This document discusses x-ray crystallography techniques including Bragg's law and applications such as determining molecular arrangements of atoms. It explains key concepts like diffraction, constructive and destructive interference patterns, and how a crystal's perfection impacts the clarity of patterns. Bragg's law is derived and its importance explained. Techniques like isomorphous replacement that use heavy atoms to solve phase problems are covered. Considerations for selecting gas chromatography/mass spectrometry or liquid chromatography/mass spectrometry are presented. The importance of animals in biomedical research and various animal models are discussed. Alternatives to animal testing including cell culture and computer models are also summarized.

1. X-Ray CRYSTALLOGRAPHY : Bragg’s law and applications
Diffraction refers to various phenomena which occur when a wave encounters an obstacle
or a slit.
It is defined as the bending of light around the corners of an obstacle or aperture into the
region of geometrical shadow of the obstacle.
What is the Significance of the Diffracted Pattern?
First we need to understand how the diffracted pattern is actually constructed and what
figure out what it tells us. Only then will we fully understand how and why it is a valuable
tool to understanding the molecular arrangements of atoms.
DESTRUCTIVE INTERFERENCE Destructive interference occurs when there is a collision of
photons in such a way that their intensities cancel out.
CONSTRUCTIVE INTERFERENCE: Constructive interference occurs when there is a collision
of protons in a way that their intensities combine.

2. After we observe the reflections of the photons on the detector, we change the angle at
which the x ray beam hits the crystal.
The crystal should be as perfect as possible! Why does the crystal have to be perfect?
Think of it this way. Atoms of the same type and that are in the same place within the
structure will result in an additive diffraction, which would definitely be easier to detect!
Therefore, the more perfect a crystal is, the more atoms that will be present in the same
places in the structure, which would be easier to detect.
Imperfect crystals usually result in fuzzy patterns that are hard to interpret.
We would then have to combine all the reflections together to find the three-dimensional
diffraction pattern. But why do we need to look at it at different angles????
What is Bragg’s Law and why is it Important? This equation explains why the faces of
crystals appear to reflect (diffract) X-ray beams at certain angles of incidence θ.
nλ = 2d sinθ
d : lattice interplanar spacing of the crystal θ : x-ray incidence angle (Bragg angle)
λ : wavelength of the characteristic x-rays
Deriving Bragg’s Law: Bragg's Law can easily be derived by considering the conditions
necessary to make the phases of the beams coincide when the incident angle equals and
reflecting angle.
The rays of the incident beam are always in phase and parallel up to the point at which
the top beam strikes the top layer at atom z.
The second beam continues to the next layer where it is scattered by atom B.
The second beam must travel the extra distance AB + BC if the two beams are to continue
traveling adjacent and parallel.
This extra distance must be an integral (n) multiple of the wavelength (λ) for the phases
of the two beams to be the same:
nλ = AB +BC (2).

3. Isomorphous Replacement (SIR, MIR)
To solve the phase of a protein crystal the complexity of the problem (i.e. the number of
variables) has to be reduced.
One way of doing that is to soak the crystal in a heavy atom solution and compare the
diffraction pattern of this derivative crystal to the diffraction pattern of the native crystal.
The heavy atoms in the structure contribute enormously to the diffraction which makes
their contribution easy to detect and the difference of the two diffraction patterns can be
taken
With direct methods the position of the heavy atoms (i.e. the heavy atom substructure) can
be determined.
Knowing these positions it is possible calculate the heavy atom contribution to each
structure factor.
This in turn means that for each structure factor one can determine the structure factors
of the heavy atoms (FH) and the amplitudes of the native protein (|FP|) and the heavy
atom derivative (|FPH|).
Drawing this relationship in the space of complex numbers is called the Harker
construction gives two possible solutions for the phase of each structure factor.
Solving the structure this way is called single isomorphous replacement (SIR).
By using at least one other heavy atom derivative this phase ambiguity can be solved
which is then called multiple isomporphous replacement (MIR).

4. The difference between a native structure factor (FP) and the derivative structure factor
(FPH) equals the heavy atom structure factor (FH) (left figure).
With direct methods we can solve the heavy atom structure factors using the amplitudes of
from the native and the derivative crystal.
Drawing a circle with the amplitudes of the native crystal as radius around one end of the
heavy atom structure factor and another circle with the amplitude of the derivative crystal
as radius we get two possible phases for the native crystal structure factor.
Considerations for Selecting GC/MS or LC/MS
Gas chromatography and mass spectrometry (GC/MS) are an effective combination for the
analysis of volatile chemicals.
Gas chromatography uses a carrier gas to move analytes through acoated, fused silica
capillary.
Separation occurs based on differential partition between the gas phase and the coating
inside the capillary.
GC/MS requires the analyte to be vaporized in order for migration through the capillary to
occur.
Analytes, therefore, must be volatile or amenable to chemical derivatization to render them
volatile.
Electron ionization (EI) is the most commonly used GC/MS ionization technique.
It is very robust and reproducible. It does not suffer from ion Suppression.
Electron ionization causes characteristic mass spectral fragmentation patterns.
Thus, EI spectra from unknowns can be searched against libraries of spectra to achieve
identification.
Liquid chromatography can separate metabolites that are not volatile and have not been
derivatized. As a result, LC/MS can analyze a much wider range of chemical species than
GC/MS. Electrospray ionization (ESI) is most commonly used in LC/MS.
Unlike EI for GC/MS, ion suppression can occur so co-eluting compounds may be underestimated or not
detected at all.

5. Therefore, for complex samples, greater separation is necessary for the reliable LC/MS
results.
The Importance of Animals in Biomedical Research
Throughout history, scientists have been solving medical problems, developing new
techniques and treatments, and curing diseases – all by using animals in biomedical
research
Why Use Animal Models?
 Very limited number of studies can be done on humans
 Allows for controlled experiments
 Environmental variables can be controlled
 Dosage/route of exposures can be controlled/varied
 Experiments can be replicated
 Physiology/anatomy can be matched to humans
Why are animals used in Research?
 organs and body systems similar to humans and other animals
 susceptible to the same diseases that affect humans
 short life span allows animals to be studied throughout their entire life
 environment easily controllable to keep experimental variables to a minimum
What animals are used in Research?
 Laboratory mice are used in research more often than any other animal species;
 These mice, plus other rodents such as rats and hamsters, make up more than 90% of
the total number of animals used; and
 Other animal species, including dogs, cats, rabbits, farm animals, fish, frogs, birds,
nonhuman primates, and many others, make up the remaining 10% of animals used
in research.
“Natural Models”
Good - Most related to the natural disease, most likely to portray all aspects of the disease
Bad - Restrictive in terms of time and types of animals used, also in terms of animals
developing illnesses (in aging studies).
1)Expensive 2) Long term studies 3) Restrictive at an experimental level
“Semi-Natural Models”:
Good – Usually good models to study disease because they are based on phenotypical
presentations of the disease.
Bad – Often lack appropriate controls because they are bred for a phenotype and the
genetics are unknown.
Lesion Models
Good - to study the function of a structure and how the loss of that structure affects other
cell populations
Bad - because most diseases are not specific to a structure or a cell population – High
mortality & variability
Chemical Models

6. Good - to study selective depletion of specific cell populations and examine the role of
transmitters / inducers in disease, more specific than lesions and sometimes leads to full
phenotype expression
Bad - because it is artificially induced and is incomplete with regards to phenotype
(pathology/not pathology), it does not always translate to the real population
Transgenic Models
Good - excellent to study the role of genes and genetic mutations in disease
Bad - because diseases like all biological events are a mix of environment and genetics so a
lot of times the genetic mutations do not recapitulate the disease.
Also, while some of the genetics are highly conserved a mouse/fish/fly/worm do not
respond the same way than humans do nor do animals of the same species!!!
(BACKGROUNDS!!!)
Alternative to Animals in Research
Cell culture and computer-generated models Cell Culture
 Preliminary screen to check the toxicity of substances
 Can answer fundamental questions about biology
 Cannot provide information about potential impacts on entire living organism
Alternatives to Animal Use in Research: A suitable replacement for animals should:
 Reliably predict biological phenomena
 Be at least as good (or better!) and a consistent model for risk assessment in humans
as well as in animals
 Be extensively validated, tested and accepted by regulatory agencies as suitable
substitutes
Model organisms are found among Prokaryotes, Protists , Fungi, Plants and Animals.
 Among the prokaryotes : Escherichia coli is the classical model of molecular biology,
the favorite organism to study transcription, translation, recombination, DNA repair
and the regulation of gene activity.
 The human parasite Plasmodium, the causative agent of malaria, and the genetic
model organisms such as Dictyostelium and Volvox are the most prominent in the
protists
 In the fungi group several models of great importance for genetics like
Saccharomyces cerevisiae Schizosaccharomyces pombe and Neurospora crassa.
 Yeasts, which are unicellular ascomycotan fungi, have proved to be extremely useful
organisms for the study of basic eukaryotic phenomena (genetic recombination,
macromolecular synthesis, metabolic regulation, cell cycle and general cell biology)
 The most prominent animal model organisms are fruit flies (Drosophila
melanogaster) , mouse (Mus musculus), nematode (Caenorhabditis elegans) .
 The regenerative capacity of stem cells offers unprecedented opportunities for
developing medical therapies for debilitating diseases and represents a source of
new, healthy tissue to treat or replace diseased or injured human organs.
 They are an excellent model that will help us to understand how they transform into
the myriad of specialized cells that make us what we are.

7. Flow Cytometry
Applications: The uses of flow in research has boomed since the mid-1980s, and is now the
gold standard for a variety of applications.
Immunophenotyping DNA cell cycle/tumor ploidy Membrane potential
Ion flux Cell viability Intracellular protein staining
pH changes Cell tracking and proliferation Sorting
Redox state Chromatin structure Total protein
Lipids Surface charge Membrane fusion/runover
Enzyme activity Oxidative metabolism Sulfhydryl groups/glutathione
DNA synthesis DNA degradation Gene expression
Phagocytosis Microparticle analysis
What Is Flow Cytometry? Flow ~ motion, Cyto ~ cell Metry ~ measure
Measuring both intrinsic and extrinsic properties of cells while in a moving fluid stream
What Happens in a Flow Cytometer?
• Cells in suspension flow single file
• through a focused laser where they scatter light and emit fluorescence that is
filtered,
• measured, then converted to digitized values that are stored in a file
• which can then be analyzed and interpreted within specialized software.
Fluidics Interrogation Electronics Interpretation
• The Fluidics System “Cells in suspension flow single file”
• Cells must flow one-by-one into the Cytometer to do single cell analysis
• Accomplished through a pressurized laminar flow system.
• The sample is injected into a sheath fluid as it passes through a small orifice (50µm-
300µm)
Interrogation : Light source needs to be focused on the same point where cells are focused.
Light source 99%=Lasers
Lasers : Light amplification by stimulated emission of radiation
• Lasers provide a single wavelength of light (monochromatic)
• They can provide milliwatts to watts of power
• Low divergence

8. • Provide coherent light
• Gas, dye, or solid state
Coherent: all emitting photons have same wavelength, phase and direction as stimulation
photons
END
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