This video encompasses all the steps and data produced for my graduation project in BSc in Biopharmaceutical science. During the course of the project we modified mammalian cells using Short Hairpin RNA to inhibit the correct function of the cytoskelleton. In this way we studied the importance of FMNL2 for the activation and regulation of actin fibers. Among the methods used are Flourescent microscopy, mamallian cell culture, cloning and flow cytometry.
Regulation of pten activity by its carboxyl terminal autoinhibitoryChau Chan Lao
Regulation of PTEN Activity by Its Carboxyl-terminal Autoinhibitory Domain.
Leticia Odriozola, Gobind Singh, Thuong Hoang, and Andrew M. Chan
From the Department of Oncological Sciences, Mount Sinai School of Medicine, New York, New York, 10029
THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. 282, NO. 32, pp. 23306–23315, August 10, 2007
目前已知PTEN(Phosphatase and tensin homolog)是腫瘤抑制蛋白,其由403個氨基酸組成,主要分PTPase及C2 domain,C2 domain使PTEN可與細胞膜作用連結。
PTEN之C-tail(aa 350~403)被發現具有調控PTEN自身活性之功能。前人研究指出C-tail有6個可磷酸化之位置(Thr-366、Ser-370、Ser-380、Thr-382、Thr-383及Ser-385),這些位置可調控PTEN之腫瘤抑制能力、胞內之分佈及穩定性。前人產生以上位置突變之PTEN變異株,發現這些變異株具有更強的腫瘤抑制能力,但穩定性將降低,這可能是因這些變異株具有更開放結構所致。
本報告針對研究PTEN C-tail在連結細胞膜和在其本身催化活性中扮演的功能。作者先產生一系列之PTEN磷酸化位置變異株,發現S385A會促使PTEN之membrane localization in vivo及加強phosphatase活性in vitro,而且此突變會使Ser-380/Thr-382/Thr-383 cluster的磷酸化程度降低,因此知Ser-385可透過被去磷酸化以調控PTEN。而以phosphomimic residues取代Ser-380/Thr-382/Thr-383會使上述S385A所產生之PTEN催化活性反轉。之後利用免疫沉澱方法,發現C-tail之71-amino acid region會與C2 domain上之CBR3 motif作用,暗示C-tail參與連結細胞膜之調控。最後利用合成之PTEN C-tail peptide,發現其可抑制PTEN之催化活性in vitro,而在細胞表現此peptide則會抑制PTEN之membrane localization,磷酸化之Akt量亦上升。以上實驗顯示C-tail在PTEN之membrane recruitment及PTPase活性調控中扮演Autoinhibitory domain角色。
Regulation of pten activity by its carboxyl terminal autoinhibitoryChau Chan Lao
Regulation of PTEN Activity by Its Carboxyl-terminal Autoinhibitory Domain.
Leticia Odriozola, Gobind Singh, Thuong Hoang, and Andrew M. Chan
From the Department of Oncological Sciences, Mount Sinai School of Medicine, New York, New York, 10029
THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. 282, NO. 32, pp. 23306–23315, August 10, 2007
目前已知PTEN(Phosphatase and tensin homolog)是腫瘤抑制蛋白,其由403個氨基酸組成,主要分PTPase及C2 domain,C2 domain使PTEN可與細胞膜作用連結。
PTEN之C-tail(aa 350~403)被發現具有調控PTEN自身活性之功能。前人研究指出C-tail有6個可磷酸化之位置(Thr-366、Ser-370、Ser-380、Thr-382、Thr-383及Ser-385),這些位置可調控PTEN之腫瘤抑制能力、胞內之分佈及穩定性。前人產生以上位置突變之PTEN變異株,發現這些變異株具有更強的腫瘤抑制能力,但穩定性將降低,這可能是因這些變異株具有更開放結構所致。
本報告針對研究PTEN C-tail在連結細胞膜和在其本身催化活性中扮演的功能。作者先產生一系列之PTEN磷酸化位置變異株,發現S385A會促使PTEN之membrane localization in vivo及加強phosphatase活性in vitro,而且此突變會使Ser-380/Thr-382/Thr-383 cluster的磷酸化程度降低,因此知Ser-385可透過被去磷酸化以調控PTEN。而以phosphomimic residues取代Ser-380/Thr-382/Thr-383會使上述S385A所產生之PTEN催化活性反轉。之後利用免疫沉澱方法,發現C-tail之71-amino acid region會與C2 domain上之CBR3 motif作用,暗示C-tail參與連結細胞膜之調控。最後利用合成之PTEN C-tail peptide,發現其可抑制PTEN之催化活性in vitro,而在細胞表現此peptide則會抑制PTEN之membrane localization,磷酸化之Akt量亦上升。以上實驗顯示C-tail在PTEN之membrane recruitment及PTPase活性調控中扮演Autoinhibitory domain角色。
REGULATION OF
GENE EXPRESSION
IN PROKARYOTES & EUKARYOTES .
This presentation is enriched with lots of information of gene expression with many pictures so that anyone can understand gene expression easily.
Gene expression is the process by which the information encoded in a gene is used to direct the assembly of a protein molecule.
Gene expression is explored through a study of protein structure and function, transcription and translation, differentiation and stem cells.
It is the process by which information from a gene is used in the synthesis of a functional gene product.
These products are often proteins, but in non-protein coding genes such as ribosomal RNA (rRNA), transfer RNA (tRNA) or small nuclear RNA (snRNA) genes, the product is a functional RNA.
The process of gene expression is used by all known life - eukaryotes (including multicellular organisms), prokaryotes (bacteria and archaea)
Regulation of gene expression:
Regulation of gene expression includes a wide range of mechanisms that are used by cells to increase or decrease the production of specific gene products (protein or RNA).
Gene regulation is essential for viruses, prokaryotes and eukaryotes as it increases the versatility and adaptability of an organism by allowing the cell to express protein when needed.
CLASSIFICATION OF GENE WITH RESPECT TO THEIR EXPRESSION:
Constitutive ( house keeping) genes:
Are expressed at a fixed rate, irrespective to the cell condition.
Their structure is simpler.
Controllable genes:
Are expressed only as needed. Their amount may increase or decrease with respect to their basal level in different condition.
Their structure is relatively complicated with some response elements.
TYPES OF REGULATION OF GENE:
positive & negative regulation.
Steps involving gene regulation of prokaryotes & eukaryotes.
Operon-structure,classification of mechanisms- lac operon,tryptophan operon ,
and many things related to gene expression.
This is a video slide so anyone can understand this topic easily by seeing pictures included in this slide.
A new effector pathway links ATM kinase with the DNA damage responseCostas Demonacos
The related kinases ATM (ataxia-telangiectasia mutated) and ATR (ataxia-telangiectasia and Rad3-related) phosphorylate a limited number of downstream protein targets in response to DNA damage. Here we report a new pathway in which ATM kinase signals the DNA damage response by targeting the transcriptional cofactor Strap. ATM phosphorylates Strap at a serine residue, stabilizing nuclear Strap and facilitating formation of a stress-responsive co-activator complex. Strap activity enhances p53 acetylation, and augments the response to DNA damage. Strap remains localized in the cytoplasm in cells derived from ataxia telangiectasia individuals with defective ATM, as well as in cells expressing a Strap mutant that cannot be phosphorylated by ATM. Targeting Strap to the nucleus reinstates protein stabilization and activates the DNA damage response. These results indicate that the nuclear accumulation of Strap is a critical regulator in the damage response, and argue that this function can be assigned to ATM through the DNA damage-dependent phosphorylation of Strap.
1 At least 2 questions from this section will be on the .docxmercysuttle
1
At least 2 questions from this section will be on the final exam
SAMPLE QUESTIONS FOR THE FINAL EXAM
Question 1. Ferritin is a protein involved in the storage of iron inside cells. To prevent toxic accumulation of
too much iron inside cells, the intracellular level of ferritin is tightly regulated. To study the regulation of
ferritin synthesis, mammalian cells are grown with or without iron in the culture medium. Note that iron in the
culture medium is rapidly transported inside cells.
a) Upon addition of iron to the culture medium, the intracellular concentration of ferritin mRNA is unchanged
but the concentration of ferritin protein increases. How do you think ferritin expression is regulated? Briefly
explain.
The regulatory sequence given below is found in the ferritin mRNA between the cap structure and the start
codon.
5’-GGGUUUCCGUUCAACAGUGCUUGGACGGAAACCC-3’
Mutations within in this sequence are used to study the regulation of ferritin expression. The following
observation are made:
• ferritin expression is high, independent of the iron concentration, when (i) the entire region is deleted, or
(ii) the region located upstream of the underlined sequence is deleted or (iii) the underlined sequence is
replaced with a random sequence.
• ferritin expression remains iron-dependent when this region is replaced by the following sequence:
5’-GGGCUCAGGUUCAACAGUGCUUGGACCUGAGCCC-3’.
Note that the sequence differences are indicated in bold.
b) Explain why these observations suggest that both sequence and structure of the 5’ end of ferritin mRNA are
important for the regulation of ferritin expression.
c) Ferritin translation becomes iron-independent when the regulatory sequence is moved from the 5’ side
(upstream of the open reading frame) to the 3’ side (downstream of the open reading frame) of ferritin mRNA.
Which step of ferritin translation do you think is affected by the intracellular level of iron?
d) IRP is a protein involved in the regulation of ferritin expression. Anti-IRP antibodies attached to sepharose
beads are added to a cell extract, then the extract is centrifuged to separate the pellet fraction (containing the
sepharose beads ) from the supernatant fraction.
If the cells are cultured in the absence of iron, ferritin mRNA is found together with IRP in the pellet. In
contrast when cells are cultured in the presence of iron ferritin mRNA remains in the supernatant fraction while
IRP alone is found in the pellet. Briefly explain the likely role of IRP in the regulation of ferritin expression.
Question 2. You are studying the development of a newly discovered insect. Like drosophila, it undergoes a
stage in early larval development where the eve gene is expressed in a pattern of 7 stripes. You are particularly
interested in stripes 2 and 5. The following figures show the organization of the cis-acting elements that control
the expression o ...
REGULATION OF
GENE EXPRESSION
IN PROKARYOTES & EUKARYOTES .
This presentation is enriched with lots of information of gene expression with many pictures so that anyone can understand gene expression easily.
Gene expression is the process by which the information encoded in a gene is used to direct the assembly of a protein molecule.
Gene expression is explored through a study of protein structure and function, transcription and translation, differentiation and stem cells.
It is the process by which information from a gene is used in the synthesis of a functional gene product.
These products are often proteins, but in non-protein coding genes such as ribosomal RNA (rRNA), transfer RNA (tRNA) or small nuclear RNA (snRNA) genes, the product is a functional RNA.
The process of gene expression is used by all known life - eukaryotes (including multicellular organisms), prokaryotes (bacteria and archaea)
Regulation of gene expression:
Regulation of gene expression includes a wide range of mechanisms that are used by cells to increase or decrease the production of specific gene products (protein or RNA).
Gene regulation is essential for viruses, prokaryotes and eukaryotes as it increases the versatility and adaptability of an organism by allowing the cell to express protein when needed.
CLASSIFICATION OF GENE WITH RESPECT TO THEIR EXPRESSION:
Constitutive ( house keeping) genes:
Are expressed at a fixed rate, irrespective to the cell condition.
Their structure is simpler.
Controllable genes:
Are expressed only as needed. Their amount may increase or decrease with respect to their basal level in different condition.
Their structure is relatively complicated with some response elements.
TYPES OF REGULATION OF GENE:
positive & negative regulation.
Steps involving gene regulation of prokaryotes & eukaryotes.
Operon-structure,classification of mechanisms- lac operon,tryptophan operon ,
and many things related to gene expression.
This is a video slide so anyone can understand this topic easily by seeing pictures included in this slide.
A new effector pathway links ATM kinase with the DNA damage responseCostas Demonacos
The related kinases ATM (ataxia-telangiectasia mutated) and ATR (ataxia-telangiectasia and Rad3-related) phosphorylate a limited number of downstream protein targets in response to DNA damage. Here we report a new pathway in which ATM kinase signals the DNA damage response by targeting the transcriptional cofactor Strap. ATM phosphorylates Strap at a serine residue, stabilizing nuclear Strap and facilitating formation of a stress-responsive co-activator complex. Strap activity enhances p53 acetylation, and augments the response to DNA damage. Strap remains localized in the cytoplasm in cells derived from ataxia telangiectasia individuals with defective ATM, as well as in cells expressing a Strap mutant that cannot be phosphorylated by ATM. Targeting Strap to the nucleus reinstates protein stabilization and activates the DNA damage response. These results indicate that the nuclear accumulation of Strap is a critical regulator in the damage response, and argue that this function can be assigned to ATM through the DNA damage-dependent phosphorylation of Strap.
1 At least 2 questions from this section will be on the .docxmercysuttle
1
At least 2 questions from this section will be on the final exam
SAMPLE QUESTIONS FOR THE FINAL EXAM
Question 1. Ferritin is a protein involved in the storage of iron inside cells. To prevent toxic accumulation of
too much iron inside cells, the intracellular level of ferritin is tightly regulated. To study the regulation of
ferritin synthesis, mammalian cells are grown with or without iron in the culture medium. Note that iron in the
culture medium is rapidly transported inside cells.
a) Upon addition of iron to the culture medium, the intracellular concentration of ferritin mRNA is unchanged
but the concentration of ferritin protein increases. How do you think ferritin expression is regulated? Briefly
explain.
The regulatory sequence given below is found in the ferritin mRNA between the cap structure and the start
codon.
5’-GGGUUUCCGUUCAACAGUGCUUGGACGGAAACCC-3’
Mutations within in this sequence are used to study the regulation of ferritin expression. The following
observation are made:
• ferritin expression is high, independent of the iron concentration, when (i) the entire region is deleted, or
(ii) the region located upstream of the underlined sequence is deleted or (iii) the underlined sequence is
replaced with a random sequence.
• ferritin expression remains iron-dependent when this region is replaced by the following sequence:
5’-GGGCUCAGGUUCAACAGUGCUUGGACCUGAGCCC-3’.
Note that the sequence differences are indicated in bold.
b) Explain why these observations suggest that both sequence and structure of the 5’ end of ferritin mRNA are
important for the regulation of ferritin expression.
c) Ferritin translation becomes iron-independent when the regulatory sequence is moved from the 5’ side
(upstream of the open reading frame) to the 3’ side (downstream of the open reading frame) of ferritin mRNA.
Which step of ferritin translation do you think is affected by the intracellular level of iron?
d) IRP is a protein involved in the regulation of ferritin expression. Anti-IRP antibodies attached to sepharose
beads are added to a cell extract, then the extract is centrifuged to separate the pellet fraction (containing the
sepharose beads ) from the supernatant fraction.
If the cells are cultured in the absence of iron, ferritin mRNA is found together with IRP in the pellet. In
contrast when cells are cultured in the presence of iron ferritin mRNA remains in the supernatant fraction while
IRP alone is found in the pellet. Briefly explain the likely role of IRP in the regulation of ferritin expression.
Question 2. You are studying the development of a newly discovered insect. Like drosophila, it undergoes a
stage in early larval development where the eve gene is expressed in a pattern of 7 stripes. You are particularly
interested in stripes 2 and 5. The following figures show the organization of the cis-acting elements that control
the expression o ...
The Matrix metalloproteinase-9 is involved in several pathologies. Its strong presence in ocular pathologies explains our interest for its genetic variation in cataract, glaucoma and retinoblastoma in Senegal. MMP9 is highly polymorphic with cataract and glaucoma. 77 mutations were noted with 21 haplotypes for the entire population. The haplotype diversity Hd is 0.831 and the nucleotide diversity Pi is 0.016; k = 17.395. The polymorphism of the Matrix metalloproteinase-9 gene is associated with all three diseases and SNP 3918249 is found in both cataract and glaucoma.
DNA damage repair Neil3 gene Knockout in MOLT-4iosrjce
RNAi is superannuated cellular mechanism that protect organism against viruses that replicate
through double- stranded RNA. RNAi can be used to diminish gene expression from plasmid expressing and
inserted sequence repeat. A stable harpin would be expressed after the vector was integrated into the genome.
In this paper a shiRNA expressing vector for Neil3 was designed and developed which is capable of replication
in MOLT-4. This shiRNA vector had the ability to arose the RNAi pathway, and reduce the gene expression of
Neil3. This was assessed by using pSilence 4.1CMV as a vector, and Gapdh as positive control.
In this research paper from the Spring 2015 semester, I described my analysis of certain genome scaffolds, or gaps within the Malaclemys terrapin genome. I examined seven of these scaffolds and determined their approximate sizes through Polymerase Chain Reaction (PCR) and Gel Electrophoresis. The DNA was then prepped to be sent for sequencing by an external source. The resulting chromatograms gave inconclusive results on the exact sequences of these scaffolds.
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
Ethanol (CH3CH2OH), or beverage alcohol, is a two-carbon alcohol
that is rapidly distributed in the body and brain. Ethanol alters many
neurochemical systems and has rewarding and addictive properties. It
is the oldest recreational drug and likely contributes to more morbidity,
mortality, and public health costs than all illicit drugs combined. The
5th edition of the Diagnostic and Statistical Manual of Mental Disorders
(DSM-5) integrates alcohol abuse and alcohol dependence into a single
disorder called alcohol use disorder (AUD), with mild, moderate,
and severe subclassifications (American Psychiatric Association, 2013).
In the DSM-5, all types of substance abuse and dependence have been
combined into a single substance use disorder (SUD) on a continuum
from mild to severe. A diagnosis of AUD requires that at least two of
the 11 DSM-5 behaviors be present within a 12-month period (mild
AUD: 2–3 criteria; moderate AUD: 4–5 criteria; severe AUD: 6–11 criteria).
The four main behavioral effects of AUD are impaired control over
drinking, negative social consequences, risky use, and altered physiological
effects (tolerance, withdrawal). This chapter presents an overview
of the prevalence and harmful consequences of AUD in the U.S.,
the systemic nature of the disease, neurocircuitry and stages of AUD,
comorbidities, fetal alcohol spectrum disorders, genetic risk factors, and
pharmacotherapies for AUD.
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
ARTIFICIAL INTELLIGENCE IN HEALTHCARE.pdfAnujkumaranit
Artificial intelligence (AI) refers to the simulation of human intelligence processes by machines, especially computer systems. It encompasses tasks such as learning, reasoning, problem-solving, perception, and language understanding. AI technologies are revolutionizing various fields, from healthcare to finance, by enabling machines to perform tasks that typically require human intelligence.
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.
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We specializes in exporting high quality Research chemical, medical intermediate, Pharmaceutical chemicals and so on. Products are exported to USA, Canada, France, Korea, Japan,Russia, Southeast Asia and other countries.
MANAGEMENT OF ATRIOVENTRICULAR CONDUCTION BLOCK.pdfJim Jacob Roy
Cardiac conduction defects can occur due to various causes.
Atrioventricular conduction blocks ( AV blocks ) are classified into 3 types.
This document describes the acute management of AV block.
Prix Galien International 2024 Forum ProgramLevi Shapiro
June 20, 2024, Prix Galien International and Jerusalem Ethics Forum in ROME. Detailed agenda including panels:
- ADVANCES IN CARDIOLOGY: A NEW PARADIGM IS COMING
- WOMEN’S HEALTH: FERTILITY PRESERVATION
- WHAT’S NEW IN THE TREATMENT OF INFECTIOUS,
ONCOLOGICAL AND INFLAMMATORY SKIN DISEASES?
- ARTIFICIAL INTELLIGENCE AND ETHICS
- GENE THERAPY
- BEYOND BORDERS: GLOBAL INITIATIVES FOR DEMOCRATIZING LIFE SCIENCE TECHNOLOGIES AND PROMOTING ACCESS TO HEALTHCARE
- ETHICAL CHALLENGES IN LIFE SCIENCES
- Prix Galien International Awards Ceremony
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
Anti ulcer drugs and their Advance pharmacology ||
Anti-ulcer drugs are medications used to prevent and treat ulcers in the stomach and upper part of the small intestine (duodenal ulcers). These ulcers are often caused by an imbalance between stomach acid and the mucosal lining, which protects the stomach lining.
||Scope: Overview of various classes of anti-ulcer drugs, their mechanisms of action, indications, side effects, and clinical considerations.
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
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!
2. Introduction - Actin Cytoskeleton Remodelling
2
Actin filamentation is important for the formation filopodia
and lamellipodia (Breitsprecher & Goode, 2013).
Formation of these cellular protrusions is very organized
and highly controlled. Regulation involves GTPases, the
Arp ⅔ complex and formin proteins (Grobe,
Wüstenhagen, Baarlink, Grosse, & Grikscheit, 2018).
Understanding mechanisms surrounding actin
cytoskeleton modelling has important applications in
understanding cell differentiation and cancer metastasis
(Grikscheit, Frank, Wang, & Grosse, 2015).
Research gate
(“The different facets of actin cytoskeleton remodeling in migrating
T... | Download Scientific Diagram,” n.d.)
3. Introduction - FMNL2 Function in Actin Remodelling
The Formin-related proteins are the largest group of Rho
GTPase effectors (Grikscheit, Frank, Wang, & Grosse,
2015).
- What are their functions?
Formin-like 2 (FMNL2) is a member of the
diaphanous-related formins (DRFs). FMNL2 has a critical
role in junctional actin assembly in cell-cell contact formation
(Péladeau, Heibein, Maltez, Copeland, & Copeland, 2016).
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4. Introduction - RNAi for Gene Suppression
RNA interference is a regulatory process that
controls post-transcriptional gene silencing.
Vector-based short hairpin RNA (shRNA) is a type
of RNAi useful for studying gene functions by knock
down of cytosolic mRNA (Moore, Guthrie, Huang, &
Taxman, 2010).
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(“shRNA Process and shRNA Diagram | Sigma-Aldrich,” n.d.)
5. Introduction - Research Goal
The goal of this semester's research project was to generate an shRNA specific for formin-like 2 (FMNL2)
and to observe its impact on gene expression.
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6. Result - Which temperature is Better?
Fig 1. PCR products from primer specificity evaluation.
Five different PCR reactions and a negative control were used to study the effect of temperature in PCR amplification as well as the
specificity of the primer; 1% agarose gel was stained with ethidium bromide to visualize the DNA lanes. The temperature for each
PCR is stated on top of each lane. All lanes show a specific product of 330 bp long.
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Ladder
68.9°C
50.0 °C
54.1°C
58.8°C
62.7°C
400bp
300bp
ShRNA
7. Result - Bacterial Transformation with pDrive.
Blue white screening was used to confirm the transformation success of one colony. Ω number of colonies with no insert, which
appears blue in culture; α number of colonies with insert, which appeared white in culture; λ ratio between α/ Ω. λ was calculate by
dividing Ω over α. The bacteria were transformed using recombinant p Drive vector.
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8. Fig 2. Screening pDrive vector for presence of shRNA-expressing construct.
After a dirty digest of 6 white colonies, the vector pDrive was screened for the presence of an shRNA-expressing DNA construct. The digest
products were analyzed by of ethidium bromide stained gel electrophoresis. (A) The results of group W1. (B) Digestion results borrowed from
group W3. First a 10 kb DNA ladder was loaded to the gel, followed by six DNA amplicon digestion experiments isolated from bacterial
colonies. 3 of the DNA amplicons were present in pDrive, bands present at roughly 400 bp.
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Result - Was DNA Construct Successfully Inserted into pDrive?
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Fig 3. Densitometry results of for DNA construct and vector concentration.
Densitometry was used to determine concentration of DNA insert and pEGFP-C3 vector. Insert and vector products were analyzed by ethidium
bromide stained gel electrophoresis. Concentration was measured using AlphaView by comparing band intensity to the ladder. A) The DNA insert
is located on the gel at roughly 400 bp. Vector is not present in panel A as it was not loaded. (B) Results of ethidium bromide stained gel
electrophoresis of ligation of digested DNA construct into pEGFP-C3 vector. Y corresponds to the DNA construct of roughly 400 bp. The vector
and 1/3 diluted vector can be visualized at approximately 5000 bp. Results were borrowed from group W8. The results were quantitatively
measured using the AlphaView, comparing the DNA ladder to the intensity of the bands.
400 Bp
5000 Bp
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Result - Determination of DNA Concentration for Ligation
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DNA Origin DNA Mass (ng)
Insert 7.47
Vector 26.6
Diluted Vector
3X
8.86
10. Result - Bacterial Transformation of pEGFP-C3
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Vector (CFU) Vector + Insert (CFU) Ratio of Vector to Vector+Insert
38 36 1:1
Table 2. Yield of DNA construct insertion into vector during transformation.
Two plates containing TOP 10 E. coli cells were transformed using the pEGFP-C3 vector. Presented in the table are the
colony counts. Ω is a ratio of the vector to vector plus ratio. Being calculated as vector+insert/ vector, ratio was rounded to the
nearest whole number. Cells were plated on agar plates containing kanamycin as a selective plate. The DNA insert was a
specific shRNA for FMNL2.
11. Result - Which colonies were transformed?
Fig 4. PCR screening of transformed cells with vector pEGFP-C3 containing ShRNA construct.
A transformation reaction was made using vector with specific ShRNA against FMNL2. LB Bacterial broth was used to culture six random
colonies from plate where the transformed cells where grown. After PCR, the products where visualized in a 1% agarose gel electrophoresis
stained with ethidium bromide.
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Ns
Sp
TL FM
Fig 5. Microscopy examination of P19 transfected cells for EGFP expression.
Two P19 cell cultures where transfected with DNA constructs, one with shRNA specific (Sp) for FMNL2 and a second DNA construct with a
non-specific (Ns) shRNA. Both DNA constructs contained shRNA and EGFP. Transfected cells were visualized and analyzed using transmitted
light (TL) and fluorescent microscopy (FM).
Result - Were the Cells Successfully Transfected with pEGFP-C3?
13. Results - Flow cytometry
Fig 6. Flow cytometry histogram of Non specific construct and specific construct.
A culture of P19 transfected cells, with vectors containing DNA sequence to transcribe ShRNA against FMNL2 RNA. Non-specific ShRNA was used
as a control. The X-axis represents the logarithm of EGFP fluorescence intensity from individual cells; 100
is the threshold to discriminate between
background fluorescence and transfected cells expressing EGFP. The percentage represents the total count which were considered successfully
transfected cells. 13
13.81% 12.60%
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Fig 7. RT-PCR for semi-quantitative analysis of FMNL2 expression.
RT-PCR was used to semi-quantitatively measure the expression of FMNL2 under specific and non-specific shRNA regulation in P19 cell lines.
The PCR products were analyzed using ethidium bromide stained gel electrophoresis. (A) Gel electrophoresis results of group W1. (B) Gel
electrophoresis results of group W7. RT-PCR was used to semi-quantitatively measure expression of FMNL2 in specific (Sp) and non-specific (Ns)
shRNA experiments. A negative control without a cDNA construct was also added, as well as a positive control containing a prepared cDNA.
FMNL2 expression was visualized between 600 and 700 bp, while GAPDH expression was measured as a loading control at roughly 300 bp.
Ratio of non-specific vs specific calculated using W7 at 25 cycles, standard deviation (std) generated using W2, W4, W7, W8 and W10 results.
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Sample FMNL2/GAPDH
Non specific 1
Specific ± std 0.89 ± 0.15
A. B.
15. Discussion - Summary
Was it possible to interfere FMNL2 RNA with a ShRNA construct, using a PCR-based strategy ?
● High primer specificity
● Transfection efficiency = 12.6%
● Decrease in FMNL2 RNA = 11%
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16. Discussion - Related Research
Average transfection efficiency for P19 cells.
● 30 to 50 % transfection efficiency (Yu et al, 2005)
● Our transfection efficiency 12.6 %.
Decrease in expression of FMNL2
● Knockdown 87.13 % of FMNL2 in TIME cells(Mumal I., 2016)
● Our expression decrease is 11%.
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17. Discussion - Future Research
Rescue experiment
○ Supplement cells with exogenous FMNL2
○ Is there some other protein working with FMNL2
More in depth into altered morphology of cell contact.
○ Fluorescence microscopy
○ Polymer specific dye
17
18. References
Breitsprecher, D., & Goode, B. L. (2013, January 1). Formins at a glance. Journal of Cell Science, Vol. 126, pp. 1–7. https://doi.org/10.1242/jcs.107250
FMNL2 formin like 2 [Homo sapiens (human)] - Gene - NCBI. (n.d.). Retrieved November 14, 2019, from https://www.ncbi.nlm.nih.gov/gene/114793
Grikscheit, K., Frank, T., Wang, Y., & Grosse, R. (2015). Junctional actin assembly is mediated by Formin-like 2 downstream of Rac1. Journal of Cell Biology, 209(3), 367–376. https://doi.org/10.1083/jcb.201412015
Grobe, H., Wüstenhagen, A., Baarlink, C., Grosse, R., & Grikscheit, K. (2018). A Rac1-FMNL2 signaling module affects cellcell contact formation independent of Cdc42 and membrane protrusions. PLoS ONE, 13(3).
https://doi.org/10.1371/journal.pone.0194716
Kühn, S. ;, Erdmann, ;, Kage, F. ;, Block, J. ;, Schwenkmezger, L. ;, Steffen, A. ;, … Geyer, M. (2015). The structure of FMNL2-Cdc42 yields insights into the mechanism of lamellipodia and filopodia formation. Item type Article.
Nature Communications. https://doi.org/10.1038/ncomms8088
Moore, C. B., Guthrie, E. H., Huang, M. T. H., & Taxman, D. J. (2010). Short hairpin RNA (shRNA): design, delivery, and assessment of gene knockdown. Methods in Molecular Biology (Clifton, N.J.), 629, 141–158.
https://doi.org/10.1007/978-1-60761-657-3_10
Mumal I. (2016) The Role of Formins in Endothelial Adherens Junction Regulation( Master dissertation) University of Ottawa, Ottawa, Canada
dx.dio.org/10.20381/ruor-5310
Péladeau, C., Heibein, A., Maltez, M. T., Copeland, S. J., & Copeland, J. W. (2016). A specific FMNL2 isoform is up-regulated in invasive cells. BMC Cell Biology, 17(1). https://doi.org/10.1186/s12860-016-0110-z
shRNA Process and shRNA Diagram | Sigma-Aldrich. (n.d.). Retrieved November 16, 2019, from https://www.sigmaaldrich.com/life-science/functional-genomics-and-rnai/shrna/library-information/shrna-process-diagram.html
The different facets of actin cytoskeleton remodeling in migrating T... | Download Scientific Diagram. (n.d.). Retrieved November 18, 2019, from
https://www.researchgate.net/figure/The-different-facets-of-actin-cytoskeleton-remodeling-in-migrating-T-cells-Represented_fig3_284228226
Yu et al, (2005) Use of Short Hairpin RNA Expression Vectors to Study Mammalian Neural Development. Methods in Enzymology, Academic Press, 392, 86-199.
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