how to read a paper + visualization + diseases

premise 1 of paper

nociceptors transmit information regarding tissue damage to pain processing centers in the brain and spinal chord

premise 2 of paper

some nociceptors are excited by capsaicin

premise 3 of paper

capsaicin triggers burning pain sensation and hypersensitivity

approach step 1 of paper

Generate a cDNA library for expression cloning

approach step 2 of paper

use calcium imaging to functionally screen for capsaicin-sensitive cDNAs

approach step 3 of paper

test cDNAs by isolating cells that display capsaicin-evoked calcium influx and sequence them

key results of paper

capsaicin activates sensory neurons and is predicted to be an ion channel based on sequence similarities and topography

TRMP8

method and cold receptor

how are capsaicin-sensitive cDNAs screen for?

cells are loaded with calcium-sensitive fluorescent dye(Fura-2) and excited at a wavelength, cells that express function TRPV1 receptors will exhibit rapid increase in intracellular calcium levels(detected by change in fluorescence)

Transient Receptor Potential Vanilloid 1 (TRPV1)

ion channel predominantly expressed in sensory neurons such as nociceptors

capsaicin

known agonist of TRPV1. Opens the ion channel, allowing influx of calcium ions into the neuron

electron microscopy(EM0

• high resolution images

• fixation → preparation → imaging

scanning EM (SEM)

typically of tissues, cells

transmission EM (TEM)

typically of subcellular structures

immunofluorescence staining (IF)

• can be used on tissue or cells in culture

• fixation → permeabilization → staining

• primary antibody + secondary antibody

use of immunostaining

use antibodies from different species to visualize the spatial relationship between different proteins on the same sample

two-photon microscopy

• allows imaging of deeper structures

• window can image for days, weeks, months

• transgenic animals tend to expressed GFP-tagged protein

cholera

• toxin binds to cell surface receptor of intestinal epithelial cells

• this activates a series of intravcellular signaling events

• elevated levels of cAMP open many ion channels

• extracellular environment becomes hypertonic drawing water out of the now hypotonic epithelial cell

• results in diarrhea and severe dehydration

cholera treatment

oral rehydration solution (ORS)draw

Dravet syndrome

• the SCN1A gene encodes for the alpha subunit of voltage-gated the sodium channel (Nav1.1) that is responsible for the generation and propagation of action potential in neurons

• SCN1A loss-of- function mutations reduce activity of Nav1.1 channels

• this can lead to seizures which can conflict with critical period of brain development during infancy

Botulinum Toxin(Botox)

• cleaves SNAREs

• lack of SNAREs prevents acetylcholine release, casing paralysis in the neuron

• extremely potent: 75 ng ti kill a person. 1 kg kills all humans

myasthenia graves

• autoimmune disorder targering nAChRs

• muscle weakness, droopy eyelid, double vision, trouble breathing/swallowing, and impaired speech

cocaine overdose

• blocks dopamine transporter

• dopamine swells in synaptic cleft

• increased postsynaptic action potentials

• dopamine excessive release in PNS

why do some people think parsley and cilantro tastes like soap

variant of OR6A2 (olfactory receptor)

severe combined immunodeficiency (SCID)

• mutations in genes involved in development and function of immune cells

• ORAI1 is involved in CRAC channel in the plasma membrane

  • a mutation in ORAI1 causes an immune deficiency by halting CRAC channel function

causes of autoimmunity

• failure of clonal deletion or inactivaton

• normal proteins are modified after drugs or environmental chemicals

• neoantigens: new proteins yield by genetic mutations

• molecular mimicry: good proteins that are similar to microbe proteins

• more likely in women

  • hormones

  • pregnancy

  • X-inactivation

multiple sclerosis

• CNS demyelinating disease

• breakdown of blood-brain barrier allows immune cells into the brain

• immune responses to EBV mistakenly target myelin due to similarities