1. The document reports on a study investigating the role of insulin signaling in pancreatic β cells using Cre-loxP mediated recombination to generate βIRKO mice lacking the insulin receptor in β cells.
2. Results showed βIRKO mice exhibited impaired glucose tolerance and insulin secretion compared to controls. Expression of genes CENP-A and PLK1 were decreased in βIRKO cells.
3. Further experiments demonstrated insulin stimulates FoxM1 binding to the CENP-A and PLK1 genes, and CENP-A expression is reduced in type 2 diabetes patient islets. Knockout of CENP-A in β cells decreased proliferation.
Transcription factors of the nuclear factor κ B family are the paradigm for signaling dependent nuclear translocation and are ideally suited to analysis through image-based chemical genetic screening. The authors describe combining high-content image analysis with a compound screen to identify compounds affecting either nuclear import or export. Validation in silico and in vitro determined an EC50 for the nuclear export blocker leptomycin B of 2.4 ng/mL (4.4 nM). The method demonstrated high selectivity (Z′ >0.95), speed, and robustness in a screen of a compound collection. It identified the IκB protein kinase inhibitor BAY 11 7082 as an import inhibitor, the p38 mitogen-activated protein (MAP) kinase inhibitor PD98509 as an import enhancer, and phorbol ester as an export inhibitor. The results establish a robust method for identifying compounds regulating nucleocy- toplasmic import or export and also implicate MAP kinases in nuclear import of nuclear factor κ B
Transcription factors of the nuclear factor κ B family are the paradigm for signaling dependent nuclear translocation and are ideally suited to analysis through image-based chemical genetic screening. The authors describe combining high-content image analysis with a compound screen to identify compounds affecting either nuclear import or export. Validation in silico and in vitro determined an EC50 for the nuclear export blocker leptomycin B of 2.4 ng/mL (4.4 nM). The method demonstrated high selectivity (Z′ >0.95), speed, and robustness in a screen of a compound collection. It identified the IκB protein kinase inhibitor BAY 11 7082 as an import inhibitor, the p38 mitogen-activated protein (MAP) kinase inhibitor PD98509 as an import enhancer, and phorbol ester as an export inhibitor. The results establish a robust method for identifying compounds regulating nucleocy- toplasmic import or export and also implicate MAP kinases in nuclear import of nuclear factor κ B
Oncology: Spatial Localization of Ras proteinsNachiket Vartak
This is a presentation of work done at the MPI Dortmund from 2008-2013 on the mechanism through with localization of the Ras protein in generated in cells. It presents the inhibiton Palmostatin-B, which inhibits this mechanism, leading to reveral of oncogenic signaling and cancerous phenotypes.
Transgenic animal production and its applicationkishoreGupta17
A genetically modified animal with the heterologous gene of interest being inserted for the purpose of biopharming or make a diseased model to study the consequences of disease and its probable therapy
Toward Precision Medicine in Neurological Disease by David GoldsteinKnome_Inc
View the webinar at http://www.knome.com/webinar-toward-precision-medicine-neurological-disease. In this presentation, Dr. Goldstein reports progress in identifying pathogenic mutations large-scale scale studies in epilepsy, in particular focusing on identifying de novo mutations as a cause of the epileptic encephalopathies. Next he discusses how sequencing is being used to diagnose rare serious unresolved genetic conditions. Finally, Dr. Goldstein describes a number of examples in which a secure genetic diagnosis has led directly to a change in clinical management.
Kaempferol increases levels of coenzyme Q in kidney cells and serves as a biosynthetic ring precursor
Complete study available in Free Radical Biology and Medicine. 2017 Sep;110:176-187.
doi: 10.1016/j.freeradbiomed.2017.06.006. Epub 2017 Jun 9.
Intracellular Traffic and Sorting of ProteinsASHIKH SEETHY
Describes intra-cellular trafficking of proteins, protein sorting, clinical aspects of protein targeting, and vesicle transport.
Download and view in slide show mode for better viewing.
Oncology: Spatial Localization of Ras proteinsNachiket Vartak
This is a presentation of work done at the MPI Dortmund from 2008-2013 on the mechanism through with localization of the Ras protein in generated in cells. It presents the inhibiton Palmostatin-B, which inhibits this mechanism, leading to reveral of oncogenic signaling and cancerous phenotypes.
Transgenic animal production and its applicationkishoreGupta17
A genetically modified animal with the heterologous gene of interest being inserted for the purpose of biopharming or make a diseased model to study the consequences of disease and its probable therapy
Toward Precision Medicine in Neurological Disease by David GoldsteinKnome_Inc
View the webinar at http://www.knome.com/webinar-toward-precision-medicine-neurological-disease. In this presentation, Dr. Goldstein reports progress in identifying pathogenic mutations large-scale scale studies in epilepsy, in particular focusing on identifying de novo mutations as a cause of the epileptic encephalopathies. Next he discusses how sequencing is being used to diagnose rare serious unresolved genetic conditions. Finally, Dr. Goldstein describes a number of examples in which a secure genetic diagnosis has led directly to a change in clinical management.
Kaempferol increases levels of coenzyme Q in kidney cells and serves as a biosynthetic ring precursor
Complete study available in Free Radical Biology and Medicine. 2017 Sep;110:176-187.
doi: 10.1016/j.freeradbiomed.2017.06.006. Epub 2017 Jun 9.
Intracellular Traffic and Sorting of ProteinsASHIKH SEETHY
Describes intra-cellular trafficking of proteins, protein sorting, clinical aspects of protein targeting, and vesicle transport.
Download and view in slide show mode for better viewing.
APNIC Foundation, presented by Ellisha Heppner at the PNG DNS Forum 2024APNIC
Ellisha Heppner, Grant Management Lead, presented an update on APNIC Foundation to the PNG DNS Forum held from 6 to 10 May, 2024 in Port Moresby, Papua New Guinea.
1.Wireless Communication System_Wireless communication is a broad term that i...JeyaPerumal1
Wireless communication involves the transmission of information over a distance without the help of wires, cables or any other forms of electrical conductors.
Wireless communication is a broad term that incorporates all procedures and forms of connecting and communicating between two or more devices using a wireless signal through wireless communication technologies and devices.
Features of Wireless Communication
The evolution of wireless technology has brought many advancements with its effective features.
The transmitted distance can be anywhere between a few meters (for example, a television's remote control) and thousands of kilometers (for example, radio communication).
Wireless communication can be used for cellular telephony, wireless access to the internet, wireless home networking, and so on.
This 7-second Brain Wave Ritual Attracts Money To You.!nirahealhty
Discover the power of a simple 7-second brain wave ritual that can attract wealth and abundance into your life. By tapping into specific brain frequencies, this technique helps you manifest financial success effortlessly. Ready to transform your financial future? Try this powerful ritual and start attracting money today!
Multi-cluster Kubernetes Networking- Patterns, Projects and GuidelinesSanjeev Rampal
Talk presented at Kubernetes Community Day, New York, May 2024.
Technical summary of Multi-Cluster Kubernetes Networking architectures with focus on 4 key topics.
1) Key patterns for Multi-cluster architectures
2) Architectural comparison of several OSS/ CNCF projects to address these patterns
3) Evolution trends for the APIs of these projects
4) Some design recommendations & guidelines for adopting/ deploying these solutions.
# Internet Security: Safeguarding Your Digital World
In the contemporary digital age, the internet is a cornerstone of our daily lives. It connects us to vast amounts of information, provides platforms for communication, enables commerce, and offers endless entertainment. However, with these conveniences come significant security challenges. Internet security is essential to protect our digital identities, sensitive data, and overall online experience. This comprehensive guide explores the multifaceted world of internet security, providing insights into its importance, common threats, and effective strategies to safeguard your digital world.
## Understanding Internet Security
Internet security encompasses the measures and protocols used to protect information, devices, and networks from unauthorized access, attacks, and damage. It involves a wide range of practices designed to safeguard data confidentiality, integrity, and availability. Effective internet security is crucial for individuals, businesses, and governments alike, as cyber threats continue to evolve in complexity and scale.
### Key Components of Internet Security
1. **Confidentiality**: Ensuring that information is accessible only to those authorized to access it.
2. **Integrity**: Protecting information from being altered or tampered with by unauthorized parties.
3. **Availability**: Ensuring that authorized users have reliable access to information and resources when needed.
## Common Internet Security Threats
Cyber threats are numerous and constantly evolving. Understanding these threats is the first step in protecting against them. Some of the most common internet security threats include:
### Malware
Malware, or malicious software, is designed to harm, exploit, or otherwise compromise a device, network, or service. Common types of malware include:
- **Viruses**: Programs that attach themselves to legitimate software and replicate, spreading to other programs and files.
- **Worms**: Standalone malware that replicates itself to spread to other computers.
- **Trojan Horses**: Malicious software disguised as legitimate software.
- **Ransomware**: Malware that encrypts a user's files and demands a ransom for the decryption key.
- **Spyware**: Software that secretly monitors and collects user information.
### Phishing
Phishing is a social engineering attack that aims to steal sensitive information such as usernames, passwords, and credit card details. Attackers often masquerade as trusted entities in email or other communication channels, tricking victims into providing their information.
### Man-in-the-Middle (MitM) Attacks
MitM attacks occur when an attacker intercepts and potentially alters communication between two parties without their knowledge. This can lead to the unauthorized acquisition of sensitive information.
### Denial-of-Service (DoS) and Distributed Denial-of-Service (DDoS) Attacks
1. NGUYEN THI NHI - Master’s student
Department of Physiology
Keimyung University School of Medicine
Progress report
2. Finished works in 8th week
1. Practice
Media making, cell thawing, cell culture, cell passage
Tissue extraction
2. Theory
Main principle of Cre-loxP System, Chromatin immunoprecipitation
(ChIP) assays, and EdU Cell Proliferation Assay
4. Cre-loxP System
• To generate the spatiotemporally controlled mutant mice, two
elements are needed in the Cre-loxP system.
• First, Cre-driver strain is generated in which Cre recombinase is
expressed by a promoter that specifically targets the cell or
tissue of interest.
• Second, loxP flanked (floxed) DNA containing mouse strain is
needed to be generated.
Conditional knockout mice are then generated by breeding the
Cre-driver strain with a floxed mouse strain.
Mouse Cre-LoxP system: general principles to determine tissue-specific roles of target genes
DOI: https://doi.org/10.5625/lar.2018.34.4.147
5. Mechanism of Cre-loxP system
(A) An overview of Cre-loxP system. 38 kDa Cre recombinase recognizes the loxP sites of
specific 34 bp DNA sequences.
(B) General breeding strategy for conditional mutation using loxP and Cre driving mouse line.
In principle, one mouse must have a tissue-specific driven cre gene and another mouse have
loxP flanked (floxed) alleles of interest gene Y. Expression of Cre recombinase excises floxed loci
and inactivates the gene Y.
7. • Protein and associated chromatin in living
cells or tissues are temporarily bonded. The
DNA–protein complexes are then sheared into
∼500 bp DNA fragments.
• Cross-linked DNA fragments associated with
the proteins are selectively
immunoprecipitated from using appropriate
protein-specific antibody.
• Then the associated DNA fragments are
purified and their sequence is determined.
The DNA undergoes PCR amplification using
primers targeting a particular genomic locus.
• These DNA sequences can be subjected to a
series of downstream analysis techniques.
Song, C., Zhang, S., and Huang, H. (2015). Choosing a suitable
method for the identification of replication origins in microbial
genomes. Front. Microbiol.
Principle
8. There are two general procedures for carrying out ChIP experiments:
native ChIP (N-ChIP) and cross-linking ChIP (X-ChIP)
N-ChIP X-ChIP
Native chromatin is used as the
substrate (proteins are not cross-
linked to the DNA).
Proteins are cross-linked to the DNA.
Fragmentation of the chromatin is
achieved by micrococcal nuclease
digestion, resulting in a nucleosome
based resolution.
Cross-linking is typically achieved
using formaldehyde and chromatin
is fragmented by sonication, creating
random fragments.
N-ChIP is restricted to proteins that
are very tightly associated with
chromatin, typically limiting this
type of ChIP to histones and their
modifications.
As the proteins are cross-linked to
the DNA a broad range of chromatin
associated factors can be analyzed
using this technique.
10. EdU Cell Proliferation Assay
Cells grown in the presence of 5-EdU incorporate the compound at
thymidine bases during S-phase. Fluorophore-labeled azide reacts with
the incorporated EdU to allow detection by microscopy or flow
cytometry.
11. EdU Cell Proliferation Assay process
Detection of actively proliferating
cells can be performed using
detection protocols for either image
based or flow cytometric analysis
12.
13. • Objective: to determine the role of insulin signal at β cell level.
• They created βIRKO mice by Cre-loxP mediated recombination
technique. They compared result between βIRKO mice and 3 controls
groups (IRlox, Rip-Cre, and WT).
- IRlox: to rule out the effect of introduction of loxP sites in the
insulin promoter
- Rip-Cre: mice carrying only the Cre transgene on a rat insulin, to
rule out any effect of Cre expression on β cell function
- WT
14. Result
They assessed the effect of β cell insulin receptor KO on glucose
homeostasis, glucose and insulin level in the fasted and random-fed
states.
• The fasting insulin levels in βIRKO mice increased compared with 3
other groups.
• βIRKO mice exhibited a decrease in acute insulin release response to
Glucose.
• After 30min of glucose challenge, insulin levels did gradually rise
suggest the retention of second phase insulin secretion in βIRKO
mice .
15. Result
• They assessed the impact of altered 1st phase insulin release on the
ability of glucose tolerance. (intraperitoneal injection)
βIRKO mice showed a significant higher glucose levels than
controls, and glucose tolerance continued worsen with age.
• They evaluated morphology of β cell by electron microscopy and
revealed well-preserved structure of cell with no apparent
differences in the cell membrane, endoplasmic reticulum, Golgi, …
• Immunohistochemical study showed a decrease (20-40%) in islet size
in 4-month-old βIRKO, but the distribution of GLUT2 remained
unchanged. Besides, the insulin content in this age was smaller than
controls.
16.
17. Insulin Regulates CENP-A and PLK1 Gene
Expression in Pancreatic β Cells
• The expression of CENP-A and PLK1 was significantly decreased in
primary β cells obtained from βIRKO mice compared to control.
• CENP-A protein’s peak expression was delayed 6-12 hr, and PLK1
was virtually undetectable throughout the cell cycle.
• Insulin binding to its receptor enhances CENP-A and PLK1
expression.
These results indicate the effect of glucose on CENP-A and PLK1
expression is dependent on the insulin receptor
18. Insulin-Stimulated FoxM1 Binding to CENP-A
and PLK1- Associated DNA in the Nucleus
• In mouse, FoxM1 was mostly localized to the nucleus in β cells. The
MAPK/ERK kinase (MEK)1/2 inhibitor (U0126) promoted nuclear
export of FoxM1.
• In human, FoxM1 was mainly localized to the cytosol in β cells of
cultured human islets after starvation. Insulin stimulation
facilitated nuclear localization of FoxM1.
(by immunocytochemistry and immunoblotting)
• FoxM1 was recruited to CENP-A and PLK1 genomic regions in
control β cells. And the binding was significantly reduced in βIRKO.
(using chromatin immunoprecipitation)
19. Insulin-Induced CENP-A Expression in
Human Islets Is Impaired in Type 2 Diabetes
• RT-PCR and qPCR analysis revealed significantly low levels of CENP-
A, and a trend to decreased expression of PLK1.
• The number of CENP-A expressing β cells was significantly
attenuated in the islets from T2DM donors. The expression of
CENP-A and PLK1 was increased by exogenous insulin treatment of
islets from non-DM donors.
Reduced CENP-A expression is decreased in islets from patients
with T2DM and it is associated with insulin stimulation.
20. β Cell-Specific CENP-A Knockout Mice
Demonstrate Reduced β Cell Proliferation
• β CenpaKO mice exhibited genomic recombination of Cenpa alleles and
consequent diminished expression of CENP-A only in islets and not in the
hypothalamus, liver, or spleen.
• The knockouts developed glucose intolerance, impaired insulin secretion,
decreased phospho-histone H3 (pHH3)+ mitotic and bromodeoxyuridine
(BrdU)+ proliferating β cells, and β cell mass by 24 weeks of age.
• CENP-A-deficient β cells from β CenpaKO mice demonstrated a
significant decrease in cell proliferation in response to a glucokinase
activator.
β CenpaKO mice exhibited a significant decrease in serum insulin levels,
β cell mass, mitosis, and proliferation compared with controls. CENP-A
plays an important role in the regulation of proliferation and the
maintenance of β cell mass.
21. Role of CENP-A and PLK1 in β Cell Proliferation
and Survival
• Knockdown of CENP-A reduced viability and metabolic activity in
control, IRS1KO, or IRS2KO β cells, but not in β IRKO cells. CENP-A
knockdown significantly increased G2/M-phase cells, suggesting
G2/M cell-cycle arrest.
• PLK1 knockdown reduced cell proliferation in control and β IRKO β
cells (evaluated by both the MTT and EdU incorporation assays).
• The expression of PLK1 was reduced in CENP-A knockdown cells,
while conversely CENP-A expression was diminished in PLK1
knockdown cells.
22. PLK1-Dependent CENP-A Deposition to
Centromere in β Cells
• CENP-A deposition at the mouse centromere marker was
attenuated in both FoxM1 inhibitor-treated control β cells and
intact βIRKO cells (in ChIP analysis).
• PLK1 knockdown and treatment with the PLK1-specific inhibitor (BI-
2536) repressed CENP-A binding at the centromere site.
• The process of CENP-A binding to the centromere was reduced in
human islets when using the IR and IGF1R dual inhibitor, the PI3K
inhibitor, or the PLK1 inhibitor.
23. Schematic of the regulation of CENP-A expression and its deposition to centromere in b cells.