The Nobel Prize in Physiology or Medicine 2009

The Nobel Prize in Physiology or Medicine 2009 was awarded jointly to Elizabeth H. Blackburn, Carol W. Greider and Jack W. Szostak "for the discovery of how chromosomes are protected by telomeres and the enzyme telomerase".

Nobelist	Born	Affiliation at the time of the award
Elizabeth H. Blackburn	26 November 1948, Hobart, Tasmania, Australia	University of California, San Francisco, CA, USA
Carol W. Greider	15 April 1961, San Diego, CA, USA	Johns Hopkins University School of Medicine, Baltimore, MD, USA
Jack W. Szostak	9 November 1952, London, United Kingdom	Harvard Medical School, Boston, MA, USA, Massachusetts General Hospital, Boston, MA, USA, Howard Hughes Medical Institute

Summary

This year's Nobel Prize in Physiology or Medicine is awarded to three scientists who have solved a major problem in biology: how the chromosomes can be copied in a complete way during cell divisions and how they are protected against degradation. The Nobel Laureates have shown that the solution is to be found in the ends of the chromosomes – the telomeres – and in an enzyme that forms them – telomerase.

The long, thread-like DNA molecules that carry our genes are packed into chromosomes, the telomeres being the caps on their ends. Elizabeth Blackburn and Jack Szostak discovered that a unique DNA sequence in the telomeres protects the chromosomes from degradation. Carol Greider and Elizabeth Blackburn identified telomerase, the enzyme that makes telomere DNA. These discoveries explained how the ends of the chromosomes are protected by the telomeres and that they are built by telomerase.

If the telomeres are shortened, cells age. Conversely, if telomerase activity is high, telomere length is maintained, and cellular senescence is delayed. This is the case in cancer cells, which can be considered to have eternal life. Certain inherited diseases, in contrast, are characterized by a defective telomerase, resulting in damaged cells. The award of the Nobel Prize recognizes the discovery of a fundamental mechanism in the cell, a discovery that has stimulated the development of new therapeutic strategies.

More details, please click The 2009 Nobel Prize in Physiology or Medicine.

Abbkine IHC-PARAFFIN Protocol (IHC-P)

Immunohistochemical (IHC) Staining allows you to detect antigens in a tissue fixed on a glass slide. The basic steps of the IHC-P protocol are as follows:
1. Fixing and embedding the tissue
2. Cutting and mounting the section
3. Deparaffinizing and rehydrating the section
4. Antigen retrieval
5. Immunohistochemical staining
6. Counterstaining (if desired)
7. Dehydrating and stabilizing with mounting medium
8. Viewing the staining under the microscope

A: Fixation
Proper fixation is key for the success of immunohistochemistry. 10% neutral buffered formalin (NBF) is most commonly used. Other fixatives such as paraformaldehyde (PFA) or Bouin solution (formalin/picric acid) are used less frequently. The ideal fixation time will depend on the size of the tissue block and the type of tissue, but fixation between 18-24 hours seems to be ideal for most applications. Under-fixation can lead to edge staining, with strong signal on the edges of the section and no signal in the middle; over-fixation can mask the epitope. Antigen retrieval can help overcome this masking, but if the tissue has been fixed for a long period of time (i.e. over a weekend), there may be no signal even after antigen retrieval.
After fixation, the tissue block is embedded in paraffin, then cut in a microtome to the desired thickness (approximately 5 microns is ideal for IHC) and affixed onto the slide. Tissue sections are best mounted on positively charged or APES (amino-propyl-tri-ethoxy-silane) coated slides. Once mounted, the slides should be dried to remove any water that may be trapped under the section. This can be done by leaving the slide at room temperature overnight. If there is a problem with the section adhering to the slide, you may also incubate the slide at 60ºC for a few hours.

B: Deparaffinization
Before proceeding with the staining protocol, the slides must be deparaffinized and rehydrated. Incomplete removal of paraffin can cause poor staining of the section.

Materials and reagents
• Xylene
• 100% ethanol
• 95% ethanol
Method
Place the slides in a rack, and perform the following washes:
1. Xylene: 2 x 3 minutes
2. Xylene 1:1 with 100% ethanol: 3 minutes
3. 100% ethanol: 2 x 3 minutes
4. 95% ethanol: 3 minutes
5. 70 % ethanol: 3 minutes
6. 50 % ethanol: 3 minutes
7. Running cold tap water to rinse
Keep the slides in the tap water until ready to perform antigen retrieval. At no time from this point onwards should the slides be allowed to dry. Drying out will cause non-specific antibody binding and therefore high background staining.

C. Antigen retrieval
Most formalin-fixed tissue requires an antigen retrieval step before immunohistochemical staining can proceed. This is due to the formation of methylene bridges during fixation, which cross-link proteins and therefore mask antigenic sites. The two methods of antigen retrieval are heat-mediated (also know as heat-induced epitope retrieval, or HIER) and enzymatic. Both antigen retrieval methods serve to break the methylene bridges and expose the antigenic sites in order to allow the antibodies to bind.
Antigen retrieval with Tris/EDTA pH 9.0 buffer is suitable for most antigens. Sodium citrate Ph 6.0 is also widely used. Heat-induced epitope retrieval is most often performed using a pressure cooker, a microwave, or a vegetable steamer. Additionally, some labs will use a water bath set to 60°C and incubate the slides in retrieval solution overnight. The optimal method for each antigen must be found experimentally.

C1. Buffer solutions for heat-induced epitope retrieval
The following solutions are three of the more popular buffers for HIER. In the absence of advice from other researchers for a particular antibody, choice of retrieval buffer is best accomplished by experiment.

• Sodium Citrate Buffer (10mM Sodium Citrate, 0.05% Tween 20, pH 6.0)
Tri-sodium citrate (dihydrate) 2.94 g
Distilled water 1000 ml
Mix to dissolve. Adjust pH to 6.0 with 1N HCl.
Add 0.5 ml of Tween 20 and mix well. Store at room temperature for 3 months or at 4°C for longer storage.

• 1 mM EDTA, adjusted to pH 8.0
EDTA 0.37 g
Distilled water 1000 ml
Store at room temperature for 3 months.

• Tris-EDTA Buffer (10mM Tris Base, 1mM EDTA Solution, 0.05% Tween 20, pH 9.0)
Tris 1.21 g
EDTA 0.37 g
Distilled water 1000 ml (100 ml to make 10x, 50 ml to make 20x)
Mix to dissolve. pH is usually at 9.0.
Add 0.5 ml of Tween 20 and mix well. Store at room temperature for 3 months or at 4C for longer storage.

C2. Heat-induced epitope retrieval methods

Slides should be placed in a metal rack for this procedure.

Materials and reagents
• Domestic stainless steel pressure cooker
• Hot plate
• Vessel with slide rack to hold approximately 400-500 ml
• Antigen retrieval buffer (i.e. Tris/EDTA pH 9.0, sodium citrate pH 6.0)

Method
1. Add the appropriate antigen retrieval buffer to the pressure cooker. Place the pressure cooker on the hotplate and turn it on full power. Do not secure the lid of the pressure cooker at this point, simply rest it on top. While waiting for the pressure cooker to come to a boil, deparaffinize and rehydrate the sections as above.
2. Once boiling, transfer the slides from the tap water to the pressure cooker. Secure the pressure cooker lid as in the manufacturer’s instructions.
3. As soon as the cooker has reached full pressure time 3 minutes (Three minutes is only suggested as a starting point antigen retrieval time).
4. When 3 minutes has elapsed, turn off the hotplate and place the pressure cooker in an empty sink.
5. Activate the pressure release valve (see the manufacturer’s instructions) and run cold water over the cooker. Once de-pressurized, open the lid and run cold water into the cooker for 10 minutes.
6. Continue with the immunohistochemical staining protocol.
Note: Other tools like microwave also can be used according to your actual situation.

D. Immunohistochemical staining

General guidelines
All incubations should be carried out in a humidified chamber to avoid drying of the tissue. Drying at any stage will lead to non-specific binding and ultimately high background staining. A shallow, plastic box with a sealed lid and wet tissue paper in the bottom is an adequate chamber, just as long as the slides are kept off the paper and can lay flat so that the reagents don’t drain off! A good solution is to cut a plastic serological pipette into lengths to fit your incubation chamber. Glue them in pairs to the bottom of the chamber, with the 2 individual pipette tubes of each pair being placed about 4.0 cm apart. This provides a level and raised surface for the slides to rest on away from the wet tissue paper. For enzymatic methods, horseradish peroxidase (HRP) or alkaline phosphatase (AP) are the most commonly used enzymes.

Protocol
Day 1
1. (If using an HRP conjugate for detection, blocking of endogenous peroxidase can be performed here but we recommend waiting until after the primary antibody incubation. See Day 2, step 2.).
2. Wash the slides 2 x 5 minutes in TBS plus 0.025% Triton X-100 with gentle agitation.
3. Block in 10% normal serum with 1% BSA in TBS for 2 hours at room temperature.
4. Drain slides for a few seconds (do not rinse) and wipe around the sections with tissue paper.
5. Apply primary antibody diluted in TBS with 1% BSA.
6. Incubate overnight at 4°C.

Day 2
1. Rinse 2 x 5min TBS 0.025% Triton with gentle agitation.
2. If using an HRP conjugate for detection, incubate the slides in 0.3% H2O2 in TBS for 15 min.
3a. For enzymatic detection (HRP or AP secondary conjugates):
Apply enzyme-conjugated secondary antibody to the slide diluted to the concentration recommended by the manufacturer in TBS with 1% BSA, and incubate for 1 hour at room temperature.
3b. For fluorescent detection:
Apply fluorophore-conjugated secondary antibody to the slide diluted to the concentration recommended by the manufacturer in TBS with 1% BSA, and incubate for 1 hour at room temperature. This step should be done in the dark to avoid photobleaching.
4. Rinse 3 x 5min TBS (If using fluorescent detection, end at this step and coverslip with mounting medium; If visualizing the protein with a chromogen, continue with the following steps.).
5. Develop with chromogen for 10 min at room temperature.
6. Rinse in running tap water for 5 min.
7. Counterstain (if required).
8. Dehydrate, clear and mount.

Controls
To estimate the contribution of the non-specific interaction and Fc receptor binding, staining protocols using an antibody directed to an irrelevant antigen having the same isotype as the antibody of interest may be analyzed in parallel with the antibody of interest. The antibody directed to the irrelevant antigen is known as the isotype control. For whole serum antibodies, use normal serum from an unimmunized animal of the same species as the primary antibody. If an isotype control is not available, a negative antibody control is recommended. Simply replace the primary antibody with antibody diluent. A positive tissue control is strongly recommended to ensure that the antibody is performing as expected.
Depending on the experiment, it may also be useful to include a negative tissue control: a tissue in which the protein of interest is not expected to be found.

Abbkine IHC-PARAFFIN Protocol (IHC-P)

Immunohistochemical (IHC) Staining allows you to detect antigens in a tissue fixed on a glass slide. The basic steps of the IHC-P protocol are as follows:
1. Fixing and embedding the tissue
2. Cutting and mounting the section
3. Deparaffinizing and rehydrating the section
4. Antigen retrieval
5. Immunohistochemical staining
6. Counterstaining (if desired)
7. Dehydrating and stabilizing with mounting medium
8. Viewing the staining under the microscope

A: Fixation
Proper fixation is key for the success of immunohistochemistry. 10% neutral buffered formalin (NBF) is most commonly used. Other fixatives such as paraformaldehyde (PFA) or Bouin solution (formalin/picric acid) are used less frequently. The ideal fixation time will depend on the size of the tissue block and the type of tissue, but fixation between 18-24 hours seems to be ideal for most applications. Under-fixation can lead to edge staining, with strong signal on the edges of the section and no signal in the middle; over-fixation can mask the epitope. Antigen retrieval can help overcome this masking, but if the tissue has been fixed for a long period of time (i.e. over a weekend), there may be no signal even after antigen retrieval.
After fixation, the tissue block is embedded in paraffin, then cut in a microtome to the desired thickness (approximately 5 microns is ideal for IHC) and affixed onto the slide. Tissue sections are best mounted on positively charged or APES (amino-propyl-tri-ethoxy-silane) coated slides. Once mounted, the slides should be dried to remove any water that may be trapped under the section. This can be done by leaving the slide at room temperature overnight. If there is a problem with the section adhering to the slide, you may also incubate the slide at 60ºC for a few hours.

B: Deparaffinization
Before proceeding with the staining protocol, the slides must be deparaffinized and rehydrated. Incomplete removal of paraffin can cause poor staining of the section.

Materials and reagents
• Xylene
• 100% ethanol
• 95% ethanol
Method
Place the slides in a rack, and perform the following washes:
1. Xylene: 2 x 3 minutes
2. Xylene 1:1 with 100% ethanol: 3 minutes
3. 100% ethanol: 2 x 3 minutes
4. 95% ethanol: 3 minutes
5. 70 % ethanol: 3 minutes
6. 50 % ethanol: 3 minutes
7. Running cold tap water to rinse
Keep the slides in the tap water until ready to perform antigen retrieval. At no time from this point onwards should the slides be allowed to dry. Drying out will cause non-specific antibody binding and therefore high background staining.

C. Antigen retrieval
Most formalin-fixed tissue requires an antigen retrieval step before immunohistochemical staining can proceed. This is due to the formation of methylene bridges during fixation, which cross-link proteins and therefore mask antigenic sites. The two methods of antigen retrieval are heat-mediated (also know as heat-induced epitope retrieval, or HIER) and enzymatic. Both antigen retrieval methods serve to break the methylene bridges and expose the antigenic sites in order to allow the antibodies to bind.
Antigen retrieval with Tris/EDTA pH 9.0 buffer is suitable for most antigens. Sodium citrate Ph 6.0 is also widely used. Heat-induced epitope retrieval is most often performed using a pressure cooker, a microwave, or a vegetable steamer. Additionally, some labs will use a water bath set to 60°C and incubate the slides in retrieval solution overnight. The optimal method for each antigen must be found experimentally.

C1. Buffer solutions for heat-induced epitope retrieval
The following solutions are three of the more popular buffers for HIER. In the absence of advice from other researchers for a particular antibody, choice of retrieval buffer is best accomplished by experiment.

• Sodium Citrate Buffer (10mM Sodium Citrate, 0.05% Tween 20, pH 6.0)
Tri-sodium citrate (dihydrate) 2.94 g
Distilled water 1000 ml
Mix to dissolve. Adjust pH to 6.0 with 1N HCl.
Add 0.5 ml of Tween 20 and mix well. Store at room temperature for 3 months or at 4°C for longer storage.

• 1 mM EDTA, adjusted to pH 8.0
EDTA 0.37 g
Distilled water 1000 ml
Store at room temperature for 3 months.

• Tris-EDTA Buffer (10mM Tris Base, 1mM EDTA Solution, 0.05% Tween 20, pH 9.0)
Tris 1.21 g
EDTA 0.37 g
Distilled water 1000 ml (100 ml to make 10x, 50 ml to make 20x)
Mix to dissolve. pH is usually at 9.0.
Add 0.5 ml of Tween 20 and mix well. Store at room temperature for 3 months or at 4C for longer storage.

C2. Heat-induced epitope retrieval methods

Slides should be placed in a metal rack for this procedure.

Materials and reagents
• Domestic stainless steel pressure cooker
• Hot plate
• Vessel with slide rack to hold approximately 400-500 ml
• Antigen retrieval buffer (i.e. Tris/EDTA pH 9.0, sodium citrate pH 6.0)

Method
1. Add the appropriate antigen retrieval buffer to the pressure cooker. Place the pressure cooker on the hotplate and turn it on full power. Do not secure the lid of the pressure cooker at this point, simply rest it on top. While waiting for the pressure cooker to come to a boil, deparaffinize and rehydrate the sections as above.
2. Once boiling, transfer the slides from the tap water to the pressure cooker. Secure the pressure cooker lid as in the manufacturer’s instructions.
3. As soon as the cooker has reached full pressure time 3 minutes (Three minutes is only suggested as a starting point antigen retrieval time).
4. When 3 minutes has elapsed, turn off the hotplate and place the pressure cooker in an empty sink.
5. Activate the pressure release valve (see the manufacturer’s instructions) and run cold water over the cooker. Once de-pressurized, open the lid and run cold water into the cooker for 10 minutes.
6. Continue with the immunohistochemical staining protocol.
Note: Other tools like microwave also can be used according to your actual situation.

D. Immunohistochemical staining

General guidelines
All incubations should be carried out in a humidified chamber to avoid drying of the tissue. Drying at any stage will lead to non-specific binding and ultimately high background staining. A shallow, plastic box with a sealed lid and wet tissue paper in the bottom is an adequate chamber, just as long as the slides are kept off the paper and can lay flat so that the reagents don’t drain off! A good solution is to cut a plastic serological pipette into lengths to fit your incubation chamber. Glue them in pairs to the bottom of the chamber, with the 2 individual pipette tubes of each pair being placed about 4.0 cm apart. This provides a level and raised surface for the slides to rest on away from the wet tissue paper. For enzymatic methods, horseradish peroxidase (HRP) or alkaline phosphatase (AP) are the most commonly used enzymes.

Protocol
Day 1
1. (If using an HRP conjugate for detection, blocking of endogenous peroxidase can be performed here but we recommend waiting until after the primary antibody incubation. See Day 2, step 2.).
2. Wash the slides 2 x 5 minutes in TBS plus 0.025% Triton X-100 with gentle agitation.
3. Block in 10% normal serum with 1% BSA in TBS for 2 hours at room temperature.
4. Drain slides for a few seconds (do not rinse) and wipe around the sections with tissue paper.
5. Apply primary antibody diluted in TBS with 1% BSA.
6. Incubate overnight at 4°C.

Day 2
1. Rinse 2 x 5min TBS 0.025% Triton with gentle agitation.
2. If using an HRP conjugate for detection, incubate the slides in 0.3% H2O2 in TBS for 15 min.
3a. For enzymatic detection (HRP or AP secondary conjugates):
Apply enzyme-conjugated secondary antibody to the slide diluted to the concentration recommended by the manufacturer in TBS with 1% BSA, and incubate for 1 hour at room temperature.
3b. For fluorescent detection:
Apply fluorophore-conjugated secondary antibody to the slide diluted to the concentration recommended by the manufacturer in TBS with 1% BSA, and incubate for 1 hour at room temperature. This step should be done in the dark to avoid photobleaching.
4. Rinse 3 x 5min TBS (If using fluorescent detection, end at this step and coverslip with mounting medium; If visualizing the protein with a chromogen, continue with the following steps.).
5. Develop with chromogen for 10 min at room temperature.
6. Rinse in running tap water for 5 min.
7. Counterstain (if required).
8. Dehydrate, clear and mount.

Controls
To estimate the contribution of the non-specific interaction and Fc receptor binding, staining protocols using an antibody directed to an irrelevant antigen having the same isotype as the antibody of interest may be analyzed in parallel with the antibody of interest. The antibody directed to the irrelevant antigen is known as the isotype control. For whole serum antibodies, use normal serum from an unimmunized animal of the same species as the primary antibody. If an isotype control is not available, a negative antibody control is recommended. Simply replace the primary antibody with antibody diluent. A positive tissue control is strongly recommended to ensure that the antibody is performing as expected.
Depending on the experiment, it may also be useful to include a negative tissue control: a tissue in which the protein of interest is not expected to be found.

Optimizing drug combinations against multiple myeloma using a quadratic phenotypic optimization platform (QPOP)

Content introduction:

• Optimizing drug combinations against multiple myeloma using a quadratic phenotypic optimization platform (QPOP)


• Laminin 511 is a target antigen in autoimmune pancreatitis


• Thy-1 (CD90) promotes bone formation and protects against obesity


• Profiling the origin, dynamics, and function of traction force in B cell activation


• G protein signaling–biased agonism at the κ-opioid receptor is maintained in striatal neurons

1. Optimizing drug combinations against multiple myeloma using a quadratic phenotypic optimization platform (QPOP)
Multiple myeloma is an incurable hematological malignancy that relies on drug combinations for first and secondary lines of treatment. The inclusion of proteasome inhibitors, such as bortezomib, into these combination regimens has improved median survival. Resistance to bortezomib, however, is a common occurrence that ultimately contributes to treatment failure, and there remains a need to identify improved drug combinations. Masturah Bte Mohd Abdul Rashid at National University of Singapore in Singapore and his colleagues developed the quadratic phenotypic optimization platform (QPOP) to optimize treatment combinations selected from a candidate pool of 114 approved drugs. QPOP uses quadratic surfaces to model the biological effects of drug combinations to identify effective drug combinations without reference to molecular mechanisms or predetermined drug synergy data. Applying QPOP to bortezomib-resistant multiple myeloma cell lines determined the drug combinations that collectively optimized treatment efficacy. They found that these combinations acted by reversing the DNA methylation and tumor suppressor silencing that often occur after acquired bortezomib resistance in multiple myeloma. Successive application of QPOP on a xenograft mouse model further optimized the dosages of each drug within a given combination while minimizing overall toxicity in vivo, and application of QPOP to ex vivo multiple myeloma patient samples optimized drug combinations in patient-specific contexts.



Read more, please click http://stm.sciencemag.org/content/10/453/eaan0941

2. Laminin 511 is a target antigen in autoimmune pancreatitis
Autoimmune pancreatitis (AIP), a major manifestation of immunoglobulin G4–related disease (IgG4-RD), is an immune-mediated disorder, but the target autoantigens are still unknown. Masahiro Shiokawa at Kyoto University Graduate School of Medicine in Kyoto, Japan and his colleagues previously reported that IgG in patients with AIP induces pancreatic injuries in mice by binding the extracellular matrix (ECM). In the current study, they identified an autoantibody against laminin 511-E8, a truncated laminin 511, one of the ECM proteins, in patients with AIP. Anti–laminin 511-E8 IgG was present in 26 of 51 AIP patients (51.0%), but only in 2 of 122 controls (1.6%), by enzyme-linked immunosorbent assay. Because truncated forms of other laminin family members in other organs have been reported, they confirmed that truncated forms of laminin 511 also exist in human and mouse pancreas. Histologic studies with patient pancreatic tissues showed colocalization of patient IgG and laminin 511. Immunization of mice with human laminin 511-E8 induced antibodies and pancreatic injury, fulfilling the pathologic criteria for human AIP. Four of 25 AIP patients without laminin 511-E8 antibodies had antibodies against integrin α6β1, a laminin 511 ligand. AIP patients with laminin 511-E8 antibodies exhibited distinctive clinical features, as the frequencies of malignancies or allergic diseases were significantly lower in patients with laminin 511-E8 antibodies than in those without. The discovery of these autoantibodies should aid in the understanding of AIP pathophysiology and possibly improve the diagnosis of AIP.

Read more, please click http://stm.sciencemag.org/content/10/453/eaaq0997

3. Thy-1 (CD90) promotes bone formation and protects against obesity
Osteoporosis and obesity result from disturbed osteogenic and adipogenic differentiation and present emerging challenges for our aging society. Because of the regulatory role of Thy-1 in mesenchyme-derived fibroblasts, Ann-Kristin Picke at Technische Universität Dresden in Dresden, Germany and his colleagues investigated the impact of Thy-1 expression on mesenchymal stem cell (MSC) fate between osteogenic and adipogenic differentiation and consequences for bone formation and adipose tissue development in vivo. MSCs from Thy-1–deficient mice have decreased osteoblast differentiation and increased adipogenic differentiation compared to MSCs from wild-type mice. Consistently, Thy-1–deficient mice exhibited decreased bone volume and bone formation rate with elevated cortical porosity, resulting in lower bone strength. In parallel, body weight, subcutaneous/epigonadal fat mass, and bone fat volume were increased. Thy-1 deficiency was accompanied by reduced expression of specific Wnt ligands with simultaneous increase of the Wnt inhibitors sclerostin and dickkopf-1 and an altered responsiveness to Wnt. They demonstrated that disturbed bone remodeling in osteoporosis and dysregulated adipose tissue accumulation in patients with obesity were mirrored by reduced serum Thy-1 concentrations. Their findings provide new insights into the mutual regulation of bone formation and obesity and open new perspectives to monitor and to interfere with the dysregulated balance of adipogenesis and osteogenesis in obesity and osteoporosis.

Read more, please click http://stm.sciencemag.org/content/10/453/eaao6806

4. Profiling the origin, dynamics, and function of traction force in B cell activation
B lymphocytes use B cell receptors (BCRs) to recognize membrane-bound antigens to further initiate cell spreading and contraction responses during B cell activation. Junyi Wang at Tsinghua University in Beijing, China and his colleagues combined traction force microscopy and live-cell imaging to profile the origin, dynamics, and function of traction force generation in these responses. They showed that B cell activation required the generation of 10 to 20 nN of traction force when encountering antigens presented by substrates with stiffness values from 0.5 to 1 kPa, which mimic the rigidity of antigen-presenting cells in vivo. Perturbation experiments revealed that F-actin remodeling and myosin- and dynein-mediated contractility contributed to traction force generation and B cell activation. Moreover, membrane-proximal BCR signaling molecules (including Lyn, Syk, Btk, PLC-γ2, BLNK, and Vav3) and adaptor molecules (Grb2, Cbl, and Dok-3) linking BCR microclusters and motor proteins were also required for the sustained generation of these traction forces. They found a positive correlation between the strength of the traction force and the mean fluorescence intensity of the BCR microclusters. Furthermore, they demonstrated that isotype-switched memory B cells expressing immunoglobulin G (IgG)–BCRs generated greater traction forces than did mature naïve B cells expressing IgM-BCRs during B cell activation. Last, they observed that primary B cells from patients with rheumatoid arthritis generated greater traction forces than did B cells from healthy donors in response to antigen stimulation. Together, these data delineate the origin, dynamics, and function of traction force during B cell activation.

Read more, please click http://stke.sciencemag.org/content/11/542/eaai9192

5. G protein signaling–biased agonism at the κ-opioid receptor is maintained in striatal neurons
Biased agonists of G protein–coupled receptors may present a means to refine receptor signaling in a way that separates side effects from therapeutic properties. Several studies have shown that agonists that activate the κ-opioid receptor (KOR) in a manner that favors G protein coupling over β-arrestin2 recruitment in cell culture may represent a means to treat pain and itch while avoiding sedation and dysphoria. Although it is attractive to speculate that the bias between G protein signaling and β-arrestin2 recruitment is the reason for these divergent behaviors, little evidence has emerged to show that these signaling pathways diverge in the neuronal environment. Jo-Hao Ho at The Scripps Research Institute in Jupiter, USA and his colleagues further explored the influence of cellular context on biased agonism at KOR ligand–directed signaling toward G protein pathways over β-arrestin–dependent pathways and found that this bias persists in striatal neurons. These findings advance our understanding of how a G protein–biased agonist signal differs between cell lines and primary neurons, demonstrate that measuring [35S]GTPγS binding and the regulation of adenylyl cyclase activity are not necessarily orthogonal assays in cell lines, and emphasize the contributions of the environment to assessing biased agonism.

Read more, please click http://stke.sciencemag.org/content/11/542/eaar4309

Optimizing drug combinations against multiple myeloma using a quadratic phenotypic optimization platform (QPOP)

Content introduction:

• Optimizing drug combinations against multiple myeloma using a quadratic phenotypic optimization platform (QPOP)


• Laminin 511 is a target antigen in autoimmune pancreatitis


• Thy-1 (CD90) promotes bone formation and protects against obesity


• Profiling the origin, dynamics, and function of traction force in B cell activation


• G protein signaling–biased agonism at the κ-opioid receptor is maintained in striatal neurons

1. Optimizing drug combinations against multiple myeloma using a quadratic phenotypic optimization platform (QPOP)
Multiple myeloma is an incurable hematological malignancy that relies on drug combinations for first and secondary lines of treatment. The inclusion of proteasome inhibitors, such as bortezomib, into these combination regimens has improved median survival. Resistance to bortezomib, however, is a common occurrence that ultimately contributes to treatment failure, and there remains a need to identify improved drug combinations. Masturah Bte Mohd Abdul Rashid at National University of Singapore in Singapore and his colleagues developed the quadratic phenotypic optimization platform (QPOP) to optimize treatment combinations selected from a candidate pool of 114 approved drugs. QPOP uses quadratic surfaces to model the biological effects of drug combinations to identify effective drug combinations without reference to molecular mechanisms or predetermined drug synergy data. Applying QPOP to bortezomib-resistant multiple myeloma cell lines determined the drug combinations that collectively optimized treatment efficacy. They found that these combinations acted by reversing the DNA methylation and tumor suppressor silencing that often occur after acquired bortezomib resistance in multiple myeloma. Successive application of QPOP on a xenograft mouse model further optimized the dosages of each drug within a given combination while minimizing overall toxicity in vivo, and application of QPOP to ex vivo multiple myeloma patient samples optimized drug combinations in patient-specific contexts.



Read more, please click http://stm.sciencemag.org/content/10/453/eaan0941

2. Laminin 511 is a target antigen in autoimmune pancreatitis
Autoimmune pancreatitis (AIP), a major manifestation of immunoglobulin G4–related disease (IgG4-RD), is an immune-mediated disorder, but the target autoantigens are still unknown. Masahiro Shiokawa at Kyoto University Graduate School of Medicine in Kyoto, Japan and his colleagues previously reported that IgG in patients with AIP induces pancreatic injuries in mice by binding the extracellular matrix (ECM). In the current study, they identified an autoantibody against laminin 511-E8, a truncated laminin 511, one of the ECM proteins, in patients with AIP. Anti–laminin 511-E8 IgG was present in 26 of 51 AIP patients (51.0%), but only in 2 of 122 controls (1.6%), by enzyme-linked immunosorbent assay. Because truncated forms of other laminin family members in other organs have been reported, they confirmed that truncated forms of laminin 511 also exist in human and mouse pancreas. Histologic studies with patient pancreatic tissues showed colocalization of patient IgG and laminin 511. Immunization of mice with human laminin 511-E8 induced antibodies and pancreatic injury, fulfilling the pathologic criteria for human AIP. Four of 25 AIP patients without laminin 511-E8 antibodies had antibodies against integrin α6β1, a laminin 511 ligand. AIP patients with laminin 511-E8 antibodies exhibited distinctive clinical features, as the frequencies of malignancies or allergic diseases were significantly lower in patients with laminin 511-E8 antibodies than in those without. The discovery of these autoantibodies should aid in the understanding of AIP pathophysiology and possibly improve the diagnosis of AIP.

Read more, please click http://stm.sciencemag.org/content/10/453/eaaq0997

3. Thy-1 (CD90) promotes bone formation and protects against obesity
Osteoporosis and obesity result from disturbed osteogenic and adipogenic differentiation and present emerging challenges for our aging society. Because of the regulatory role of Thy-1 in mesenchyme-derived fibroblasts, Ann-Kristin Picke at Technische Universität Dresden in Dresden, Germany and his colleagues investigated the impact of Thy-1 expression on mesenchymal stem cell (MSC) fate between osteogenic and adipogenic differentiation and consequences for bone formation and adipose tissue development in vivo. MSCs from Thy-1–deficient mice have decreased osteoblast differentiation and increased adipogenic differentiation compared to MSCs from wild-type mice. Consistently, Thy-1–deficient mice exhibited decreased bone volume and bone formation rate with elevated cortical porosity, resulting in lower bone strength. In parallel, body weight, subcutaneous/epigonadal fat mass, and bone fat volume were increased. Thy-1 deficiency was accompanied by reduced expression of specific Wnt ligands with simultaneous increase of the Wnt inhibitors sclerostin and dickkopf-1 and an altered responsiveness to Wnt. They demonstrated that disturbed bone remodeling in osteoporosis and dysregulated adipose tissue accumulation in patients with obesity were mirrored by reduced serum Thy-1 concentrations. Their findings provide new insights into the mutual regulation of bone formation and obesity and open new perspectives to monitor and to interfere with the dysregulated balance of adipogenesis and osteogenesis in obesity and osteoporosis.

Read more, please click http://stm.sciencemag.org/content/10/453/eaao6806

4. Profiling the origin, dynamics, and function of traction force in B cell activation
B lymphocytes use B cell receptors (BCRs) to recognize membrane-bound antigens to further initiate cell spreading and contraction responses during B cell activation. Junyi Wang at Tsinghua University in Beijing, China and his colleagues combined traction force microscopy and live-cell imaging to profile the origin, dynamics, and function of traction force generation in these responses. They showed that B cell activation required the generation of 10 to 20 nN of traction force when encountering antigens presented by substrates with stiffness values from 0.5 to 1 kPa, which mimic the rigidity of antigen-presenting cells in vivo. Perturbation experiments revealed that F-actin remodeling and myosin- and dynein-mediated contractility contributed to traction force generation and B cell activation. Moreover, membrane-proximal BCR signaling molecules (including Lyn, Syk, Btk, PLC-γ2, BLNK, and Vav3) and adaptor molecules (Grb2, Cbl, and Dok-3) linking BCR microclusters and motor proteins were also required for the sustained generation of these traction forces. They found a positive correlation between the strength of the traction force and the mean fluorescence intensity of the BCR microclusters. Furthermore, they demonstrated that isotype-switched memory B cells expressing immunoglobulin G (IgG)–BCRs generated greater traction forces than did mature naïve B cells expressing IgM-BCRs during B cell activation. Last, they observed that primary B cells from patients with rheumatoid arthritis generated greater traction forces than did B cells from healthy donors in response to antigen stimulation. Together, these data delineate the origin, dynamics, and function of traction force during B cell activation.

Read more, please click http://stke.sciencemag.org/content/11/542/eaai9192

5. G protein signaling–biased agonism at the κ-opioid receptor is maintained in striatal neurons
Biased agonists of G protein–coupled receptors may present a means to refine receptor signaling in a way that separates side effects from therapeutic properties. Several studies have shown that agonists that activate the κ-opioid receptor (KOR) in a manner that favors G protein coupling over β-arrestin2 recruitment in cell culture may represent a means to treat pain and itch while avoiding sedation and dysphoria. Although it is attractive to speculate that the bias between G protein signaling and β-arrestin2 recruitment is the reason for these divergent behaviors, little evidence has emerged to show that these signaling pathways diverge in the neuronal environment. Jo-Hao Ho at The Scripps Research Institute in Jupiter, USA and his colleagues further explored the influence of cellular context on biased agonism at KOR ligand–directed signaling toward G protein pathways over β-arrestin–dependent pathways and found that this bias persists in striatal neurons. These findings advance our understanding of how a G protein–biased agonist signal differs between cell lines and primary neurons, demonstrate that measuring [35S]GTPγS binding and the regulation of adenylyl cyclase activity are not necessarily orthogonal assays in cell lines, and emphasize the contributions of the environment to assessing biased agonism.

Read more, please click http://stke.sciencemag.org/content/11/542/eaar4309

Optimizing drug combinations against multiple myeloma using a quadratic phenotypic optimization platform (QPOP)

Content introduction:

• Optimizing drug combinations against multiple myeloma using a quadratic phenotypic optimization platform (QPOP)


• Laminin 511 is a target antigen in autoimmune pancreatitis


• Thy-1 (CD90) promotes bone formation and protects against obesity


• Profiling the origin, dynamics, and function of traction force in B cell activation


• G protein signaling–biased agonism at the κ-opioid receptor is maintained in striatal neurons

1. Optimizing drug combinations against multiple myeloma using a quadratic phenotypic optimization platform (QPOP)
Multiple myeloma is an incurable hematological malignancy that relies on drug combinations for first and secondary lines of treatment. The inclusion of proteasome inhibitors, such as bortezomib, into these combination regimens has improved median survival. Resistance to bortezomib, however, is a common occurrence that ultimately contributes to treatment failure, and there remains a need to identify improved drug combinations. Masturah Bte Mohd Abdul Rashid at National University of Singapore in Singapore and his colleagues developed the quadratic phenotypic optimization platform (QPOP) to optimize treatment combinations selected from a candidate pool of 114 approved drugs. QPOP uses quadratic surfaces to model the biological effects of drug combinations to identify effective drug combinations without reference to molecular mechanisms or predetermined drug synergy data. Applying QPOP to bortezomib-resistant multiple myeloma cell lines determined the drug combinations that collectively optimized treatment efficacy. They found that these combinations acted by reversing the DNA methylation and tumor suppressor silencing that often occur after acquired bortezomib resistance in multiple myeloma. Successive application of QPOP on a xenograft mouse model further optimized the dosages of each drug within a given combination while minimizing overall toxicity in vivo, and application of QPOP to ex vivo multiple myeloma patient samples optimized drug combinations in patient-specific contexts.



Read more, please click http://stm.sciencemag.org/content/10/453/eaan0941

2. Laminin 511 is a target antigen in autoimmune pancreatitis
Autoimmune pancreatitis (AIP), a major manifestation of immunoglobulin G4–related disease (IgG4-RD), is an immune-mediated disorder, but the target autoantigens are still unknown. Masahiro Shiokawa at Kyoto University Graduate School of Medicine in Kyoto, Japan and his colleagues previously reported that IgG in patients with AIP induces pancreatic injuries in mice by binding the extracellular matrix (ECM). In the current study, they identified an autoantibody against laminin 511-E8, a truncated laminin 511, one of the ECM proteins, in patients with AIP. Anti–laminin 511-E8 IgG was present in 26 of 51 AIP patients (51.0%), but only in 2 of 122 controls (1.6%), by enzyme-linked immunosorbent assay. Because truncated forms of other laminin family members in other organs have been reported, they confirmed that truncated forms of laminin 511 also exist in human and mouse pancreas. Histologic studies with patient pancreatic tissues showed colocalization of patient IgG and laminin 511. Immunization of mice with human laminin 511-E8 induced antibodies and pancreatic injury, fulfilling the pathologic criteria for human AIP. Four of 25 AIP patients without laminin 511-E8 antibodies had antibodies against integrin α6β1, a laminin 511 ligand. AIP patients with laminin 511-E8 antibodies exhibited distinctive clinical features, as the frequencies of malignancies or allergic diseases were significantly lower in patients with laminin 511-E8 antibodies than in those without. The discovery of these autoantibodies should aid in the understanding of AIP pathophysiology and possibly improve the diagnosis of AIP.

Read more, please click http://stm.sciencemag.org/content/10/453/eaaq0997

3. Thy-1 (CD90) promotes bone formation and protects against obesity
Osteoporosis and obesity result from disturbed osteogenic and adipogenic differentiation and present emerging challenges for our aging society. Because of the regulatory role of Thy-1 in mesenchyme-derived fibroblasts, Ann-Kristin Picke at Technische Universität Dresden in Dresden, Germany and his colleagues investigated the impact of Thy-1 expression on mesenchymal stem cell (MSC) fate between osteogenic and adipogenic differentiation and consequences for bone formation and adipose tissue development in vivo. MSCs from Thy-1–deficient mice have decreased osteoblast differentiation and increased adipogenic differentiation compared to MSCs from wild-type mice. Consistently, Thy-1–deficient mice exhibited decreased bone volume and bone formation rate with elevated cortical porosity, resulting in lower bone strength. In parallel, body weight, subcutaneous/epigonadal fat mass, and bone fat volume were increased. Thy-1 deficiency was accompanied by reduced expression of specific Wnt ligands with simultaneous increase of the Wnt inhibitors sclerostin and dickkopf-1 and an altered responsiveness to Wnt. They demonstrated that disturbed bone remodeling in osteoporosis and dysregulated adipose tissue accumulation in patients with obesity were mirrored by reduced serum Thy-1 concentrations. Their findings provide new insights into the mutual regulation of bone formation and obesity and open new perspectives to monitor and to interfere with the dysregulated balance of adipogenesis and osteogenesis in obesity and osteoporosis.

Read more, please click http://stm.sciencemag.org/content/10/453/eaao6806

4. Profiling the origin, dynamics, and function of traction force in B cell activation
B lymphocytes use B cell receptors (BCRs) to recognize membrane-bound antigens to further initiate cell spreading and contraction responses during B cell activation. Junyi Wang at Tsinghua University in Beijing, China and his colleagues combined traction force microscopy and live-cell imaging to profile the origin, dynamics, and function of traction force generation in these responses. They showed that B cell activation required the generation of 10 to 20 nN of traction force when encountering antigens presented by substrates with stiffness values from 0.5 to 1 kPa, which mimic the rigidity of antigen-presenting cells in vivo. Perturbation experiments revealed that F-actin remodeling and myosin- and dynein-mediated contractility contributed to traction force generation and B cell activation. Moreover, membrane-proximal BCR signaling molecules (including Lyn, Syk, Btk, PLC-γ2, BLNK, and Vav3) and adaptor molecules (Grb2, Cbl, and Dok-3) linking BCR microclusters and motor proteins were also required for the sustained generation of these traction forces. They found a positive correlation between the strength of the traction force and the mean fluorescence intensity of the BCR microclusters. Furthermore, they demonstrated that isotype-switched memory B cells expressing immunoglobulin G (IgG)–BCRs generated greater traction forces than did mature naïve B cells expressing IgM-BCRs during B cell activation. Last, they observed that primary B cells from patients with rheumatoid arthritis generated greater traction forces than did B cells from healthy donors in response to antigen stimulation. Together, these data delineate the origin, dynamics, and function of traction force during B cell activation.

Read more, please click http://stke.sciencemag.org/content/11/542/eaai9192

5. G protein signaling–biased agonism at the κ-opioid receptor is maintained in striatal neurons
Biased agonists of G protein–coupled receptors may present a means to refine receptor signaling in a way that separates side effects from therapeutic properties. Several studies have shown that agonists that activate the κ-opioid receptor (KOR) in a manner that favors G protein coupling over β-arrestin2 recruitment in cell culture may represent a means to treat pain and itch while avoiding sedation and dysphoria. Although it is attractive to speculate that the bias between G protein signaling and β-arrestin2 recruitment is the reason for these divergent behaviors, little evidence has emerged to show that these signaling pathways diverge in the neuronal environment. Jo-Hao Ho at The Scripps Research Institute in Jupiter, USA and his colleagues further explored the influence of cellular context on biased agonism at KOR ligand–directed signaling toward G protein pathways over β-arrestin–dependent pathways and found that this bias persists in striatal neurons. These findings advance our understanding of how a G protein–biased agonist signal differs between cell lines and primary neurons, demonstrate that measuring [35S]GTPγS binding and the regulation of adenylyl cyclase activity are not necessarily orthogonal assays in cell lines, and emphasize the contributions of the environment to assessing biased agonism.

Read more, please click http://stke.sciencemag.org/content/11/542/eaar4309

53BP1-RIF1-shieldin counteracts DSB resection through CST-and Polα-dependent fill-in

Content introduction:

• 53BP1–RIF1–shieldin counteracts DSB resection through CST- and Polα-dependent fill-in


• Leukaemia hijacks a neural mechanism to invade the central nervous system


• Thymic tuft cells promote an IL-4-enriched medulla and shape thymocyte development


• The shieldin complex mediates 53BP1-dependent DNA repair


• Glucose-regulated phosphorylation of TET2 by AMPK reveals a pathway linking diabetes to cancer

1. 53BP1–RIF1–shieldin counteracts DSB resection through CST- and Polα-dependent fill-in

In DNA repair, the resection of double-strand breaks dictates the choice between homology-directed repair—which requires a 3′ overhang—and classical non-homologous end joining, which can join unresected ends. BRCA1-mutant cancers show minimal resection of double-strand breaks, which renders them deficient in homology-directed repair and sensitive to inhibitors of poly(ADP-ribose) polymerase 1 (PARP1). When BRCA1 is absent, the resection of double-strand breaks is thought to be prevented by 53BP1, RIF1 and the REV7–SHLD1–SHLD2–SHLD3 (shieldin) complex, and loss of these factors diminishes sensitivity to PARP1 inhibitors. Here Zachary Mirman at Rockefeller University in New York, USA and his colleagues address the mechanism by which 53BP1–RIF1–shieldin regulates the generation of recombinogenic 3′ overhangs. They report that CTC1–STN1–TEN1 (CST), a complex similar to replication protein A that functions as an accessory factor of polymerase-α (Polα)–primase, is a downstream effector in the 53BP1 pathway. CST interacts with shieldin and localizes with Polα to sites of DNA damage in a 53BP1- and shieldin-dependent manner. As with loss of 53BP1, RIF1 or shieldin, the depletion of CST leads to increased resection. In BRCA1-deficient cells, CST blocks RAD51 loading and promotes the efficacy of PARP1 inhibitors. In addition, Polα inhibition diminishes the effect of PARP1 inhibitors. These data suggest that CST–Polα-mediated fill-in helps to control the repair of double-strand breaks by 53BP1, RIF1 and shieldin.



Read more, please click https://www.nature.com/articles/s41586-018-0324-7

2. Leukaemia hijacks a neural mechanism to invade the central nervous system

Acute lymphoblastic leukaemia (ALL) has a marked propensity to metastasize to the central nervous system (CNS). In contrast to brain metastases from solid tumours, metastases of ALL seldom involve the parenchyma but are isolated to the leptomeninges, which is an infrequent site for carcinomatous invasion. Although metastasis to the CNS occurs across all subtypes of ALL, a unifying mechanism for invasion has not yet been determined. Here Hisayuki Yao at Duke University in Durham, USA and his colleagues show that ALL cells in the circulation are unable to breach the blood–brain barrier in mice; instead, they migrate into the CNS along vessels that pass directly between vertebral or calvarial bone marrow and the subarachnoid space. The basement membrane of these bridging vessels is enriched in laminin, which is known to coordinate pathfinding of neuronal progenitor cells in the CNS. The laminin receptor α6 integrin is expressed in most cases of ALL. They found that α6 integrin–laminin interactions mediated the migration of ALL cells towards the cerebrospinal fluid in vitro. Mice with ALL xenografts were treated with either a PI3Kδ inhibitor, which decreased α6 integrin expression on ALL cells, or specific α6 integrin-neutralizing antibodies and showed significant reductions in ALL transit along bridging vessels, blast counts in the cerebrospinal fluid and CNS disease symptoms despite minimally decreased bone marrow disease burden. Their data suggest that α6 integrin expression, which is common in ALL, allows cells to use neural migratory pathways to invade the CNS.

Read more, please click https://www.nature.com/articles/s41586-018-0342-5

3. Thymic tuft cells promote an IL-4-enriched medulla and shape thymocyte development

The thymus is responsible for generating a diverse yet self-tolerant pool of T cells. Although the thymic medulla consists mostly of developing and mature AIRE+ epithelial cells, recent evidence has suggested that there is far greater heterogeneity among medullary thymic epithelial cells than was previously thought. Here Corey N. Miller at University of California in San Francisco, USA and his colleagues describe in detail an epithelial subset that is remarkably similar to peripheral tuft cells that are found at mucosal barriers. Similar to the periphery, thymic tuft cells express the canonical taste transduction pathway and IL-25. However, they are unique in their spatial association with cornified aggregates, ability to present antigens and expression of a broad diversity of taste receptors. Some thymic tuft cells pass through an Aire-expressing stage and depend on a known AIRE-binding partner, HIPK2, for their development. Notably, the taste chemosensory protein TRPM5 is required for their thymic function through which they support the development and polarization of thymic invariant natural killer T cells and act to establish a medullary microenvironment that is enriched in the type 2 cytokine, IL-4. These findings indicate that there is a compartmentalized medullary environment in which differentiation of a minor and highly specialized epithelial subset has a non-redundant role in shaping thymic function.

Read more, please click https://www.nature.com/articles/s41586-018-0345-2

4. The shieldin complex mediates 53BP1-dependent DNA repair

53BP1 is a chromatin-binding protein that regulates the repair of DNA double-strand breaks by suppressing the nucleolytic resection of DNA termini. This function of 53BP1 requires interactions with PTIP and RIF1, the latter of which recruits REV7 (also known as MAD2L2) to break sites. How 53BP1-pathway proteins shield DNA ends is currently unknown, but there are two models that provide the best potential explanation of their action. In one model the 53BP1 complex strengthens the nucleosomal barrier to end-resection nucleases, and in the other 53BP1 recruits effector proteins with end-protection activity. Here Sylvie M. Noordermeer at Mount Sinai Hospital in Toronto, Ontario, Canada and his colleagues identify a 53BP1 effector complex, shieldin, that includes C20orf196 (also known as SHLD1), FAM35A (SHLD2), CTC-534A2.2 (SHLD3) and REV7. Shieldin localizes to double-strand-break sites in a 53BP1- and RIF1-dependent manner, and its SHLD2 subunit binds to single-stranded DNA via OB-fold domains that are analogous to those of RPA1 and POT1. Loss of shieldin impairs non-homologous end-joining, leads to defective immunoglobulin class switching and causes hyper-resection. Mutations in genes that encode shieldin subunits also cause resistance to poly(ADP-ribose) polymerase inhibition in BRCA1-deficient cells and tumours, owing to restoration of homologous recombination. Finally, they show that binding of single-stranded DNA by SHLD2 is critical for shieldin function, consistent with a model in which shieldin protects DNA ends to mediate 53BP1-dependent DNA repair.

Read more, please click https://www.nature.com/articles/s41586-018-0340-7

5. Glucose-regulated phosphorylation of TET2 by AMPK reveals a pathway linking diabetes to cancer

Diabetes is a complex metabolic syndrome that is characterized by prolonged high blood glucose levels and frequently associated with life-threatening complications. Epidemiological studies have suggested that diabetes is also linked to an increased risk of cancer. High glucose levels may be a prevailing factor that contributes to the link between diabetes and cancer, but little is known about the molecular basis of this link and how the high glucose state may drive genetic and/or epigenetic alterations that result in a cancer phenotype. Here Di Wu at Fudan University in Shanghai, China and his colleagues show that hyperglycaemic conditions have an adverse effect on the DNA 5-hydroxymethylome. They identify the tumour suppressor TET2 as a substrate of the AMP-activated kinase (AMPK), which phosphorylates TET2 at serine 99, thereby stabilizing the tumour suppressor. Increased glucose levels impede AMPK-mediated phosphorylation at serine 99, which results in the destabilization of TET2 followed by dysregulation of both 5-hydroxymethylcytosine (5hmC) and the tumour suppressive function of TET2 in vitro and in vivo. Treatment with the anti-diabetic drug metformin protects AMPK-mediated phosphorylation of serine 99, thereby increasing TET2 stability and 5hmC levels. These findings define a novel ‘phospho-switch’ that regulates TET2 stability and a regulatory pathway that links glucose and AMPK to TET2 and 5hmC, which connects diabetes to cancer. Their data also unravel an epigenetic pathway by which metformin mediates tumour suppression. Thus, this study presents a new model for how a pernicious environment can directly reprogram the epigenome towards an oncogenic state, offering a potential strategy for cancer prevention and treatment.

Read more, please click https://www.nature.com/articles/s41586-018-0350-5