Organism-level systems biology in mammals aims to identify, analyze, control, and design molecular and cellular networks executing various biological functions in mammals. In particular, system-level identification and analysis of molecular and cellular networks can be accelerated by next-generation mammalian genetics. Mammalian genetics without crossing, where all production and phenotyping studies of genome-edited animals are completed within a single generation drastically reduce the time, space, and effort of conducting the systems research. Next-generation mammalian genetics is based on recent technological advancements in genome editing and developmental engineering. The process begins with introduction of double-strand breaks into genomic DNA by using site-specific endonucleases, which results in highly efficient genome editing in mammalian zygotes or embryonic stem cells. By using nuclease-mediated genome editing in zygotes, or ~100% embryonic stem cell-derived mouse technology, whole-body knock-out and knock-in mice can be produced within a single generation. These emerging technologies allow us to produce multiple knock-out or knock-in strains in high-throughput manner. In this review, we discuss the basic concepts and related technologies as well as current challenges and future opportunities for next-generation mammalian genetics in organism-level systems biology. After identification of key genes by classical forward and reverse genetics, systems biology in mammals has been further accelerated by a series of genome projects, especially at the molecular-to-cellular levels, where in vitro cell culture systems allow system-level identification, analysis, control, and design of molecular networks. On the other hand, organism-level systems biology in mammals still remains an important challenge in biology.4Systems Biology is a natural extension of molecular and cellular biology,In order to identify and analyze molecular networks and/or cellular circuits in organisms, gene knock-out (KO) or knock-in (KI) are powerful technologies often used in mammalian reverse genetics. However, this classical genetics requires several generations of crosses to produce mutant animals of sufficient quality and quantity for phenotype analysis. The time consuming conventional methods for producing KO or KI mice usually involve targeting-vector construction , the introduction of target mutations into embryonic stem cells (ESCs) by homologous recombination (a few weeks), and the injection of the mutant ESCs into wild-type blastocysts to produce chimera mice (~3 weeks). If the mutant ESCs contribute to the germ-line of the newborn chimera mice, their next-generation offspring will possess a heterozygous mutation (~3 months). Further crossings of the offspring  will produce mice with completely homozygous KO or KI mutations on an inbred genomic background, which is required for reliable phenotype analysis. Thus, conventional methods require substantial amounts of time, space, and effort to knock out or knock in even a single gene. Therefore, to comprehensively identify and quantitatively analyze molecular networks and/or cellular circuits in organisms in an efficient manner will require next-generation genetics, i.e., genetic alterations without crossing. In this review, we discuss the basic concepts and related technologies as well as current challenges and future opportunities for next-generation mammalian genetics in organism-level systems biology.7 Various genetic tools can be also introduced by transgenic (Tg) mice techniques.8Mammalian genetics (particularly in mice) has been widely exploited in order to investigate complex and dynamic biological processes executed by molecular networks and/or cellular circuits in organisms. Forward genetics (germline mutagenesis and gene-trap) and reverse genetics (targeted KO or KI) are available in mouse genetics as in other model organisms such as yeast, nematode and fly. Especially, developmental engineering based on the establishment of cultured ESCs was often used to generate KO and/or KI mice.9 the generated strains additionally need to be backcrossed to a ‘standard’ inbred strain such as C57BL/6 (hereafter denoted as B6) several times. This labor-intensive step is practically required in most cases because F1 hybrid strains or 129 strain-derived ESCs are commonly used in Tg zygote production or targeting in ESCs, respectively due to their higher viability or efficient germ-line transmission in F0 chimera.However, the production of genome-edited mice has been generally low-throughput, and needed huge time and effort in the conventional ways Fig. . For exa12 and for systematic gene function studies.14 The gene-trap strategy has more recently been applied to such forward-genetics approaches, and >100,000 of trapped ESC lines have been established and kept in international organizations .15 Other systematic international efforts to collect, prepare and maintain mutant mice and ESCs have also been performed, such as the International Knockout Mouse Consortium/International Mouse Phenotype Consortium (http://www.mousephenotype.org).19 Multiple Cre Tg/KI strains have also been established by individual researchers, institutes and international consortiums.22 However, to carry out organism-level systems biology, these large-scale efforts should be scaled down to the single-laboratory scale or even to the single-researcher scale. To address this technological challenge, next-generation mammalian genetics without crossing is proposed here to allow completion of KO or KI mouse production and phenotyping analysis within the F0 generation  non-homologous end joining (NHEJ), where the broken ends are directly reconnected with a frequent insertion or deletion of a random number of bases (denoted as “indels”),24 (3) microhomology-mediated end joining (MMEJ), where a small microhomology fragment is used for the end-joining.25 Induced DSB and the following repair processes enable highly efficient genome editing (both KO or KI) at the broken locus. Although the genome editing induced by DSBs was previously investigated by introduction of I-SceI  into cultured mammalian cells, which stimulated extrachromosomal homologous recombination,26 DSB-induced genome editing has been practically used in recent years. We review such efficient genome editing methods using site-specific nucleases, which can accelerate the production of KO and KI mice via relatively simple steps and thus help realize next-generation mammalian genetics  lead to several DNA repair pathways, such as (1) homology-directed repair (HDR) where a homologous DNA sequence is used for recombination or annealing,27 zinc-finger nuclease (ZFN),28 transcription activator-like effector nuclease (TALEN)29 and clustered regularly interspaced short palindromic repeats (CRISPR)-associated Protein9 (Cas9).30 ZFN and TALEN are categorized into customizable endonucleases because they are composed of a customizable sequence-specific DNA-binding domain fused to a nonspecific DNA catalytic domain of FokI endonuclease.31 On the other hand, Cas9 is a RNA-guided endonuclease and recruited to specific DNA sequences by a short RNA guide molecule that recognizes target DNA via base-pairing.32Three major classes of site-specific endonucleases have been used for genome-editing,34 The individual zinc finger recognizes 3 bp in the major groove of DNA.35 Selected zinc-finger modules that recognize nearly all of the 64 possible nucleotide triplets have been developed. Therefore, an assembled custom array of six zinc fingers can be constructed to recognize a unique 18 bp sequence.38 This length of target sequence covers a possible 68 billion unique DNA sequences.The DNA-binding domain of ZFN contains 3–6 arrays of Cys2-His2 zinc finger motif.Xanthomonas.40 The individual TALE repeat recognizes a single base pair via two hypervariable amino acid residues inside the repeat (repeat-variable di-residues or RVDs). The four most common RVDs  are known to recognize each of the four nucleotides . Therefore, the tandem TALE-repeat is usually constructed with approximately 18 TALE repeats of different base pair-binding specificities, under consideration of its limitation that TALE-binding sites should start with a T base. The TALE repeat domain generally gives similar DNA-binding specificity and more flexibly when compared with ZFNs.41TALEN contains another type of customizable DNA-binding domain, which is composed of a tandem 33–35-amino acid repeat (TALE repeat) derived from the plant pathogenic bacteria genus FokI endonuclease catalytic domain is essential for cleavage of DNA by ZFN and TALEN.31 This means that two ZFN or TALEN molecules must bind on both right and left sides of the target site with an appropriate orientation and spacing. Therefore, the dimer recognizes 2-fold longer sequence at the target site than single ZFN or TALEN molecules. This molecular property gives higher specificity and reduced off-target effect.Dimerization of the 32 the CRISPR RNA (crRNA) which anneals with the target sequence, and the trans-activating crRNA (tracrRNA) which is partially complementary to the crRNA and anneals to the crRNA. This two-component RNA system was further simplified to synthetic single-guide RNA (sgRNA) consisting of a fusion of crRNA and tracrRNA.42 The target sequence in the CRISPR/Cas9 system can be readily changed by simply re-designing a part (around 20 bp) of the crRNA or sgRNA. This simplicity is in contrast to the much more burdensome procedures in ZFN and TALEN vector construction. This simplicity endows the CRISPR/Cas9 system with a significant advantage for use as a site-specific endonuclease for various genome editing purposes, including multiple gene KO,44 or even genome-wide gene perturbations.46Unlike the former molecules, Cas9 is an RNA-guided DNA endonuclease derived from the type II bacterial adaptive immune system CRISPR, and is recruited to specific target sequences by two short RNA molecules:47 PAM sequence varies according to the CRISPR-based systems and organisms, and restricts the flexibility of the target sequence. However, the sequence dependency of the Cas9 molecule can be artificially modified and such PAM engineering can expand the target range of the system.49 Other modification of Cas9 molecules contribute to off-target suppression. Cas9 induces DSBs at approximately three bases upstream of the PAM by two endonuclease domains, a RuvC-like endonuclease domain (RuvC domain) and a HNH-like endonuclease domain (HNH domain), which are located at the amino terminus and the mid-region of the Cas9, respectively.50 The RuvC domain cleaves the non-complementary strand while the HNH domain cleaves the crRNA-complementary strand. Inactivation of these endonuclease domains via point mutations can convert Cas9 endonuclease into a DNA “nickase” that creates a single-stranded break, which reduces off-target activity by 50-fold to 1500-fold in cell lines and zygotes without sacrificing on-target cleavage efficiency.52 Others have tried FokI-dCas9 fusion protein as a dimer to improve targeting specificity by their recognition of distinct sites.54 Use of truncated sgRNA can also suppress undesired off-target activity by >5000-fold without sacrificing on-target genome editing efficiency, possibly by decreasing the sgRNA-DNA interface.55Many studies have tried to increase the flexibility and decrease any off-target effect of the CRISPR/Cas9 system for practical use. The DNA cleavage activity of Cas9 molecules is dependent on the presence of a short (around 2–6 nucleotides) protospacer adjacent motif (PAM), which is located beside the complemental sequence of crRNA/sgRNA-targeted region.25 . Potential off-target effects can be also excluded by using the second set of triple sgRNAs that covers ~70% of all mouse genes.62DSBs induced by site-specific endonucleases activate an internal DSB-repair pathway, which is exploited for efficient genome editing. Among them, NHEJ-dependent indel insertion is the simplest and the most effective method for gene KOTargeted insertion or KI of a DNA fragment with mutated sequence, short functional sequence , or functional expression cassette can be also facilitated via HDR, NHEJ and MMEJ by co-transfer of linear or circular donor vector, PCR fragment or single-stranded oligo DNA nucleotide (ssODN) together with the site-specific endonucleases Fig. .69 The targeting rate is relatively low but efficiently enriched by antibiotic drug selection in the culture. A shorter functional sequence or small mutation can be more simply introduced by using ssODN.75Homologous recombination (HR)-dependent targeting is mediated by a form of HDR. This pathway has been widely used for a large fragment insertion or KI both in cultured cells and zygotes by using a donor targeting vector with long homology arms.77 In the NHEJ-mediated insertion, both the donor plasmid and the target genome loci are digested simultaneously. And then, the digested donor plasmid is integrated into the digested genome loci. A PCR fragment or double-stranded ODN can be also applied as an integrated fragment without digestion. This pathway works not only in cultured mammalian cells (including ESCs) but also in zebrafish, and does not necessarily require antibiotic selection.81 In addition, there is no need to prepare a targeting vector with long homology arms, which is generally a time-consuming process. On the other hand, it is of note that the direction of the inserted fragment is not controllable, and indels are usually introduced at the junction site. Therefore, the method is inappropriate for some KI purposes, such as in-frame KI of an exogenous ORF into an endogenous gene.NHEJ-mediated fragment insertion/KI is easier and more efficient than the HR pathway, because the NHEJ-repair reaction is thought to predominate over the HR reaction for DSB repair.83 Therefore, the editing pathway potentially overcomes problems in HR-mediated or NHEJ-mediated KI.MMEJ-mediated editing provides more simplified KI strategy with precise direction and junction sequence. Instead of the conventionally used long homology arms for HR-mediated KI, this pathway uses only extremely short microhomology sequences (10–40 bp) for the precise fragment insertion. MMEJ-mediated KI also works in mammalian cells, and the inserted fragment can be supplied as an in vivo digested plasmid or a PCR fragment.87The compelling advantages of the site-specific endonucleases in efficient genome-editing has been examined in recent years. In particular, zygotic genome editing enables one-step production of genome-edited animals, skipping the in vitro targeting step in ESCs. Introduction of components into one-cell zygotes are relatively simple and easy, particularly for the CRISPR/Cas9 system, which just requires cytoplasmic microinjection or electroporation.57 where up to 75% of live-born F0 founders were harboring mutations. TALEN has been similarly tested,88 while CRISPR/Cas9 was mainly used in the most recent studies, since 2013.90 This method accelerates the generation of KO animals via the co-injection of RNA encoding the Cas9 protein and target-locus-specific guide RNAs into embryos. Long deletions of a genomic region (10–100 kb) were induced by using two sgRNAs.92 Others reported F0 phenotyping of CRISPR/Cas9 KO animals,93 suggesting the potential of this method for use in next-generation genetics schemes. Several modifications of the CRISPR/Cas9 system have been also introduced to improve the efficiency and specificity of targeted mutations in a genome.61 However, two problems have remained: (1) first-generation mice often contain a mosaic of wild-type and KO cells, and (2) the rate of whole-body bi-allelic mutant mice generated is relatively low . Therefore, the highly efficient (>90%) production of whole-body bi-allelic KO in a single generation remained a fundamental challenge for next-generation mammalian genetics. To realize this vision of next-generation mammalian genetics, the triple-CRISPR method significantly improved bi-allelic modification efficiency and further elicited almost perfect (~100%) whole-body bi-allelic KO mice62 , short functional sequences or even a large reporter cassette were tested mainly by using CRISPR/Cas9 system.101 In contrast to the improved KO rates in the one-step production scheme, zygotic KI by HDR still remains inefficient, particularly in the case of long fragment insertion by homologous recombination . Several studies have tried to improve the genome-editing (KI) rate. For example, inhibition of the NHEJ pathway by administration of DNA ligase IV inhibitor (Scr7) gives a 2 to 4-fold increase of HDR rate in mouse zygotes,97 although another study debated the capacity of this inhibitor in human models.102 Similarly, the treatment with an actin polymerization inhibitor  increases the HDR targeting rate presumably due to the delayed DSB repair.99 The use of Cas9 protein rather than synthesized mRNA also increases HDR rate.98 One of these studies showed an increase of the genome-editing (KI) rate, up to ~45% KI efficiency of live-born pups by injecting Cas9 protein complex with synthesized dual-crRNA:tracrRNA into pronuclei.98 The use of Cas9 protein also reduces mosaicism when introduced with proper timing in early zygotes.101 Another study reported the generation of two KI newborns out of 123 injected embryos, where one was heterozygous KI while another was homozygous KI.92 ssODN-mediated KI, in which the cut sites of the targeted genome and the inserted fragment are ligated with 80-bp ssODNs homologous to the two cut ends 103 although testing this in zygotes still remains to be done. Please note however, that the reproducibility of these recent studies should be further examined because some of the conclusions are based on small number of experiments. Further improvements in the targeting rate and specificity will be needed for one-step generation of KI mice.On the other hand, one-step production of KI mice (zygotic KI) is still under development. An earlier study using ZFN reported that the KI mice were generated in one-step manner at the production rate of 1.7–4.5% .105 In recent studies, an HDR targeting rate over 10% was achieved in ESCs even using very short (0.5 kb) homologous arms together with CRISPR/Cas9.104 Multiple KO ESCs were also prepared in one-step manner, at the rate of approximately 20% in a triple KO experiment.59 The other modern genome-editing methods discussed above may potentially to be applicable as well.Although one-step generation of KI mice would be ideal for next-generation mammalian genetics, a number of issue have yet to be overcome, specifically inefficient editing and live-birth rate (particularly with a large fragment KI) and undesirable indel insertion and mosaicism in resultant animals. Alternatively, two-step generation of KI mice by almost completely ESC-derived mouse (“ES mouse”) is an attractive alternative at present. The advantages of using ESCs are in its selectivity of sex, easier storage and flexibility for more complex genome editing (e.g. multi-gene KO and KI) in in vitro culture. Furthermore, genome editing in ESCs is becoming easier by the site-specific nucleases.113 However, several possible drawbacks of the method are known. First, substantial contamination of host cells was often observed in chimera mice produced by this method, which can cause developmental abnormalities.115 Second, the genetic heterozygosity of both tetraploid embryos and ESCs seems to be crucial for survival of the resultant ES mice,114 which means that the use of inbred ESCs does not seem possible and further backcrossing is required. In addition, preparing hundreds of tetraploid embryos every time does not seem practical for routine generation of many ES mice. For these reasons, few reports have used tetraploid complementation in a large-scale phenotyping assay of ES mice.Validity of ES mouse production and phenotyping analysis within a single generation was first proposed and tested by using the tetraploid complementation method.118 The eight-cell injection/aggregation, in which totipotent host cells and ESCs as epiblast precursors are used, produces almost completely ESC-derived mice with ~0.1% contamination of host embryo cells. Furthermore, it is striking that ES mice from inbred strains, such as B6 and BALB/C were efficiently generated at the live-birth rates of 11~29% (ES mice/embryos transferred), which was comparable to the 129 strain (9~15%) and hybrid ES mice (6~40%) and significantly higher than in blastocyst injection in any case .117 Therefore, F0 phenotyping of inbred ES mice generated with 8-cell injection/aggregation was considered plausible in the study. Possible drawbacks would be that the ES mouse production rate might depend on the ‘quality’ of cultured ESCs. A further optimized method to stably maintain ESCs in their naive pluripotent state was thus required.Alternatively, ES mice can be generated by ESCs injected into or aggregated with eight-cell embryos rather than the conventionally used blastocyst embryos.119 and others). Historically, the leukemia inhibitory factor–signal transducer and activator of transcription 3 (LIF/Stat3) pathway has been found to be indispensable for maintaining pluripotency and self-renewal ability of ESC.123 More recently, additional pathways essential for ESC maintenance or differentiation were uncovered. One is the Wnt–β-catenin pathway which supports ESC propagation and maintenance of the pluripotent naive state, and is antagonized by glycogen synthase kinase-3 (Gsk3)–Tcf3 .128 The other is fibroblast growth factor 4–mitogen-activated protein kinase (Mek)–mitogen-activated protein kinase (Mapk or Erk) pathway which leads ESC differentiation and thus its inhibition suppresses ESC differentiation.130 Therefore, naive pluripotent ESCs can be stably maintained by shielding the cells from these differentiation triggers, and by addition of inhibitors  and in serum-free conditions.133 3i/2i-cultured ESCs exhibit restricted expressions of lineage-affiliated genes and stabilized gene expressions involving a naive pluripotent state via epigenetic modulations and appropriate control of the pluripotency factors.139Secretory regulatory factors and their downstream mechanisms for maintaining ESC’s naive pluripotency have been well studied 141  are similar to, or even lesser than conventional chimera mouse production.The administration of 3i/2i enabled more efficient creation and maintenance of ESCs from even inbred mouse strains (including B6) or rat,141 Fig. . In addi149 Aneuploidy detection in cell culture populations is also important for ESC’s quality control. This can be performed by not only a conventional karyotyping but also by a droplet digital PCR-based screening.150 Furthermore, additional chemical treatments can possibly ameliorate ESC culture conditions. So far, a variety of chemicals including ROCK inhibitor,152 PKC inhibitor,154 ERK/p38 inhibitor,155 HDAC inhibitors  may potentially contribute to improved potency of ESCs. Therefore, applications of these chemicals and routine quality control may help accelerate next-generation genetics based on ES mouse production.To further improve the efficiency of ES mouse production, better culture methods and ESC quality control will be needed. Genomic instability, in particular, should be avoided during culture because chromosomal aneuploidy can cause embryonic death. Telomere extension seems important for maintaining normal karyotype of ESCs, and frequent activation of telomere maintenance factor Zscan4 restores and maintains the ESC’s potency in long-term culture.161 are developing rapidly in recent years, high-throughput genome-edited mouse production is required for their in vivo implementation. Next-generation mammalian genetics potentially enables a single laboratory or a single researcher to generate, maintain and analyze multiple genome-edited strains rather than institutes or consortiums for production, deposit and distribution of various strains and ESCs. Because sgRNAs for targeted sites can be readily designed and prepared, the CRISPR/Cas9 system, in particular, makes such large-scale genetics feasible. Indeed, a recent study generated 31 novel CRISPR-KO mice lacking testis-expressing genes.162 Since whole-body bi-allelic KO rates were not sufficiently high for the next-generation scheme, the authors performed the crossing of selected F0 founders based on sequence and PCR screening data. In order to realize almost perfect (~100%) whole-body bi-allelic KO rate for next-generation mammalian genetics, we recently performed triple-CRISPR-based large-scale reverse genetics for sleep research. To identify genes involving neural electrophysiological activities during sleep or wake, we first developed an average neuron model in silico and found that genes involved in intracellular Ca2+ regulation  are important for electrophysiological slow-wave-oscillation patterns during sleep. To further assess the roles of these genes in vivo, we next produced KO mice for 33 genes with the triple-CRISPR methods and eventually identified 8 genes important for regulating sleep duration.163As discussed above, high-throughput KO or KI mouse production is pivotal for accelerating system-level identification, and analysis of molecular networks and cellular circuits in organisms. Given that various genetic tools, such as optogenetics and chemogenetic tools164 In this experiment, a 3i + LIF-cultured ESC clone derived from a double-KO mouse lacking two core clock genes (Cry1 and Cry2) was established. A rescue expression cassette of the wild-type or mutated Cry1 gene was then homologously knocked-in in the ESC clone . Double-KO ES mice and KO-rescue ES mice were then generated and used for F0 phenotyping to measure in vivo 24 h rhythmicity. As explicitly indicated by these examples, next-generation mammalian genetics enables large-scale organism-level experiments within reasonable time, space and labor.ES mouse technology can deal with more complex genome editing, which would be difficult, if not impossible, with the conventional crossing-based genetics. For example, we also produced ~20 KO-rescue ES mice in order to perform system-level analysis of circadian clock-gene circuits in organisms.62 the yields of bi-allelic KO mice lacking tyrosinase gene (judged by the white coat color) were 17% on average, and 36% in the best case, of the injected and transferred B6 zygotes. Therefore, between 60 (average) and 30 (best case) of host embryos would be enough for generating a sufficient number (around 10) of bi-allelic KO mice. The rate of bi-allelic tyrosinase KO mice among the F0 littermates was 97.5% on average and 100% at best. Similarly, at least in our ES-mouse experiments of Cry1 rescue in the Cry1/Cry2 DKO background,164 the yield of ES mice available for phenotyping was 5.5% on average, and 35% in the best case, of the injected 8-cell embryos. Therefore, between 170 (average) and 30 (best case) of host embryos would be enough for generating a sufficient number (around 10) of ES mice. The rate of ES mice among the F0 littermates was 43% as average and 91% at the best. Only the littermates of embryonic lethal, non-KO or non-ES mice were sacrificed and no further animals are needed. The number of animals used is thus much smaller than the conventional methods, in which a similar number of host embryos are used for injection, and only a part of the founders or chimera mice are used for further crossing. In the conventional case, dozens of littermates are produced and sacrificed during crossing to select mice with an expected genotype. With conventional methods the number needed exponentially increases when a more complicated genetic background  is desired, while with next-generation genetics the number of used animals is not dependent on genetic complexity.Next-generation genetics is also important for improving animal welfare and 3R principles, particularly contributing to “reduction” of animal use. In our triple-CRISPR experiments,164 suggesting that mutational variations (triple-CRISPR) or undetectable contamination of wild-type cells (ES mouse) do not seem problematic at lease in these cases. To further exclude the possibility of artifact phenotypes due to mutational variations or undetectable contamination of wild-type cells, we recommend that researchers independently generate a whole-body bi-allelic KO mice by using a second set of triple-CRISPR for the same gene, or to independently generate a whole-body bi-allelic KI mice using an independent clone of ES cells. Such stringent criteria (the production of independent KO or KI mice to confirm the observed phenotype) is sometimes difficult to fulfill with conventional mouse genetics because it takes a couple of years for another round of production. On the other hand, this step is feasible with next-generation genetics because it only takes a couple of months. In this sense, the quality of scientific studies using next-generation genetics should exceed that of scientific studies using conventional genetics.On the other hand, researchers need to take special care regarding some issues with the use of F0 animals for phenotype studies. In particular, researchers should carefully consider to what extent potential mosaicism  would affect the final results of a scientific study. In our above experiments, the phenotypic variations of F0 mice were comparable with those in wild-type or suitable control animals,164 and tyrosinase triple-CRISPR KO mice for a sleep study62), (2) evaluation of genetic composition of the F0 animals with strict criteria . In addition, it is also useful, if necessary, to evaluate undesirable mutations in the coding sequences by exon sequencing163 of triple-CRISPR KO mice. It is also useful to avoid cumulative mutations165 by using ESCs with minimal passage numbers for the production of ES mice. Moreover, these experimental procedures should be described according to a general guideline for animal experiments . Further efforts to minimize problems from these issues should be expanded in future studies.Another point that researchers need to consider carefully is experimental design. This includes (1) preparation of appropriate control animals without any obvious defects in the focused phenotype (e.g., ES mice from wild-type ESCs or ESCs without genome editing for a clock gene rescue study,Next-generation mammalian genetics will facilitate system-level analysis of molecular and cellular circuits in organisms. To further improve the throughput for genome-edited animal production, additional developments of new technologies related to next-generation mammalian genetics will be required. For example, one-step production of whole-body bi-allelic KI mice by more efficient genome editing of zygotes still remains unachieved. For the two-step production of KI mice using ES mouse technology, the preparation of host embryos and surrogate mothers are still labor intensive and pose limitations on its throughput. New technologies which overcome these limitations will further accelerate next-generation mammalian genetics and also reduce the number of experimental animals used to obtain the same information.167 to systematically survey the functions of molecular networks in organisms. Although most large-scale organism-level phenotyping projects are usually labor-intensive, the development of more facile alternatives is possible. Recently, whole-body clearing and imaging methods with single-cell resolution have been developed169 and started to provide comprehensive and quantitative experimental data at the cell-to-organism level, further facilitating organism-level systems biology. The development of non-invasive phenotyping will be also an attractive direction. For example, sleep phenotyping represents such a recent attempt where a non-invasive sleep phenotyping system was used instead of a conventional invasive EEG/EMG-based measurement system. In fact, these non-invasive methods have already enabled sleep phenotyping of dozens of triple-CRISPR KO mice.163 Organism-level systems biology is thus coming to fruition with next-generation mammalian genetics, whole-body clearing and imaging with single-cell resolution, and non-invasive and quantitative phenotyping methods. Organism-level systems biology based on such new technologies will accelerate our understanding of complex and dynamic molecular and cellular circuits in the near future.Next-generation mammalian genetics together with efficient, quantitative and non-invasive phenotyping methods will provide an attractive platform for investigating the organism-level functions of the molecular and cellular circuits of interests. Genome-wide phenome analysis has been performed in international KO mouse phenotyping efforts


Drosophila visual system. The exact mechanism by which Bdl mediates axon-glia recognition, however, remains unknown. Here, we present evidence showing that Bdl interacts with the Ig transmembrane protein Turtle (Tutl). Tutl is specifically expressed in photoreceptor axons. Loss of tutl in photoreceptors, like loss of bdl in WG, disrupts glial extension and axon ensheatment. Epistasis analysis shows that Tutl interacts genetically with Bdl. Tutl interacts with Bdl in trans in cultured cells. We propose that Tutl interacts with Bdl in mediating axon-glia recognition for WG extension and axon ensheathment.Proper recognition between axons and glial processes is required for the establishment of axon ensheathment in the developing nervous system. Recent studies have begun to reveal molecular events underlying developmental control of axon-glia recognition. In our previous work, we showed that the transmembrane protein Borderless (Bdl) is specifically expressed in wrapping glia (WG), and is required for the extension of glial processes and the ensheathment of photoreceptor axons in the developing  The ensheathment of axons by glial processes is required for insulating axons for propagating action potentials . The estDrosophila adult visual system reveals several important genes in the control of glial development and axon ensheathment. For instance, the FGF signaling pathway has been implicated in regulating glial proliferation, migration and differentiation [Recent data from genetic dissection of the development of the ntiation . At the ntiation , 4. Uponntiation .The mechanisms underlying axon-glia recognition for R-cell axon ensheathment remains unclear. In our previous study , we identurtle (tutl) gene encoding for a transmembrane protein, a member of the conserved IgSF9/Dasm1/Tutl subfamily of the Ig superfamily [bdl-like WG extension and axon ensheathment phenotype. Further molecular and genetic analysis support that WG-specific Bdl interacts with Tutl on R-cell axons in mediating axon-glia recognition for WG extension and axon ensheathment.To determine the exact mechanism by which Bdl mediates axon-glia recognition, we set out to identify R-cell axon-surface factors that interact with Bdl on WG in the developing fly visual system. We found that mutants defective in the erfamily –10, dispIn our previous study , we showtutl eye-mosaic mutants in which the majority of eye tissues were homozygous for tutl mutations. Since tutl mutant clones were generated by eye-specific mitotic recombination, the tutl gene was selectively removed in R cells, but not removed in glia. We found that anti-Tutl staining was absent in tutl mutant R cells in the eye disc, and was also missing in subretial space where tutl mutant axons projected into, while R-cell differentiation remains normal in tutl eye-mosaic tissues  of homozygous tutl tissues were generated in the developing eye. This allows the removal of the tutl gene in R cells, but not in WG. The patterns of R-cell axonal projection and WG extension in tutl eye-mosaic mutants were then analyzed. In wild type, developing R cells in the eye disc project their axons basally into subretinal space, and then migrate posteriorly into the optic stalk , the extension of WG in the lamina was severely disrupted  displayed a mild WG extension phenotype . This result suggests a role for Htl in WG extension that is different from that of Tutl and Bdl.Previous studies by us and others show that the FGF8 receptor Heartless (Htl) is required for WG extension and axonal ensheathment , 5. Howe and htl , 11, wasbdl disrupted WG extension and the ensheathment of R-cell axons. To determine if tutl mutations cause a similar phenotype in R-cell axonal wrapping, we performed ultrastructural analysis to examine R-cell axon ensheathment in the optic stalk at third-instar larval stage.In our previous study , we showtutl in axon ensheathment, we performed eye-specific genetic mosaic analysis. Large clones (>90% of eye tissues) of homozygous tutl eye tissues were generated, which removed tutl in R cells, but not in glia. Interestingly, we found that like loss of bdl in WG [tutl in R-cell axons caused defective entheathment of R-cell axons in the optic lobe. Compared to that in wild type, the number of bundles completely wrapped by glial processes decreased significantly in tutl eye-mosaic animals . In our previous study [To test if Bdl is capable of interacting with Tutl in trans on cell surface, we performed cell-cell co-aggregation studies. Bdl and Tutl were expressed in us study , we showtutl in R-cell axons, like loss of bdl in WG, disrupts WG extension and axon ensheathment. Tutl displays trans interactions with Bdl in cultured cells, and interacts genetically with Bdl in the control of WG extension and axon ensheathment in the developing visual system. We propose that Bdl on WG interacts with Tutl on R-cell axons in mediating axon-glia recognition in the developing visual system.Our previous study suggests that Bdl functions as a WG-specific cell surface receptor in mediating WG extension and axon ensheathment by interacting with an unknown ligand on R-cell axons . In thistutl disrupts WG extension and R-cell axon ensheathment, but does not affect R-cell differentiation and axonal projections. In our previous study [Our results identify a novel and important role for Tutl in mediating glial extension and axon ensheathment at third-instar larval stage. Loss of us study , we showus study –14.tutl enhanced the phenotype in flies with mild Bdl overexpression , tutlK14703 (BDSC#10451), UAS-htl. DN (BDSC#5366) were obtained from the Bloomington Drosophila Stock Center. UAS-tutl-RNAi transgene and the tutl23 allele (i.e. Δtutl) in which most of the tutl genomic sequence was deleted, were generated in our previous study [bdlEX2 and tutl/bdl double deletion  alleles were generated in our previous study [tutl01085 or tutl23 mutant tissues were generated in an otherwise heterozygous or wild-type eye by eye-specific mitotic recombination using the eyFlp/FRT system [tutl in R cells, UAS-tutl-RNAi flies were crossed with flies carrying the eye-specific driver GMR-GAL4. To overexpress Bdl in WG, flies carrying the WG-specific driver Mz97-GAL4 were crossed with flies carrying the mild UAS-bdl transgene generated in our previous study [htl. DN flies were crossed with flies carrying the WG-specific driver Mz97-GAL4. BDSC#1099, tutlKN BDSC#536 were obT system . For knotutl-GFP and UAS-bdl-RFP were generated in our previous studies [UAS- studies , 7. Bdl  studies , includiWhole mount eye–brain complexes from third-instar larvae were dissected and stained as described previously , 19. AntEye-brain complexes from late third-instar larvae were dissected, fixed and sectioned similarly as described in our previous study . Images Drosophila S2 cells (kindly provided by Dr. David Hipfner at IRCM) were transfected with plasmid DNA (0.2 μg actin-GAL4 and 1.8 μg UAS construct) for the expression of GFP, Tutl-GFP or Bdl-RFP similarly as described in our previous studies [ studies , 7. Co-a studies . A cell p value is <0.05.Two-tailed t-tests were used for analysis of two paired samples. One-way ANOVA were used for multiple comparisons. The difference is considered as significant when a


Accumulating evidence shows that tumours contain a distinct subpopulation of cancer stem cells (CSCs) which are capable of self-renewal, differentiation, and tumour-initiation,2. PreviIn recent years, multiple studies have demonstrated that aldehyde dehydrogenase (ALDH) can serve as a specific marker for cancer stem cells in a variety of cancers–10. We fFor a vaccine to be clinically relevant, it needs to be examined in the therapeutic models. Since solid tumours harbor only a small number of cancer stem cells,12, therTo explore the mechanism (s) underlying the antitumour effect of the CSC-DC vaccine, we assessed the systemic immune responses elicited by DC-based cancer stem cell vaccination. Our results showed that CSC-DC vaccination conferred host CSC-specific CTL activity and antibody responses, resulting in significantly reduced population of ALDH(high) CSCs in treated tumours. In addiProgrammed death ligand 1 (PD-L1) is a critical molecule found on the surface of tumour cells. PD-L1 molecules render tumour-reactive T cell tolerance to tumour cells by binding to programmed death-1 (PD-1) expressed on activated T cells,19. AntiIn spite of the fact that solid tumours account for the major cancer burden, over 90% of mortality in cancer patients were attributed to the subsequent spread of cancer cells to distant tissues. CSCs meThe involvement of chemokines and their receptors in cancer development, particularly metastasis, has been established over the last decade. We examTogether, these experiments have offered direct evidence that CSC-DC vaccine could induce significant antitumour effect by immunologically targeting cancer stem cells. Combination of CSC-DC vaccine strategy with traditional therapy, e.g. surgery, chemotherapy and/or radiation therapy; simultaneously blockage of immune checkpoint–mediated immune suppression; investigation and application of novel adjuvant, and understanding of the mechanism(s) underlining the CSC-based vaccine represent critical respects in future research and application of CSC-targeted cancer immunotherapy.


Hematopoietic stem cell transplantation is a potent form of immunotherapy, potentially life-saving for many malignant hematologic diseases. However, donor lymphocytes infused with the graft while exerting a graft versus malignancy effect can also cause potentially fatal graft versus host disease (GVHD). Our group has previously validated the inducible caspase-9 suicide gene in the haploidentical stem cell transplant setting, which proved successful in reversing signs and symptoms of GVHD within hours, using a non-therapeutic dimerizing agent. Cellular death pathways such as apoptosis and necroptosis are important processes in maintaining healthy cellular homeostasis within the human body. Here, we review two of the most widely investigated cell death pathways active in T-cells (apoptosis and necroptosis), as well as the emerging strategies that can be exploited for the safety of T-cell therapies. Furthermore, such strategies could be exploited for the safety of other cellular therapeutics as well. HSV-TK) suicide gene [ΔiC9) suicide gene, the latter validated by our group [ΔiC9 suicide gene activates the apoptotic pathway in gene modified cells after administration of an otherwise inert agent inducible of dimerization [Effector T-cells are key players of adaptive cellular immune responses, protecting the host from infectious pathogens, cancer, and other cells foreign to the body. In the context of allogeneic hematopoietic stem cell transplantation , engrafted T-cells of donor origin also contribute to the so-called graft-versus-tumor effect, which together with the conditioning regimen, aids in tumor surveillance and/or eradication . Howeveride gene  and the ur group ,7,8. Whirization . Importarization ,7,8, witThis review will summarize the molecular pathways of programmed cell death and the in vitro and in vivo investigations of suicide gene strategies for the safety of T-cell therapies with a focus on prevention/treatment of GVHD. Such strategies could be exported to other T-cell therapies as well as to other cellular therapeutics.T-cell survival is influenced by the signals the cell receives through the (i) T-cell receptor (TCR), co-stimulatory molecules, including CD28; (ii) adhesion molecules; (iii) cytokines and (iv) other pro- or anti-apoptotic molecules. All of these factors are important for the optimal function of T-cells. In fact, in the absence of appropriate survival signals, T-cells undergo activated cell-autonomous death (ACAD) , whereasCaspase-dependent apoptosis can be activated through the extrinsic cell-death-receptors pathway, and/or through the intrinsic mitochondrial pathway .In the extrinsic apoptotic pathway, cell-death-receptor-adaptor molecules ) deliver pro-apoptotic signals  that areThe intrinsic apoptotic pathway is triggered by TCR stimulation, DNA damage, endoplasmic reticulum (ER) stress, hormones, or cytokine deprivation. The extrinsic and intrinsic apoptotic pathways converge at the level of the effector caspases, as activated caspase-8 is also able to cause cleavage of the B-cell lymphoma 2(BCL-2)-family protein BH3-interacting-domain death agonist (BID) to generate truncated BID (tBID). tBID induces the pro-apoptotic functions of the mitochondria by causing the oligomerization of BAX (BCL-2-associated X protein) and/or BAK (BCL-2 antagonist/killer). Pro-apoptotic proteins of the BCL-2 family can be classified according to the number of BCL-22 homology (BH1-4) domains in their sequence. BAX and BAK, for example, are multi-domain proteins containing the BH1, BH2 and BH3 domains. The oligomerization of effectors  on the mitochondrial outer membrane (MOM) provokes its permeabilization , with reLife or death pathways in T-cells are dictated by the balance of anti-apoptotic factors and pro-apoptotic factors. Examples of anti-apoptotic factors blocking downstream caspases activation include the caspase-8 (FLICE)-like inhibitory protein (FLIP) for the extrinsic pathway . FLIP, aThe intrinsic apoptotic pathway is also regulated by the balance of pro-apoptotic and anti-apoptotic members of the BCL-2 family, by other mitochondria derived molecules, and by other caspase inhibitors. BCL-2 inhibitor of apoptosis inhibits the apoptotic process in several steps, by controlling calcium flux  and bindThe same molecule can act as either pro-apoptotic or anti-apoptotic depending on the type of activation signal involved. For example, the activation of caspases-8, -3 and/or -7 through TCR stimulation rather than cell death pathways can exert a pro-survival function ,36. EarlThe regulation of life and death of T-cells uses molecular mechanisms that may be different in distinct T-cell populations, depending on their state of activation. Studies in cell lines and primary human and murine T-cells show that while full length hematopoietic progenitor kinase 1 (HPK1) leads to activation of the NF-κB complex upon TCR stimulation, whereas its proteolytic fragment HPK1-C blocks NF-κB activation after TCR ligation . The autApoptosis is a clean process without inflammation or tissue destruction because apoptotic cells are rapidly engulfed by phagocytes (efferocytosis), due to the translocation of phosphatidylserine from the inner leaflet to the outer leaflet of the plasma membrane as a consequence of caspases activation. After being engulfed by the phagocytes, apoptotic cells are transported to lysosomes, where their components are degraded into building units for re-use . InternaSeveral laboratories have tested the possibility of promoting antigen specific tolerance for the therapy of GVHD , by developing methodologies that mimic the mechanisms by which the immune system maintains peripheral tolerance in the steady state. The principle is that administration of apoptotic donor cells would target host APCs and down-regulate its immunogenic function to control GVHD . InteresSome investigated strategies aimed at blocking the interaction between molecules associated with T-cell function, potentially resulting in reduced GVHD without compromised graft-versus-tumor (GVT) activity. One such strategy included blocking fibroblast growth factor-inducible 14 (FN14) interaction with tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK). This investigation was performed in Balb/c mice (H-2d) engrafted with allogeneic bone marrow and T-cells from black 6 (B6) donor mice (H-2b), as well as murine lymphoma cells. Tweak-Fn14 inhibition was accomplished using an anti-FN14-IgG1 antibody variant with compromised antibody-dependent cellular cytotoxicity (ADCC) activity. This inhibition reduced the incidence and severity of gastrointestinal GVHD. Importantly, Fn14 blockade showed no negative effect on GVT activity . To date+ T-cells led to 100% survival of mice with lymphoma and minimal GVHD, while mice receiving control T-cells all succumbed to lymphoma and GVHD. This positive effect was reproduced with human T-cells with TRAIL overexpression leading to enhanced cytolysis of tumor cells and alloreactive T-cells, as compared with control T-cells. This combination of decreased GVHD and increased GVT effect makes this genetic engineering of donor T-cells in the allo-HSCT setting a potentially promising cell therapy [Another strategy involved the infusion of TRAIL overexpressing donor T-cells in mice with resulting increased induced apoptosis of alloreactive T-cells with reduced GVHD in a death receptor 5 (DR5) dependent manner, with an unexpected increase of in vitro and in vivo GVT effect. TRAIL therapy .+ T-cells immediately after allo-HSCT using a single dose of human anti-CD4 monoclonal antibody IT1208, currently under clinical investigation for treatment of advanced solid tumors and in vivo murine leukemia and lymphoma models. This approach led to increased interferon gamma production and Programmed death-ligand 1 (PD-L1) upregulation of PD-L1 on recipient GVHD-targeted tissues. The interaction with PD-1 expressing CD8+ T-cells and PD-L1+ target tissues of GVHD resulted in T-cell exhaustion and apoptosis, thereby preventing acute GVHD but not chronic GVHD, which was associated with reconstitution of donor CD4+ T-cells beginning by day 21 post HSCT. However, three doses of anti-CD4 monoclonal antibody on days 0, 14, and 28 prevented both acute and chronic GVHD, and subsequently recovered donor CD4+ T-cells did not cause chronic GVHD. Conversely, PD-L1 upregulation on donor CD8+ T-cell and subsequent interaction with CD80 on host lymphoid tissues led to increased CD8+ T-cell survival and GVT effect [Ni et al. reported on the temporary depletion of donor CD4T effect .Leclerc et al. have demonstrated that blocking TNF/Tumor Necrosis Factor Receptor 2(TNF-R2) interaction on T-regulatory cells (T-regs) prevented GVHD without inhibiting the GVT effect. This was accomplished infusing a TNF-R2 mAb to C57BL/6 mice allografted with bone, marrow, T-cells, with or without HY-antigen specific T-regs from sex mismatched cells from sex mismatched C57 black 6 (C57BL/6) mice . Targeting TNF/TNF-R2 interaction represents an opportunity to efficiently modulate alloreactivity in allo-HSCT to either exacerbate it for a powerful antileukemic effect or reduce it to control GVHD .Mizraih et al. found that ex vivo exposure of mobilized peripheral blood hematopoietic progenitor cells to FAS-ligand (FASL) and TNF-α apoptotic ligands could inhibit GVHD without impairing GVT. This was investigated in non-obese diabetic mice with severe combined immunodeficiency disease (NOD.SCID) engrafted with human mobilized peripheral blood hematopoietic progenitor cells and the human colon carcinoma HT29 cell line, where functional assays revealed that the death receptors’ modulated graft composition as compared with incubation in medium, without detectable quantitative variations. Apparently, pre-treating mobilized peripheral blood hematopoietic progenitor cells with toxic doses of FASL and TNF-α did not affect their proliferative and engraftment potential . All in The deeper understanding of the molecular pathways of apoptosis in T-cells may open the door to innovative therapeutic strategies to prevent or eliminate GVHD, autoimmunity, and eventually overcome the inhibitory effects of the tumor on T-cell mediated immune responses.Spencer and colleagues, using cell permeable synthetic ligands that can bind FK506 Binding Protein 12 (FKBP12) , demonstIn order eliminate the immunosuppressive and toxic effect of calcineurin inhibition , FK506 vThe first reports of dimerization-induced apoptosis involved inducible Fas receptors (iFas) ,57. OtheIn designing a gene therapy strategy, it is important to consider the effect of the over-expression of the pro-apoptotic candidate gene on cell survival, as, for example, mere overexpression of the wild type BAX molecule has been demonstrated to significantly limit cell viability . MoreoveDifferent requirements on cleavage and dimerization for each molecule, together with the different design of the transgene constructs, may explain why enforced dimerization of some molecules, such as caspase-3, resulted in inducible cell death in some experiences ,53, but Caspase-9 is part of the intrinsic apoptotic pathway. It is activated by the release of cytochrome C from damaged mitochondria through its interaction with apoptotic protease-activating factor 1 (APAF-1). Caspase-9 can then activate caspase-3 and the other terminal effector molecules. The optimized ΔiC9 molecule consists of one FKBP12-F36V binding domain linked, via a Ser-Gly-Gly-Gly-Ser linker, to a caspase-9 molecule. The caspase recruitment domain (CARD) is removed from ΔiC9 because its physiological function of effecting caspase-9 dimerization is now superfluous .ΔiC9 gene modified donor T-cells has resulted in significant in vivo expansion and persistence, both in patients who developed GVHD and in patients who did not. Administration of a single dose of a non-therapeutic dimerizing agent resulted in ≥90% gene modified cell elimination within hours, with resolution of acute GVHD within 24 h [ΔiC9 suicide gene modified T-cells after allo-HSCT has resulted in effective control of GVHD, and, although the number of patients treated thus far is low, it has the potential of reducing GVHD-related morbidity and mortality.Infusion of hin 24 h . Intereshin 24 h ,8. The ihin 24 h , epigenehin 24 h , or selehin 24 h . The emehin 24 h . DespiteConsidering the sigmoid dose-response curve of the homodimerizer on ΔiC9, one current research interest involves dose reduction of the dimerizer to activate the ΔiC9 switch, in order to control GVHD without abrogating it, thereby potentially preserving anti-tumor immunity to the highest degree.The ΔiC9 strategy is now being investigated in different settings, such as in patients with relapsed hematologic malignancies after related transplants , as well as add-back after haploidentical transplants in patients with non-malignant conditions (NCT02065869 13 February 2014).ΔiC9 suicide gene, the HSV-TK, being almost completely human derived, proved immunogenic, especially in patients with a higher degree of T-cell immune reconstitution, with limited persistence of HSV-TK cells [Contrary to the TK cells . AdditioTK cells . In bothTK cells ,77,78, lTK cells . The lacTK cells ,7.Necroptosis is a form of caspase-independent cell death in T-cells occurring especially during viral or bacterial infections, with the purpose to kill pathogen-infected cells before the invading pathogen proliferates. Necroptosis is a form of molecular programmed necrosis characterized by swelling of the organelles and plasma membrane rupture , triggered by a variety of stimuli, including infectious agents, intracellular disturbances , cell surface death receptors’ activation, such as toll-like receptors 3 and 4, or TNFR1, and other factors as reviewed in detail elsewhere . Unlike TNF produced by macrophages has the ability to induce apoptosis or necrosis, especially in the presence of inhibitors of protein and DNA or RNA synthesis. TNF kills cells by necroptosis when the apoptosis pathway is inhibited; in fact, activated caspase-8 exerts a blocking effect on necroptosis. When cells are infected by a virus or bacteria, their transcriptional or translational machinery is inhibited; in addition, viruses and bacteria often encode molecules that inhibit apoptosis, thereby sensitizing the target cells to TNF-induced necroptosis.Recruitment of TRADD by stimulation of TNFR1, as described in the previous paragraphs, also attracts receptor-interacting protein kinase 1 (RIPK1), forming TRADD-dependent receptor complex I. RIPK1 is then subject to Lys63 polyubiquitylation by IAP1 or cIAP2, activating NF-κB, with resulting upregulation of anti-apoptotic genes such as FLIPL ,82. A moNecroptosis is involved in lymphocyte homeostasis , as wellSeveral groups have designed inducible dimerization systems in order to more fully understand the molecular mechanisms leading to activation of necroptosis . This syContrarily, Wu et al.  demonstrEnforced dimerization of MLKL through addition of a gyrase domain also resulted in potent inducible cell death in vitro . Being aInducible necroptosis could be exploited for the safety of T-cells or other cellular therapeutics, albeit dedicated validation experiments would need to be accomplished for this goal.Models of inducible necroptosis and inducible apoptosis also offer a framework for studying the increasingly recognized interactions between the two processes. A better understanding of this relationship could open the door to future research aiming at exploiting these important cell death pathways for cellular therapy, as well as anti-cancer treatments. Although enforced dimerization of inducible proteins of the apoptotic pathway or the necroptotic pathway led to impressive inducible cell elimination, in order to grant complete elimination of the gene modified cells, one potential strategy involves the combination of two inducible molecules. Perhaps, combining one inducible molecule from the apoptotic pathway and one inducible molecule from the necroptotic pathway could counteract immune-evasion strategies characteristic of each death process.Suicide gene strategies have the potential to increase the safety and the clinical applicability of novel cellular therapies. The infusion of suicide gene modified T-cells after allo-HSCT has resulted in effective control of GVHD, which can substantially reduce GVHD-related non-relapse morbidity and mortality. Current research strategies aim to maximize the positive impact on both non-relapse and relapse mortality in this setting.Suicide gene application can also be beneficial for the safety of other T-cell applications, such as the infusion of chimeric antigen receptor (CAR) redirected T-cells ,95,96, aConsidering that there are many human diseases associated with abnormal cell death processes, especially autoimmune, inflammatory conditions, and cancer, detailed findings about cell death pathways will also contribute to the understanding of disease pathogenesis, with the potential of developing novel forms of treatment. As a matter of fact, many agents are under active investigation targeting anti-apoptotic factors and cell survival proteins for the treatment of patients with cancer, with the goal of inhibiting cancer cell survival and potentially reversing chemoresistance .In summary, investigations regarding the balance between cell survival and cell death represent a fascinating chapter of investigative medicine, with multiple potential clinical applications.


We report the development of multiplexed cellular super-resolution imaging using DNA-barcoded binders. in situ in fixed cells.Recent advances in super-resolution fluorescence imaging allow researchers to overcome the classical diffraction limit of light, and are already starting to make an impact in biology. However, a key challenge for traditional super-resolution methods is their limited multiplexing capability, which prevents a systematic understanding of multi-protein interactions on the nanoscale. Exchange-PAINT, a recently developed DNA-based multiplexing approach, in theory facilitates spectrally-unlimited multiplexing by sequentially imaging target molecules using orthogonal dye-labeled ‘imager’ strands. While this approach holds great promise for the bioimaging community, its widespread application has been hampered by the availability of DNA-conjugated ligands for protein labeling. Herein, we report a universal approach for the creation of DNA-barcoded labeling probes for highly multiplexed Exchange-PAINT imaging, using a variety of affinity reagents such as primary and secondary antibodies, nanobodies, and small molecule binders. Furthermore, we extend the availability of orthogonal imager strands for Exchange-PAINT to over 50 and assay their orthogonality in a novel DNA origami-based crosstalk assay. Using our optimized conjugation and labeling strategies, we demonstrate nine-color super-resolution imaging  Compared to complementary characterization methods such as electron microscopy,1 fluorescence imaging allows the efficient and specific detection of targets like proteins or nucleic acids using affinity labeling reagents such as antibodies.2 However, the spatial resolution of conventional fluorescence microscopy is limited, by the diffraction limit of light, to ∼200 nm. Large efforts have been devoted to overcome this limitation, resulting in a number of so-called super-resolution methods that can nowadays readily achieve sub-20 nm resolution in cells.3 Most super-resolution microscopy techniques, such as Structured Illumination Microscopy (SIM),4 Stimulated Emission Depletion (STED) microscopy,5 (fluorescent) Photo-Activated Localization Microscopy ((f)PALM)7 and (direct) Stochastic Optical Reconstruction Microscopy ((d)STORM),9 to this date rely on target labeling using static or fixed fluorescent tags. This labeling is usually achieved via either genetically encoded fusion proteins (PALM) or immunolabeling using dye-conjugated antibodies . While these super-resolution approaches have already enabled new biological findings, some limitations persist. Two of the major limitations of single-molecule localization-based techniques such as PALM or STORM are the hard-to-control photophysical properties of fluorophores and the limited photon budget of fixed target labels.Fluorescence microscopy has become a standard method for 10 In this technique, fluorescently labeled ligands freely diffuse in solution and bind either statically or transiently to targets of interest.11 This binding is detected as an apparent “blinking” of the target molecule or structure of interest. This enables the decoupling of blinking from the photophysical dye switching properties and thus alleviates one issue of STORM or PALM. However, the binding of diffusing ligands to their targets is achieved by electrostatic or hydrophobic interactions and is thus hard to program for different target species in a single cell, thus preventing easy-to-implement multiplexed detection. DNA-PAINT,17 a variation of PAINT, achieves stochastic switching of fluorescence signals between the ON- and OFF-states by the repetitive, transient binding of short fluorescently labeled oligonucleotides (“imager” strands) to complementary “docking” strands that are conjugated to targets  technique. targets . Upon bi23 On the other hand, the large sizes of these complexes leads to steric hindrance in the labeling process, which impedes the achievable labeling density and efficiency. Both of these effects can reduce the achievable spatial resolution.The original Exchange-PAINT study demonstrated sequential 4-color imaging of cellular protein targets labeled with DNA-modified antibodies using different imager strands conjugated with a single-color dye. While successful, this labeling approach was based on biotinylated primary antibodies in combination with streptavidin and biotinylated docking strands to form an ‘antibody-streptavidin-DNA’ sandwich. This labeling procedure leads to two disadvantages; on one hand, the ‘linkage-error’, that is, the distance between the true target and labeled DNA docking site, is increased due to the addition of streptavidin, which ultimately leads to a localization offset from the true target position.Here, we introduce a general framework for labeling protein targets using DNA-PAINT docking strands, which are directly coupled to various labeling probes, thus addressing the aforementioned issues. First, we design and evaluate the performance and orthogonality of 52 DNA sequences for Exchange-PAINT. Next, we directly conjugate DNA oligonucleotides to antibodies, avoiding the biotin–streptavidin sandwich, and then extend the platform to small-sized binders, including nanobodies and small molecules, to further enhance the achievable labeling density and spatial accuracy. Finally, we successfully use our labeling platform to demonstrate nine-target super-resolution imaging in fixed biological samples.15 to theoretically enable 52-plex super-resolution imaging. We started with strand design using the “CANADA” software,24 employing the following conditions: the length of the docking site is 9 base pairs (bp), the GC-content is 40% (3 out of these 9), and there should be no sequence homology with more than 3 bases. To ensure the experimental orthogonality of the designed sequences and to test their performances in DNA-PAINT (e.g. achievable resolution), we conducted a series of 52 in vitro experiments . As an example, the result for the experiment with the imager sequence P40 is depicted in To extend the multiplexing capabilities of Exchange-PAINT, we designed 37 sequences in addition to the previously published 15 strands,eriments . We desieriments , right, in situ, we next describe the synthesis of barcoded DNA-antibody conjugates for DNA-PAINT imaging. Antibodies (Abs) are the most widely used labeling probes. High specificity and affinity for target antigens coupled with the large repertoire of commercially available antibodies make them an integral component in life science research.To translate this large multiplexing capability They are routinely used in diverse immunoassay applications, including immunofluorescence (IF) imaging and immunohistochemistry (IHC). These attributes prompted us to first adopt the antibody-labeling platform for DNA-PAINT imaging and develop a general DNA conjugation approach that builds upon the vast array of available antibodies to complement the high multiplexing capability of Exchange-PAINT.via SM(PEG)2  cyclohexane-1-carboxylate) crosslinkers 2 crosslinkers. In this step, the N-hydroxysuccinimide (NHS) ester group of SM(PEG)2 reacts with the amine groups present on the lysine residues and anchors the maleimide group on the antibody surface. After removing the excess cross-linker using size-exclusion chromatography, maleimide-functionalized antibodies were reacted with thiol-modified DNA oligonucleotides to form stable DNA-antibody conjugates. The antibody conjugates were purified using a molecular weight cut-off filter (100 kDa). The successful conjugation of DNA strands to antibodies was verified using matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). We have optimized the protocols to yield conjugates with close to 1 DNA label/Ab .antibody . Next, D26 This underlines a substantial improvement of the labeling density and size over previously published DNA-PAINT cell data,15 where biotin–streptavidin-mediated DNA conjugated antibodies failed to resolve this hollow tubular structure. To semi-quantitatively assess the achievable resolution, we measured the cross-sectional profile of localizations of a “hollow” microtubule structure. As depicted in 27 We also tested our direct DNA-conjugated antibodies for dual-color super-resolution imaging , only ∼13 kDa in MW and have high affinity for their target molecule.28 Previous reports have demonstrated the enhanced resolving power of nanobodies for super-resolution imaging of microtubules.30IgG antibodies, typically used in immunofluorescence studies, are ∼150 kDa in MW and ∼10 nm in size. Although the large commercially available repertoire of antibodies is advantageous for their use in highly multiplexed imaging, their rather large sizes are ultimately a concern when highly accurate localization of the target is necessary or when high density labelingtrans-cyclooctene (TCO) to couple DNA-PAINT docking strands to a model anti-GFP nanobody (6 (Ms)–1, quantitative and can proceed in physiological conditions, which helps to rapidly and efficiently conjugate DNA while preserving the functionality of the nanobodies.31 In brief, a TCO-NHS ester was used to react with the primary amine groups of lysine residues on nanobodies in PBS (pH = 8) for 3 hours. Simultaneously, amine-modified DNA-PAINT docking strands were reacted with Tz and subsequently purified using HPLC. The TCO-modified nanobodies were then coupled with the Tz-modified DNA-PAINT docking strands during a reaction in PBS (pH = 7.4) for 3 hours. As for the case of antibody conjugation discussed above, we have optimized the protocols to yield conjugates with close to 1 DNA label/nanobody  and nanobody . The str32 and the expression of GFP was detected after 2 days of transfection . HeLa cells were transfected with a baculoviral vector containing mitochondrial leader sequence-fused GFP (BacMam2.0),sfection . The trasfection  shows a sfection  correlatsfection . We noteA step-by-step protocol for nanobody-DNA conjugation is described in ESI Protocol 3.33 Actin filaments are usually present in high density in cells with individual fiber diameters as small as 5–10 nm.34 Imaging of the actin cytoskeleton structure using DNA-conjugated phalloidin probes will not only allow investigation of the compatibility of small molecule probes with DNA-PAINT, but also demonstrate the benefit of employing a smaller targeting agent to resolve high density sub-10 nm structures using DNA-based imaging.Small molecule binders represent another important class of targeting reagents for high-density protein labeling. To test the compatibility of DNA-PAINT imaging with small molecule probes, we selected phalloidin, a bicyclic heptapeptide, to selectively target F-actin.To create DNA-conjugated phalloidin probes, we used the Tz and TCO-based conjugation method , similar35We tested the performance of our DNA-phalloidin probes in HeLa cells. To preserve the cytoskeleton ultrastructure, we fixed HeLa cells using 0.1% glutaraldehyde along with 3% PFA. Staining of the actin filaments was achieved by incubating cells with 1 μM of DNA-phalloidin probes . After rA step-by-step protocol for Phalloidin-DNA conjugation is described in ESI Protocol 4.Protein interaction networks mediate cellular responses to various environmental stimuli. It is increasingly evident that the spatial heterogeneity of protein distribution in cells leads to intracellular functionality differences among distinct compartments and intercellular variance among cells located in different regions. Mapping the heterogeneity of protein networks is challenging for three reasons: (1) the location information of proteins needs to be well preserved; (2) comprehensive studies probing multiple protein targets need to be performed in order to understand the whole network; (3) high spatial accuracy is required to achieve subcellular mapping, rendering conventional diffraction-limited fluorescence imaging unsuitable.in situ. By synergistically combining optimized DNA probe design and improved DNA-antibody conjugation, we here report the thus far unprecedented nine-target super-resolution imaging in biological samples.The development of Exchange-PAINT imaging enables highly multiplexed super-resolution detection in single cells and is hence desirable for protein network mapping directly 36 The nuclear pore complex signal was present specifically in the nucleus which was indicated by DAPI staining of the nucleus.Given that indirect immunostaining approaches are most widely used and present a cost-effective method for labeling protein targets, we first tested the multiplexed super-resolution imaging using DNA-conjugated secondary antibodies. Here, we stained fixed HeLa cells with phalloidin and primary antibodies from seven different species followed by DNA-conjugated secondary antibodies from the donkey species. Seven rounds of probe exchange were performed to image all eight targets. The results showed that eight cellular structures could be clearly visualized with Exchange-PAINT, and there was minimal-to-no crosstalk of signals among the different antibodies . It can The use of secondary antibodies for multiplexed detection, however, is limited by the availability of primary antibodies from different species. Therefore, we next used directly DNA-labeled primary antibodies and small molecule binders, and achieved nine-target super-resolution visualization . NuclearDetailed information regarding the primary and secondary antibodies and imager sequences can be found in ESI Tables 3–7.In summary, we have developed a versatile labeling platform for the conjugation of DNA oligonucleotides to various labeling probes for DNA-PAINT and Exchange-PAINT with high labeling density, spatial accuracy and achievable resolution. We also demonstrated the use of our labeled probes for highly multiplexed imaging in biological samples with nanoscale resolution. The conjugation method is efficient and simple to implement, and should be easily adopted in common biological labs. We anticipate that the conjugation methods developed here can make Exchange-PAINT accessible to a broader scientific community, and will consequently be used to solve more complex biological questions.


Saccharomyces cerevisiae. This pattern has been shown to bedepolarized in glucose-depleted cells after formaldehyde fixation and stainingwith rhodamine-tagged phalloidin. Loss of actin cables in mother cells wasremarkable. Here we extend our knowledge on actin in live glucose-depleted cellsco-expressing the marker of actin patches (Abp1-RFP) with the marker of actincables (Abp140-GFP). Glucose depletion resulted in appearance of actin patchesalso in mother cells. However, even after 80 min of glucose deprivation thesecells showed a clear network of actin cables labeled with Abp140-GFP in contrastto previously published data. In live cells with a mitochondrial dysfunction(rho0 cells), glucose depletion resulted in almost immediateappearance of Abp140-GFP foci partially overlapping with Abp1-RFP patches inmother cells. Residual actin cables were clustered in patch-associated bundles.A similar overlapping “patchy” pattern of both actin markers was observed upontreatment of glucose-deprived rho+ cells with FCCP (the inhibitor ofoxidative phosphorylation) and upon treatment with formaldehyde. While theformaldehyde-targeted process stays unknown, our results indicate that publisheddata on yeast actin cytoskeleton obtained from glucose-depleted cells afterfixation should be considered with caution.Actin filaments form cortical patches and emanating cables in fermenting cells of Saccharomyces cerevisiae,the actin cytoskeleton is primarily composed of two morphologically distinctstructures: cortical actin patches and actin cables 11S. cerevisiae came from the studies offormaldehyde-fixed cells labeled with fluorescently-tagged phalloidin  The actin cytoskeleton has extensively been studied in various types of eukaryoticcells. It plays a key role in various essential processes including cellularmovement, protein trafficking and secretion, cell division, cell growth and plasmamembrane remodeling. In the budding yeast act1 null mutant. It was suggested that these fusions are notincorporated into actin cables S. cerevisiae cells 18S. cerevisiae cells  wild type cellsdepleted for glucose for 80 minutes still display a complex network of actin cables(marker Abp140-GFP) and depolarized pattern of actin patches (marker Abp1-RFP). Whenthe cells were treated with the mitochondrial uncoupler FCCP (carbonylcyanidep-trifluoromethoxyphenylhydrazone) simultaneously with glucose deprivation, thepopulation consisted of a large number of cells with destabilized actin cablesvisualized by Abp140-GFP. Several accumulations of Abp140-GFP partially overlappedwith the Abp1-RFP signal in cortical patches. A similar pattern was found inglucose-deprived rho+ cells after formaldehyde fixation and in liveglucose-deprived cells with mitochondrial dysfunction (rho0 cells). Weassume that stability of actin cables reflects the metabolic status of the cell.Based on comparison of live and formaldehyde-fixed cells, our data suggest thatformaldehyde affects respiration before fixation and this uneven signaling resultsin destabilization of actin cables in glucose-deprived cells.In this study, we demonstrate that live rhoS. cerevisiae cells arrest translation andafter formaldehyde fixation display a depolarized F-actin distribution patternlabelled with rhodamine-tagged phalloidin + (respiring) cells thatwere grown in a glucose rich medium displayed polarized distribution of actinpatches, usually localized to the cell cortex of daughter cells and actin cablesemanating into mother cells . The F-actin distribution pattern wascompletely different in cells starved for glucose for 30 minutes andsubsequently fixed with formaldehyde in the absence of glucose. In these cells,only a depolarized pattern of F-actin chunks/accumulations labeled withRh-phalloidin was observed .Glucose-depleted + (respiring) cellsexpressing established fluorescence markers of the two different F-actinstructures patches and cables (Abp1-RFP and Abp140-GFP). Both the glucose-grownand the glucose-depleted cells were fixed with 3.7 % formaldehyde for 30minutes, and changes in distribution of both markers Abp1-RFP and Abp140-GFPwere analyzed . The pattern of actin cables (Abp140-GFP) and actinpatches (Abp1-RFP) was not affected when the cells were fixed in the presence ofglucose , but the filamentous pattern of Abp140-GFP and polarizeddistribution of Abp1-RFP almost dissipated in the cells starved for glucose for30 minutes prior to fixation . The fluorescence signal ofAbp140-GFP was accumulated in small dots. Prolonged glucose starvation up to 80minutes resulted in appearance of chunks of both F-actin markers  In contrast, our experiments on live glucose-depleted rho+cells expressing both actin markers (Abp140-GFP and Abp1-RFP) revealed differentactin pattern compared to formaldehyde-fixed rho+ cells. As expected,Abp1-RFP was localized to actin patches accumulated in buds, and Abp140-GFPlabelled the actin cables emanating from the buds in cells exponentially growingin high glucose medium . A 30-minute- or a prolonged 80-minute-glucose deprivation led to a changed distribution pattern of actin patches andled to the appearance of patches also in mother cells . Whereasthe polarized pattern of actin patches was lost, we did not observe any obviousloss of the actin cable integrity in these cells. These cells still displaybundles of actin cables. The pattern of F-actin cables destabilization shape wasstudied in detailed time course glucose deprivation . Whereas liveglucose-depleted cells displayed actin cables , glucose deprivation for10 minutes was critical for the stability of actin cables in formaldehyde-fixedglucose-depleted cells , including those labeled with Rh-phalloidin.To compare our findings with published data on actin distribution in fixed 24We conclude that a glucose depletion up to 80 minutes does not lead to loss ofactin cables and that formaldehyde fixation affects distribution of actincytoskeleton in these cells.The effect of formaldehyde fixation on the mitochondrial network was examined instrains co-expressing Abp140-GFP from the chromosomal site with theplasmid-derived RFP-tagged mitochondrial marker MITO-RFP (plasmidpYX142-mtRFPm). In live cells grown on glucose both, actin cables andmitochondria, were intact  and in glucose-deprived live cells themitochondrial network was even more branched and tubular . Informaldehyde-fixed fermenting cells , Abp140-GFP-labeled actincables were preserved and the mitochondrial network was not obviously alteredcompared to live cells. In contrast and consistent with our previous data, theformaldehyde fixation of glucose-depleted cells resulted in destabilization ofboth, actin cables and the mitochondrial network .Thus, as shown by live imaging analyses, glucose starvation by itself did notaffect the integrity of either the cable pattern or the mitochondrial network inlive cells. However, we showed here that the combined effect of glucosestarvation and formaldehyde fixation resulted in destabilization of both, themitochondrial network and actin cables. Hence, it can be speculated, thatformaldehyde fixation affects integrity and/or function of mitochondria andconsequently, the integrity of actin cables is affected.0 cellsco-expressing Abp140-GFP (actin cables) and Abp1-RFP (actin patches) fromchromosomal sites. These exponentially growing rho0 cells displayedthe polarized actin pattern consisting of cortical actin patches accumulated inbuds (Abp1-RFP) and associated actin cables (Abp140-GFP) emanating into themother cells . In contrast to rho+ (wild type) cells, glucose starvation of rho0 cells for 80 minutesresulted in an obvious loss of actin cables . Abp1-RFP wasre-localized from buds also into the mother cells and both actin markers,Abp140-GFP and Abp1-RFP, were accumulated in enlarged and partially overlappingchunks or bodies. Detailed time course analyses revealed that destabilized actincables appeared in most of the rho0 cells in the population after a15 minute glucose deprivation .We speculate that the effect of formaldehyde fixation on the actin cableintegrity in glucose-deprived cells might be a consequence of mitochondrialdysfunction. Therefore live-cell imaging analyses were performed withrespiratory-deficient ethidium bromide-induced rho+cells co-expressing Abp140-GFP and Abp1-RFP with the proton-ionophore FCCP . The drug was added to cultures of exponentially growing cells alreadycultivated in either glucose-free or 3 % glycerol-containing media for 80minutes. After a 20 minutes treatment with FCCP, most of the cells displayedonly chunks of accumulated Abp140-GFP overlapping with chunks of Abp1-RFP. Weconclude that mitochondrial dysfunction affects the integrity of actin cables inglucose-depleted cells.To confirm our previous data that changes in mitochondrial respiration affect thestability of actin cables in glucose-depleted cells we treated rhoS. cerevisiae cells Microscopic analyses of the actin cytoskeleton based on the Rh-phalloidin staining offormaldehyde-fixed etal.et al.It is evident that dynamics of actin is directly linked to growth and life cyclecontrols 10111112mdm20Δ tpm1Δ whi2Δ 0 cells , the patternof F-actin distribution in formaldehyde-fixed cells should be viewed with caution. A remarkable loss of actin cables in formaldehyde-fixed cells has been usuallyinterpreted as a consequence of various stresses including osmotic stress 0) F-actin patches as well as F-actin cablesare immediately collapsed. In this respect, our data are in consistence withprevious conclusions that respiration is needed for actin repolarization 9ras signaling and theysuggested that the cell death occurs within colonies formed from these cofilinmutants. However, it could be suggested that a similar failure ofras signaling may happen only as a consequence of theformaldehyde fixation in the absence of glucose. This still has to be elucidated. As we document here, the formaldehyde fixation affects distribution of both, actinstructures and the mitochondrial network in the absence of glucose. This closeinterconnection between intact functional mitochondria and F-actin cables is furthersupported by our observations in the absence of glucose that in cells withcompromised mitochondria  A question remains whether this detrimental effect of formaldehyde could beprevented. Details remain to be elucidated. However, based on our experience fromour earlier F-actin studies in fungi 441424344S. cerevisiae strains used in this study are listed in Table 1.Yeast cultures were grown in YPD medium  or SC medium  at 30°C. Correspondingsolid media contained 2 % agar. Standard methods were used for all DNAmanipulations TM micromanipulator. For glucosedeprivation, exponentially growing cells were transferred to glucose-freesynthetic (SC) medium and incubated under shaking for additional 30-80 min. Therho0 strain was prepared by the treatment with ethidium bromideas described elsewhere Cells grown in presence of glucose and glucose-depleted yeast cells were fixedwith formaldehyde  added to the medium for 30 minutesand stained for F-actin with rhodamine-tagged phalloidin  as described elsewhere 1


Chronic kidney disease (CKD) is a significant worldwide healthcare problem. Regardless of the initial injury, renal fibrosis is the common final pathway leading to end stage renal disease. Although the underlying mechanisms are not fully defined, evidence indicates that besides inflammation, oxidative stress plays a crucial role in the etiology of renal fibrosis. Oxidative stress results from an imbalance between the production of free radicals that are often increased by inflammation and mitochondrial dysfunction, and reduced anti-oxidant defenses. Several studies have demonstrated that oxidative stress may occur secondary to activation of transforming growth factor β1 (TGF-β1) activity, consistent with its role to increase nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) activity. A number of other oxidative stress-related signal pathways have also been identified, such as nuclear factor erythroid-2 related factor 2 (Nrf2), the nitric oxide (NO)-cyclic guanosine monophosphate (cGMP)-cGMP-dependent protein kinase 1-phosphodiesterase (cGMP-cGK1-PDE) signaling pathway, and the peroxisome proliferator-activated receptor gamma (PPARγ) pathway. Several antioxidant and renoprotective agents, including cysteamine bitartrate, epoxyeicosatrienoic acids (EETs), and cytoglobin (Cygb) have demonstrated ameliorative effects on renal fibrosis in preclinical or clinical studies. The mechanism of action of many traditional Chinese medicines used to treat renal disorders is based on their antioxidant properties, which could form the basis for new therapeutic approaches. This review focuses on the signaling pathways triggered by oxidative stress that lead to renal fibrosis and provides an update on the development of novel anti-oxidant therapies for CKD. The associated poor outcomes result in increased costs to the healthcare system  continues to increase world-wide, even though some studies suggest that the overall prevalence may be tapering off in some developed countries during the recent decade -mediated hypertension  is the most abundant TGF-β1 membrane binding protein. It potentiates TGF-β1 binding to the type I and type II signaling receptors. A synthetic peptide (P144), representing the membrane-proximal ligand-binding domain of betaglycan, effectively blocks TGF-β1-induced effects in various animal models. For example, Baltanas and colleagues reported that P144 prevents oxidative stress and inhibits NADPH oxidases in kidneys of spontaneously hypertensive rats  regulates expression of many genes that oppose inflammatory and oxidative damage, including genes that encode thiol molecules, detoxifying enzymes, antioxidant proteins, and stress response proteins. Accumulating evidence indicates that Nrf2 signaling is protective in various models of renal disease  is the ester prodrug of mycophenolic acid (MPA), a non-competitive, highly selective, and reversible inhibitor of inosine monophosphate dehydrogenase. Recent evidence indicates that MMF protects from renal damage and reduces oxidative stress and renal inflammation secondary to immunosuppression -cGMP-dependent protein kinase 1 (cGMP-cGK1) signaling pathway is involved in renal fibrosis , thereby improving endothelial function and attenuating inflammation  may contribute to the renoprotective effects of relaxin. Relaxin's antifibrotic effects were found to depend on heterodimerization of AT2 and RXFP-1   and diacylglycerol kinase, two upstream inhibitors directly induced by PPARγ agonists.Pioglitazone attenuates fibrosis in part by reducing mitochondrial ROS. It affects mitochondrial function by several means, including (a) by increasing mtDNA; (b) by affecting coupling-uncoupling dynamics and increasing glucose use, thus reducing ROS production; (c) by promoting mitochondrial biogenesis along with PPAR coactivator 1α and nuclear respiratory factor 1; (d) by increasing expression of mitochondrial enzymes involved in eliminating or producing ROS; (e) by reducing cytochrome C oxidase activity and subsequent phosphorylation of p66via PPARγ-independent mechanisms  in the FONT phase 1 trial. At 16 months of follow-up, 71% of participants showed stable glomerular filtration rate and reduced proteinuria. However, the trial was halted after the release of the warnings by FDA about rosiglitazone's adverse cardiovascular effects in elderly type 2 diabetics. Nevertheless, it should be noted that no specific safety concerns were identified in children involved in the FSGS FONT trial  are copper-containing enzymes that oxidatively deaminate primary amines, thereby producing ammonium, hydrogen peroxide, and aldehydes , MMPs, and apoptosis-related proteins. Soluble epoxide hydrolase (sEH) also plays an essential role in CKD by hydrolyzing EETs to the corresponding inactive dihydroxyeicosatrienoic acids. Inhibiting sEH with 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA) reduced EMT associated E-cadherin suppression, phenotypic transition, α-SMA elevation, PI3K-Akt activation, and GSK-3β phosphorylation -ureido]cyclohexyloxy) benzoic acid (t-TUCB) in the UUO model. sEH inhibition enhanced levels of EET regioisomers and inhibited UUO-induced upregulated TGF-β1/Smad3 and NF-κB signaling, oxidative stress, tubular injury, and apoptosis, and also downregulated antifibrotic factors , which are abundant in the kidney, are cytochrome P-450-dependent derivatives of arachidonic acid with anti-inflammatory, anti-oxidant, and fibrinolytic functions , a novel member of the globin superfamily, is expressed by fibroblasts in various organs. Using the remnant kidney model, Mimura et al.  investigA number of traditional Chinese medicines with anti-oxidant properties have been shown to have beneficial actions in treating CKD in preclinical models, some of which are listed in Table Trigonella foenum-graecum L. (TFG) and Psoralea corylifolia L., is used to treat kidney deficiency. HLBW was recently shown to improve hyperglycemia, hyperlipidemia, and proteinuria in type 2 diabetic rats induced by high fat diet and streptozotocin treatment , a Chinese herbal prescription composed of Salvia miltiorrhiza (commonly known as Danshen) attenuates oxidative stress in the streptozotocin-treated mouse model of diabetic nephropathy via upregulated expression of Nrf2 , was found to downregulate expression of oxidative carbonyl proteins in the streptozotocin-treated rat model of diabetic nephropathy , extracted from the traditional Chinese herb root of Angelica sinensis, Cimicifuga heracleifolia, and Lignsticum chuangxiong, has well known anti-platelet, antithrombotic, and antioxidant actions. SF was reported to protect the kidney of streptozotocin-induced diabetic rats by inhibiting the expression of TGF-β1 and collagen IV , extracted from several plants including Rosa laevigata Michx. (RLM) fruit were found to attenuate ischemia-reperfusion injury in the rat, which was associated with upregulation of Nrf2, as well as silent information regulator factor 2-related enzyme 1 (Sirt1) and heme oxygenase-1 (HO-1)  from Salviae miltiorrhizae radix, is a scavenger of superoxide anions and hydroxyl radicals , as well as NF-κB p65, cyclooxygenase-2, phospho-p38 MAPK, and inducible NOS. MLP was similarly protective against renal injury in streptozotocin-induced diabetic rats , extracted from In conclusion, oxidative stress plays an important role in the pathophysiology of renal fibrosis. Increased ROS formation is not only a consequence of renal fibrosis, but also positively impacts on pro-fibrotic signaling. A better understanding of the signaling pathways by which oxidative stress induces renal fibrosis may lead to the development of novel therapeutic strategies. The mechanism of action of many traditional Chinese medicines with proven therapeutic value in treating kidney disorders is based on their anti-oxidant properties. A better understanding of the cellular and molecular mechanisms behind their actions will undoubtedly lead to new drugs that effectively prevent or reverse renal fibrosis. It is our contention that further studies on the mechanisms underlying the renoprotective effect of antioxidant agents in traditional Chinese medicines will provide new insights that propel the development of novel therapies to slow the progression of CKD.WL conceived the idea and helped research and write the manuscript; GB helped research and write the manuscript; FF helped write the manuscript and offered insight into traditional Chinese medicines; YW helped write the manuscript; RR helped research, write, and edit the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


By contrast, the ZnO-CdS nanorods conducted with the postannealing procedure exhibited superior photoactivity and photoelectrochemical performance; the substantial changes in the microstructures and optical properties of the composite nanorods following postannealing in this study might account for the observed results.The ZnO-CdS core-shell composite nanorods with CdS shell layer thicknesses of 5 and 20 nm were synthesized by combining the hydrothermal growth of ZnO nanorods with the sputtering thin-film deposition of CdS crystallites. The microstructures and optical properties of the ZnO-CdS nanorods were associated with the CdS shell layer thickness. A thicker CdS shell layer resulted in a rougher surface morphology, more crystal defects, and a broader optical absorbance edge in the ZnO-CdS rods. The ZnO-CdS (20 nm) nanorods thus engaged in more photoactivity in this study. When they were further subjected to a postannealing procedure in ambient Ar/H T. To) i.eee times g. From F2, respectively, under visible-light illumination. The reduction-oxidation reactions in the electrolyte during the PEC measurements involved the electron-hole pairs that presented in the CdS shell layers upon exposure to visible-light irradiation  composite nanorods exhibited a rougher surface and more surface crystal defects compared with the ZnO-CdS (5 nm) nanorods. These differences engendered the ZnO-CdS (20 nm) nanorods with higher photoactivity and PEC performance. Further postannealing the ZnO-CdS (20 nm) nanorods in ambient Ar/H


Using the differently expressed AD genes, we identified significant KEGG pathways related to metabolism and immune processes. Using the up- and down- regulated AD gene list, we further found up-regulated AD gene were significantly enriched in immune and metabolic pathways. We further compare the immune and metabolic KEGG pathways from the expression dataset with those from previous GWAS datasets, and found that most of these pathways are shared in both GWAS and expression datasets.In recent years, several pathway analyses of genome-wide association studies reported the involvement of metabolic and immune pathways in Alzheimer's disease (AD). Until now, the exact mechanisms of these pathways in AD are still unclear. Here, we conducted a pathway analysis of a whole genome AD case-control expression dataset ( To investigate Alzheimer's disease (AD) genetic risk factors, several genome-wide association studies (GWAS) have identified several new AD susceptibility loci in European populations, and some of these loci were successfully replicated in other studies -5. To den = 41, 25 AD cases and 16 controls) from the human temporal cortex tissue, and conducted a pathway analysis using all the differently expressed AD genes, up-regulated AD genes, and down-regulated AD genes. We further compare the AD risk pathways from the expression dataset with those from previous GWAS datasets to address the potential mechanisms.We think that the different expression (up-regulation or down-regulation) of genes in these pathways may contribute to AD susceptibility. Here, we selected a whole genome AD case-control expression dataset , the most significant signal from the pathway analysis of all differently expressed AD genes  using human temporal cortex tissue [P < 0.05 after false discovery rate (FDR) correction [The expression dataset came from a whole genome AD case-control expression study  and 593 significantly down-regulated (AD cases vs. controls) transcripts, which correspond to 710 and 654 unique genes (3 transcripts are both up- and down-regulated in AD cases and controls). 599 of 710 up-regulated genes were successfully mapped to 599 unique Entrez Gene IDs in WebGestalt database [The total of 1196 significantly differently expressed transcripts include 603 significantly up-regulated (AD cases database . Other 1database . Other 7vs. controls) and down-regulated genes (AD cases vs. controls). We selected KEGG pathways including 10-300 genes for subsequent pathway analysis [p-value below 0.05 [We selected the WebGestalt database for pathway analysis, and the hypergeometric test was used to identify the significant KEGG pathways . We firsanalysis . The ent