Thrombopoietin receptor–independent stimulation of hematopoietic stem cells by eltrombopag

Content introduction:

• Thrombopoietin receptor–independent stimulation of hematopoietic stem cells by eltrombopag


• Sarcoplasmic reticulum calcium leak contributes to arrhythmia but not to heart failure progression


• Evaluation of mutant huntingtin and neurofilament proteins as potential markers in Huntington’s disease


• ANP32A regulates ATM expression and prevents oxidative stress in cartilage, brain, and bone


• Phosphatidylinositol 4-phosphate is a major source of GPCR-stimulated phosphoinositide production

1. Thrombopoietin receptor–independent stimulation of hematopoietic stem cells by eltrombopag
Eltrombopag (EP), a small-molecule thrombopoietin receptor (TPO-R) agonist and potent intracellular iron chelator, has shown remarkable efficacy in stimulating sustained multilineage hematopoiesis in patients with bone marrow failure syndromes, suggesting an effect at the most immature hematopoietic stem and multipotent progenitor level. Although the functional and molecular effects of EP on megakaryopoiesis have been studied in the past, mechanistic insights into its effects on the earliest stages of hematopoiesis have been limited. Yun-Ruei Kao at Albert Einstein College of Medicine in Bronx, USA and his colleagues investigated the effects of EP treatment on hematopoietic stem cell (HSC) function using purified primary HSCs in separation-of-function mouse models, including a TPO-R–deficient strain, and stem cells isolated from patients undergoing TPO-R agonist treatment. Their mechanistic studies showed a stimulatory effect on stem cell self-renewal independently of TPO-R. Human and mouse HSCs responded to acute EP treatment with metabolic and gene expression alterations consistent with a reduction of intracellular labile iron pools that are essential for stem cell maintenance. Iron preloading prevented the stem cell stimulatory effects of EP. Moreover, comparative analysis of stem cells in the bone marrow of patients receiving EP showed a marked increase in the number of functional stem cells compared to patients undergoing therapy with romiplostim, another TPO-R agonist lacking an iron-chelating ability. Together, their study demonstrates that EP stimulates hematopoiesis at the stem cell level through iron chelation–mediated molecular reprogramming and indicates that labile iron pool–regulated pathways can modulate HSC function.



Read more, please click http://stm.sciencemag.org/content/10/458/eaas9563

2. Sarcoplasmic reticulum calcium leak contributes to arrhythmia but not to heart failure progression
Increased sarcoplasmic reticulum (SR) Ca2+ leak via the cardiac ryanodine receptor (RyR2) has been suggested to play a mechanistic role in the development of heart failure (HF) and cardiac arrhythmia. Mice treated with a selective RyR2 stabilizer, rycal S36, showed normalization of SR Ca2+ leak and improved survival in pressure overload (PO) and myocardial infarction (MI) models. The development of HF, measured by echocardiography and molecular markers, showed no difference in rycal S36– versus placebo-treated mice. Reduction of SR Ca2+ leak in the PO model by the rycal-unrelated RyR2 stabilizer dantrolene did not mitigate HF progression. Development of HF was not aggravated by increased SR Ca2+ leak due to RyR2 mutation (R2474S) in volume overload, an SR Ca2+ leak–independent HF model. Arrhythmia episodes were reduced by rycal S36 treatment in PO and MI mice in vivo and ex vivo in Langendorff-perfused hearts. Isolated cardiomyocytes from murine failing hearts and human ventricular failing and atrial nonfailing myocardium showed reductions in delayed afterdepolarizations, in spontaneous and induced Ca2+ waves, and in triggered activity in rycal S36 versus placebo cells, whereas the Ca2+ transient, SR Ca2+ load, SR Ca2+ adenosine triphosphatase function, and action potential duration were not affected. Rycal S36 treatment of human induced pluripotent stem cells isolated from a patient with catecholaminergic polymorphic ventricular tachycardia could rescue the leaky RyR2 receptor. These results suggest that SR Ca2+ leak does not primarily influence contractile HF progression, whereas rycal S36 treatment markedly reduces ventricular arrhythmias, thereby improving survival in mice.

Read more, please click http://stm.sciencemag.org/content/10/458/eaan0724

3. Evaluation of mutant huntingtin and neurofilament proteins as potential markers in Huntington’s disease
Huntington’s disease (HD) is a genetic progressive neurodegenerative disorder, caused by a mutation in the HTT gene, for which there is currently no cure. The identification of sensitive indicators of disease progression and therapeutic outcome could help the development of effective strategies for treating HD. Lauren M. Byrne at University College London (UCL) Institute of Neurology in London, UK and his colleagues assessed mutant huntingtin (mHTT) and neurofilament light (NfL) protein concentrations in cerebrospinal fluid (CSF) and blood in parallel with clinical evaluation and magnetic resonance imaging in premanifest and manifest HD mutation carriers. Among HD mutation carriers, NfL concentrations in plasma and CSF correlated with all nonbiofluid measures more closely than did CSF mHTT concentration. Longitudinal analysis over 4 to 8 weeks showed that CSF mHTT, CSF NfL, and plasma NfL concentrations were highly stable within individuals. In their cohort, concentration of CSF mHTT accurately distinguished between controls and HD mutation carriers, whereas NfL concentration, in both CSF and plasma, was able to segregate premanifest from manifest HD. In silico modeling indicated that mHTT and NfL concentrations in biofluids might be among the earliest detectable alterations in HD, and sample size prediction suggested that low participant numbers would be needed to incorporate these measures into clinical trials. These findings provide evidence that biofluid concentrations of mHTT and NfL have potential for early and sensitive detection of alterations in HD and could be integrated into both clinical trials and the clinic.

Read more, please click http://stm.sciencemag.org/content/10/458/eaat7108

4. ANP32A regulates ATM expression and prevents oxidative stress in cartilage, brain, and bone
Osteoarthritis is the most common joint disorder with increasing global prevalence due to aging of the population. Current therapy is limited to symptom relief, yet there is no cure. Its multifactorial etiology includes oxidative stress and overproduction of reactive oxygen species, but the regulation of these processes in the joint is insufficiently understood. Frederique M. F. Cornelis at Skeletal Biology and Engineering Research Center in KU Leuven, Belgium and his colleagues report that ANP32A protects the cartilage against oxidative stress, preventing osteoarthritis development and disease progression. ANP32A is down-regulated in human and mouse osteoarthritic cartilage. Microarray profiling revealed that ANP32A protects the joint by promoting the expression of ATM, a key regulator of the cellular oxidative defense. Antioxidant treatment reduced the severity of osteoarthritis, osteopenia, and cerebellar ataxia features in Anp32a-deficient mice, revealing that the ANP32A/ATM axis discovered in cartilage is also present in brain and bone. Their findings indicate that modulating ANP32A signaling could help manage oxidative stress in cartilage, brain, and bone with therapeutic implications for osteoarthritis, neurological disease, and osteoporosis.

Read more, please click http://stm.sciencemag.org/content/10/458/eaar8426

5. Phosphatidylinositol 4-phosphate is a major source of GPCR-stimulated phosphoinositide production

Phospholipase C (PLC) enzymes hydrolyze the plasma membrane (PM) lipid phosphatidylinositol 4,5-bisphosphate (PI4,5P2) to generate the second messengers inositol trisphosphate (IP3) and diacylglycerol (DAG) in response to receptor activation in almost all mammalian cells. Rafael Gil de Rubio at University of Rochester in Rochester, USA and his colleagues previously found that stimulation of G protein–coupled receptors (GPCRs) in cardiac cells leads to the PLC-dependent hydrolysis of phosphatidylinositol 4-phosphate (PI4P) at the Golgi, a process required for the activation of nuclear protein kinase D (PKD) during cardiac hypertrophy. They hypothesized that GPCR-stimulated PLC activation leading to direct PI4P hydrolysis may be a general mechanism for DAG production. They measured GPCR activation–dependent changes in PM and Golgi PI4P pools in various cells using GFP-based detection of PI4P. Stimulation with various agonists caused a time-dependent reduction in PI4P-associated, but not PI4,5P2-associated, fluorescence at the Golgi and PM. Targeted depletion of PI4,5P2 from the PM before GPCR stimulation had no effect on the depletion of PM or Golgi PI4P, total inositol phosphate (IP) production, or PKD activation. In contrast, acute depletion of PI4P specifically at the PM completely blocked the GPCR-dependent production of IPs and activation of PKD but did not change the abundance of PI4,5P2. Acute depletion of Golgi PI4P had no effect on these processes. These data suggest that most of the PM PI4,5P2 pool is not involved in GPCR-stimulated phosphoinositide hydrolysis and that PI4P at the PM is responsible for the bulk of receptor-stimulated phosphoinositide hydrolysis and DAG production.

Read more, please click http://stke.sciencemag.org/content/11/547/eaan1210

The Nobel Prize in Physiology or Medicine 2003

The Nobel Prize in Physiology or Medicine 2003 was awarded jointly to Paul C. Lauterbur and Sir Peter Mansfield "for their discoveries concerning magnetic resonance imaging."

Nobelist	Born	Died	Affiliation at the time of the award
Paul C. Lauterbur	6 May 1929, Sidney, OH, USA	27 March 2007, Urbana, IL, USA	University of Illinois, Urbana, IL, USA
Sir Peter Mansfield	9 October 1933, London, United Kingdom	8 February 2017	University of Nottingham, School of Physics and Astronomy, Nottingham, United Kingdom

Summary
Imaging of human internal organs with exact and non-invasive methods is very important for medical diagnosis, treatment and follow-up. This year’s Nobel Laureates in Physiology or Medicine have made seminal discoveries concerning the use of magnetic resonance to visualize different structures. These discoveries have led to the development of modern magnetic resonance imaging, MRI, which represents a breakthrough in medical diagnostics and research.

Atomic nuclei in a strong magnetic field rotate with a frequency that is dependent on the strength of the magnetic field. Their energy can be increased if they absorb radio waves with the same frequency (resonance). When the atomic nuclei return to their previous energy level, radio waves are emitted. These discoveries were awarded the Nobel Prize in Physics in 1952. During the following decades, magnetic resonance was used mainly for studies of the chemical structure of substances. In the beginning of the 1970s, this year’s Nobel Laureates made pioneering contributions, which later led to the applications of magnetic resonance in medical imaging.



More details, please click The 2003 Nobel Prize in Physiology or Medicine.

The Nobel Prize in Physiology or Medicine 2003

The Nobel Prize in Physiology or Medicine 2003 was awarded jointly to Paul C. Lauterbur and Sir Peter Mansfield "for their discoveries concerning magnetic resonance imaging."

Nobelist	Born	Died	Affiliation at the time of the award
Paul C. Lauterbur	6 May 1929, Sidney, OH, USA	27 March 2007, Urbana, IL, USA	University of Illinois, Urbana, IL, USA
Sir Peter Mansfield	9 October 1933, London, United Kingdom	8 February 2017	University of Nottingham, School of Physics and Astronomy, Nottingham, United Kingdom

Summary
Imaging of human internal organs with exact and non-invasive methods is very important for medical diagnosis, treatment and follow-up. This year’s Nobel Laureates in Physiology or Medicine have made seminal discoveries concerning the use of magnetic resonance to visualize different structures. These discoveries have led to the development of modern magnetic resonance imaging, MRI, which represents a breakthrough in medical diagnostics and research.

Atomic nuclei in a strong magnetic field rotate with a frequency that is dependent on the strength of the magnetic field. Their energy can be increased if they absorb radio waves with the same frequency (resonance). When the atomic nuclei return to their previous energy level, radio waves are emitted. These discoveries were awarded the Nobel Prize in Physics in 1952. During the following decades, magnetic resonance was used mainly for studies of the chemical structure of substances. In the beginning of the 1970s, this year’s Nobel Laureates made pioneering contributions, which later led to the applications of magnetic resonance in medical imaging.



More details, please click The 2003 Nobel Prize in Physiology or Medicine.

Thrombopoietin receptor–independent stimulation of hematopoietic stem cells by eltrombopag

Content introduction:

• Thrombopoietin receptor–independent stimulation of hematopoietic stem cells by eltrombopag


• Sarcoplasmic reticulum calcium leak contributes to arrhythmia but not to heart failure progression


• Evaluation of mutant huntingtin and neurofilament proteins as potential markers in Huntington’s disease


• ANP32A regulates ATM expression and prevents oxidative stress in cartilage, brain, and bone


• Phosphatidylinositol 4-phosphate is a major source of GPCR-stimulated phosphoinositide production

1. Thrombopoietin receptor–independent stimulation of hematopoietic stem cells by eltrombopag
Eltrombopag (EP), a small-molecule thrombopoietin receptor (TPO-R) agonist and potent intracellular iron chelator, has shown remarkable efficacy in stimulating sustained multilineage hematopoiesis in patients with bone marrow failure syndromes, suggesting an effect at the most immature hematopoietic stem and multipotent progenitor level. Although the functional and molecular effects of EP on megakaryopoiesis have been studied in the past, mechanistic insights into its effects on the earliest stages of hematopoiesis have been limited. Yun-Ruei Kao at Albert Einstein College of Medicine in Bronx, USA and his colleagues investigated the effects of EP treatment on hematopoietic stem cell (HSC) function using purified primary HSCs in separation-of-function mouse models, including a TPO-R–deficient strain, and stem cells isolated from patients undergoing TPO-R agonist treatment. Their mechanistic studies showed a stimulatory effect on stem cell self-renewal independently of TPO-R. Human and mouse HSCs responded to acute EP treatment with metabolic and gene expression alterations consistent with a reduction of intracellular labile iron pools that are essential for stem cell maintenance. Iron preloading prevented the stem cell stimulatory effects of EP. Moreover, comparative analysis of stem cells in the bone marrow of patients receiving EP showed a marked increase in the number of functional stem cells compared to patients undergoing therapy with romiplostim, another TPO-R agonist lacking an iron-chelating ability. Together, their study demonstrates that EP stimulates hematopoiesis at the stem cell level through iron chelation–mediated molecular reprogramming and indicates that labile iron pool–regulated pathways can modulate HSC function.



Read more, please click http://stm.sciencemag.org/content/10/458/eaas9563

2. Sarcoplasmic reticulum calcium leak contributes to arrhythmia but not to heart failure progression
Increased sarcoplasmic reticulum (SR) Ca2+ leak via the cardiac ryanodine receptor (RyR2) has been suggested to play a mechanistic role in the development of heart failure (HF) and cardiac arrhythmia. Mice treated with a selective RyR2 stabilizer, rycal S36, showed normalization of SR Ca2+ leak and improved survival in pressure overload (PO) and myocardial infarction (MI) models. The development of HF, measured by echocardiography and molecular markers, showed no difference in rycal S36– versus placebo-treated mice. Reduction of SR Ca2+ leak in the PO model by the rycal-unrelated RyR2 stabilizer dantrolene did not mitigate HF progression. Development of HF was not aggravated by increased SR Ca2+ leak due to RyR2 mutation (R2474S) in volume overload, an SR Ca2+ leak–independent HF model. Arrhythmia episodes were reduced by rycal S36 treatment in PO and MI mice in vivo and ex vivo in Langendorff-perfused hearts. Isolated cardiomyocytes from murine failing hearts and human ventricular failing and atrial nonfailing myocardium showed reductions in delayed afterdepolarizations, in spontaneous and induced Ca2+ waves, and in triggered activity in rycal S36 versus placebo cells, whereas the Ca2+ transient, SR Ca2+ load, SR Ca2+ adenosine triphosphatase function, and action potential duration were not affected. Rycal S36 treatment of human induced pluripotent stem cells isolated from a patient with catecholaminergic polymorphic ventricular tachycardia could rescue the leaky RyR2 receptor. These results suggest that SR Ca2+ leak does not primarily influence contractile HF progression, whereas rycal S36 treatment markedly reduces ventricular arrhythmias, thereby improving survival in mice.

Read more, please click http://stm.sciencemag.org/content/10/458/eaan0724

3. Evaluation of mutant huntingtin and neurofilament proteins as potential markers in Huntington’s disease
Huntington’s disease (HD) is a genetic progressive neurodegenerative disorder, caused by a mutation in the HTT gene, for which there is currently no cure. The identification of sensitive indicators of disease progression and therapeutic outcome could help the development of effective strategies for treating HD. Lauren M. Byrne at University College London (UCL) Institute of Neurology in London, UK and his colleagues assessed mutant huntingtin (mHTT) and neurofilament light (NfL) protein concentrations in cerebrospinal fluid (CSF) and blood in parallel with clinical evaluation and magnetic resonance imaging in premanifest and manifest HD mutation carriers. Among HD mutation carriers, NfL concentrations in plasma and CSF correlated with all nonbiofluid measures more closely than did CSF mHTT concentration. Longitudinal analysis over 4 to 8 weeks showed that CSF mHTT, CSF NfL, and plasma NfL concentrations were highly stable within individuals. In their cohort, concentration of CSF mHTT accurately distinguished between controls and HD mutation carriers, whereas NfL concentration, in both CSF and plasma, was able to segregate premanifest from manifest HD. In silico modeling indicated that mHTT and NfL concentrations in biofluids might be among the earliest detectable alterations in HD, and sample size prediction suggested that low participant numbers would be needed to incorporate these measures into clinical trials. These findings provide evidence that biofluid concentrations of mHTT and NfL have potential for early and sensitive detection of alterations in HD and could be integrated into both clinical trials and the clinic.

Read more, please click http://stm.sciencemag.org/content/10/458/eaat7108

4. ANP32A regulates ATM expression and prevents oxidative stress in cartilage, brain, and bone
Osteoarthritis is the most common joint disorder with increasing global prevalence due to aging of the population. Current therapy is limited to symptom relief, yet there is no cure. Its multifactorial etiology includes oxidative stress and overproduction of reactive oxygen species, but the regulation of these processes in the joint is insufficiently understood. Frederique M. F. Cornelis at Skeletal Biology and Engineering Research Center in KU Leuven, Belgium and his colleagues report that ANP32A protects the cartilage against oxidative stress, preventing osteoarthritis development and disease progression. ANP32A is down-regulated in human and mouse osteoarthritic cartilage. Microarray profiling revealed that ANP32A protects the joint by promoting the expression of ATM, a key regulator of the cellular oxidative defense. Antioxidant treatment reduced the severity of osteoarthritis, osteopenia, and cerebellar ataxia features in Anp32a-deficient mice, revealing that the ANP32A/ATM axis discovered in cartilage is also present in brain and bone. Their findings indicate that modulating ANP32A signaling could help manage oxidative stress in cartilage, brain, and bone with therapeutic implications for osteoarthritis, neurological disease, and osteoporosis.

Read more, please click http://stm.sciencemag.org/content/10/458/eaar8426

5. Phosphatidylinositol 4-phosphate is a major source of GPCR-stimulated phosphoinositide production

Phospholipase C (PLC) enzymes hydrolyze the plasma membrane (PM) lipid phosphatidylinositol 4,5-bisphosphate (PI4,5P2) to generate the second messengers inositol trisphosphate (IP3) and diacylglycerol (DAG) in response to receptor activation in almost all mammalian cells. Rafael Gil de Rubio at University of Rochester in Rochester, USA and his colleagues previously found that stimulation of G protein–coupled receptors (GPCRs) in cardiac cells leads to the PLC-dependent hydrolysis of phosphatidylinositol 4-phosphate (PI4P) at the Golgi, a process required for the activation of nuclear protein kinase D (PKD) during cardiac hypertrophy. They hypothesized that GPCR-stimulated PLC activation leading to direct PI4P hydrolysis may be a general mechanism for DAG production. They measured GPCR activation–dependent changes in PM and Golgi PI4P pools in various cells using GFP-based detection of PI4P. Stimulation with various agonists caused a time-dependent reduction in PI4P-associated, but not PI4,5P2-associated, fluorescence at the Golgi and PM. Targeted depletion of PI4,5P2 from the PM before GPCR stimulation had no effect on the depletion of PM or Golgi PI4P, total inositol phosphate (IP) production, or PKD activation. In contrast, acute depletion of PI4P specifically at the PM completely blocked the GPCR-dependent production of IPs and activation of PKD but did not change the abundance of PI4,5P2. Acute depletion of Golgi PI4P had no effect on these processes. These data suggest that most of the PM PI4,5P2 pool is not involved in GPCR-stimulated phosphoinositide hydrolysis and that PI4P at the PM is responsible for the bulk of receptor-stimulated phosphoinositide hydrolysis and DAG production.

Read more, please click http://stke.sciencemag.org/content/11/547/eaan1210

The Nobel Prize in Physiology or Medicine 2003

The Nobel Prize in Physiology or Medicine 2003 was awarded jointly to Paul C. Lauterbur and Sir Peter Mansfield "for their discoveries concerning magnetic resonance imaging."

Nobelist	Born	Died	Affiliation at the time of the award
Paul C. Lauterbur	6 May 1929, Sidney, OH, USA	27 March 2007, Urbana, IL, USA	University of Illinois, Urbana, IL, USA
Sir Peter Mansfield	9 October 1933, London, United Kingdom	8 February 2017	University of Nottingham, School of Physics and Astronomy, Nottingham, United Kingdom

Summary
Imaging of human internal organs with exact and non-invasive methods is very important for medical diagnosis, treatment and follow-up. This year’s Nobel Laureates in Physiology or Medicine have made seminal discoveries concerning the use of magnetic resonance to visualize different structures. These discoveries have led to the development of modern magnetic resonance imaging, MRI, which represents a breakthrough in medical diagnostics and research.

Atomic nuclei in a strong magnetic field rotate with a frequency that is dependent on the strength of the magnetic field. Their energy can be increased if they absorb radio waves with the same frequency (resonance). When the atomic nuclei return to their previous energy level, radio waves are emitted. These discoveries were awarded the Nobel Prize in Physics in 1952. During the following decades, magnetic resonance was used mainly for studies of the chemical structure of substances. In the beginning of the 1970s, this year’s Nobel Laureates made pioneering contributions, which later led to the applications of magnetic resonance in medical imaging.



More details, please click The 2003 Nobel Prize in Physiology or Medicine.

The Nobel Prize in Physiology or Medicine 2004

The Nobel Prize in Physiology or Medicine 2004 was awarded jointly to Richard Axel and Linda B. Buck "for their discoveries of odorant receptors and the organization of the olfactory system."

Nobelist	Born	Affiliation at the time of the award
Richard Axel	2 July 1946, New York, NY, USA	Columbia University, New York, NY, USA
Linda B. Buck	29 January 1947, Seattle, WA, USA	Fred Hutchinson Cancer Research Center, Seattle, WA, USA

Summary

The sense of smell long remained the most enigmatic of our senses. The basic principles for recognizing and remembering about 10,000 different odours were not understood. This year’s Nobel Laureates in Physiology or Medicine have solved this problem and in a series of pioneering studies clarified how our olfactory system works. They discovered a large gene family, comprised of some 1,000 different genes (three per cent of our genes) that give rise to an equivalent number of olfactory receptor types. These receptors are located on the olfactory receptor cells, which occupy a small area in the upper part of the nasal epithelium and detect the inhaled odorant molecules.

Each olfactory receptor cell possesses only one type of odorant receptor, and each receptor can detect a limited number of odorant substances. Our olfactory receptor cells are therefore highly specialized for a few odours. The cells send thin nerve processes directly to distinct micro domains, glomeruli, in the olfactory bulb, the primary olfactory area of the brain. Receptor cells carrying the same type of receptor send their nerve processes to the same glomerulus. From these micro domains in the olfactory bulb the information is relayed further to other parts of the brain, where the information from several olfactory receptors is combined, forming a pattern. Therefore, we can consciously experience the smell of a lilac flower in the spring and recall this olfactory memory at other times.



More details, please click The 2004 Nobel Prize in Physiology or Medicine.

The Nobel Prize in Physiology or Medicine 2004

The Nobel Prize in Physiology or Medicine 2004 was awarded jointly to Richard Axel and Linda B. Buck "for their discoveries of odorant receptors and the organization of the olfactory system."

Nobelist	Born	Affiliation at the time of the award
Richard Axel	2 July 1946, New York, NY, USA	Columbia University, New York, NY, USA
Linda B. Buck	29 January 1947, Seattle, WA, USA	Fred Hutchinson Cancer Research Center, Seattle, WA, USA

Summary

The sense of smell long remained the most enigmatic of our senses. The basic principles for recognizing and remembering about 10,000 different odours were not understood. This year’s Nobel Laureates in Physiology or Medicine have solved this problem and in a series of pioneering studies clarified how our olfactory system works. They discovered a large gene family, comprised of some 1,000 different genes (three per cent of our genes) that give rise to an equivalent number of olfactory receptor types. These receptors are located on the olfactory receptor cells, which occupy a small area in the upper part of the nasal epithelium and detect the inhaled odorant molecules.

Each olfactory receptor cell possesses only one type of odorant receptor, and each receptor can detect a limited number of odorant substances. Our olfactory receptor cells are therefore highly specialized for a few odours. The cells send thin nerve processes directly to distinct micro domains, glomeruli, in the olfactory bulb, the primary olfactory area of the brain. Receptor cells carrying the same type of receptor send their nerve processes to the same glomerulus. From these micro domains in the olfactory bulb the information is relayed further to other parts of the brain, where the information from several olfactory receptors is combined, forming a pattern. Therefore, we can consciously experience the smell of a lilac flower in the spring and recall this olfactory memory at other times.



More details, please click The 2004 Nobel Prize in Physiology or Medicine.

The Nobel Prize in Physiology or Medicine 2004

The Nobel Prize in Physiology or Medicine 2004 was awarded jointly to Richard Axel and Linda B. Buck "for their discoveries of odorant receptors and the organization of the olfactory system."

Nobelist	Born	Affiliation at the time of the award
Richard Axel	2 July 1946, New York, NY, USA	Columbia University, New York, NY, USA
Linda B. Buck	29 January 1947, Seattle, WA, USA	Fred Hutchinson Cancer Research Center, Seattle, WA, USA

Summary

The sense of smell long remained the most enigmatic of our senses. The basic principles for recognizing and remembering about 10,000 different odours were not understood. This year’s Nobel Laureates in Physiology or Medicine have solved this problem and in a series of pioneering studies clarified how our olfactory system works. They discovered a large gene family, comprised of some 1,000 different genes (three per cent of our genes) that give rise to an equivalent number of olfactory receptor types. These receptors are located on the olfactory receptor cells, which occupy a small area in the upper part of the nasal epithelium and detect the inhaled odorant molecules.

Each olfactory receptor cell possesses only one type of odorant receptor, and each receptor can detect a limited number of odorant substances. Our olfactory receptor cells are therefore highly specialized for a few odours. The cells send thin nerve processes directly to distinct micro domains, glomeruli, in the olfactory bulb, the primary olfactory area of the brain. Receptor cells carrying the same type of receptor send their nerve processes to the same glomerulus. From these micro domains in the olfactory bulb the information is relayed further to other parts of the brain, where the information from several olfactory receptors is combined, forming a pattern. Therefore, we can consciously experience the smell of a lilac flower in the spring and recall this olfactory memory at other times.



More details, please click The 2004 Nobel Prize in Physiology or Medicine.

The Nobel Prize in Physiology or Medicine 2005

The Nobel Prize in Physiology or Medicine 2005 was awarded jointly to Barry J. Marshall and J. Robin Warren "for their discovery of the bacterium Helicobacter pylori and its role in gastritis and peptic ulcer disease."

Nobelist	Born	Affiliation at the time of the award
Barry J. Marshall	30 September 1951, Kalgoorlie, Australia	NHMRC Helicobacter pylori Research Laboratory, Australia
J. Robin Warren	11 June 1937, Adelaide, Australia	Perth, Australia

Summary

This year’s Nobel Laureates in Physiology or Medicine made the remarkable and unexpected discovery that inflammation in the stomach (gastritis) as well as ulceration of the stomach or duodenum (peptic ulcer disease) is the result of an infection of the stomach caused by the bacterium Helicobacter pylori.

Robin Warren (born 1937), a pathologist from Perth, Australia, observed small curved bacteria colonizing the lower part of the stomach (antrum) in about 50% of patients from which biopsies had been taken. He made the crucial observation that signs of inflammation were always present in the gastric mucosa close to where the bacteria were seen.

Barry Marshall (born 1951), a young clinical fellow, became interested in Warren’s findings and together they initiated a study of biopsies from 100 patients. After several attempts, Marshall succeeded in cultivating a hitherto unknown bacterial species (later denoted Helicobacter pylori) from several of these biopsies. Together they found that the organism was present in almost all patients with gastric inflammation, duodenal ulcer or gastric ulcer. Based on these results, they proposed that Helicobacter pylori is involved in the aetiology of these diseases.

Even though peptic ulcers could be healed by inhibiting gastric acid production, they frequently relapsed, since bacteria and chronic inflammation of the stomach remained. In treatment studies, Marshall and Warren as well as others showed that patients could be cured from their peptic ulcer disease only when the bacteria were eradicated from the stomach. Thanks to the pioneering discovery by Marshall and Warren, peptic ulcer disease is no longer a chronic, frequently disabling condition, but a disease that can be cured by a short regimen of antibiotics and acid secretion inhibitors.



More details, please click The 2005 Nobel Prize in Physiology or Medicine.

The Nobel Prize in Physiology or Medicine 2005

The Nobel Prize in Physiology or Medicine 2005 was awarded jointly to Barry J. Marshall and J. Robin Warren "for their discovery of the bacterium Helicobacter pylori and its role in gastritis and peptic ulcer disease."

Nobelist	Born	Affiliation at the time of the award
Barry J. Marshall	30 September 1951, Kalgoorlie, Australia	NHMRC Helicobacter pylori Research Laboratory, Australia
J. Robin Warren	11 June 1937, Adelaide, Australia	Perth, Australia

Summary

This year’s Nobel Laureates in Physiology or Medicine made the remarkable and unexpected discovery that inflammation in the stomach (gastritis) as well as ulceration of the stomach or duodenum (peptic ulcer disease) is the result of an infection of the stomach caused by the bacterium Helicobacter pylori.

Robin Warren (born 1937), a pathologist from Perth, Australia, observed small curved bacteria colonizing the lower part of the stomach (antrum) in about 50% of patients from which biopsies had been taken. He made the crucial observation that signs of inflammation were always present in the gastric mucosa close to where the bacteria were seen.

Barry Marshall (born 1951), a young clinical fellow, became interested in Warren’s findings and together they initiated a study of biopsies from 100 patients. After several attempts, Marshall succeeded in cultivating a hitherto unknown bacterial species (later denoted Helicobacter pylori) from several of these biopsies. Together they found that the organism was present in almost all patients with gastric inflammation, duodenal ulcer or gastric ulcer. Based on these results, they proposed that Helicobacter pylori is involved in the aetiology of these diseases.

Even though peptic ulcers could be healed by inhibiting gastric acid production, they frequently relapsed, since bacteria and chronic inflammation of the stomach remained. In treatment studies, Marshall and Warren as well as others showed that patients could be cured from their peptic ulcer disease only when the bacteria were eradicated from the stomach. Thanks to the pioneering discovery by Marshall and Warren, peptic ulcer disease is no longer a chronic, frequently disabling condition, but a disease that can be cured by a short regimen of antibiotics and acid secretion inhibitors.



More details, please click The 2005 Nobel Prize in Physiology or Medicine.

Immunofluorescence Staining

Immunofluorescence is a technique allowing the visualization of a specific protein or antigen in cells or tissue sections by binding a specific antibody chemically conjugated with a fluorescent dye such as fluorescein isothiocyanate (FITC). There are two major types of immunofluorescence staining methods: 1) direct immunofluorescence staining in which the primary antibody is labeled with fluorescence dye, and 2) indirect immunofluorescence staining in which a secondary antibody labeled with fluorochrome is used to recognize a primary antibody. Immunofluorescence staining can be performed on cells fixed on slides and tissue sections and examined under a fluorescence microscope or confocal microscope.

The method relies on proper fixation of cells to retain cellular distribution of antigen and to preserve cellular morphology. After fixation, the cells are exposed to primary antibody directed against the protein of interest, in the prescence of permeabilizing reagents to ensure antibody access to the epitope. Following incubation with the primary antibody, the unbound primary antibody is removed and the bound primary antibody is then labeled by incubation with a fluorescently tagged secondary antibody directed against the primary antibody host species.

Materials:

1XPhosphate Buffered Saline (PBS)
4% Formaldehyde: methanol free, use fresh and store opened vials at 4°C in dark. Dilute with 1X PBS to make a 4% formaldehyde solution.
0.1%Triton X-100 (prepared with 1× PBS)
Blocking Buffer: 1X PBS/5% normal serum (Sharing the same or similar species with secondary antibodies)
Antibody Dilution Buffer: To prepare 10 ml, add 30 µl Triton X-100 to 10 ml 1X PBS. Mix well then add 0.1g BSA.
Mounting medium: 50% glycerol with 0.1%(w/v) p-phenylenediamine in PBS or use Fluormount G

A. Sample Preparation

Cultured Cell Lines (IF-IC)
NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.

1. Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in warm PBS.
NOTE: Formaldehyde is toxic, use only in a fume hood.

2. Allow cells to fix for 15 min at room temperature.

3. Aspirate fixative, rinse three times in 1X PBS for 5 min each.

4. Permeate the cells at room temperature for 15 minutes with 0.1%Triton X-100 (prepared with 1× PBS)

5. Wash the cover glass with 1× PBS for 5 minutes and repeat 3 times.
Proceed with Immunostaining (Section B).

Frozen/Cryostat Sections (IF-F)

1. For fixed frozen tissue proceed with Immunostaining (Section B).

2. For fresh, unfixed frozen tissue, fix immediately, as follows:

a. Cover sections with 4% formaldehyde diluted in warm 1X PBS.
b. Allow sections to fix for 15 min at room temperature.
c. Rinse slides three times in PBS for 5 min each.
d. Permeate the cells at room temperature for 15 minutes with 0.1%Triton X-100 (prepared with 1× PBS)
e. Rinse slides three times in PBS for 5 min each.
Proceed with Immunostaining (Section B).

B. Immunostaining

NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.

1. Block specimen in blocking buffer for 30 min.
2. Aspirate blocking solution, apply diluted primary antibody.
3. Incubate overnight at 4°C.
4. Rinse three times in 1X PBS for 5 min each.
NOTE: If using a fluorochrome-conjugated primary antibody, then skip to Step 7.

5. Incubate secondary antibody diluted in antibody dilution buffer for 1–2 hr at room temperature in the dark.
6. Rinse three times in 1X PBS for 5 min each.
7. Invert Coverslip slides cell-side-down, on one drop mounting medium. Gently blot with paper towel, then seal edge by painting with nail polish. Let dry.
8. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C protected from light (6-12 month).

Immunofluorescence Staining

Immunofluorescence is a technique allowing the visualization of a specific protein or antigen in cells or tissue sections by binding a specific antibody chemically conjugated with a fluorescent dye such as fluorescein isothiocyanate (FITC). There are two major types of immunofluorescence staining methods: 1) direct immunofluorescence staining in which the primary antibody is labeled with fluorescence dye, and 2) indirect immunofluorescence staining in which a secondary antibody labeled with fluorochrome is used to recognize a primary antibody. Immunofluorescence staining can be performed on cells fixed on slides and tissue sections and examined under a fluorescence microscope or confocal microscope.

The method relies on proper fixation of cells to retain cellular distribution of antigen and to preserve cellular morphology. After fixation, the cells are exposed to primary antibody directed against the protein of interest, in the prescence of permeabilizing reagents to ensure antibody access to the epitope. Following incubation with the primary antibody, the unbound primary antibody is removed and the bound primary antibody is then labeled by incubation with a fluorescently tagged secondary antibody directed against the primary antibody host species.

Materials:

1XPhosphate Buffered Saline (PBS)
4% Formaldehyde: methanol free, use fresh and store opened vials at 4°C in dark. Dilute with 1X PBS to make a 4% formaldehyde solution.
0.1%Triton X-100 (prepared with 1× PBS)
Blocking Buffer: 1X PBS/5% normal serum (Sharing the same or similar species with secondary antibodies)
Antibody Dilution Buffer: To prepare 10 ml, add 30 µl Triton X-100 to 10 ml 1X PBS. Mix well then add 0.1g BSA.
Mounting medium: 50% glycerol with 0.1%(w/v) p-phenylenediamine in PBS or use Fluormount G

A. Sample Preparation

Cultured Cell Lines (IF-IC)
NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.

1. Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in warm PBS.
NOTE: Formaldehyde is toxic, use only in a fume hood.

2. Allow cells to fix for 15 min at room temperature.

3. Aspirate fixative, rinse three times in 1X PBS for 5 min each.

4. Permeate the cells at room temperature for 15 minutes with 0.1%Triton X-100 (prepared with 1× PBS)

5. Wash the cover glass with 1× PBS for 5 minutes and repeat 3 times.
Proceed with Immunostaining (Section B).

Frozen/Cryostat Sections (IF-F)

1. For fixed frozen tissue proceed with Immunostaining (Section B).

2. For fresh, unfixed frozen tissue, fix immediately, as follows:

a. Cover sections with 4% formaldehyde diluted in warm 1X PBS.
b. Allow sections to fix for 15 min at room temperature.
c. Rinse slides three times in PBS for 5 min each.
d. Permeate the cells at room temperature for 15 minutes with 0.1%Triton X-100 (prepared with 1× PBS)
e. Rinse slides three times in PBS for 5 min each.
Proceed with Immunostaining (Section B).

B. Immunostaining

NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.

1. Block specimen in blocking buffer for 30 min.
2. Aspirate blocking solution, apply diluted primary antibody.
3. Incubate overnight at 4°C.
4. Rinse three times in 1X PBS for 5 min each.
NOTE: If using a fluorochrome-conjugated primary antibody, then skip to Step 7.

5. Incubate secondary antibody diluted in antibody dilution buffer for 1–2 hr at room temperature in the dark.
6. Rinse three times in 1X PBS for 5 min each.
7. Invert Coverslip slides cell-side-down, on one drop mounting medium. Gently blot with paper towel, then seal edge by painting with nail polish. Let dry.
8. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C protected from light (6-12 month).

Immunofluorescence Staining

Immunofluorescence is a technique allowing the visualization of a specific protein or antigen in cells or tissue sections by binding a specific antibody chemically conjugated with a fluorescent dye such as fluorescein isothiocyanate (FITC). There are two major types of immunofluorescence staining methods: 1) direct immunofluorescence staining in which the primary antibody is labeled with fluorescence dye, and 2) indirect immunofluorescence staining in which a secondary antibody labeled with fluorochrome is used to recognize a primary antibody. Immunofluorescence staining can be performed on cells fixed on slides and tissue sections and examined under a fluorescence microscope or confocal microscope.

The method relies on proper fixation of cells to retain cellular distribution of antigen and to preserve cellular morphology. After fixation, the cells are exposed to primary antibody directed against the protein of interest, in the prescence of permeabilizing reagents to ensure antibody access to the epitope. Following incubation with the primary antibody, the unbound primary antibody is removed and the bound primary antibody is then labeled by incubation with a fluorescently tagged secondary antibody directed against the primary antibody host species.

Materials:

1XPhosphate Buffered Saline (PBS)
4% Formaldehyde: methanol free, use fresh and store opened vials at 4°C in dark. Dilute with 1X PBS to make a 4% formaldehyde solution.
0.1%Triton X-100 (prepared with 1× PBS)
Blocking Buffer: 1X PBS/5% normal serum (Sharing the same or similar species with secondary antibodies)
Antibody Dilution Buffer: To prepare 10 ml, add 30 µl Triton X-100 to 10 ml 1X PBS. Mix well then add 0.1g BSA.
Mounting medium: 50% glycerol with 0.1%(w/v) p-phenylenediamine in PBS or use Fluormount G

A. Sample Preparation

Cultured Cell Lines (IF-IC)
NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.

1. Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in warm PBS.
NOTE: Formaldehyde is toxic, use only in a fume hood.

2. Allow cells to fix for 15 min at room temperature.

3. Aspirate fixative, rinse three times in 1X PBS for 5 min each.

4. Permeate the cells at room temperature for 15 minutes with 0.1%Triton X-100 (prepared with 1× PBS)

5. Wash the cover glass with 1× PBS for 5 minutes and repeat 3 times.
Proceed with Immunostaining (Section B).

Frozen/Cryostat Sections (IF-F)

1. For fixed frozen tissue proceed with Immunostaining (Section B).

2. For fresh, unfixed frozen tissue, fix immediately, as follows:

a. Cover sections with 4% formaldehyde diluted in warm 1X PBS.
b. Allow sections to fix for 15 min at room temperature.
c. Rinse slides three times in PBS for 5 min each.
d. Permeate the cells at room temperature for 15 minutes with 0.1%Triton X-100 (prepared with 1× PBS)
e. Rinse slides three times in PBS for 5 min each.
Proceed with Immunostaining (Section B).

B. Immunostaining

NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.

1. Block specimen in blocking buffer for 30 min.
2. Aspirate blocking solution, apply diluted primary antibody.
3. Incubate overnight at 4°C.
4. Rinse three times in 1X PBS for 5 min each.
NOTE: If using a fluorochrome-conjugated primary antibody, then skip to Step 7.

5. Incubate secondary antibody diluted in antibody dilution buffer for 1–2 hr at room temperature in the dark.
6. Rinse three times in 1X PBS for 5 min each.
7. Invert Coverslip slides cell-side-down, on one drop mounting medium. Gently blot with paper towel, then seal edge by painting with nail polish. Let dry.
8. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C protected from light (6-12 month).

The Tandem Duplicator Phenotype Is a Prevalent Genome-Wide Cancer Configuration Driven by Distinct Gene Mutations

Content introduction:

• The Tandem Duplicator Phenotype Is a Prevalent Genome-Wide Cancer Configuration Driven by Distinct Gene Mutations


• Comprehensive Analysis of Alternative Splicing Across Tumors from 8,705 Patients


• Synthetic Lethal and Convergent Biological Effects of Cancer-Associated Spliceosomal Gene Mutations


• Apoptotic Cell-Derived Extracellular Vesicles Promote Malignancy of Glioblastoma Via Intercellular Transfer of Splicing Factors


• Autocrine Mfge8 Signaling Prevents Developmental Exhaustion of the Adult Neural Stem Cell Pool

1. The Tandem Duplicator Phenotype Is a Prevalent Genome-Wide Cancer Configuration Driven by Distinct Gene Mutations
The tandem duplicator phenotype (TDP) is a genome-wide instability configuration primarily observed in breast, ovarian, and endometrial carcinomas. Here, Francesca Menghi at The Jackson Laboratory for Genomic Medicine in Farmington, USA and his colleagues stratify TDP tumors by classifying their tandem duplications (TDs) into three span intervals, with modal values of 11 kb, 231 kb, and 1.7 Mb, respectively. TDPs with ∼11 kb TDs feature loss of TP53 and BRCA1. TDPs with ∼231 kb and ∼1.7 Mb TDs associate with CCNE1 pathway activation and CDK12 disruptions, respectively. They demonstrate that p53 and BRCA1 conjoint abrogation drives TDP induction by generating short-span TDP mammary tumors in genetically modified mice lacking them. Lastly, they show how TDs in TDP tumors disrupt heterogeneous combinations of tumor suppressors and chromatin topologically associating domains while duplicating oncogenes and super-enhancers.



Read more, please click https://www.cell.com/cancer-cell/fulltext/S1535-6108(18)30265-4

2. Comprehensive Analysis of Alternative Splicing Across Tumors from 8,705 Patients
Comprehensive analysis of alternative splicing across 32 The Cancer Genome Atlas cancer types from 8,705 patients detects alternative splicing events and tumor variants by reanalyzing RNA and whole-exome sequencing data. Tumors have up to 30% more alternative splicing events than normal samples. Association analysis of somatic variants with alternative splicing events confirmed known trans associations with variants in SF3B1 and U2AF1 and identified additional trans-acting variants (e.g., TADA1, PPP2R1A). Many tumors have thousands of alternative splicing events not detectable in normal samples; on average, André Kahles at ETH Zurich in Zurich, Switzerland and his colleagues identified ≈930 exon-exon junctions (“neojunctions”) in tumors not typically found in GTEx normals. From Clinical Proteomic Tumor Analysis Consortium data available for breast and ovarian tumor samples, they confirmed ≈1.7 neojunction- and ≈0.6 single nucleotide variant-derived peptides per tumor sample that are also predicted major histocompatibility complex-I binders (“putative neoantigens”).

Read more, please click https://www.cell.com/cancer-cell/fulltext/S1535-6108(18)30306-4

3. Synthetic Lethal and Convergent Biological Effects of Cancer-Associated Spliceosomal Gene Mutations
Mutations affecting RNA splicing factors are the most common genetic alterations in myelodysplastic syndrome (MDS) patients and occur in a mutually exclusive manner. The basis for the mutual exclusivity of these mutations and how they contribute to MDS is not well understood. Here Stanley Chun-Wei Lee at Memorial Sloan Kettering Cancer Center in New York, USA and his colleagues report that although different spliceosome gene mutations impart distinct effects on splicing, they are negatively selected for when co-expressed due to aberrant splicing and downregulation of regulators of hematopoietic stem cell survival and quiescence. In addition to this synthetic lethal interaction, mutations in the splicing factors SF3B1 and SRSF2 share convergent effects on aberrant splicing of mRNAs that promote nuclear factor κB signaling. These data identify shared consequences of splicing-factor mutations and the basis for their mutual exclusivity.

Read more, please click https://www.cell.com/cancer-cell/fulltext/S1535-6108(18)30308-8

4. Apoptotic Cell-Derived Extracellular Vesicles Promote Malignancy of Glioblastoma Via Intercellular Transfer of Splicing Factors
Aggressive cancers such as glioblastoma (GBM) contain intermingled apoptotic cells adjacent to proliferating tumor cells. Nonetheless, intercellular signaling between apoptotic and surviving cancer cells remain elusive. In this study, Marat S. Pavlyukov at University of Alabama at Birmingham, Wallace Tumor Institute in Birmingham, USA and his colleagues demonstrate that apoptotic GBM cells paradoxically promote proliferation and therapy resistance of surviving tumor cells by secreting apoptotic extracellular vesicles (apoEVs) enriched with various components of spliceosomes. apoEVs alter RNA splicing in recipient cells, thereby promoting their therapy resistance and aggressive migratory phenotype. Mechanistically, they identified RBM11 as a representative splicing factor that is upregulated in tumors after therapy and shed in extracellular vesicles upon induction of apoptosis. Once internalized in recipient cells, exogenous RBM11 switches splicing of MDM4 and Cyclin D1 toward the expression of more oncogenic isoforms.

Read more, please click https://www.cell.com/cancer-cell/fulltext/S1535-6108(18)30226-5

5. Autocrine Mfge8 Signaling Prevents Developmental Exhaustion of the Adult Neural Stem Cell Pool
Adult neurogenesis, arising from quiescent radial-glia-like neural stem cells (RGLs), occurs throughout life in the dentate gyrus. How neural stem cells are maintained throughout development to sustain adult mammalian neurogenesis is not well understood. Here, Yi Zhou at Perelman School of Medicine, University of Pennsylvania in Philadelphia, USA and his colleagues show that milk fat globule-epidermal growth factor (EGF) 8 (Mfge8), a known phagocytosis factor, is highly enriched in quiescent RGLs in the dentate gyrus. Mfge8-null mice exhibit decreased adult dentate neurogenesis, and furthermore, adult RGL-specific deletion of Mfge8 leads to RGL overactivation and depletion. Similarly, loss of Mfge8 promotes RGL activation in the early postnatal dentate gyrus, resulting in a decreased number of label-retaining RGLs in adulthood. Mechanistically, loss of Mfge8 elevates mTOR1 signaling in RGLs, inhibition of which by rapamycin returns RGLs to quiescence. Together, their study identifies a neural-stem-cell-enriched niche factor that maintains quiescence and prevents developmental exhaustion of neural stem cells to sustain continuous neurogenesis in the adult mammalian brain.

Read more, please click https://www.cell.com/cell-stem-cell/fulltext/S1934-5909(18)30390-4

Immunofluorescence Staining

Immunofluorescence is a technique allowing the visualization of a specific protein or antigen in cells or tissue sections by binding a specific antibody chemically conjugated with a fluorescent dye such as fluorescein isothiocyanate (FITC). There are two major types of immunofluorescence staining methods: 1) direct immunofluorescence staining in which the primary antibody is labeled with fluorescence dye, and 2) indirect immunofluorescence staining in which a secondary antibody labeled with fluorochrome is used to recognize a primary antibody. Immunofluorescence staining can be performed on cells fixed on slides and tissue sections and examined under a fluorescence microscope or confocal microscope.

The method relies on proper fixation of cells to retain cellular distribution of antigen and to preserve cellular morphology. After fixation, the cells are exposed to primary antibody directed against the protein of interest, in the prescence of permeabilizing reagents to ensure antibody access to the epitope. Following incubation with the primary antibody, the unbound primary antibody is removed and the bound primary antibody is then labeled by incubation with a fluorescently tagged secondary antibody directed against the primary antibody host species.

Materials:

1XPhosphate Buffered Saline (PBS)
4% Formaldehyde: methanol free, use fresh and store opened vials at 4°C in dark. Dilute with 1X PBS to make a 4% formaldehyde solution.
0.1%Triton X-100 (prepared with 1× PBS)
Blocking Buffer: 1X PBS/5% normal serum (Sharing the same or similar species with secondary antibodies)
Antibody Dilution Buffer: To prepare 10 ml, add 30 µl Triton X-100 to 10 ml 1X PBS. Mix well then add 0.1g BSA.
Mounting medium: 50% glycerol with 0.1%(w/v) p-phenylenediamine in PBS or use Fluormount G

A. Sample Preparation

Cultured Cell Lines (IF-IC)
NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.

1. Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in warm PBS.
NOTE: Formaldehyde is toxic, use only in a fume hood.

2. Allow cells to fix for 15 min at room temperature.

3. Aspirate fixative, rinse three times in 1X PBS for 5 min each.

4. Permeate the cells at room temperature for 15 minutes with 0.1%Triton X-100 (prepared with 1× PBS)

5. Wash the cover glass with 1× PBS for 5 minutes and repeat 3 times.
Proceed with Immunostaining (Section B).

Frozen/Cryostat Sections (IF-F)

1. For fixed frozen tissue proceed with Immunostaining (Section B).

2. For fresh, unfixed frozen tissue, fix immediately, as follows:

a. Cover sections with 4% formaldehyde diluted in warm 1X PBS.
b. Allow sections to fix for 15 min at room temperature.
c. Rinse slides three times in PBS for 5 min each.
d. Permeate the cells at room temperature for 15 minutes with 0.1%Triton X-100 (prepared with 1× PBS)
e. Rinse slides three times in PBS for 5 min each.
Proceed with Immunostaining (Section B).

B. Immunostaining

NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.

1. Block specimen in blocking buffer for 30 min.
2. Aspirate blocking solution, apply diluted primary antibody.
3. Incubate overnight at 4°C.
4. Rinse three times in 1X PBS for 5 min each.
NOTE: If using a fluorochrome-conjugated primary antibody, then skip to Step 7.

5. Incubate secondary antibody diluted in antibody dilution buffer for 1–2 hr at room temperature in the dark.
6. Rinse three times in 1X PBS for 5 min each.
7. Invert Coverslip slides cell-side-down, on one drop mounting medium. Gently blot with paper towel, then seal edge by painting with nail polish. Let dry.
8. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C protected from light (6-12 month).

Immunofluorescence Staining

Immunofluorescence is a technique allowing the visualization of a specific protein or antigen in cells or tissue sections by binding a specific antibody chemically conjugated with a fluorescent dye such as fluorescein isothiocyanate (FITC). There are two major types of immunofluorescence staining methods: 1) direct immunofluorescence staining in which the primary antibody is labeled with fluorescence dye, and 2) indirect immunofluorescence staining in which a secondary antibody labeled with fluorochrome is used to recognize a primary antibody. Immunofluorescence staining can be performed on cells fixed on slides and tissue sections and examined under a fluorescence microscope or confocal microscope.

The method relies on proper fixation of cells to retain cellular distribution of antigen and to preserve cellular morphology. After fixation, the cells are exposed to primary antibody directed against the protein of interest, in the prescence of permeabilizing reagents to ensure antibody access to the epitope. Following incubation with the primary antibody, the unbound primary antibody is removed and the bound primary antibody is then labeled by incubation with a fluorescently tagged secondary antibody directed against the primary antibody host species.

Materials:

1XPhosphate Buffered Saline (PBS)
4% Formaldehyde: methanol free, use fresh and store opened vials at 4°C in dark. Dilute with 1X PBS to make a 4% formaldehyde solution.
0.1%Triton X-100 (prepared with 1× PBS)
Blocking Buffer: 1X PBS/5% normal serum (Sharing the same or similar species with secondary antibodies)
Antibody Dilution Buffer: To prepare 10 ml, add 30 µl Triton X-100 to 10 ml 1X PBS. Mix well then add 0.1g BSA.
Mounting medium: 50% glycerol with 0.1%(w/v) p-phenylenediamine in PBS or use Fluormount G

A. Sample Preparation

Cultured Cell Lines (IF-IC)
NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.

1. Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in warm PBS.
NOTE: Formaldehyde is toxic, use only in a fume hood.

2. Allow cells to fix for 15 min at room temperature.

3. Aspirate fixative, rinse three times in 1X PBS for 5 min each.

4. Permeate the cells at room temperature for 15 minutes with 0.1%Triton X-100 (prepared with 1× PBS)

5. Wash the cover glass with 1× PBS for 5 minutes and repeat 3 times.
Proceed with Immunostaining (Section B).

Frozen/Cryostat Sections (IF-F)

1. For fixed frozen tissue proceed with Immunostaining (Section B).

2. For fresh, unfixed frozen tissue, fix immediately, as follows:

a. Cover sections with 4% formaldehyde diluted in warm 1X PBS.
b. Allow sections to fix for 15 min at room temperature.
c. Rinse slides three times in PBS for 5 min each.
d. Permeate the cells at room temperature for 15 minutes with 0.1%Triton X-100 (prepared with 1× PBS)
e. Rinse slides three times in PBS for 5 min each.
Proceed with Immunostaining (Section B).

B. Immunostaining

NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.

1. Block specimen in blocking buffer for 30 min.
2. Aspirate blocking solution, apply diluted primary antibody.
3. Incubate overnight at 4°C.
4. Rinse three times in 1X PBS for 5 min each.
NOTE: If using a fluorochrome-conjugated primary antibody, then skip to Step 7.

5. Incubate secondary antibody diluted in antibody dilution buffer for 1–2 hr at room temperature in the dark.
6. Rinse three times in 1X PBS for 5 min each.
7. Invert Coverslip slides cell-side-down, on one drop mounting medium. Gently blot with paper towel, then seal edge by painting with nail polish. Let dry.
8. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C protected from light (6-12 month).

Immunofluorescence Staining

Immunofluorescence is a technique allowing the visualization of a specific protein or antigen in cells or tissue sections by binding a specific antibody chemically conjugated with a fluorescent dye such as fluorescein isothiocyanate (FITC). There are two major types of immunofluorescence staining methods: 1) direct immunofluorescence staining in which the primary antibody is labeled with fluorescence dye, and 2) indirect immunofluorescence staining in which a secondary antibody labeled with fluorochrome is used to recognize a primary antibody. Immunofluorescence staining can be performed on cells fixed on slides and tissue sections and examined under a fluorescence microscope or confocal microscope.

The method relies on proper fixation of cells to retain cellular distribution of antigen and to preserve cellular morphology. After fixation, the cells are exposed to primary antibody directed against the protein of interest, in the prescence of permeabilizing reagents to ensure antibody access to the epitope. Following incubation with the primary antibody, the unbound primary antibody is removed and the bound primary antibody is then labeled by incubation with a fluorescently tagged secondary antibody directed against the primary antibody host species.

Materials:

1XPhosphate Buffered Saline (PBS)
4% Formaldehyde: methanol free, use fresh and store opened vials at 4°C in dark. Dilute with 1X PBS to make a 4% formaldehyde solution.
0.1%Triton X-100 (prepared with 1× PBS)
Blocking Buffer: 1X PBS/5% normal serum (Sharing the same or similar species with secondary antibodies)
Antibody Dilution Buffer: To prepare 10 ml, add 30 µl Triton X-100 to 10 ml 1X PBS. Mix well then add 0.1g BSA.
Mounting medium: 50% glycerol with 0.1%(w/v) p-phenylenediamine in PBS or use Fluormount G

A. Sample Preparation

Cultured Cell Lines (IF-IC)
NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.

1. Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in warm PBS.
NOTE: Formaldehyde is toxic, use only in a fume hood.

2. Allow cells to fix for 15 min at room temperature.

3. Aspirate fixative, rinse three times in 1X PBS for 5 min each.

4. Permeate the cells at room temperature for 15 minutes with 0.1%Triton X-100 (prepared with 1× PBS)

5. Wash the cover glass with 1× PBS for 5 minutes and repeat 3 times.
Proceed with Immunostaining (Section B).

Frozen/Cryostat Sections (IF-F)

1. For fixed frozen tissue proceed with Immunostaining (Section B).

2. For fresh, unfixed frozen tissue, fix immediately, as follows:

a. Cover sections with 4% formaldehyde diluted in warm 1X PBS.
b. Allow sections to fix for 15 min at room temperature.
c. Rinse slides three times in PBS for 5 min each.
d. Permeate the cells at room temperature for 15 minutes with 0.1%Triton X-100 (prepared with 1× PBS)
e. Rinse slides three times in PBS for 5 min each.
Proceed with Immunostaining (Section B).

B. Immunostaining

NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.

1. Block specimen in blocking buffer for 30 min.
2. Aspirate blocking solution, apply diluted primary antibody.
3. Incubate overnight at 4°C.
4. Rinse three times in 1X PBS for 5 min each.
NOTE: If using a fluorochrome-conjugated primary antibody, then skip to Step 7.

5. Incubate secondary antibody diluted in antibody dilution buffer for 1–2 hr at room temperature in the dark.
6. Rinse three times in 1X PBS for 5 min each.
7. Invert Coverslip slides cell-side-down, on one drop mounting medium. Gently blot with paper towel, then seal edge by painting with nail polish. Let dry.
8. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C protected from light (6-12 month).

Immunofluorescence Staining

Immunofluorescence is a technique allowing the visualization of a specific protein or antigen in cells or tissue sections by binding a specific antibody chemically conjugated with a fluorescent dye such as fluorescein isothiocyanate (FITC). There are two major types of immunofluorescence staining methods: 1) direct immunofluorescence staining in which the primary antibody is labeled with fluorescence dye, and 2) indirect immunofluorescence staining in which a secondary antibody labeled with fluorochrome is used to recognize a primary antibody. Immunofluorescence staining can be performed on cells fixed on slides and tissue sections and examined under a fluorescence microscope or confocal microscope.

The method relies on proper fixation of cells to retain cellular distribution of antigen and to preserve cellular morphology. After fixation, the cells are exposed to primary antibody directed against the protein of interest, in the prescence of permeabilizing reagents to ensure antibody access to the epitope. Following incubation with the primary antibody, the unbound primary antibody is removed and the bound primary antibody is then labeled by incubation with a fluorescently tagged secondary antibody directed against the primary antibody host species.

Materials:

1XPhosphate Buffered Saline (PBS)
4% Formaldehyde: methanol free, use fresh and store opened vials at 4°C in dark. Dilute with 1X PBS to make a 4% formaldehyde solution.
0.1%Triton X-100 (prepared with 1× PBS)
Blocking Buffer: 1X PBS/5% normal serum (Sharing the same or similar species with secondary antibodies)
Antibody Dilution Buffer: To prepare 10 ml, add 30 µl Triton X-100 to 10 ml 1X PBS. Mix well then add 0.1g BSA.
Mounting medium: 50% glycerol with 0.1%(w/v) p-phenylenediamine in PBS or use Fluormount G

A. Sample Preparation

Cultured Cell Lines (IF-IC)
NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.

1. Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in warm PBS.
NOTE: Formaldehyde is toxic, use only in a fume hood.

2. Allow cells to fix for 15 min at room temperature.

3. Aspirate fixative, rinse three times in 1X PBS for 5 min each.

4. Permeate the cells at room temperature for 15 minutes with 0.1%Triton X-100 (prepared with 1× PBS)

5. Wash the cover glass with 1× PBS for 5 minutes and repeat 3 times.
Proceed with Immunostaining (Section B).

Frozen/Cryostat Sections (IF-F)

1. For fixed frozen tissue proceed with Immunostaining (Section B).

2. For fresh, unfixed frozen tissue, fix immediately, as follows:

a. Cover sections with 4% formaldehyde diluted in warm 1X PBS.
b. Allow sections to fix for 15 min at room temperature.
c. Rinse slides three times in PBS for 5 min each.
d. Permeate the cells at room temperature for 15 minutes with 0.1%Triton X-100 (prepared with 1× PBS)
e. Rinse slides three times in PBS for 5 min each.
Proceed with Immunostaining (Section B).

B. Immunostaining

NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.

1. Block specimen in blocking buffer for 30 min.
2. Aspirate blocking solution, apply diluted primary antibody.
3. Incubate overnight at 4°C.
4. Rinse three times in 1X PBS for 5 min each.
NOTE: If using a fluorochrome-conjugated primary antibody, then skip to Step 7.

5. Incubate secondary antibody diluted in antibody dilution buffer for 1–2 hr at room temperature in the dark.
6. Rinse three times in 1X PBS for 5 min each.
7. Invert Coverslip slides cell-side-down, on one drop mounting medium. Gently blot with paper towel, then seal edge by painting with nail polish. Let dry.
8. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C protected from light (6-12 month).

Immunofluorescence Staining

Immunofluorescence is a technique allowing the visualization of a specific protein or antigen in cells or tissue sections by binding a specific antibody chemically conjugated with a fluorescent dye such as fluorescein isothiocyanate (FITC). There are two major types of immunofluorescence staining methods: 1) direct immunofluorescence staining in which the primary antibody is labeled with fluorescence dye, and 2) indirect immunofluorescence staining in which a secondary antibody labeled with fluorochrome is used to recognize a primary antibody. Immunofluorescence staining can be performed on cells fixed on slides and tissue sections and examined under a fluorescence microscope or confocal microscope.

The method relies on proper fixation of cells to retain cellular distribution of antigen and to preserve cellular morphology. After fixation, the cells are exposed to primary antibody directed against the protein of interest, in the prescence of permeabilizing reagents to ensure antibody access to the epitope. Following incubation with the primary antibody, the unbound primary antibody is removed and the bound primary antibody is then labeled by incubation with a fluorescently tagged secondary antibody directed against the primary antibody host species.

Materials:

1XPhosphate Buffered Saline (PBS)
4% Formaldehyde: methanol free, use fresh and store opened vials at 4°C in dark. Dilute with 1X PBS to make a 4% formaldehyde solution.
0.1%Triton X-100 (prepared with 1× PBS)
Blocking Buffer: 1X PBS/5% normal serum (Sharing the same or similar species with secondary antibodies)
Antibody Dilution Buffer: To prepare 10 ml, add 30 µl Triton X-100 to 10 ml 1X PBS. Mix well then add 0.1g BSA.
Mounting medium: 50% glycerol with 0.1%(w/v) p-phenylenediamine in PBS or use Fluormount G

A. Sample Preparation

Cultured Cell Lines (IF-IC)
NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.

1. Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in warm PBS.
NOTE: Formaldehyde is toxic, use only in a fume hood.

2. Allow cells to fix for 15 min at room temperature.

3. Aspirate fixative, rinse three times in 1X PBS for 5 min each.

4. Permeate the cells at room temperature for 15 minutes with 0.1%Triton X-100 (prepared with 1× PBS)

5. Wash the cover glass with 1× PBS for 5 minutes and repeat 3 times.
Proceed with Immunostaining (Section B).

Frozen/Cryostat Sections (IF-F)

1. For fixed frozen tissue proceed with Immunostaining (Section B).

2. For fresh, unfixed frozen tissue, fix immediately, as follows:

a. Cover sections with 4% formaldehyde diluted in warm 1X PBS.
b. Allow sections to fix for 15 min at room temperature.
c. Rinse slides three times in PBS for 5 min each.
d. Permeate the cells at room temperature for 15 minutes with 0.1%Triton X-100 (prepared with 1× PBS)
e. Rinse slides three times in PBS for 5 min each.
Proceed with Immunostaining (Section B).

B. Immunostaining

NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.

1. Block specimen in blocking buffer for 30 min.
2. Aspirate blocking solution, apply diluted primary antibody.
3. Incubate overnight at 4°C.
4. Rinse three times in 1X PBS for 5 min each.
NOTE: If using a fluorochrome-conjugated primary antibody, then skip to Step 7.

5. Incubate secondary antibody diluted in antibody dilution buffer for 1–2 hr at room temperature in the dark.
6. Rinse three times in 1X PBS for 5 min each.
7. Invert Coverslip slides cell-side-down, on one drop mounting medium. Gently blot with paper towel, then seal edge by painting with nail polish. Let dry.
8. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C protected from light (6-12 month).

The Tandem Duplicator Phenotype Is a Prevalent Genome-Wide Cancer Configuration Driven by Distinct Gene Mutations

Content introduction:

• The Tandem Duplicator Phenotype Is a Prevalent Genome-Wide Cancer Configuration Driven by Distinct Gene Mutations


• Comprehensive Analysis of Alternative Splicing Across Tumors from 8,705 Patients


• Synthetic Lethal and Convergent Biological Effects of Cancer-Associated Spliceosomal Gene Mutations


• Apoptotic Cell-Derived Extracellular Vesicles Promote Malignancy of Glioblastoma Via Intercellular Transfer of Splicing Factors


• Autocrine Mfge8 Signaling Prevents Developmental Exhaustion of the Adult Neural Stem Cell Pool

1. The Tandem Duplicator Phenotype Is a Prevalent Genome-Wide Cancer Configuration Driven by Distinct Gene Mutations
The tandem duplicator phenotype (TDP) is a genome-wide instability configuration primarily observed in breast, ovarian, and endometrial carcinomas. Here, Francesca Menghi at The Jackson Laboratory for Genomic Medicine in Farmington, USA and his colleagues stratify TDP tumors by classifying their tandem duplications (TDs) into three span intervals, with modal values of 11 kb, 231 kb, and 1.7 Mb, respectively. TDPs with ∼11 kb TDs feature loss of TP53 and BRCA1. TDPs with ∼231 kb and ∼1.7 Mb TDs associate with CCNE1 pathway activation and CDK12 disruptions, respectively. They demonstrate that p53 and BRCA1 conjoint abrogation drives TDP induction by generating short-span TDP mammary tumors in genetically modified mice lacking them. Lastly, they show how TDs in TDP tumors disrupt heterogeneous combinations of tumor suppressors and chromatin topologically associating domains while duplicating oncogenes and super-enhancers.



Read more, please click https://www.cell.com/cancer-cell/fulltext/S1535-6108(18)30265-4

2. Comprehensive Analysis of Alternative Splicing Across Tumors from 8,705 Patients
Comprehensive analysis of alternative splicing across 32 The Cancer Genome Atlas cancer types from 8,705 patients detects alternative splicing events and tumor variants by reanalyzing RNA and whole-exome sequencing data. Tumors have up to 30% more alternative splicing events than normal samples. Association analysis of somatic variants with alternative splicing events confirmed known trans associations with variants in SF3B1 and U2AF1 and identified additional trans-acting variants (e.g., TADA1, PPP2R1A). Many tumors have thousands of alternative splicing events not detectable in normal samples; on average, André Kahles at ETH Zurich in Zurich, Switzerland and his colleagues identified ≈930 exon-exon junctions (“neojunctions”) in tumors not typically found in GTEx normals. From Clinical Proteomic Tumor Analysis Consortium data available for breast and ovarian tumor samples, they confirmed ≈1.7 neojunction- and ≈0.6 single nucleotide variant-derived peptides per tumor sample that are also predicted major histocompatibility complex-I binders (“putative neoantigens”).

Read more, please click https://www.cell.com/cancer-cell/fulltext/S1535-6108(18)30306-4

3. Synthetic Lethal and Convergent Biological Effects of Cancer-Associated Spliceosomal Gene Mutations
Mutations affecting RNA splicing factors are the most common genetic alterations in myelodysplastic syndrome (MDS) patients and occur in a mutually exclusive manner. The basis for the mutual exclusivity of these mutations and how they contribute to MDS is not well understood. Here Stanley Chun-Wei Lee at Memorial Sloan Kettering Cancer Center in New York, USA and his colleagues report that although different spliceosome gene mutations impart distinct effects on splicing, they are negatively selected for when co-expressed due to aberrant splicing and downregulation of regulators of hematopoietic stem cell survival and quiescence. In addition to this synthetic lethal interaction, mutations in the splicing factors SF3B1 and SRSF2 share convergent effects on aberrant splicing of mRNAs that promote nuclear factor κB signaling. These data identify shared consequences of splicing-factor mutations and the basis for their mutual exclusivity.

Read more, please click https://www.cell.com/cancer-cell/fulltext/S1535-6108(18)30308-8

4. Apoptotic Cell-Derived Extracellular Vesicles Promote Malignancy of Glioblastoma Via Intercellular Transfer of Splicing Factors
Aggressive cancers such as glioblastoma (GBM) contain intermingled apoptotic cells adjacent to proliferating tumor cells. Nonetheless, intercellular signaling between apoptotic and surviving cancer cells remain elusive. In this study, Marat S. Pavlyukov at University of Alabama at Birmingham, Wallace Tumor Institute in Birmingham, USA and his colleagues demonstrate that apoptotic GBM cells paradoxically promote proliferation and therapy resistance of surviving tumor cells by secreting apoptotic extracellular vesicles (apoEVs) enriched with various components of spliceosomes. apoEVs alter RNA splicing in recipient cells, thereby promoting their therapy resistance and aggressive migratory phenotype. Mechanistically, they identified RBM11 as a representative splicing factor that is upregulated in tumors after therapy and shed in extracellular vesicles upon induction of apoptosis. Once internalized in recipient cells, exogenous RBM11 switches splicing of MDM4 and Cyclin D1 toward the expression of more oncogenic isoforms.

Read more, please click https://www.cell.com/cancer-cell/fulltext/S1535-6108(18)30226-5

5. Autocrine Mfge8 Signaling Prevents Developmental Exhaustion of the Adult Neural Stem Cell Pool
Adult neurogenesis, arising from quiescent radial-glia-like neural stem cells (RGLs), occurs throughout life in the dentate gyrus. How neural stem cells are maintained throughout development to sustain adult mammalian neurogenesis is not well understood. Here, Yi Zhou at Perelman School of Medicine, University of Pennsylvania in Philadelphia, USA and his colleagues show that milk fat globule-epidermal growth factor (EGF) 8 (Mfge8), a known phagocytosis factor, is highly enriched in quiescent RGLs in the dentate gyrus. Mfge8-null mice exhibit decreased adult dentate neurogenesis, and furthermore, adult RGL-specific deletion of Mfge8 leads to RGL overactivation and depletion. Similarly, loss of Mfge8 promotes RGL activation in the early postnatal dentate gyrus, resulting in a decreased number of label-retaining RGLs in adulthood. Mechanistically, loss of Mfge8 elevates mTOR1 signaling in RGLs, inhibition of which by rapamycin returns RGLs to quiescence. Together, their study identifies a neural-stem-cell-enriched niche factor that maintains quiescence and prevents developmental exhaustion of neural stem cells to sustain continuous neurogenesis in the adult mammalian brain.

Read more, please click https://www.cell.com/cell-stem-cell/fulltext/S1934-5909(18)30390-4

The Tandem Duplicator Phenotype Is a Prevalent Genome-Wide Cancer Configuration Driven by Distinct Gene Mutations

Content introduction:

• The Tandem Duplicator Phenotype Is a Prevalent Genome-Wide Cancer Configuration Driven by Distinct Gene Mutations


• Comprehensive Analysis of Alternative Splicing Across Tumors from 8,705 Patients


• Synthetic Lethal and Convergent Biological Effects of Cancer-Associated Spliceosomal Gene Mutations


• Apoptotic Cell-Derived Extracellular Vesicles Promote Malignancy of Glioblastoma Via Intercellular Transfer of Splicing Factors


• Autocrine Mfge8 Signaling Prevents Developmental Exhaustion of the Adult Neural Stem Cell Pool

1. The Tandem Duplicator Phenotype Is a Prevalent Genome-Wide Cancer Configuration Driven by Distinct Gene Mutations
The tandem duplicator phenotype (TDP) is a genome-wide instability configuration primarily observed in breast, ovarian, and endometrial carcinomas. Here, Francesca Menghi at The Jackson Laboratory for Genomic Medicine in Farmington, USA and his colleagues stratify TDP tumors by classifying their tandem duplications (TDs) into three span intervals, with modal values of 11 kb, 231 kb, and 1.7 Mb, respectively. TDPs with ∼11 kb TDs feature loss of TP53 and BRCA1. TDPs with ∼231 kb and ∼1.7 Mb TDs associate with CCNE1 pathway activation and CDK12 disruptions, respectively. They demonstrate that p53 and BRCA1 conjoint abrogation drives TDP induction by generating short-span TDP mammary tumors in genetically modified mice lacking them. Lastly, they show how TDs in TDP tumors disrupt heterogeneous combinations of tumor suppressors and chromatin topologically associating domains while duplicating oncogenes and super-enhancers.



Read more, please click https://www.cell.com/cancer-cell/fulltext/S1535-6108(18)30265-4

2. Comprehensive Analysis of Alternative Splicing Across Tumors from 8,705 Patients
Comprehensive analysis of alternative splicing across 32 The Cancer Genome Atlas cancer types from 8,705 patients detects alternative splicing events and tumor variants by reanalyzing RNA and whole-exome sequencing data. Tumors have up to 30% more alternative splicing events than normal samples. Association analysis of somatic variants with alternative splicing events confirmed known trans associations with variants in SF3B1 and U2AF1 and identified additional trans-acting variants (e.g., TADA1, PPP2R1A). Many tumors have thousands of alternative splicing events not detectable in normal samples; on average, André Kahles at ETH Zurich in Zurich, Switzerland and his colleagues identified ≈930 exon-exon junctions (“neojunctions”) in tumors not typically found in GTEx normals. From Clinical Proteomic Tumor Analysis Consortium data available for breast and ovarian tumor samples, they confirmed ≈1.7 neojunction- and ≈0.6 single nucleotide variant-derived peptides per tumor sample that are also predicted major histocompatibility complex-I binders (“putative neoantigens”).

Read more, please click https://www.cell.com/cancer-cell/fulltext/S1535-6108(18)30306-4

3. Synthetic Lethal and Convergent Biological Effects of Cancer-Associated Spliceosomal Gene Mutations
Mutations affecting RNA splicing factors are the most common genetic alterations in myelodysplastic syndrome (MDS) patients and occur in a mutually exclusive manner. The basis for the mutual exclusivity of these mutations and how they contribute to MDS is not well understood. Here Stanley Chun-Wei Lee at Memorial Sloan Kettering Cancer Center in New York, USA and his colleagues report that although different spliceosome gene mutations impart distinct effects on splicing, they are negatively selected for when co-expressed due to aberrant splicing and downregulation of regulators of hematopoietic stem cell survival and quiescence. In addition to this synthetic lethal interaction, mutations in the splicing factors SF3B1 and SRSF2 share convergent effects on aberrant splicing of mRNAs that promote nuclear factor κB signaling. These data identify shared consequences of splicing-factor mutations and the basis for their mutual exclusivity.

Read more, please click https://www.cell.com/cancer-cell/fulltext/S1535-6108(18)30308-8

4. Apoptotic Cell-Derived Extracellular Vesicles Promote Malignancy of Glioblastoma Via Intercellular Transfer of Splicing Factors
Aggressive cancers such as glioblastoma (GBM) contain intermingled apoptotic cells adjacent to proliferating tumor cells. Nonetheless, intercellular signaling between apoptotic and surviving cancer cells remain elusive. In this study, Marat S. Pavlyukov at University of Alabama at Birmingham, Wallace Tumor Institute in Birmingham, USA and his colleagues demonstrate that apoptotic GBM cells paradoxically promote proliferation and therapy resistance of surviving tumor cells by secreting apoptotic extracellular vesicles (apoEVs) enriched with various components of spliceosomes. apoEVs alter RNA splicing in recipient cells, thereby promoting their therapy resistance and aggressive migratory phenotype. Mechanistically, they identified RBM11 as a representative splicing factor that is upregulated in tumors after therapy and shed in extracellular vesicles upon induction of apoptosis. Once internalized in recipient cells, exogenous RBM11 switches splicing of MDM4 and Cyclin D1 toward the expression of more oncogenic isoforms.

Read more, please click https://www.cell.com/cancer-cell/fulltext/S1535-6108(18)30226-5

5. Autocrine Mfge8 Signaling Prevents Developmental Exhaustion of the Adult Neural Stem Cell Pool
Adult neurogenesis, arising from quiescent radial-glia-like neural stem cells (RGLs), occurs throughout life in the dentate gyrus. How neural stem cells are maintained throughout development to sustain adult mammalian neurogenesis is not well understood. Here, Yi Zhou at Perelman School of Medicine, University of Pennsylvania in Philadelphia, USA and his colleagues show that milk fat globule-epidermal growth factor (EGF) 8 (Mfge8), a known phagocytosis factor, is highly enriched in quiescent RGLs in the dentate gyrus. Mfge8-null mice exhibit decreased adult dentate neurogenesis, and furthermore, adult RGL-specific deletion of Mfge8 leads to RGL overactivation and depletion. Similarly, loss of Mfge8 promotes RGL activation in the early postnatal dentate gyrus, resulting in a decreased number of label-retaining RGLs in adulthood. Mechanistically, loss of Mfge8 elevates mTOR1 signaling in RGLs, inhibition of which by rapamycin returns RGLs to quiescence. Together, their study identifies a neural-stem-cell-enriched niche factor that maintains quiescence and prevents developmental exhaustion of neural stem cells to sustain continuous neurogenesis in the adult mammalian brain.

Read more, please click https://www.cell.com/cell-stem-cell/fulltext/S1934-5909(18)30390-4