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I recently read Sherlock Holmes and in the book A Study In Scarlet, Holmes says to Watson that he has discovered a reagent that can only be precipitated by Haemoglobin and nothing else. I know about the Guiacum test but is there any other test like I have discussed above as the Sherlock Holmes test.
This very point is well addressed by Laura J Snyder in Sherlock Homes: Scientific Detective (a great read) where the author challenges the view that Conan Doyle is the 'father of scientific crime detection' and argues that the new science of forensics actually influenced Conan Doyles' writing, rather than his novels being the inspiration for the new emerging discipline.
Furthermore, not all his claims were scientifically valid, and the 'infallible test for bloodstains' is a good example.
The relevant passage is as follows:
Let us examine, for example, the 'infallible' test for bloodstains Holmes is presented as having 'invented'. By1887, when A Study in Scarlet was published, many researchers already shared the desire for such a test; this desire was not satisfied until the turn of the 20th century,when the spectroscopic method was developed.
A modern chemist has noted that the method Holmes describes - one which would precipitate a brownish dust and change the color of blood in water to mahogany - would need an acid to increase the oxidation rate, as well as a material to be oxidized. By examining the possibilities for the 'few white crystals' and the 'drop of transparent fluid' that Holmes uses, this chemist suggests that the 'Sherlock Holmes test' would probably have had a sensitivity similar to the guaiacum test that Holmes derides as being 'clumsy and uncertain'.
Moreover, Holmes' test does not distinguish between human blood and the blood of animals- a problem that, by 1887, was considered an even larger concern than the sensitivity of the blood tests currently in use. A solution to this problem did not arise until the work of Paul Uhlenhuth in 1901 .
The two references quoted in the above passage are:
 Gerber, S.M. (1983) A study in scarlet: blood identification in 1875. In Chemistry and Crime: From Sherlock Holmes to Today's Courtroom (Gerber, S.M. ed.), pp. 31-35, American Chemical Society (Columbus,OH, USA)
 Thorwald, J. (1965) The Century of the Detective (Winston, R. and Winston, C. trans.), Harcourt, Brace and World
Interestingly, random proteinoids stewed up in modern laboratories under fake primeval Earth conditions will carry out some simple enzyme reactions ( Table 29.04 ). They are far slower and less accurate than enzymes made by real cells, but nonetheless they can perform recognizable enzymatic reactions. For example, random proteinoids can often remove carbon dioxide from molecules like pyruvate or oxaloacetate and split organic esters. About 50% of all modern enzymes contain metal ions as co-factors and the addition of metal ions greatly extends the enzyme activities of random proteinoids. The presence of traces of copper promotes reactions involving amino groups and iron mediates oxidation-reduction reactions. Incorporation of zinc allows the breakdown of ATP, which is used by modern cells, both as a precursor of nucleic acids and as an energy carrier. Most modern enzymes that process nucleic acids possess a zinc atom as co-factor.
Table 29.04 . Enzyme Activities of Random Proteinoids
|Amination and deamination||Cu 2+||α-Ketoglutarate ↔ glutamate|
|Peroxidase and catalase||Heme, basic proteinoids||H2O2 and H-donors such as hydroquinone or NADH|
|Decarboxylation||Basic or acidic proteinoids||Oxaloacetate, pyruvate|
Artificial random proteinoids show inefficient but detectable enzyme activities.
Chemistry Chapter Four:
The balanced chemical equation shows that 16 CO2 molecules are produced for every 2 molecules of octane burned. We can extend this numerical relationship between molecules to the amounts in moles as follows: The coefficients in a chemical reaction specify the relative amounts in moles of each of the substances involved in the reaction.
The recipe shows the numerical relationships between the pizza ingredients. It says that
if we have 2 cups of cheese—and enough of everything else—we can make 1 pizza. We
can write this relationship as a ratio between the cheese and the pizza.
What if we have 6 cups of cheese? Assuming that we have enough of everything else, we
can use the above ratio as a conversion factor to calculate the number of pizzas.
Six cups of cheese are sufficient to make 3 pizzas. The pizza recipe contains numerical
ratios between other ingredients as well, including the following:
2 mol C8H18(l) : 16 mol CO2(g)
We can use this ratio to determine how many moles of CO2 are produced for a given
number of moles of C8H18 burned. Suppose that we burn 22.0 moles of C8H18 how many
moles of CO2 are produced? We use the ratio from the balanced chemical equation in the same way that we used the ratio from the pizza recipe. This ratio acts as a conversion fac-
tor allowing us to convert from the amount in moles of the reactant (C8H18) to the amount in moles of the product (CO2):
22.0 mol C8H18 * 16 mol CO2
2 mol C8H18 = 176 mol CO2
Mass A ---> Amount A (in moles) --> Amount B (in moles) ---> Mass B
where A and B are two different substances involved in the reaction. We use the molar mass of A to convert from the mass of A to the amount of A (in moles). We use the appropriate
ratio from the balanced chemical equation to convert from the amount of A (in moles) to the amount of B (in moles). And finally, we use the molar mass of B to convert from the amount of B (in moles) to the mass of B. To calculate the mass of CO2 emitted upon the combustion of 3.7 * 1015 g of octane, therefore, we use the following conceptual plan:
g C8H18 ---> mol C8H18 ---> mol CO2 --> g CO2
We follow the conceptual plan to solve the problem, beginning with g C8H18 and canceling units to arrive at g CO2:
3.7 * 10^15 g C8H18 * 1 mol C8H18/114.22 g C8H18
*16 mol CO2/2 mol C8H18 * 44.01 g CO2/1 mol CO2
The world's petroleum combustion produces 1.1 * 1016 g CO2 (1.1 * 1013 kg) per year. In comparison, volcanoes produce about 2.0 * 1011 kg CO2 per year.*
1 crust + 5 ounces tomato sauce + 2 cups cheese S 1 pizza
Suppose that we have 4 crusts, 10 cups of cheese, and 15 ounces of tomato sauce. How many pizzas can we make?
We have enough crusts to make:
4 crusts * 1 Pizza/1 crust = 4 pizza
10 cups cheese x 1 pizza/ 2 cups cheese = 5 pizzas
We have enough crusts for 4 pizzas, enough cheese for 5 pizzas, but enough tomato sauce for only 3 pizzas. Consequently, unless we get more ingredients, we can make only 3 pizzas. The tomato sauce limits how many pizzas we can make. If the pizza recipe were a chemical reaction, the tomato sauce would be the limiting reactant, the reactant that limits the amount of product in a chemical reaction. Notice that the limiting reactant is simply the reactant that makes the least amount of product. Reactants that do not limit the amount of product—such as the crusts and the cheese in this example—are said to be in excess. If this were a chemical reaction, 3 pizzas would be the theoretical yield, the amount of product that can be made in a chemical reaction based on the amount of limiting reactant.
Let us carry this analogy one step further. Suppose we go on to cook our pizzas and accidentally burn one of them. Even though we theoretically have enough ingredients for 3 pizzas, we end up with only 2. If this were a chemical reaction, the 2 pizzas would be our actual yield, the amount of product actually produced by a chemical reaction. (The actual yield is always equal to or less than the theoretical yield because at least a small amount of product is usually lost to other reactions or does not form during a reaction.) Finally, we calculate our percent yield, the percentage of the theoretical yield that was actually attained, as follows:
Percent Yield = Actual Yield/Theoretical Yield x 100%
Summarizing: Limiting Reactant and Yield
▶ The limiting reactant (or limiting reagent) is the reactant that is completely consumed in a chemical reaction and limits the amount of product.
▶ The reactant in excess is any reactant that occurs in a quantity greater than is
required to completely react with the limiting reactant.
▶ The theoretical yield is the amount of product that can be made in a chemical reaction based on the amount of limiting reactant.
▶ The actual yield is the amount of product actually produced by a chemical reaction.
▶ The percent yield is calculated as actual yield/theoretical yield
We can apply these concepts to a chemical reaction. Recall from Section 3.10 our
balanced equation for the combustion of methane:
We have developed antibody micropatterns into a novel two-hybrid assay for the detailed investigation of conformation-specific in cis interactions of our model protein, MHC I. The versatility of our assay with extracellular HA and intracellular GFP fusions supports a broad range of applications, especially in the study of specific protein-protein interactions that require investigation in the native environment of live cells. This two-hybrid assay circumvents the disadvantages of employing two different fluorescent proteins on the cytoplasmic tails of the proteins of interest (for example for FRET microscopy) that may be biased through unspecific interactions and aggregation of the fluorescent tags.
The example of MHC I proteins demonstrates the challenges of the functional analysis of protein-protein interactions and the limitations of conventional methods, which yield no information on the spatial resolution or the distinction of different protein conformations. Previous experiments with FRET have revealed cluster formation of antibody-labelled MHC I proteins at the cells surface, but the involvement of free heavy chains was only indirectly shown (Matko et al., 1994). Other studies involved co-immunoprecipitation experiments that revealed the existence of free heavy chain-dimers of different murine MHC I allotypes by pull-down with conformation-specific antibodies. However, it could not be excluded that the detected interactions were enhanced, or indeed caused, by detergents after cell lysis. Additional pulse-chase experiments confirmed that MHC I proteins associate after they have traversed the medial Golgi, but could not localize them to the cell surface (Capps et al., 1993).
Our own co-immunoprecipitation experiments confirm these observations for the murine K b allotype, but they also lack spatial resolution ( Figure 4 ). The differential co-immunoprecipitation of surface-biotinylated MHC I proteins finally confirms the presence of protein-protein associations at the cell surface ( Figure 4𠅏igure supplement 1 ), but even this method cannot entirely exclude the involvement of intracellular MHC I proteins.
Our two-hybrid assay finally solves the questions of generation and location for MHC I protein-protein interactions. The assay principle has allowed us to establish a system in which we generate defined conformations of MHC I proteins. The results demonstrate that MHC I protein association depends on the generation of free heavy chains, and together with our previous work (Montealegre et al., 2015), they suggest that these free heavy chains originate from the captured empty K b HC/β2m dimers at the cell surface upon dissociation of β2m. By holding the dimers in the anti-HA patterns and triggering the dissociation of β2m with the 37ଌ shift, we were able to show that in cis interactions are indeed happening at the cell surface ( Figure 3B ). Of course, it is possible that in addition, free heavy chains are generated elsewhere in the cell by β2m dissociation, for example in endosomes, and that they might associate in these locations also.
Is it possible that more than one kind of MHC I protein-protein interaction exists in cells? The MHC I clusters identified by the Yewdell and Edidin groups (Yewdell, 2006 Matko et al., 1994) contain peptide-bound MHC I proteins. Since our cells were TAP-deficient and thus contained no, or few, peptides for binding to MHC I proteins, we would not have seen the clusters observed by them. If, for example, the Yewdell clusters are formed in the ER then they would not even have formed in our system upon addition of external peptide.
The spatial organization of bait proteins in the plasma membrane allows for the quantification of co-captured proteins into the antibody pattern elements ( Figure 3C ). In our setup, we determined the distribution of prey proteins in control experiments as biological background (this corresponds to our background ratio of 1.1). This background includes also MHC I proteins that are co-captured on the antibody pattern elements before the temperature shift. Whether this background corresponds to pre-formed protein-protein interactions or a heterogeneous surface distribution was not tested and requires detailed analysis.
For MHC I in cis interactions at the cell surface, various functional roles have been proposed. They might be a means of accelerated disposal for free heavy chains, preventing re-binding of β2m and peptide and perhaps leading to enhanced internalization and degradation in lysosomes (Montealegre et al., 2015). This hypothesis is supported by our finding that associated MHC I proteins do not bind peptide well ( Figure 3𠅏igure supplement 2 ) and therefore probably do not interact with TCRs. They might be in trans ligands for NK cell receptors or similar proteins, perhaps signaling stress or activation states (Garcia-Beltran et al., 2016 Burian et al., 2016). We cannot entirely exclude that the associated MHC I proteins contain some K b HC/β2m dimers that are peptide-receptive, as has been suggested for human MHC I oligomers (Bodnár et al., 2003), but free heavy chains are clearly essential for in cis interactions, since single-chain K b HC/β2m dimers do not associate ( Figure 3A ). Another possibility is that the associated free heavy chain might influence the surface levels of other proteins with which MHC I proteins are known to interact, such as APLP or insulin receptor, thereby mediating non-immunological functions of MHC I proteins (Tuli et al., 2008 Shatz, 2009 Dixon-Salazar et al., 2014). Our assay is a promising tool to extend the interaction studies for MHC I proteins by the proposed interaction partners.
We have shown here the formation of homotypic in cis interactions of the murine MHC I allotype K b . Interestingly, previous work suggests that the tendency of in cis interactions varies among MHC I allotypes (Capps et al., 1993). Thus, it was hypothesized that those MHC I allotypes that do not associate are not internalized (by the accelerated disposal mechanism proposed above) and that they will bind exogenous peptides to provoke autoimmune reactions (Capps et al., 1993). This might be interesting in the case of those subtypes of HLA-B27 that are implicated in inflammatory autoimmune diseases such as spondyloarthropathies (Chen et al., 2017 Allen et al., 1999). For HLA-B*27:05, formation of heavy chain dimers at the cell surface, or in early endocytic compartments, was demonstrated to occur through a disulfide bond between the unpaired cysteine-67 residues. Since K b does not have an unpaired cysteine in the extracellular domain, this type of dimerization is not possible for K b . Still, the interesting possibility exists that the inis heavy chain associationsꃞscribed by usਏor K b might also occur with B*27:05 and might cause the formation of theovalent B*27:05 dimers. We look forward to future investigations.
Due its versatility, our assay allows for the development of a screen to test for the tendency of individual MHC Iਊllotypes to associate with themselves, and with other allotypes. This may be extended to human MHC I proteins, whose empty dimers can be enriched at the cell surface by incubation with low-affinity dipeptide ligands (Saini et al., 2015), and even to the empty forms of HLA-F that were recently discovered to bind NK cell receptors (Garcia-Beltran et al., 2016 Burian et al., 2016). Consequently, by its application to the human system, this screening tool can be developed to investigate the correlation between cell surface protein-protein interactions and human autoimmune disease. Generation of anti-HA antibody micropatterns by microcontact printing on conventional glass coverslips makes this assay especially suitable for such high-throughput approaches.
In addition to its demonstrated application in the detection of conformation-dependent in cis interactions, the assay can be further developed towards moreꃞtailed analysis. One possibility is the integration of conventional immunostaining for the identification of other proteins involved in the redistribution of co-captured proteins. For MHC I proteins, forxample, observing the accumulation of adaptor proteins involved in endocytic processes (e.g. Rab proteins) on the pattern elements under condition of co-capture will contribute to understand the nature of MHCI protein endocytosis and the functional role of in cis interactions.
Another possible technical development is to combine the assay with fluorescence revovery after photobleaching (FRAP) measurements to test the dynamics of the interactions (i.e. dissociation and re-association). Such a combined two-hybrid-FRAP assay is potentially superior to conventional (FRET) experiments, since the enrichment of proteins in the pattern elements increases the abundance of the interaction partners and might thus enable the detection of very weak interactions.
We show here that asparagine is critical for malaria parasite survival, in line with the known remarkable abundance of asparagine in Plasmodium proteins. While the presence of asparagine repeats in low complexity regions exists as a hallmark of P. falciparum proteins 34,35 , asparagine serves as one of the most abundant amino acids in proteins of other Plasmodium species as well 18 . In comparison with prokaryotic and eukaryotic proteomes with an asparagine frequency of ∼ 4–5% (ref. 36), the frequency of asparagine calculated from the complete coding sequences of Plasmodium species was found to fall within a range of 7–14% (P. falciparum ∼ 14%, P. knowlesi ∼ 8%, P. vivax ∼ 7%, P. berghei ∼ 13%, P. yoelii ∼ 13% and P. chaubadi ∼ 12%) with P. falciparum presenting the highest degree of asparagine content 18 . While the functional significance of asparagine repeats in P. falciparum still remains unclear 37,38 , it has been proposed that such asparagine repeats may serve as transfer RNA (tRNA) sponges that can facilitate protein folding by serving as inherent chaperones and control stage-specific expressions by modulating the translational rate 19 . Also, the parasite genome encodes a putative AS despite lacking canonical pathways for amino acid biosynthesis 15,16 . In the present study, we have characterized the AS of malaria parasites, generated KO parasites in P. berghei to address the essentiality of AS in the entire life cycle and determined the importance of extracellular asparagine in the survival of PbASKO parasites by using asparaginase treatment to deplete asparagine.
While ammonia-dependent AS-A is known to be present in prokaryotes 29,39,40 and in certain protozoan parasites such as Leishmania and Trypanosoma 41 , AS-B which utilizes glutamine as its preferred amino group donor is found in E. coli 31 , yeasts 42 and higher eukaryotes 32,43 . Here we use sequence comparison together with the enzyme assays performed for recombinant PbAS and total parasite lysates to reveal that the parasite enzyme belongs to the AS-B family 30,31,32 . The catalytic efficiency of the recombinant parasite enzyme with respect to glutamine (kcat/Km=2.7 × 10 3 M −1 s −1 ) was found to be at least 14-fold higher in comparison with ammonia (kcat/Km=0.19 × 10 3 M −1 s −1 ). Gene KO carried out for AS in P. berghei and subsequent comparisons made between the PbASKO phenotype and PbWT for the entire life cycle showed a significant delay in the mortality of mice infected with PbASKO asexual-stage parasites. These results serve as evidence of the synthesis of asparagine, suggesting that asparagine derived from haemoglobin degradation and extracellular sources 17,26 may not support optimal blood-stage development. More importantly, the sexual stage development of PbASKO parasites was drastically affected, with a nearly 69% reduction in the formation of sporozoites observed. We also found a significant decline in the formation of PbASKO EEFs, which led to a delay in the appearance of asexual stages and subsequent mortality when PbASKO sporozoites were injected intravenously to initiate liver-stage development in mice. Thus, asparagine synthesis mediated by endogenous AS is crucial for the optimal virulence of malaria parasites especially in the sexual and liver stages where asparagine availabilities in the mosquito host and mouse hepatocytes may prove severely limiting.
Unlike asexual stages wherein haemoglobin degradation serves as an intrinsic source of amino acids 17,26 , free-living sexual-stage parasites depend primarily on the mosquito haemolymph, and liver-stage parasites must subvert the metabolically active host hepatocytes for extracellular sources 44 . Given this context, we were interested to determine whether the absence of endogenous asparagine synthesis is compensated through the utilization of extracellular asparagine and this is in turn responsible for the suboptimal survival of PbASKO sexual and liver stages. To deplete the extracellular asparagine, PbASKO-infected mice were injected with three doses of L -asparaginase (50 IU per mouse), conforming to the ALL treatment regimens in children and adults that involve multiple doses of L -asparaginase (E. coli, Erwinia or pegylated) 22,23 administered over several days (5,000–10,000 IU m −2 for E. coli asparaginase) 22,23,45 . Although asparaginase treatment did not have significant effect on PbASKO asexual-stage development per se, sexual-stage development was fully inhibited in mosquitoes, as the PbASKO male gametocytes failed to exflagellate.
While asparagine depletion can affect protein synthesis in general, the expression of a particular subset of proteins related to exflagellation may be hindered severely. In addition, given limitations associated with female gametocyte functionality assessments, abnormalities associated with female gametocytes cannot be ruled out at this stage, and this would require extensive cross-fertilization studies 46 . Interestingly, asparagine content seems to be higher in gametocyte and sporozoite proteins of P. falciparum when compared with the asexual stages 19 . Unlike the asexual stages, which are completed within 24 h (for P. berghei) followed by the release of merozoites that invade fresh RBCs/reticulocytes, gametocytes reside within the same host cell for several days until gametes form in the mosquito host 47,48 . Therefore, their amino acid requirements may differ from those of asexual stages, and especially when the host haemoglobin reservoir is depleted during later stages of maturation. Asparagine depletion was found to inhibit the liver-stage development of PbASKO sporozoites in mice, suggesting the key role played by asparagine during the liver stages as well. The absence of EEFs and detectable parasite RNA in hepatocytes isolated from mice infected with PbASKO sporozoites suggests that asparagine depletion may lead to the prevention of PbASKO sporozoite invasion or to an early arrest of liver-stage development. Further investigations are needed to decipher the molecular events underlying the inhibition of sexual- and liver-stage development as well as the significance of these findings with other Plasmodium species, P. vivax in particular.
Our next goal was to examine whether asparagine supplementation can rescue effects of asparaginase treatment on PbASKO parasites. While in vitro supplementation had no effect, exflagellation could be partially restored by reducing asparaginase treatment in mice to 2 days and by supplementing them in vivo with asparagine, suggesting that asparagine is vital for the functional maturation of male gametocytes rather being required transiently for exflagellation. However, a similar in vivo supplementation failed to restore the liver-stage development of PbASKO sporozoites in asparaginase-treated mice. Considering the development of liver stages in metabolically active hepatocytes and the robust activity of asparaginase utilized in ALL treatment 22,23,45 , supplemented asparagine may become insufficient and/or less accessible for liver stages. This also denotes that asparagine requirement can serve as an effective target for preventing liver infections.
Altogether our findings lay the foundation for the examination of amino acid requirements in malaria parasites as a versatile therapeutic target for multiple stages (Fig. 8). As malaria parasites are auxotrophic to most of their amino acids 15,17,26 , depleting their extracellular sources would effectively interfere with the development of sexual stages in mosquitoes and liver stages in vertebrate hosts. The notion of extracellular amino acid depletion has thus far been pursued only in cancer therapies 22,23,49 , and the present study highlights its relevance to malaria treatment. For amino acids such as asparagine synthesized in the parasite, this approach may be combined with inhibitors specific to parasite enzymes. It would be interesting to examine whether adenylated sulfoximine derivatives (transition-state analogues capable of inhibiting AS and thus capable of suppressing the proliferation of asparaginase-resistant leukaemia cell lines 21,24 ) may be combined with asparaginase treatment to prevent the sexual- and liver-stage development of PbWT parasites. Furthermore, asparaginase resistance leading to subsequent relapse in patients with ALL has been mainly attributed to the increased expression of AS and to the reduced efflux of asparagine in leukaemia cells 21,24 that are mediated by various signalling events involving amino acid response, survival-related MEK/ERK and mTORC pathways 21,50 . However, the existence of a rudimentary amino acid response pathway lacking key homologues of downstream transcriptional factors that control the starvation response together with atypical kinase cascades and the absence of TORC and TORC-associated nutrient-sensing mechanisms in Plasmodium 17,51 suggest that the parasite responses in terms of resistance development may be different from those of cancer cells, and this issue will require further investigation.
Therapeutic options of depleting extracellular sources, targeting biosynthetic enzymes, transporters and AaRS are depicted in red. Brown box highlights the approaches relevant for sexual and liver stages. The approach of targeting AaRS is highlighted in green box. AAs, amino acids AaRS, aminoacyl-tRNA synthetases.
Transporters that facilitate the uptake of amino acids in malaria parasites need to be explored for new targets. Interestingly, the physiological relevance of transporters in malaria parasites remains unclear, with only one putative amino acid/auxin permease transporter annotated in the parasite genome. It appears that malaria parasites have evolved with a divergent set of transporters for amino acid uptake and that probable candidates may be those belonging to the major facilitator superfamily and ATP-binding cassette superfamily 52,53 . Aminoacyl-tRNA synthetases (AaRS) of malaria parasites may serve as another set of targets. As AaRS inhibitors are known for their anti-bacterial and anti-fungal properties, international efforts have been dedicated to the development of new compounds with better efficacy levels 54,55 . It has recently been shown that analogues of borrelidin-inhibiting threonyl-tRNA synthetase can offer 100% protection in P. yoelii-infected mice 28 . Inhibitors are also available for asparaginyl-tRNA synthetases, of which tirandamycin B from Streptomyces sp. 17,944 was shown to exhibit in vitro antifilarial activity against Brugia malayi, a parasitic nematode that causes elephantiasis 56 . It would be of interest to examine the antimalarial potential of asparaginyl-tRNA synthetase inhibitors in malaria parasites. Thus, a combination of strategies that deplete extracellular amino acids while targeting biosynthetic enzymes, transporters and AaRS in parasites may be explored for the development of a single-exposure radical cure with prophylaxis and chemoprotection 2,11,12 . Over the last few years, a collective endeavour spearheaded by the Medicines for Malaria Venture (MMV a not for profit public–private partnership) in collaboration with academic entities and pharmaceutical companies led to the identification of more than 25,000 compounds with submicromolar IC50 values via high-throughput phenotypic screening tests performed against asexual stages of the malaria parasite. Approximately 400 compounds were selected based on their drug- and probe-like properties and were made available through the ‘Open Access Malaria Box’ 2,3 . It would be therefore worthwhile to screen the already existing MMV portfolio for possible leading candidates that may target the aforementioned aspects of asparagine requirement in malaria parasites. To conclude, targeting the asparagine requirement in malaria parasites offers new therapeutic options to combat malaria.
When a solution of a protein is boiled, the protein frequently becomes insoluble—i.e., it is denatured—and remains insoluble even when the solution is cooled. The denaturation of the proteins of egg white by heat—as when boiling an egg—is an example of irreversible denaturation. The denatured protein has the same primary structure as the original, or native, protein. The weak forces between charged groups and the weaker forces of mutual attraction of nonpolar groups are disrupted at elevated temperatures, however as a result, the tertiary structure of the protein is lost. In some instances the original structure of the protein can be regenerated the process is called renaturation.
Denaturation can be brought about in various ways. Proteins are denatured by treatment with alkaline or acid, oxidizing or reducing agents, and certain organic solvents. Interesting among denaturing agents are those that affect the secondary and tertiary structure without affecting the primary structure. The agents most frequently used for this purpose are urea and guanidinium chloride. These molecules, because of their high affinity for peptide bonds, break the hydrogen bonds and the salt bridges between positive and negative side chains, thereby abolishing the tertiary structure of the peptide chain. When denaturing agents are removed from a protein solution, the native protein re-forms in many cases. Denaturation can also be accomplished by reduction of the disulfide bonds of cystine—i.e., conversion of the disulfide bond (―S―S―) to two sulfhydryl groups (―SH). This, of course, results in the formation of two cysteines. Reoxidation of the cysteines by exposure to air sometimes regenerates the native protein. In other cases, however, the wrong cysteines become bound to each other, resulting in a different protein. Finally, denaturation can also be accomplished by exposing proteins to organic solvents such as ethanol or acetone. It is believed that the organic solvents interfere with the mutual attraction of nonpolar groups.
Some of the smaller proteins, however, are extremely stable, even against heat for example, solutions of ribonuclease can be exposed for short periods of time to temperatures of 90 °C (194 °F) without undergoing significant denaturation. Denaturation does not involve identical changes in protein molecules. A common property of denatured proteins, however, is the loss of biological activity—e.g., the ability to act as enzymes or hormones.
Although denaturation had long been considered an all-or-none reaction, it is now thought that many intermediary states exist between native and denatured protein. In some instances, however, the breaking of a key bond could be followed by the complete breakdown of the conformation of the native protein.
Although many native proteins are resistant to the action of the enzyme trypsin, which breaks down proteins during digestion, they are hydrolyzed by the same enzyme after denaturation. The peptide bonds that can be split by trypsin are inaccessible in the native proteins but become accessible during denaturation. Similarly, denatured proteins give more intense colour reactions for tyrosine, histidine, and arginine than do the same proteins in the native state. The increased accessibility of reactive groups of denatured proteins is attributed to an unfolding of the peptide chains.
If denaturation can be brought about easily and if renaturation is difficult, how is the native conformation of globular proteins maintained in living organisms, in which they are produced stepwise, by incorporation of one amino acid at a time? Experiments on the biosynthesis of proteins from amino acids containing radioactive carbon or heavy hydrogen reveal that the protein molecule grows stepwise from the N terminus to the C terminus in each step a single amino acid residue is incorporated. As soon as the growing peptide chain contains six or seven amino acid residues, the side chains interact with each other and thus cause deviations from the straight or β-chain configuration. Depending on the nature of the side chains, this may result in the formation of an α-helix or of loops closed by hydrogen bonds or disulfide bridges. The final conformation is probably frozen when the peptide chain attains a length of 50 or more amino acid residues.
Autopoiesis literally means &lsquoself-making&rsquo (from the Greek auto for self, and the verb poiéō meaning &lsquoI make&rsquo or &lsquoI do&rsquo) and it refers to the unique ability of a living organism to continually repair and maintain itself&mdashultimately to the point of reproducing itself&mdashusing energy and raw materials from its environment. In contrast, an allopoietic system (from the Greek allo for other) such as a car factory, uses energy and raw materials to produce an organized structure (a car) which is something other than itself (a factory). 9
Autopoiesis is a unique and amazing property of life&mdashthere is nothing else like it in the known universe. It is made up of a hierarchy of irreducibly structured levels. These include: (i) components with perfectly pure composition, (ii) components with highly specific structure, (iii) components that are functionally integrated, (iv) comprehensively regulated information-driven processes, and (v) inversely-causal meta-informational strategies for individual and species survival (these terms will be explained shortly). Each level is built upon, but cannot be explained in terms of, the level below it. And between the base level (perfectly pure composition) and the natural environment, there is an unbridgeable abyss. The enormously complex details are still beyond our current knowledge and understanding, but I will illustrate the main points using an analogy with a vacuum cleaner.
A vacuum cleaner analogy
My mother was excited when my father bought our first electric vacuum cleaner in 1953. It consisted of a motor and housing, exhaust fan, dust bag, and a flexible hose with various end pieces. Our current machine uses a cyclone filter and follows me around on two wheels rather than on sliders as did my mother&rsquos original one. My next version might be the small robotic machine that runs around the room all by itself until its battery runs out. If I could afford it, perhaps I might buy the more expensive version that automatically senses battery run-down and returns to its induction housing for battery recharge.
Notice the hierarchy of control systems here. The original machine required an operator and some physical effort to pull the machine in the required direction. The transition to two wheels allows the machine to trail behind the operator with little effort, and the cyclone filter eliminates the messy dust bag. The next transition to on-board robotic control requires no effort at all by the operator, except to initiate the action to begin with and to take the machine back to the power source for recharge when it has run down. And the next transition to automatic sensing of power run-down and return-to-base control mechanism requires no effort at all by the operator once the initial program is set up to tell the machine when to do its work.
If we now continue this analogy to reach the living condition of autopoiesis, the next step would be to install an on-board power generation system that could use various organic, chemical or light sources from the environment as raw material. Next, install a sensory and information processing system that could determine the state of both the external and internal environments (the dirtiness of the floor and the condition of the vacuum cleaner) and make decisions about where to expend effort and how to avoid hazards, but within the operating range of the available resources. Then, finally, the pièce de résistance, to install a meta-information (information about information) facility with the ability to automatically maintain and repair the life system, including the almost miraculous ability to reproduce itself&mdashautopoiesis.
Notice that each level of structure within the autopoietic hierarchy depends upon the level below it, but it cannot be explained in terms of that lower level. For example, the transition from out-sourced to on-board power generation depends upon there being an electric motor to run. An electric vacuum cleaner could sit in the cupboard forever without being able to rid itself of its dependence upon an outside source of power&mdashit must be imposed from the level above, for it cannot come from the level below. Likewise, autopoiesis is useless if there is no vacuum cleaner to repair, maintain and reproduce. A vacuum cleaner without autopoietic capability could sit in the cupboard forever without ever attaining to the autopoietic stage&mdashit must be imposed from the level above, as it cannot come from the level below.
The autopoietic hierarchy is therefore structured in such a way that any kind of naturalistic transition from one level to a higher level would constitute a Polanyi impossibility. That is, the structure at level i is dependent upon the structure at level i-1 but cannot be explained by the structure at that level. So the structure at level i must have been imposed from level i or above.
The naturalistic abyss
Most origin-of-life researchers agree (at least in the more revealing parts of their writings) 10 that there is no naturalistic experimental evidence directly demonstrating a pathway from non-life to life. They continue their research, however, believing that it is just a matter of time before we discover that pathway. But by using the vacuum cleaner analogy, we can give a solid demonstration that the problem is a Polanyi impossibility right at the foundation&mdashlife is separated from non-life by an unbridgeable abyss.
Dirty, mass-action environmental chemistry
The &lsquosimple&rsquo structure of the early vacuum cleaner is not simple at all. It is made of high-purity materials (aluminium, plastic, fabric, copper wire, steel plates etc) that are specifically structured for the job in hand and functionally integrated to achieve the designed task of sucking up dirt from the floor. Surprisingly, the dirt that it sucks up contains largely the same materials that the vacuum cleaner itself is made of&mdashaluminium, iron and copper in the mineral grains of dirt, fabric fibres in the dust, and organic compounds in the varied debris of everyday home life. However, it is the difference in form and function of these otherwise similar materials that distinguishes the vacuum cleaner from the dirt on the floor. In the same way, it is the amazing form and function of life in a cell that separates it from the non-life in its environment.
Naturalistic chemistry is invariably &lsquodirty chemistry&rsquo while life uses only &lsquoperfectly-pure chemistry&rsquo. I have chosen the word &lsquodirty chemistry&rsquo not in order to denigrate origin-of-life research, but because it is the term used by Nobel Prize winner Professor Christian de Duve, a leading atheist researcher in this field. 11 Raw materials in the environment, such as air, water and soil, are invariably mixtures of many different chemicals. In &lsquodirty chemistry&rsquo experiments, contaminants are always present and cause annoying side reactions that spoil the hoped-for outcomes. As a result, researchers often tend to fudge the outcome by using artificially purified reagents. But even when given pure reagents to start with, naturalistic experiments typically produce what a recent evolutionist reviewer variously called &lsquomuck&rsquo, &lsquogoo&rsquo and &lsquogunk&rsquo 12 &mdashwhich is actually toxic sludge. Even our best industrial chemical processes can only produce reagent purities in the order of 99.99%. To produce 100% purity in the laboratory requires very highly specialized equipment that can sort out single molecules from one another.
Another crucial difference between environmental chemistry and life is that chemical reactions in a test tube follow the Law of Mass Action. 13 Large numbers of molecules are involved, and the rate of a reaction, together with its final outcome, can be predicted by assuming that each molecule behaves independently and each of the reactants has the same probability of interacting. In contrast, cells metabolize their reactants with single-molecule precision, and they control the rate and outcome of reactions, using enzymes and nano-scale-structured pathways, so that the result of a biochemical reaction can be totally different to that predicted by the Law of Mass Action.
Publication in Nature ( 5 ): ‘Plasma thromboplastin’
Rosemary described her further examination of the diluted plasma of this patient, who had very little prothrombin. She saw how his plasma might provide evidence of a blood-thromboplastin. A few minutes after recalcification she removed an aliquot and used it in lieu of brain in a one-stage prothrombin test. The aliquot was as powerful as brain. At the same time a separate aliquot added to fibrinogen showed only trace amounts of thrombin.
Rosemary also used, as a crude preparation of antihaemophilic globulin (AHG) (see above), the ammonium sulphate precipitate made by 33% saturation of adsorbed normal plasma. A preparation of platelets could be made by differential centrifugation of citrated blood, i.e. by obtaining first a platelet-rich plasma, and then, after further centrifugation, separating and washing the platelets in saline. 53 ) had shown that factor V was mainly precipitated by the second ammonium precipitate at 50% saturation. Rosemary adsorbed and eluted from normal serum a preparation containing the activity then known as factor VII. When the AHG preparation, the serum eluate, platelets and calcium were added together, there was a powerful thromboplastin, clotting normal plasma in 13 s. This ‘thromboplastic’ strength was bettered later in our work.
Lupus-type circulating anticoagulants had not been discovered by 1952 and it seems likely, and with the wisdom of hindsight, that this patient was an example where prothrombin was deficient. Adsorbed normal plasma (a source of factor V) and normal serum (a source of factor VII) failed to shorten the patient's one-stage test. Many years later I appreciated that this type of anticoagulant was easily diluted out, and it was this property that allowed powerful blood-thromboplastin to be discovered (see above). Some years afterwards the patient's blood was re-examined by the late Roger Hardisty, and he reported finding a circulating anticoagulant. It is likely he was correct.
Tips & Tricks for Sonicating Different Sample Types
As previously explained, every step in the ChIP workflow, including sonication, must be tested for each cell type and target to achieve the best results.
ChIP assays with standard cell culture samples require between 1,000 and 5 million cells, depending on the level expression of the target of interest and the nature of its association with chromatin. As a general rule, the more cells you can have, the better the ChIP experiment will work. That some cells are harder to lyse than others and give a very low chromatin yield, and that different proteins have different expression levels in different cell types are also very important considerations when getting started with ChIP assays.
Performing ChIP with a Low Amount of Starting Material
If you work with patient samples, tissue biopsies, or sorted cell populations, you might have only barely enough cells to perform ChIP assays. In these cases, you can slightly modify the standard ChIP protocol to improve your results, avoid material loss, and in general reduce the number of steps.
The use of siliconized tubes during fixation avoids material loss by preventing the material from sticking to the tube wall. Additionally, the nuclear isolation step can be skipped when the sample amount is extremely limited. For the immunoprecipitation, you need to ensure to use a very sensitive and specific antibody that is validated to work well in ChIP-Seq assays.
A final consideration is that the library preparation method is very important for successful ChIP-Seq assays when performing experiments with limited amounts of starting material. The risk when performing ChIP with a low amount of chromatin is that PCR duplicates will be created when the immunoprecipitated chromatin is amplified during the library preparation step, and the sequencing of PCR duplicates cannot be discriminated from the signal generated from the immunoprecipitated chromatin. To solve this problem, Active Motif developed an NGS library preparation kit where the adapters contain unique molecular identifiers that allows the removal of PCR duplicates during the bioinformatic analyses.
Performing ChIP Assays with PBMCs
Peripheral blood mononuclear cells (PBMCs) are well known to be difficult to lyse, which results in low chromatin yields and poor-quality chromatin in some cases. To improve cell lysis, we recommend using a swelling buffer as well as a detergent in the sonication buffer and processing the samples with stronger sonication conditions than what is used with cell line samples.
To avoid sample loss, we don&rsquot recommend performing nuclear isolation with a Dounce homogenizer and performing longer sonication to complete the PBMC lysis and chromatin fragmentation. To help you perform ChIP assays with PBMCs, Active Motif offers the ChIP-IT® PBMC kit that contains all the buffers needed for efficient cell lysis and a good chromatin yield from PBMC samples.
How to Do ChIP Assays with FFPE Tissue Samples
Many clinical researchers have accumulated a lot of FFPE tissue samples associated with the medical characteristics of their patients. This kind of resource is very valuable for retrospective studies, but unfortunately, it is often difficult to obtain good chromatin yield and quality from the paraffin blocks because the material is in low quantity and the fixation and embedding protocol of the tissue blocks can be either unknown or very variable from sample to sample. Moreover, the harsh fixation conditions used when preparing FFPE tissue samples can destroy protein epitopes in some cases.
When performing ChIP with FFPE tissue samples, the first step is to carefully remove the paraffin and rehydrate the tissue section. To extract and shear the chromatin, you will need to treat the tissue with a lysis buffer and sometimes perform enzymatic shearing in addition to sonication since over-fixation is common with FFPE tissues, which makes the chromatin harder to solubilize and fragment.
Before starting the immunoprecipitation and the downstream sequencing, the chromatin quality and fragmentation efficiency need to be carefully checked. Because this poses a significant challenge to most researchers, Active Motif developed the ChIP-IT® FFPE II kit to help facilitate the extraction of the highest quality chromatin from FFPE samples.
A Quik Way Around Partial Restriction Digests
No matter how many times you look at it, it’s not going to change. You are planning your next cloning experiment, but there’s a problem. The only restriction enzyme that cuts in a suitable position on your plasmid vector also, as luck would have it, cuts in another position elsewhere in the vector so you&hellip