Glioblastoma (GBM) represents the most frequent primary brain tumor in adults and carries a dismal prognosis despite aggressive, multimodal treatment regimens involving maximal resection, radiochemotherapy, and maintenance chemotherapy. Histologically, GBMs are characterized by a high degree of VEGF-mediated vascular proliferation. In consequence, new targeted anti-angiogenic therapies, such as the monoclonal anti-VEGF-A antibody bevacizumab, have proven effective in attenuating tumor (neo)angiogenesis and were shown to possess therapeutic activity in several phase II trials. However, the role of bevacizumab in the context of multimodal therapy approaches appears to be rather complex. This review will give insights into current concepts, limitations, and controversies regarding the molecular mechanisms and the clinical benefits of bevacizumab treatment in combination with radio(chemo)therapy - particularly in face of the results of recent phase III trials, which failed to demonstrate convincing improvements in overall survival (OS). GBMs are highly vascularized tumors that critically depend on the generation of tumor-associated blood vessels , 2. TheyCurrently, the most prominent VEGF targeting drug is bevacizumab (BEV), a recombinant humanized monoclonal antibody that binds to human VEGF-A. Several groups have investigated the use of BEV with an already proven efficacy in metastatic colon, breast, and lung cancer for patients with recurrent malignant gliomas , 13. BEVRecent prospective phase III trials (AVAglio & RTOG 0825) were designed to prove the efficacy of TMZ-based radiochemotherapy in combination with BEV as first-line therapy for GBM. While the RTOG 0825 trial failed to show significant benefits in terms of progression-free (PFS) and overall survival (OS), the AVAglio study demonstrated a significant prolongation of PFS by 4.4 months in the BEV arm. However, this PFS benefit did not translate into an improvement in OS , 34. In Anti-angiogenic therapy targeting VEGF or its receptors is considered to impact on glioma growth through several mechanisms, some of which are only poorly understood . The iniIn principle, the term “vascular normalization” might be misleading as some tortuous, large-diameter vessels will not immediately disappear upon administration of BEV . But there is another reason, which supports the notion that RT and BEV treatment can act synergistically. The term “window of opportunity” as introduced by Jain et al. appears to describe the physiological effects that can be observed during RT and BEV treatment more precisely . It doesin vivo FET-PET). Several reports have described obvious characteristics following BEV treatment, but so far no data on combined modality approaches are available [In summary, the aforementioned novel neuroradiological findings, which are associated with anti-angiogenic therapy and have not been previously observed under standard radiochemotherapeutic regimens, fuel the still ongoing debate about pseudo-progression and/or pseudo-response . This divailable -72.The treatment of malignant brain tumors has been subject to a variety of clinical studies combining RT with BEV with or without TMZ . In 2014The phase II GLARIUS trial explored the efficacy of BEV plus irinotecan compared to standard TMZ in the first-line radiochemotherapy of GBM patients with non-methylated O-6-methylguanine-DNA methyltransferase (MGMT) promoter. In the BEV/irinotecan arm, PFS was significantly prolonged from a median of 5.9 months (95% CI 2.7-6.2 months) to 9.7 months . Nevertheless, no prolongation of the median OS and no improvement in the quality of life were observed . In summOne might come to discordant conclusions when considering the available clinical phase II/III results . ApparenThe decision of the EMA (European Medicines Agency) against BEV for the treatment of newly diagnosed GBM was based on the results of both RTOG 0825 and AVAglio. However, the observations of initial attempts to use BEV in recurrent glioma appear promising and inspired an ongoing second line chemotherapy trial , which might lead to the approval of BEV for the treatment of recurrent malignant glioma. This trial is based on the efficacy of BEV and lomustine within the BELOB phase II trial . AccordiWe have learned from the treatment of high grade gliomas with BEV in both preclinical models and clinical settings that anti-angiogenic therapies considerably change tumorbiological properties, including vascularization and tumor cell metabolism. These effects can be clearly demonstrated by means of neuroradiological and neuro¬pathological analyses. Nevertheless, although the morphological and physiological changes in the tumor were associated with prolonged PFS in several phase II/III trials the OS remained virtually unchanged. Which are the putative reasons for this and how should they be addressed in the future?The concept of vascular normalization implies increased pericyte coverage as well as reduced basement membrane thickness of tumor vessels, and led to the hypothesis that these changes could favor tumor oxygenation, which in turn would improve treatment effects and thus patient survival , 78. Alr+ circulating endothelial cells (CECs) identified a subgroup of patients with longer PFS and OS, which also encompassed more long-term responders [Despite encouraging results using anti-angiogenic therapy in GBM it appears that only a subset of patients experiences survival benefits when receiving BEV treatment, and striking differences in response rates and long-term tumor control have been observed. Biomarkers able to identify patients, who would specifically benefit from anti-angiogenic therapy in combination with radiochemotherapy still remain an unmet need not only in neurooncology , 84. In sponders . Additiosponders . FurtherMolecular genetic advances have contributed to a better understanding of GBM pathophysiology and might have the potential for disease stratification. The most recent, clinically relevant classification has been provided by The Cancer Genome Atlas consortium (TCGA). On the basis of gene expression profiling with respect to p53, epidermal growth factor (EGFR), neurofibromin (NFI), platelet-derived growth factor alpha (PDGFRA) and isocitrate dehydrogenase 1 (IDH1) four distinct molecular subtypes of GBMs  were defined . ImportaIn summary, for both phase III trials considerable efforts have been undertaken to define patient subgroups who would particularly profit from BEV treatment plus radiochemotherapy. Different strategies were followed. Whereas the RTOG 0825 consortium started with the Colman signature and subsequently defined a 9-/10-gene set of mesenchymal GBM , the AVAThe interaction between VEGF signaling and RT has been addressed in several preclinical studies , 95-100.The clinical efficacy of BEV application in combination with RT as assessed by PFS and OS is currently being extensively discussed. However, one aspect remains commonly disregarded in this context: The beneficial impact of BEV on attenuating radiation-induced cerebral necrosis. To our knowledge, there has been only one small prospective randomized trial addressing this issue, and it provided clear evidence for the successful treatment of radiogenic brain necrosis with BEV . This isThe increased risk of radiation necrosis was an initial concern when the concept of re-irradiation of malignant glioma emerged. However, in recent years this treatment option has been adopted as safe and effective -121. ForOne further concern about anti-angiogenic treatment in the context of RT is an increased stimulation of glioma cell migration and invasiveness. Results from preclinical studies have shown that (perivascular) invasion increases during anti-VEGF therapy and might thereby limit therapeutic efficacy , 128-130Finally, a major caveat of BEV application in combination with RT in GBM remains: detrimental neurocognitive effects. Unfortunately, RTOG 0825 and AVAglio, do not provide conclusive results in this regard. From a physiological point of view, this hypothesis cannot be declined, since VEGF is a relevant neuroprotector and its targeting might give rise to possible negative side effects of RT. Available literature on this topic delineates a similar picture in animal models, where VEGF apart from stimulating microvascular proliferation and angiogenesis - enhances neuronal differentiation, protection, and regeneration , 136. ThOverall, it remains unclear how the therapeutic alliance of RT and BEV application for GBM will develop. In our opinion, there are several open questions, which need to be addressed in the future.Are there subgroups of patients who are specifically eligible or not suitable for BEV/RT treatment?Having predictive biomarkers would be highly advisable, and a validation of already defined subgroups is of utmost importance.What are the molecular mechanisms of BEV-mediated radioprotection of normal brain parenchyma and might dose-escalation make sense when detrimental effects on the normal tissue can be avoided?It is relevant to delineate in how far dose escalation with concomitant BEV treatment is feasible.After RT/BEV treatment, how long should maintenance BEV therapy be performed?Some of the adverse effects could be related to sustained BEV treatment alone and not to the combined RT/BEV regimen.Clearly, future trials need to focus on patient subgroups for whom real benefits in terms of PFS and OS can be expected as depicted in Figure One facet of BEV treatment, which is currently moving into the focus of interest, and has not been intensively discussed here, are the immunological consequences of anti-angiogenic therapy. The tumor microenvironment and particularly the tumor endothelium appear to strongly impact on the regulation immune mechanisms and thus contribute to the establishment of an immunosuppressive milieu. VEGF has been attributed an essential role in this regard. Accordingly, interfering with VEGF function is known to have local as well as systemic immunological effects, which - not only due to the advent of immunotherapeutic approaches - certainly will be further examined in future studies , 141.


Viruses cause epidemics on all major cultures of agronomic importance, representing a serious threat to global food security. As strict intracellular pathogens, they cannot be controlled chemically and prophylactic measures consist mainly in the destruction of infected plants and excessive pesticide applications to limit the population of vector organisms. A powerful alternative frequently employed in agriculture relies on the use of crop genetic resistances, approach that depends on mechanisms governing plant–virus interactions. Hence, knowledge related to the molecular bases of viral infections and crop resistances is key to face viral attacks in fields. Over the past 80 years, great advances have been made on our understanding of plant immunity against viruses. Although most of the known natural resistance genes have long been dominant R genes (encoding NBS-LRR proteins), a vast number of crop recessive resistance genes were cloned in the last decade, emphasizing another evolutive strategy to block viruses. In addition, the discovery of RNA interference pathways highlighted a very efficient antiviral system targeting the infectious agent at the nucleic acid level. Insidiously, plant viruses evolve and often acquire the ability to overcome the resistances employed by breeders. The development of efficient and durable resistances able to withstand the extreme genetic plasticity of viruses therefore represents a major challenge for the coming years. This review aims at describing some of the most devastating diseases caused by viruses on crops and summarizes current knowledge about plant–virus interactions, focusing on resistance mechanisms that prevent or limit viral infection in plants. In addition, I will discuss the current outcomes of the actions employed to control viral diseases in fields and the future investigations that need to be undertaken to develop sustainable broad-spectrum crop resistances against viruses. As obligatory intracellular parasites, plant viruses depend on the host machinery to multiply and invade their hosts. In their simplest form, viruses consist of a DNA or RNA genomic segment encoding only few genes and encapsided into a protein shell, called the capsid. Because of natural physical barriers , viruses are delivered into plant cells through wounds or through the action of vectors  that feed on or infect the plants. Following entry into a host cell and genome decapsidation, the infectious cycle includes translation and replication of the viral genome, assembly of progeny virus particles, generalized invasion of the host through cell-to-cell and long-distance movements of viral particles or ribonucleoprotein complexes and finally, transmission to new hosts by vectors. In some cases, transmission to the following generation of host plant is also observed as a result of seed infection. In 2012, the International Committee on Taxonomy of Viruses reported 92 genera of plant viruses of which 82 were assigned in 21 different families .Plant infection by viruses causes physiological disorders responsible for plant diseases of economic and agronomic significance in many crops. Widely employed in fields, the use of plant varieties carrying genetic resistances constitutes the most effective, economical and ecological measure to control viral infections. The last decades have seen substantial advances on the molecular dialog between viral pathogens and their plant hosts, bringing new strategies directly exploitable in crop improvement programs. Nevertheless, the spread of crop viral pests has increased dramatically in recent years. Globalization, trade and climate change, as well as reduced resilience in production systems due to decades of agricultural intensification have all played a part.This review intends: (i) to provide a brief overview of severe virus-associated plant diseases and their impact on crop production, (ii) to summarize prophylactic strategies employed to control viral epidemics in fields, (iii) to bring an update on current knowledge about plant resistances against viruses, (iv) to present the technical approaches currently employed in crop improvement programs, and (v) to discuss how antiviral mechanisms based on PAMP-triggered immunity (PTI) could be the source of novel plant resistances in fields.Management of plant virus diseases is a matter of vital importance and concern to farmers, horticulturists, foresters, manufacturers, as well as consumers. It is well-established that virus diseases in different crops cause enormous losses all over the world in terms of quantity and/or quality of products. Although it’s very difficult to put a clear figure on the financial impact of plant viruses in agriculture, the yield losses that can be ascribed to plant viruses are estimated to cost worldwide more than $30 billion annually .Cassava mosaic begomoviruses cause more than 25 million tons of losses per year in Africa, India, and Sri Lanka  were estimated to over 100 million trees worldwide ] since the 1970s have exceeded 10 billion euros .Because viral agents are obligate intracellular parasites, curative treatments of virus infections are impossible, making viral diseases very difficult to control in fields. Prophylactic control measures are therefore crucial in combating epidemics on crops. They consist mainly on combining cultural practices, biosecurity measures and organism-vector management  for their plant-to-plant spread. Hence, viral diseases can be efficiently controlled by limiting the populations of their vectors with the applications of appropriate pesticides. The use of non-host “trap plants” may be also considered to attract vectors to reduce the number of individuals feeding on the crop of interest and thus, the transmission of the disease .Epidemics often arise from new viruses or new variants of classic viruses that spilled over from reservoir species to crops. Although this phenomenon results from a complex evolutionary process in which the main players are ecological factors, virus genetic plasticity and host factors, viral diseases can be controlled by managing the spatial structure and composition of field parcels, which impacts resistance durability .Minimizing viral epidemics involves the respect of international legislations concerning worldwide trade of virus-free plant material, which applies to any development stage of a plant that can be carrier of viruses , as well as manipulation of decontaminated horticultural tools.The use of genetically resistant plants is one of the most efficient, sustainable and frequently employed strategies to control virus infections in fields. For centuries, it has involved plants selected by breeders for their agronomic proprieties combined to the absence of disease symptoms. However, from the middle of the 20th century, plant improvement programs capitalize strongly on the knowledge associated to plant–virus interactions to develop resistant varieties exploitable in agriculture.Figure 2).Faced with viral attacks, plants defend themselves through several resistance layers, that are complementary in terms of defense timing (at early or late infection steps), location (in the first infected leaf or in systemic tissues) and targeting the virus-derived molecules  identified in plant–virus interactions belong to the nucleotide binding site-leucine-rich repeat (NBS-LRR) class, that specifically recognize the viral avirulence (avr) gene products, through the establishment of the so-called “gene-for-gene” interaction. Although a direct physical interaction between the avr and R gene products had originally been suggested, the current understanding favors the more sophisticated “guard hypothesis” model  through the recognition of the PVX capsid (CP). Rx1 CC domain has been shown to form a heterodimer with the cellular ranGTPase-activating protein 2 (ranGAP2), interaction required for Rx1 function , in an ATP dependent manner , HSP90 (Heat shock protein), and RAR1 , a phenomenon involving the programmed death of the infected and neighboring cells, with the consequences to confine the pathogen in the hypersensitive lesion and to prevent any further pathogen spread in the plant. A functional module that mediates HR against viruses  requires the interaction of two lipases, EDS1 (Enhanced disease susceptibility 1) and PAD4  with the protein SAG101 , a mechanism that has been demonstrated for both N and Rx1 genes  production, salicylic acid (SA) accumulation, and extensive transcriptional reprogramming. In addition, the activation of rmovirus . Recent rmovirus . Meanwhix1 genes  and thatR genes, the RTM genes (for Restricted TEV Movement) were the first cloned non-NBS-LRR dominant resistance genes against viruses. Genetic characterization of natural Arabidopsis accessions and mutants showed that at least five dominant RTM genes are involved in resistance to Tobacco etch potyvirus (TEV), Lettuce mosaic potyvirus (LMV), and PPV , the translation elongation factors 1A and 1B (eEF1A and eEF1B) and the poly(A)-binding proteins (PABPs) 2, 4, and 8  belonging to the eIF4E and eIF4G families  protein in the recessive resistance to bymoviruses (ra blocking vascular transport of Potato potyvirus A (PVA) was genetically characterized in potato  contributing to systemic resistance against PPV  has been recognized as an evolutionarily conserved process in most eukaryotes, that is triggered by double-stranded RNAs (dsRNAs). These dsRNAs are processed by ribonuclease III-type DICER-like (DCL) enzymes into small RNAs (sRNAs), 21–24 nucleotides in length, that are incorporated into an RNA-induced cytoplasmic silencing complex (RISC), whose key catalytic component corresponds to one member of the ARGONAUTE (AGO) protein family. Once integrated into the RISC, sRNAs base-pair to their target-mRNA and induce their cleavage . In planFigure 2C). What initially appeared counter-intuitive, infection from DNA viruses produce also dsRNAs, most likely via bidirectional convergent transcription  during the replication step. These molecules are targeted by the RNA-silencing machinery to produce virus-derived small RNAs  encoding a methyltransferase that protects siRNA and miRNA duplexes from degradation (Ty-1 and Ty-3 conferring resistance to Tomato yellow leaf curl begomovirus (TYLCV) has been recently shown to encode a tomato RDR. Although resistant plants do not show symptoms upon a challenge with TYLCV, low levels of virus are still detectable, a phenomenon characteristic of a virus tolerance more than a real resistance . In the case of TMV-triggered HR, the response persists up to 3 weeks during the time plants are protected against not only TMV but also other pathogens (N. benthamiana (During the R-mediated resistance activation (see the Section “Dominant Resistances” in this review), cellular responses elicited at the infection site are emitted to distant non-infected tissues, resulting in a resistance or in reduced susceptibility state that can remain efficient during several weeks . This phathogens . How SARathogens . Moreoveathogens . The sigathogens . Recent athogens . Jasmoniathogens  but is ethamiana . It is lthamiana . Interesthamiana . Althougthamiana , ethylenthamiana , and brathamiana .Ultimately, the main objective of research on plant–virus interactions consists on the implementation of efficient antiviral resistances in crop plants. For long, genetics-mediated resistance strategies have involved exploiting plant natural variability by introgression of resistance genes through a classical breeding process. However, the onset of evolved pathogens able to overcome these resistance, sometimes very rapidly after resistance deployment , raised de novo whole genome shotgun (WGS) sequencing of reference genotypes and whole genome resequencing (WGRS) of several cultivars, land races, and wild relatives , restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), and simple sequence repeats (SSRs) contributed, through what is called “Marker-assisted selection (MAS),” to make substantially easier, quicker, and more accurate the selection of resistant genotypes during the introgression steps . Importaelatives . Despiteelatives , MAS hasPotato potyvirus Y . Initially reported in transgenic tobacco plants expressing the TMV CP  plants resistant to viral attacks are grouped into three major categories. First of all, resistance genes with a dominant determinism can be introduced to another plant species. The best examples in plant–virus interactions involve the successful transfer of d plants  but it sd plants . The oppe TMV CP , the rese TMV CP . These oe TMV CP . In the e TMV CP . In spitA. thaliana, both TILLING and ecoTILLING have spread rapidly to other model plants  and major crops . It seems now clear that these strategies are emerging as major crop improvement tools, with especially successful examples of recent applications to antiviral protection  method, that consists of a classical mutagenesis step followed by the targeted search for plants carrying a mutation in a gene of interest . By creaotection .S-methyl, that leads to plant protection against many pathogens including viruses  is able to infect a broad range of herbaceous crops  and fruit trees  without causing any symptoms, this virus was identified as an excellent candidate for VIGS-mediated vaccination, that could be simply applied widely by replacing the insert with a sequence derived from a different virus. This strategy has been recently illustrated , and that triggers virus-induced gene silencing (VIGS) targeting both primary and challenge viruses . Primaryin Japan . Moreovein Japan . Howeverin Japan . Given Austrated  and reprIn an attempt to increase resistance durability in fields, crop improvement requires a continuous pipeline of new resistance genes. Given the importance of PAMP-triggered immunity (PTI) in the field of pathology over the last decades, PTI against plant viruses represents an unexplored question that needs to be addressed.Over the last decades, a concept revolutionizing the understanding of immunity emerged in plant pathology. This new concept stems from the ability for each organism to discriminate between self and non-self molecules through the action of pattern recognition receptors (PRRs) perceiving specific microbial molecular signatures, named pathogen-associated molecular patterns (PAMPs). Perception of PAMPs by these immune receptors induces a downstream signaling cascade including PRR association with positive regulators, phosphorylation events, successive activation of cytoplasmic kinases (including the MAP kinases) and defense-related transcription factors, as well as specific defense genes expression. This range of fast, efficient and multi-layered defense responses is referred as PTI . To counTo date, most current models about plant innate immunity exclude viruses, as they are not generally viewed as encoding typical PAMPs and effectors that would trigger PTI and ETI responses . The facR gene-based immunity in plant–virus interactions involves specific recognition of viral avirulence factors by host-encoded NBS-LRR proteins and leads to HR and SAR (described previously in this review), recent hypotheses advance that it may correspond to antiviral ETI mechanisms  and MDA5 (Melanoma differentiation-associated gene 5), both highly activated by dsRNAs, as well as 5′ triphosphate-RNAs for RIG-I , a family of a dozen type I-transmembrane proteins containing a domain with LRRs and a tail that contains a conserved region called Toll/IL-1 receptor (TIR) domain , and shaor RIG-I . In the or RIG-I .Bean pod mottle comovirus (Arabidopsis resistance to plant viruses (Arabidopsis defense against the Cabbage leaf curl geminivirus (CaLCuV). Strikingly, NIK1 is cleaved by the viral protein NSP during the infection, suppressing its kinase activity and thus enhancing susceptibility to CaLCuV infection  colonization strategies employed by plant and animal viruses inside host cells are highly similar , (ii) plFigure 3), that does not invalidate previous models but instead tries (i) to bring the concept of PTI as a novel plant antiviral mechanism, and (ii) to integrate the concept of innate (PTI and ETI) and adaptive (gene silencing) antiviral immunities, both triggered by similar virus-derived elicitor molecules, in order to address the important question of plant defense against viruses in a more holistic way.Thus, strongly based on the understanding of plant immune pathways and the knowledge on antiviral mechanisms from the animal field, I propose here a model of plant immunity against viruses . Successful viruses encode specialized effectors that are able to suppress this defense layer. In turn, specific R gene products interact with these effectors (playing then the role of avirulence factors), leading to ETI. In the case of RNAi-based adaptive immunity, the viral elicitor molecules (mostly dsRNAs) are recognized by DCLs. This leads to virus degradation at the nucleic acid level, a process that can be suppressed by a second class of virus-encoded effectors, commonly named silencing suppressors. Interestingly, Thus, following the virus entry into a plant cell, viral nucleic acids are perceived as PAMPs by specific intracellular PRRs triggering PTI pathways  are highly conserved within virus families and differ radically from prokaryotic or eukaryotic nucleic acids. It is these typical viral features that are perceived by specialized PRRs in animals , triggering very fast and very efficient immune responses. Because the innate pathway is solely programmed by structural and nucleotide sequence genomic features, plant antiviral PTI should be a remarkably versatile mechanism because it could respond virtually to any plant virus. Moreover, timeliness of defense responses is a key parameter in resistance success, especially in the case of viruses, given their tremendous adaptation abilities. And yet, the first measurable PTI responses are within minutes after PAMP perception , making In the world agriculture context, intensification of cultural practices, climate alterations, and extensive exchanges affecting global markets are associated with an increased incidence of plant viral diseases. In consequence, management of viral epidemics is a matter of global food security. Nowadays, antiviral measures employed in fields benefits greatly from the latest knowledge highlighted by plant virologists, geneticists and molecular biologists, as well as engineers and breeders, whose coordinated effort enables a deployment in crop improvement programs. This notwithstanding, the situation in fields remains worrying, given the emergence of numerous crop diseases ascribed to viruses, a statement that could be aggravated in the future through climate changes that might affect strongly virus and organism-vectors populations in terms of magnitude, locations and dynamics . In a coThe author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Entotheonella’ further underlining the vast bioactive underestimated potential in microorganisms  products far surpasses anything possible by chemical synthesis. Hence, drug discovery has returned to nature, especially microorganisms, as a source of novel chemistry. Recently, genome sequencing and mining has demonstrated that bioactive compounds hitherto believed to be produced by a eukaryotic organism (a marine sponge) were indeed synthesized by one of the dominant (uncultured) prokaryotes, ‘et al., antiSMASH or NaPDoS, which can identify characteristic structures of polyketide and non-ribosomal peptide synthetases , however, genome mining reveals approximately 15 gene clusters likely encoding for pathways likely producing bioactives .Classical bioassay guided fractionation has been used for decades as the major bioprospecting path, however, this is often a laborious process, and rediscovery of known compounds is a major challenge despite developments in de-replication strategies. The rapidly developing sequencing techniques are introducing sequencing of microbial genomes in almost any microbial research environment, and hence so-called genome mining has become a useful tool in discovery of novel microbial products  and bioassays. A vast amount of studies have demonstrated that the production of secondary metabolites in microorganisms is dependent on growth substrate and environment. Stress signals, such as antibiotics  with chemical (metabolomic molecular networking), genome and metagenome-based analyses is indeed likely to enhance our bio-discovery hit rate and provide novel understanding of the ecology and natural interactions of microorganisms.Authors have no conflict of interest to declare.


Neurodegenerative diseases (ND) are characterized by the progressive loss of specific neuronal populations with consequent deterioration of brain's function. While some ND have monogenic causes, the vast majority is sporadic. Accordingly, etiology varies greatly between these conditions; the pathogenesis, however, shares important common traits. Trophic deprivation, oxidative stress, accumulation of abnormal protein aggregates, and bioenergetics defects have been in fact described in most, if not all, ND. To counterbalance these noxious stimuli cells deploy intrinsic neuroprotective responses, at least during early pathogenesis. Adaptation includes strategies to optimize energetic resources, for instance reduction of rRNA synthesis to repress translation, suppression of transcription, and bioenergetics and metabolic redesign. Additional mechanisms include potentiation of antioxidant capacity, induction of endoplasmic reticulum (ER) stress, and activation of protein quality control systems and autophagy. Strategies to potentiate these naturally occurring processes might provide foundation to devise new experimental treatments.This e-book contains a collection of reviews and original articles summarizing the state-of-the-art knowledge on protective responses sustaining neuronal survival and activity. The ultimate goal of this work is to inspire novel studies elucidating strategies to contrast these incurable disorders.Francelle et al.). In line with this concept, Stilling and colleagues show how aging is associated with changes in gene expression and in RNA splicing, and with the upregulation of immune system functions in the hippocampus . Interestingly, affected genes are involved in synaptic function, therefore suggesting a potential pathogenic link between age-dependent transcriptional alterations and late onset of Alzheimer's disease (AD) .The first article reviews transcriptional deregulation in Huntington's disease (HD). Francelle and colleagues point out that while transcriptional changes are directly caused by mutant huntingtin, they may also represent a response to secondary stress conditions triggered by dysfunctional transcriptional machineries. They speculate that transient expression changes typical of HD may represent a self-defense mechanism because some of these altered genes activate pro-survival functions, which may be impaired during aging. Hence, the investigation of gene expression changes with aging in the striatum, the brain region mostly affected in HD, might help to develop strategies to modify mutant huntingtin toxicity .A second original research article illustrates how two transcription factors important for the development of dopaminergic neurons also play a role in their maintenance in adulthood. Previous studies indicated that haploinsufficiency of the transcription factor Foxa 2 leads to abnormalities in motor behavior in old age and an associated progressive loss of dopaminergic neurons reminiscent of Parkinson's disease (PD) .A third original article addresses neuronal self defense mechanisms based on the regulation of rRNA synthesis. Downregulation of rRNA synthesis has been associated with neuronal loss in several ND, although disruption of nucleolar activity may trigger context-specific neuroprotective responses (Chan).Edwin Chan reviews aberrant RNA species—an emerging mechanism of toxicity in ND—particularly in polyQ diseases linked to nucleotide repeat expansion and in frontotemporal dementia/ALS (FTD/ALS). The piece discusses the impact of these mutant RNAs and proposes that toxicity may be ascribed to alternative splicing of RNAs, hairpin, and double-stranded CAG repeat RNAs, sequestration of cellular proteins, nucleolar stress, repeat associated non-ATG translation (RAN-translation) of CAG expansion transcripts . In the context of this post-transcriptional regulation of protein synthesis, this group also proposes in a second original article the use of antisense long noncoding RNAs in neuronal cell lines to activate translation in a gene specific manner . Finally, Amadio and colleagues contributed with another original article on post-transcriptional regulation, proposing that ELAV  proteins to heme-oxygenase-1 mRNA—a gene activated to counteract oxidative stress—to increase its expression and elicit neuroprotection with beneficial effects .The role of antisense long noncoding RNAs (lncRNAs) as regulators of neuronal gene expression is rapidly emerging . In this context the minireview by Canal and colleagues focuses on the mTOR inhibitor RTP801/REDD1, a protein encoded by the stress responsive gene DNA damage inducible transcript 4. REDD1 expression is regulated during development and in response to DNA damage. In addition, REDD1 is induced by stressors, such as hypoxia and ER stress. Interestingly REDD1 is also found upregulated in PD. The authors propose a model in which REDD1 may have either a protective or toxic function in cell survival depending on its protein level that changes at different disease stages. At low level, REDD1 might be a protective compensatory response at early disease stage .The mammalian (or mechanistic) target of rapamycin (mTOR) acts as a central regulator of cell homeostasis, and its activity is tightly regulated . SG are dynamic cellular compartments that contribute to the arrest of mRNA translation in response to stress insults, such as UV-irradiation, oxidative stress. SG are also linked to amyloidogenic proteins and irreversible protein aggregates perturb their normal dynamics. Alteration of SG dynamics however may be due to irreversible protein aggregates. The articles also explores the nexus between heat shock proteins and proteolytic pathways, and specifically focuses on the crosstalk between different protein degradation pathways such as ubiquitin-proteasome system, chaperone mediated autophagy and macroautophagy. The work reveals that these mechanism share a certain degree of redundancy and the function of the unaffected one can rescue malfunction of the other pathways .The next group of articles deals with accumulation of protein aggregates, which constitutes another shared mechanism of neurodegeneration and represents a promising therapeutic target. The review by Angelika and Fabio Falsone focuses on different regulators of protein aggregates, including stress granules (SG) . PDI is upregulated in ALS and this upregulation is shown early in the disease progression in ALS rat models. PDI inactivation increases ER stress, defined as disruption of the ER linked to accumulation of misfolded proteins, while at early stages PDI could prevent aggregation of superoxide dismutase 1 (SOD1). However, at a later stage increased UPR can result in increased PDI activation that may result in neurotoxicity by increasing superoxide production or according to a second model by increasing hydroperoxyde . A third mini-review on this topic, contributed by Ujval Anilkumar and Jochen Prehn, discusses non-cell death related functions of anti-apoptotic proteins such as the pro-survival BCL-2 in mitochondrial physiology and ER homeostasis (Anilkumar and Prehn). J proteins, which belong to the chaperone family, may exert neuroprotective effects by facilitating proteostasis, as also discussed in the mini-review by Koutras and Braun.Jaronen and coworkers focus in their review on the role of ER stress and unfolded protein response (UPR) on the protein disulfide isomerase (PDI), a chaperone regulator of misfolded protein degradation, in ALS (Sugaya and Nakano). This article considers a “one-hit model,” i.e., a mathematical model in which the risk of cell death remains constant. The next review article by Fiebich and colleagues, instead, formulates a two-hit hypothesis that includes also neuroinflammation . Anti-neuroinflammatory approaches represent a promising strategy against neurodegeneration and this review discusses the role of P2 receptors, which can bind ATP released by injured cells. This represents a second detrimental event synergizing with aggregation and the possibility of decreasing neuroinflammation by targeting P2 receptors is accordingly discussed .Sugaya and Nagano propose an alternative hypothesis to explain the variability in the onset of familial ALS (fALS) despite same SOD1 genetic mutation. Based on available data for SOD1-linked fALS and a risk-based modeling approach, the authors suggest a SOD1 prion-like model of propagation. Spread of toxic aggregates through cell-to-cell transmission has been observed in several ND. However, the “protective aggregation hypothesis” according to which misfolded protein aggregates may represent a defensive response to protect cells from more toxic oligomeric species is hypothesized also for SOD1-linked fALS .In the context of neuroprotective strategies aiming at potentiating the cell antioxidant capacity, the inhibition of NMDA receptor and the calcium dependent enzyme nitric oxide synthase (nNOS) activities is discussed by 2+ overload is a pathogenic feature of sporadic and fALS, characterized by degeneration of motor neurons (MNs). In this original article by Mühling and colleagues, pharmacological stimulation of Ca2+ transporters in this vulnerable neuronal population is presented as a potential neuroprotective strategy. The authors show that, in SOD1G93A mice, mitochondrial and plasma membrane Ca2+ transporters are expressed at higher levels in MNs dissected from the hypoglossal nucleus. These findings are in line with an activity dependent Ca2+ clearance deficit and could represent a compensatory response to the disease .CaKreiner).In the last review closing this e-book, Grzegorz Kreiner elaborates on the difficulties of modeling ND and arguments that, despite the unavoidable differences between human and rodents, compensatory mechanisms shown in mouse models could help developing new hypotheses to devise neuroprotective strategies  to RP is here acknowledged.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 ability to develop cost-effective, scalable and robust bioprocesses for human pluripotent stem cells (hPSCs) will be key to their commercial success as cell therapies and tools for use in drug screening and disease modelling studies. This review outlines key process economic drivers for hPSCs and progress made on improving the economic and operational feasibility of hPSC bioprocesses. Factors influencing key cost metrics, namely capital investment and cost of goods, for hPSCs are discussed. Step efficiencies particularly for differentiation, media requirements and technology choice are amongst the key process economic drivers identified for hPSCs. Progress made to address these cost drivers in hPSC bioprocessing strategies is discussed. These include improving expansion and differentiation yields in planar and bioreactor technologies, the development of xeno-free media and microcarrier coatings, identification of optimal bioprocess operating conditions to control cell fate and the development of directed differentiation protocols that reduce reliance on expensive morphogens such as growth factors and small molecules. These approaches offer methods to further optimise hPSC bioprocessing in terms of its commercial feasibility. Human pluripotent stem cells (hPSCs) have opened fresh avenues in modern medicine that have the potential to revolutionise healthcare, particularly since the first derivation of induced pluripotent stem cells (iPSCs) , 2. HumahPSCs can provide a cornerstone of the regenerative medicine industry via the provision of cell therapies for diseases with unmet clinical needs. hiPSCs in particular might provide a diagnostic tool capable of assuaging the high late-phase failure rate of NCEs in clinical trials , 8. The 4 cells for indications such as macular degeneration to 108 cells for diseases such as diabetes .–30.28–30When examining process options for hPSC manufacture, it is important to consider not only the operational performance but also the consequences on the economics, quality, regulatory compliance, safety and flexibility. These key considerations are summarised in Reimbursement pressures have resulted in an increased awareness of the importance of estimating and improving manufacturing costs for stem cell products. This section discusses factors that influence two key cost metrics: fixed capital investment (FCI) and cost of goods (COG).The FCI represents the cost to build a manufacturing facility ready for start-up. It includes the cost of the building with the fixed (non-disposable) equipment, piping, instrumentation and utilities installed. Estimates of facility costs are often made using factorial estimates. These are well established for traditional stainless steel biopharmaceutical facilities using the Lang Factor method , which iThe COG represents the cost to manufacture a stem cell product and comprises direct  and indirect (e.g. maintenance) costs. Simaria et al.  summarisDecision-support software can aid the design of cost-effective bioprocesses. However, to date, few published cost studies exist for stem cell bioprocessing. Commercially available flowsheeting software packages have been employed to cost stem cell process designs at fixed scales . SimariaIn addition to considering the costs of stem cell bioprocessing alternatives, it is also useful to capture the impact of uncertainties such as lot-to-lot variability and contamination risks, particularly when manual processing techniques are employed . StochasIn order to achieve cost-effective bioprocesses for hPSCs, efforts need to focus on increasing the overall productivity and/or decreasing the overall production costs. Hence, critical process economic drivers for hPSC processes include expansion and differentiation yields as well as the cost of materials and labour. The remainder of this paper therefore focuses on progress made on the expansion and differentiation yields in planar and bioreactor technologies as well as the development of media and cell-anchorage materials for these unit operations.Until recently, planar hPSC culture platforms relied heavily on the use of xenogeneic growth substrates and non-human feeder layers, which risk contamination of hPSC-derived products. Furthermore, feeder layers differentially secrete signalling factors, resulting in poorly defined culture conditions that are unsuitable for empirical study of hPSC expansion . The devThe labour-intensive nature of T-flasks limits their throughput and applicability to larger-scale processes , 25. SysAutomated, closed-process systems such as the CompacT SelecT , capable of handling 90 × T175 flasks simultaneously, and the Nunc Automatic Cell Factory Manipulator (ThermoFisher Scientific), capable of manipulating 4 × 40 layer vessels, may help increase the throughput of 2D hPSC bioprocess strategies  during ePlanar processing platforms will continue to have a place in commercial hPSC bioprocesses. This is particularly likely for the production of autologous cell therapies and patient-specific hPSC-derived cells for personalised medicine drug screening that necessitate a scale-out, rather than a scale-up approach to bioprocess design .There are two main methods of 3D hPSC culture; the use of suspended microcarriers as adherent surfaces for stem cell growth , or growMicrocarriers are small beads or discs, which permit propagation or directed differentiation of hPSCs within a 3D bioreactor. hPSC studies investigating microcarriers have recently achieved expansion folds as high as 28-fold over 6 days . MicrocaA critical property of microcarriers is their high surface area to volume ratio, on which large populations of hPSC cells may be cultured in a relatively small vessel, alleviating the costs associated with expensive media and supplements necessary for hPSC bioprocessing , 56. MicThe common use of microcarrier coatings that contain animal-derived components, which are unsuitable for use in the manufacture of cell therapies, is a significant challenge to culture of hPSCs on microcarriers. Serum-free and feeder-free microcarrier platforms are available for hPSC-based processing, although many of these utilise the microcarrier coating, Matrigel, which is derived from murine origins . RecombiXeno-free microcarrier coatings utilise polymers to mimic Matrigel and feeder layer properties in order to encourage attachment and self-renewal of hPSCs on microcarriers. Expansion folds on xeno-free microcarriers comparable to those coated with Matrigel have been reported , 63. It Harvesting of cells from microcarriers is usually carried out using enzymatic separation, which can add to material costs associated with microcarrier-based culture of hPSCs. Microcarriers coated in thermo-sensitive polymers that allow detachment of seeded cells obviate the need for dissociation enzymes, although studies in this area are still in their preliminary phases in this area .Parallel to developing GMP-based hPSC expansion protocols, research has focused on optimising bioreactor conditions for dynamic hPSC expansion processes so as to increase achievable expansion folds and cell concentrations. This will help reduce COG associated with manufacture of hPSCs. Controlling the dissolved oxygen levels has been found to be critical during hPSC culture on microcarriers in SUBs; 2.5 higher expansion folds and ∼85% improvements in maximum cell concentrations were reported in a hypoxic environment when compared to uncontrolled conditions . FurtherhPSCs can be cultured as suspended aggregates in bioreactors. When hPSCs are grown as aggregates the rho-associated protein kinase inhibitor (ROCKi), Y-27632, is used to protect single cells from dissociation-induced apoptosis . Each ceAgitation rates can be used to successfully modulate uniform aggregate size in order to improve expansion of hPSCs as aggregates and reduce cell loss due to shear forces , 68. Thi5 cells/mL were found to maximise viability of hPSCs [6 hPSCs/mL have been achieved using dynamic, aggregate-based culture techniques [6 cells/mL) [The importance of cell inoculation concentration has been demonstrated during aggregate culture of hPSCs in dynamic bioreactor conditions; seeding concentrations of 2–3 × 10of hPSCs , 76. Sinof hPSCs . Cell coells/mL) .A few aggregate hPSC expansion processes combine xeno-free conditions with defined media , 69, 71.Traditional stem cell differentiation protocols were designed around bench-scale research paradigms and little effort was made to incorporate reproducibility and process robustness into these experiments . DirecteThe use of small molecules within differentiation protocols has helped to improve their reproducibility via reduced use of recombinant growth factors , 86. SevConcentrated research into bioreactor-based differentiation strategies has stemmed from the need to translate differentiation from a lab-scale area of research into processes capable of producing industrially relevant cell numbers in a reproducible manner. A number of studies in which hPSCs have been successfully differentiated in bioreactor conditions have been carried out, either attached to microcarriers  or in thhPSCs can be differentiated towards a number of clinically relevant lineages in SUBs including cardiac , haematoOne question arising from the birth of SUB-based hPSC differentiation is how a dynamic, controlled environment might be harnessed to augment processing strategies in this area . One of Novel, microfluidic systems could be a key tool in elucidating the effects of specific environmental parameters on hPSC propagation and differentiation. Microfluidic bioreactors provide an ultra-scale down, high throughput platform by which to study single cells or colonies in strictly defined conditions –107. TheThe effect of the biochemical properties of microcarriers on hPSC fate determination is poorly understood. It has also been suggested that the mechanical properties of microcarriers, such as their stiffness and size can also be investigated and optimised for specific purposes . RationaNovel, integrated hPSC bioprocesses, whereby multiple unit operations are carried out using continuous culture strategies, are being explored as an alternative to segregated bioprocess strategies. Integrated bioprocesses negate the need for labour-intensive processes that usually take place following hPSC expansion such as harvest and transfer of cells prior to differentiation. Processing hPSCs in this manner can help to avoid process bottlenecks and increase throughputs of hPSC product manufacture. Additionally, integrated iPSC bioprocess protocols offer greater containment capabilities, reducing the potential for contamination within the bioprocess. Several studies in which hPSC expansion and differentiation are carried out as a single unit operation using continuous culture strategies exist , 74, 77.Early investigations incorporating expansion and differentiation focused on overcoming the technical hurdles of carrying out two different operations in one integrated process step; as such only modest yields of target cells were achieved . Recent To date, no studies have produced an integrated process for hPSCs from derivation all the way through to differentiation. This has been achieved with mouse iPSCs (miPSCs) in a SUB . To our The fulfilment of the clinical potential of hPSCs depends on the development of scalable, robust, GMP-compliant bioprocesses. Concentrated efforts to develop rigorously designed bioprocesses with a greater emphasis placed on QbD will continue to enhance process understanding with respects to defining critical quality attributes and identifying key process variables that must be controlled. hPSC-derived products are subject to strict economic boundaries owing to the nature of global healthcare systems. Future work must build upon the promise of works reviewed in this paper in order to make delivering such products on budget an achievable goal. Novel approaches discussed in this review offer methods to further optimise hPSC bioprocessing platforms.Michael J. Jenkins is a doctoral research student in Biochemical Engineering at University College London (UCL) in the UK. Michael's project, in collaboration with Neusentis Ltd., focuses on cost-effective bioprocess design for hPSC-derived cell products including cell therapies and drug screening tools. His research involves the creation of novel decisional tools that combine process economics analysis, bioprocess optimisation and uncertainty analyses of hPSC bioprocesses. He obtained his Master's degree in Biochemical Engineering at UCL, with a year as an exchange student within the Biotechnology Department at Lund University, Sweden.Suzanne S. Farid is Professor in Bioprocess Systems Engineering at the Advanced Centre for Biochemical Engineering at University College London (UCL) in the UK and Co-Director of the EPSRC Centre for Innovative Manufacturing in Emergent Macromolecular Therapies. She leads research on ‘Bioprocess Decisional Tools’ that has pioneered the development of algorithms at the process–business interface to facilitate cost-effective bioprocess design, capacity planning and R&D portfolio management. She sits on the UK BioIndustry Association Manufacturing Advisory Committee and is a Fellow of the IChemE. She obtained her Bachelor's and Ph.D. degrees in Biochemical Engineering with UCL and Lonza Biologics.


Gua sha (press-stroking) and Ba guan (cupping) by Nielsen et al. deserves a passionate welcome, even though it seems to have room for improvement, especially in the field of cupping [This is a comment on “Addendum: Safety Standards for Gua Sha (Press-Stroking) and Ba Guan (Cupping)” . Interve cupping .Compromising with reality inevitably, recently published addendum of this guideline added a process for disinfection or sterilization of cupping devices, which might retract clinical importance of this guideline . Althoughttp://www.hira.or.kr/). From our experience of clinical practice and trials, these single-use cups can be utilized very safely and any specific adverse events hardly occur [Korean National Health Insurance Service has reimbursed the material costs of single-use, disposable cupping cups for wet-cupping treated by doctors of Korean Medicine (KM) abiding by the decision of National Health Insurance Policy Deliberative Committee, Ministry of Health & Welfare Since 2012. Presently, there are seven companies producing disposable cups after obtaining approval from the ministry . Any licly occur .Because there is not yet any survey result on the current state of utilization of disposable cupping in Korea, future related study will be necessary on the real usage of disposable cupping cups in KM clinics. But it seems to be that more and more doctors of KM move to using disposable cupping cups because they are safer than reused ones, and doctors do not need to pay for them. Patients who are prejudiced against the wet-cupping therapy for its unsanitary nature will participate in cupping treatment willingly in this situation. As several times used, sterilized blunt needles were thrown out by single-use, disposable needles for acupuncture treatment currently, single-use, disposable cupping cups will be a market leader in future. Government policy can also promote this process as it is working in Korea.


Neonatal hypoxic ischemic encephalopathy (HIE) is a devastating disease that primarily causes neuronal and white matter injury and is among the leading cause of death among infants. Currently there are no well-established treatments; thus, it is important to understand the pathophysiology of the disease and elucidate complications that are creating a gap between basic science and clinical translation. In the development of neuroprotective strategies and translation of experimental results in HIE, there are many limitations and challenges to master based on an appropriate study design, drug delivery properties, dosage, and use in neonates. We will identify understudied targets after HIE, as well as neuroprotective molecules that bring hope to future treatments such as melatonin, topiramate, xenon, interferon-beta, stem cell transplantation. This review will also discuss some of the most recent trials being conducted in the clinical setting and evaluate what directions are needed in the future. Neonatal hypoxic ischemic encephalopathy (HIE) is a devastating disease that primarily causes neuronal and white matter injury. HIE has tremendous detrimental effects on the developing brain and is among the leading causes of death among infants, as well as the major underlying cause of seizures in term infants ,2,3. AltNeonatal HIE can also be characterized as an injury that occurs in the immature brain, resulting in delayed cell death via excitotoxicity, inflammation, and oxidative stress . These aBefore the advent of hypothermia, clinicians were not able to provide much care to neonates suffering from HIE besides systemic supportive care . It is nNeonatal hypoxic ischemic encephalopathy is of great importance since it is the major cause and contributor to global infant mortality and morbidity . The incNeonatal hypoxic ischemic encephalopathy usually presents clinically in the earliest days of life in a term infant and can be characterized by difficulty initiating and maintaining respiration, depression of tone and reflexes, subnormal levels of consciousness, and multiple seizures .Since HIE is the major underlying cause of seizures in term infants, a large variety of seizures may present . Subtle,In order to classify neonatal HIE and the degree of injury, the Sarnat 3 stage grading system is used and widely accepted. This system consists of three stages ranging from mild, moderate, and severe, all based on clinical symptoms described above, along with electroencephalogram evaluation .Presently, there are no well-established effective therapies for neonatal HIE . HypotheSince the pathophysiology of HIE injury is quite complex, there are a myriad of potential interventional targets following HIE where prevention of cellular damage can occur  Figure . TargetiThe pathophysiology involving neonatal HIE consists of multiple phases . One theThe next phase that follows is a primary energy failure phase that occurs at the cellular level a. Since A second energy failure phase also occurs 6–48 h after an episode of hypoxia ischemia  Figure b. The seA third phase where deleterious factors cause further damage and potentiates injury and worsens outcomes has recently been proposed c. This tThe neurovascular unit, also referred to as the blood-brain barrier (BBB), consists of the basement membrane, capillary endothelial cells, tight junctions, pericytes, and astrocytes . It is bIt is believed that cytokines, nitric oxide, and vascular endothelial growth factor (VEGF) help regulate mechanisms that lead to tight junction disruption and increased BBB permeability . Increasin vitro and in vivo studies have revealed that VEGF and matrix metalloproteinases (MMP) are involved in the initial opening of the BBB within hours of an hypoxic-ischemic event [After HIE it is postulated that there are biphasic temporal patterns of BBB opening. Numerous ic event . While tic event . Once thAlthough many adverse outcomes occur once the BBB is permeable, there are experimental studies that show that stabilizing the BBB and reducing inflammatory markers after hypoxic ischemic events as well as HIE may be beneficial ,38,39,40Similarly another study utilizing a neonatal rat model administered plasminogen activator inhibitor-1 following an HIE insult. This group was able to demonstrate that inhibition of tissue plasminogen activator (tPA) was able to prevent MMP activation, prevent HIE-tPA induced opening of the BBB, and decrease tPA-converted plasmin activities as a protease that normally degrades the extracellular matrix and the BBB .Some strides have been made, yet further investigations are needed to better understand BBB integrity after HIE . It has Angiogenesis is characterized as the process involving the growth of new vessels that extend the existing blood circulation into avascular regions. The angiogenesis process consists of vascular basal lamina formation, proliferation and migration, and tubular formation of migrating cells. Some findings suggest that angiogenesis is activated after acute insult in the neonatal brain . One stuet al. observed increases of doublecortin, a microtubule-associated protein only expressed in immature neurons, with immunostaining in the subventricular zone ipsilateral to HIE-induced lesioning one to three weeks post injury. However, after the fourth week there were no identifiable newly formed mature striatal neurons, suggesting that there was limited neurogenesis at this time period. These results also suggest depletion of the neuronal progenitor pool since they are more vulnerable to HIE than SVZ stem cells or that the environment is not conducive to the maturation and survival of newly formed neurons since there maybe a lack of trophic support [Neurogenesis is comprised of cell proliferation, migration and differentiation . It is b support . Thus, a support ,51.et al. demonstrated that bone marrow derived mesenchymal stem cells treatment after HIE was able to increase neurogenesis and formation of new oligodendrocytes, evidenced by NeuN-positive and olig2-positive cells [There have been a number of promising stem cell therapy experiments that promote neurogenesis after injury. For example, van Velthoven ve cells . Again, ve cells ,53.After HIE, there is evidence of differential cellular death mechanisms often occurring in different cells. One cellular death mechanism that has increased scientific interests and has been recently implicated in HIE has a potential therapeutic target is autophagy ,54. Autoet al. demonstrated that brain-derived neurotrophic factor (BDNF) with a concentration range of 50 to 200 ng/mL protects neurons from hypoxia injury in vitro via the induction of autophagy [in vivo model of neonatal HIE brain injury found that lithium was able to inhibit post-ischemic autophagy after 72 h. This reduction of autophagy was indicated by decreased number of LC3-positive cells in lithium-treated animals 72 h post-HIE. Given these results, it is unclear if a reduction of autophagy occurred as a secondary effect to lithium induced neuroprotection since there is less cellular debris and damage contributing to autophagy processing or if lithium directly inhibited autophagy [For example, Chen utophagy . It was utophagy . Conversutophagy . Mixed rutophagy . Over thutophagy .The distinct mechanisms of injury-induced damage in immature brain is frequently studied, yet remains poorly understood, especially at early stages of development. Also, a thorough evaluation method of the developmental and behavioral sequelae after brain injury needs to be established .in vitro and in vivo models of experimental studies. In-vitro studies are relatively inexpensive and often precede a following in vivo analysis, yet it has its limitations of treating cells outside of their normal environment. Therefore, their results may differ from in vivo effects [in vivo tests can confirm in vitro results, the informative value of the results is more significant. In most cases, drug studies proven to be effective in vitro, show ineffective in animal models. The underlying reason may be drug delivery issues, toxicity, or other problems [In general, there are  effects . If in vproblems  Even thoproblems ,62,63.Especially since HIE and neonatal stroke are very heterogeneous entities, therefore finding the ideal method to study this disorder remains a difficult task . Ashwal Other new promising neonatal hypoxic-ischemic models have been introduced recently, like a perinatal global ischemic brain injury model in rats providing intrapartum hypoxia in rats . AnotherThe great variety of established models and promising new designs points out the importance of carefully choosing the appropriate model, especially for testing possible treatment strategies . A few kAlthough much progress in experimental development and technology has been made in the past few decades, the issue of discovering disease models, that can mimic human pathophysiology sufficiently, remains an ongoing challenge ,77,78.Treatment strategies for central nervous system (CNS) diseases are challenged by the drug delivery mechanisms for crossing the BBB . The undIn a recent publication, rapid and transient BBB disruptions were described after HIE in neonatal mice associated with alteration of tight-junction proteins. This response is often followed by activation of tightening compensatory mechanisms. More understanding of this phenomenon might provide an opportunity window for compounds to access the brain tissue easier after HIE .Additionally, in cases of neonatal HIE the clinical translation is more difficult when compared to adult drug administrations, since most of the pharmacological developments are approved only for adults and the regulations for pediatric and neonatal use are highly restricted. Due to the small size and huge variability of pediatric patients, neonatal pharmacotherapies are limited to mostly off label usage of compounds developed for adults to extrapolate dosages ,85,86. AProgressive research in neonates might improve and optimize future age-appropriate treatment demanding tailored, personalized clinical pharmacological and physiological understanding ,88,89,90Over the past decade, clinical and basic science research in neonates has achieved huge progress in neuroprotective and neurointensive care, and established hypothermia as the standard of care treatment for neonatal HIE. Although there has been much progress, further investigation is needed to discover adjuvant neuroprotective strategies . This isOsteopontin (OPN) is a mutifunctional glycoprotein with increased upregulation in the brain after neonatal HIE. OPN has both pro- and anti- inflammatory properties; thus, its exact role in injury is not well predicted ,134,135.Inflammation plays an important role in the pathology of HIE, interventions at the inflammation portion of the disease can potentially be beneficial . Thus, ic-Jun N-terminal kinases (JNKs) activation is associated with an assortment of environmental stressors and for that reason they are known as stress activated protein kinases ,142. ThrThe TAT-JBD peptide may serve as a treatment option for neonatal HIE due to its promising results in reducing neuronal damage and loss of mitochondrial activity through early JNK inhibition, with overall improvements in anatomical outcomes, therefore improving cognitive and behavioral results post-HIE. The present study shows that early JNK inhibition by the short-lived TAT-JBD peptide may be a promising therapy for neonatal HIE by conferring long-term anatomical and behavioral improvements .A retrospective study by Donald F. Meyn, Jr. and colleagues  analyzedTherapeutic interventions enabling prevention or reduction in hypoxia-induced brain damage before or during an earlier stage of free-radical production will require continued investigation for optimal effectiveness . These rN-acetyl-5-methoxytryptamine) is an endogenous indolamine and another scavenger that has shown promising effects in the treatment of HIE. It has antioxidant, anti-inflammatory, and anti-apoptotic properties [Melatonin (operties . Melatonoperties  or in cooperties . Aly andoperties . Intraveoperties . With itoperties  it holdsoperties  and resuEdaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) is a free scavenger that is thought to be useful for the treatment of acute cerebral stroke. Edaravone is believed to interact with peroxyl and hydroxyl radicals creating a radical intermediate that forms stable oxidation products ,106. Ni Erythropoietin (Epo) is a 34 kilodalton glycoprotein with pleotropic properties. Epo has been reported to have effects on a variety of receptor-mediated and cell-specific mechanisms that are beneficial and essential after HIE. Epo has been associated with anti-inflammatory, anti-excitotoxic, anti-oxidative, and anti-apoptotic effects as well as promoting neurogenesis and angiogenesis ,109,110.et al. demonstrated that a single dose of Epo (1000 U/kg i.p.) immediately after neonatal hypoxic–ischemic insult diminished long-term spatial memory deficits. In addition, a treatment group that received Epo but did not undergo HIE, showed no differential effects concerning learning or memory from the treatment [Hypoxia-inducible factor-1 mediates the increase of Epo after HIE in the brain, leading not only to an increase in Epo expression but also an increase in the Epo receptor in neurons, astrocytes, and microglia ,112. Eporeatment . One clireatment . Furtherreatment .There have been several completed clinical trials concerning Epo after HIE injury ,158,159.in vitro but also to be essential for neurogenesis and cell fate commitment [Epo exerts neuroprotection through phosphorylation of its receptor and Janus Kinase 2, which provides a docking complex for intracellular signaling proteins including PI3K as well as Akt, signal transducer and activator of transcription 5 (STAT5), and the extracellular signal-regulated kinase (ERK). Activating these pathways leads to alteration of cell proliferation, survival, and differentiation by affecting a number of downstream targets. For example, Akt limits inflammation  and decrmmitment ,163.Allopurinol is a xanthine oxidase inhibitor that lowers uric acid concentration in patients with gout and neoplastic diseases. In addition, allopurinol functions as a chelator of non-protein bound iron as well as a direct scavenger of hydroxyl radicals, suggesting it may serve in neuroprotection . Indeed,post hoc analysis revealed a potential benefit in treatment of females (NCT00189007) [In 2014, the same group published the results of another clinical trial. Maternal treatment with allopurinol during fetal hypoxia did not significantly lower neuronal damage markers in umbilical cord blood. However, 0189007) . There iXenon is a potent anesthetic with a low gas partition coefficient. It crosses the BBB easily and guarantees rapid induction of anesthesia. As an anesthetic it has proven to be safe in adults and well tolerated . HoweverImportant neuroprotective effects of Xenon have been demonstrated in adult rats in transient brain ischemia . In neonXenon was reported to be safe for use in a phase II randomized study outcomes after demonstrating to have similar results as cooling therapy alone . An ongo+ channels [Topiramate (TPM) is an anticonvulsant agent with multiple mechanisms of action ,175, impchannels , high vochannels , carbonichannels , and mitchannels .Even though no clinical studies have been published to prove an additive or synergistic action of TPM in concert with hypothermia in newborns, ongoing clinical trials (NCT01765218), Topiramate in Neonates Receiving Whole Body Cooling for Hypoxic Ischemic Encephalopathy, are investigating whether topiramate improves the outcomes of babies with neonatal hypoxic encephalopathy who are receiving whole body cooling. This trial is to be completed in 2017.TPM in concert with melatonin decreases infarcted volume and apoptosis in neonatal HIE rat model . In addiMelatonin and topiramate, acting on different stages of HIE, used alone or in combination, significantly decreased the percent infarcted area, and apoptotic cell death in neonatal HIE rat model. It is necessary to investigate different doses and application times of these agents as combination therapy in order to provide more effective neuroprotection. Furthermore, an ongoing trial: The NeoNATI trial (NCT01241019) will evaluate neurological outcomes at 6, 12, and 18 months of life and help clarify questions as to whether the administration of TPM in newborns with HIE potentiates the neuroprotective effect of treatment with hypothermia. They hypothesize that the combination treatment with moderate whole-body hypothermia associated with TPM administration is safe and enhances the neuroprotective properties of hypothermia for the treatment of neonatal HIE4) has gained a lot of interest in the research community due to its ability to alleviate excitotoxic damage in vitro by binding to the magnesium site on N-methyl-d-aspartate (NMDA) glutamate channel [Magnesium Sulfate  [Additionally, cord blood (CB) represents a rich source of stem cells used in several animal models of neurological diseases ,132,185.em cells . Studiesem cells ,186,187.0593242) ,188.ex vivo modulations, way of administration, and choice of stem cells [Due to the lack of imaging diagnostic difficulties to detect HIE in premature newborns and insufficient data, present stem cell therapy trials are restricted to full term infants ,190. Furem cells . It shouem cells , and gliem cells .As reviewed above, the complex etiology of HIE requires treatment that will act on multiple processes . There iAlthough there have been significant strides in the basic sciences to create novel neuroprotective and therapeutic strategies to combat HIE, there is still much more research needed to be conducted in order to translate potential therapies. Some gaps in our knowledge concerning the pathophysiology and the timing of important endogenous neuroprotective and neuroregenerative mechanisms still exist. In order to make basic science results more clinically relevant and translational, combinational therapies with hypothermia should be considered and studied. There is also a need for more biomarker studies that can be used along with the brain imaging, and long-term neuro-assessments ,194. Thu


Over a century ago, it was found that cancer cells often have extra chromosomes; that is, normal human cells contain 46 chromosomes, whereas cancer cells contain abnormal numbers of chromosomes with cell-to-cell variability. Polyploid giant cancer cells (PGCCs) refer to a special subpopulation of cancer cells with giant and multinuclei and contribute to solid tumor heterogeneity. PGCCs differ from normal cells and even other cancer cells in cell size, morphology, proliferation pattern, expression of cell differentiation markers, and chromosome numbers and contribute to tumor formation and chemoradioresistance. The shape of PGCC nuclei is usually irregular and the size is at least three to five times larger than those of regular diploid cancer cells. PGCCs are the key contributor to the heterogeneity of human solid cancers and chromosome structural abnormalities, such as inversions, deletions, duplications, and translocations.Mechanistically, PGCCs could be formed through end reduplication or cell fusion, reverting to regular cancer cells through splitting, budding, or burst-like mechanisms. PGCCs are divided asymmetrically and cycled slowly to form a dynamic population. However, these giant cells can also revert to regular-sized cancer cells through a reductive division, named as depolyploidization. Asymmetric cell division of giant cancer cells by meiosis-like depolyploidization had been previously proposed to explain the unexpected life cycle of these cells. In this special issue, D. Zhang et al. reported the asymmetric cell division in polyploid giant cancer cells and low eukaryotic cells and revealed the similarities in the budding process between yeast and PGCCs. This mechanism of PGCCs initialed the daughter cell generation which has also been reported in the normal growth of skeletal muscle and osteoclasts and in cells infected by virus or in vitro cell culture. Moreover, PGCCs were able to express certain normal and cancer stem cell markers and differentiate into the adipose tissue, cartilage, and bone. Single PGCC was able to form cancer spheroids in vitro and generate tumor xenograft in immunodeficient mice, indicating that these PGCCs had remarkable biologic features of cancer stem cells.Furthermore, PGCCs are able to generate erythrocytes in vitro and in vivo besides their cancer stem cell properties. The difference of erythrocytes generated by bone marrow and PGCCs is the different forms of hemoglobin (see below). In human body, erythrocytes are produced in the bone marrow with a process known as hematopoiesis. The bone marrow stroma contains mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs), which give rise to erythrocytes, leukocytes, and platelets. In adults, bone marrow is generally considered the main source of erythrocytes. However, PGCCs have an ability to generate erythrocytes in vitro and in vivo.α plays an important role in the VM formation, while L. Zhang et al. provided the evidence that erythroid cells were localized in the cytoplasm of or around the PGCCs in serous ovarian carcinoma tissues and cancer cells in the VM structures and that these erythroid cells expressed hemoglobin-β/γ/ε/δ and hemoglobin-ζ detected by immunostaining. Thus, these VM structures can be formed by PGCCs or other cancer cells and their newly generated fetal erythrocytes with high O2 binding affinity.During cancer development, tumor cells undergo avascular growth. However, after a tumor mass reached a certain size, vasculogenic mimicry (VM) will connect with endothelium dependent vessels to obtain sufficient blood and oxygen supply to support further growth of tumor cells and support tumor invasion and metastasis. Accumulating evidence has demonstrated that different types of cancer utilize VM to form a blood supply network to support their growth, invasion, and metastasis and, clinically, such a tumor is usually associated with poor prognosis. However, the source of erythrocytes in VM remains unclear. PGCCs can be induced by treatment of cancer cells with cobalt chloride (a hypoxia mimic) in vitro and hypoxia will increase self-renewal of cancer stem cells and promote the stem cell-like phenotype besides induction of PGCCs formation. Moreover, hypoxia also promotes the formation of vasculogenic mimicry (VM). B. Sun et al. showed that hypoxia inducible factor-1In addition, in this special issue, W. Wang et al. and L. Yao et al. demonstrated that epithelial-mesenchymal transition and Wnt signaling pathway could regulate the VM formation. Thus, elucidation of the molecular mechanisms of PGCC and VM formation could provide a novel insight into research in embryology, stem cells, and tumorigenesis. Identification of the PGCCs and tumor-derived erythrocytes could be a survival mechanism in hypoxia and targeting of PGCCs might be further developed as a potential therapeutic strategy for human cancers. Research focus on VM-targeted therapies could include dendritic cell vaccine and cytokine-induced killer cell therapy to conquer the recurrence and metastasis of aggressive cancers.Shiwu ZhangXiaochun XuSiwei ZhuJun Liu


Hypoxic-ischemic (HI) injury to developing brain results from birth asphyxia in neonates and from cardiac arrest in infants and children. It is associated with varying degrees of neurologic sequelae, depending upon the severity and length of HI. Global HI triggers a series of cellular and biochemical pathways that lead to neuronal injury. One of the key cellular pathways of neuronal injury is inflammation. The inflammatory cascade comprises activation and migration of microglia – the so-called “brain macrophages,” infiltration of peripheral macrophages into the brain, and release of cytotoxic and proinflammatory cytokines. In this article, we review the inflammatory and immune mechanisms of secondary neuronal injury after global HI injury to developing brain. Specifically, we highlight the current literature on microglial activation in relation to neuronal injury, proinflammatory and anti-inflammatory/restorative pathways, the role of peripheral immune cells, and the potential use of immunomodulators as neuroprotective compounds. Regions of the developing brain become vulnerable to hypoxic-ischemic (HI) as the neurons develop operating voltage-dependent ion channels, neurotransmitter receptors, synaptic connections, and increased mitochondria to supply the ATP for the consequent increase in energy demand . Global A cascade of cellular and biochemical responses to the initial HI insult can lead to secondary neuronal injury after reoxygenation. One of the crucial but understudied mechanisms of secondary neuronal injury after global HI is inflammation . It is cTherapeutic hypothermia has been shown to protect the brain after cardiac arrest in adults and after HI in term newborns –11; it iIn this article, we review current understanding about the inflammatory and immune mechanisms involved in acute HI injury of developing brain and the neuroprotective agents that can curtail inflammation and immune dysregulation. New anti-inflammatory targets continue to be identified and constitute an important area for translational medicine –22. OverA wealth of information on HI brain injury and repair is available through translational adult stroke research. The immature brain differs from the adult in its capability to use metabolic fuels, vulnerability to glutamate excitotoxicity, and oxidative stress . There iGlobal HI of brain initiates a cascade of excitotoxicity and oxidative damage that in turn causes microvascular injury, blood–brain barrier (BBB) dysfunction, and postischemic inflammation. These events all exacerbate the initial injury and can lead to permanent cerebral damage. The modes of secondary neuronal loss after HI are apoptosis, autophagy, programed necrosis, and unregulated necrosis, which arise from cell swelling and bursting of nuclear and cell membranes . ApoptosFor many years, the brain was considered an immune-privileged organ, but advances in neuroimmunology have challenged this dogma and helped to expand our understanding of the immune processes that occur in healthy and diseased brain . It is wMicroglia are resident macrophages of brain and are known to actively remove cellular debris during normal development and under pathologic conditions. They were first identified as innate cells of the central nervous system (CNS), distinct from neurons and other glia, by Nissl in 1899. Many years later, in the early part of the 20th century, del Rio Hortega confirmed this distinction using silver carbonate staining methods  and propAlthough microglia are highly active during pathologic conditions, they are also active under physiologic conditions as they survey the microenvironment and the status of neurons and participate in housekeeping and remodeling within the CNS . In deveN-methyl-d-aspartate (NMDA)-mediated excitotoxicity. These mechanisms not only scavenge the HI damaged neurons but they can also affect viable neurons. Much of our current knowledge of inflammation, particularly microglial activation, after HI injury of developing brain comes from the Vannucci model, in which HI is induced in postnatal rats and mice by exposure to low oxygen and carotid ligation -9 and breakdown of collagen and laminin in the vascular basement membrane  levels rise significantly , a neurotrophic factor with neuroprotective properties . In a roMicroglia are kept under check through neuronal–glial cross talk. Chemokine receptors and corresponding ligands allow interactions between neurons and microglia and control proinflammatory responses of microglia under physiologic conditions . CD200/CStudies from adult rodent models of focal HI suggest that, in addition to innate inflammation, the peripheral immune system may have a role in the etiology of neuronal damage. More precisely, several studies have shown that acute brain injury from focal ischemia is associated with a massive activation of the peripheral immune system, with rapid mobilization of immune effector cells from the spleen , 124. ThPreconditioning, salvaging, and repair are three main modes of achieving neuroprotection .Hypothermia is now standard of care for term HI encephalopathy, so studies focused on adjunct therapies will be added to that treatment . As our Cannabinoids are diverse chemical compounds, which are either endogenously produced in body (endocannabinoids) or derived from cannabis and related plants (phytocannabinoids) or prepared chemically (synthetic cannabinoids). CB compounds act on cell surface CB receptors to exert a variety of effects, including potent anti-inflammatory and immunomodulatory effects. There are two major subtypes of CB receptors – CB1 and CB2. The CB1 receptors present on neurons produce the psychoactive effects of non-selective CBs like tetrahydrocannabinol . CB2 recCannabidiol is a major constituent of the cannabis plant, representing up to 40% in plant extracts. CBD has shown neuroprotective effects in adult rodent models of stroke –144. CBDTNF-α is a potent proinflammatory cytokine that plays a key role in neurotoxicity after ischemia. Anti-TNF-α has been shown to be neuroprotective in rodent models of focal cerebral ischemia , 147. ThRecombinant human IL-1 receptor antagonist (rhIL-1ra) has been shown to protect against focal cerebral ischemia in the rat through its actions on microglia . A randoMinocycline is a tetracycline derivative that has been shown to be safe and effective as an antibiotic and anti-inflammatory drug for treating systemic inflammatory conditions. Minocycline crosses the BBB and has demonstrated neuroprotective qualities in experimental models of post-arrest global HI , traumatIbudilast is mainly a phosphodiesterase (PDE) inhibitor, but it also affects the function of lymphocytes, endothelial cells, and glial cells. In a neuronal-microglial co-culture study, ibudilast suppressed neuronal necrosis that was induced by LPS and interferon-γ activation of microglia . IdibulaRecent evidence supports the involvement of vitamin D3 in immunologic processes that protect the nervous system . In the Steroid molecules, including sex steroids, inhibit microglia and prevent release of proinflammatory cytokines. Steroid molecules also offer neuroprotection through release of neurotrophic factors from microglia . HoweverStatins have been shown to reduce infarct size in experimental animal models of stroke. Statins attenuate the inflammatory cytokine responses that occur after cerebral ischemia, and their antioxidant properties ameliorate ischemic oxidative stress in the brain. Additionally, statins upregulate endothelial nitric oxide synthase and inhibit iNOS, effects that are potentially neuroprotective . CurrentPropentofylline acts by blocking the uptake of adenosine and inhibiting the PDE enzyme system , 175. AdIn preclinical models of term brain HI, melatonin markedly decreased microglial activation in association with neuroprotection . A studyEpo and its receptor (EpoR) are expressed in the developing CNS and are required for normal brain development . Epo inhXenon is an odorless, dense noble gas with anesthetic properties. Xenon’s neuroprotective properties have been demonstrated in cell culture , a rodenN-acetyl-l-cysteine (NAC) to microglia to suppress neuroinflammation, using much lower concentrations than are needed with systemic dosing (Anti-inflammatory and immunomodulator agents like CBD are hydrophobic and, therefore, delivery across BBB becomes an important consideration for optimal neuroprotective effects within a safe dose-range. A novel approach is to use alternative delivery methods like nanoparticles, targeting the inflammatory system. Nanoparticles, such as polyamidoamine dendrimers have been shown to concentrate in activated microglia and astrocytes in the brains of newborn rabbits with cerebral palsy, but not healthy controls. This nanotechnology approach has shown excellent results to deliver dendrimer-bound c dosing –195.Neuroinflammatory and neuroimmune dysregulation play a key role in secondary neuronal damage after global HI injury in the developing brain. Microglia activated by HI, initiate a cascade of inflammatory reactions that lead to neuronal damage. Indeed, a growing number of anti-inflammatory and immunomodulatory compounds have shown promising neuroprotective effects in preclinical settings. Some of these compounds have even entered clinical trials for adult victims of stroke. However, substantial work is needed to improve our understanding of neuroinflammation after global HI injury in developing brain. The specific issues yet to be determined in relation to inflammatory and immunologic mechanisms after global HI include the temporal, topographic, and gender pattern of neuroinflammation, neuronal–glial regulatory interactions, and their contribution to secondary neuronal injury, the role of the peripheral immune system in potentiating secondary brain injury after multi-organ ischemia, and the development of anti-inflammatory and immunomodulatory compounds as neuroprotective agents after HI injury of developing brain.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.