Validation of endotoxin-core antibodies in dried blood spots as a measure of environmental enteropathy and intestinal permeability.

OBJECTIVE: To validate a method for measuring endotoxin-core antibodies (EndoCAb) from dried blood spots (DBS)-drops of capillary whole blood collected and dried on filter paper-as an indicator of environmental enteropathy (EE) in infancy and early childhood. METHODS: A commercially available enzyme immunoassay kit was adapted for use with DBS, with optimized steps for sample elution. Technical validation included analysis of linearity/recovery, precision and reliability, lower limit of detection, and correspondence between matched plasma and DBS samples. Validation in a field-based setting was implemented with samples from Peruvian infants (n = 82; age = 2-33 months) collected at two time points six months apart. RESULTS: A high correspondence between plasma and DBS levels of EndoCAb was observed (R2 = 0.93, P < .001). The lower limit of detection was found to be 0.01 GMU/mL. Interassay coefficient of variation (CV) was 10.9% and 8.06% for low and high controls, respectively. Mean intra-assay CVs were 3.22% and 1.83%, respectively. In a sample of Peruvian infants, EndoCAb levels increased with age as expected (P < .001). Age explained nearly 34.6% of the variance in EndoCAb across the sample. CONCLUSION: These findings demonstrate the validity and feasibility of measuring EndoCAb in remote field settings using minimally invasive DBS sampling.

A Structure-Activity Analysis for Probing the Mechanism of Processive Double-Stranded DNA Digestion by λ Exonuclease Trimers.

λ exonuclease (λexo) is an ATP-independent 5'-to-3' exonuclease that binds to double-stranded DNA (dsDNA) ends and processively digests the 5'-strand into mononucleotides. The crystal structure of λexo revealed that the enzyme forms a ring-shaped homotrimer with a central funnel-shaped channel for tracking along the DNA. On the basis of this structure, it was proposed that dsDNA enters the open end of the channel, the 5'-strand is digested at one of the three active sites, and the 3'-strand passes through the narrow end of the channel to emerge out the back. This model was largely confirmed by the structure of the λexo-DNA complex, which further revealed that the enzyme unwinds the DNA by 2 bp prior to cleavage, to thread the 5'-end of the DNA into the active site. On the basis of this structure, an "electrostatic ratchet" model was proposed, in which the enzyme uses a hydrophobic wedge to insert into the base pairs to unwind the DNA, a two-metal mechanism for nucleotide hydrolysis, a positively charged pocket to bind to the terminal 5'-phosphate generated after each round of cleavage, and an arginine residue (Arg-45) to bind to the minor groove of the downstream end of the DNA. To test this model, in this study we have determined the effects of 11 structure-based mutations in λexo on DNA binding and exonuclease activities in vitro, and on DNA recombination in vivo. The results are largely consistent with the model for the mechanism that was proposed on the basis of the structure and provide new insights into the roles of particular residues of the protein in promoting the reaction. In particular, a key role for Arg-45 in DNA binding is revealed.

Crystal structures of Escherichia coli exonuclease I in complex with single-stranded DNA provide insights into the mechanism of processive digestion.

Escherichia coli Exonuclease I (ExoI) digests single-stranded DNA (ssDNA) in the 3'-5' direction in a highly processive manner. The crystal structure of ExoI, determined previously in the absence of DNA, revealed a C-shaped molecule with three domains that form a central positively charged groove. The active site is at the bottom of the groove, while an extended loop, proposed to encircle the DNA, crosses over the groove. Here, we present crystal structures of ExoI in complex with four different ssDNA substrates. The structures all have the ssDNA bound in essentially the predicted manner, with the 3'-end in the active site and the downstream end under the crossover loop. The central nucleotides of the DNA form a prominent bulge that contacts the SH3-like domain, while the nucleotides at the downstream end of the DNA form extensive interactions with an 'anchor' site. Seven of the complexes are similar to one another, but one has the ssDNA bound in a distinct conformation. The highest-resolution structure, determined at 1.95 Å, reveals an Mg(2+) ion bound to the scissile phosphate in a position corresponding to Mg(B) in related two-metal nucleases. The structures provide new insights into the mechanism of processive digestion that will be discussed.

Crystal structures of lambda exonuclease in complex with DNA suggest an electrostatic ratchet mechanism for processivity.

The λ exonuclease is an ATP-independent enzyme that binds to dsDNA ends and processively digests the 5'-ended strand to form 5' mononucleotides and a long 3' overhang. The crystal structure of λ exonuclease revealed a toroidal homotrimer with a central funnel-shaped channel for tracking along the DNA, and a mechanism for processivity based on topological linkage of the trimer to the DNA was proposed. Here, we have determined the crystal structure of λ exonuclease in complex with DNA at 1.88-Å resolution. The structure reveals that the enzyme unwinds the DNA prior to cleavage, such that two nucleotides of the 5'-ended strand insert into the active site of one subunit of the trimer, while the 3'-ended strand passes through the central channel to emerge out the back of the trimer. Unwinding of the DNA is facilitated by several apolar residues, including Leu78, that wedge into the base pairs at the single/double-strand junction to form favorable hydrophobic interactions. The terminal 5' phosphate of the DNA binds to a positively charged pocket buried at the end of the active site, while the scissile phosphate bridges two active site Mg(2+) ions. Our data suggest a mechanism for processivity in which wedging of Leu78 and other apolar residues into the base pairs of the DNA restricts backward movement, whereas attraction of the 5' phosphate to the positively charged pocket drives forward movement of the enzyme along the DNA at each cycle of the reaction. Thus, processivity of λ exonuclease operates not only at the level of the trimer, but also at the level of the monomer.

Malnutrition, illness, poverty, and infant growth: A test of a syndemic hypothesis in Nuñoa, Peru.

The concept of syndemics provides an important framework for understanding the complex interactions of biological and social conditions. Its use in public health and epidemiological research has increased substantially in the past ten years. Many syndemic analyses rely on the use of a sum score and subsequently fail to demonstrate biological interaction, leading some scholars to question the utility of the syndemic approach. Here, we utilize data from 86 mother/infant pairs from the rural district of Nuñoa, Peru to test a potential syndemic relationship among infection, malnutrition and infant growth. Between 2014 and 2015, surveys were conducted to assess household wealth, sanitation, dietary diversity, and reported illness, while anthropometric measures of mothers and infants were conducted to assess nutritional status via height-for-age and weight-for-height z-scores. Ethnographic insight was used in the selection of key economic variables including the development of an agricultural wealth index. We then assessed whether this constellation of health outcomes met the criteria for a syndemic by performing a quantitative analysis in which we tested for (1) an association between economic marginalization and high-risk environments; (2) the concentration of malnutrition, poor growth, and infection; and (3) biological interaction among these health outcomes. We found that economic measures were associated with pathogenic and nutritional risk, and that these in turn were associated with infectious disease, nutritional status, and growth. However, we did not find evidence that the proposed syndemic met criteria (2) or (3). We conclude that, despite being both socially and biologically plausible, a syndemic of malnutrition, poor growth, and infection did not exist in this context. This analysis moves syndemic research forward by demonstrating that such hypotheses are falsifiable, thus presenting a process by which they may be tested and lending support to the use of syndemic theory as an effective analytic framework.