A reading report for <A Secreted Slit2 Fragment Regulates Adipose Tissue Thermogenesis and Metabolic Function
>, only for private study use, please do not use it for profit or public.
Epidermal Growth Factor use in Diabetic Foot UlcersG H PRABHU
EGF is used by many diabetologists to treat Diabetic Foot Ulcers. If used early EGF can prevent progression of DFU resulting in amputations and related morbidity and mortality.
Epidermal Growth Factor use in Diabetic Foot UlcersG H PRABHU
EGF is used by many diabetologists to treat Diabetic Foot Ulcers. If used early EGF can prevent progression of DFU resulting in amputations and related morbidity and mortality.
Cell Signaling is an important facet of biological life. It allows cells to perceive and respond to the extracellular environment allowing development, growth, immunity, etc. Additionally, errors in cell signaling may result in cancer growth, diabetes. ... The inducer does not diffuse from the cell producing it.
Cell Signaling is an important facet of biological life. It allows cells to perceive and respond to the extracellular environment allowing development, growth, immunity, etc. Additionally, errors in cell signaling may result in cancer growth, diabetes. ... The inducer does not diffuse from the cell producing it.
Cloning and expression of Human glutamic acid decarboxylase (GAD 65) gene in ...Open Access Research Paper
Diabetes is a chronic autoimmune disease characterized by the inability of body to produce or respond to insulin a hormone required by body to burn glucose for energy. Type I Diabetes mellitus, also known as Insulin Dependent Diabetes mellitus is a most frequent chronic disease of childhood, afflicts 0.2-0.3% of human individuals due to auto immune destruction of insulin secreting pancreatic β cells. GAD65 is the major auto antigen in Insulin Dependent Diabetes Mellitus (IIDM). Thus, this project is aimed at expression of GAD65 in E. coli. GAD65 gene was cloned into pET-28a bacterial expression vector and expression was studied in BL21 DE3 cells. Different parameters of induction like isopropyl-β-D-thiogalactopyranoside (IPTG), temperature, time interval were standardized. The recombinant clones induced with 2 μM of IPTG at 30oC for 4 h at flask level produced the protein upto 537μg/ml. Furthermore, the specificity of the purified recombinant protein was confirmed by western blot analysis using monoclonal antibodies. This work establishes a strategy in E. coli for the expression of GAD65 with optimized parameters.
El lunes y martes 20 y 21 de noviembre coordinamos un simposio internacional en la Fundación Ramón Areces, sobre los defectos del transporte de aminoácidos.
El lunes y martes 20 y 21 de noviembre coordinamos un simposio internacional en la Fundación Ramón Areces, sobre los defectos del transporte de aminoácidos.
Extraction and purification of product from fermentation is known as Downstream Processing ( DSP) or Product Recovery
It is an essential step in the manufacture of pharmaceuticals product
Cost of the product is determined by the DSP involved
By using flow cytometry, staining dyes are needed. Creative Bioarray can choose different dyes to perform the assays, including propidium iodide (PI), BrdU, 7-amino actinomycin-D (7-AAD), Hoechst 33342 and 33258, and 4’6’-diamidino-2-phenylindole (DAPI), based on the customer’s applications or requirements.
https://www.creative-bioarray.com/cell-cycle-assays.htm
Cell cycle refers to the set of events through which a cell grows, replicates its genome, and ultimately divides into two daughter cells through the process of mitosis.
https://www.creative-bioarray.com/cell-cycle-assays.htm
By using flow cytometry, staining dyes are needed. Creative Bioarray can choose different dyes to perform the assays, including propidium iodide (PI), BrdU, 7-amino actinomycin-D (7-AAD), Hoechst 33342 and 33258, and 4’6’-diamidino-2-phenylindole (DAPI), based on the customer’s applications or requirements.
https://www.creative-bioarray.com/cell-cycle-assays.htm
Generation of MRP2 Efflux Transporter Knock-Out in HepaRG Cell Linemdmitc
MilliporeSigma's Jennifer Pratt recently presented a poster at the 2016 AAPS/ITC Transporter Workshop demonstrating the utility of HepaRG MRP2 Knockout cells for investigating drug-transporter interactions in the liver involving MRP2.
A reading report for <Phosphoproteomics Identifies CK2 as a Negative Regula...星云 王
A reading report for <Phosphoproteomics Identifies CK2 as a Negative Regulator of Beige Adipocyte Thermogenesis and Energy Expenditure>, only for private study use, please do not use it for profit or public.
Tom Selleck Health: A Comprehensive Look at the Iconic Actor’s Wellness Journeygreendigital
Tom Selleck, an enduring figure in Hollywood. has captivated audiences for decades with his rugged charm, iconic moustache. and memorable roles in television and film. From his breakout role as Thomas Magnum in Magnum P.I. to his current portrayal of Frank Reagan in Blue Bloods. Selleck's career has spanned over 50 years. But beyond his professional achievements. fans have often been curious about Tom Selleck Health. especially as he has aged in the public eye.
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Introduction
Many have been interested in Tom Selleck health. not only because of his enduring presence on screen but also because of the challenges. and lifestyle choices he has faced and made over the years. This article delves into the various aspects of Tom Selleck health. exploring his fitness regimen, diet, mental health. and the challenges he has encountered as he ages. We'll look at how he maintains his well-being. the health issues he has faced, and his approach to ageing .
Early Life and Career
Childhood and Athletic Beginnings
Tom Selleck was born on January 29, 1945, in Detroit, Michigan, and grew up in Sherman Oaks, California. From an early age, he was involved in sports, particularly basketball. which played a significant role in his physical development. His athletic pursuits continued into college. where he attended the University of Southern California (USC) on a basketball scholarship. This early involvement in sports laid a strong foundation for his physical health and disciplined lifestyle.
Transition to Acting
Selleck's transition from an athlete to an actor came with its physical demands. His first significant role in "Magnum P.I." required him to perform various stunts and maintain a fit appearance. This role, which he played from 1980 to 1988. necessitated a rigorous fitness routine to meet the show's demands. setting the stage for his long-term commitment to health and wellness.
Fitness Regimen
Workout Routine
Tom Selleck health and fitness regimen has evolved. adapting to his changing roles and age. During his "Magnum, P.I." days. Selleck's workouts were intense and focused on building and maintaining muscle mass. His routine included weightlifting, cardiovascular exercises. and specific training for the stunts he performed on the show.
Selleck adjusted his fitness routine as he aged to suit his body's needs. Today, his workouts focus on maintaining flexibility, strength, and cardiovascular health. He incorporates low-impact exercises such as swimming, walking, and light weightlifting. This balanced approach helps him stay fit without putting undue strain on his joints and muscles.
Importance of Flexibility and Mobility
In recent years, Selleck has emphasized the importance of flexibility and mobility in his fitness regimen. Understanding the natural decline in muscle mass and joint flexibility with age. he includes stretching and yoga in his routine. These practices help prevent injuries, improve posture, and maintain mobilit
Lung Cancer: Artificial Intelligence, Synergetics, Complex System Analysis, S...Oleg Kshivets
RESULTS: Overall life span (LS) was 2252.1±1742.5 days and cumulative 5-year survival (5YS) reached 73.2%, 10 years – 64.8%, 20 years – 42.5%. 513 LCP lived more than 5 years (LS=3124.6±1525.6 days), 148 LCP – more than 10 years (LS=5054.4±1504.1 days).199 LCP died because of LC (LS=562.7±374.5 days). 5YS of LCP after bi/lobectomies was significantly superior in comparison with LCP after pneumonectomies (78.1% vs.63.7%, P=0.00001 by log-rank test). AT significantly improved 5YS (66.3% vs. 34.8%) (P=0.00000 by log-rank test) only for LCP with N1-2. Cox modeling displayed that 5YS of LCP significantly depended on: phase transition (PT) early-invasive LC in terms of synergetics, PT N0—N12, cell ratio factors (ratio between cancer cells- CC and blood cells subpopulations), G1-3, histology, glucose, AT, blood cell circuit, prothrombin index, heparin tolerance, recalcification time (P=0.000-0.038). Neural networks, genetic algorithm selection and bootstrap simulation revealed relationships between 5YS and PT early-invasive LC (rank=1), PT N0—N12 (rank=2), thrombocytes/CC (3), erythrocytes/CC (4), eosinophils/CC (5), healthy cells/CC (6), lymphocytes/CC (7), segmented neutrophils/CC (8), stick neutrophils/CC (9), monocytes/CC (10); leucocytes/CC (11). Correct prediction of 5YS was 100% by neural networks computing (area under ROC curve=1.0; error=0.0).
CONCLUSIONS: 5YS of LCP after radical procedures significantly depended on: 1) PT early-invasive cancer; 2) PT N0--N12; 3) cell ratio factors; 4) blood cell circuit; 5) biochemical factors; 6) hemostasis system; 7) AT; 8) LC characteristics; 9) LC cell dynamics; 10) surgery type: lobectomy/pneumonectomy; 11) anthropometric data. Optimal diagnosis and treatment strategies for LC are: 1) screening and early detection of LC; 2) availability of experienced thoracic surgeons because of complexity of radical procedures; 3) aggressive en block surgery and adequate lymph node dissection for completeness; 4) precise prediction; 5) adjuvant chemoimmunoradiotherapy for LCP with unfavorable prognosis.
Report Back from SGO 2024: What’s the Latest in Cervical Cancer?bkling
Are you curious about what’s new in cervical cancer research or unsure what the findings mean? Join Dr. Emily Ko, a gynecologic oncologist at Penn Medicine, to learn about the latest updates from the Society of Gynecologic Oncology (SGO) 2024 Annual Meeting on Women’s Cancer. Dr. Ko will discuss what the research presented at the conference means for you and answer your questions about the new developments.
Flu Vaccine Alert in Bangalore Karnatakaaddon Scans
As flu season approaches, health officials in Bangalore, Karnataka, are urging residents to get their flu vaccinations. The seasonal flu, while common, can lead to severe health complications, particularly for vulnerable populations such as young children, the elderly, and those with underlying health conditions.
Dr. Vidisha Kumari, a leading epidemiologist in Bangalore, emphasizes the importance of getting vaccinated. "The flu vaccine is our best defense against the influenza virus. It not only protects individuals but also helps prevent the spread of the virus in our communities," he says.
This year, the flu season is expected to coincide with a potential increase in other respiratory illnesses. The Karnataka Health Department has launched an awareness campaign highlighting the significance of flu vaccinations. They have set up multiple vaccination centers across Bangalore, making it convenient for residents to receive their shots.
To encourage widespread vaccination, the government is also collaborating with local schools, workplaces, and community centers to facilitate vaccination drives. Special attention is being given to ensuring that the vaccine is accessible to all, including marginalized communities who may have limited access to healthcare.
Residents are reminded that the flu vaccine is safe and effective. Common side effects are mild and may include soreness at the injection site, mild fever, or muscle aches. These side effects are generally short-lived and far less severe than the flu itself.
Healthcare providers are also stressing the importance of continuing COVID-19 precautions. Wearing masks, practicing good hand hygiene, and maintaining social distancing are still crucial, especially in crowded places.
Protect yourself and your loved ones by getting vaccinated. Together, we can help keep Bangalore healthy and safe this flu season. For more information on vaccination centers and schedules, residents can visit the Karnataka Health Department’s official website or follow their social media pages.
Stay informed, stay safe, and get your flu shot today!
New Directions in Targeted Therapeutic Approaches for Older Adults With Mantl...i3 Health
i3 Health is pleased to make the speaker slides from this activity available for use as a non-accredited self-study or teaching resource.
This slide deck presented by Dr. Kami Maddocks, Professor-Clinical in the Division of Hematology and
Associate Division Director for Ambulatory Operations
The Ohio State University Comprehensive Cancer Center, will provide insight into new directions in targeted therapeutic approaches for older adults with mantle cell lymphoma.
STATEMENT OF NEED
Mantle cell lymphoma (MCL) is a rare, aggressive B-cell non-Hodgkin lymphoma (NHL) accounting for 5% to 7% of all lymphomas. Its prognosis ranges from indolent disease that does not require treatment for years to very aggressive disease, which is associated with poor survival (Silkenstedt et al, 2021). Typically, MCL is diagnosed at advanced stage and in older patients who cannot tolerate intensive therapy (NCCN, 2022). Although recent advances have slightly increased remission rates, recurrence and relapse remain very common, leading to a median overall survival between 3 and 6 years (LLS, 2021). Though there are several effective options, progress is still needed towards establishing an accepted frontline approach for MCL (Castellino et al, 2022). Treatment selection and management of MCL are complicated by the heterogeneity of prognosis, advanced age and comorbidities of patients, and lack of an established standard approach for treatment, making it vital that clinicians be familiar with the latest research and advances in this area. In this activity chaired by Michael Wang, MD, Professor in the Department of Lymphoma & Myeloma at MD Anderson Cancer Center, expert faculty will discuss prognostic factors informing treatment, the promising results of recent trials in new therapeutic approaches, and the implications of treatment resistance in therapeutic selection for MCL.
Target Audience
Hematology/oncology fellows, attending faculty, and other health care professionals involved in the treatment of patients with mantle cell lymphoma (MCL).
Learning Objectives
1.) Identify clinical and biological prognostic factors that can guide treatment decision making for older adults with MCL
2.) Evaluate emerging data on targeted therapeutic approaches for treatment-naive and relapsed/refractory MCL and their applicability to older adults
3.) Assess mechanisms of resistance to targeted therapies for MCL and their implications for treatment selection
These simplified slides by Dr. Sidra Arshad present an overview of the non-respiratory functions of the respiratory tract.
Learning objectives:
1. Enlist the non-respiratory functions of the respiratory tract
2. Briefly explain how these functions are carried out
3. Discuss the significance of dead space
4. Differentiate between minute ventilation and alveolar ventilation
5. Describe the cough and sneeze reflexes
Study Resources:
1. Chapter 39, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 34, Ganong’s Review of Medical Physiology, 26th edition
3. Chapter 17, Human Physiology by Lauralee Sherwood, 9th edition
4. Non-respiratory functions of the lungs https://academic.oup.com/bjaed/article/13/3/98/278874
Title: Sense of Smell
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the primary categories of smells and the concept of odor blindness.
Explain the structure and location of the olfactory membrane and mucosa, including the types and roles of cells involved in olfaction.
Describe the pathway and mechanisms of olfactory signal transmission from the olfactory receptors to the brain.
Illustrate the biochemical cascade triggered by odorant binding to olfactory receptors, including the role of G-proteins and second messengers in generating an action potential.
Identify different types of olfactory disorders such as anosmia, hyposmia, hyperosmia, and dysosmia, including their potential causes.
Key Topics:
Olfactory Genes:
3% of the human genome accounts for olfactory genes.
400 genes for odorant receptors.
Olfactory Membrane:
Located in the superior part of the nasal cavity.
Medially: Folds downward along the superior septum.
Laterally: Folds over the superior turbinate and upper surface of the middle turbinate.
Total surface area: 5-10 square centimeters.
Olfactory Mucosa:
Olfactory Cells: Bipolar nerve cells derived from the CNS (100 million), with 4-25 olfactory cilia per cell.
Sustentacular Cells: Produce mucus and maintain ionic and molecular environment.
Basal Cells: Replace worn-out olfactory cells with an average lifespan of 1-2 months.
Bowman’s Gland: Secretes mucus.
Stimulation of Olfactory Cells:
Odorant dissolves in mucus and attaches to receptors on olfactory cilia.
Involves a cascade effect through G-proteins and second messengers, leading to depolarization and action potential generation in the olfactory nerve.
Quality of a Good Odorant:
Small (3-20 Carbon atoms), volatile, water-soluble, and lipid-soluble.
Facilitated by odorant-binding proteins in mucus.
Membrane Potential and Action Potential:
Resting membrane potential: -55mV.
Action potential frequency in the olfactory nerve increases with odorant strength.
Adaptation Towards the Sense of Smell:
Rapid adaptation within the first second, with further slow adaptation.
Psychological adaptation greater than receptor adaptation, involving feedback inhibition from the central nervous system.
Primary Sensations of Smell:
Camphoraceous, Musky, Floral, Pepperminty, Ethereal, Pungent, Putrid.
Odor Detection Threshold:
Examples: Hydrogen sulfide (0.0005 ppm), Methyl-mercaptan (0.002 ppm).
Some toxic substances are odorless at lethal concentrations.
Characteristics of Smell:
Odor blindness for single substances due to lack of appropriate receptor protein.
Behavioral and emotional influences of smell.
Transmission of Olfactory Signals:
From olfactory cells to glomeruli in the olfactory bulb, involving lateral inhibition.
Primitive, less old, and new olfactory systems with different path
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Ve...kevinkariuki227
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Verified Chapters 1 - 19, Complete Newest Version.pdf
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Verified Chapters 1 - 19, Complete Newest Version.pdf
Ozempic: Preoperative Management of Patients on GLP-1 Receptor Agonists Saeid Safari
Preoperative Management of Patients on GLP-1 Receptor Agonists like Ozempic and Semiglutide
ASA GUIDELINE
NYSORA Guideline
2 Case Reports of Gastric Ultrasound
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micro teaching on communication m.sc nursing.pdfAnurag Sharma
Microteaching is a unique model of practice teaching. It is a viable instrument for the. desired change in the teaching behavior or the behavior potential which, in specified types of real. classroom situations, tends to facilitate the achievement of specified types of objectives.
Explore natural remedies for syphilis treatment in Singapore. Discover alternative therapies, herbal remedies, and lifestyle changes that may complement conventional treatments. Learn about holistic approaches to managing syphilis symptoms and supporting overall health.
9. Backgrounds
Secreted factor Slit2, a member of the family of Slit homolog proteins, is highly
expressed in adipose tissues under the control of PRDM16 and cold exposure.
Slits are best known for their role in axon guidance (Brose and Tessier-Lavigne,
2000) and have previously not been described to have any role in adipose tissue.
10. Backgrounds
AP2-PRDM16 transgenic mice show enhanced “browning” of their subcutaneous adipose
depots, leading to augmented energy expenditure, reduced weight gain on high-fat diet, and
improved glucose and insulin homeostasis (Seale et al., 2011).
The various metabolic benefits of activating brown or beige fat have raised great interest in
the discovery of hormones and secreted proteins that can act on fat tissue locally or
systemically to induce browning.
Interestingly, beige fat development occurs in distinct pockets of cells, consistent with the
possibility of a paracrine regulatory factor at work.
White adipose tissues secrete many proteins factors (adipokines) that influence local and
systemic metabolism, including adipsin, adiponectin, leptin, and TNFα (Blüher and
Mantzoros, 2015 and Rosen and Spiegelman, 2014).
Whether beige adipocytes also secrete factors that can influence the function of fat cells or
other organs has not been well studied.
11. Results
Beige and brown markers: Eva1, Ear2 ( Wu et al., 2012)
Beige-enriched mitochondrial marker: Gatm ( Kazak et al., 2015)
Slit2 Is a Factor Secreted from Beige Adipose Cells
12. Results Slit2 Is a Factor Secreted from Beige Adipose Cells
一、变化倍数 >1.3
1,260 /5,360 proteins ( 2 对 2 )
aP2-PRDM16 by more than >1.3-fold versus the wild-type adipocytes
无血清培养基 , 原代培养 wt 、 aP2-PRDM16 附睾脂肪细胞上清——串联质谱标签
(tandemmasstag,TMT) 检测肽类
二、含有信号肽
presence of a signal peptide in the annotated gene
三、可能受 PRDM16 调节
regulation by PRDM16 in tissues
共 13 个符合要求的前体蛋白
13. Results Slit2 Is a Factor Secreted from Beige Adipose Cells
分析肽的来源:在条件性培养基中检测到来自同一家族来源的多肽片段
14. Results Slit2 Is a Factor Secreted from Beige Adipose Cells
The Slit family in mice and humans comprises three members: Slit1, Slit2, and Slit3.
These are all extracellular matrix proteins of 180–200 kDa with a 29 amino acid signal
peptide for classical secretion.
To investigate the function of the Slit members in fat, we first analyzed their expression
and regulation in various adipose tissues.
15. Results Slit2 Is a Factor Secreted from Beige Adipose Cells
Tissue mRNA expression of Slit2 in 6 weeks C57/b6 mice.
16. Results Slit2 Is a Factor Secreted from Beige Adipose Cells
B-C. Normalized mRNA expression of Slit2, Slit3 and Prdm16 in brown fat tissue (BAT) from (B) aP2-
PRDM16 mice and (C) adipocyte-specific knockout of PRDM16 (prdm16Adipo-KO)
17. Results Slit2 Is a Factor Secreted from Beige Adipose Cells
Normalized mRNA expression of Slit2 and Ucp1 in iWAT, eWAT and BAT after 3 days treatment with daily
injections of CL 316,243 (1 mg/kg).
BAT
18. Results Slit2 Is a Factor Secreted from Beige Adipose Cells
Moreover, the mRNA expression of Slit2, but not Slit3, is also inducible in fat by acute, but
not long-term, cold exposure in BAT and iWAT 。
19. Results Slit2 Is a Factor Secreted from Beige Adipose Cells
The expression of Slit2 is suppressed in iWAT in diet-induced obese (DIO) mice that also
present very low Ucp1 and Adipsin mRNA levels .
Slit2 mRNA is also downregulated in epididymal WAT (eWAT)
20. Results Slit2 Is a Factor Secreted from Beige Adipose Cells
But not in classical BAT , suggesting distinct mechanisms of transcriptional regulation.
Slit2 is induced in inguinal cells upon stimulation with the cyclic AMP-activator forskolin .
21. Results Slit2 Is a Factor Secreted from Beige Adipose Cells
These data point to a physiologic regulation of Slit2 in adipose cells and
tissues and are suggestive of a link between Slit2 and thermogenic function.
Focused on Slit2
22. Results Slit2 Promotes a Thermogenic Program in Cells and in Mice
Other 13 high-priority
candidates (as
commercially available
recombinant proteins) did
not produce a thermogenic
response
Commercial recombinant Slit2
treatment induced an increase of 3-
fold in Ucp1 mRNA, as well as
large increases in expression of
other genes associated with
thermogenesis, including Dio2 and
Cidea 1μg/ml
Western blotting
Slit2 (180 kDa)
Several additional cleavage
products, 50 kDa and 37 kDa
23. Results Slit2 Promotes a Thermogenic Program in Cells and in Mice
Ectopic expression of Slit2 robustly induced a thermogenic gene program, with an 8-fold
increase in Ucp1 mRNA and 2- to 5-fold elevations in Dio2, Elovl3, and Cidea
24. Results Slit2 Promotes a Thermogenic Program in Cells and in Mice
We generated primary adipocytes from Slit2flox/flox mice and deleted both the full-length and the
cleaved 50 kDa form of Slit2, using adenovirus-mediated Cre expression
25. Results Slit2 Promotes a Thermogenic Program in Cells and in Mice
This resulted in a reduction in thermogenic gene expression and expression of mitochondrial
genes in both primary inguinal fat cells and primary brown fat cells
26. Results Slit2 Promotes a Thermogenic Program in Cells and in Mice
In primary brown fat cells, loss of Slit2 results in reduced oxygen consumption .
寡霉素: ATP 合酶抑制剂
FCCP :解偶联剂—线粒体最大耗氧能力
27. To understand the molecular relevance of Slit2 in vivo, we used injection of Cre
recombinase driven by an AAV vector (AAV-8-CRE) for 3 weeks, which reduced
endogenous Slit2 levels in the brown fat by 70%
28. Results Slit2 Promotes a Thermogenic Program in Cells and in Mice
This resulted in a significant reduction in Ucp1 expression and also reductions in expression of
several other mitochondrial genes in this tissue.
Without any difference in weight loss between the groups
29. Results Slit2 Promotes a Thermogenic Program in Cells and in Mice
Together these experiments suggest that Slit2 is
involved in regulation of thermogenic gene
expression in vivo.
30. Results Slit2 Promotes a Thermogenic Program in Cells and in Mice
LacZ or Slit2 by intravenous delivery
of adenoviral vectors to lean mice.
This protocol results in robust
expression and secretion of Slit2 from
the liver .
Multiple Slit2 fragments
secreted into the
circulation, including a
prominent 50 kDa fragment
similar or identical to the
50 kDa band also observed
in cultured cells
No changes in lipolysis
or lipogenesis gene
expression were seen in
the liver
31. Results Slit2 Promotes a Thermogenic Program in Cells and in Mice
. In skeletal muscle, no gene expression changes in
glucose transporters Glut1 and Glut4 or the
inflammatory gene Tnfα were observed
32. Results Slit2 Promotes a Thermogenic Program in Cells and in Mice
Circulating Slit2 induced a
thermogenic gene-expression
program in the iWAT, with a 2.5-
fold induction of Ucp1 in iWAT and
1.5-fold induction of Prdm16
White fat selective genes,
including Leptin and
Resistin, were strongly
suppressed by circulating
Slit2
35. Results Slit2 Promotes a Thermogenic Program in Cells and in Mice
Circulating Slit2 did not change any of the vascular and neuronal markers in fat or in
skeletal muscle
36. Results Slit2 Promotes a Thermogenic Program in Cells and in Mice
Taken together, these results demonstrate that ectopic-expressed
Slit2 in circulation can promote a thermogenic program in cultured
adipocytes and adipose tissues.
37. Results Identification and Characterization of a Slit2 Cleavage Fragment
Western blot: Detected with a Slit2 antibody (left) and an anti-FLAG antibody (right).
Boxed immunoreactive bands were analyzed using mass spectrometry (see Experimental
Procedures).
38. Results Identification and Characterization of a Slit2 Cleavage Fragment
In contrast, peptides identified from the full-length 180 kDa band mapped to all portions of
the Slit2 protein.
Taken together, these results demonstrate that the smaller 50 kDa fragment of Slit2 from fat
cells contains the entire C-terminal region of Slit2. The same or a similar cleavage product
has been observed previously (Nguyen Ba-Charvet et al., 2001) but has no established
function.
39. Results Identification and Characterization of a Slit2 Cleavage Fragment
adenoviral constructs
Slit2-C: a signal peptide for secretion, and a C-terminal V5-tag
Slit2-N: N-terminal portion of Slit2 immediately upstream of the Slit2-C sequence
40. Results Identification and Characterization of a Slit2 Cleavage Fragment
Transduced with LacZ-, Slit2-N-, and Slit2-C-expressing viruses on day 2, and the cells were
harvested on day 6.
Both Slit2-N and Slit2-C proteins were efficiently expressed in adipocytes at the predicted
molecular sizes: 140 kDa and 50 kDa, respectively .
Interestingly, only Slit2-C, but not Slit2-N, was efficiently secreted into the blood following
intravenous delivery of adenoviruses into mice , despite efficient hepatic transduction for both
constructs.
41. Results Identification and Characterization of a Slit2 Cleavage Fragment
Based on these data, we have focused exclusively on the
biological effects of Slit2-C in subsequent experiments in
vitro and in vivo.
42. Results Slit2-C Is Sufficient to Recapitulate the Thermogenic Activity of Full-Length
Slit2
Slit2-C induced a thermogenic gene expression comparable to full-length Slit2 in primary
inguinal cells, while primary brown fat cells responded stronger to Slit2-C
43. Results Slit2-C Is Sufficient to Recapitulate the Thermogenic Activity of Full-Length
Slit2
In the iWAT, Ucp1 mRNA was significantly induced 3-fold, and expression of other
mitochondrial genes also showed a modest but significant 1.5- to 2-fold increase .
The classical brown fat showed significant changes in the transcriptional regulators Prdm16,
Nrf1, and Errα.
In addition, there was also an upregulation of expression of several mitochondrial genes such as
Atp5b, Uqcrb, Atp6v0d2, Atp9b, and Cox5a, suggestive of an activation of BAT.
44. Results Slit2-C Is Sufficient to Recapitulate the Thermogenic Activity of Full-Length
Slit2
In the BAT, UCP1 staining was similar between the
two groups;
however, the tissue in Slit2-C-treated animals had a
more dense-looking appearance with smaller lipid
droplets
Clark electrode
O2 consumption was significantly
elevated in BAT from mice that
received Slit2-C adenovirus versus
LacZ control.
45. Results Increased Circulating Slit2-C Augments Whole-Body Energy Expenditure and
Improves Glucose Homeostasis in Obese Mice
(A) O2 consumption
(B) RER(respiratory exchange ratio ), (C) locomotor activity, (D)
accumulated food intake
Whole-body energy expenditure
Averaged oxygen
consumption at days 5–7
in mice with no
significant difference in
body weight between
the groups.
46. Results Increased Circulating Slit2-C Augments Whole-Body Energy Expenditure and
Improves Glucose Homeostasis in Obese Mice
The elevated whole-body oxygen consumption in the Slit2-C-treated mice was
accompanied by a reduction in the mass of the brown and inguinal, but not
epididymal, depots
47. Results Increased Circulating Slit2-C Augments Whole-Body Energy Expenditure and
Improves Glucose Homeostasis in Obese Mice
Importantly, circulating Slit2-C was found to dramatically improve glucose tolerance in DIO mice
48. Results Slit2-C Induces a Thermogenic Program through the PKA Signaling Pathway in
Adipocytes
Phosphoarrays to identify the intracellular signaling pathways
activated in primary inguinal adipocytes transduced with Slit2-C
versus LacZ adenovirus
Slit2-C fragment 缺少 slit2 的受体 ROBO 结合的结构域;提示存在其他受体。
ErbB 家族的两个蛋白被特异性的磷酸化 , 其含有四个受体酪氨酸激酶,在结构上与表皮
生长因子受体( EGFR )类似。
49. Results Slit2-C Induces a Thermogenic Program through the PKA Signaling Pathway in
Adipocytes
Of the 39 receptor tyrosine kinases and intracellular kinases tested in these initial assays, robust
phosphorylation changes were observed in only two proteins,
phospho-EGFR and phospho-ERK1/2, together with changes in total EGFR upon Slit2-C
overexpression
50. Results Slit2-C Induces a Thermogenic Program through the PKA Signaling Pathway in
Adipocytes
antagonizing the EGFR and ERK pathways with specific inhibitors
51. Results Slit2-C Induces a Thermogenic Program through the PKA Signaling Pathway in
Adipocytes
Failed to reverse the Slit2-C-induced thermogenic gene expression.
These data suggest that the EGFR and ERK pathways are activated by, but not required for,
the thermogenic activity of Slit2-C.
52. Results Slit2-C Induces a Thermogenic Program through the PKA Signaling Pathway in
Adipocytes
We therefore next turned to protein kinase A (PKA) signaling, a pathway known to be involved
in the canonical thermogenic activation of fat cells
To exclude potential intracellular effects of adenoviral overexpression, we also generated
serum-free conditioned media from cells expressing LacZ, Slit2-FL, or Slit2-C.
53. Results Slit2-C Induces a Thermogenic Program through the PKA Signaling Pathway in
Adipocytes
As a comparison, activation of protein kinase C (PKC)
substrates and ATGLS406 by Slit2-C was minimal
54. Results Slit2-C Induces a Thermogenic Program through the PKA Signaling Pathway in
Adipocytes
Under the same conditions, Slit2-C also increases the protein levels of UCP1, confirming the
gene-expression levels upon Slit2-C overexpression .
55. Results Slit2-C Induces a Thermogenic Program through the PKA Signaling Pathway in
Adipocytes
Propranolol, a pan-β-receptor antagonist, did not inhibit Slit2-C-induced thermogenesis,
indicating that β-adrenergic signaling is not required for Slit2-C activity.
However, an inhibitor of PKA (H89) significantly reduced PKA substrate phosphorylation and
also blunted Slit2-C-induced Ucp1 and Dio2 gene expression
56. Results Slit2-C Induces a Thermogenic Program through the PKA Signaling Pathway in
Adipocytes
Similar effects were seen using the adenylyl-cyclase inhibitor SQ-22536, which inhibits the
formation of intracellular cAMP .
Together these data indicate that the generation of cAMP and activation of PKA signaling
are important for the thermogenic activity of Slit2-C.
57. Results Slit2-C Induces a Thermogenic Program through the PKA Signaling Pathway in
Adipocytes
The purity and quantification of the protein
content (compared with an albumin standard of
known concentration) were verified by silver-
stained SDS-gel electrophoresis .
This shows a 50 kDa band as well as
a single FLAG-reactive and Slit2-
reactive band on a western blot
58. Results Slit2-C Induces a Thermogenic Program through the PKA Signaling Pathway in
Adipocytes
Importantly, binding of nanomolar concentrations of purified Slit2-C to the cell surface
on live adipocytes incubated at 4°C was observed, suggesting the presence of a Slit2-C
cell-surface receptor on adipocytes.
59. Results Slit2-C Induces a Thermogenic Program through the PKA Signaling Pathway in
Adipocytes
Importantly, similarly to the virus-overexpression experiments, a subset of PKA-substrate
phosphorylations was increased after Slit2-C protein treatment in a time-dependent and
dose-dependent manner similar to norepinephrine.
primary inguinal cells
60. Results Slit2-C Induces a Thermogenic Program through the PKA Signaling Pathway in
Adipocytes
In contrast with NE,
which induces a full
response by 5 min of
treatment, Slit2-C
induces PKA
phosphorylation at a
slightly delayed time
that peaks around 60
to 90 min (Figures 6L
and S6H)
61. Results Slit2-C Induces a Thermogenic Program through the PKA Signaling Pathway in
Adipocytes
The purified protein also induces subsequent changes in thermogenic gene expression in both
white and brown adipocytes in culture 2 hr after protein treatment.
62. Conclusions
Slit2 is a secreted protein regulated by Prdm16 and by obesity.
Slit2 is posttranslationally cleaved by adipocytes
A circulating Slit2-C fragment improves glucose homeostasis in mice
Slit2-C protein activates a thermogenic PKA pathway in adipocytes
在棕色脂肪组织中:CL刺激3天后,slit2表达略有升高但无统计学意义。,高脂饮食16周,slit2,3未受抑制。
并未得到振奋人心的结果。
This might be explained by a rapid-desensitization mechanism upon long-term activation of cAMP, similar to the transient upregulation of Slit2 mRNA seen upon cold exposur
There was a trend to an increase in Slit2 gene expression in iWAT after 3 days of treatment with the β-adrenergic agonist CL316, 243, but this did not reach statistical significance
相关性
(D) Normalized thermogenic mRNA expression in primary inguinal cells overexpressing Slit2-FL or LacZ.y
原代腹股沟脂肪组织过表达
cAMP效应元件Cre
Nevertheless, we generated primary adipocytes from Slit2flox/flox mice and deleted both the full-length and the cleaved 50 kDa form of Slit2, using adenovirus-mediated Cre expression (Figure 2E).
因为商用抗体的活性并不足够将上清中slit2所有结合纯化。作者用过表达slit2腺病毒蛋白后加了C端标签
Unfortunately, the antibodies commercially available for Slit2 were not effective for immunoaffinity purification of Slit2 from the conditioned media. As an alternative strategy, we generated adenoviruses that express full-length Slit2 with a FLAG tag at the C terminus (Slit2-CTF). Primary inguinal cultures were transduced with Slit2-CTF on day 2, and serum-free conditioned media was collected between days 6 and 7. Western blotting of conditioned media from Slit2-CTF-transduced adipocytes showed secretion of full-length Slit2 (∼180 kDa), as well as fragments corresponding to ∼140 kDa and ∼50 kDa when using an anti-Slit2 antibody
Matched peptides to Slit2-FL or Slit2-C (bold blue) using C-terminal, FLAG-tagged Slit2-overexpression conditioned medium.
Subjected immunoaffinity-purified, FLAG-tagged Slit2-CTF bands to mass spectrometry.
Peptides identified from the 50 kDa FLAG-reactive fragment mapped exclusively to the C terminus of Slit2
in DIO C57/b6 mice under 6 days after injection with LacZ or Slit2-C adenovirus
comprehensive laboratory animal monitoring system (CLAMS)全身实验动物检测系统
SLIT2-C诱发全身耗氧量与呼吸交换率(RER),运动活性,食物摄取量(图5A-5E),或体重(图5G)没有差别。
耗氧量增加伴随着棕色、腹股沟脂肪组织减少,但是附睾脂肪无明显变化
共识
The Slit2-C fragment as defined here completely lacks this ROBO interaction domain, suggesting that other receptors might be involved in signaling from this protein in adipocytes.
CM:conditioned medium
Western blot analysis for PKA-substrate phosphorylation upon acute treatment (30 min) with conditioned medium from cells expressing LacZ, Slit2-FL, or
Slit2-C.