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Lecture 1
Principles of Cell Engineering: Overview Unlike people or animals, cells have very few behaviors – choices: Divide, Differentiate or Die-Apoptose.
- Cell’s choices are never strange and are regulated at multiple redundant levels
- Understanding such regulation is needed for deliberate control of cell behavior, aka successful cell engineering
- Deliberate control of cell behavior is predicted to reverse and even prevent most-all diseases Proliferation is deficient Example: Dyskeratosis Congenita Bottlenecks: scalability (every cell in the body), delivery vehicles (AAV will work only once. Multiple viral vectors would be needed. Side effects of DNA DSB → Delivery vehicles) Q: Why is Cas9 enzymatically inactive in CRISPR-TA? Cas9 is rendered enzymatically inactive in CRISPR-TA to avoid inducing double-strand breaks in DNA, allowing for targeted gene regulation without altering the genetic sequence. This modification prevents the Cas9 protein from cleaving the DNA, ensuring precise control over gene expression levels by guiding the inactive Cas9 protein (dCas9) to specific genomic locations using the guide RNA. The inactivation of Cas9 in CRISPR-TA enables researchers to manipulate gene activity without causing permanent changes to the DNA sequence, facilitating the study of gene function and regulation. Q: Why is dDNA needed for correcting mutations in dyskerin? dDNA (donor DNA) is needed for correcting mutations in dyskerin because dyskeratosis congenita (DC), caused by mutations in the dyskerin gene, leads to dysfunctional telomeres and defective telomerase activity. Telomeres protect chromosome ends and are maintained by telomerase. The introduction of correct donor DNA helps to repair the mutated dyskerin gene, restoring proper telomerase function, stabilizing telomeres, and potentially mitigating the symptoms and complications associated with DC. This strategy aims to replace the faulty genetic sequence in dyskerin, promoting the production of functional dyskerin and improving telomerase activity to alleviate DC-related issues. Example: X-SCID (Lack of differentiation in blood lineage – lymphocytes) Example: DMD – mature muscle fibers are deficient in a key protein – dystrophin Q: is SCID curable because only HSC, but not other tissue stem cells self-renew or because of systemic nature of the HSC?
Severe Combined Immunodeficiency (SCID) is considered curable primarily due to the unique nature of Hematopoietic Stem Cells (HSCs) found in the bone marrow. HSCs possess the ability to self-renew and differentiate into all types of blood cells, including immune cells. When treated through bone marrow or stem cell transplantation, healthy HSCs are introduced into the body, allowing them to repopulate the immune system, thereby restoring normal immune function. This curative potential is specific to HSCs and their systemic nature, enabling the reconstitution of a functional immune system, unlike many other tissue-specific stem cells that do not possess the same capacity for self-renewal or systemic impact. No cure: Problems of scalability and lack of systemic delivery; viral toxicity, exon skipping does not provide a cure. Apoptosis is excessive – degenerative pathologies Q: Why is apoptosis in CNS highly consequential? Apoptosis in the central nervous system (CNS) is highly consequential due to several reasons. Firstly, the CNS has limited regenerative capacity compared to other tissues, making cell loss through apoptosis more critical and potentially irreversible. Secondly, neurons, the primary cell type in the CNS, are typically long-lived cells, and their loss via apoptosis can lead to permanent functional deficits and neurodegenerative conditions. Additionally, excessive or uncontrolled apoptosis in the CNS can contribute to various neurological disorders, impacting cognitive function, motor skills, and other essential neurological processes. Lastly, the intricate and delicate connections between neurons and their specific roles in neural circuits make the consequences of apoptosis in the CNS especially severe, potentially disrupting complex brain functions and leading to neurological dysfunction. Apoptosis is dysfunctional – senescence and cancer Q: Are cancers caused just by hyper-proliferation? Summarize yes of no logic. No, cancers are not solely caused by hyper-proliferation. While increased cell proliferation is a hallmark of cancer, the development of cancer is a complex, multifaceted process involving various genetic, environmental, and lifestyle factors. Cancer arises from a combination of genetic mutations, alterations in cell signaling pathways, evasion of normal regulatory mechanisms, immune system evasion, angiogenesis (formation of new blood vessels), tissue invasion, and metastasis (spread to other parts of the body). Hyper-proliferation is one aspect of cancer but not the sole cause; it's part of a broader set of processes that contribute to the development and progression of cancer. Barcoding: single cell analysis in complex biological environments Identifying drug-resistant cancer cells from a patient involves analyzing tumor samples to determine which cells survive or proliferate despite exposure to specific chemotherapy drugs. These cells often possess genetic mutations or adaptive changes that confer resistance to the drugs used. Assessing their susceptibility to alternative chemotherapeutic agents involves testing these cells against different drugs or treatment regimens to evaluate if they remain sensitive or exhibit cross-resistance, which is when resistance to one drug confers resistance to others due to shared mechanisms or pathways of action. Understanding the spectrum of drug
Q: what becomes different between the second and the seventh decade of human life: (a) the rate of cell damage, (b) the rate of cell repair, (c) both? Between the second and the seventh decade of human life, both the rate of cell damage and the rate of cell repair tend to change. Typically, there's an increase in the rate of cell damage and a decline in the rate of cell repair during this period, contributing to age-related physiological changes and the onset of various age-related diseases. Q: What is unethical research and why do some engage in it Pressure, opportunity, rationalization
Lecture 2
Genetic control of cell fate and behavior. Editing genomes. CRSPR (Clustered Regularly Interspaced Short Palindromic Repeats): Bacterial system that has evolved to eliminate bacteriophage integration and is now adopted for mammalian genome editing and regulation of gene expression. The gRNA/Cas9 complex is recruited to the target sequence by the base-pairing between the gRNA sequence and the complement to the target sequence in the genomic DNA. For successful binding of Cas9, the genomic target sequence must also contain the correct Protospacer Adjacent Motiff (PAM) sequence immediately following the target sequence (learn more about PAM sequences). The binding of the gRNA/Cas9 complex localizes the Cas9 to the genomic target sequence so that the wild-type Cas9 can cut both strands of DNA causing a Double Strand Break (DSB). A DSB can be repaired through one of two general repair pathways: (1) the Non-Homologous End Joining (NHEJ) DNA repair pathway or (2) the Homology Directed Repair (HDR) pathway. The NHEJ repair pathway often results in inserts/deletions (InDels) at the DSB site that can lead to frameshifts and/or premature stop codons, effectively disrupting the open reading frame (ORF) of the targeted gene (Learn more about using CRISPR technology to disrupt a gene). The HDR pathway requires the presence of a repair template, which is used to fix the DSB. HDR faithfully copies the sequence of the repair template to the cut target sequence. Specific nucleotide changes can be introduced into a targeted gene by the use of HDR with a repair template Q: Does PAM sequence need to be inserted into genome for CRISPR to work? Yes, for the CRISPR-Cas9 system to function effectively, a Protospacer Adjacent Motif (PAM) sequence must naturally exist near the target DNA region within the genome. The PAM sequence is recognized by the Cas9 protein and is essential for initiating the DNA cleavage process. Without a suitable PAM sequence adjacent to the target site, the Cas9 enzyme won't bind and induce double-strand breaks, hindering the CRISPR-Cas9 system's ability to edit the specific genomic region.
During NHEJ repair, InDels (insertions/deletions) may occur as a small number of nucleotides are either inserted or deleted at random at the DSB site. InDels alter the Open Reading Frame (ORF) of the target gene, which may significantly change the amino acid sequence downstream of the DSB. Additionally, InDels could also introduce a premature stop codon either by creating one at the DSB or by shifting the reading frame to create one downstream of the DSB. Any of these outcomes of the NHEJ repair pathway can be leveraged by scientists to disrupt their target gene. Q: Will CRISPR cause complete gene KO or a hemizygosity? CRISPR technology can induce both complete gene knockout (KO) and hemizygosity, depending on the specific alterations introduced. A complete gene knockout occurs when both alleles of a gene are effectively disrupted or deleted, resulting in the loss of function. Hemizygosity, however, involves the modification of one allele, potentially leading to a partial loss of gene function while the other allele remains unaltered. Q: What are CRISPR off-target effects and why do we try to limit them? Off target effects In order to introduce nucleotide modifications to genomic DNA, a DNA repair template containing the desired sequence must be present during HDR. The DNA template is normally transfected into the cell along with the gRNA/Cas9 and must have a high degree of homology to the sequence immediately upstream and downstream of the DSB. The length and binding position of each homology arm is dependent on the size of the change being introduced. In the presence of a suitable template, HDR can faithfully introduce specific nucleotide changes at the Cas9 induced DSB. Q: Will CRISPR work for most/all cells in a patient? CRISPR's effectiveness can vary among different cell types in a patient, but it generally has the capability to function in a wide range of cells, with variations in efficiency and specificity depending on the particular cell type and the delivery method used. Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements. Nat Biotechnol. 2018; 36:765-771. NHEJ: Efficient in G1, but occurs in all phases of a cell cycle Likely shift of ORF; Prevalent in progenitor cells; inaccurate, but fast and can lead to cancers. HDR: occurs in S- phase when daughter chromatids are available No shift of ORF; Prevalent in tissue stem cells; Accurate, but slow. Tissue stem cells do not turn into cancers. DSBà cell cycle arrest and apoptosis; or genomic instability, which can lead to cancers. The dCas9 can be utilized as a platform for DNA transcriptional regulators to activate or repress gene expression by fusing the inactive enzyme (that retains its gRNA-binding ability) to known
Q: Do these approaches cure DMD or turn it into another milder disease? Exon skipping and Dystrophin mini-gene therapies for Duchenne muscular dystrophy (DMD) aim to alleviate the symptoms and slow disease progression rather than cure it completely. These approaches do not turn DMD into another milder disease, but they have the potential to partially restore dystrophin production, improving muscle function and potentially delaying the onset or progression of some symptoms associated with DMD. However, they do not address the underlying genetic cause of the condition, and their effects may vary among individuals. Therapeutic viral delivery vectors MDX viral titer: 10^14 for <1g mouse neonate Not very feasible; human baby is 3-4kg. Q: How many infectious particles (titer) would be needed to accomplish the degree of mouse AAV therapy in a human? How does that number compare with the total mRNA in such human (assuming 10^3 mRNAs per cell) To achieve a similar degree of AAV therapy in a human compared to a mouse with an MDX viral titer of 10^14 for a <1g mouse neonate, scaling up for a human baby weighing 3-4kg would require a significantly higher number of infectious particles. A rough estimation could suggest a need for viral titers in the range of several orders of magnitude higher, potentially in the range of 10^18 to 10^20 infectious particles or more for effective therapeutic impact in a human. Comparing this to the total mRNA in a human (assuming 10^3 mRNAs per cell), which could be in the range of tens of trillions to hundreds of trillions of mRNA molecules, the amount of viral particles needed for such therapeutic delivery would be extremely substantial, highlighting the significant challenge of scaling up viral therapies for larger organisms like humans. NP CRISPR delivery: CRISPR-nano Q: Why are off target effects minimized in this method, as compared to the recombinant Cas9 in AAV? Off-target effects are minimized in CRISPR-nano due to its localized and controlled delivery of the CRISPR-Cas system, ensuring precise targeting and reduced exposure to unintended cellular regions. In contrast, recombinant Cas9 in AAV-mediated delivery may exhibit higher off-target effects as it can disseminate widely throughout the body, potentially interacting with unintended genomic loci and increasing the likelihood of off-target modifications. CRISPR-nano effectively performs HDR in vivo Work in progress: delivery from circulation and targeting CRISPR to tissue stem cells Q: List two key advantages of targeting CRISPR to tissue stem cells. Targeting CRISPR to tissue stem cells offers the advantage of potentially achieving long-term therapeutic effects by altering the genetic makeup of these cells, leading to sustained changes in tissue regeneration and repair. Additionally, focusing CRISPR on tissue stem cells may allow
for more precise and specific modifications, reducing off-target effects and enhancing the therapeutic potential of the treatment.
Lecture 3
Reporters, Transgenes, Knock-outs,- ins, Cre-Lox and barcoding. Mouse genetics is an important intermediate in bio-engineering and biomedicine. Amyotrophic lateral sclerosis, is a progressive neurodegenerative disease: motor neurons in the brain and the spinal cord apoptose. There is sporadic and familial (genetic) ALS. The first gene shown to be mutated in FALS encodes the enzyme superoxide dismutase 1 (SOD1), which is mitochondrial enzyme that is getting rid of reactive oxygen species (ROS). ALS patients can have one of ~150 pathogenic mutations in SOD1. Mutant SOD1 transgenic mice recapitulate many features of ALS. Q: What would be the best way to recapitulate human ALS in a mouse? Insert an additional copy of SOD1 that has human mutation and is under human promoter. ROSAβgeo26 (ROSA26) locus: A transgene will be for sure expressed from mouse Chromosome 6 and it will not interfere with the rest of that locus. Q: What to do if your transgene interferes with embryonic development or early life viability? If your transgene interferes with embryonic development or early life viability, one potential strategy is to conditionally regulate the transgene's expression using inducible promoters or genetic switches, allowing control over the timing or level of transgene expression. Another approach is to modify the transgene construct or use tissue-specific promoters to restrict its expression to specific cells or developmental stages, minimizing adverse effects on embryonic development or early life viability. SOD1 transgene will be expressed in only in neurons and only when tamoxifen (Estrogen homolog) is added. Knock-In Reporter: time and space of gene expression Reporter of interest: Green Fluorescent Protein (GFP), Luciferase, etc. DNA is interchangeable between species. Q: What is the key advantage of Knock-in reporters over Ros26 transgenes? The key advantage of knock-in reporters over Rosa26 transgenes lies in their integration at specific genomic loci, ensuring consistent and predictable expression patterns as they utilize the endogenous regulatory elements, thus providing more accurate representation and regulation of gene expression in comparison to the potentially variable expression seen with Rosa transgenes that integrate randomly within the genome.
ScarTrace, GESTALT1, MEMOIR systems hgRNA CRISPR for Barcoding Q: How can a location of cells relative to each other be deduced from their barcodes? Cells' relative locations can be inferred from their barcodes by examining the similarities or differences in the barcode sequences, where identical or closely related barcodes suggest spatial proximity or common lineage, indicating cells that were in close proximity or derived from a common ancestor within a tissue or spatial context. Analysis of barcode diversity or similarity among cells helps deduce their spatial organization or physical proximity within a tissue or structure.
Lecture 4
Master-switch of Epigenetics What is epigenetics? Q: What is different between neuron and muscle cell or between proliferating versus differentiated cell: A). Genomic DNA sequence? B). Chromatin? C). Transcriptome? D). Proteome? Chromatin: Neurons and muscle cells, as well as proliferating versus differentiated cells, exhibit differences in their chromatin structure and organization. Chromatin undergoes changes in accessibility, compaction, and modifications, influencing gene expression and cellular identity in distinct cell types or states. What is chromatin? Q: The size of the nucleus is ~1micron; What is the metric length of genomic DNA that is packed into the nucleus? The genomic DNA in a human nucleus, if linearly stretched out, would be several feet long. On average, the length of genomic DNA in a human cell is approximately 2 meters, whereas the diameter of the nucleus is around 5-10 micrometers. This incredible packaging is achieved by tightly coiling and folding the DNA around histone proteins, condensing it into a highly compact structure within the limited space of the nucleus. Q: Which state of the chromatin (condensed vs. decondensed) is permissive for a) CRISPR, b) transcription, c) gene expression? Decondensed: A decondensed or open chromatin state allows accessibility to DNA sequences, facilitating the binding of CRISPR components for gene editing, enabling transcription factors to access gene sequences for transcription, and promoting the overall gene expression by allowing regulatory elements and transcriptional machinery to interact with DNA.
Gene activation/inactivation starts at DNA – through DNA methylation. DNA methylation at a CpG island located in the promoter region of a gene. Methylation inhibits transcription either by directly preventing transcription factor binding, or indirectly by binding regulatory proteins to methyl binding domain (MBD). The cytosine moiety of cytosine–guanine (CpG) dinucleotides in mammalian DNA can be methylated at carbon 5 to form 5ʹ methylcytosine. The methyl group for this chemical modification of the DNA is donated by SAM. This reaction is catalyzed by a family of DNMTs. Of these, DNMT3A and 3B primarily perform de novo methyl transfer, whereas DNMT1 mainly acts as maintenance DNMT with greater affinity for partially methylated (i.e., hemi- methylated) DNA. Methylation of cytosine in CpG-rich regions (i.e., CpG islands) located in or near gene promoters results in gene silencing. SAM- S-Adenosine Methionine. Q: Can a high copy expression of a TF (from Rosa26 transgene, for example) open chromatin on a promoter for which this TF is specific? Yes, a high copy expression of a transcription factor (TF) from a Rosa26 transgene, if specific to a particular promoter, can potentially open chromatin at that promoter. Elevated levels of the TF can increase its binding frequency to the target promoter, facilitating chromatin remodeling and leading to a more accessible and permissive chromatin state for gene expression. Achieving the epigenetic permissiveness state for desired cell fate, responses, etc. Methylation patterns are initially established by the de novo DNA methyltransferases DNMT3A and DNMT38. When DNA replication and cell division occur, these methyl marks are maintained in daughter cells by the maintenance methyltransferase, DNMT1, which has a preference for hemimethylated DNA. If DNMT1 is inhibited or absent when the cell divides, the round of cell division will result in passive demethylation. TET-mediated active DNA demethylation. a DNA methyltransferases (DNMTs) convert unmodified cytosine to 5-methylcytosine (5mC). 5mC can be converted back to unmodified cytosine by TET-mediated oxidation to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), followed by excision of 5fC or 5caC mediated by thymine DNA glycosylase (TDG) coupled with base excision repair (BER). Currently, there is no known enzyme that is able to remove the strong covalent bond of the methyl group from the cytosine residue. Instead, the methylated cytosine is thought to undergo a series of further modifications that ultimately change the 5mC into a thymine. This elicits a base mismatch and activates the base excision repair to replace the residue with a new cytosine. Q: Why would proteins like cMyc that promote cell proliferation facilitate reprogramming from one cell fate to another?
could provide a complete CMV promoter sequence, which might be more suitable for the repressive effects of SynR repressors by maintaining the normal regulatory elements of the promoter.
Lecture 5
Organelles, Sub-cellular probes. Subcellular probes Small molecules under 1 kDa freely diffuse through the cell- membrane system; anchoring probes for lysosomes or other acidic organelles are equipped with lipophilic weakly basic moieties, that is, lipophilic amines. Once the probes diffuse inside the acidic organelles, the amines are protonated and the probes become positively charged and trapped inside the organelles. A strong negative membrane potential as high as 180-200 mV within the matrix. Cations with strong lipophilicity move across the phospholipid bilayer of the inner membrane and accumulate inside the mitochondrial matrix 1:10 as compared to other organelles. Q: Why would one use sub-cellular probes? Sub-cellular probes are utilized in real-life applications to precisely study and visualize biological processes within specific cellular compartments, enabling a detailed understanding of localized events, interactions, and dynamics. These probes offer insights into sub-cellular structures, organelles, and molecular activities, crucial for deciphering cellular functions, disease mechanisms, and developing targeted therapeutic interventions. Utilizing CRISPR for sub-cellular fluorescent probing. The first use of dCas9 for genomic imaging was published by Chen et al. in 2013. In this work, the authors genetically fused dCas9 and EGFP, and demonstrated the feasibility of using dCas9-EGFP with one sgRNA to image the highly repetitive elements of the telomere, as well as to image non-repetitive regions of the MUC4 gene through the use of an array of at least 26 different sgRNAs (Figure 2A). To improve gene detection, other researchers have tagged dCas with more FP molecules, such as through the use of the supernova tagging system (SunTag), which is poly(GCN4) peptide scaffold that enables recruitment of up to 24 FPs through interactions between GCN4 and the single-chain variable fragment (scFv) of the antibody against GCN4 (Figure 2B). Q: How can dCas9-FP be used to determine epigenetic and transcriptional status of a given gene? dCas9-FP can be employed in chromatin immunoprecipitation (ChIP) assays by fusing it with a fluorescent protein and chromatin-associated protein to visualize the epigenetic state (e.g., histone modifications) of a specific gene locus via fluorescence microscopy. Additionally, coupling dCas9-FP with RNA detection techniques like RNA FISH (Fluorescence In Situ
Hybridization) allows the visualization of transcriptional activity by tracking RNA transcripts at the gene locus. Nucleus The nuclear envelope surrounds the nucleus. Heterochromatin is located near the periphery and euchromatin is located near the center. Shown here is only one chromosome territory however, each chromosome would have its own territory. Nuclear pores are specific portals of entry and exit to and from the nucleus. Transcription factories are areas where transcriptional complexes assemble with DNA and where transcription occurs. The nuclear matrix is a network of several proteins that add structure and support to the nucleus and also provide places for attachment of other proteins and DNA. The nucleolus is the ribosomal manufacturing site. Promyelocytic leukemia bodies (PML body) have been associated with protein storage, transcription, DNA repair, viral defense, stress, cell cycle, regulation, proteolysis, and apoptosis. Cajal bodies are centers of enzymatic activity and DNA modification. They may also be sites of assembly or modification of the transcription machinery and may also contain structures such as the methylation and acetylation machinery and the spliceosome. The nuclear matrix: the network of lamins and their associated nuclear envelope proteins and matrix attachment region proteins (MAR). This structure adds support and rigidity to the nucleus. The chromosome territory binds to lamin proteins of the nuclear matrix to maintain its position within the nuclear three-dimensional space. The normal nuclear lamina is shown on the left and an LMNA mutant is depicted on the right. The proteins of the LINC (LInks the Nucleus to the Cytoplasm) complex are indicated and include the nesprins (light blue), SUN proteins (pink), emerin (green) and lamins (maroon). Chromatin (black) interacts with the lamina and active transcription complexes (orange) in normal cells, but is relocalized to the interior in the LMNA mutant cells and therefore cannot interact with transcription complexes. Mitochondrion Schematic diagram representing a mitochondrion and its electron transport chain (ETC) complex I, complex II, complex III, complex IV, and complex V. Reducing equivalents (NADH) derived from tricarboxylic acid cycle (TCA cycle) are utilized by ETC to generate proton (H+) gradient in the intermembrane space (IMS) across the inner membrane (IM) that is used for the synthesis of ATP by complex V. Complex I and complex III are the main sites for formation of superoxide (O2·-). In addition, O2·- formation can also occur by α- ketoglutarate dehydrogenase (a-KGDH) of TCA cycle. Q: Will ROS damage mitochondrial DNA? Yes, Reactive Oxygen Species (ROS) can damage mitochondrial DNA (mtDNA). The close proximity of mtDNA to the electron transport chain, where ROS are generated, makes mtDNA vulnerable to oxidative damage, leading to mutations and impairments in mitochondrial function. Q: Will ROS damage adjacent cells?
mitochondrial cytochrome c release and assembly of the apoptosome (comprising ~7 molecules of apoptotic protease-activating factor-1 (APAF1) and the same number of caspase- homodimers). In the intrinsic pathway (route 2), diverse stimuli that provoke cell stress or damage typically activate one or more members of the BH3-only protein family. BH3-only protein activation above a crucial threshold overcomes the inhibitory effect of the anti-apoptotic B-cell lymphoma-2 (BCL-2) family members and promotes the assembly of BAK-BAX oligomers within mitochondrial outer membranes. These oligomers permit the efflux of intermembrane space proteins, such as cytochrome c, into the cytosol. On release from mitochondria, cytochrome c can seed apoptosome assembly. Active caspase-9 then propagates a proteolytic cascade of further caspase activation events. The granzyme B-dependent route to caspase activation (route 3) involves the delivery of this protease into the target cell through specialized granules that are released from cytotoxic T lymphocytes (CTL) or natural killer (NK) cells. CTL and NK granules contain numerous granzymes as well as a pore- forming protein, perforin, which oligomerizes in the membranes of target cells to permit entry of the granzymes. Granzyme B, similar to the caspases, also cleaves its substrates after Asp residues and can process BID as well as caspase-3 and -7 to initiate apoptosis. BAD, BCL-2 antagonist of cell death; BAK, BCL-2- antagonist/killer-1; BAX, BCL-2- associated X protein; BID, BH3-interacting domain death agonist; BIK, BCL-2-interacting killer; BIM, BCL-2-like-11; BMF, BCL-2 modifying factor; HRK, harakiri (also known as death protein-5); PUMA, BCL-2 binding component-3. Q: Name two general classes of mitochondrial pathologies. Two general classes of mitochondrial pathologies include mitochondrial DNA (mtDNA) mutations, which can lead to inherited mitochondrial diseases affecting oxidative phosphorylation and cellular energy production, and mitochondrial dysfunction arising from defects in nuclear DNA-encoded mitochondrial proteins, causing disruptions in various mitochondrial functions, such as metabolism and reactive oxygen species (ROS) production. Q: If apoptosis is excessive, how would you engineer cells to be more resistant to it? To render cells more resistant to excessive apoptosis, one could engineer cells by overexpressing anti-apoptotic proteins from the BCL-2 family, such as BCL-2, BCL-XL, or MCL-1, which inhibit the mitochondrial pathway by preventing BAK-BAX oligomerization, thereby reducing cytochrome c release and apoptosome assembly. Additionally, interfering with upstream death receptor signaling, such as downregulating caspase-8 or blocking the activation of caspase-3 and -7, could be a strategy to mitigate excessive apoptotic responses. Lysosome ion channels and transporters. The lysosomal lumen contains a battery of soluble hydrolytic enzymes that degrade proteins (proteases), triglycerides (lysosomal acid lipase; LAL), glycoproteins (glycosidases), heparan sulphate (sulfatases), and nucleic acids (nucleases, RNAase and DNAse). Lysosomal ion channels and transporters include vATPase; vacuolar H+-ATPase (proton pump), chloride channel (ClC); ClC family that exchanges cytosolic Cl− for lysosomal H+ (ClC-6 and ClC-7), K+ channels (BK). Non-selective cation channels include Transient Receptor Potential Cation Channel (TRPML); permeable to Ca2+, Na+, K+, Zn2+ and Fe2+, P2X4 channel; permeable to
Ca2+, Na+ and K+ and two-pore channels (TPC); permeable to H+, Ca2+ and Na+. Ion transporters include Na+/H+ exchangers (NHEs) and Ca2+/H+ exchangers (CHX). Endosomes, lysosomes and autophagy Lysosomes harbor specific enzymes that breakdown proteins, sugars and lipids into simple products that the cell then utilizes to build renewal these substances. Each of these lysosomal enzymes has specific substances that they are capable of degrading. In the case of deficient enzyme of one of these enzymes, buildup of one and other substances occur resulting initially in dysfunctional lysosomes and then the whole cell. This is reflected in the malfunction of different organs and systems resulting in serious and progressive health problems. Upon contact with foreign biomolecules, the cell plasma membrane actively internalizes the biomolecules in the form of vesicles, which are then transferred to the mildly acidic early endosomes with a pH value of 6.5. Within the early endosomes, the biomolecules are categorized and labeled for their respective destinations. The late endosomes act as transfer stations, receiving both obsolete biomolecules from early endosomes and acid hydrolases from the trans-Golgi network of the Golgi apparatus. The acid hydrolases are tagged by mannose-6-phosphate (M6P) receptors in the cis-Golgi and further protected by M6P receptor (M6PR) protein in the trans-Golgi, because, once released, the acid hydrolases could cause cellular damage. From the trans-Golgi network, the vesicles containing acid hydrolases labeled with M6PR are selectively transferred to the late endosomes. Within the medium acidic environment of late endosomes with pH values around 5.5. The obsolete biomolecules and acid hydrolases are transferred from the late endosomes to lysosomes. The low pH value (ca. 4.5) of lysosomes, as well as endosomes, is mediated by a series of proton pumps, such as the vacuolar- type H+-ATPase, which utilizes energy from ATP to supply the import of protons into the lysosome or endosome membrane. When the acid hydrolases enter the lysosomes, they are activated by the low pH value and degrade the bio-macromolecules to low-molecular-weight materials for biosynthesis. Angew Chem Int Ed Engl. 2016 Aug 29. doi: 10.1002/anie.201510721. PMID: 27571316 Q: Fasting induces autophagy; why is this healthy? Fasting induces autophagy, a cellular process that clears damaged organelles and recycles cellular components, which can promote cellular renewal and maintain cellular health. This process helps in removing dysfunctional molecules and organelles, supporting cellular homeostasis and potentially reducing the risk of various diseases. Organelle Engineering А. Micro fluidic chip was used to encapsulate cells in w/o droplets encased in a lipid monolayer. An aqueous phase containing cells was passed through a flow focusing junction where It met an oil phase → droplet generation. B. Schematic depicting the transformation of cells-in-droplets to cells- in- vesicles. e droplets descended through the column under gravity. As droplets transferred into the aqueous phase the interfacial monolayer wrapped around them, transforming them into vesicles with cells encapsulated inside. The aqueous channel contained cells in medium (L-15 with 125 mM sucrose), and the oil phase contained dissolved
Q: what is the effector of Notch pathway? The effector of the Notch pathway is the transcription factor CSL (CBF1/RBPJκ/Su(H)/LAG1), which interacts with the intracellular domain of the Notch receptor upon activation, leading to the regulation of target gene expression involved in various cellular processes such as development, differentiation, and cell fate determination. Cell responses are regulated by the intensity and timing of interactive signaling pathways - 4D control. A simplified overview of the main components of Notch signaling. Upon Notch ligand binding, a two-step proteolysis cleavage process (small arrows) within the juxtamembrane region and transmembrane domain of the Notch receptor is catalyzed by a member of the disintegrin and metalloproteases (ADAMS) family and the γ-secretase containing complex, respectively, then the Notch intracellular domain (NICD) is released from the membrane and translocates to the nucleus, where it forms a transcriptional activation complex with CSL and coactivators (CoA), thereby inducing the transcription of target genes. DOI:10.1186/2040-2384-1- Oscillatory expression of DIl1, Hes1, and MyoD controls self- renewal and the timing of differentiation. a DIl1, Hes1, and MyoD are dynamically expressed in activated muscle stem cells. Hes1 is no longer expressed, and DIl1 and MyoD expression are sustained when muscle stem cells differentiate. MSC muscle stem cell. Nature Communications (2021)12: https://doi.org/10.1038/s41467-021-21631- Organogenic properties of TGF-beta Thalidomide is best known as a major teratogen that caused birth defects in up to 12, children in the 1960s; it was prescribed to treat morning sickness in pregnant women. Clin Ther. 2003 Feb;25(2):342-95. Q: Why was there no significant side-effects in women who took thalldomide? Women who took thalidomide during pregnancy did not experience significant side effects because the drug's teratogenic effects, causing birth defects like limb malformations, primarily occurred during a specific window of fetal development (around 20-36 days post-conception) when most women might not have been exposed due to different stages of pregnancy or variability in drug metabolism. Therefore, the critical period for thalidomide-induced birth defects likely did not coincide with the timing of drug exposure for many women. Interactome: morphogens, focal adhesions and mechano-sensing.
- GF interaction with ECM (fibronectin) activates it and promotes activation of integrins;
- in turn, focal adhesion and cytoskeletal organization promote signal transduction;
- One outcome of this signaling is to remodel ECM, hence changing the intensity of signaling pathways, cell adhesion and biomechanics. Q: Would GF signaling facilitate or inhibit cell adhesion?
Growth factor (GF) signaling would typically facilitate cell adhesion by activating integrins through interaction with the extracellular matrix (ECM), leading to the formation of focal adhesions and cytoskeletal organization that promote stronger cell-substrate interactions and adhesion. This signaling cascade supports cell adhesion, which is essential for various cellular processes including cell migration, proliferation, and tissue organization. Q: Why would key regulators of cell adhesion be anti-cancer drug targets? Key regulators of cell adhesion are potential anti-cancer drug targets because their manipulation can disrupt tumor progression by inhibiting cancer cell migration, invasion, and metastasis. Targeting these regulators may impede the ability of cancer cells to interact with the extracellular matrix, hamper their invasive properties, and potentially reduce their capacity to spread to other tissues, thereby limiting cancer progression and metastatic potential. Integrins exist at the plasma membrane in a resting, inactive state. When cells encounter a mechanically rigid matrix or are exposed to an exogenous force integrins become activated, which favors integrin oligomerization or clustering, talin 1 and p130Cas protein unfolding, vinculin–talin association, and Src and focal adhesion kinase (FAK) stimulation of RhoGTPase-dependent actomyosin contractility and actin remodelling. Mature FAs vary in size between 1 to 5 μm and to date, more than 80 types of proteins (~150 proteins) have been located in the FA plaque although not all interactions have been proven in vivo. Nature reviews Cancer, 2009, V9, p108. Journal of Cell Science 2009 122: 1059-1069 Experimental Cell Research 343 (2016) 14– Mechano-biology and growth factor signaling. Co-signaling of integrins and growth factor receptors has been shown to trigger a synergistic effect that increases and prolongs growth factor signaling. The recruitment of common molecules from both signaling cascade induces an enhanced effect of growth factor. Exploiting this synergistic signaling permits to lower the effective dose of growth factors in wound healing therapies. Q: would adhesion promote higher intensity of MAPK and AKT in the presence of ligands of these pathways? How about Delta/Notch: would it become more activated in adherent cells and if yes, why? Yes, adhesion can potentially promote a higher intensity of MAPK and AKT signaling in the presence of ligands for these pathways. Integrin-mediated adhesion can amplify growth factor signaling, leading to increased activation of MAPK and AKT pathways due to the co-signaling effects between integrins and growth factor receptors. Delta/Notch signaling might become more activated in adherent cells because cell-cell adhesion can facilitate the localization and interaction of Delta and Notch proteins, enhancing their cleavage and activation at the cell membrane, thereby promoting Notch signaling in adherent cells. Q: Why would one use the EMC stiffness gradient or sheer stress to fine-tune cell fate? Utilizing the extracellular matrix (ECM) stiffness gradient or shear stress helps fine-tune cell fate by mimicking the physiological microenvironment, where varying mechanical cues influence
