Lost in Translation.

"Scleroderma," the rheumatologist said after examining my stiff swollen arms and legs. "Unfortunately, given your biomarkers, it's likely to get worse before it gets better, but you never know." She gave a quick rundown of what I might expect-rapidly progressive skin and joint tightening, GI symptoms, high likelihood of multi-organ involvement…. "Let's hope for the best." She paused, then asked if I had any questions.

Double Talk.

In this series of short essays, stories, poems, and personal observations, Robert A. Burton, neurologist and writer, uses both fiction and nonfiction to explore many paradoxes and contradictions inherent in scientific inquiry. A novelist as well as author of On Being Certain and A Skeptic's Guide to the Mind, Burton brings story to science and science to story.

Plague Journal.

Given a strong family history of early heart attacks, the future has always been an iffy proposition. Miraculously, I have bypassed the early off-ramps and find myself approaching 80, stents in place, considering the very real but previously unimaginable possibility of still more. But what kind of more? With dopamine on the wane and no longer supercharged by the push and shove of unbridled ambition and pride, bigger and grander are out of the question. Tired clichés poke through the widening cracks in my thinking to become uninvited bulletins of compromise and consolation. Be grateful. Relax, reminisce, enjoy sunsets, learn the backyard birds' names, maybe even sing to them, and count blessings.

When Will the News be Bad Enough?

The cardiac rehab nurse calls out each of our group's blood pressures and pulse rates. It is my first posthospitalization class and I am relieved to be in the middle of the pack. Although fully aware that numbers are not fate, I cannot help wondering if the worst performers will fully satisfy the dark needs of heart disease statistics. I presume that others are making similar calculations, yet wince at the ugly direction of my mind. Maybe it is not necessary to do better than another; if we take our meds, eat wisely, and exercise to the max, it is possible that our entire group will do well.

Substrate binding to Src: A new perspective on tyrosine kinase substrate recognition from NMR and molecular dynamics.

Most signal transduction pathways in humans are regulated by protein kinases through phosphorylation of their protein substrates. Typical eukaryotic protein kinases are of two major types: those that phosphorylate-specific sequences containing tyrosine (~90 kinases) and those that phosphorylate either serine or threonine (~395 kinases). The highly conserved catalytic domain of protein kinases comprises a smaller N lobe and a larger C lobe separated by a cleft region lined by the activation loop. Prior studies find that protein tyrosine kinases recognize peptide substrates by binding the polypeptide chain along the C-lobe on one side of the activation loop, while serine/threonine kinases bind their substrates in the cleft and on the side of the activation loop opposite to that of the tyrosine kinases. Substrate binding structural studies have been limited to four families of the tyrosine kinase group, and did not include Src tyrosine kinases. We examined peptide-substrate binding to Src using paramagnetic-relaxation-enhancement NMR combined with molecular dynamics simulations. The results suggest Src tyrosine kinase can bind substrate positioning residues C-terminal to the phosphoacceptor residue in an orientation similar to serine/threonine kinases, and unlike other tyrosine kinases. Mutagenesis corroborates this new perspective on tyrosine kinase substrate recognition. Rather than an evolutionary split between tyrosine and serine/threonine kinases, a change in substrate recognition may have occurred within the TK group of the human kinome. Protein tyrosine kinases have long been therapeutic targets, but many marketed drugs have deleterious off-target effects. More accurate knowledge of substrate interactions of tyrosine kinases has the potential for improving drug selectivity.

Factors Associated with the Rapid and Durable Decline in Malaria Incidence in El Salvador, 1980-2017.

A decade after the Global Malaria Eradication Program, El Salvador had the highest burden of malaria in Mesoamerica, with approximately 20% due to Plasmodium falciparum. A resurgence of malaria in the 1970s led El Salvador to alter its national malaria control strategy. By 1995, El Salvador recorded its last autochthonous P. falciparum case with fewer than 20 Plasmodium vivax cases annually since 2011. By contrast, its immediate neighbors continue to have the highest incidences of malaria in the region. We reviewed and evaluated the policies and interventions implemented by the Salvadoran National Malaria Program that likely contributed to this progress toward malaria elimination. Decentralization of the malaria program, early regional stratification by risk, and data-driven stratum-specific actions resulted in the timely and targeted allocation of resources for vector control, surveillance, case detection, and treatment. Weekly reporting by health workers and volunteer collaborators-distributed throughout the country by strata and informed via the national surveillance system-enabled local malaria teams to provide rapid, adaptive, and focalized program actions. Sustained investments in surveillance and response have led to a dramatic reduction in local transmission, with most current malaria cases in El Salvador due to importation from neighboring countries. Additional support for systematic elimination efforts in neighboring countries would benefit the region and may be needed for El Salvador to achieve and maintain malaria elimination. El Salvador's experience provides a relevant case study that can guide the application of similar strategies in other countries committed to malaria elimination.

Colorimetric tests for diagnosis of filarial infection and vector surveillance using non-instrumented nucleic acid loop-mediated isothermal amplification (NINA-LAMP).

Accurate detection of filarial parasites in humans is essential for the implementation and evaluation of mass drug administration programs to control onchocerciasis and lymphatic filariasis. Determining the infection levels in vector populations is also important for assessing transmission, deciding when drug treatments may be terminated and for monitoring recrudescence. Immunological methods to detect infection in humans are available, however, cross-reactivity issues have been reported. Nucleic acid-based molecular assays offer high levels of specificity and sensitivity, and can be used to detect infection in both humans and vectors. In this study we developed loop-mediated isothermal amplification (LAMP) tests to detect three different filarial DNAs in human and insect samples using pH sensitive dyes for enhanced visual detection of amplification. Furthermore, reactions were performed in a portable, non-instrumented nucleic acid amplification (NINA) device that provides a stable heat source for LAMP. The efficacy of several strand displacing DNA polymerases were evaluated in combination with neutral red or phenol red dyes. Colorimetric NINA-LAMP assays targeting Brugia Hha I repeat, Onchocerca volvulus GST1a and Wuchereria bancrofti LDR each exhibit species-specificity and are also highly sensitive, detecting DNA equivalent to 1/10-1/5000th of one microfilaria. Reaction times varied depending on whether a single copy gene (70 minutes, O. volvulus) or repetitive DNA (40 min, B. malayi and W. bancrofti) was employed as a biomarker. The NINA heater can be used to detect multiple infections simultaneously. The accuracy, simplicity and versatility of the technology suggests that colorimetric NINA-LAMP assays are ideally suited for monitoring the success of filariasis control programs.

Single-use, electricity-free amplification device for detection of HIV-1.

Early and accurate diagnosis of HIV is key for the reduction of transmission and initiation of patient care. The availability of a rapid nucleic acid test (NAT) for use at the point-of-care (POC) will fill a gap in HIV diagnostics, improving the diagnosis of acute infection and HIV in infants born to infected mothers. In this study, we evaluated the performance of non-instrumented nucleic acid amplification, single-use disposable (NINA-SUD) devices for the detection of HIV-1 in whole blood using reverse-transcription, loop-mediated isothermal amplification (RT-LAMP) with lyophilized reagents. The NINA-SUD heating device harnesses the heat from an exothermic chemical reaction initiated by the addition of saline to magnesium iron powder. Reproducibility was demonstrated between NINA-SUD units and comparable, if not superior, performance for detecting clinical specimens was observed as compared to the thermal cycler. The stability of the lyophilized HIV-1 RT-LAMP reagents was also demonstrated following storage at -20, 4, 25, and 30°C for up to one month. The single-use, disposable NAT minimizes hands-on time and has the potential to facilitate HIV-1 testing in resource-limited settings or at the POC.

NINA-LAMP compared to microscopy, RDT, and nested PCR for the detection of imported malaria.

Microscopy and field adaptable rapid diagnostic tests (RDTs) are not sensitive and specific in certain conditions such as poor training of microscopists, lack of electricity, or lower sensitivity in the detection of non-falciparum malaria. More sensitive point-of-care testing (POCT) would reduce delays in diagnosis and initiation of therapy. In the current study, we have evaluated the efficacy of noninstrumented nucleic acid amplification (NINA) coupled with loop-mediated isothermal amplification (LAMP) for detection of traveler's malaria (n=140) in comparison with microscopy, nested PCR, and the only Food and Drug Administration-approved rapid diagnostic test. NINA-LAMP was 100% sensitive and 98.6% specific when compared to nested PCR. For non-falciparum detection, NINA-LAMP sensitivity was 100% sensitive compared to nested PCR, whereas RDT sensitivity was 71%. LAMP is highly sensitive and specific for symptomatic malaria diagnosis regardless of species.

Structure, stability, and interaction of the fibrin(ogen) alphaC-domains.

Our recent study established the NMR structure of the recombinant bAalpha406-483 fragment corresponding to the NH(2)-terminal half of the bovine fibrinogen alphaC-domain and revealed that at increasing concentrations this fragment forms oligomers (self-associates). The major goals of the study presented here were to determine the structure and self-association of the full-length human fibrinogen alphaC-domains. To accomplish these goals, we prepared a recombinant human fragment, hAalpha425-503, homologous to bovine bAalpha406-483, and demonstrated using NMR, CD, and size-exclusion chromatography that its overall fold and ability to form oligomers are similar to those of bAalpha406-483. We also prepared recombinant hAalpha392-610 and bAalpha374-568 fragments corresponding to the full-length human and bovine alphaC-domains, respectively, and tested their structure, stability, and ability to self-associate. Size-exclusion chromatography revealed that both fragments form reversible oligomers in a concentration-dependent manner. Their oligomerization was confirmed in sedimentation equilibrium experiments, which also established the self-association affinities of these fragments and revealed that the addition of each monomer to assembling alphaC-oligomers substantially increases the stabilizing free energy. In agreement, unfolding experiments monitored by CD established that self-association of both fragments results in a significant increase in their thermal stability. Analysis of CD spectra of both fragments revealed that alphaC self-association results in an increase in the level of regular structure, implying that the COOH-terminal half of the alphaC-domain adopts an ordered conformation in alphaC-oligomers and that this domain contains two independently folded subdomains. Altogether, these data further clarify the structure of the human and bovine alphaC-domains and the molecular mechanism of their self-association into alphaC-polymers in fibrin.

Tyr130 phosphorylation triggers Syk release from antigen receptor by long-distance conformational uncoupling.

The Syk protein-tyrosine kinase plays a major role in signaling through the B cell receptor for antigen (BCR). Syk binds the receptor via its tandem pair of SH2 domains interacting with a doubly phosphorylated immunoreceptor tyrosine-based activation motif (dp-ITAM) of the BCR complex. Upon phosphorylation of Tyr-130, which lies between the two SH2 domains distant to the phosphotyrosine binding sites, Syk dissociates from the receptor. To understand the structural basis for this dissociation, we investigated the structural and dynamic characteristics of the wild type tandem SH2 region (tSH2) and a variant tandem SH2 region (tSH2(pm)) with Tyr-130 substituted by Glu to permanently introduce a negative charge at this position. NMR heteronuclear relaxation experiments, residual dipolar coupling measurements and analytical ultracentrifugation revealed substantial differences in the hydrodynamic behavior of tSH2 and tSH2(pm). Although the two SH2 domains in tSH2 are tightly associated, the two domains in tSH2(pm) are partly uncoupled and tumble in solution with a faster correlation time. In addition, the equilibrium dissociation constant for the binding of tSH2(pm) to dp-ITAM (1.8 microM) is significantly higher than that for the interaction between dp-ITAM and tSH2 but is close to that for a singly tyrosine-phosphorylated peptide binding to a single SH2 domain. Experimental data and hydrodynamic calculations both suggest a loss of domain-domain contacts and change in relative orientation upon the introduction of a negative charge on residue 130. A long-distance structural mechanism by which the phosphorylation of Y130 negatively regulates the interaction of Syk with immune receptors is proposed.

NMR solution structure, stability, and interaction of the recombinant bovine fibrinogen alphaC-domain fragment.

According to the existing hypothesis, in fibrinogen, the COOH-terminal portions of two Aalpha chains are folded into compact alphaC-domains that interact intramolecularly with each other and with the central region of the molecule; in fibrin, the alphaC-domains switch to an intermolecular interaction resulting in alphaC-polymers. In agreement, our recent NMR study identified within the bovine fibrinogen Aalpha374-538 alphaC-domain fragment an ordered compact structure including a beta-hairpin restricted at the base by a 423-453 disulfide linkage. To establish the complete structure of the alphaC-domain and to further test the hypothesis, we expressed a shorter alphaC-fragment, Aalpha406-483, and performed detailed analysis of its structure, stability, and interactions. NMR experiments on the Aalpha406-483 fragment identified a second loose beta-hairpin formed by residues 459-476, yielding a structure consisting of an intrinsically unstable mixed parallel/antiparallel beta-sheet. Size-exclusion chromatography and sedimentation velocity experiments revealed that the Aalpha406-483 fragment forms soluble oligomers whose fraction increases with an increase in concentration. This was confirmed by sedimentation equilibrium analysis, which also revealed that the addition of each monomer to an assembling alphaC-oligomer substantially increases its stabilizing free energy. In agreement, unfolding experiments monitored by CD established that oligomerization of Aalpha406-483 results in increased thermal stability. Altogether, these experiments establish the complete NMR solution structure of the Aalpha406-483 alphaC-domain fragment, provide direct evidence for the intra- and intermolecular interactions between the alphaC-domains, and confirm that these interactions are thermodynamically driven.

Residue-specific 13C' CSA tensor principal components for ubiquitin: correlation between tensor components and hydrogen bonding.

The residue-specific 13C' CSA tensor principal components, sigma(11), sigma(22), sigma(33), and the tensor orientation defined by the rotation angles beta and gamma have been determined by solution NMR for uniformly labeled ubiquitin partially aligned in four different media. Spurious chemical shift deviations due to solvent effects were corrected with an offset calculated by linear regression of the residual dipolar couplings and chemical shifts at increasing alignment strengths. Analysis of this effect revealed no obvious correlation to solvent exposure. Data obtained in solution from a protein offer a better sampling of 13C' CSA for different amino acid types in a complex heterogeneous environment, thereby allowing for the evaluation of structural variables that would be challenging to achieve by other methods. The 13C' CSA principal components cluster about the average values previously determined, and experimental correlations observed between sigma(11), sigma(22) tensorial components and C'O...H(N) hydrogen bonding are discussed. The inverse association of sigma(11) and sigma(22) exemplify the calculated and solid-state NMR observed effect on the tensor components by hydrogen bonding. We also show that 13C' CSA tensors are sensitive to hydrogen-bond length but not hydrogen-bond angle. This differentiation was previously unavailable. Similarly, hydrogen bonding to the conjugated NH of the same peptide plane has no detectable effect. Importantly, the observed weak correlations signify the presence of confounding influences such as nearest-neighbor effects, side-chain conformation, electrostatics, and other long-range factors to the 13C' CSA tensor. These analyses hold future potential for exploration provided that more accurate data from a larger number of proteins and alignments become available.

B-myc: N-terminal recognition of myc binding proteins.

B-Myc is an endogenous, N-terminal homologue of transcription factor c-Myc that lacks the C-terminal DNA binding and protein dimerization domain of c-Myc. Clinical mutations in the c-Myc N-terminal region, and the subsequent misregulation of Myc, are implicated in the development of numerous human cancers. Myc functions to both activate and repress transcription by associating with multiple binding partners. We investigated the structural and dynamical properties of B-Myc, free or associated with the transactivation inhibitor, MM-1, and the activator, TBP, using NMR spectroscopy. B-Myc has no persistent tertiary structure, yet regions corresponding to Myc homology boxes 1 and 2 (MBI and MBII, respectively) have molten globule-like characteristics. B-Myc binds to MM-1 in a specific manner without becoming highly structured. The local regions of B-Myc involved in binding differ for MM-1 and TBP, and regions not identified by mutagenesis are found to be involved in MM-1 binding. The results provide new insights into Myc N-terminal protein-protein interactions. We propose a model for Myc regulation through differential involvement of MBI and MBII in the binding of Myc interacting proteins.

Determination of the residue-specific 15N CSA tensor principal components using multiple alignment media.

The individual components of the backbone (15)N CSA tensor, sigma(11), sigma(22), sigma(33), and the orientation of sigma(11) relative to the NH bond described by the angle beta have been determined for uniformly labeled (15)N, (13)C ubiquitin from partial alignment in phospholipid bicelles, Pf1 phage, and poly(ethylene glycol) by measuring the residue-specific residual dipolar couplings and chemical shift deviations. No strong correlation between any of the CSA tensor components is observed with any single structural feature. However, the experimentally determined tensor components agree with the previously determined average CSA principal components [Cornilescu and Bax (2000) J. Am. Chem. Soc. 122, 10143-10154]. Significant deviations from the averages coincide with residues in beta-strand or extended regions, while alpha-helical residue tensor components cluster close to the average values.

Identification of an ordered compact structure within the recombinant bovine fibrinogen alphaC-domain fragment by NMR.

The NMR solution structure of the bovine fibrinogen alphaC-domain fragment, including residues Aalpha374-538, reveals a type-I' beta-hairpin, restricted at the base by a C423-C453 disulfide linkage and a short turn preceding C423. Although both faces of the hairpin are formed mainly by hydrophilic residues, one of them is uncharged while the other has a characteristic pattern of charged residues which are highly conserved among vertebrate species. Chemical shift indexing and relaxation data indicate the presence of a collapsed hydrophobic region next to the hairpin that includes approximately 30 residues with slower concerted motion and higher content of nonpolar residues and, according to a previous study (Tsurupa, G., Tsonev, L., and Medved, L. (2002) Biochemistry 41, 6449-6459), may cooperate with the hairpin to form a compact cooperative unit (domain). Structure and relaxation data show that the region between C423 and C453 is populated by both random coil and beta-structure, suggesting that the cooperative structure in the isolated alphaC-domain is intrinsically unstable. This observation is in agreement with a very low energy of stabilization of the Aalpha374-538 fragment determined in unfolding experiments. The low stability of the alphaC-domain suggests a possible explanation for the previously observed intra- and intermolecular interactions of these domains in fibrinogen and fibrin.