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Contents
Contributing Authors vii General Acknowledgments xiii

Associate Authors viii How to Contribute xv
Faculty Advisors ix How to Use This Book xvii
Preface xi Selected USMLE Laboratory Values xviii
Special Acknowledgments xii First Aid Checklist for the USMLE Step 1 xx
` SECTION I G U I D E TO E F F I C I E N T E X A M P R E PA R AT I O N 1
Introduction 2 Test-Taking Strategies 19

USMLE Step 1—The Basics 2 Clinical Vignette Strategies 21
Learning Strategies 11 If You Think You Failed 22
Timeline for Study 14 Testing Agencies 22
Study Materials 18 References 23
` SECTION I SUPPLEMENT S P E C I A L S I T UAT I O N S 25
` SECTION II HIGH-YIELD GENERAL PRINCIPLES 27
How to Use the Database 28 Pathology 203

Biochemistry 31 Pharmacology 229
Immunology 93 Public Health Sciences 257
Microbiology 121
FAS1_2022_00_Frontmatter.indd 5 11/10/21 10:50 AM

` SECTION III H I G H - Y I E L D O R G A N S YS T E M S 281
Approaching the Organ Systems 282 Neurology and Special Senses 503
Cardiovascular 285 Psychiatry 575
Endocrine 331 Renal 601
Gastrointestinal 365 Reproductive 635
Hematology and Oncology 411 Respiratory 683
Musculoskeletal, Skin, and Connective Tissue 453 Rapid Review 713
` SECTION IV TO P - R AT E D R E V I E W R E S O U R C E S 7 37
How to Use the Database 738 Biochemistry 742

Question Banks 740 Cell Biology and Histology 742
Web and Mobile Apps 740 Microbiology and Immunology 742
Comprehensive 741 Pathology 743
Anatomy, Embryology, and Neuroscience 741 Pharmacology 743
Behavioral Science 742 Physiology 744
`
Abbreviations and Symbols 745 Index 771
Image Acknowledgments 753 About the Editors 828
vi
FAS1_2022_00_Frontmatter.indd 6 11/10/21 10:50 AM

HIGH-YIELD PRINCIPLES IN
Biochemistry
“The nitrogen in our DNA, the calcium in our teeth, the iron in our blood, ` Molecular 32
the carbon in our apple pies were made in the interiors of collapsing stars.
We are made of starstuff.” ` Cellular 44
—Carl Sagan
` Laboratory Techniques 50
“Biochemistry is the study of carbon compounds that crawl.”
—Mike Adams ` Genetics 54
“We think we have found the basic mechanism by which life comes from ` Nutrition 63
life.”
—Francis H. C. Crick ` Metabolism 71
DNA was the first three-dimensional Xerox machine.

—Kenneth Ewart Boulding
This high-yield material includes molecular biology, genetics, cell

biology, and principles of metabolism (especially vitamins, cofactors,
minerals, and single-enzyme-deficiency diseases). When studying
metabolic pathways, emphasize important regulatory steps and enzyme
deficiencies that result in disease, as well as reactions targeted by
pharmacologic interventions. For example, understanding the defect
in Lesch-Nyhan syndrome and its clinical consequences is higher yield
than memorizing every intermediate in the purine salvage pathway.
Do not spend time learning details of organic chemistry, mechanisms, or

physical chemistry. Detailed chemical structures are infrequently tested;
however, many structures have been included here to help students
learn reactions and the important enzymes involved. Familiarity with
the biochemical techniques that have medical relevance—such as
ELISA, immunoelectrophoresis, Southern blotting, and PCR—is
useful. Review the related biochemistry when studying pharmacology or
genetic diseases as a way to reinforce and integrate the material.
31
FAS1_2022_01-Biochem.indd 31 11/4/21 11:59 AM

32 SEC TION II Biochemistry  BIOCHEMISTRY—Molecular
` BIOCHEMISTRY—MOLECULAR
Chromatin structure DNA exists in the condensed, chromatin form to

fit into the nucleus. DNA loops twice around a
histone octamer to form a nucleosome (“beads
DNA double-helix on a string”). H1 binds to the nucleosome
and to “linker DNA,” thereby stabilizing the
chromatin fiber.
H1 histone DNA has ⊝ charge from phosphate groups.
(linker)
DNA Histones are large and have ⊕ charge from
lysine and arginine.
In mitosis, DNA condenses to form
Nucleosome Euchromatin Supercoiled chromosomes. DNA and histone synthesis
(H2A, H2B, structure occurs during S phase.
H3, H4) 2 Heterochromatin Mitochondria have their own DNA, which is
circular and does not utilize histones.
Metaphase
chromosome
Heterochromatin Condensed, appears darker on EM (labeled H Heterochromatin = highly condensed.

A
in A ; Nu, nucleolus). Sterically inaccessible, Barr bodies (inactive X chromosomes) may be
E thus transcriptionally inactive. methylation, visible on the periphery of nucleus.
H acetylation.
Nu
Euchromatin Less condensed, appears lighter on EM (labeled Eu = true, “truly transcribed.”

E in A ). Transcriptionally active, sterically Euchromatin is expressed.
accessible.
DNA methylation Changes the expression of a DNA segment DNA is methylated in imprinting.
without changing the sequence. Involved with Methylation within gene promoter (CpG islands)
aging, carcinogenesis, genomic imprinting, typically represses (silences) gene transcription.
transposable element repression, and X CpG methylation makes DNA mute.
chromosome inactivation (lyonization). Dysregulated DNA methylation is implicated in
Fragile X syndrome.
Histone methylation Usually causes reversible transcriptional Histone methylation mostly makes DNA mute.
suppression, but can also cause activation Lysine and arginine residues of histones can be
depending on location of methyl groups. methylated.
Histone acetylation Removal of histone’s ⊕ charge relaxed DNA Thyroid hormone receptors alter thyroid
coiling transcription. hormone synthesis by acetylation.
Histone acetylation makes DNA active.
Histone deacetylation Removal of acetyl groups tightened DNA Histone deacetylation may be responsible for the
coiling transcription. altered gene expression in Huntington disease.

Biochemistry  BIOCHEMISTRY—Molecular SEC TION II 33
Nucleotides Nucleoside = base + (deoxy)ribose (sugar).

Nucleotide = base + (deoxy)ribose + phosphate; 5′ end of incoming nucleotide bears the
linked by 3′-5′ phosphodiester bond. triphosphate (energy source for the bond).
α-Phosphate is target of 3′ hydroxyl attack.
Purines (A,G)—2 rings. Pure As Gold.

Pyrimidines (C,U,T)—1 ring. CUT the pyramid.
Thymine has a methyl.
Deamination reactions: C-G bond (3 H bonds) stronger than A-T bond
Cytosine uracil (2 H bonds). C-G content melting
Adenine hypoxanthine temperature of DNA. “C-G bonds are like
Guanine xanthine Crazy Glue.”
5-methylcytosine thymine
Amino acids necessary for purine synthesis (cats
Uracil found in RNA; thymine in DNA. purr until they GAG):
Methylation of uracil makes thymine. Glycine
Aspartate
Glutamine
Purine (A, G) Pyrimidine (C, U, T)
Nucleoside
CO2 Carbamoyl Aspartate
Aspartate Glycine phosphate
C N C
Phosphate
N C N C
C N10–Formyl- O-
O P O-
C C tetrahydrofolate C C
N N N N O
N10–Formyl- Nitrogenous base CH₂

Glutamine
tetrahydrofolate
Deoxyribose sugar
Nucleotide

De novo pyrimidine Various immunosuppressive, antineoplastic, and antibiotic drugs function by interfering with
and purine synthesis nucleotide synthesis:
Pyrimidine base production Purine base production or Pyrimidine synthesis:
(requires aspartate) Ribose 5-P reuse from salvage pathway Leflunomide: inhibits dihydroorotate
(de novo requires aspartate, dehydrogenase
Glutamine + CO2 glycine, glutamine, and THF)
2 ATP
5-fluorouracil (5-FU) and its prodrug
CPS2 (carbamoyl
phosphate capecitabine: form 5-F-dUMP, which inhibits
2 ADP + Pi + synthetase II) PRPP (phosphoribosyl
Glutamate pyrophosphate) synthetase thymidylate synthase ( dTMP)
Purine synthesis:
Carbamoyl 6-mercaptopurine (6-MP) and its prodrug
phosphate
Aspartate azathioprine: inhibit de novo purine
Leflunomide
6-MP, synthesis; azathioprine is metabolized via
PRPP
Orotic azathioprine purine degradation pathway and can lead to
acid
immunosuppression when administered with
UMP synthase UMP Mycophenolate, xanthine oxidase inhibitor
(impaired in IMP ribavirin
orotic aciduria) UDP Mycophenolate and ribavirin: inhibit inosine
ctas ide
reduucleot
Hydroxyurea AMP GMP monophosphate dehydrogenase

e
n
Purine and pyrimidine synthesis:

Ribo
dUDP CTP
Hydroxyurea: inhibits ribonucleotide
reductase
N5N10- dUMP
Methotrexate (MTX), trimethoprim (TMP),
Thymidylate
methylene THF
synthase
5-FU, and pyrimethamine: inhibit dihydrofolate

THF capecitabine
Dihydrofolate
DHF reductase ( deoxythymidine monophosphate
reductase dTMP [dTMP]) in humans (methotrexate),
bacteria (trimethoprim), and protozoa
MTX, TMP,
pyrimethamine (pyrimethamine)
CPS1 = m1tochondria, urea cycle, found in liver

and kidney cells
CPS2 = cytwosol, pyrimidine synthesis, found in
most cells

Purine salvage deficiencies
Nucleic acids Ribose 5-phosphate Nucleic acids

PRPP synthetase De novo synthesis
Nucleotides GMP IMP AMP
Cladribine, pentostatin
Lesch-Nyhan
syndrome
ADA
HGPRT APRT
Nucleosides Guanosine Inosine Adenosine
SCID
PRPP
Free bases Guanine PRPP Hypoxanthine Adenine
XO
Allopurinol
Xanthine
Febuxostat Degradation and salvage
XO
Uric acid
Urate oxidase (rasburicase)
Allantoin Excretion
ADA, adenosine deaminase; APRT, adenine phosphoribosyltransferase; HGPRT, hypoxanthine guanine phosphoribosyltransferase, XO, xanthine
oxidase; SCID, severe combined immune deficiency (autosomal recessive inheritance)
Adenosine deaminase ADA is required for degradation of adenosine One of the major causes of autosomal recessive
deficiency and deoxyadenosine. ADA dATP SCID.
ribonucleotide reductase activity
DNA precursors in cells lymphocytes.
Lesch-Nyhan Defective purine salvage due to absent HGPRT, HGPRT:
syndrome which converts hypoxanthine to IMP and Hyperuricemia
guanine to GMP. Compensatory in purine Gout
synthesis ( PRPP amidotransferase activity) Pissed off (aggression, self-mutilation)
excess uric acid production. X-linked Red/orange crystals in urine
recessive. Tense muscles (dystonia)
Findings: intellectual disability, self-mutilation, Treatment: allopurinol, febuxostat.
aggression, hyperuricemia (red/orange “sand”
[sodium urate crystals] in diaper), gout,
dystonia, macrocytosis.
Genetic code features

Unambiguous Each codon specifies only 1 amino acid.
Degenerate/ Most amino acids are coded by multiple codons. Exceptions: methionine (AUG) and tryptophan
redundant Wobble—codons that differ in 3rd (“wobble”) (UGG) encoded by only 1 codon.
position may code for the same tRNA/amino
acid. Specific base pairing is usually required
only in the first 2 nucleotide positions of
mRNA codon.
Commaless, Read from a fixed starting point as a continuous Exceptions: some viruses.
nonoverlapping sequence of bases.
Universal Genetic code is conserved throughout Exception in humans: mitochondria.
evolution.

DNA replication Occurs in 5′ 3′ direction (“5ynth3sis”) in continuous and discontinuous (Okazaki fragment) fashion.
Semiconservative. More complex in eukaryotes than in prokaryotes, but shares analogous enzymes.
Origin of Particular consensus sequence in genome AT-rich sequences (such as TATA box regions)
replication A where DNA replication begins. May be single are found in promoters and origins of
(prokaryotes) or multiple (eukaryotes). replication.
Replication fork B Y-shaped region along DNA template where
leading and lagging strands are synthesized.
Helicase C Unwinds DNA template at replication fork. Helicase halves DNA.
Deficient in Bloom syndrome (BLM gene
mutation).
Single-stranded Prevent strands from reannealing or degradation
binding proteins D by nucleases.
DNA Creates a single- (topoisomerase I) or double- In eukaryotes: irinotecan/topotecan inhibit
topoisomerases E (topoisomerase II) stranded break in the helix topoisomerase (TOP) I, etoposide/teniposide
to add or remove supercoils (as needed due to inhibit TOP II.
underwinding or overwinding of DNA). In prokaryotes: fluoroquinolones inhibit TOP II
(DNA gyrase) and TOP IV.
Primase F Makes an RNA primer on which DNA
polymerase III can initiate replication.
DNA polymerase III G Prokaryotes only. Elongates leading strand DNA polymerase III has 5′ 3′ synthesis and
by adding deoxynucleotides to the 3′ end. proofreads with 3′ 5′ exonuclease.
Elongates lagging strand until it reaches Drugs blocking DNA replication often have a
primer of preceding fragment. modified 3′ OH, thereby preventing addition of
the next nucleotide (“chain termination”).
DNA polymerase I H Prokaryotes only. Degrades RNA primer; Same functions as DNA polymerase III, also
replaces it with DNA. excises RNA primer with 5′ 3′ exonuclease.
DNA ligase I Catalyzes the formation of a phosphodiester Joins Okazaki fragments.
bond within a strand of double-stranded DNA. Ligase links DNA.
Telomerase Eukaryotes only. A reverse transcriptase (RNA- Upregulated in progenitor cells and also often in
dependent DNA polymerase) that adds DNA cancer; downregulated in aging and progeria.
(TTAGGG) to 3′ ends of chromosomes to avoid Telomerase TAGs for Greatness and Glory.
loss of genetic material with every duplication.
G 3'
E
DNA polymerase III 5'
Topoisomerase
C A
Helicase Origin of replication
Leading strand
B
Replication fork Lagging strand 3'
Okazaki fragment 5'
D
A
Area of interest Single-stranded RNA primer
Origin of replication binding protein
Leading strand Lagging strand I
F DNA ligase
Fork Fork Primase
movement movement
G
DNA polymerase III H
Lagging strand Leading strand
DNA polymerase I

DNA repair
Double strand
Nonhomologous end Brings together 2 ends of DNA fragments to 5´
Double strand break
3´ 5´
Double strand break
joining repair double-stranded breaks. 3´ 5´ 3´

5´
3´
Homology not required. Part of the DNA may be
lost or translocated. Nonhomologous end joining
Homologous Requires 2 homologous DNA duplexes. DoubleAstrand break Double strand break
5´ 3´ 5´ 3´
recombination strand from damaged dsDNA 3´ is repaired 5´ 3´
5´
5´
3´
using a complementary strand from intact 3´ 5´
homologous dsDNA as a template. Homologous recombination

Nonhomologous end joining
Defective in breast/ovarian cancers with BRCA1
or BRCA2 mutations and in Fanconi anemia.
Restores duplexes accurately without loss of
nucleotides. Homologous recombination
Single strand
Nucleotide excision Specific endonucleases remove the Occurs in G1 phase of cell cycle.
repair oligonucleotides containing damaged bases; Defective in xeroderma pigmentosum
DNA polymerase and ligase fill and reseal the (inability to repair DNA pyrimidine dimers
gap, respectively. Repairs bulky helix-distorting caused by UV exposure). Presents with dry
lesions (eg, pyrimidine dimers). skin, photosensitivity, skin cancer.
Base excision repair Base-specific Glycosylase removes altered base Occurs throughout cell cycle.
and creates AP site (apurinic/apyrimidinic). Important in repair of spontaneous/toxic
One or more nucleotides are removed by deamination.
AP-Endonuclease, which cleaves 5′ end. AP- “GEL Please.”
Lyase cleaves 3′ end. DNA Polymerase-β fills
the gap and DNA ligase seals it.
Mismatch repair Mismatched nucleotides in newly synthesized Occurs predominantly in S phase of cell cycle.
strand are removed and gap is filled and Defective in Lynch syndrome (hereditary
resealed. nonpolyposis colorectal cancer [HNPCC]).
UV exposure Pyrimidine dimer Deaminated C

T T
U G
A A G A
AP U
site
TT Endonucleases remove Glycosylase removes base Mismatched segment
G
damaged segment G removed
(AP site)
A A A
Endonuclease and lyase
G remove backbone segment
Newly replaced segment
T T C T
A A G A
Nucleotide excision repair Base excision repair Mismatch repair

Mutations in DNA Degree of change: silent << missense < nonsense < frameshift. Single nucleotide substitutions are
repaired by DNA polymerase and DNA ligase. Types of single nucleotide (point) mutations:
Transition—purine to purine (eg, A to G) or pyrimidine to pyrimidine (eg, C to T).
Transversion—purine to pyrimidine (eg, A to T) or pyrimidine to purine (eg, C to G).
Single nucleotide substitutions
Silent mutation Codes for same (synonymous) amino acid; often involves 3rd position of codon (tRNA wobble).
Missense mutation Results in changed amino acid (called conservative if new amino acid has similar chemical
structure). Examples: sickle cell disease (substitution of glutamic acid with valine).
Nonsense mutation Results in early stop codon (UGA, UAA, UAG). Usually generates nonfunctional protein. Stop the
nonsense!
Other mutations
Frameshift mutation Deletion or insertion of any number of nucleotides not divisible by 3 misreading of all
nucleotides downstream. Protein may be shorter or longer, and its function may be disrupted or
altered. Examples: Duchenne muscular dystrophy, Tay-Sachs disease.
Splice site mutation Retained intron in mRNA protein with impaired or altered function. Examples: rare causes of
cancers, dementia, epilepsy, some types of β-thalassemia, Gaucher disease, Marfan syndrome.
Original Silent Missense Nonsense Frameshift Frameshift
sequence mutation mutation mutation insertion deletion
T G
Coding DNA
5´
GAG GAA GTG TAG GA G GA C 3´
mRNA codon
5´
GAG GAA GUG UAG GAU GAC 3´
Amino acid Glu Glu Val Stop Asp Asp
Altered amino acids
Lac operon Classic example of a genetic response to an environmental change. Glucose is the preferred
metabolic substrate in E coli, but when glucose is absent and lactose is available, the lac operon is
activated to switch to lactose metabolism. Mechanism of shift:
Low glucose adenylate cyclase activity generation of cAMP from ATP activation of
catabolite activator protein (CAP) transcription.
High lactose unbinds repressor protein from repressor/operator site transcription.
Genes
CAP
Adenylate Lacl site P O LacZ LacY LacA
CAP cAMP cyclase Glucose DNA 5′ 3′
AUG AUG AUG

Messenger RNA
Binds CAP site, ATP
induces transcription
RNA
Lac operon STATE CAP polymerase
Low glucose
Lactose available
Lac genes strongly expressed
Lacl CAP site Promoter Operator LacZ LacY LacA Repressor protein
High glucose
o r, Lactose unavailable
e ra t o n Lac genes not expressed
Binds opnscripti
blocks tra
Low glucose
Repressor Lactose unavailable Lac genes not expressed
protein CAP
High glucose
site P O
Lactose available Very low (basal) expression
Allolactose Inactivated
(inducer) repressor

Functional Enhancer/
silencer Promoter 5´ UTR Open reading frame 3´ UTR Silencer
organization of a
eukaryotic gene Exon Intron Exon Intron Exon
DNA 5´ CAAT TATAAA GT AG GT AG AATAAA 3´

(coding strand)
CAAT Box TATA Box
Polyadenylation signal
Transcription start
Transcription
Exon Intron Exon Intron Exon
Pre-mRNA GU AG GU AG AAUAAA
Splicing
5´ cap
Protein coding region
Mature AAUAAA AAAAAA
mRNA
AUG start codon Stop
Poly-A tail
Translation
Protein
Regulation of gene expression

Promoter Site where RNA polymerase II and multiple Promoter mutation commonly results in
other transcription factors bind to DNA dramatic in level of gene transcription.
upstream from gene locus (AT-rich upstream
sequence with TATA and CAAT boxes, which
differ between eukaryotes and prokaryotes).
Enhancer DNA locus where regulatory proteins Enhancers and silencers may be located close to,
(“activators”) bind, increasing expression of a far from, or even within (in an intron) the gene
gene on the same chromosome. whose expression they regulate.
Silencer DNA locus where regulatory proteins
(“repressors”) bind, decreasing expression of a
gene on the same chromosome.
Epigenetics Changes made to gene expression (heritable Primary mechanisms of epigenetic change
mitotically/meiotically) without a change in include DNA methylation, histone
underlying DNA sequence. modification, and noncoding RNA.
RNA processing Initial transcript is called heterogeneous nuclear mRNA is transported out of nucleus to be
(eukaryotes) RNA (hnRNA). hnRNA is then modified and translated in cytosol.
becomes mRNA. mRNA quality control occurs at cytoplasmic
The following processes occur in the nucleus: processing bodies (P-bodies), which contain
Cap Coding
5'
Capping of 5′ end (addition of exonucleases, decapping enzymes, and
Gppp 7-methylguanosine cap; cotranscriptional) microRNAs; mRNAs may be degraded or
3' Polyadenylation of 3′ end (∼200 A’s poly-A stored in P-bodies for future translation.
HO-AAAAA
Tail
tail; posttranscriptional) Poly-A polymerase does not require a template.
Splicing out of introns (posttranscriptional) AAUAAA = polyadenylation signal. Mutation
Capped, tailed, and spliced transcript is called in polyadenylation signal early degradation
mRNA. prior to translation.

RNA polymerases
Eukaryotes RNA polymerase I makes rRNA, the most I, II, and III are numbered in the same order
common (rampant) type; present only in that their products are used in protein
nucleolus. synthesis: rRNA, mRNA, then tRNA.
RNA polymerase II makes mRNA (massive), α-amanitin, found in Amanita phalloides (death
microRNA (miRNA), and small nuclear RNA cap mushrooms), inhibits RNA polymerase II.
(snRNA). Causes dysentery and severe hepatotoxicity if
RNA polymerase III makes 5S rRNA, tRNA ingested.
(tiny). Dactinomycin inhibits RNA polymerase in both
No proofreading function, but can initiate prokaryotes and eukaryotes.
chains. RNA polymerase II opens DNA at
promoter site.
Prokaryotes 1 RNA polymerase (multisubunit complex) Rifamycins (rifampin, rifabutin) inhibit DNA-
makes all 3 kinds of RNA. dependent RNA polymerase in prokaryotes.
Splicing of pre-mRNA Part of process by which precursor mRNA (pre-mRNA) is transformed into mature mRNA. Introns
typically begin with GU and end with AG. Alterations in snRNP assembly can cause clinical
disease; eg, in spinal muscular atrophy, snRNP assembly is affected due to SMN protein
congenital degeneration of anterior horns of spinal cord symmetric weakness (hypotonia, or
“floppy baby syndrome”).
snRNPs are snRNA bound to proteins (eg, Smith [Sm]) to form a spliceosome that cleaves pre-
mRNA. Anti-U1 snRNP antibodies are associated with SLE, mixed connective tissue disease,
other rheumatic diseases.
Primary transcript combines with 5′ splice site U1 snRNP Branch point 3′ splice site
small nuclear ribonucleoproteins
U2 snRNP
(snRNPs) and other proteins to
form spliceosome. 5′ O P GU A AG P O 3′
Exon 1 Intron Exon 2
Spliceosome
Cleavage at 5′ splice site; lariat-

shaped (loop) intermediate is
generated. UG
P A
OH 3′ AG P O
Exon 1 Exon 2
Mature mRNA
Cleavage at 3′ splice site; lariat
is released to precisely remove
intron and join 2 exons.
P +
Exon 1 Exon 2 UG
P A
AG OH 3′

Introns vs exons Exons contain the actual genetic information Introns are intervening sequences and stay
coding for protein or functional RNA. in the nucleus, whereas exons exit and are
Introns do not code for protein, but are expressed.
important in regulation of gene expression. Alternative splicing—can produce a variety
Different exons are frequently combined by of protein products from a single hnRNA
alternative splicing to produce a larger number (heterogenous nuclear RNA) sequence (eg,
of unique proteins. transmembrane vs secreted Ig, tropomyosin
variants in muscle, dopamine receptors in the
brain, host defense evasion by tumor cells).
Exon 1 Exon 2 Exon 3 Exon 4 Exon 5 Exon 6

5′ 3′
DNA 3′ 5′
Transcription
hnRNA 5′ 3′
1 2 3 4 5 6
Splicing Alternative splicing
mRNA 5′ 3′ 5′ 3′ 5′ 3′
1 2 4 5 6 1 3 5 6 1 3 4 5 6
Translation
1 5 1 5 1 5
Proteins 6 6 6
4 4
2 3 3

tRNA
Structure 75–90 nucleotides, 2º structure, cloverleaf form, anticodon end is opposite 3′ aminoacyl end. All
tRNAs, both eukaryotic and prokaryotic, have CCA at 3′ end along with a high percentage of
chemically modified bases. The amino acid is covalently bound to the 3′ end of the tRNA. CCA
Can Carry Amino acids.
T-arm: contains the TΨC (ribothymidine, pseudouridine, cytidine) sequence necessary for tRNA-
ribosome binding. T-arm Tethers tRNA molecule to ribosome.
D-arm: contains Dihydrouridine residues necessary for tRNA recognition by the correct aminoacyl-
tRNA synthetase. D-arm allows Detection of the tRNA by aminoacyl-tRNA synthetase.
Attachment site: 3′-ACC-5′ is the amino acid ACCeptor site.
Charging Aminoacyl-tRNA synthetase (uses ATP; 1 unique enzyme per respective amino acid) and
binding of charged tRNA to the codon are responsible for the accuracy of amino acid selection.
Aminoacyl-tRNA synthetase matches an amino acid to the tRNA by scrutinizing the amino acid
before and after it binds to tRNA. If an incorrect amino acid is attached, the bond is hydrolyzed.
A mischarged tRNA reads the usual codon but inserts the wrong amino acid.
Structure Charging Pairing
(aminoacylation) (codon-anticodon)
Amino acid Amino acid
O O
Attachment site OH 3´ 3´ 3´
A A A
C C C
C C C
5´ 5´ 5´
IF2
T-arm
D-arm ATP AMP + PPi (initiation factor)
D D D
C Ψ T
Aminoacyl-tRNA C Ψ T C Ψ T
D D D
synthetase
Variable arm
Anticodon
loop U A C U A C Anticodon (5´-CAU-3´) U A C
Wobble
position C C C A U G A U A C
mRNA
Codon
(5´-AUG-3´)
Start and stop codons

mRNA start codon AUG. AUG inAUGurates protein synthesis.
Eukaryotes Codes for methionine, which may be removed
before translation is completed.
Prokaryotes Codes for N-formylmethionine (fMet). fMet stimulates neutrophil chemotaxis.
mRNA stop codons UGA, UAA, UAG. UGA = U Go Away.
Recognized by release factors. UA A = U Are Away.
UAG = U Are Gone.

Protein synthesis
Initiation 1. Eukaryotic initiation factors (eIFs) identify Eukaryotes: 40S + 60S 80S (even).
the 5′ cap. Prokaryotes: 30S + 50S 70S (prime).
2. eIFs help assemble the 40S ribosomal Synthesis occurs from N-terminus to
subunit with the initiator tRNA. C-terminus.
3. eIFs released when the mRNA and the
ATP—tRNA Activation (charging).
ribosomal 60S subunit assemble with the
GTP—tRNA Gripping and Going places
complex. Requires GTP.
(translocation).
Elongation Aminoacyl-tRNA binds to A site (except for
initiator methionine, which binds the P site), Think of “going APE”:
requires an elongation factor and GTP. A site = incoming Aminoacyl-tRNA.
rRNA (“ribozyme”) catalyzes peptide bond P site = accommodates growing Peptide.
formation, transfers growing polypeptide to E site = holds Empty tRNA as it Exits.
amino acid in A site. Elongation factors are targets of bacterial toxins
Ribosome advances 3 nucleotides toward 3′ (eg, Diphtheria, Pseudomonas).
end of mRNA, moving peptidyl tRNA to P
site (translocation). Shine-Dalgarno sequence—ribosomal binding
site in prokaryotic mRNA. Recognized by 16S
Termination Eukaryotic release factors (eRFs) recognize the
RNA in ribosomal subunit. Enables protein
stop codon and halt translation completed
synthesis initiation by aligning ribosome with
polypeptide is released from ribosome.
start codon so that code is read correctly.
Requires GTP.
60/50S
40/30S
M
R
M
Initiation M M H
Initiator tRNA
U A C
U A C 5´ A U G C A U G A U 3´ U A C G U A
mRNA
E P A
5´ A U G C A U G A U 3´
E P A
S Ribosome moves left to
right along mRNA
M Elongation M
H
U G A
G U A U A C
U A C Q G U A
Termination
A U G C A U G A U A U G C A U G A U
5´ 3´ 5´ 3´
E P A E P A
Posttranslational modifications
Trimming Removal of N- or C-terminal propeptides from zymogen to generate mature protein (eg,
trypsinogen to trypsin).
Covalent alterations Phosphorylation, glycosylation, hydroxylation, methylation, acetylation, and ubiquitination.
Chaperone protein Intracellular protein involved in facilitating and maintaining protein folding. In yeast, heat
shock proteins (eg, HSP60) are constitutively expressed, but expression may increase with high
temperatures, acidic pH, and hypoxia to prevent protein denaturing/misfolding.

44 SEC TION II Biochemistry  BIOCHEMISTRY—Cellular
` BIOCHEMISTRY—CELLULAR
Cell cycle phases Checkpoints control transitions between phases of cell cycle. This process is regulated by cyclins,
cyclin-dependent kinases (CDKs), and tumor suppressors. M phase (shortest phase of cell cycle)
includes mitosis (prophase, prometaphase, metaphase, anaphase, telophase) and cytokinesis
(cytoplasm splits in two). G1 is of variable duration.
REGULATION OF CELL CYCLE
Cyclin-dependent Constitutively expressed but inactive when not bound to cyclin.
kinases
Cyclin-CDK complexes Cyclins are phase-specific regulatory proteins that activate CDKs when stimulated by growth
factors. The cyclin-CDK complex can then phosphorylate other proteins (eg, Rb) to coordinate
cell cycle progression. This complex must be activated/inactivated at appropriate times for cell
cycle to progress.
Tumor suppressors p53 p21 induction CDK inhibition Rb hypophosphorylation (activation) G1-S
progression inhibition. Mutations in tumor suppressor genes can result in unrestrained cell
division (eg, Li-Fraumeni syndrome).
Growth factors (eg, insulin, PDGF, EPO, EGF) bind tyrosine kinase receptors to transition the cell
from G1 to S phase.
CELL TYPES
Permanent Remain in G0, regenerate from stem cells. Neurons, skeletal and cardiac muscle, RBCs.
Stable (quiescent) Enter G1 from G0 when stimulated. Hepatocytes, lymphocytes, PCT, periosteal cells.
Labile Never go to G0, divide rapidly with a short G1. Bone marrow, gut epithelium, skin, hair
Most affected by chemotherapy. follicles, germ cells.
Cell cycle arrest

p21 Cyclin
GO
Cyclin
CDK CDK
DNA damage Rb, p53 modulate G1
p21
G1 restriction point
th
ow
Gr
is
M
es
Li-Fraumeni syndrome
kin
to
(loss of function) Cy
Mito
p53
sis
HPV E6
IN TERPH
BCL-2 BCL-XL
Rb P
DNA
P
P
BAX/BAK
AS
Sy n
Rb E2F S
E
t he
is
s
Caspase activation E2F G2

Gene transcription
Apoptosis
(intrinsic pathway)

Biochemistry  BIOCHEMISTRY—Cellular SEC TION II 45
Rough endoplasmic Site of synthesis of secretory (exported) proteins N-linked glycosylation occurs in the
reticulum and of N-linked oligosaccharide addition to eNdoplasmic reticulum.
lysosomal and other proteins. Mucus-secreting goblet cells of small intestine
Nissl bodies (RER in neurons)—synthesize and antibody-secreting plasma cells are rich in
peptide neurotransmitters for secretion. RER.
Free ribosomes—unattached to any membrane; Proteins within organelles (eg, ER, Golgi bodies,
site of synthesis of cytosolic, peroxisomal, and lysosomes) are formed in RER.
mitochondrial proteins.
Smooth endoplasmic Site of steroid synthesis and detoxification of Hepatocytes and steroid hormone–producing
reticulum drugs and poisons. Lacks surface ribosomes. cells of the adrenal cortex and gonads are rich
Location of glucose-6-phosphatase (last step in in SER.
both glycogenolysis and gluconeogenesis).
Cell trafficking Golgi is distribution center for proteins and lipids from ER to vesicles and plasma membrane.
Posttranslational events in GOlgi include modifying N-oligosaccharides on asparagine, adding
O-oligosaccharides on serine and threonine, and adding mannose-6-phosphate to proteins for
lysosomal and other proteins.
Endosomes are sorting centers for material from outside the cell or from the Golgi, sending it to
lysosomes for destruction or back to the membrane/Golgi for further use.
I-cell disease (inclusion cell disease/mucolipidosis type II)—inherited lysosomal storage disorder
(autosomal recessive); defect in N-acetylglucosaminyl-1-phosphotransferase failure of the
Golgi to phosphorylate mannose residues ( mannose-6-phosphate) on glycoproteins enzymes
secreted extracellularly rather than delivered to lysosomes lysosomes deficient in digestive
enzymes buildup of cellular debris in lysosomes (inclusion bodies). Results in coarse facial
features, gingival hyperplasia, corneal clouding, restricted joint movements, claw hand deformities,
kyphoscoliosis, and plasma levels of lysosomal enzymes. Symptoms similar to but more severe
than Hurler syndrome. Often fatal in childhood.
Key: Signal recognition particle (SRP)—abundant,
brane
cytosolic ribonucleoprotein that traffics
mem polypeptide-ribosome complex from the
Clathrin sma
Pla
Secretory cytosol to the RER. Absent or dysfunctional
vesicle
COPI Late Early SRP accumulation of protein in cytosol.
endosome endosome
COPII Vesicular trafficking proteins

Lysosome COPI: Golgi Golgi (retrograde); cis-Golgi
Retrograde trans ER.
Anterograde COPII: ER cis-Golgi (anterograde). “Two
Golgi (COPII) steps forward (anterograde); one
apparatus (COPI) step back (retrograde).”
Clathrin: trans-Golgi lysosomes; plasma
membrane endosomes (receptor-mediated
cis
endocytosis [eg, LDL receptor activity]).
Rough
endoplasmic
reticulum
Nuclear envelope

46 SEC TION II Biochemistry  BIOCHEMISTRY—Cellular
Peroxisome Membrane-enclosed organelle involved in:

β-oxidation of very-long-chain fatty acids (VLCFA) (strictly peroxisomal process)
α-oxidation of branched-chain fatty acids (strictly peroxisomal process)
Catabolism of amino acids and ethanol
Synthesis of bile acids and plasmalogens (important membrane phospholipid, especially in
white matter of brain)
Zellweger syndrome—autosomal recessive disorder of peroxisome biogenesis due to mutated PEX
genes. Hypotonia, seizures, jaundice, craniofacial dysmorphia, hepatomegaly, early death.
Refsum disease—autosomal recessive disorder of α-oxidation buildup of phytanic acid due
to inability to degrade it. Scaly skin, ataxia, cataracts/night blindness, shortening of 4th toe,
epiphyseal dysplasia. Treatment: diet, plasmapheresis.
Adrenoleukodystrophy—X-linked recessive disorder of β-oxidation due to mutation in ABCD1
gene VLCFA buildup in adrenal glands, white (leuko) matter of brain, testes. Progressive
disease that can lead to adrenal gland crisis, progressive loss of neurologic function, death.
Proteasome Barrel-shaped protein complex that degrades ubiquitin-tagged proteins. Defects in the ubiquitin-
proteasome system have been implicated in some cases of Parkinson disease.
Cytoskeletal elements A network of protein fibers within the cytoplasm that supports cell structure, cell and organelle
movement, and cell division.
TYPE OF FILAMENT PREDOMINANT FUNCTION EXAMPLES
Microfilaments Muscle contraction, cytokinesis Actin, microvilli.
Intermediate Maintain cell structure Vimentin, desmin, cytokeratin, lamins, glial
filaments fibrillary acidic protein (GFAP), neurofilaments.
Microtubules Movement, cell division Cilia, flagella, mitotic spindle, axonal trafficking,
centrioles.
Microtubule Cylindrical outer structure composed of a Drugs that act on microtubules (microtubules
Positive
helical array of polymerized heterodimers get constructed very terribly):
end (+) of α- and β-tubulin. Each dimer has 2 GTP Mebendazole (antihelminthic)
Heterodimer bound. Incorporated into flagella, cilia, mitotic Griseofulvin (antifungal)
spindles. Also involved in slow axoplasmic Colchicine (antigout)
transport in neurons. Vinca alkaloids (anticancer)
Molecular motor proteins—transport cellular Taxanes (anticancer)
cargo toward opposite ends of microtubule. Negative end near nucleus.
Protofilament Retrograde to microtubule (+ −)—dynein. Positive end points to periphery.
Anterograde to microtubule (− +)—kinesin.
Negative Clostridium tetani toxin, herpes simplex virus, Ready? Attack!
end (–) poliovirus, and rabies virus use dynein for
retrograde transport to the neuronal cell body.

Cilia structure Motile cilia consist of 9 doublet + 2 singlet arrangement of microtubules (axoneme) A .
Basal body (base of cilium below cell membrane) consists of 9 microtubule triplets B with no
central microtubules.
Nonmotile (primary) cilia work as chemical signal sensors and have a role in signal transduction
and cell growth control. Dysgenesis may lead to polycystic kidney disease, mitral valve prolapse,
or retinal degeneration.
Axonemal dynein—ATPase that links peripheral 9 doublets and causes bending of cilium by
differential sliding of doublets.
Gap junctions enable coordinated ciliary movement.
A B
Dynein
arm
Microtubule
A
Microtubule
B
Nexin
Doublets
Triplets
Primary ciliary Also called Kartagener syndrome. Autosomal recessive. Dynein arm defect immotile cilia
dyskinesia dysfunctional ciliated epithelia.
Developmental abnormalities due to impaired migration and orientation (eg, situs inversus A , hearing
A
loss due to dysfunctional eustachian tube cilia); recurrent infections (eg, sinusitis, ear infections,
R L bronchiectasis due to impaired ciliary clearance of debris/pathogens); infertility ( risk of ectopic
pregnancy due to dysfunctional fallopian tube cilia, immotile spermatozoa).
Lab findings: nasal nitric oxide (used as screening test).
Sodium-potassium Na+/K+-ATPase is located in the plasma 2 strikes? K, you’re still in. 3 strikes? Nah, you’re
pump membrane with ATP site on cytosolic side. For out!
each ATP consumed, 2 K+ go in to the cell Cardiac glycosides (digoxin and digitoxin)
(pump dephosphorylated) and 3 Na+ go out of directly inhibit Na+/K+-ATPase indirect
the cell (pump phosphorylated). inhibition of Na+/Ca2+ exchange [Ca2+]i
cardiac contractility.
Extracellular
3Na+ 2K+
space
Plasma
membrane
P
Cytosol 2K+
3Na+ ATP ADP P

48 SECTION II Biochemistry  BIOCHEMISTRY—Cellular
Collagen Most abundant protein in the human body. Type I - Skeleton

Extensively modified by posttranslational Type II - Cartilage
modification. Type III - Arteries
Organizes and strengthens extracellular matrix. Type IV - Basement membrane
Types I to IV are the most common types in SCAB
humans.
Type I Most common (90%)—Bone (made by Type I: bone, tendone.
osteoblasts), Skin, Tendon, dentin, fascia, production in osteogenesis imperfecta type I.
cornea, late wound repair.
Type II Cartilage (including hyaline), vitreous body, Type II: cartwolage.
nucleus pulposus.
Type III Reticulin—skin, blood vessels, uterus, fetal Type III: deficient in vascular type of Ehlers-
tissue, early wound repair. Danlos syndrome (threE D).
Type IV Basement membrane/basal lamina (glomerulus, Type IV: under the floor (basement membrane).
cochlea), lens. Defective in Alport syndrome; targeted by
autoantibodies in Goodpasture syndrome.
Myofibroblasts are responsible for secretion
(proliferative stage) and wound contraction.
Collagen synthesis and structure
Fibroblast Preprocollagen S ynthesis—translation of collagen α

Pro α-chain backbone (Gly-X-Y) chains (preprocollagen)—usually Gly-X-Y
Nucleus Hydroxylation of proline and
(X is often proline or lysine and Y is often
OH
OH lysine (requires vitamin C) hydroxyproline or hydroxylysine). Collagen is
Collagen mRNA Sugar
Glycosylation
1/3 glycine; glycine content of collagen is less
Cytoplasm OH
OH variable than that of lysine and proline.
RER
Hydroxylation—hydroxylation
Triple helix formation
Procollagen (“hydroxCylation”) of specific proline
Golgi and lysine residues. Requires vitamin C;
deficiency scurvy.
Exocytosis
Glycosylation—glycosylation of pro-α-chain
Extracellular
space hydroxylysine residues and formation of
Cleavage of procollagen
C- and N-terminals procollagen via hydrogen and disulfide bonds
Tropocollagen (triple helix of 3 collagen α chains). Problems
forming triple helix osteogenesis imperfecta.
Self assembly into
collagen fibrils Exocytosis—exocytosis of procollagen into
extracellular space.
Proteolytic processing—cleavage of
disulfide-rich terminal regions of procollagen
Formation of cross-links
(stabilized by lysyl oxidase) insoluble tropocollagen.
Assembly and alignment—collagen assembles
Collagen fiber
in fibrils and aligns for cross-linking.
Cross-linking—reinforcement of staggered
tropocollagen molecules by covalent lysine-
hydroxylysine cross-linkage (by copper-
containing lysyl oxidase) to make collagen
fibrils. Cross-linking of collagen with age.
Problems with cross-linking Menkes disease.
FAS1_2022_01-Biochem.indd 48 11/8/21 1:04 PM

Osteogenesis Genetic bone disorder (brittle bone May be confused with child abuse.
imperfecta disease) caused by a variety of gene defects Treat with bisphosphonates to fracture risk.
A
(most commonly COL1A1 and COL1A2). Patients can’t BITE:
Most common form is autosomal dominant Bones = multiple fractures
with production of otherwise normal type I I (eye) = blue sclerae
collagen (altered triple helix formation). Teeth = dental imperfections
Upper Manifestations include: Ear = hearing loss
extremity Multiple fractures and bone deformities
B
(arrows in A ) after minimal trauma (eg,
during birth)
Blue sclerae B due to the translucent
connective tissue over choroidal veins
Some forms have tooth abnormalities,
including opalescent teeth that wear easily
due to lack of dentin (dentinogenesis
imperfecta)
Conductive hearing loss (abnormal ossicles)
Ehlers-Danlos Faulty collagen synthesis causing A B

syndrome hyperextensible skin A , hypermobile joints B ,
and tendency to bleed (easy bruising).
Multiple types. Inheritance and severity vary.
Can be autosomal dominant or recessive. May
be associated with joint dislocation, berry and
aortic aneurysms, organ rupture.
Hypermobility type (joint instability): most
common type.
Classical type (joint and skin symptoms):
caused by a mutation in type V collagen (eg,
COL5A1, COL5A2).
Vascular type (fragile tissues including vessels
[eg, aorta], muscles, and organs that are prone
to rupture [eg, gravid uterus]): mutations in
type III procollagen (eg, COL3A1).
Menkes disease X-linked recessive connective tissue disease caused by impaired copper absorption and transport
due to defective Menkes protein ATP7A (Absent copper), vs ATP7B in Wilson disease (copper
Buildup). Leads to activity of lysyl oxidase (copper is a necessary cofactor) defective collagen
cross-linking. Results in brittle, “kinky” hair, growth and developmental delay, hypotonia, risk of
cerebral aneurysms.

50 SEC TION II Biochemistry  Biochemistry—Laboratory Techniques
Elastin Stretchy protein within skin, lungs, large arteries, elastic ligaments, vocal cords, epiglottis,
ligamenta flava (connect vertebrae relaxed and stretched conformations).
Rich in nonhydroxylated proline, glycine, and lysine residues, vs the hydroxylated residues of
collagen.
Single Tropoelastin with fibrillin scaffolding.
elastin Stretch
Relax Cross-link
Cross-linking occurs extracellularly via lysyl oxidase and gives elastin its elastic properties.
molecule
Broken down by elastase, which is normally inhibited by α1-antitrypsin.
α1-Antitrypsin deficiency results in unopposed elastase activity, which can cause COPD.
Marfan syndrome—autosomal dominant (with variable expression) connective tissue disorder
A
affecting skeleton, heart, and eyes. FBN1 gene mutation on chromosome 15 (fifteen) results in
defective fibrillin-1, a glycoprotein that forms a sheath around elastin and sequesters TGF-β.
Findings: tall with long extremities; chest wall deformity (pectus carinatum [pigeon chest] or
pectus excavatum A ); hypermobile joints; long, tapering fingers and toes (arachnodactyly); cystic
medial necrosis of aorta; aortic root aneurysm rupture or dissection (most common cause of
death); mitral valve prolapse; risk of spontaneous pneumothorax.
Homocystinuria—most commonly due to cystathionine synthase deficiency leading to
homocysteine buildup. Presentation similar to Marfan syndrome with pectus deformity, tall
stature, arm:height ratio, upper:lower body segment ratio, arachnodactyly, joint hyperlaxity,
skin hyperelasticity, scoliosis.
Marfan syndrome Homocystinuria
INHERITANCE Autosomal dominant Autosomal recessive
INTELLECT Normal Decreased
VASCULAR COMPLICATIONS Aortic root dilatation Thrombosis
LENS DISLOCATION Upward/temporal (Marfan fans out) Downward/nasal
` BIOCHEMISTRY—LABORATORY TECHNIQUES
Polymerase chain Molecular biology lab procedure used to amplify a desired fragment of DNA. Useful as a diagnostic
reaction tool (eg, neonatal HIV, herpes encephalitis).
5' 3' 5' 3' 5' 3'
5' 3' 3' 5'

DNA primer
dNTP
Repeat
3' 5' 5' 3'
Double-stranded DNA
3' 5' 3' 5' 3' 5'
enaturation—DNA template, DNA primers, a heat-stable DNA polymerase, and

D
deoxynucleotide triphosphates (dNTPs) are heated to ~ 95ºC to separate the DNA strands.
Annealing—sample is cooled to ~ 55ºC. DNA primers anneal to the specific sequence to be
amplified on the DNA template.
Elongation—temperature is increased to ~ 72ºC. DNA polymerase adds dNTPs to the strand to
replicate the sequence after each primer.
Heating and cooling cycles continue until the amount of DNA is sufficient.

Biochemistry  Biochemistry—Laboratory Techniques SEC TION II 51
CRISPR/Cas9 A genome editing tool derived from bacteria. Consists of a guide RNA (gRNA) , which is
complementary to a target DNA sequence, and an endonuclease (Cas9), which makes a single- or
double-strand break at the target site . Imperfectly cut segments are repaired by nonhomologous
end joining (NHEJ) accidental frameshift mutations (“knock-out”) , or a donor DNA
sequence can be added to fill in the gap using homology-directed repair (HDR) .
Potential applications include removing virulence factors from pathogens, replacing disease-causing
alleles of genes with healthy variants (in clinical trials for sickle cell disease), and specifically targeting
tumor cells.
Cas9 gRNA
Donor DNA
NHEJ 3A 3B HDR
+
Frameshift/inactivation Edited sequence

(”knock-out”) (”knock-in”)
Blotting procedures
Southern blot 1. DNA sample is enzymatically cleaved into I: Parents
smaller pieces, which are separated on a gel
PEDIGREE
by electrophoresis, and then transferred to a

membrane.
II: Children
2. Membrane is exposed to labeled DNA probe
that anneals to its complementary strand. Aa Aa aa Aa AA Genotype
3. Resulting double-stranded, labeled piece
SOUTHERN BLOT
of DNA is visualized when membrane is Mutant
exposed to film or digital imager. Normal
Northern blot Similar to Southern blot, except that an RNA

sample is electrophoresed. Useful for studying SNoW DRoP:
mRNA levels, which are reflective of gene Southern = DNA
expression. Northern = RNA
Western = Protein
Western blot Sample protein is separated via gel electrophoresis
Northern blots detect splicing errors.
and transferred to a membrane. Labeled
antibody is used to bind to relevant protein.
Southwestern blot Identifies DNA-binding proteins (eg, c-Jun, Southern (DNA) + Western (protein) =
c-Fos [leucine zipper motif]) using labeled Southwestern (DNA-binding protein).
double-stranded DNA probes.

52 SEC TION II Biochemistry  Biochemistry—Laboratory Techniques
Flow cytometry Laboratory technique to assess size, granularity, Commonly used in workup of hematologic
and protein expression (immunophenotype) of abnormalities (eg, leukemia, paroxysmal
individual cells in a sample. nocturnal hemoglobinuria, fetal
RBCs in pregnant person’s blood) and
immunodeficiencies (eg, CD4+ cell count in
HIV).
Cells are tagged with antibodies specific to Fluorescent

label
surface or intracellular proteins. Antibodies
Antibody
are then tagged with a unique fluorescent
dye. Sample is analyzed one cell at a time by
Anti-CD3 Ab Cell
focusing a laser on the cell and measuring
light scatter and intensity of fluorescence.
Anti-CD8 Ab
Laser
Fluorescence
is detected; Laser makes
tor
labeled cells tec label fluoresce
De
are counted
Data are plotted either as histogram (one
measure) or scatter plot (any two measures, as
shown). In illustration: 104
Cells in left lower quadrant ⊝ for both CD8
and CD3. 103
Cells in right lower quadrant ⊕ for CD8
and ⊝ for CD3. In this example, right
CD3
102
lower quadrant is empty because all
CD8-expressing cells also express CD3. 101
Cells in left upper quadrant ⊕ for CD3 and
⊝ for CD8. 100
100 101 102 103 104
Cells in right upper quadrant ⊕ for both
CD8
CD8 and CD3.
Microarrays Array consisting of thousands of DNA oligonucleotides arranged in a grid on a glass or silicon chip.
The DNA or RNA samples being compared are attached to different fluorophores and hybridized
to the array. The ratio of fluorescence signal at a particular oligonucleotide reflects the relative
amount of the hybridizing nucleic acid in the two samples.
Used to compare the relative transcription of genes in two RNA samples. Can detect single
nucleotide polymorphisms (SNPs) and copy number variants (CNVs) for genotyping, clinical
genetic testing, forensic analysis, and cancer mutation and genetic linkage analysis when DNA is
used.
Enzyme-linked Immunologic test used to detect the presence of either a specific antigen or antibody in a patient’s
immunosorbent assay blood sample. Detection involves the use of an antibody linked to an enzyme. Added substrate
reacts with the enzyme, producing a detectable signal. Can have high sensitivity and specificity,
but is less specific than Western blot. Often used to screen for HIV infection.

Biochemistry  Biochemistry—Laboratory Techniques SEC TION II 53
Karyotyping Colchicine is added to cultured cells to halt A

chromosomes in metaphase. Chromosomes
are stained, ordered, and numbered according
to morphology, size, arm-length ratio, and
banding pattern (arrows in A point to extensive
abnormalities in a cancer cell).
Can be performed on a sample of blood, bone
marrow, amniotic fluid, or placental tissue.
Used to diagnose chromosomal imbalances
(eg, autosomal trisomies, sex chromosome
disorders).
Fluorescence in situ Fluorescent DNA or RNA probe binds A

hybridization to specific gene or other site of interest
on chromosomes (arrows in A point to
abnormalities in a cancer cell; each fluorescent
color represents a chromosome-specific probe).
Used for specific localization of genes and direct
visualization of chromosomal anomalies.
Microdeletion—no fluorescence on a
chromosome compared to fluorescence at
the same locus on the second copy of that
chromosome.
Translocation—fluorescence signal that
corresponds to one chromosome is found in
a different chromosome (two white arrows in
A show fragments of chromosome 17 that
have translocated to chromosome 19).
Duplication—a second copy of a
chromosome, resulting in a trisomy or
tetrasomy (two blue arrows in A duplicated
chromosomes 8, resulting in a tetrasomy).
Molecular cloning Production of a recombinant DNA molecule in a bacterial host. Useful for production of human
proteins in bacteria (eg, human growth hormone, insulin).
Steps:
1. Isolate eukaryotic mRNA (post-RNA processing) of interest.
2. Add reverse transcriptase (an RNA-dependent DNA polymerase) to produce complementary
DNA (cDNA, lacks introns).
3. Insert cDNA fragments into bacterial plasmids containing antibiotic resistance genes.
4. Transform (insert) recombinant plasmid into bacteria.
5. Surviving bacteria on antibiotic medium produce cloned DNA (copies of cDNA).

54 SEC TION II Biochemistry  BIOCHEMISTRY—Genetics
Gene expression Transgenic strategies in mice involve: Knock-out = removing a gene, taking it out.
modifications Random insertion of gene into mouse Knock-in = inserting a gene.
genome
Targeted insertion or deletion of gene Random insertion—constitutive expression.
through homologous recombination with Targeted insertion—conditional expression.
mouse gene
RNA interference Process whereby small non-coding RNA molecules target mRNAs to inhibit gene expression.
MicroRNA Naturally produced by cell as hairpin structures. Abnormal expression of miRNAs contributes
Loose nucleotide pairing allows broad to certain malignancies (eg, by silencing an
targeting of related mRNAs. When miRNA mRNA from a tumor suppressor gene).
binds to mRNA, it blocks translation of mRNA
and sometimes facilitates its degradation.
Small interfering Usually derived from exogenous dsRNA source Can be produced by transcription or
RNA (eg, virus). Once inside a cell, siRNA requires chemically synthesized for gene “knockdown”
complete nucleotide pairing, leading to highly experiments.
specific mRNA targeting. Results in mRNA
cleavage prior to translation.
` BIOCHEMISTRY—GENETICS
Genetic terms
TERM DEFINITION EXAMPLE
Codominance Both alleles contribute to the phenotype of the Blood groups A, B, AB; α1-antitrypsin
heterozygote. deficiency; HLA groups.
Variable expressivity Patients with the same genotype have varying Two patients with neurofibromatosis type 1 (NF1)
phenotypes. may have varying disease severity.
Incomplete Not all individuals with a disease show the BRCA1 gene mutations do not always result in
penetrance disease. breast or ovarian cancer.
% penetrance × probability of inheriting
genotype = risk of expressing phenotype.
Pleiotropy One gene contributes to multiple phenotypic Untreated phenylketonuria (PKU) manifests with
effects. light skin, intellectual disability, musty body odor.
Anticipation Increased severity or earlier onset of disease in Trinucleotide repeat diseases (eg, Huntington
succeeding generations. disease).
Loss of heterozygosity If a patient inherits or develops a mutation in Retinoblastoma and the “two-hit hypothesis,”
a tumor suppressor gene, the wild type allele Lynch syndrome (HNPCC), Li-Fraumeni
must be deleted/mutated/eliminated before syndrome.
cancer develops. This is not true of oncogenes.
Epistasis The allele of one gene affects the phenotypic Albinism, alopecia.
expression of alleles in another gene.
Aneuploidy An abnormal number of chromosomes; due to Down syndrome, Turner syndrome,
chromosomal nondisjunction during mitosis oncogenesis.
or meiosis.

Biochemistry  BIOCHEMISTRY—Genetics SEC TION II 55
Genetic terms (continued)

TERM DEFINITION EXAMPLE
Dominant negative Exerts a dominant effect. A heterozygote A single mutated p53 tumor suppressor gene
mutation produces a nonfunctional altered protein that results in a protein that is able to bind DNA
also prevents the normal gene product from and block the wild type p53 from binding to the
functioning. promoter.
Linkage Tendency for certain alleles to occur in close
disequilibrium proximity on the same chromosome more or
less often than expected by chance. Measured
in a population, not in a family, and often
varies in different populations.
Mosaicism Presence of genetically distinct cell lines in the McCune-Albright syndrome—due to Gs-protein
A
same individual. activating mutation. Presents with unilateral
Somatic mosaicism—mutation arises from café-au-lait spots A with ragged edges,
mitotic errors after fertilization and propagates polyostotic fibrous dysplasia (bone is replaced
through multiple tissues or organs. by collagen and fibroblasts), and at least one
Germline (gonadal) mosaicism—mutation only endocrinopathy (eg, precocious puberty).
in egg or sperm cells. If parents and relatives Lethal if mutation occurs before fertilization
do not have the disease, suspect gonadal (or (affecting all cells), but survivable in patients
germline) mosaicism. with mosaicism.
Locus heterogeneity Mutations at different loci result in the same Albinism, retinitis pigmentosa, familial
disease. hypercholesteremia.
Allelic heterogeneity Different mutations in the same locus result in β-thalassemia.
the same disease.
Heteroplasmy Presence of both normal and mutated mtDNA passed from mother to all children.
mtDNA, resulting in variable expression in
mitochondrially inherited disease.
Uniparental disomy Offspring receives 2 copies of a chromosome from Uniparental is euploid (correct number of
1 parent and no copies from the other parent. chromosomes). Most occurrences of uniparental
HeterodIsomy (heterozygous) indicates a meiosis disomy (UPD) normal phenotype. Consider
I error. IsodIsomy (homozygous) indicates a isodisomy in an individual manifesting a
meiosis II error or postzygotic chromosomal recessive disorder when only one parent is a
duplication of one of a pair of chromosomes, carrier. Examples: Prader-Willi and Angelman
and loss of the other of the original pair. syndromes.
Hardy-Weinberg If p and q represent the frequencies of alleles Hardy-Weinberg law assumptions include:
population genetics A and a, respectively, in a population, then No mutation occurring at the locus
p + q = 1: Natural selection is not occurring
A (p) a (q)
p2 = frequency of homozygosity for allele A Completely random mating
AA Aa
A (p)
(p2) (pq) q2 = frequency of homozygosity for allele a No net migration
Aa aa
2pq = frequency of heterozygosity (carrier Large population
a (q) frequency, if an autosomal recessive disease) If a population is in Hardy-Weinberg
(pq) (q2)
Therefore, the sum of the frequencies of these equilibrium, then the values of p and q remain
genotypes is p2 + 2pq + q2 = 1. constant from generation to generation.
The frequency of an X-linked recessive disease
in males = q and in females = q2.

56 SEC TION II Biochemistry  BIOCHEMISTRY—Genetics
Disorders of imprinting Imprinting—one gene copy is silenced by methylation, and only the other copy is expressed
parent-of-origin effects. The expressed copy may be mutated, may not be expressed, or may be
deleted altogether.
Prader-Willi syndrome Angelman syndrome
WHICH GENE IS SILENT? Maternally derived genes are silenced Paternally derived UBE3A is silenced
Disease occurs when the paternal allele is deleted Disease occurs when the maternal allele is
or mutated deleted or mutated
SIGNS AND SYMPTOMS Hyperphagia, obesity, intellectual disability, Seizures, Ataxia, severe Intellectual disability,
hypogonadism, hypotonia inappropriate Laughter
Set SAIL for Angel Island
CHROMOSOMES INVOLVED Chromosome 15 of paternal origin UBE3A on maternal copy of chromosome 15
NOTES 25% of cases are due to maternal uniparental 5% of cases are due to paternal uniparental
disomy disomy
POP: Prader-Willi, Obesity/overeating, Paternal MAMAS: Maternal allele deleted, Angelman
allele deleted syndrome, Mood, Ataxia, Seizures
P = Paternal
M = Maternal
Normal Mutation
Active gene
P M P M
Silenced gene (imprinting)
Gene deletion/mutation
Prader-Willi syndrome
Angelman syndrome

Biochemistry  BIOCHEMISTRY—Genetics SEC TION II 57
Modes of inheritance
Autosomal dominant Often due to defects in structural genes. Many Often pleiotropic (multiple apparently unrelated
generations, both males and females are affected. effects) and variably expressive (different
A a between individuals). Family history crucial
to diagnosis. With one affected (heterozygous)
a Aa aa
parent, each child has a 50% chance of being
a Aa aa
affected.
Autosomal recessive With 2 carrier (heterozygous) parents, on average: Often due to enzyme deficiencies. Usually seen
each child has a 25% chance of being affected, in only 1 generation. Commonly more severe
50% chance of being a carrier, and 25% chance than dominant disorders; patients often present
of not being affected nor a carrier. in childhood.
A a risk in consanguineous families.
Unaffected individual with affected sibling has
A AA Aa
2/3 probability of being a carrier.
a Aa aa
X-linked recessive Sons of heterozygous mothers have a 50% Commonly more severe in males. Females
chance of being affected. No male-to-male usually must be homozygous to be affected.
carrier transmission. Skips generations.
X X X X
X XX XX X XX XX
Y XY XY Y XY XY
X-linked dominant Transmitted through both parents. Children of Examples: fragile X syndrome, Alport syndrome,
affected mothers each have a 50% chance of hypophosphatemic rickets (also called X-linked
being affected. 100% of daughters and 0% of hypophosphatemia)—phosphate wasting at
sons of affected fathers will be affected. proximal tubule ricketslike presentation.
X X X X
X XX XX X XX XX
Y XY XY Y XY XY
Mitochondrial Transmitted only through the mother. All Caused by mutations in mtDNA.
inheritance offspring of affected females may show signs of Examples: mitochondrial myopathies, Leber
disease. hereditary optic neuropathy.
Variable expression in a population or even
within a family due to heteroplasmy.
= unaffected male; = affected male; = unaffected female; = affected female.

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5. Away then with all desponding thoughts. To undertake
vigorously, and rely confidently on the divine assistance, is more
than half the conquest: Let us arise and be doing, and the Lord will
be with us ¹. It is true, religion in the souls of men is the immediate
work of God, and all our natural endeavours can neither produce it
alone, nor merit those supernatural aids by which it must be wrought.
The Holy Ghost must come upon us, and the power of the Highest
overshadow us, before that holy thing can be begotten, and Christ
formed in us. But yet we must not expect that this work should be
done without any endeavours of our own; we must not lie loitering in
the ditch, and wait till omnipotence pull us thence; no, no, we must
bestir ourselves, and actuate these powers which we have already
received. We must put forth ourselves to our utmost capacities, and
then our labour shall not be vain in the Lord ². All the art and industry
of man cannot form the smallest herb, or make a stalk of corn to
grow in the field. It is the energy of nature, and the influences of
heaven, which produce this effect. It is God who causeth the grass to
grow, and herb for the service of man ³; and yet nobody will say that
the labours of the husbandman are useless or unnecessary. So
likewise the human soul is immediately created by God; it is he who
both formeth and enliveneth the child, and yet he hath appointed the
marriage-bed as the ordinary means for the propagation of mankind:
and so, though there must intervene a stroke of omnipotence to
effect this mighty change in our souls; yet ought we to do what we
can, that we may be more ready to receive the seeds of grace and
the dew of heaven. It is true, God hath been found of some who
sought him not; he hath cast himself in their way who were quite out
of his; he hath laid hold upon them, and stopt their course on a
sudden; for so was St. Paul converted in his journey to Damascus.
But certainly this is not God’s ordinary method of dealing with men:
though he hath not tied himself to means, yet he hath tied us to the
use of them; and we have never more reason to expect the divine
assistance, than when we are doing our utmost endeavours. It shall
therefore be my next work to shew what course we may take for
attaining that blessed temper I have described.
¹ 1 Chronicles xxii. 16. ² 1 Corinthians xv. 58.
³ Psalms civ. 14.
We must shun all manner of sin.
6. Now if we desire to have our souls moulded to this holy frame,

and have Christ formed in our hearts, we must carefully avoid all
sinful practices. There can be no treaty of peace, till we lay down
these weapons of rebellion wherewith we fight against heaven; nor
can we expect to have our distempers cured, if we be daily feeding
on poison. Every wilful sin gives a mortal wound to the soul, and puts
it at a greater distance from God. And we can never hope to have
our hearts purified from corrupt affections, till our hands are cleansed
from vicious actions.
We must know what things are sinful.

7. And, first, Let us inform ourselves well what those sins are
from which we ought to abstain. And here we must not take our
measures from the maxims of the world, or the practices of those
whom in charity, we account good men. Most people have very light
apprehensions of these things, and are not sensible of any fault,
unless it be gross. And those who are more serious, many times
allow themselves too great latitude. Alas! how much pride, and
vanity, and passion; how much weakness and folly doth every day
show itself in their converse and behaviour! It may be they are
humbled for it, and striving against it, but the progress is so small,
and their failings so many, that we had need to chuse an exacter
pattern. Every one of us must answer for himself, and the practice of
others will never warrant and secure us. It is the highest folly to
regulate our actions by any other standard, than that by which they
must be judged. If ever therefore we would cleanse our way, it must
be by taking heed thereto according to the word of God ¹. And that
word which is quick and powerful, and sharper than any two-edged
sword, piercing even to the dividing asunder of soul and spirit, and of
the joints and marrow ², will certainly discover many things to be
sinful, which pass for very innocent in the eyes of the world. Let us
therefore imitate the psalmist, who saith, Concerning the works of
men, by the words of thy lips, I have kept myself from the path of the
destroyer ³. Let us acquaint ourselves well with the holy laws of our
religion: let us consider the discourses of our blessed Saviour,
(especially that divine sermon on the mount) and the writings of his
holy apostles; where an unbiassed mind may clearly discern those
bounds by which our actions ought to be confined: and then let us
never look upon any sin as light and inconsiderable, but be fully
persuaded, that the smallest is infinitely heinous in the sight of God,
and prejudicial to the souls of men: and that if we had the right sense
of things, we should be as deeply affected with the least
irregularities, as now we are with the greatest crimes.
¹ Psalms cxix. 9. ² Hebrews iv. 12.
³ Psalms xvii. 4.
We must resist temptations.
8. Among those things which we discover to be sinful, there will

be some to which, through our nature, or long custom, we are so
wedded, that it will be like cutting of the right-hand, or pulling out the
right-eye, to abandon them. But must we therefore sit down and wait
till all difficulties be over, and every temptation be gone? This were to
imitate the fool in the poet, who stood the whole day at the river-side,
till all the water should run by. We must not indulge our inclinations,
as we do little children, till they grow weary of the thing they are
unwilling to let go. We must not continue our sinful practices, in
hopes that the divine grace will one day over-power us.
9. If the heinous nature of sin cannot affect us, at least we may
be frighted by its dreadful consequences. That selfish principle which
pusheth us forward to sinful pleasures, may make us loath to buy
them at the rate of everlasting misery. Let us therefore accustom
ourselves to consider seriously what a fearful thing it must be to
offend that infinite Being, on whom we depend every moment; who
needs but withdraw his mercies to make us miserable, or his
assistance to make us nothing. Let us remember the shortness and
uncertainty of our lives, and that after we have taken a few turns
more in the world, and conversed a little longer amongst men, we
must all go down to the dark and silent grave, and carry nothing
along with us but anguish and regret for all our sinful enjoyments.
What horror must then seize the guilty soul, to find itself naked and
all alone before the impartial judge of the world, to render an exact
account, not only of its more considerable transactions, but of every
word that the tongue hath uttered, and the most secret thought that
ever passed through the mind? Let us represent to ourselves the
terrors of that dreadful day, when the foundations of the earth shall
be shaken, the heavens shall pass away with a great noise, and the
elements shall melt with fervent heat ¹. The present frame of nature
shall be dissolved, and our eyes shall behold the blessed Jesus,
(who came once into the world in all humility to visit us, to purchase
pardon for us, and beseech us to accept of it) now appearing in the
majesty of his glory, and descending from heaven in flaming fire, to
take vengeance on those that have despised his mercy. Then all the
hidden things of darkness shall be brought to light, and the counsels
of the heart made manifest ². Then those secret impurities and subtle
frauds whereof the world did never suspect us, shall be exposed and
laid open to public view; and many thousand actions which we never
dreamed to be sinful, shall be charged home upon our consciences,
with such evident convictions of guilt, that we shall neither be able to
deny, or excuse them. Then shall all the angels in heaven, and all
the saints that ever lived on the earth, approve that dreadful
sentence which shall be passed on wicked men; and those who
loved and esteemed them when in the world, shall look upon them
with indignation and abhorrence.
¹ 2 Peter iii. 10. ² 1 Corinthians iv. 5.
10. ’Tis true, this is a melancholy subject; there is horror in the

consideration of it: but sure it must be infinitely more dreadful to
endure it; and such thoughts as these may be useful to fright us from
the courses that would lead us thither. How fond soever we may be
of sinful pleasures, we shall startle when pressed with that question,
Who can dwell with everlasting burnings ¹.
¹ Isaiah xxxiii. 14.
We must keep a constant watch over ourselves.

11. But it will not suffice to consider those things once and again,
nor to form some resolutions of abandoning our sins, unless we
maintain a constant guard, and be constantly watching against them.
Sometimes the mind is awakened, and we resolve to reform: but
alas! it presently falleth asleep, and we lose that prospect which we
had, and then temptations take the advantage; they solicit us
continually, and frequently engage our consent before we are aware.
It is the folly and ruin of most people to live at adventure, seldom
considering what they are about to say or do. If we would have our
resolutions take effect, we must take heed unto our ways, set a
watch before the door of our lips, and examine the motions that arise
in our heart, whence they come, and whither they go; whether it be
pride or passion, or any corrupt humour, that prompteth us to any
design, and whether God will be pleased with it? And if we have no
time for long reasonings, let us at least turn our eyes towards God,
and place ourselves in his presence, to ask his leave and
approbation for what we do. Let us consider ourselves under the all-
seeing eye of that divine majesty, as in the midst of an infinite globe
of light; which compasseth us about both behind and before, and
pierceth to the inmost corners of our soul. The sense of the divine
presence is a ready means, both to discover what is unlawful, and to
restrain us from it. There are some things a person could make a
shift to defend, and yet he dares not look God in the face, and
adventure upon them. If we look unto him we shall be lightned; if we
set him always before us, he will guide us by his eye, and instruct us
in the way wherein we ought to walk.
We must often examine our actions.

12. This care and watchfulness over our actions, must be
seconded by frequent and serious reflections upon them; not only
that we may obtain the divine mercy; but that we may strengthen our
resolutions, and learn to decline or resist temptations. It is an advice
worthy of a Christian, though it first dropped from a Heathen pen,
that before we betake ourselves to rest, we renew and examine all
the passages of the day, that we may redress what we find to have
been amiss, and make the shipwrecks of one day be as marks to
direct our course in another. But, withal, we must not forget to
implore the divine assistance, especially against those sins that most
easily beset us: and though our hearts are not yet moulded into that
spiritual frame, yet methinks such considerations as have been
proposed may stir us up to some seriousness, and make our prayers
against it as earnest, at least, as they are wont to be against other
calamities; and I doubt not but God, who heareth the cry of the
ravens, will have some regard even to such petitions as proceed
from those natural passions which himself hath implanted in us.
It is fit to restrain ourselves in many lawful things.

13. Thus we are to make the first essay for recovering the divine
life, by restraining the natural inclinations, that they break not out into
sinful practices. But Christian prudence will teach us to abstain from
gratifications that are not simply unlawful; and that not only that we
may secure our innocence, which would be in continual hazard, if we
should strain our liberty to the utmost point; but also that we may
teach our appetites to obey, as prudent parents deal with their
children, who cross their wills in many little things, to make them
manageable in more considerable instances. He who would mortify
the pride and vanity of his spirit, should stop his ears to the most
deserved praises, and sometimes forbear his just vindication, from
the censures and aspersions of others. He who would check a
revengeful humour, would do well to deny himself the satisfaction of
representing to others the injuries he hath sustained. And if we
would so take heed to our ways, that we sin not with our tongue, we
must accustom ourselves to solitude and silence. Thus we may
make our appetites more moderate in their cravings, by accustoming
them to frequent refusals; but it is not enough to have them under
violence and restraint.
We must strive to put ourselves out of love with the world.

14. Our next essay must be to possess our minds with a deep
persuasion of the vanity and emptiness of worldly enjoyments. This
is an ordinary theme, but alas! how few understand and believe what
they say? These notions float in our brains, and come sliding off our
tongues, but we have no deep impression of them on our spirits. We
feel not the truth which we pretend to believe. We can tell that all the
glory and splendor, all the pleasures of the world, are vanity and
nothing; and yet these nothings take up all our thoughts, and
engross all our affections. Perhaps sometimes we resolve to be no
longer deluded with them; but these thoughts seldom outlive the next
temptation. And after we have been frustrated a thousand times, we
must continually be repeating the experiment. The least difference of
circumstances is enough to make us expect that satisfaction in one
thing, which we missed in another. But had we once a real contempt
of worldly things, this were a considerable advancement in our way.
The soul of man is of a vigorous and active nature, and hath in it an
unextinguishable thirst, an immaterial kind of fire, always catching at
some object or other, in conjunction wherewith it thinks to be happy:
and were it once rent from the world, it would search after some
higher object, to satisfy its importunate cravings. The love of the
world and the love of God, are like the scales of a balance, as the
one falleth the other doth rise. It therefore nearly concerns us to be
convinced of the emptiness and vanity of creature enjoyments. Let
us seriously consider what our reason and faith, our own experience,
and the observation of others suggest. Amidst all our pursuits and
designs, let us stop and ask ourselves, for what end is this? At what
do I aim? Can the gross pleasures of sense, or a heap of white or
yellow earth, or the esteem of silly creatures like myself, satisfy an
immortal soul? Have I not tried these things already? Will they have
a higher relish, and yield me more contentment to-morrow than
yesterday, or the next year than they did the last? There may be
some little difference between that which I am now pursuing, and
that which I enjoyed before: but sure my former enjoyments did shew
as pleasant, and promise as fair before I attained them. Like the
rainbow, they ♦ looked very glorious at a distance, but when I
approached, I found nothing but emptiness and vapour. O what a
poor thing would the life of man be, if it were capable of no higher
enjoyments!
♦ “look” replaced with “looked” per Errata
We must do those outward actions that are commanded.
15. When our inclinations towards worldly things are in some

measure subdued, we must proceed conscientiously to perform
those duties which religion ♦ requires. If we cannot get our inward
dispositions presently changed, let us study at least to regulate our
outward deportment: if our hearts be not yet inflamed with divine
love, let us however ♠own our allegiance to that infinite Majesty, by
attending his service, and listening to his word; by speaking
reverently of his name; and praising his goodness, and exhorting
others to serve and obey him. If we want that charity, and those
bowels of compassion which we ought to have towards our
neighbours, yet must we not omit any occasion of doing them good.
If our hearts be haughty and proud, we must nevertheless study a
modest and humble deportment. These external performances are of
little value in themselves, yet may they help us forward to better
things. It is always good to be doing what we can, for then God is
wont to assist our feeble endeavours. Nor need we fear the
imputation of hypocrisy, though our actions thus somewhat out-run
our affections, seeing they still proceed from a sense of our duty, and
our design is not to appear better than we are, but that we may really
become so.
♦ “require” replaced with “requires” per Errata
♠ “owe” replaced with “own” per Errata
We must endeavour to form internal acts of devotion,

charity, &c.
16. Moreover, let us be often lifting up our hearts to God; and if
we do not say that we love him above all things, let us at least
acknowledge that it is our duty, and would be our happiness so to
do. Let us lament the dishonour done him by sinful men, and
applaud the praises that are given him by that glorious company
above. Let us yield ourselves up to him a thousand times, to be
governed by his laws, and disposed of at his pleasure: and though
our stubborn heart start back, yet let us tell him we are convinced
that his will is always just and good; and therefore desire him to do
with us whatsoever he pleaseth, whether we will or not.
Thus should we exercise ourselves unto godliness: and when we

are employing the powers that we have, the Spirit of God is wont to
strike in, and elevate these acts of our soul beyond the pitch of
nature, and give them a divine impression.
Consideration a great instrument of religion.

17. I shall mention but two other helps; and the first is, deep and
serious consideration. *The assent which is ordinarily given to divine
truths, is very faint and languid. Men are unwilling to quarrel with the
religion of their country; but are seldom at the pains to consider what
they profess to believe; and thence it is, that they have so little
influence on their practice. Those spiritless and paralytic thoughts
(as one rightly terms them) are not able to move the will, and direct
the hand. We must therefore labour for a full persuasion of divine
truths, a sense and feeling of spiritual things. Let us urge forward our
spirits, and make them approach the invisible world, and fix our mind
upon immaterial things, till we clearly perceive that these are no
dreams; nay, that all things are dreams and shadows besides them.
When we look about us, and behold the beauty and magnificence of
this goodly frame, the order and harmony of the whole creation, let
our thoughts from thence take their flight toward that omnipotent
wisdom and goodness which did at first produce, and doth still
uphold the same. When we reflect upon ourselves, let us consider
that we are not a mere piece of organized matter, a curious and well
contrived engine; that there is more in us than flesh, and blood, and
bones, even a divine spark, capable to know, and love, and enjoy
our Maker. And though it be now exceedingly clogged with its dull
and lumpish companion; yet ere long it shall be delivered, and can
subsist without the body, as well as that can do without the cloaths,
which we throw off at our pleasure. Let us often withdraw our
thoughts from this earth, this scene of misery, and folly, and sin, and
raise them towards that glorious world; whose innocent and blessed
inhabitants solace themselves eternally in the divine presence, and
know no other passion, but an unmixed joy, and an unbounded love:
and then consider how the blessed Son of God came down to this
lower world to live among us, and die for us, that he might bring us to
a portion of the same felicity; and think how he hath overcome the
sharpness of death, and opened the kingdom of heaven to all
believers, and is now set down on the right-hand of the Majesty on
high ¹; and yet is not the less mindful of us, but receiveth our prayers,
and presenteth them unto his Father, and is daily visiting his church
with the influences of his Spirit, as the sun reacheth us with his
beams.
¹ Hebrews i. 3.
We should consider the excellency of the divine nature.
18. Let me further suggest some particular subjects of meditation.

And first, if we would love God, let us consider the excellency of his
nature, and his love and kindness towards us. It is little we know of
the divine perfections; and yet that little may fill our souls with
admiration and love. If it be the understanding that directs the
affections, certainly the excellencies of the divine nature (the traces
whereof we cannot but discover in every thing we behold) should not
fail to engage our hearts. Shall we not be infinitely more transported
with that almighty wisdom and goodness, which fills the universe,
and displays itself in all the parts of the creation, which establisheth
the frame of nature, and turneth the mighty wheels of providence,
and keepeth the world from disorder and ruin, than with the faint rays
of the same perfections which we meet with in our fellow creatures?
Shall we doat on the scattered pieces of a rude and imperfect
picture, and never be affected with the original beauty? This were an
unaccountable stupidity and blindness. Whatever we find lovely in a
friend, or in a saint, ought not to engross, but to elevate our affection:
we should conclude with ourselves, that if there be so much
sweetness in a drop, there must be infinitely more in the fountain. If
there be so much splendor in a ray, what must the sun be in its
glory?
19. Nor can we pretend the remoteness of the object, as if God
were at too great a distance for our converse or love: he is not far
from every one of us; for in him we live, and move, and have our
being. ¹ We cannot open our eyes, but we must behold some
footsteps of his glory; and we cannot turn them toward him, but we
shall be sure to find his intent upon us, waiting as it were to catch a
look, ready to entertain the most intimate communion with us. Let us
therefore endeavour to raise our minds to the clearest conceptions of
the divine nature. Let us consider all that his works declare, or his
word discovers of him unto us; and let us especially contemplate that
visible representation of him which was made in our own nature by
his Son, who was the brightness of his glory, and the express image
of his person, ² and who appeared in the world to discover at once
what God is, and what we ought to be. Let us represent him unto our
minds as we find him described in the gospel, and there we shall
behold the perfections of the divine nature, tho’ covered with the veil
of human infirmities. And while we contemplate a Being, infinite in
power, in wisdom, and goodness, the author and fountain of all
perfections, let us pray that our eyes may affect our heart, ³ and while
we are musing the fire may burn. ⁴
¹ Acts xvii. 27. ² Hebrews i. 3.
³ Lamentations iii. 51. ⁴ Psalms xxxix. 3.
We should meditate on his goodness and love.

20. Hereunto add the consideration of God’s favour and goodwill
towards us. Now as the word of God is full of the expressions of his
love towards man, so all his works loudly proclaim it. He gave us our
being, and by preserving us in it, doth renew the donation every
moment. He hath placed us in a rich and well-furnished world, and
liberally provided for all our necessities. He raineth down blessings
from heaven upon us, and causeth the earth to bring forth our
provision. He giveth us our food and raiment; and while we are
spending the productions of one year, he is preparing for us against
another. He sweetneth our lives with innumerable comforts, and
gratifieth every faculty with suitable objects. The eye of his
providence is always upon us, and he watcheth for our safety when
we are fast asleep, neither minding him nor ourselves. But lest we
should think these testimonies of his kindness less considerable,
because they are the easy issues of his omnipotent power, and do
not put him to any trouble or pain, he hath taken a more wonderful
method to endear himself to us. He hath testified his affection to us,
by suffering as well as by doing; and because he could not suffer in
his own nature, he assumed ours. The eternal Son of God cloathed
himself with the infirmities of our flesh, and left the company of those
blessed spirits, who knew well how to love and adore him, that he
might dwell among men, and wrestle with the obstinacy of that
rebellious race to reduce them to their allegiance, and to offer
himself up as a sacrifice for them. I remember one of the poets hath
an ingenious fancy to express the passion wherewith he found
himself overcome after a long resistance, That the God of love had
shot all his golden arrows at him, but could never pierce his heart, till
at length he put himself into the bow, and darted himself straight into
his breast. Methinks this doth someway adumbrate God’s method of
dealing with men: he had long contended with a stubborn world, and
thrown down many a blessing upon them; and when all his other
gifts could not prevail, he at last made a gift of himself. The account
which we have of our Saviour’s life in the gospel doth all along
present us with the story of his love. All the pains that he took, and
the troubles that he endured, were the wonderful effects, and
uncontroulable evidences of it. But, O that last, that dismal scene! Is
it possible to remember it, and question his kindness, or deny him
ours? Here, here it is we should fix our most serious thoughts, that
Christ may dwell in our hearts by faith; that we being rooted and
grounded in love, may be able to comprehend with all saints, what is
the breadth and length, and depth and height; and to know the love
of Christ, which passeth knowledge, that we may be filled with all the
fulness of God! ¹
¹ Ephesians iii. 17, 18, 19.
21. We ought also frequently to reflect on those particular tokens

of love, which God hath bestowed on ourselves; how long he hath
borne with our follies and sins, and waited to be gracious unto us;
wrestling, as it were, with the stubbornness of our hearts, and
essaying every method to reclaim us. We should keep a register in
our minds of all the eminent blessings and deliverances we have met
with; some whereof have been so conveyed, that we might clearly
perceive they were not the issues of chance, but the gracious effects
of the divine favour, and the signal returns of our prayers.
As a help to charity, we must remember that all men are

nearly related to God.
22. If we would love all men, let us consider the relation wherein
they stand to God, and the impresses of his image, which are
stamped upon them. They are not only his creatures, the
workmanship of his hands, but such of whom he taketh special care,
and for whom he hath a very tender regard; having laid the designs
of their happiness before the foundations of the world; and being
willing to live and converse with them to all eternity. The meanest
and most contemptible person whom we behold, is the offspring of
heaven, one of the children of the Most High; and, however unworthy
he may behave himself of that relation to God, so long as God hath
not disowned himself by a final sentence, he will have us
acknowledge him as one of his; and, as such, embrace him with a
cordial affection. What a concern are we wont to have for those that
any ways belong to the person whom we love? How gladly do we lay
hold on every opportunity to gratify the child or servant of a friend?
And sure our love towards God would as naturally spring forth in
charity towards men, did we mind the interest that he is pleased to
take in them; and consider that every soul is dearer to him than the
material world; and that he did not account the blood of his Son too
great a price for their redemption.
That they carry his image upon them.

23. Again, as all men stand in a near relation to God, so they
have still so much of his image stamped on them, as may excite us
to love them. In some this image is more conspicuous, and we can
discern the lovely tracts of wisdom and goodness. And though, in
others, it be miserably sullied and defaced, yet it is not altogether
erased. Some lineaments still remain: all men are endowed with
rational and immortal souls, with understandings and wills capable of
the most excellent things. And if they be at present disordered and
put out of tune by wickedness and folly, this may indeed move our
compassion, but ought not to extinguish our love. When we see a
person of a rugged humour and perverse disposition, full of malice
and dissimulation, very foolish, and very proud, it is hard to fall in
love with an object that presents itself unto us, under an idea so little
grateful and lovely. But when we consider these evil qualities as the
diseases of a soul, which in itself is capable of all that wisdom and
goodness, wherewith the best of saints have ever been adorned, this
will turn our aversion into pity, and make us behold him with such
resentments, as we have when we look on a beautiful body that is
mangled with wounds, or disfigured by some loathsome disease.
And however we hate the vices we shall not cease to love the man.
Prayer another instrument of religion; the advantages of

mental prayer.
24. There remains yet another help; and that is, fervent prayer.
Holiness is the gift of God; indeed the greatest gift he doth bestow,
or we are capable to receive, and he hath promised his Holy Spirit to
those that ask it of him. In prayer we make the nearest approaches
to God, and lie open to the influences of heaven: then it is that the
sun of righteousness doth visit us with his directest rays, and
dissipateth our darkness, and imprinteth his image on our souls. I
cannot now insist on the advantages of this exercise, or the
dispositions wherewith it ought to be performed; I shall only tell you,
that as there is one sort of prayer, wherein we make use of the voice,
which is necessary in public; and may sometimes have its advantage
in private; and another wherein, though we utter no sound, yet we
conceive the expressions, and form the words in our mind; so there
is a third kind of prayer, wherein the soul takes a higher flight, and
having collected all its forces, by long and serious meditation, it
darteth itself (if I may so speak) towards God, in sighs and groans,
and thoughts too big for expression.
This mental prayer is one of the most powerful instruments of the

divine life; and it may be the apostle hath a peculiar respect unto it,
when he saith, that the Spirit helpeth our infirmities, making
intercession for us with groanings that cannot be uttered.
The use of the holy sacrament.

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Contributing Authors vii General Acknowledgments xiii

Introduction 2 Test-Taking Strategies 19

` SECTION I SUPPLEMENT S P E C I A L S I T UAT I O N S 25

` SECTION II HIGH-YIELD GENERAL PRINCIPLES 27

How to Use the Database 28 Pathology 203

FAS1_2022_00_Frontmatter.indd 5 11/10/21 10:50 AM

How to Use the Database 738 Biochemistry 742

FAS1_2022_00_Frontmatter.indd 6 11/10/21 10:50 AM

DNA was the first three-dimensional Xerox machine.

This high-yield material includes molecular biology, genetics, cell

Do not spend time learning details of organic chemistry, mechanisms, or

FAS1_2022_01-Biochem.indd 31 11/4/21 11:59 AM

Chromatin structure DNA exists in the condensed, chromatin form to

Heterochromatin Condensed, appears darker on EM (labeled H Heterochromatin = highly condensed.

Euchromatin Less condensed, appears lighter on EM (labeled Eu = true, “truly transcribed.”

FAS1_2022_01-Biochem.indd 32 11/4/21 11:59 AM

Nucleotides Nucleoside = base + (deoxy)ribose (sugar).

Purines (A,G)—2 rings. Pure As Gold.

N10–Formyl- Nitrogenous base CH₂

FAS1_2022_01-Biochem.indd 33 11/4/21 11:59 AM

Hydroxyurea AMP GMP monophosphate dehydrogenase

Purine and pyrimidine synthesis:

5-FU, and pyrimethamine: inhibit dihydrofolate

CPS1 = m1tochondria, urea cycle, found in liver

FAS1_2022_01-Biochem.indd 34 11/4/21 11:59 AM

Purine salvage deficiencies

Nucleic acids Ribose 5-phosphate Nucleic acids

Genetic code features

FAS1_2022_01-Biochem.indd 35 11/4/21 11:59 AM

FAS1_2022_01-Biochem.indd 36 11/4/21 11:59 AM

joining repair double-stranded breaks. 3´ 5´ 3´

homologous dsDNA as a template. Homologous recombination

UV exposure Pyrimidine dimer Deaminated C

Newly replaced segment

Nucleotide excision repair Base excision repair Mismatch repair

FAS1_2022_01-Biochem.indd 37 11/4/21 11:59 AM

Altered amino acids

AUG AUG AUG

FAS1_2022_01-Biochem.indd 38 11/4/21 11:59 AM

DNA 5´ CAAT TATAAA GT AG GT AG AATAAA 3´

Regulation of gene expression

FAS1_2022_01-Biochem.indd 39 11/4/21 11:59 AM

Cleavage at 5′ splice site; lariat-

FAS1_2022_01-Biochem.indd 40 11/4/21 11:59 AM

Exon 1 Exon 2 Exon 3 Exon 4 Exon 5 Exon 6

FAS1_2022_01-Biochem.indd 41 11/4/21 11:59 AM

Start and stop codons

FAS1_2022_01-Biochem.indd 42 11/4/21 11:59 AM

FAS1_2022_01-Biochem.indd 43 11/4/21 11:59 AM

Cell cycle arrest

Caspase activation E2F G2

FAS1_2022_01-Biochem.indd 44 11/4/21 11:59 AM

COPII Vesicular trafficking proteins

FAS1_2022_01-Biochem.indd 45 11/4/21 11:59 AM

Peroxisome Membrane-enclosed organelle involved in:

FAS1_2022_01-Biochem.indd 46 11/4/21 11:59 AM

Fibroblast Preprocollagen S ynthesis—translation of collagen α

enaturation—DNA template, DNA primers, a heat-stable DNA polymerase, and