recruit numerous host proteins and other factors for genomic RNA replication. Overall, the replication process depends on virus–plant protein-protein, RNA–protein and protein–lipid interactions. Recent publications provide strong evidence for the important role of chloroplasts in viral RNA synthesis. The chloroplast is considered to be a multifunctional organelle responsible for photosynthesis and for the generation of plant defense signaling molecules. High-throughput technologies (genomics and proteomics), and electron microscopy, including three-dimensional tomography, have revealed that several groups of plant RNA viruses utilize chloroplast membranes to assemble viral replication complexes (VRCs). Moreover, some chloroplast-related proteins reportedly interact with both viral proteins and their genomic RNAs and participate in trafficking these molecules to the chloroplast, where replication occurs. Here, we present the current knowledge on the important role of chloroplasts in the viral replication process. Positive-sense, single-stranded [(+)ssRNA] viruses are the main agents of many serious diseases of agriculturally important crops. Despite their relatively small genomes, these viruses that encode only a few proteins can efficiently replicate in their host cells . Virusesvia a neck-like opening and single- and/or double-membrane systems ; chloroplasts are also crucial for interorganellar signaling . Zhao etIn this review, we describe chloroplast–virus interactions on the basis of recent evidence highlighting the direct function of this organelle in virus RNA synthesis. We also list the most useful molecular approaches for studying viral RNA replication in this context.Alfamovirus genus, are also associated with the chloroplast envelope. However, further studies performed by Arabidopsis thaliana protoplasts showed that AMV replicates on tonoplast membranes.Although ss(+)RNA viruses display various genetic organization and expression strategies, they all replicate in a similar manner. All known plant ss(+)RNA viruses encode RNA-dependent RNA polymerase (RdRp) and ancillary replication factors. These viral proteins, which are produced in the early stage of infection, are involved in the assembly of VRCs and help to stabilize and facilitate optimal conditions for RNA synthesis. Viral and host proteins are selectively bound to the ss(+)RNA template and target subcellular membranes, which are later reorganized around the favorable microenvironment to form VRCs . Within The replication complex of the aforementioned virus families is associated with the outer chloroplast membrane, particularly with peripheral vesicles and cytoplasmic invaginations (CIs) on the surface. Among recently identified host factors participating in the viral replication process are several chloroplast and chloroplast-related proteins Table . Virus-iRecently published data indicate the role of the 140K/98K protein domains of TYMV, which possess two amphipathic helices (αA and αB) with a 41-residue internal sequence described as the chloroplast envelope-targeting domain (CTD) . Based ovia vesicles from ER-exit sites (ERESs) to the outer chloroplast membrane  attachment protein receptor) that are essential host factors for successful virus infection. For example, the cellular SNARE protein Syp71 directly mediates the fusion of vesicles with chloroplasts  showed that viral RNA and TGB3, which possesses a chloroplast-targeting signal, primarily accumulate near the surface of chloroplast membranes , with VRCs adjacent to the chloroplast membrane , and exonuclease XRN4, which all play a crucial role in the early stage of viral replication (Arabidopsis (via enzymatic activity by glutathione transferases (GSTs), and the 3′UTR-associated protein NbGSTU4 that in the presence of glutathione is able to neutralize oxidative stress inside chloroplasts, enabling efficient minus-strand RNA synthesis , followed by mass spectrometry (Tombusviridae family), potyviruses TuMV and TEV, and Bromoviridae, as exemplified by brome mosaic virus (in situ mapping are powerful tools that provide essential architectural information regarding VRC biogenesis and the function of these structures (Identification of host factors involved in virus replication may be achieved trometry . For ideic virus ,b. All tructures .In summary, the abovementioned research studies broaden our knowledge of chloroplasts and their important direct and indirect roles in the replication of several plant virus species. In recent years, increasing numbers of chloroplast and chloroplast-related proteins have been shown to be involved in the formation of membranous replication factories and RNA synthesis. However, it is worth noting that in many cases the mechanism of interactions between viral and the identified plant factors as well as their influence on the regulation of replication remains unknown. Importantly, it should be noted that despite being replication sites, chloroplasts are primarily implicated in plant defense against viruses. Therefore, molecular insights into pathways exploited by viral pathogens and the implementation of innovative powerful imaging techniques to study plant–virus interactions is of great importance. The results obtained might contribute to the development of new and effective antiviral strategies against existing and emerging plant virus diseases.MB and AO-S conceived the review. MB wrote a large part of the manuscript. AO-S critically reviewed and complemented the review. Both authors have made a substantial, direct and intellectual contributions to the work, and approved it for publication.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.