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Incoming links: Amino acid
A DNA library synthesis method determines which variants can be reached, how often they are sampled, and which evolutionary paths remain available. The nominal number of DNA molecules is less informative than the distribution of functional amino acid sequences presented to the assay. directed evolution couples diversification to a genotype-phenotype linkage, screening or selection, and recovery of successful genotypes. Construction controls the accessible moves through protein sequence space and therefore interacts with assay capacity, starting sequence and epistasis; it is part of the evolutionary strategy rather than an interchangeable preliminary step.
error-prone PCR explores substitutions throughout an existing gene without requiring prior structural knowledge. R. Craig Cadwell and Gerald F. Joyce demonstrated controlled PCR mutagenesis using a ribozyme sequence, establishing a general route for generating variant templates.[ref]
Randomization of genes by PCR mutagenesis by R. Craig Cadwell and Gerald F. Joyce. Altered PCR fidelity and distribution of mutations
For protein evolution, polymerase preferences, sequence context and the genetic code make amino acid changes uneven: a substitution requiring two nucleotide changes is less accessible than a single-base neighbour. Increasing the mutation rate reaches more distant variants but often increases inactive products and makes attribution difficult. Insertions and deletions can also change the reading frame. Existing useful sequence combinations are usually retained at a low mutation load, but extensive remodelling is hard to reach by successive selections if intermediate genotypes are deleterious.An idealized Poisson distribution with mean mutation count makes this trade-off quantitative:At , about 37% of molecules have no change and 37% have one; at , only about 5% are unchanged and about 80% contain two or more changes. This is a planning model, not a guarantee that a real PCR DNA library is unbiased or independent at every site. Early PCR events can generate many related descendants, and the number of nucleotide mutations is not the number of distinct amino acid substitutions.
Oligonucleotide-directed saturation mutagenesis concentrates the assay budget at specified sites, for example an active site pocket or an interface. degenerate codons are convenient but biased. NNK and NNS each encode 32 codons, including one stop codon, with unequal multiplicities for different amino acids. At independently synthesized sites the ideal fraction lacking these introduced stops is , even before assembly errors or deleterious substitutions. A DNA library of amino acid combinations can therefore contain many redundant codons and uneven representation. Trinucleotide mixtures assemble complete chosen codons and can exclude stops, equalize amino acid sampling, or deliberately favour a chemically relevant subset. Bernhard Virnekas and colleagues developed such trinucleotide phosphoramidites for mixed-oligonucleotide synthesis.[ref]
Trinucleotide phosphoramidites: ideal reagents for the synthesis of mixed oligonucleotides for random mutagenesis by Bernhard Virnekas and others. Whole-codon synthesis for controlled amino-acid mixtures
Controlled diversity is valuable, but restricting sites according to an incomplete structural model can miss beneficial remote mutations.DNA shuffling offers a different move: genetic recombination among already useful or homologous sequences. Random fragmentation followed by annealing and polymerase extension reconstructs genes from overlapping parental fragments. Willem P. C. Stemmer demonstrated reassembly and genetic recombination, including chimeric sequences, providing a practical route to combine sequence blocks rather than relying only on isolated substitutions.[ref]
DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution by Willem P. C. Stemmer. Fragmentation, gene reassembly and recombinant products
This can unite improvements acquired in separate lineages and remove unwanted changes by genetic recombination with a parent. However, crossovers favour compatible homologous regions, closely related parents may add little diversity, and breaking a coadapted combination can lower fitness. Positive or negative epistasis means the fitness of a recombinant cannot be predicted by adding the effects of its parental substitutions.Designed synthetic genes and pooled oligonucleotide assembly can specify more extensive combinations, deletions, insertions or carefully chosen paths between distant sequences. This can test combinations inaccessible to single-base steps and explicitly balance beneficial candidates against exploratory variants. The cost is that synthesis, assembly and amplification introduce errors and abundance differences; a designed sequence is not necessarily a correctly constructed or well represented molecule. Long constructs may need assembly from overlapping fragments, and different fragment yields can skew the DNA library. A finite screen also prevents exhaustive exploration simply by purchasing a larger pool.
Sampling makes these differences concrete. If a target variant has DNA library frequency , screening independent clones finds it with probabilityFor equally frequent variants, the expected number of distinct variants observed is . Screening 1000 clones from a uniform two-site amino acid DNA library with visits about 92% of the variants on average; from a three-site DNA library with , it visits only about 12%. These are expected fractions, not probabilities that every variant has been sampled. Roughly clones give a 95% chance of observing one specified rare variant when is small. Synthesis bias and clonal duplication can make the effective coverage substantially worse.
The course of evolution should therefore be planned alongside screening. Begin with a mutation load or site set that the assay can actually cover, sequence the unselected DNA library to measure mutation spectra, stop frequency, parental carry-over and representation, and compare those frequencies with selected output. Use matched expression conditions and retain genotype-phenotype linkage: selection for growth, expression or display can otherwise enrich a variant that performs poorly as a purified enzyme. Reconstruct hits independently, sequence the complete gene, measure soluble protein and validate the desired activity or specificity at controlled protein concentration. Include parental and known inactive controls and, where relevant, more than one substrate or environmental condition. Subsequent rounds can recombine verified hits, adjust mutation load or target sites suggested by the first round. The best synthesis method maximizes useful, testable diversity under the assay budget and enables appropriate paths through the fitness landscape; maximal random diversity alone is not the objective.
During amino acid starvation, cells reduce bulk biological translation while selectively promoting an adaptive response. It has both a direct substrate effect on elongation and regulated effects on initiation. Their balance depends on which amino acid is limiting, how severely it is depleted, the organism and the duration of deprivation.
The immediate substrate problem is reduced charging of the relevant transfer RNAs by aminoacyl-tRNA synthetases. A ribosome encountering a cognate codon waits longer for an appropriate charged adaptor, potentially creating queues or ribosome collisions. The response is not necessarily identical at every synonymous codon, because transfer RNA isoacceptors, their charging and their competition differ. In mammalian biological cells, comparative ribosome profiling under particular amino acid limitations demonstrated selective codon-associated pausing rather than an invariant slowdown at every sense codon.[ref]
Translational control through differential ribosome pausing during amino acid limitation in mammalian cells by Alicia M. Darnell and others. Summary and Results: amino-acid-specific pausing, tRNA charging and translational responses
Persistent stalls can engage ribosome-associated quality control to rescue stalled machinery and dispose of incomplete products. Increased ribosome occupancy at a site therefore need not mean increased productive synthesis.In eukaryotes, GCN2 links amino acid limitation to the integrated stress response. Deacylated transfer RNA and signals from starved ribosomes, supported by GCN1, promote GCN2 activation.[ref]
Activation of GCN2 by the ribosomal P-stalk by Alison J. Inglis and others. Abstract and Results: ribosome-dependent activation and relationship to translational stress
Its protein kinase activity phosphorylates the alpha subunit of eIF2 at serine 51. Normal eIF2 brings initiating methionyl-tRNA to the small ribosomal subunit as part of a ternary complex:After initiation, eIF2 must exchange bound GDP for GTP, a reaction promoted by eIF2B. Phosphorylated eIF2 inhibits this recycling activity, reducing ternary-complex supply and initiation on many messenger RNAs. This conserves scarce substrates and limits addition of further ribosomes to already stressed templates. Genetic experiments established that GCN2-dependent regulation can repress bulk synthesis while increasing production of ATF4.[ref]Regulated translation initiation controls stress-induced gene expression in mammalian cells by Heather P. Harding and others. Abstract and Results: eIF2 kinase dependence and selective ATF4 expression
The apparent paradox is resolved by upstream open reading frames. In the canonical mammalian ATF4 leader, a short permissive upstream open reading frame is followed by an inhibitory one that overlaps the ATF4 coding region in a different reading frame. When ternary complex is plentiful, scanning small ribosomal subunits that have completed the first short frame reacquire it rapidly and initiate at the inhibitory frame. After eIF2 phosphorylation, reacquisition is delayed: some scanning subunits pass that inhibitory start and become competent before the downstream ATF4 start. Reduced initiation capacity can therefore increase initiation at a particular downstream coding region. Experiments using upstream open reading frame mutations and reporter assays directly tested this reinitiation mechanism.[ref]
Reinitiation involving upstream ORFs regulates ATF4 mRNA translation in mammalian cells by Krishna M. Vattem and Ronald C. Wek. Abstract and Results: uORF mutations, spacing and delayed reinitiation
The increase is not unlimited: if ternary complex becomes too scarce, initiation at ATF4 also fails.ATF4 then changes transcription of adaptive genes, including those supporting amino acid supply, transport and stress management. Yeast uses the related GCN2–eIF2 logic to increase GCN4 production through a leader with several upstream open reading frames; it should not be drawn as the identical two-frame ATF4 arrangement. The response can recover after replenishment through restoration of charging and dephosphorylation of eIF2. Prolonged, unresolved deprivation can instead favor damage and apoptosis. These later gene expression changes complement the initial translational response rather than explaining its earliest onset.
A parallel mammalian pathway regulates mTORC1. In nutrient-replete conditions, mTORC1 promotes cap-dependent initiation partly by phosphorylating 4E-BP proteins and activating S6 kinase. Phosphorylated 4E-BP proteins release eIF4E, allowing it to associate with eIF4G and assemble an active eIF4F initiation complex. amino acid starvation reduces mTORC1 activity, allowing hypophosphorylated 4E-BP proteins to bind eIF4E and restrain initiation. A concrete upstream sensor is Sestrin2: loss of leucine promotes its inhibitory interaction with GATOR2, influencing Rag GTPases and nutrient-dependent recruitment of mTORC1 to the lysosome.[ref]
Sestrin2 is a leucine sensor for the mTORC1 pathway by Rachel L. Wolfson and others. Abstract and Results: leucine binding, sensor mutants and GATOR2 interactions
This route measures nutrient sufficiency through a different mechanism from the GCN2 pathway; neither should be reduced to the other.Reduced mTORC1 signaling also helps limit investment in ribosome production and permits autophagy, which can recycle cellular material to supply amino acids. These adaptations operate on different timescales from waiting for a missing charged transfer RNA. The outcome is selective redistribution of translational capacity, not simply uniform cessation of every protein synthesis event. Some cap-dependent transcripts are especially sensitive to initiation-factor availability, while stress-regulated leaders can favor others.
To distinguish the mechanisms, compare a defined single-amino acid withdrawal with matched complete medium and replenishment. Control residual amino acids in supplements and measure viability. Measure global nascent protein synthesis, transfer RNA charging, eIF2 phosphorylation, ATF4 synthesis, and mTORC1 substrate phosphorylation over time. Combine ribosome profiling with RNA sequencing and external normalization; otherwise globally reduced synthesis can disappear through library normalization. Compare normal biological cells with GCN2 loss or a nonphosphorylatable eIF2 variant, and use ATF4 leader reporters with separately mutated upstream open reading frames. Independently perturb 4E-BP proteins or mTORC1 signaling. Recovery of initiation signaling without recovery of elongation would show that substrate shortage still limits output. A loss of large polysomes suggests reduced initiation, whereas maintained or increased occupancy can accompany stalled elongation; neither pattern alone measures productive protein synthesis.
In bacteria, a related sensing principle feeds a different control system: in Escherichia coli, RelA recognizes starved ribosomes containing deacylated transfer RNA and synthesizes (p)ppGpp, initiating the stringent response. Structural work explains how the starved ribosome activates RelA.[ref]
Ribosome-dependent activation of stringent control by Alan Brown and others. Abstract and structural Results: deacylated tRNA, RelA activation and alarmone synthesis
The stringent response reallocates gene expression and restrains investment in growth machinery; it is not an eIF2-based mechanism. Across these systems, the common strategy is to lower demand while preserving or increasing the capacity to restore amino acid supply.A time-calibrated phylogenetic tree combines evidence about ancestry with evidence about elapsed time. First identify homologous RNA or protein sequences, preferably orthologous loci whose splits reflect the species divergences of interest. A multiple sequence alignment proposes corresponding ancestral positions. Conserved ribosomal RNA can compare distant lineages, whereas faster-evolving regions help resolve recent splits; amino acid alignments can retain useful deep information after many nucleotide sites have changed repeatedly.
A phylogenetics model estimates a topology and branch lengths in substitutions per site, using explicit substitution probabilities rather than treating every observed mismatch as one historical change. Repeated substitutions, unequal rates among sites, selection, and compositional bias can make observed mismatch fractions underestimate the amount of change. Multiple loci help test consistency; a single gene tree can disagree with the species history through duplication, incomplete lineage sorting, or horizontal transfer.
A molecular clock converts branch change to time through an evolutionary rate. For two equally evolving lineages, an expected distance and rate give a divergence time , but sequence information alone usually identifies a rate-time product, not its two factors separately. A fossil securely assigned to a descendant clade supplies a dated constraint: the ancestral split must be at least as old as that fossil. The oldest known fossil is normally a minimum age, not proof that the lineage originated exactly then.
Several independent fossil constraints, their dating and placement uncertainty, and a relaxed molecular clock permit rates to vary while estimating divergence times with uncertainty intervals. Geological absence alone is a weak upper bound when preservation or sampling is poor. Conflicting loci, uncertain alignments, saturation, model misspecification, and uncertain fossil assignments should be investigated rather than hidden by a precise-looking time axis. The resulting phylogenetic tree is a joint inference from sequences, evolutionary assumptions, and geological evidence.
A point mutation changes a nucleotide at one genomic position. Replication errors and chemical damage can generate substitutions; repair and other proofreading processes influence the retained mutation rate. A coding change may be synonymous or alter an amino acid, while a noncoding change can affect regulation. Its evolutionary effect depends on context, so mutation, functional consequence and eventual fixation are distinct events.
Mutation supplies new variants, while genetic drift, selection and population history determine their persistence. Under a simple neutral model, increasing population size raises the number of new variants but lowers each one's fixation probability; the neutral substitution rate is approximately the mutation rate. Selective constraint, generation time and lineage-specific processes can therefore complicate a universal molecular clock. Molecular evolution is not directed towards anticipated needs.
To infer a phylogeny, compare homologous positions in appropriately chosen orthologous sequences and estimate the tree and branch lengths under a substitution model. Shared derived characters support common ancestry, but reversals and parallel substitutions mean raw mismatches underestimate evolutionary distance. In a simple equal-rates four-state illustration, a mismatch fraction gives corrected pairwise distance . The formula's strong divergence as reflects saturation and loss of reliable historical information, not arbitrarily precise evidence for an ancient split.
The molecular-clock calibration and rate-time identifiability issue is fundamental: branch lengths first measure expected substitutions per site. A clock relates them to time by ; for two equal-rate lineages separated for time , pairwise distance is , so . Absolute times require external information, such as independently dated fossils or geological constraints, or informative sampling dates when the data contain a temporal signal. Sequence differences alone generally identify only rate-times-time, not both quantities separately.
Time-calibrated inference should propagate uncertainty in topology, substitution rates, ancestral-state reconstruction and calibration bounds. Relaxed clocks permit different lineage rates. Fossil minima are not automatically exact split dates; orthology mistakes, incomplete lineage sorting, recombination, horizontal transfer and selection can cause discordance between one gene tree and species history. Multiple independent loci and well-justified calibrations make a stronger historical argument than a single uncalibrated sequence comparison. No unique numerical tree or date can be constructed here without actual sequence observations and calibration data.
The AFM pulls a tethered molecule through its cantilever. Deflection, multiplied by a calibrated cantilever spring constant, measures force; support motion must be corrected for that deflection and any linker compliance to obtain molecular extension. Initially folded structure can resist extension, then contacts break and more polypeptide polymer contour length becomes available. The readable trace rises with increasing extension, with fluctuations and a strongly rising large-extension part. It should not be interpreted as a direct image of individual amino acids or as establishing a unique microscopic unfolding pathway.
An ideal inextensible freely jointed chain with the stated segment count and length hasIts mean extension is strictly below at every finite force. Thus the quoted maximum does not establish ideal FJC behaviour: if both numbers are taken literally, it exceeds the model's polymer contour length by about . Finite segment stretch, inaccurate effective segment parameters, additional linkers or an extension-zero/calibration error can account for such a difference. If the numerical descriptions are approximate, the discrepancy may be within their uncertainty; even exact agreement with a polymer contour length would not prove freely jointed statistics. Low-force fluctuations and the full force–extension relation are separate tests.
Past exam of the chemistry B course of the University of Cambridge 2024 Paper 1 B5 c Solution 2026-10-20
View the backbone along N–C to display in a Newman projection. The front amide nitrogen carries the preceding carbonyl and H, while the back C carries the next carbonyl, H and side chain. Rotation changes the contacts among these groups. A corresponding view along C–C displays . The peptide C–N bond itself is approximately planar and is not the freely rotating bond in either definition.
glycine replaces the side chain by a second hydrogen. Removing C removes many contacts that exclude backbone arrangements for other residues, so its allowed region is much broader and includes substantial positive- space. Its alpha carbon is also achiral, so the steric map is approximately mirror-symmetric between positive and negative torsions in the simple isolated-residue model.
proline closes a five-membered ring from C through three methylenes back to nitrogen. In the N–C projection, the nitrogen's side-chain arm and C's C arm are tied together, preventing unrestricted relative rotation. Its is consequently confined near the usual negative- region rather than exploring the broad range of an acyclic amino acid. proline also lacks a backbone N–H donor when incorporated into an ordinary internal peptide. Ring closure restricts strongly, but does not freeze every backbone or ring degree of freedom. These differences are naturally represented by distinct Ramachandran plots; the original torsion/steric analysis established the basis for such comparisons.[ref]
Stereochemistry of polypeptide chain configurations by G. N. Ramachandran and others. 1963, pp. 95–99, backbone torsions and steric restrictions
A BLOSUM substitution matrix is estimated from conserved, ungapped blocks of related protein sequences. Closely related sequences are clustered to reduce overrepresentation, and aligned amino acid pairs from different clusters are counted. Let be their normalized observed pair frequency, and the frequency expected from independent draws at the background amino acid frequencies. With unordered pairs, and for .
For BLOSUM62, scores are rounded half-bit logarithms of these odds:A positive entry means that the aligned pair occurs more often in related sequences than the independent-background model predicts; a negative entry means less often. Adding these scores accumulates evidence across aligned positions. A score is neither a percentage identity nor a direct substitution probability.[ref]
The statistics of sequence similarity scores by National Center for Biotechnology Information. The choice of substitution scores; The PAM and BLOSUM amino acid substitution matrices
Use local sequence alignment, such as the Smith-Waterman algorithm, to compare the globin fragment with the matching region of each full-length target. A global sequence alignment would penalize long target flanks absent from the fragment. For a linear gap penalty the dynamic programming recurrence includes a restart option:Initialize boundary entries to zero, start traceback at the largest entry anywhere, and stop at zero. If the entire query is known to be valid and must be retained, fitting sequence alignment is a suitable refinement: align the whole fragment while leaving target flanks unpenalized.
First check the sequence alphabet: the prompt calls the targets “genes”. If the FASTA format file contains DNA rather than protein, translate the relevant coding sequences into amino acids before a protein-to-protein sequence alignment. If coding frames are unknown, search translated frames. A file in FASTA format specifies storage syntax, not whether the contents are nucleotides or residues.
Use the supplied BLOSUM62 entry for every diagonal move in the Smith-Waterman algorithm, while retaining the stipulated linear gap penalty of . If and are the current amino acids, the dynamic programming recurrence isThe zero option permits a new local start. The following original implementation also records the traceback coordinates needed in part (c); its tie ordering is left, up, diagonal, and an equal overall maximum replaces the previous best cell, as in the supplied convention:
def local_alignment(query,reference,alphabet,scores,gap=-1):
lookup={aa:i for i,aa in enumerate(alphabet)}
table=np.asarray(scores,dtype=int)
height,width=len(query)+1,len(reference)+1
H=np.zeros((height,width),dtype=int)
back=np.zeros((height,width),dtype=np.int8)
best=(0,0,0)
for i,a in enumerate(query,1):
for j,b in enumerate(reference,1):
# Order implements the printed LEFT, UP, DIAGONAL tie priority.
candidates=(0,H[i,j-1]+gap,H[i-1,j]+gap,
H[i-1,j-1]+int(table[lookup[a],lookup[b]]))
value=max(candidates);H[i,j]=value
back[i,j]=0 if value==0 else candidates.index(value)
if value>=best[0]:best=(value,i,j)
score,i,j=best;query_end,ref_end=i,j
aq=[];ar=[]
while H[i,j]>0:
step=back[i,j]
if step==1:aq.append('-');ar.append(reference[j-1]);j-=1
elif step==2:aq.append(query[i-1]);ar.append('-');i-=1
elif step==3:aq.append(query[i-1]);ar.append(reference[j-1]);i-=1;j-=1
qa=''.join(reversed(aq));ra=''.join(reversed(ar));length=len(qa)
identities=sum(a==b and a!='-' for a,b in zip(qa,ra))
similarities=sum(a!='-' and b!='-' and table[lookup[a],lookup[b]]>0 for a,b in zip(qa,ra))
gaps=sum(a=='-' or b=='-' for a,b in zip(qa,ra))
return dict(query=qa,reference=ra,score=int(score),query_start=i+1,query_end=query_end,
reference_start=j+1,reference_end=ref_end,query_length=len(query),reference_length=len(reference),
alignment_length=length,identities=int(identities),similarities=int(similarities),gaps=int(gaps),
identity_percent=100*identities/length if length else 0.,
similarity_percent=100*similarities/length if length else 0.,gap_percent=100*gaps/length if length else 0.)
queries = {"baboon": baboon, "squirrel_monkey": squirrel_monkey,
"dingo": dingo, "goldfish": goldfish}
alignments = {
name: local_alignment(seq, reference_epo_receptor_human, amino_acids, bmat)
for name, seq in queries.items()
}
for name, result in alignments.items():
print(name, "score", result["score"])
print(result["query"])
print(result["reference"])The resulting local sequence alignments, with the query above the reference, are:
baboon; score 314
SLLTPSDLDPLILTLSLILVLIVVLLTVLALLSHRRALKQKIWPGIPSPESEFEGLFTTHKG-FQL
SLLTPSDLDPLILTLSLILVVILVLLTVLALLSHRRALKQKIWPGIPSPESEFEGLFTTHKGNFQLsquirrel monkey; score 278
VCFWEEAASAGMGPDNYSFSYQL-DNEPWKR-CRLHQTPTARGVVRFWCSLPRADTSS
VCFWEEAASAGVGPGNYSFSYQLED-EPWK-LCRLHQAPTARGAVRFWCSLPTADTSSdingo; score 301
MNHLW--THLWPG-VGSLCLLLAGAAWASLPKP-L-DPKFESKAALLAARAPEELLCFTERLEDLVCFWEE
MDHL-GAS-LWP-QVGSLCLLLAGAAWAP-P-PNLPDPKFESKAALLAARGPEELLCFTERLEDLVCFWEEgoldfish; score 190
L--LVLSIGLILC--L-LSLTVI-L-SHQKFLLK-KLWPDIPTPEHKFPGLFTVYKGDFKE-WMSH-NNGSM
LDPLILTLSLILVVILVL-LTVLALLSHRR-ALKQKIWPGIPSPESEFEGLFTTHKGNF-QLWL-YQNDGCLThe several short gaps are a consequence of the specified inexpensive linear gap penalty; substituting a different default or an affine gap penalty would solve a different scoring problem.
For each match state, count the nongap amino acids and divide by the number of nongap observations in that column. A gap is not one of the twenty emitted amino acids; including it in the denominator would make the emission probabilities sum to less than one:
def emission_counts(column):
counts = Counter(str(x) for x in column if x != ".")
n = sum(counts.values())
if n == 0:
raise ValueError("an all-gap column has no empirical emission distribution")
return counts, n
match_emissions = {}
for state, j in enumerate(match_columns, 1):
counts, n = emission_counts(A[:, j])
match_emissions[state] = {aa: count/n for aa, count in counts.items()}
print(state, j+1, match_emissions[state])The table gives an exact compact representation of every emission probability: divide each displayed count by the denominator in its row; all unlisted amino acids have probability zero. The state number follows the order of match states, while the column refers to the original multiple sequence alignment.
| State | Alignment column | Nongap counts | |
|---|---|---|---|
| 1 | 1 | 9 | I:1, K:1, R:1, S:2, V:3, Y:1 |
| 2 | 2 | 9 | D:1, E:2, G:1, I:1, L:1, Q:2, S:1 |
| 3 | 3 | 9 | E:2, I:1, K:1, Q:1, R:3, T:1 |
| 4 | 4 | 9 | L:8, M:1 |
| 5 | 5 | 9 | E:3, G:1, L:2, N:1, Q:1, S:1 |
| 6 | 6 | 9 | A:1, D:2, G:1, K:1, N:1, S:2, T:1 |
| 7 | 7 | 9 | A:1, E:3, I:2, L:1, M:1, V:1 |
| 8 | 8 | 9 | I:2, L:7 |
| 9 | 9 | 9 | D:3, E:1, K:1, L:2, N:1, Q:1 |
| 10 | 10 | 9 | D:1, E:3, G:1, N:1, P:1, S:2 |
| 11 | 11 | 9 | A:5, D:1, G:1, P:1, T:1 |
| 12 | 12 | 9 | I:1, L:7, Y:1 |
| 13 | 13 | 9 | E:1, F:1, G:1, H:2, M:1, Q:2, T:1 |
| 14 | 14 | 9 | F:9 |
| 15 | 15 | 9 | F:1, L:8 |
| 16 | 16 | 9 | E:1, I:1, L:1, N:3, Q:1, R:1, Y:1 |
| 17 | 17 | 9 | D:6, E:2, S:1 |
| 18 | 18 | 9 | C:7, F:1, S:1 |
| 19 | 19 | 9 | K:1, N:6, R:2 |
| 20 | 20 | 9 | E:1, F:1, I:5, L:1, V:1 |
| 21 | 21 | 9 | K:3, P:1, Q:1, S:4 |
| 22 | 29 | 7 | K:4, P:1, R:1, S:1 |
| 23 | 30 | 7 | E:2, F:1, G:1, I:1, K:1, V:1 |
| 24 | 31 | 7 | D:3, E:1, G:1, K:1, S:1 |
| 25 | 32 | 8 | D:1, E:1, G:1, K:1, N:1, R:3 |
| 26 | 33 | 8 | D:7, N:1 |
| 27 | 34 | 8 | A:1, P:6, S:1 |
| 28 | 35 | 9 | C:1, H:1, I:1, K:1, M:1, R:1, T:3 |
| 29 | 36 | 9 | E:1, F:1, G:1, L:1, Q:3, W:1, Y:1 |
| 30 | 37 | 9 | I:5, L:1, T:2, Y:1 |
| 31 | 38 | 9 | A:1, G:1, P:3, S:4 |
| 32 | 39 | 9 | I:1, K:6, L:1, R:1 |
| 33 | 40 | 9 | F:1, N:1, Q:4, T:2, V:1 |
| 34 | 41 | 9 | A:1, F:1, I:1, V:6 |
| 35 | 42 | 9 | I:1, L:7, V:1 |
| 36 | 43 | 9 | A:1, C:1, E:1, N:1, R:1, S:3, V:1 |
| 37 | 44 | 9 | D:3, E:5, Q:1 |
| 38 | 45 | 9 | A:1, C:7, I:1 |
| 39 | 46 | 9 | K:2, L:3, R:4 |
| 40 | 47 | 9 | A:3, E:1, L:1, N:3, T:1 |
| 41 | 48 | 9 | I:4, L:1, T:1, V:3 |
| 42 | 49 | 9 | L:9 |
| 43 | 50 | 9 | D:1, E:1, H:1, Q:1, S:1, T:1, V:1, Y:2 |
| 44 | 51 | 9 | E:1, K:1, Q:2, R:1, V:4 |
| 45 | 52 | 9 | L:9 |
| 46 | 53 | 9 | G:9 |
| 47 | 54 | 9 | P:8, T:1 |
| 48 | 55 | 9 | W:9 |
| 49 | 56 | 9 | A:2, C:7 |
| 50 | 57 | 9 | A:6, C:1, L:1, T:1 |
| 51 | 58 | 9 | A:1, D:3, F:1, S:2, V:1, Y:1 |
| 52 | 59 | 9 | K:4, R:2, S:3 |
| 53 | 60 | 9 | A:2, F:1, I:2, L:2, V:2 |
| 54 | 61 | 9 | A:7, G:1, Y:1 |
| 55 | 62 | 9 | E:3, G:3, Q:3 |
| 56 | 63 | 9 | I:3, M:3, T:1, Y:2 |
| 57 | 64 | 9 | F:3, L:2, M:3, N:1 |
| 58 | 65 | 9 | H:1, I:3, L:1, V:4 |
| 59 | 66 | 9 | K:1, M:1, P:1, R:4, S:1, T:1 |
| 60 | 67 | 9 | D:1, E:3, Q:4, Y:1 |
| 61 | 68 | 9 | I:1, L:7, M:1 |
| 62 | 69 | 9 | D:1, E:4, Q:4 |
| 63 | 70 | 9 | K:7, R:2 |
| 64 | 71 | 9 | A:1, I:2, M:1, N:1, R:1, Y:3 |
| 65 | 72 | 9 | A:1, C:1, D:1, E:2, I:2, V:2 |
| 66 | 73 | 9 | K:9 |
| 67 | 74 | 9 | H:4, L:1, Q:2, S:1, V:1 |
| 68 | 75 | 9 | E:8, Q:1 |
| 69 | 76 | 9 | Q:3, R:1, S:2, T:3 |
| 70 | 77 | 9 | C:2, E:3, G:1, R:1, S:1, V:1 |
| 71 | 78 | 9 | A:1, D:2, E:3, I:1, K:1, Y:1 |
| 72 | 79 | 9 | I:2, L:3, M:1, N:1, Q:1, V:1 |
These are unregularized maximum-likelihood estimates of the emission probabilities. No additional artificial observations are added. Although only emissions are requested here, a full profile hidden Markov model would also require transitions and deletion mechanisms to represent the gaps that remain in some match columns.
For each alignment column, ignore gaps, form the empirical distribution of its amino acids, and calculate its Shannon entropy in bits. The requested alignment column information content isThus it is the relative entropy from the column distribution to the uniform distribution on twenty amino acids, not the ordinary Shannon entropy itself. A column containing one residue type has the maximum value bits.
information = []
for j in range(A.shape[1]):
counts, n = emission_counts(A[:, j])
p = np.array(list(counts.values()), dtype=float) / n
H = -np.sum(p * np.log2(p))
information.append(float(np.log2(20) - H))
print(np.round(information, 4))The values for all 79 alignment columns are as follows, in bits. Each line lists its one-based column interval followed by the corresponding values in order:
01–10: 1.9025, 1.5964, 1.9025, 3.8187, 1.9025, 1.5964, 1.9025, 3.5577, 1.9025, 1.9025
11–20: 2.4420, 3.3355, 1.5964, 4.3219, 3.8187, 1.6803, 3.0975, 3.3355, 3.0975, 2.4420
21–30: 2.5692, 2.8219, 3.5106, 3.3219, 4.3219, 4.3219, 2.8000, 2.0703, 2.6574, 1.8003
31–40: 2.1938, 1.9163, 3.7784, 3.2606, 1.6803, 1.6803, 2.6642, 2.5692, 2.8753, 2.2631
41–50: 2.8753, 3.3355, 1.6803, 2.9703, 3.3355, 2.7914, 2.2086, 2.5692, 4.3219, 1.3742
51–60: 2.2631, 4.3219, 4.3219, 3.8187, 4.3219, 3.5577, 2.8753, 1.9025, 2.7914, 2.0409
61–70: 3.3355, 2.7370, 2.4309, 2.4309, 2.5692, 2.0409, 2.5692, 3.3355, 2.9298, 3.5577
71–79: 1.9025, 1.8187, 4.3219, 2.2631, 3.8187, 2.4309, 1.9025, 1.9025, 1.9025A high score indicates conservation among the observed nongap letters, but does not itself imply low gap fraction. For example, an insertion column represented by only one observed nongap residue has maximal information by this calculation; its tiny sample size still makes that estimate uncertain.
Columns 22–28 form one run between alignment columns 21 and 29, corresponding to match states and . A self-repeating insertion state can emit a variable number of residues, so these seven alignment columns require one populated insertion state, not seven successive mandatory states.
Pool all nongap residues in this run to estimate its emission probabilities:
insert_columns = np.flatnonzero(~match_mask)
runs = np.split(insert_columns, np.flatnonzero(np.diff(insert_columns) > 1) + 1)
insert_emissions = []
for run in runs:
if len(run):
counts, n = emission_counts(A[:, run].ravel())
insert_emissions.append({aa: count/n for aa, count in counts.items()})
print(run+1, n, insert_emissions[-1])There are 24 emitted residues, givingAll other amino acids have probability zero in this empirical emission probabilities. A conventional full architecture may provide an optional insertion state at every boundary, including the two ends, giving possible insertion positions; only one has an observed insertion here and is required by this alignment's populated insertion region.
A food supplies material or usable chemical energy that contributes to the organism's maintenance, growth or normal function after ingestion and physiological processing. It must be usable in the amount and form supplied, without its harmful effects defeating that nutritive role. Mere combustibility, a pleasant taste, or the presence of carbon and nitrogen is insufficient.
An edible material may need digestion before its constituents can be absorbed. A protein need not enter the blood unchanged to nourish: hydrolysis supplies absorbable amino acids. Conversely, a substance can burn in a laboratory yet furnish little accessible energy to human metabolism. The nutritional value of a mixture depends on availability, proportions and the organism consuming it. No requirement says that each separate food must contain every constituent of a complete diet.
A useful nineteenth-century division separates nitrogenous or plastic foods from non-nitrogenous or respiratory foods. The first includes animal and plant proteins, which supply amino acids for tissue renewal. The second includes carbohydrates and fats, whose oxidation supplies much of the body's energy. But the division is functional rather than absolute: proteins can also be oxidized, and lipids and carbohydrates supply material for cellular structures as well as fuel. The modern account follows anabolism and catabolism rather than assigning each class one exclusive destination.[ref]
24.1 Overview of Metabolic Reactions. Anabolic and Catabolic Reactions: synthesis, breakdown and energy transfer
A fuller classification includes proteins; carbohydrates, including sugars and starch; lipids, including fats and essential fatty components; water; and required mineral constituents. Water provides solvent, transport medium and a participant in many reactions. Mineral constituents supply, among other needs, ions for electrical and osmotic functions and material for mineralized tissues. Neither class has to supply combustible energy to be indispensable. Meat, milk, grain and legumes are mixtures spanning several classes, rather than chemically pure examples of just one.
In modern classification, vitamins and other required micronutrients must also be included. Their specific deficiency roles and present classification should not be attributed wholesale to 1864. Dietary fibre is not equivalent to digestible starch; it can have important intestinal functions, and fermentable fractions can contribute metabolites through the microbiota. The adequacy of a diet concerns the whole combination of nutrients and the person's requirements, not a single measure of heat released on burning it.[ref]
24.7 Nutrition and Diet. Macronutrients; Vitamins; Minerals: energy-yielding and non-energy-yielding nutritional requirements
Oxygen is essential to ordinary aerobic life, but in the usual dietary sense it is a respiratory input rather than an ingested food class. Likewise, a drug that changes appetite or stimulates a physiological response is not thereby a substitute for nutritive material. A convincing experiment on food value must distinguish failure to eat, failure to digest or absorb, and a genuine missing dietary constituent; maintaining equal gross food mass alone does not control those differences.
Plants generally incorporate combined nitrogen into organic compounds, whereas animals eliminate the surplus nitrogen of metabolized compounds chiefly as Ammonia, urea or uric acid. The two processes connect nitrogen chemistry to the organism's carbon supply, water economy and environment.
Roots take up nitrate and ammonium from their surroundings. Nitrate must be reduced before its nitrogen can be incorporated in the usual amino form: nitrate reductase produces nitrite, and nitrite reductase produces ammonium. The balanced reduction, written without implying a single elementary reaction, isReducing equivalents and energy must be supplied by metabolism; nitrate is not simply deposited unchanged inside every protein.
In the modern biochemical account, glutamine synthetase incorporates ammonium into glutamine, using ATP; glutamate synthase transfers that nitrogen to a carbon skeleton derived from 2-Oxoglutarate, producing glutamate. Further transfer and synthesis distribute nitrogen into other amino acids, proteins, nucleotides and other cell constituents. Carbon metabolism therefore provides both energy and skeletons for nitrogen assimilation. These named enzymes and reactions explain the process today; they were not a completed molecular scheme in 1864.[ref]
Nitrogen uptake, assimilation and remobilization in plants. Nitrogen Assimilation: nitrate reduction and the GS/GOGAT cycle
Ordinary higher-plant cells do not themselves reduce atmospheric molecular nitrogen merely because the gas surrounds the leaves. Biological nitrogen fixation is performed by particular microorganisms; symbiotic associations can supply fixed nitrogen to a host plant. The later elucidation of root-nodule symbiosis should be distinguished from what the examiner could have expected. Organic nitrogen can also be taken up in suitable circumstances, so nitrate and ammonium are important routes rather than an exhaustive prohibition on every other source.[ref]
46.3 Biogeochemical Cycles. The Nitrogen Cycle: fixation, ammonification, nitrification and return to the atmosphere
A useful assimilation experiment grows comparable seedlings in a defined medium containing all required minerals except combined nitrogen, with parallel nitrate- or ammonium-supplied cultures. Initial seed reserves, contaminating nitrogen, water and microbial growth must be allowed for. Growth and final nitrogen content are more informative than green appearance alone. A plant initially growing in a nitrogen-free solution may simply be consuming seed reserves; that observation does not demonstrate fixation of atmospheric nitrogen.
Animals obtain most of their nitrogen from organic food. Amino acid metabolism separates nitrogen handling from oxidation or reuse of the carbon skeleton. Free Ammonia is toxic at high concentrations, and mammals convert much of their waste nitrogen into urea in the liver; the kidneys then eliminate it in urine. Uric acid is also produced from purine breakdown. Renal ammonium excretion has a separate important connection with acid-base balance. Urea formation and urinary excretion are different processes in different organs.[ref]
Many aquatic animals can release Ammonia directly into abundant surrounding water. Birds, many reptiles and many insects eliminate much nitrogen as relatively insoluble uric acid, conserving water; mammals generally depend more on soluble urea. These are broad physiological strategies with exceptions, not a rule that all animals have human urine. Faecal nitrogen includes unabsorbed food as well as endogenous material, while smaller losses can occur through skin and secretions. For a stated interval the accounting identity isPositive retention during growth and negative balance during tissue loss need not be errors of excretion. Nitrogen is redistributed or lost in compounds; it is not simply breathed out as all the atmospheric nitrogen passing through the lungs.
These names refer to several unrelated chemical classes: blood and bile pigments, a photosynthetic pigment family, an amino sulfonic acid, a disputed historical faecal extract, and storage polysaccharides or proteins. Similar colour or a historical name does not establish chemical identity.
Haematocrystallin denotes the crystallizable colouring substance of red blood, now identified with hemoglobin. It combines a globin protein with iron-containing haem groups. Within intact red cells it reversibly binds oxygen; oxygenated and deoxygenated forms have different absorption spectra. Crystals obtained from blood differ with species and preparation. This is not the same thing as a crystal formed from an old tissue haemorrhage. The word does not mean that all circulating blood normally consists of free pigment crystals.[ref]
Haematoidin is the yellow-orange to reddish pigment characteristically encountered in old extravasated blood, sometimes as rhomboidal crystals or radiating clusters. It is associated with bilirubin and lacks the iron retained in haemosiderin. The historical inference that any reddish blood-derived crystal must be the same substance as haematocrystallin is therefore wrong. In a tissue preparation the local matrix and other constituents can complicate an exact chemical identification from appearance alone.[ref]
Hematoidin crystals in sputum smears: Cytopathology and clinical associations. Results; Discussion: crystal morphology, bilirubin association and absence of iron
Haematin is a dark iron-containing derivative of haem, obtained when the globin association and the iron's chemical environment are altered. In modern chemical usage, hematin is the ferric hydroxide form of the haem pigment; hemin is the corresponding chloride form. Historical preparations and terminology were less consistently separated. Neither should be equated with iron-free bilirubin, and a haem-derived crystal test is not the crystallization of intact hemoglobin.[ref]
Hematin in PubChem. Identity and chemical description: hydroxyl ferric protoporphyrin
[ref]Chlorophyll is the green pigment system of photosynthetic organisms, present in organized pigment-protein assemblies in chloroplasts in green plants. Modern chemistry distinguishes several chlorophylls: chlorophyll a, for example, has a magnesium-containing tetrapyrrolic ring system and a hydrophobic side chain. It absorbs light used in photosynthetic energy conversion. Extraction into alcohol can reveal the green pigment, but a leaf extract is a mixture rather than a chemically pure universal chlorophyll. Its green colour must not be attributed to iron in the same way as haem.[ref]
Biliverdin is a green, open-chain tetrapyrrolic pigment. In modern haem catabolism, ring cleavage by haem oxygenase releases iron and forms biliverdin; reduction then produces bilirubin. The pigments contribute to changing colours of some resolving haemorrhages and to bile-pigment chemistry. This sequence concerns degradation of the haem group, not conversion of the entire globin protein directly into one green molecule.[ref]
Taurin, now taurine, is a colourless crystalline amino sulfonic acid with the structure . It contains sulfur and nitrogen and can form conjugates with bile acids, including taurocholic acid. It is not itself a green bile pigment, an ordinary carboxylic amino acid, or a standard amino acid directly inserted into proteins by translation. The chemical classification and formula distinguish it from neighbouring names in the list.[ref]
Excretin was the name William Marcet gave to a crystalline material extracted from human faeces. His contemporary account describes treatment with hot alcohol, removal of material with lime, extraction with ether, and formation of silky needle-like crystals; the material was insoluble in water, soluble in ether and hot alcohol, and melted in the low-to-mid 90s Celsius. His 1859 account reports carbon, hydrogen, oxygen and sulfur and gives an equivalent-weight formula based on an assumed sulfur content. These are historical analytical claims, not a modern authenticated molecular specification. Later work questioned the purity and chemical individuality of excretin, so that old name alone does not establish a definite modern structure.[ref]
Excretin in A Text-book of Physiological Chemistry (1914). p. 523: later doubts about identity and possible impure sterol material
[ref]Glycogen is an animal glucose-storage polysaccharide, particularly important in the liver and skeletal muscles. Its highly branched chains contain alpha-(1→4) links with alpha-(1→6) branch points. Preparations form opalescent dispersions and commonly show a reddish-brown iodine reaction rather than the intense blue associated with long amylose chains. Hydrolysis yields glucose. Liver glycogen can support the regulation of circulating glucose, whereas muscle glycogen mainly supports local muscle metabolism; a modern account must distinguish those physiological destinations.[ref]
Biochemistry, Glycogen (StatPearls). Introduction; Cellular Level: glucose storage, branching and distinct liver/muscle roles
Gluten is the cohesive protein-rich material left when wheat dough is washed to remove much of its starch. It is not a single small molecule: modern analysis distinguishes gliadin and glutenin families whose associations contribute extensibility and elasticity. It is poorly soluble in water as a dough network, contains nitrogen, and on full hydrolysis supplies amino acids. Its nutritive role is that of a plant protein mixture, not a nitrogen-free carbohydrate.[ref]
Changes in the rheology and aggregation behavior of wheat glutenin and gliadin mediated by low-sodium salt. Introduction; experimental comparison of the rheology of the two protein fractions
Legumin is a seed storage globulin associated particularly with peas and related legumes. A classical preparation extracts protein into suitable dilute salt solution and precipitates it under changed conditions. Modern legumins are 11S globulin assemblies, often hexamers formed from processed subunits. They are nitrogen-containing proteins, chemically distinct from starch despite occurring beside it in a seed. The older name vegetable casein indicates an analogy of precipitation or nutritive role; it does not establish identity with milk casein.[ref]
Pea Legumin Overexpressed in Wheat Endosperm Assembles into an Ordered Paracrystalline Matrix. Abstract; results on precursor processing and 11S hexamer assembly
Chyle is intestinal lymph enriched with absorbed dietary lipids; its usual route ends in the venous circulation at the left jugulosubclavian junction. The milky appearance after a fat-containing meal is chiefly due to suspended lipid-rich particles, now called chylomicrons.
Formation begins with lipid digestion in the small intestine. Pancreatic lipase helps hydrolyse dietary triglycerides; bile salts help the digestion products reach the intestinal absorptive surface in mixed micelles. Enterocytes take up the products, reconstruct much of the long-chain triglyceride, and package it with other lipids and apolipoproteins into chylomicrons. These leave the basolateral side of the enterocytes and enter the central lacteals of the intestinal villi. Thus the particles carried in chyle are not simply intact globules of a meal passing through an open hole from the intestinal lumen.[ref]
From the lacteals, chyle enters lymphatic channels in the intestinal wall and then collecting vessels in the mesentery. These vessels pass through mesenteric lymph nodes, with afferent vessels entering and efferent vessels leaving the nodes. Successive collectors join the intestinal lymphatic trunk or trunks. They usually communicate with the cisterna chyli, an expanded origin of the thoracic duct in the upper lumbar region, near the bodies of L1–L2 and to the right of the aorta. The cisterna chyli also receives lymph from lumbar trunks; the stream beyond it therefore contains material from more than the intestine. A conspicuous sac is not present in every individual.
The thoracic duct passes through the aortic hiatus of the respiratory diaphragm and ascends in the posterior mediastinum, typically between the aorta and azygos vein, behind the oesophagus. It crosses towards the left in the middle or upper thorax, continues to the root of the neck and arches to its terminal venous opening. The usual endpoint is the left venous angle, where the left internal jugular vein joins the left subclavian vein; terminal branches and the precise opening show anatomical variation. It is not normally the right venous angle or a direct entrance into the portal vein.[ref]
Anatomy, Thorax, Thoracic Duct (StatPearls). Structure and Course: cisterna chyli, thoracic course, left cervical arch and variable venous termination
The flow is promoted by intrinsic contraction of collecting lymphatic vessels, movements of surrounding muscles and respiratory changes of pressure. Lymphatic valves limit backward movement. Lymph nodes are interposed biological structures, not pumps that propel every particle independently.[ref]
21.1 Anatomy of the Lymphatic and Immune Systems. Lymphatic Capillaries; Larger Lymphatic Vessels, Trunks, and Ducts; lymph transport
This route applies particularly to the long-chain dietary lipids carried by chylomicrons. Most absorbed glucose and amino acids, and much shorter-chain fatty acid material, follow portal vascular routes instead. Confusing these pathways would obscure the reason for asking separately about chyle. In a classical anatomical demonstration, the mesenteric channels of an appropriately fed animal appear white, and an injected or dissected preparation can trace their continuity towards the thoracic duct. The timing after a fatty meal matters: an empty-looking channel in a fasted preparation does not prove absence of a lymphatic vessel. The molecular account of chylomicrons is a modern explanation of the older gross observation.
The defensible core of William Odling's statement is a transfer of energy: sunlight helps build reduced food material, and its subsequent oxidation can supply animal heat and work. Here solar force means solar energy, and eremacausis means slow oxidation, rather than a special vital agent. The historical wording, from his 1866 lectures, must be qualified where it identifies the fuel with the substance of the working muscle or contrasts plant and animal metabolism too absolutely.[ref]
Lectures on Animal Chemistry by William Odling. Lecture IV, discussion of solar energy and animal oxidation; see also the concluding recapitulation
In oxygenic photosynthesis, light absorbed by chlorophyll supports conversion of carbon dioxide and water into reduced organic material, with release of oxygen. An overall account, written for one glucose unit, isThis summarizes several stages, rather than describing a single elementary chemical reaction. The evolved oxygen comes from water. The product can furnish starch, other carbohydrates and precursors for fat and protein; the latter additionally requires a source of nitrogen. A human obtains these materials directly from plants or through animals that have eaten plants. Thus a grain meal and the flesh of a grain-fed animal can trace much of their chemical energy to the same original sunlight.[ref]
8.1 Overview of Photosynthesis. Main Structures and Summary of Photosynthesis; photosynthetic products and their passage through food chains
The reverse overall balance for complete aerobic utilization of glucose isSimilarly a representative fatty acid, palmitic acid, has the complete oxidation balanceBoth equations conserve each chemical element. The relevant energy difference belongs to the complete set of reactants and products, including oxygen; merely breaking a bond does not supply net energy. In modern terms, controlled cellular respiration couples part of the available chemical free energy to ATP formation. ATP hydrolysis can then support muscle contraction, active transport and biosynthesis; some energy is transferred to heat. The process occurs through enzyme-mediated steps, without a flame, although complete combustion in a calorimeter can help measure the corresponding chemical energy change.
Glands and muscles continually replace constituents, and their proteins can be degraded. Amino acids can contribute carbon skeletons to energy metabolism, while their nitrogen is disposed of largely as urea. But ordinary muscle contraction does not require proportional destruction and rebuilding of the contractile protein at every stroke. Carbohydrates and fats are major fuels for muscle, and a gland can use those fuels to support secretion. The historical picture of burning the organ's own substance is therefore an inadequate general explanation.[ref]
This correction was already an experimental issue before the exam. Edward Frankland's 1866 discussion compared the mechanical work of climbing with the energy that could be assigned to protein destruction estimated from urinary nitrogen. The latter was insufficient to account for all the work. His combustion measurements also distinguished the heat from burning a sample completely from the smaller amount available when nitrogenous material leaves the animal as still-combustible urea. Measuring external work, urinary products and calorific values gives the hypothesis a quantitative test; it is more informative than simply pointing out that an active animal gets warm.[ref]
On the Source of Muscular Power by Edward Frankland. pp. 394–395, criticism of tissue-consumption doctrine; pp. 401–406, calorific measurements and comparison with climbing work, 1866
Finally, plants themselves carry out cellular respiration, and not every ecosystem obtains its input from sunlight: chemolithotrophy is an alternative. The plant-animal contrast is useful for following energy through an ordinary food chain, but it is not an exhaustive division of biochemical capabilities. Conservation of energy also requires the bookkeeping formfor an idealized interval in which is the metabolizable food input, with energy lost in excreta already subtracted and other material-energy exchanges neglected. Here is external work, is net outward heat transfer, and includes changes in stored chemical and other energy. The living body transforms energy; it does not create it.