Development of a synthetic live bacterial therapeutic for the human metabolic disease phenylketonuria

Content introduction:

• Development of a synthetic live bacterial therapeutic for the human metabolic disease phenylketonuria


• A linked organ-on-chip model of the human neurovascular unit reveals the metabolic coupling of endothelial and neuronal cells


• Targeted delivery of a PD-1-blocking scFv by CAR-T cells enhances anti-tumor efficacy in vivo


• Cas9-mediated allelic exchange repairs compound heterozygous recessive mutations in mice


• A standardized bacterial taxonomy based on genome phylogeny substantially revises the tree of life

1. Development of a synthetic live bacterial therapeutic for the human metabolic disease phenylketonuria

Phenylketonuria (PKU) is a genetic disease that is characterized by an inability to metabolize phenylalanine (Phe), which can result in neurotoxicity. To provide a potential alternative to a protein-restricted diet, Vincent M Isabella at Synlogic Inc. in Cambridge, Massachusetts, USA and his colleagues engineered Escherichia coli Nissle to express genes encoding Phe-metabolizing enzymes in response to anoxic conditions in the mammalian gut. Administration of our synthetic strain, SYNB1618, to the Pahenu2/enu2 PKU mouse model reduced blood Phe concentration by 38% compared with the control, independent of dietary protein intake. In healthy Cynomolgus monkeys, they found that SYNB1618 inhibited increases in serum Phe after an oral Phe dietary challenge. In mice and primates, Phe was converted to trans-cinnamate by SYNB1618, quantitatively metabolized by the host to hippurate and excreted in the urine, acting as a predictive biomarker for strain activity. SYNB1618 was detectable in murine or primate feces after a single oral dose, permitting the evaluation of pharmacodynamic properties. Their results define a strategy for translation of live bacterial therapeutics to treat metabolic disorders.



Read more, please click https://www.nature.com/articles/nbt.4222

2. A linked organ-on-chip model of the human neurovascular unit reveals the metabolic coupling of endothelial and neuronal cells

The neurovascular unit (NVU) regulates metabolic homeostasis as well as drug pharmacokinetics and pharmacodynamics in the central nervous system. Metabolic fluxes and conversions over the NVU rely on interactions between brain microvascular endothelium, perivascular pericytes, astrocytes and neurons, making it difficult to identify the contributions of each cell type. Here Ben M Maoz at Harvard University in Cambridge, Massachusetts, USA and his colleagues model the human NVU using microfluidic organ chips, allowing analysis of the roles of individual cell types in NVU functions. Three coupled chips model influx across the blood–brain barrier (BBB), the brain parenchymal compartment and efflux across the BBB. They used this linked system to mimic the effect of intravascular administration of the psychoactive drug methamphetamine and to identify previously unknown metabolic coupling between the BBB and neurons. Thus, the NVU system offers an in vitro approach for probing transport, efficacy, mechanism of action and toxicity of neuroactive drugs.

Read more, please click https://www.nature.com/articles/nbt.4226

3. Targeted delivery of a PD-1-blocking scFv by CAR-T cells enhances anti-tumor efficacy in vivo

The efficacy of chimeric antigen receptor (CAR) T cell therapy against poorly responding tumors can be enhanced by administering the cells in combination with immune checkpoint blockade inhibitors. Alternatively, the CAR construct has been engineered to coexpress factors that boost CAR-T cell function in the tumor microenvironment. Sarwish Rafiq at Memorial Sloan Kettering Cancer Center in New York, USA and his colleagues modified CAR-T cells to secrete PD-1-blocking single-chain variable fragments (scFv). These scFv-secreting CAR-T cells acted in both a paracrine and autocrine manner to improve the anti-tumor activity of CAR-T cells and bystander tumor-specific T cells in clinically relevant syngeneic and xenogeneic mouse models of PD-L1+ hematologic and solid tumors. The efficacy was similar to or better than that achieved by combination therapy with CAR-T cells and a checkpoint inhibitor. This approach may improve safety, as the secreted scFvs remained localized to the tumor, protecting CAR-T cells from PD-1 inhibition, which could potentially avoid toxicities associated with systemic checkpoint inhibition.

Read more, please click https://www.nature.com/articles/nbt.4195

4. Cas9-mediated allelic exchange repairs compound heterozygous recessive mutations in mice

Dan Wang at University of Massachusetts Medical School in Worcester, Massachusetts, USA and his colleagues report a genome-editing strategy to correct compound heterozygous mutations, a common genotype in patients with recessive genetic disorders. Adeno-associated viral vector delivery of Cas9 and guide RNA induces allelic exchange and rescues the disease phenotype in mouse models of hereditary tyrosinemia type I and mucopolysaccharidosis type I. This approach recombines non-mutated genetic information present in two heterozygous alleles into one functional allele without using donor DNA templates.

Read more, please click https://www.nature.com/articles/nbt.4219

5. A standardized bacterial taxonomy based on genome phylogeny substantially revises the tree of life

Taxonomy is an organizing principle of biology and is ideally based on evolutionary relationships among organisms. Development of a robust bacterial taxonomy has been hindered by an inability to obtain most bacteria in pure culture and, to a lesser extent, by the historical use of phenotypes to guide classification. Culture-independent sequencing technologies have matured sufficiently that a comprehensive genome-based taxonomy is now possible. Donovan H Parks at University of Queensland in Queensland, Australia and his colleagues used a concatenated protein phylogeny as the basis for a bacterial taxonomy that conservatively removes polyphyletic groups and normalizes taxonomic ranks on the basis of relative evolutionary divergence. Under this approach, 58% of the 94,759 genomes comprising the Genome Taxonomy Database had changes to their existing taxonomy. This result includes the description of 99 phyla, including six major monophyletic units from the subdivision of the Proteobacteria, and amalgamation of the Candidate Phyla Radiation into a single phylum. Their taxonomy should enable improved classification of uncultured bacteria and provide a sound basis for ecological and evolutionary studies.

Read more, please click https://www.nature.com/articles/nbt.4229

Development of a synthetic live bacterial therapeutic for the human metabolic disease phenylketonuria

Content introduction:

• Development of a synthetic live bacterial therapeutic for the human metabolic disease phenylketonuria


• A linked organ-on-chip model of the human neurovascular unit reveals the metabolic coupling of endothelial and neuronal cells


• Targeted delivery of a PD-1-blocking scFv by CAR-T cells enhances anti-tumor efficacy in vivo


• Cas9-mediated allelic exchange repairs compound heterozygous recessive mutations in mice


• A standardized bacterial taxonomy based on genome phylogeny substantially revises the tree of life

1. Development of a synthetic live bacterial therapeutic for the human metabolic disease phenylketonuria

Phenylketonuria (PKU) is a genetic disease that is characterized by an inability to metabolize phenylalanine (Phe), which can result in neurotoxicity. To provide a potential alternative to a protein-restricted diet, Vincent M Isabella at Synlogic Inc. in Cambridge, Massachusetts, USA and his colleagues engineered Escherichia coli Nissle to express genes encoding Phe-metabolizing enzymes in response to anoxic conditions in the mammalian gut. Administration of our synthetic strain, SYNB1618, to the Pahenu2/enu2 PKU mouse model reduced blood Phe concentration by 38% compared with the control, independent of dietary protein intake. In healthy Cynomolgus monkeys, they found that SYNB1618 inhibited increases in serum Phe after an oral Phe dietary challenge. In mice and primates, Phe was converted to trans-cinnamate by SYNB1618, quantitatively metabolized by the host to hippurate and excreted in the urine, acting as a predictive biomarker for strain activity. SYNB1618 was detectable in murine or primate feces after a single oral dose, permitting the evaluation of pharmacodynamic properties. Their results define a strategy for translation of live bacterial therapeutics to treat metabolic disorders.



Read more, please click https://www.nature.com/articles/nbt.4222

2. A linked organ-on-chip model of the human neurovascular unit reveals the metabolic coupling of endothelial and neuronal cells

The neurovascular unit (NVU) regulates metabolic homeostasis as well as drug pharmacokinetics and pharmacodynamics in the central nervous system. Metabolic fluxes and conversions over the NVU rely on interactions between brain microvascular endothelium, perivascular pericytes, astrocytes and neurons, making it difficult to identify the contributions of each cell type. Here Ben M Maoz at Harvard University in Cambridge, Massachusetts, USA and his colleagues model the human NVU using microfluidic organ chips, allowing analysis of the roles of individual cell types in NVU functions. Three coupled chips model influx across the blood–brain barrier (BBB), the brain parenchymal compartment and efflux across the BBB. They used this linked system to mimic the effect of intravascular administration of the psychoactive drug methamphetamine and to identify previously unknown metabolic coupling between the BBB and neurons. Thus, the NVU system offers an in vitro approach for probing transport, efficacy, mechanism of action and toxicity of neuroactive drugs.

Read more, please click https://www.nature.com/articles/nbt.4226

3. Targeted delivery of a PD-1-blocking scFv by CAR-T cells enhances anti-tumor efficacy in vivo

The efficacy of chimeric antigen receptor (CAR) T cell therapy against poorly responding tumors can be enhanced by administering the cells in combination with immune checkpoint blockade inhibitors. Alternatively, the CAR construct has been engineered to coexpress factors that boost CAR-T cell function in the tumor microenvironment. Sarwish Rafiq at Memorial Sloan Kettering Cancer Center in New York, USA and his colleagues modified CAR-T cells to secrete PD-1-blocking single-chain variable fragments (scFv). These scFv-secreting CAR-T cells acted in both a paracrine and autocrine manner to improve the anti-tumor activity of CAR-T cells and bystander tumor-specific T cells in clinically relevant syngeneic and xenogeneic mouse models of PD-L1+ hematologic and solid tumors. The efficacy was similar to or better than that achieved by combination therapy with CAR-T cells and a checkpoint inhibitor. This approach may improve safety, as the secreted scFvs remained localized to the tumor, protecting CAR-T cells from PD-1 inhibition, which could potentially avoid toxicities associated with systemic checkpoint inhibition.

Read more, please click https://www.nature.com/articles/nbt.4195

4. Cas9-mediated allelic exchange repairs compound heterozygous recessive mutations in mice

Dan Wang at University of Massachusetts Medical School in Worcester, Massachusetts, USA and his colleagues report a genome-editing strategy to correct compound heterozygous mutations, a common genotype in patients with recessive genetic disorders. Adeno-associated viral vector delivery of Cas9 and guide RNA induces allelic exchange and rescues the disease phenotype in mouse models of hereditary tyrosinemia type I and mucopolysaccharidosis type I. This approach recombines non-mutated genetic information present in two heterozygous alleles into one functional allele without using donor DNA templates.

Read more, please click https://www.nature.com/articles/nbt.4219

5. A standardized bacterial taxonomy based on genome phylogeny substantially revises the tree of life

Taxonomy is an organizing principle of biology and is ideally based on evolutionary relationships among organisms. Development of a robust bacterial taxonomy has been hindered by an inability to obtain most bacteria in pure culture and, to a lesser extent, by the historical use of phenotypes to guide classification. Culture-independent sequencing technologies have matured sufficiently that a comprehensive genome-based taxonomy is now possible. Donovan H Parks at University of Queensland in Queensland, Australia and his colleagues used a concatenated protein phylogeny as the basis for a bacterial taxonomy that conservatively removes polyphyletic groups and normalizes taxonomic ranks on the basis of relative evolutionary divergence. Under this approach, 58% of the 94,759 genomes comprising the Genome Taxonomy Database had changes to their existing taxonomy. This result includes the description of 99 phyla, including six major monophyletic units from the subdivision of the Proteobacteria, and amalgamation of the Candidate Phyla Radiation into a single phylum. Their taxonomy should enable improved classification of uncultured bacteria and provide a sound basis for ecological and evolutionary studies.

Read more, please click https://www.nature.com/articles/nbt.4229

The Modes of Cell Death: Apoptosis and Necrosis

Two distinct modes of cell death, apoptosis and necrosis, can be recognized based on differences in the morphological, biochemical, and molecular changes of the dying cell. The assays of cell viability presented in this chapter are discussed in light of their applicability to differentiate between these two mechanisms.

1. Apoptosis

The terms “apoptosis,” “active cell death,” “cell suicide,” and “shrinkage necrosis” are being used, often interchangeably, to define a particular mode of cell death characterized by a specific pattern of changes in nucleus and cytoplasm. Because this mode of cell death plays a role during the programmed cell death, as originally described in embryology, the term “programmed cell death” is also being used synonymously (albeit incorrectly, in the context of denoting the mode of cell death) with apoptosis.

The role of apoptosis in embryology, endocrinology, and immunology is the subject of several reviews.The wide interest in apoptosis in oncology, so apparent in recent years, stems from the observations that this mode of cell death is triggered by a variety of antitumor drugs, radiation, or hyperthermia and that the intrinsic propensity of tumor cells to respond by apoptosis is modulated by expression of several oncogenes such as Bcl-2, c-myc, or tumor suppressor gene p53 and may be prognostic of treatment. Extensive research is underway in many laboratories to understand the mechanism of apoptosis. Knowledge of the molecular events of this process may be the basis for new antitumor strategies. Apoptosis affecting CD4+ lymphocytes of HIV-infected patients also appears to play a pivotal role in pathogenesis of AIDS. The most common feature of apoptosis is active participation of the cell in its self-annihilation. The cell mobilizes a cascade of events that lead to its disintegration and the formation of the “apoptotic bodies” which are subsequently engulfed by the neighboring cells without invoking inflammation. Increased cytoplasmic Ca2+ concentration, cell dehydration, increased lipid peroxidation, chromatin condensation originating at the nuclear periphery, activation of endonuclease which has preference to DNA at the internucleosomal (linker) sections, proteolysis, fragmentation of the nucleus, and fragmentation of the cell are the most characteristic events of apoptosis. On the other hand, even during advanced stages of apoptosis, the structural integrity and the transport function of the plasma membrane are preserved. Also preserved and functionally active are the mitochondria and lysosomes. Thus, regardless of cell type, or the nature of event which triggers apoptosis, this mode of cell death has many features in common. Some of these features can be analyzed by image or flow cytometry, and several methods have been described to identify apoptotic cells.

Mitotic death, also termed delayed reproductive death, shows some features of apoptosis and thus may represent delayed apoptosis; it occurs as a result of cell exposure to relatively low doses of drugs or radiation, which induce irreparable damage, but allow cells to complete at least one round of division.

2. Necrosis

Necrosis is an alternative to the apoptotic mechanism of cell death. Most often it is induced by an overdose of cytotoxic agents and is a cell response to a gross injury. However, certain cell types do respond even to pharmacological concentrations of some drugs or moderate doses of physical agents by necrosis rather than apoptosis and the reason for the difference in response is not entirely clear. While apoptosis requires active participation of the involved cell, often even in terms of initiation of the de novo protein synthesis, necrosis is a passive and degenerative process. In vivo, necrosis triggers the inflammatory response in the tissue, due to a release of cytoplasmic constituents to intercellular space, often resulting in scar formation. In contrast, remains of apoptotic cells are phagocytized not only by the “professional” macrophages, but also by other neighboring cells, without evoking any inflammatory reaction. The early event of necrosis is swelling of cell mitochondria, followed by rupture of the plasma membrane, and release of the cytoplasmic content.

The costimulatory molecule CD226 signals through VAV1 to amplify TCR signals and promote IL-17 production by CD4+ T cells

Content introduction:

• The costimulatory molecule CD226 signals through VAV1 to amplify TCR signals and promote IL-17 production by CD4+ T cells


• Modeling of patient virus titers suggests that availability of a vaccine could reduce hepatitis C virus transmission among injecting drug users


• TORC1 inhibition enhances immune function and reduces infections in the elderly


• Mitochondrial redox sensing by the kinase ATM maintains cellular antioxidant capacity


• IKK promotes cytokine-induced and cancer-associated AMPK activity and attenuates phenformin-induced cell death in LKB1-deficient cells

1. The costimulatory molecule CD226 signals through VAV1 to amplify TCR signals and promote IL-17 production by CD4+ T cells
The activation of T cells requires the guanine nucleotide exchange factor VAV1. Using mice in which a tag for affinity purification was attached to endogenous VAV1 molecules, Guillaume Gaud at Université de Toulouse in Toulouse, France and his colleagues analyzed by quantitative mass spectrometry the signaling complex that assembles around activated VAV1. Fifty VAV1-binding partners were identified, most of which had not been previously reported to participate in VAV1 signaling. Among these was CD226, a costimulatory molecule of immune cells. Engagement of CD226 induced the tyrosine phosphorylation of VAV1 and synergized with T cell receptor (TCR) signals to specifically enhance the production of interleukin-17 (IL-17) by primary human CD4+ T cells. Moreover, co-engagement of the TCR and a risk variant of CD226 that is associated with autoimmunity (rs763361) further enhanced VAV1 activation and IL-17 production. Thus, their study reveals that a VAV1-based, synergistic cross-talk exists between the TCR and CD226 during both physiological and pathological T cell responses and provides a rational basis for targeting CD226 for the management of autoimmune diseases.



Read more, please click http://stke.sciencemag.org/content/11/538/eaar3083

2. Modeling of patient virus titers suggests that availability of a vaccine could reduce hepatitis C virus transmission among injecting drug users
The major route of hepatitis C virus (HCV) transmission in the United States is injection drug use. Marian Major at Center for Biologics Evaluation and Research in Silver Spring, USA and her colleagues hypothesized that if an HCV vaccine were available, vaccination could affect HCV transmission among people who inject drugs by reducing HCV titers after viral exposure without necessarily achieving sterilizing immunity. To investigate this possibility, they developed a mathematical model to determine transmission probabilities relative to the HCV RNA titers of needle/syringe-sharing donors. They simulated sharing of two types of syringes fitted with needles that retain either large or small amounts of fluid after expulsion. Using previously published viral kinetics data from both naïve subjects infected with HCV and reinfected individuals who had previously cleared an HCV infection, they estimated transmission risk between pairs of serodiscordant injecting drug users, accounting for syringe type, rinsing, and sharing frequency. They calculated that the risk of HCV transmission through syringe sharing increased ~10-fold as viral titers (log10 IU/ml) increased ~25-fold. Cumulative analyses showed that, assuming sharing episodes every 7 days, the mean transmission risk over the first 6 months was >90% between two people sharing syringes when one had an HCV RNA titer >5 log10 IU/ml. For those with preexisting immunity that rapidly controlled HCV, the cumulative risk decreased to 1 to 25% depending on HCV titer and syringe type. Their modeling approach demonstrates that, even with transient viral replication after exposure during injection drug use, HCV transmission among people sharing syringes could be reduced through vaccination if an HCV vaccine were available.

Read more, please click http://stm.sciencemag.org/content/10/449/eaao4496

3. TORC1 inhibition enhances immune function and reduces infections in the elderly
Inhibition of the mechanistic target of rapamycin (mTOR) protein kinase extends life span and ameliorates aging-related pathologies including declining immune function in model organisms. The objective of this phase 2a randomized, placebo-controlled clinical trial was to determine whether low-dose mTOR inhibitor therapy enhanced immune function and decreased infection rates in 264 elderly subjects given the study drugs for 6 weeks. A low-dose combination of a catalytic (BEZ235) plus an allosteric (RAD001) mTOR inhibitor that selectively inhibits target of rapamycin complex 1 (TORC1) downstream of mTOR was safe and was associated with a significant (P = 0.001) decrease in the rate of infections reported by elderly subjects for a year after study drug initiation. In addition, Joan B. Mannick at Novartis Institutes for Biomedical Research in Cambridge, USA and his colleagues observed an up-regulation of antiviral gene expression and an improvement in the response to influenza vaccination in this treatment group. Thus, selective TORC1 inhibition has the potential to improve immune function and reduce infections in the elderly.

Read more, please click http://stm.sciencemag.org/content/10/449/eaaq1564

4. Mitochondrial redox sensing by the kinase ATM maintains cellular antioxidant capacity
Mitochondria are integral to cellular energy metabolism and ATP production and are involved in regulating many cellular processes. Mitochondria produce reactive oxygen species (ROS), which not only can damage cellular components but also participate in signal transduction. The kinase ATM, which is mutated in the neurodegenerative, autosomal recessive disease ataxia-telangiectasia (A-T), is a key player in the nuclear DNA damage response. However, ATM also performs a redox-sensing function mediated through formation of ROS-dependent disulfide-linked dimers. Yichong Zhang at Yale School of Medicine in New Haven, USA and his colleagues found that mitochondria-derived hydrogen peroxide promoted ATM dimerization. In HeLa cells, ATM dimers were localized to the nucleus and inhibited by the redox regulatory protein thioredoxin 1 (TRX1), suggesting the existence of a ROS-mediated, stress-signaling relay from mitochondria to the nucleus. ATM dimer formation did not affect its association with chromatin in the absence or presence of nuclear DNA damage, consistent with the separation of its redox and DNA damage signaling functions. Comparative analysis of U2OS cells expressing either wild-type ATM or the redox sensing–deficient C2991L mutant revealed that one function of ATM redox sensing is to promote glucose flux through the pentose phosphate pathway (PPP) by increasing the abundance and activity of glucose-6-phosphate dehydrogenase (G6PD), thereby increasing cellular antioxidant capacity. The PPP produces the coenzyme NADPH needed for a robust antioxidant response, including the regeneration of TRX1, indicating the existence of a regulatory feedback loop involving ATM and TRX1. They propose that loss of the mitochondrial ROS-sensing function of ATM may cause cellular ROS accumulation and oxidative stress in A-T.

Read more, please click http://stke.sciencemag.org/content/11/538/eaaq0702

5. IKK promotes cytokine-induced and cancer-associated AMPK activity and attenuates phenformin-induced cell death in LKB1-deficient cells
The 5′ AMP-activated protein kinase (AMPK) is an energy sensor that is activated upon phosphorylation of Thr172 in its activation loop by the kinase LKB1, CAMKK2, or TAK1. TAK1-dependent AMPK phosphorylation of Thr172 is less well characterized than phosphorylation of this site by LKB1 or CAMKK2. An important target of TAK1 is IκB kinase (IKK), which controls the activation of the transcription factor NF-κB. Ricardo J. Antonia at University of North Carolina at Chapel Hill in Chapel Hill, USA and his colleagues tested the hypothesis that IKK acted downstream of TAK1 to activate AMPK by phosphorylating Thr172. IKK was required for the phosphorylation of Thr172 in AMPK in response to treatment with the inflammatory cytokine IL-1β or TNF-α or upon TAK1 overexpression. In addition, IKK regulated basal AMPK Thr172 phosphorylation in several cancer cell types independently of TAK1, indicating that other modes of IKK activation could stimulate AMPK. They found that IKK directly phosphorylated AMPK at Thr172 independently of the tumor suppressor LKB1 or energy stress. Accordingly, in LKB1-deficient cells, IKK inhibition reduced AMPK Thr172 phosphorylation in response to the mitochondrial inhibitor phenformin. This response led to enhanced apoptosis and suggests that IKK inhibition in combination with phenformin could be used clinically to treat patients with LKB1-deficient cancers.

Read more, please click http://stke.sciencemag.org/content/11/538/eaan5850

The Modes of Cell Death: Apoptosis and Necrosis

Two distinct modes of cell death, apoptosis and necrosis, can be recognized based on differences in the morphological, biochemical, and molecular changes of the dying cell. The assays of cell viability presented in this chapter are discussed in light of their applicability to differentiate between these two mechanisms.

1. Apoptosis

The terms “apoptosis,” “active cell death,” “cell suicide,” and “shrinkage necrosis” are being used, often interchangeably, to define a particular mode of cell death characterized by a specific pattern of changes in nucleus and cytoplasm. Because this mode of cell death plays a role during the programmed cell death, as originally described in embryology, the term “programmed cell death” is also being used synonymously (albeit incorrectly, in the context of denoting the mode of cell death) with apoptosis.

The role of apoptosis in embryology, endocrinology, and immunology is the subject of several reviews.The wide interest in apoptosis in oncology, so apparent in recent years, stems from the observations that this mode of cell death is triggered by a variety of antitumor drugs, radiation, or hyperthermia and that the intrinsic propensity of tumor cells to respond by apoptosis is modulated by expression of several oncogenes such as Bcl-2, c-myc, or tumor suppressor gene p53 and may be prognostic of treatment. Extensive research is underway in many laboratories to understand the mechanism of apoptosis. Knowledge of the molecular events of this process may be the basis for new antitumor strategies. Apoptosis affecting CD4+ lymphocytes of HIV-infected patients also appears to play a pivotal role in pathogenesis of AIDS. The most common feature of apoptosis is active participation of the cell in its self-annihilation. The cell mobilizes a cascade of events that lead to its disintegration and the formation of the “apoptotic bodies” which are subsequently engulfed by the neighboring cells without invoking inflammation. Increased cytoplasmic Ca2+ concentration, cell dehydration, increased lipid peroxidation, chromatin condensation originating at the nuclear periphery, activation of endonuclease which has preference to DNA at the internucleosomal (linker) sections, proteolysis, fragmentation of the nucleus, and fragmentation of the cell are the most characteristic events of apoptosis. On the other hand, even during advanced stages of apoptosis, the structural integrity and the transport function of the plasma membrane are preserved. Also preserved and functionally active are the mitochondria and lysosomes. Thus, regardless of cell type, or the nature of event which triggers apoptosis, this mode of cell death has many features in common. Some of these features can be analyzed by image or flow cytometry, and several methods have been described to identify apoptotic cells.

Mitotic death, also termed delayed reproductive death, shows some features of apoptosis and thus may represent delayed apoptosis; it occurs as a result of cell exposure to relatively low doses of drugs or radiation, which induce irreparable damage, but allow cells to complete at least one round of division.

2. Necrosis

Necrosis is an alternative to the apoptotic mechanism of cell death. Most often it is induced by an overdose of cytotoxic agents and is a cell response to a gross injury. However, certain cell types do respond even to pharmacological concentrations of some drugs or moderate doses of physical agents by necrosis rather than apoptosis and the reason for the difference in response is not entirely clear. While apoptosis requires active participation of the involved cell, often even in terms of initiation of the de novo protein synthesis, necrosis is a passive and degenerative process. In vivo, necrosis triggers the inflammatory response in the tissue, due to a release of cytoplasmic constituents to intercellular space, often resulting in scar formation. In contrast, remains of apoptotic cells are phagocytized not only by the “professional” macrophages, but also by other neighboring cells, without evoking any inflammatory reaction. The early event of necrosis is swelling of cell mitochondria, followed by rupture of the plasma membrane, and release of the cytoplasmic content.

The costimulatory molecule CD226 signals through VAV1 to amplify TCR signals and promote IL-17 production by CD4+ T cells

Content introduction:

• The costimulatory molecule CD226 signals through VAV1 to amplify TCR signals and promote IL-17 production by CD4+ T cells


• Modeling of patient virus titers suggests that availability of a vaccine could reduce hepatitis C virus transmission among injecting drug users


• TORC1 inhibition enhances immune function and reduces infections in the elderly


• Mitochondrial redox sensing by the kinase ATM maintains cellular antioxidant capacity


• IKK promotes cytokine-induced and cancer-associated AMPK activity and attenuates phenformin-induced cell death in LKB1-deficient cells

1. The costimulatory molecule CD226 signals through VAV1 to amplify TCR signals and promote IL-17 production by CD4+ T cells
The activation of T cells requires the guanine nucleotide exchange factor VAV1. Using mice in which a tag for affinity purification was attached to endogenous VAV1 molecules, Guillaume Gaud at Université de Toulouse in Toulouse, France and his colleagues analyzed by quantitative mass spectrometry the signaling complex that assembles around activated VAV1. Fifty VAV1-binding partners were identified, most of which had not been previously reported to participate in VAV1 signaling. Among these was CD226, a costimulatory molecule of immune cells. Engagement of CD226 induced the tyrosine phosphorylation of VAV1 and synergized with T cell receptor (TCR) signals to specifically enhance the production of interleukin-17 (IL-17) by primary human CD4+ T cells. Moreover, co-engagement of the TCR and a risk variant of CD226 that is associated with autoimmunity (rs763361) further enhanced VAV1 activation and IL-17 production. Thus, their study reveals that a VAV1-based, synergistic cross-talk exists between the TCR and CD226 during both physiological and pathological T cell responses and provides a rational basis for targeting CD226 for the management of autoimmune diseases.



Read more, please click http://stke.sciencemag.org/content/11/538/eaar3083

2. Modeling of patient virus titers suggests that availability of a vaccine could reduce hepatitis C virus transmission among injecting drug users
The major route of hepatitis C virus (HCV) transmission in the United States is injection drug use. Marian Major at Center for Biologics Evaluation and Research in Silver Spring, USA and her colleagues hypothesized that if an HCV vaccine were available, vaccination could affect HCV transmission among people who inject drugs by reducing HCV titers after viral exposure without necessarily achieving sterilizing immunity. To investigate this possibility, they developed a mathematical model to determine transmission probabilities relative to the HCV RNA titers of needle/syringe-sharing donors. They simulated sharing of two types of syringes fitted with needles that retain either large or small amounts of fluid after expulsion. Using previously published viral kinetics data from both naïve subjects infected with HCV and reinfected individuals who had previously cleared an HCV infection, they estimated transmission risk between pairs of serodiscordant injecting drug users, accounting for syringe type, rinsing, and sharing frequency. They calculated that the risk of HCV transmission through syringe sharing increased ~10-fold as viral titers (log10 IU/ml) increased ~25-fold. Cumulative analyses showed that, assuming sharing episodes every 7 days, the mean transmission risk over the first 6 months was >90% between two people sharing syringes when one had an HCV RNA titer >5 log10 IU/ml. For those with preexisting immunity that rapidly controlled HCV, the cumulative risk decreased to 1 to 25% depending on HCV titer and syringe type. Their modeling approach demonstrates that, even with transient viral replication after exposure during injection drug use, HCV transmission among people sharing syringes could be reduced through vaccination if an HCV vaccine were available.

Read more, please click http://stm.sciencemag.org/content/10/449/eaao4496

3. TORC1 inhibition enhances immune function and reduces infections in the elderly
Inhibition of the mechanistic target of rapamycin (mTOR) protein kinase extends life span and ameliorates aging-related pathologies including declining immune function in model organisms. The objective of this phase 2a randomized, placebo-controlled clinical trial was to determine whether low-dose mTOR inhibitor therapy enhanced immune function and decreased infection rates in 264 elderly subjects given the study drugs for 6 weeks. A low-dose combination of a catalytic (BEZ235) plus an allosteric (RAD001) mTOR inhibitor that selectively inhibits target of rapamycin complex 1 (TORC1) downstream of mTOR was safe and was associated with a significant (P = 0.001) decrease in the rate of infections reported by elderly subjects for a year after study drug initiation. In addition, Joan B. Mannick at Novartis Institutes for Biomedical Research in Cambridge, USA and his colleagues observed an up-regulation of antiviral gene expression and an improvement in the response to influenza vaccination in this treatment group. Thus, selective TORC1 inhibition has the potential to improve immune function and reduce infections in the elderly.

Read more, please click http://stm.sciencemag.org/content/10/449/eaaq1564

4. Mitochondrial redox sensing by the kinase ATM maintains cellular antioxidant capacity
Mitochondria are integral to cellular energy metabolism and ATP production and are involved in regulating many cellular processes. Mitochondria produce reactive oxygen species (ROS), which not only can damage cellular components but also participate in signal transduction. The kinase ATM, which is mutated in the neurodegenerative, autosomal recessive disease ataxia-telangiectasia (A-T), is a key player in the nuclear DNA damage response. However, ATM also performs a redox-sensing function mediated through formation of ROS-dependent disulfide-linked dimers. Yichong Zhang at Yale School of Medicine in New Haven, USA and his colleagues found that mitochondria-derived hydrogen peroxide promoted ATM dimerization. In HeLa cells, ATM dimers were localized to the nucleus and inhibited by the redox regulatory protein thioredoxin 1 (TRX1), suggesting the existence of a ROS-mediated, stress-signaling relay from mitochondria to the nucleus. ATM dimer formation did not affect its association with chromatin in the absence or presence of nuclear DNA damage, consistent with the separation of its redox and DNA damage signaling functions. Comparative analysis of U2OS cells expressing either wild-type ATM or the redox sensing–deficient C2991L mutant revealed that one function of ATM redox sensing is to promote glucose flux through the pentose phosphate pathway (PPP) by increasing the abundance and activity of glucose-6-phosphate dehydrogenase (G6PD), thereby increasing cellular antioxidant capacity. The PPP produces the coenzyme NADPH needed for a robust antioxidant response, including the regeneration of TRX1, indicating the existence of a regulatory feedback loop involving ATM and TRX1. They propose that loss of the mitochondrial ROS-sensing function of ATM may cause cellular ROS accumulation and oxidative stress in A-T.

Read more, please click http://stke.sciencemag.org/content/11/538/eaaq0702

5. IKK promotes cytokine-induced and cancer-associated AMPK activity and attenuates phenformin-induced cell death in LKB1-deficient cells
The 5′ AMP-activated protein kinase (AMPK) is an energy sensor that is activated upon phosphorylation of Thr172 in its activation loop by the kinase LKB1, CAMKK2, or TAK1. TAK1-dependent AMPK phosphorylation of Thr172 is less well characterized than phosphorylation of this site by LKB1 or CAMKK2. An important target of TAK1 is IκB kinase (IKK), which controls the activation of the transcription factor NF-κB. Ricardo J. Antonia at University of North Carolina at Chapel Hill in Chapel Hill, USA and his colleagues tested the hypothesis that IKK acted downstream of TAK1 to activate AMPK by phosphorylating Thr172. IKK was required for the phosphorylation of Thr172 in AMPK in response to treatment with the inflammatory cytokine IL-1β or TNF-α or upon TAK1 overexpression. In addition, IKK regulated basal AMPK Thr172 phosphorylation in several cancer cell types independently of TAK1, indicating that other modes of IKK activation could stimulate AMPK. They found that IKK directly phosphorylated AMPK at Thr172 independently of the tumor suppressor LKB1 or energy stress. Accordingly, in LKB1-deficient cells, IKK inhibition reduced AMPK Thr172 phosphorylation in response to the mitochondrial inhibitor phenformin. This response led to enhanced apoptosis and suggests that IKK inhibition in combination with phenformin could be used clinically to treat patients with LKB1-deficient cancers.

Read more, please click http://stke.sciencemag.org/content/11/538/eaan5850

The Nobel Prize in Physiology or Medicine 2007

The Nobel Prize in Physiology or Medicine 2007 was awarded jointly to Mario R. Capecchi, Sir Martin J. Evans and Oliver Smithies "for their discoveries of principles for introducing specific gene modifications in mice by the use of embryonic stem cells."

Nobelist	Born	Affiliation at the time of the award
Mario R. Capecchi	6 October 1937, Verona, Italy	University of Utah, Salt Lake City, UT, USA
Sir Martin J. Evans	1 January 1941, Stroud, United Kingdom	Cardiff University, Cardiff, United Kingdom
Oliver Smithies	Born: 23 June 1925, Halifax, United Kingdom Died: 10 January 2017, Chapel Hill, NC, USA	University of North Carolina, Chapel Hill, NC, USA

Summary

This year’s Nobel Laureates have made a series of ground-breaking discoveries concerning embryonic stem cells and DNA recombination in mammals. Their discoveries led to the creation of an immensely powerful technology referred to as gene targeting in mice. It is now being applied to virtually all areas of biomedicine – from basic research to the development of new therapies.

Gene targeting is often used to inactivate single genes. Such gene “knockout” experiments have elucidated the roles of numerous genes in embryonic development, adult physiology, aging and disease. To date, more than ten thousand mouse genes (approximately half of the genes in the mammalian genome) have been knocked out. Ongoing international efforts will make “knockout mice” for all genes available within the near future.

With gene targeting it is now possible to produce almost any type of DNA modification in the mouse genome, allowing scientists to establish the roles of individual genes in health and disease. Gene targeting has already produced more than five hundred different mouse models of human disorders, including cardiovascular and neuro-degenerative diseases, diabetes and cancer.



More details, please click The 2007 Nobel Prize in Physiology or Medicine.

The Nobel Prize in Physiology or Medicine 2007

The Nobel Prize in Physiology or Medicine 2007 was awarded jointly to Mario R. Capecchi, Sir Martin J. Evans and Oliver Smithies "for their discoveries of principles for introducing specific gene modifications in mice by the use of embryonic stem cells."

Nobelist	Born	Affiliation at the time of the award
Mario R. Capecchi	6 October 1937, Verona, Italy	University of Utah, Salt Lake City, UT, USA
Sir Martin J. Evans	1 January 1941, Stroud, United Kingdom	Cardiff University, Cardiff, United Kingdom
Oliver Smithies	Born: 23 June 1925, Halifax, United Kingdom Died: 10 January 2017, Chapel Hill, NC, USA	University of North Carolina, Chapel Hill, NC, USA

Summary

This year’s Nobel Laureates have made a series of ground-breaking discoveries concerning embryonic stem cells and DNA recombination in mammals. Their discoveries led to the creation of an immensely powerful technology referred to as gene targeting in mice. It is now being applied to virtually all areas of biomedicine – from basic research to the development of new therapies.

Gene targeting is often used to inactivate single genes. Such gene “knockout” experiments have elucidated the roles of numerous genes in embryonic development, adult physiology, aging and disease. To date, more than ten thousand mouse genes (approximately half of the genes in the mammalian genome) have been knocked out. Ongoing international efforts will make “knockout mice” for all genes available within the near future.

With gene targeting it is now possible to produce almost any type of DNA modification in the mouse genome, allowing scientists to establish the roles of individual genes in health and disease. Gene targeting has already produced more than five hundred different mouse models of human disorders, including cardiovascular and neuro-degenerative diseases, diabetes and cancer.



More details, please click The 2007 Nobel Prize in Physiology or Medicine.

Protein Kinase C-β Dictates B Cell Fate by Regulating Mitochondrial Remodeling, Metabolic Reprogramming, and Heme Biosynthesis

Content introduction:

• Protein Kinase C-β Dictates B Cell Fate by Regulating Mitochondrial Remodeling, Metabolic Reprogramming, and Heme Biosynthesis


• Cancer Cells Co-opt the Neuronal Redox-Sensing Channel TRPA1 to Promote Oxidative-Stress Tolerance


• Autoimmune Th17 Cells Induced Synovial Stromal and Innate Lymphoid Cell Secretion of the Cytokine GM-CSF to Initiate and Augment Autoimmune Arthritis


• A Single-Cell Transcriptomic Atlas of Thymus Organogenesis Resolves Cell Types and Developmental Maturation


• Transcription Factor IRF8 Orchestrates the Adaptive Natural Killer Cell Response

1. Protein Kinase C-β Dictates B Cell Fate by Regulating Mitochondrial Remodeling, Metabolic Reprogramming, and Heme Biosynthesis
PKCβ-null (Prkcb−/−) mice are severely immunodeficient. Here Carlson Tsui at The Francis Crick Institute in London, UK and his colleagues show that mice whose B cells lack PKCβ failed to form germinal centers and plasma cells, which undermined affinity maturation and antibody production in response to immunization. Moreover, these mice failed to develop plasma cells in response to viral infection. At the cellular level, they have shown that Prkcb−/− B cells exhibited defective antigen polarization and mTORC1 signaling. While altered antigen polarization impaired antigen presentation and likely restricted the potential of GC development, defective mTORC1 signaling impaired metabolic reprogramming, mitochondrial remodeling, and heme biosynthesis in these cells, which altogether overwhelmingly opposed plasma cell differentiation. Taken together, their study reveals mechanistic insights into the function of PKCβ as a key regulator of B cell polarity and metabolic reprogramming that instructs B cell fate.



Read more, please click https://www.cell.com/immunity/fulltext/S1074-7613(18)30202-4

2. Cancer Cells Co-opt the Neuronal Redox-Sensing Channel TRPA1 to Promote Oxidative-Stress Tolerance
Cancer cell survival is dependent on oxidative-stress defenses against reactive oxygen species (ROS) that accumulate during tumorigenesis. Here, Nobuaki Takahashi at Harvard Medical School in Boston, USA and his colleagues show a non-canonical oxidative-stress defense mechanism through TRPA1, a neuronal redox-sensing Ca2+-influx channel. In TRPA1-enriched breast and lung cancer spheroids, TRPA1 is critical for survival of inner cells that exhibit ROS accumulation. Moreover, TRPA1 promotes resistance to ROS-producing chemotherapies, and TRPA1 inhibition suppresses xenograft tumor growth and enhances chemosensitivity. TRPA1 does not affect redox status but upregulates Ca2+-dependent anti-apoptotic pathways. NRF2, an oxidant-defense transcription factor, directly controls TRPA1 expression, thus providing an orthogonal mechanism for protection against oxidative stress together with canonical ROS-neutralizing mechanisms. These findings reveal an oxidative-stress defense program involving TRPA1 that could be exploited for targeted cancer therapies.

Read more, please click https://www.cell.com/cancer-cell/fulltext/S1535-6108(18)30183-1

3. Autoimmune Th17 Cells Induced Synovial Stromal and Innate Lymphoid Cell Secretion of the Cytokine GM-CSF to Initiate and Augment Autoimmune Arthritis
Despite the importance of Th17 cells in autoimmune diseases, it remains unclear how they control other inflammatory cells in autoimmune tissue damage. Using a model of spontaneous autoimmune arthritis, Keiji Hirota at Osaka University in Osaka, Japan and his colleagues showed that arthritogenic Th17 cells stimulated fibroblast-like synoviocytes via interleukin-17 (IL-17) to secrete the cytokine GM-CSF and also expanded synovial-resident innate lymphoid cells (ILCs) in inflamed joints. Activated synovial ILCs, which expressed CD25, IL-33Ra, and TLR9, produced abundant GM-CSF upon stimulation by IL-2, IL-33, or CpG DNA. Loss of GM-CSF production by either ILCs or radio-resistant stromal cells prevented Th17 cell-mediated arthritis. GM-CSF production by Th17 cells augmented chronic inflammation but was dispensable for the initiation of arthritis. They showed that GM-CSF-producing ILCs were present in inflamed joints of rheumatoid arthritis patients. Thus, a cellular cascade of autoimmune Th17 cells, ILCs, and stromal cells, via IL-17 and GM-CSF, mediates chronic joint inflammation and can be a target for therapeutic intervention.

Read more, please click https://www.cell.com/immunity/fulltext/S1074-7613(18)30146-8

4. A Single-Cell Transcriptomic Atlas of Thymus Organogenesis Resolves Cell Types and Developmental Maturation
Thymus development is critical to the adaptive immune system, yet a comprehensive transcriptional framework capturing thymus organogenesis at single-cell resolution is still needed. Eric M. Kernfeld at University of Massachusetts Medical School in Worcester, USA and his colleagues applied single-cell RNA sequencing (RNA-seq) to capture 8 days of thymus development, perturbations of T cell receptor rearrangement, and in vitro organ cultures, producing profiles of 24,279 cells. They resolved transcriptional heterogeneity of developing lymphocytes, and genetic perturbation confirmed T cell identity of conventional and non-conventional lymphocytes. They characterized maturation dynamics of thymic epithelial cells in vivo, classified cell maturation state in a thymic organ culture, and revealed the intrinsic capacity of thymic epithelium to preserve transcriptional regularity despite exposure to exogenous retinoic acid. Finally, by integrating the cell atlas with human genome-wide association study (GWAS) data and autoimmune-disease-related genes, they implicated embryonic thymus-resident cells as possible participants in autoimmune disease etiologies. This resource provides a single-cell transcriptional framework for biological discovery and molecular analysis of thymus organogenesis.

Read more, please click https://www.cell.com/immunity/fulltext/S1074-7613(18)30184-5

5. Transcription Factor IRF8 Orchestrates the Adaptive Natural Killer Cell Response
Natural killer (NK) cells are innate lymphocytes that display features of adaptive immunity during viral infection. Biallelic mutations in IRF8 have been reported to cause familial NK cell deficiency and susceptibility to severe viral infection in humans; however, the precise role of this transcription factor in regulating NK cell function remains unknown. Here, Nicholas M. Adams at Memorial Sloan Kettering Cancer Center in New York, USA and his colleagues show that cell-intrinsic IRF8 was required for NK-cell-mediated protection against mouse cytomegalovirus infection. During viral exposure, NK cells upregulated IRF8 through interleukin-12 (IL-12) signaling and the transcription factor STAT4, which promoted epigenetic remodeling of the Irf8 locus. Moreover, IRF8 facilitated the proliferative burst of virus-specific NK cells by promoting expression of cell-cycle genes and directly controlling Zbtb32, a master regulator of virus-driven NK cell proliferation. These findings identify the function and cell-type-specific regulation of IRF8 in NK-cell-mediated antiviral immunity and provide a mechanistic understanding of viral susceptibility in patients with IRF8 mutations.

Read more, please click https://www.cell.com/immunity/fulltext/S1074-7613(18)30187-0

The Nobel Prize in Physiology or Medicine 2008

The Nobel Prize in Physiology or Medicine 2008 was divided, one half awarded to Harald zur Hausen "for his discovery of human papilloma viruses causing cervical cancer", the other half jointly to Françoise Barré-Sinoussi and Luc Montagnier "for their discovery of human immunodeficiency virus."

Nobelist	Born	Affiliation at the time of the award
Harald zur Hausen	11 March 1936, Gelsenkirchen, Germany	German Cancer Research Center, Heidelberg, Germany
Françoise Barré-Sinoussi	30 July 1947, Paris, France	Regulation of Retroviral Infections Unit, Virology Department, Institut Pasteur, Paris, France
Luc Montagnier	18 August 1932, Chabris, France	World Foundation for AIDS Research and Prevention, Paris, France

Summary

This year’s Nobel Prize awards discoveries of two viruses causing severe human diseases.



Harald zur Hausen went against current dogma and postulated that oncogenic human papilloma virus (HPV) caused cervical cancer, the second most common cancer among women. He realized that HPV-DNA could exist in a non-productive state in the tumours, and should be detectable by specific searches for viral DNA. He found HPV to be a heterogeneous family of viruses. Only some HPV types cause cancer. His discovery has led to characterization of the natural history of HPV infection, an understanding of mechanisms of HPV-induced carcinogenesis and the development of prophylactic vaccines against HPV acquisition.

Françoise Barré-Sinoussi and Luc Montagnier discovered human immunodeficiency virus (HIV). Virus production was identified in lymphocytes from patients with enlarged lymph nodes in early stages of acquired immunodeficiency, and in blood from patients with late stage disease. They characterized this retrovirus as the first known human lentivirus based on its morphological, biochemical and immunological properties. HIV impaired the immune system because of massive virus replication and cell damage to lymphocytes. The discovery was one prerequisite for the current understanding of the biology of the disease and its antiretroviral treatment.

More details, please click The 2008 Nobel Prize in Physiology or Medicine.

Cryo-EM structure of the insect olfactory receptor Orco

Content introduction:

• Cryo-EM structure of the insect olfactory receptor Orco


• Modulating plant growth–metabolism coordination for sustainable agriculture


• Autism-like phenotype and risk gene mRNA deadenylation by CPEB4 mis-splicing


• Genetic and transcriptional evolution alters cancer cell line drug response


• 5-HT release in nucleus accumbens rescues social deficits in mouse autism model

1. Cryo-EM structure of the insect olfactory receptor Orco
The olfactory system must recognize and discriminate amongst an enormous variety of chemicals in the environment. To contend with such diversity, insects have evolved a family of odorant-gated ion channels comprised of a highly conserved co-receptor (Orco) and a divergent odorant receptor (OR) that confers chemical specificity. Here, Joel A. Butterwick at The Rockefeller University in New York, USA and his colleagues present the single-particle cryo-electron microscopy structure of an Orco homomer from the parasitic fig wasp Apocrypta bakeri at 3.5 Å resolution, providing structural insight into this receptor family. Orco possesses a novel channel architecture, with four subunits symmetrically arranged around a central pore that diverges into four lateral conduits that open to the cytosol. The Orco tetramer has few inter-subunit interactions within the membrane and is bound together by a small cytoplasmic anchor domain. The minimal sequence conservation among ORs maps largely to the pore and anchor domain, shedding light on how the architecture of this receptor family accommodates its remarkable sequence diversity and facilitates the evolution of odour tuning.



Read more, please click https://www.nature.com/articles/s41586-018-0420-8

2. Modulating plant growth–metabolism coordination for sustainable agriculture
Enhancing global food security by increasing the productivity of green revolution varieties of cereals risks increasing the collateral environmental damage produced by inorganic nitrogen fertilizers. Improvements in the efficiency of nitrogen use of crops are therefore essential; however, they require an in-depth understanding of the co-regulatory mechanisms that integrate growth, nitrogen assimilation and carbon fixation. Here Shan Li at Chinese Academy of Sciences in Beijing, China and her colleagues show that the balanced opposing activities and physical interactions of the rice GROWTH-REGULATING FACTOR 4 (GRF4) transcription factor and the growth inhibitor DELLA confer homeostatic co-regulation of growth and the metabolism of carbon and nitrogen. GRF4 promotes and integrates nitrogen assimilation, carbon fixation and growth, whereas DELLA inhibits these processes. As a consequence, the accumulation of DELLA that is characteristic of green revolution varieties confers not only yield-enhancing dwarfism, but also reduces the efficiency of nitrogen use. However, the nitrogen-use efficiency of green revolution varieties and grain yield are increased by tipping the GRF4–DELLA balance towards increased GRF4 abundance. Modulation of plant growth and metabolic co-regulation thus enables novel breeding strategies for future sustainable food security and a new green revolution.

Read more, please click https://www.nature.com/articles/s41586-018-0415-5

3. Autism-like phenotype and risk gene mRNA deadenylation by CPEB4 mis-splicing
Common genetic contributions to autism spectrum disorder (ASD) reside in risk gene variants that individually have minimal effect sizes. As environmental factors that perturb neurodevelopment also underlie idiopathic ASD, it is crucial to identify altered regulators that can orchestrate multiple ASD risk genes during neurodevelopment. Cytoplasmic polyadenylation element binding proteins 1–4 (CPEB1–4) regulate the translation of specific mRNAs by modulating their poly(A)-tails and thereby participate in embryonic development and synaptic plasticity. Here Alberto Parras at Instituto de Salud Carlos III in Madrid, Spain and his colleagues find that CPEB4 binds transcripts of most high-confidence ASD risk genes. The brains of individuals with idiopathic ASD show imbalances in CPEB4 transcript isoforms that result from decreased inclusion of a neuron-specific microexon. In addition, 9% of the transcriptome shows reduced poly(A)-tail length. Notably, this percentage is much higher for high-confidence ASD risk genes, correlating with reduced expression of the protein products of ASD risk genes. An equivalent imbalance in CPEB4 transcript isoforms in mice mimics the changes in mRNA polyadenylation and protein expression of ASD risk genes and induces ASD-like neuroanatomical, electrophysiological and behavioural phenotypes. Together, these data identify CPEB4 as a regulator of ASD risk genes.

Read more, please click https://www.nature.com/articles/s41586-018-0423-5

4. Genetic and transcriptional evolution alters cancer cell line drug response
Human cancer cell lines are the workhorse of cancer research. Although cell lines are known to evolve in culture, the extent of the resultant genetic and transcriptional heterogeneity and its functional consequences remain understudied. Here Uri Ben-David at Broad Institute of Harvard and MIT in Cambridge, USA and his colleagues use genomic analyses of 106 human cell lines grown in two laboratories to show extensive clonal diversity. Further comprehensive genomic characterization of 27 strains of the common breast cancer cell line MCF7 uncovered rapid genetic diversification. Similar results were obtained with multiple strains of 13 additional cell lines. Notably, genetic changes were associated with differential activation of gene expression programs and marked differences in cell morphology and proliferation. Barcoding experiments showed that cell line evolution occurs as a result of positive clonal selection that is highly sensitive to culture conditions. Analyses of single-cell-derived clones demonstrated that continuous instability quickly translates into heterogeneity of the cell line. When the 27 MCF7 strains were tested against 321 anti-cancer compounds, they uncovered considerably different drug responses: at least 75% of compounds that strongly inhibited some strains were completely inactive in others. This study documents the extent, origins and consequences of genetic variation within cell lines, and provides a framework for researchers to measure such variation in efforts to support maximally reproducible cancer research.

Read more, please click https://www.nature.com/articles/s41586-018-0409-3

5. 5-HT release in nucleus accumbens rescues social deficits in mouse autism model
Dysfunction in prosocial interactions is a core symptom of autism spectrum disorder. However, the neural mechanisms that underlie sociability are poorly understood, limiting the rational development of therapies to treat social deficits. Here Jessica J. Walsh at Stanford University in Stanford, USA and his colleagues show in mice that bidirectional modulation of the release of serotonin (5-HT) from dorsal raphe neurons in the nucleus accumbens bidirectionally modifies sociability. In a mouse model of a common genetic cause of autism spectrum disorder—a copy number variation on chromosome 16p11.2—genetic deletion of the syntenic region from 5-HT neurons induces deficits in social behaviour and decreases dorsal raphe 5-HT neuronal activity. These sociability deficits can be rescued by optogenetic activation of dorsal raphe 5-HT neurons, an effect requiring and mimicked by activation of 5-HT1b receptors in the nucleus accumbens. These results demonstrate an unexpected role for 5-HT action in the nucleus accumbens in social behaviours, and suggest that targeting this mechanism may prove therapeutically beneficial.

Read more, please click https://www.nature.com/articles/s41586-018-0416-4

Cryo-EM structure of the insect olfactory receptor Orco

Content introduction:

• Cryo-EM structure of the insect olfactory receptor Orco


• Modulating plant growth–metabolism coordination for sustainable agriculture


• Autism-like phenotype and risk gene mRNA deadenylation by CPEB4 mis-splicing


• Genetic and transcriptional evolution alters cancer cell line drug response


• 5-HT release in nucleus accumbens rescues social deficits in mouse autism model

1. Cryo-EM structure of the insect olfactory receptor Orco
The olfactory system must recognize and discriminate amongst an enormous variety of chemicals in the environment. To contend with such diversity, insects have evolved a family of odorant-gated ion channels comprised of a highly conserved co-receptor (Orco) and a divergent odorant receptor (OR) that confers chemical specificity. Here, Joel A. Butterwick at The Rockefeller University in New York, USA and his colleagues present the single-particle cryo-electron microscopy structure of an Orco homomer from the parasitic fig wasp Apocrypta bakeri at 3.5 Å resolution, providing structural insight into this receptor family. Orco possesses a novel channel architecture, with four subunits symmetrically arranged around a central pore that diverges into four lateral conduits that open to the cytosol. The Orco tetramer has few inter-subunit interactions within the membrane and is bound together by a small cytoplasmic anchor domain. The minimal sequence conservation among ORs maps largely to the pore and anchor domain, shedding light on how the architecture of this receptor family accommodates its remarkable sequence diversity and facilitates the evolution of odour tuning.



Read more, please click https://www.nature.com/articles/s41586-018-0420-8

2. Modulating plant growth–metabolism coordination for sustainable agriculture
Enhancing global food security by increasing the productivity of green revolution varieties of cereals risks increasing the collateral environmental damage produced by inorganic nitrogen fertilizers. Improvements in the efficiency of nitrogen use of crops are therefore essential; however, they require an in-depth understanding of the co-regulatory mechanisms that integrate growth, nitrogen assimilation and carbon fixation. Here Shan Li at Chinese Academy of Sciences in Beijing, China and her colleagues show that the balanced opposing activities and physical interactions of the rice GROWTH-REGULATING FACTOR 4 (GRF4) transcription factor and the growth inhibitor DELLA confer homeostatic co-regulation of growth and the metabolism of carbon and nitrogen. GRF4 promotes and integrates nitrogen assimilation, carbon fixation and growth, whereas DELLA inhibits these processes. As a consequence, the accumulation of DELLA that is characteristic of green revolution varieties confers not only yield-enhancing dwarfism, but also reduces the efficiency of nitrogen use. However, the nitrogen-use efficiency of green revolution varieties and grain yield are increased by tipping the GRF4–DELLA balance towards increased GRF4 abundance. Modulation of plant growth and metabolic co-regulation thus enables novel breeding strategies for future sustainable food security and a new green revolution.

Read more, please click https://www.nature.com/articles/s41586-018-0415-5

3. Autism-like phenotype and risk gene mRNA deadenylation by CPEB4 mis-splicing
Common genetic contributions to autism spectrum disorder (ASD) reside in risk gene variants that individually have minimal effect sizes. As environmental factors that perturb neurodevelopment also underlie idiopathic ASD, it is crucial to identify altered regulators that can orchestrate multiple ASD risk genes during neurodevelopment. Cytoplasmic polyadenylation element binding proteins 1–4 (CPEB1–4) regulate the translation of specific mRNAs by modulating their poly(A)-tails and thereby participate in embryonic development and synaptic plasticity. Here Alberto Parras at Instituto de Salud Carlos III in Madrid, Spain and his colleagues find that CPEB4 binds transcripts of most high-confidence ASD risk genes. The brains of individuals with idiopathic ASD show imbalances in CPEB4 transcript isoforms that result from decreased inclusion of a neuron-specific microexon. In addition, 9% of the transcriptome shows reduced poly(A)-tail length. Notably, this percentage is much higher for high-confidence ASD risk genes, correlating with reduced expression of the protein products of ASD risk genes. An equivalent imbalance in CPEB4 transcript isoforms in mice mimics the changes in mRNA polyadenylation and protein expression of ASD risk genes and induces ASD-like neuroanatomical, electrophysiological and behavioural phenotypes. Together, these data identify CPEB4 as a regulator of ASD risk genes.

Read more, please click https://www.nature.com/articles/s41586-018-0423-5

4. Genetic and transcriptional evolution alters cancer cell line drug response
Human cancer cell lines are the workhorse of cancer research. Although cell lines are known to evolve in culture, the extent of the resultant genetic and transcriptional heterogeneity and its functional consequences remain understudied. Here Uri Ben-David at Broad Institute of Harvard and MIT in Cambridge, USA and his colleagues use genomic analyses of 106 human cell lines grown in two laboratories to show extensive clonal diversity. Further comprehensive genomic characterization of 27 strains of the common breast cancer cell line MCF7 uncovered rapid genetic diversification. Similar results were obtained with multiple strains of 13 additional cell lines. Notably, genetic changes were associated with differential activation of gene expression programs and marked differences in cell morphology and proliferation. Barcoding experiments showed that cell line evolution occurs as a result of positive clonal selection that is highly sensitive to culture conditions. Analyses of single-cell-derived clones demonstrated that continuous instability quickly translates into heterogeneity of the cell line. When the 27 MCF7 strains were tested against 321 anti-cancer compounds, they uncovered considerably different drug responses: at least 75% of compounds that strongly inhibited some strains were completely inactive in others. This study documents the extent, origins and consequences of genetic variation within cell lines, and provides a framework for researchers to measure such variation in efforts to support maximally reproducible cancer research.

Read more, please click https://www.nature.com/articles/s41586-018-0409-3

5. 5-HT release in nucleus accumbens rescues social deficits in mouse autism model
Dysfunction in prosocial interactions is a core symptom of autism spectrum disorder. However, the neural mechanisms that underlie sociability are poorly understood, limiting the rational development of therapies to treat social deficits. Here Jessica J. Walsh at Stanford University in Stanford, USA and his colleagues show in mice that bidirectional modulation of the release of serotonin (5-HT) from dorsal raphe neurons in the nucleus accumbens bidirectionally modifies sociability. In a mouse model of a common genetic cause of autism spectrum disorder—a copy number variation on chromosome 16p11.2—genetic deletion of the syntenic region from 5-HT neurons induces deficits in social behaviour and decreases dorsal raphe 5-HT neuronal activity. These sociability deficits can be rescued by optogenetic activation of dorsal raphe 5-HT neurons, an effect requiring and mimicked by activation of 5-HT1b receptors in the nucleus accumbens. These results demonstrate an unexpected role for 5-HT action in the nucleus accumbens in social behaviours, and suggest that targeting this mechanism may prove therapeutically beneficial.

Read more, please click https://www.nature.com/articles/s41586-018-0416-4

Basic Techniques for Mammalian Cell Tissue Culture

Tissue culture technology has found wide application in the field of cell biology. Cell cultures are utilized in cytogenetic, biochemical, and molecular laboratories for diagnostic as well as research studies. In most cases, cells or tissues must be grown in culture for days or weeks to obtain sufficient numbers of cells for analysis. Maintenance of cells in long-term culture requires strict adherence to aseptic technique to avoid contamination and potential loss of valuable cell lines.

An important factor influencing the growth of cells in culture is the choice of tissue culture medium. Many different recipes for tissue culture media are available and each laboratory must determine which medium best suits their needs. Individual laboratories may elect to use commercially prepared medium or prepare their own. Commercially available medium can be obtained as a sterile and ready-to-use liquid, in a concentrated liquid form, or in a powdered form. Besides providing nutrients for growing cells, medium is generally supplemented with antibiotics, fungicides, or both to inhibit contamination.

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually cell death. The first step in subculturing monolayers is to detach cells from the surface of the primary culture vessel by trypsinization or mechanical means. The resultant cell suspension is then subdivided, or reseeded, into fresh cultures. Secondary cultures are checked for growth, fed periodically, and may be subsequently subcultured to produce tertiary cultures, etc. The time between passaging cells depends on the growth rate and varies with the cell line. The Basic Protocol describes subculturing of a monolayer culture grown in petri plates or flasks; the Alternate Protocol 1 describes passaging of suspension cultures.

CAUTION: When working with human blood, cells, or infectious agents, appropriate biosafety practices must be followed.
NOTE: All solutions and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.

Trypsinzing And Subculturing Cells From A Monolayer

A primary culture is grown to confluency in a 60mm petri plate or 25 cm2 tissue culture flask containing 5 ml tissue culture medium. Cells are dispersed by trypsin treatment and then reseeded into secondary cultures. The process of removing cells from the primary culture and transferring them to secondary cultures constitutes a passage, or subculture.

Materials
Primary cultures of cells

PBS/HBSS without Ca2+ and Mg2+, 37°C

Trypsin/EDTA solution , 37°C

Complete medium with serum: e.g., supplemented DMEM with 10% to 15% (v/v) FBS, 37°C

Sterile Pasteur pipets

37°C warming tray or incubator

Tissue culture plasticware or glassware including pipets and 25-cm2 flasks or 60-mm petri plates, sterile

NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO2 incubator unless otherwise specified. Some media (e.g., DMEM) may require altered levels of CO2 to maintain pH 7.4.

1. Remove all medium from primary culture with a sterile Pasteur pipet. Wash adhering cell monolayer once or twice with a small volume of 37°C PBS/HBSS without Ca2+ and Mg2+ to remove any residual FBS that may inhibit the action of trypsin.

Use a buffered salt solution that is Ca2+ and Mg2+ free to wash cells. Ca2+ and Mg2+ in the salt solution can cause cells to stick together.

If this is the first medium change, rather than discarding medium that is removed from primary culture, put it into a fresh dish or flask. The medium contains unattached cells that may attach and grow, thereby providing a backup culture.

2. Add enough 37°C trypsin/EDTA solution to culture to cover adhering cell layer.

3. Place plate on a 37°C warming tray 1 to 2 min. Tap bottom of plate on the countertop to dislodge cells. Check culture with an inverted microscope to be sure that cells are rounded up and detached from the surface.

If cells are not sufficiently detached, return plate to warming tray for an additional minute or two.

4. Add 2 ml 37°C complete medium. Draw cell suspension into a Pasteur pipet and rinse cell layer two or three times to dissociate cells and to dislodge any remaining adherent cells. As soon as cells are detached, add serum or medium containing serum to inhibit
further trypsin activity that might damage cells.

If cultures are to be split 1/3 or 1/4 rather than 1/2, add sufficient medium such that 1 ml of cell suspension can be transferred into each fresh culture vessel.

5. Add an equal volume of cell suspension to fresh plates or flasks that have been appropriately labeled.

Alternatively, cells can be counted using a hemacytometer or Coulter counter and diluted to the desired density so a specific number of cells can be added to each culture vessel. A final concentration of ∼5 × 104 cells/ml is appropriate for most subcultures.

For primary cultures and early subcultures, 60-mm petri plates or 25cm2 flasks are generally used; larger vessels (e.g., 150mm plates or 75cm2 flasks) may be used for later subcultures.

Cultures should be labeled with date of subculture and passage number.

6. Add 4 ml fresh medium to each new culture. Incubate in a humidified 37°C, 5% CO2 incubator.

If using 75cm2 culture flasks, add 9 ml medium per flask.

Some labs now use incubators with 5% CO2 and 4% O2. The low oxygen concentration is thought to simulate the in vivo environment of cells and to enhance cell growth.

For some media it is necessary to adjust the CO2 to a higher or lower level to maintain the pH at 7.4.

7. If necessary, feed subconfluent cultures after 3 or 4 days by removing old medium and adding fresh 37°C medium.

8. Passage secondary culture when it becomes confluent by repeating steps 1 to 7, and continue to passage as necessary.

Passaging Cells In Suspension Culture

A suspension culture is grown in culture flasks in a humidified 37°C, 5% CO2 incubator. Passaging of suspension cultures is somewhat less complicated than passaging of monolayer cultures. Because the cells are suspended in medium rather than attached to a surface, it is not necessary to disperse them enzymatically before passaging. However, before passaging, cells must be maintained in culture by feeding every 2 to 3 days until they reach confluency (i.e., until the cells clump together in the suspension and the medium appears turbid when the flask is swirled).

NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO2 incubator unless otherwise specified. Some media (e.g., DMEM) may require altered levels of CO2 to maintain pH 7.4.

1. Feed cells as follows every 2 to 3 days until the cultures are confluent:

a. Remove flask of suspension cells from incubator, taking care not to disturb those that have settled to the flask bottom.

b. Aseptically remove and discard about one-third of the medium from flask and replace with an equal volume of prewarmed (37°C) medium. If the cells are growing rapidly, add an additional 10% medium by volume in order to maintain optimum concentration of 1 × 106 cells/ml. Gently swirl flask to resuspend cells.

c. Return flask to incubator. If there is <15 ml of medium in the flask, incubate flask in horizontal position to enhance cell/medium contact.

At higher volumes of medium the flask can be incubated in the vertical position.

If using a 25-cm2 flask, there should be 20 to 30 ml of medium in the flask at confluency.

2. On the days cultures are not being fed, check them by swirling flask to resuspend cells and observing color changes in the medium that indicate good metabolic growth.

3. When cultures are confluent (∼2.5 × 106 cells/ml), passage culture as follows:

a. Remove flask from incubator and swirl flask so that cells are evenly distributed in the medium.

b. Aseptically remove half of the volume of cell suspension and place into a fresh flask.

c. Feed each flask with 7 to 10 ml prewarmed medium and return flask to incubator.

Some labs prefer to split the cells 1:3 or 1:4, although increasing the split ratio will result in a longer interval before subcultures reach confluency.

Basic Techniques for Mammalian Cell Tissue Culture

Tissue culture technology has found wide application in the field of cell biology. Cell cultures are utilized in cytogenetic, biochemical, and molecular laboratories for diagnostic as well as research studies. In most cases, cells or tissues must be grown in culture for days or weeks to obtain sufficient numbers of cells for analysis. Maintenance of cells in long-term culture requires strict adherence to aseptic technique to avoid contamination and potential loss of valuable cell lines.

An important factor influencing the growth of cells in culture is the choice of tissue culture medium. Many different recipes for tissue culture media are available and each laboratory must determine which medium best suits their needs. Individual laboratories may elect to use commercially prepared medium or prepare their own. Commercially available medium can be obtained as a sterile and ready-to-use liquid, in a concentrated liquid form, or in a powdered form. Besides providing nutrients for growing cells, medium is generally supplemented with antibiotics, fungicides, or both to inhibit contamination.

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually cell death. The first step in subculturing monolayers is to detach cells from the surface of the primary culture vessel by trypsinization or mechanical means. The resultant cell suspension is then subdivided, or reseeded, into fresh cultures. Secondary cultures are checked for growth, fed periodically, and may be subsequently subcultured to produce tertiary cultures, etc. The time between passaging cells depends on the growth rate and varies with the cell line. The Basic Protocol describes subculturing of a monolayer culture grown in petri plates or flasks; the Alternate Protocol 1 describes passaging of suspension cultures.

CAUTION: When working with human blood, cells, or infectious agents, appropriate biosafety practices must be followed.
NOTE: All solutions and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.

Trypsinzing And Subculturing Cells From A Monolayer

A primary culture is grown to confluency in a 60mm petri plate or 25 cm2 tissue culture flask containing 5 ml tissue culture medium. Cells are dispersed by trypsin treatment and then reseeded into secondary cultures. The process of removing cells from the primary culture and transferring them to secondary cultures constitutes a passage, or subculture.

Materials
Primary cultures of cells

PBS/HBSS without Ca2+ and Mg2+, 37°C

Trypsin/EDTA solution , 37°C

Complete medium with serum: e.g., supplemented DMEM with 10% to 15% (v/v) FBS, 37°C

Sterile Pasteur pipets

37°C warming tray or incubator

Tissue culture plasticware or glassware including pipets and 25-cm2 flasks or 60-mm petri plates, sterile

NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO2 incubator unless otherwise specified. Some media (e.g., DMEM) may require altered levels of CO2 to maintain pH 7.4.

1. Remove all medium from primary culture with a sterile Pasteur pipet. Wash adhering cell monolayer once or twice with a small volume of 37°C PBS/HBSS without Ca2+ and Mg2+ to remove any residual FBS that may inhibit the action of trypsin.

Use a buffered salt solution that is Ca2+ and Mg2+ free to wash cells. Ca2+ and Mg2+ in the salt solution can cause cells to stick together.

If this is the first medium change, rather than discarding medium that is removed from primary culture, put it into a fresh dish or flask. The medium contains unattached cells that may attach and grow, thereby providing a backup culture.

2. Add enough 37°C trypsin/EDTA solution to culture to cover adhering cell layer.

3. Place plate on a 37°C warming tray 1 to 2 min. Tap bottom of plate on the countertop to dislodge cells. Check culture with an inverted microscope to be sure that cells are rounded up and detached from the surface.

If cells are not sufficiently detached, return plate to warming tray for an additional minute or two.

4. Add 2 ml 37°C complete medium. Draw cell suspension into a Pasteur pipet and rinse cell layer two or three times to dissociate cells and to dislodge any remaining adherent cells. As soon as cells are detached, add serum or medium containing serum to inhibit
further trypsin activity that might damage cells.

If cultures are to be split 1/3 or 1/4 rather than 1/2, add sufficient medium such that 1 ml of cell suspension can be transferred into each fresh culture vessel.

5. Add an equal volume of cell suspension to fresh plates or flasks that have been appropriately labeled.

Alternatively, cells can be counted using a hemacytometer or Coulter counter and diluted to the desired density so a specific number of cells can be added to each culture vessel. A final concentration of ∼5 × 104 cells/ml is appropriate for most subcultures.

For primary cultures and early subcultures, 60-mm petri plates or 25cm2 flasks are generally used; larger vessels (e.g., 150mm plates or 75cm2 flasks) may be used for later subcultures.

Cultures should be labeled with date of subculture and passage number.

6. Add 4 ml fresh medium to each new culture. Incubate in a humidified 37°C, 5% CO2 incubator.

If using 75cm2 culture flasks, add 9 ml medium per flask.

Some labs now use incubators with 5% CO2 and 4% O2. The low oxygen concentration is thought to simulate the in vivo environment of cells and to enhance cell growth.

For some media it is necessary to adjust the CO2 to a higher or lower level to maintain the pH at 7.4.

7. If necessary, feed subconfluent cultures after 3 or 4 days by removing old medium and adding fresh 37°C medium.

8. Passage secondary culture when it becomes confluent by repeating steps 1 to 7, and continue to passage as necessary.

Passaging Cells In Suspension Culture

A suspension culture is grown in culture flasks in a humidified 37°C, 5% CO2 incubator. Passaging of suspension cultures is somewhat less complicated than passaging of monolayer cultures. Because the cells are suspended in medium rather than attached to a surface, it is not necessary to disperse them enzymatically before passaging. However, before passaging, cells must be maintained in culture by feeding every 2 to 3 days until they reach confluency (i.e., until the cells clump together in the suspension and the medium appears turbid when the flask is swirled).

NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO2 incubator unless otherwise specified. Some media (e.g., DMEM) may require altered levels of CO2 to maintain pH 7.4.

1. Feed cells as follows every 2 to 3 days until the cultures are confluent:

a. Remove flask of suspension cells from incubator, taking care not to disturb those that have settled to the flask bottom.

b. Aseptically remove and discard about one-third of the medium from flask and replace with an equal volume of prewarmed (37°C) medium. If the cells are growing rapidly, add an additional 10% medium by volume in order to maintain optimum concentration of 1 × 106 cells/ml. Gently swirl flask to resuspend cells.

c. Return flask to incubator. If there is <15 ml of medium in the flask, incubate flask in horizontal position to enhance cell/medium contact.

At higher volumes of medium the flask can be incubated in the vertical position.

If using a 25-cm2 flask, there should be 20 to 30 ml of medium in the flask at confluency.

2. On the days cultures are not being fed, check them by swirling flask to resuspend cells and observing color changes in the medium that indicate good metabolic growth.

3. When cultures are confluent (∼2.5 × 106 cells/ml), passage culture as follows:

a. Remove flask from incubator and swirl flask so that cells are evenly distributed in the medium.

b. Aseptically remove half of the volume of cell suspension and place into a fresh flask.

c. Feed each flask with 7 to 10 ml prewarmed medium and return flask to incubator.

Some labs prefer to split the cells 1:3 or 1:4, although increasing the split ratio will result in a longer interval before subcultures reach confluency.

Anti-VSV-G Tag Monoclonal antibodies

VSV-G, a vesicular stomatitis virus G (VSV-G) protein fragment, which is commonly used in biomedical research to pseudotype retroviral and lentiviral vectors. The VSV-G epitope tag is commonly engineered onto the N- or C- terminus of a protein of interest so that the tagged protein can be analyzed and visualized using immunochemical methods.

Abbkine offers below Anti-VSV-G Tag antibodies for your choice.

[table id=21 /]

About Abbkine Scientific Co., Ltd.

Abbkine Scientific Co., Ltd. is a leading biotechnology company that focuses on developing and providing innovative, high quality assay kits, recombinant proteins, antibodies and other research tools to accelerate life science fundamental research, drug discovery, etc.  Find more details, please visit the website at Abbkine.

Anti-Myc Tag antibodies

Continued from Anti-V5 Tag antibodies, today I will overview Anti-Myc Tag antibodies.

A Myc tag is a polypeptide protein tag derived from the c-Myc gene product that can be added to a protein using recombinant DNA technology. The recombinant Myc fusion protein can be recognized by a well-known high-affinity 2D5 antibody, which can specifically recognizes native and denatured forms of Myc fusion proteins, with which can meet your any requirements for Western Blot, Immunofluorescence and Immunoprecipitation assays. Under native conditions, the elution of Myc-tag proteins can be achieved by the addition of the Myc tag peptide which competes with the recombinant proteins.

Abbkine offers below anti-Myc tag antibodies for your experiments.

[table id=20 /]

About Abbkine Scientific Co., Ltd.

Abbkine Scientific Co., Ltd. is a leading biotechnology company that focuses on developing and providing innovative, high quality assay kits, recombinant proteins, antibodies and other research tools to accelerate life science fundamental research, drug discovery, etc.  Find more details, please visit the website at Abbkine.

Anti-VSV-G Tag antibodies will be continued next time.

Anti-Myc Tag antibodies

Continued from Anti-V5 Tag antibodies, today I will overview Anti-Myc Tag antibodies.

A Myc tag is a polypeptide protein tag derived from the c-Myc gene product that can be added to a protein using recombinant DNA technology. The recombinant Myc fusion protein can be recognized by a well-known high-affinity 2D5 antibody, which can specifically recognizes native and denatured forms of Myc fusion proteins, with which can meet your any requirements for Western Blot, Immunofluorescence and Immunoprecipitation assays. Under native conditions, the elution of Myc-tag proteins can be achieved by the addition of the Myc tag peptide which competes with the recombinant proteins.

Abbkine offers below anti-Myc tag antibodies for your experiments.

[table id=20 /]

About Abbkine Scientific Co., Ltd.

Abbkine Scientific Co., Ltd. is a leading biotechnology company that focuses on developing and providing innovative, high quality assay kits, recombinant proteins, antibodies and other research tools to accelerate life science fundamental research, drug discovery, etc.  Find more details, please visit the website at Abbkine.

Anti-VSV-G Tag antibodies will be continued next time.