Geographic uniformity of the Lyme disease spirochete (Borrelia burgdorferi) and its shared history with tick vector (Ixodes scapularis) in the Northeastern United States

Over 80% of reported cases of Lyme disease in the United States occur in coastal regions of northeastern and mid-Atlantic states. The genetic structure of the Lyme disease spirochete (Borrelia burgdorferi) and its main tick vector (Ixodes scapularis) was studied concurrently and comparatively by sampling natural populations of I. scapularis ticks along the East Coast from 1996 to 1998. Borrelia is genetically highly diverse at the outer surface protein ospC. Since Borrelia is highly clonal, the ospC alleles can be used to define clones. A newly designed reverse line blotting (RLB) assay shows that up to 10 Borrelia clones can infect a single tick. The clone frequencies in Borrelia populations are the same across the Northeast. On the other hand, I. scapularis populations show strong regional divergence (among northeastern, mid-Atlantic, and southern states) as well as local differentiation. The high genetic diversity within Borrelia populations and the disparity in the genetic structure between Borrelia and its tick vector are likely consequences of strong balancing selection on local Borrelia clones. Demographically, both Borrelia and I. scapularis populations in the Northeast show the characteristics of a species that has recently expanded from a population bottleneck. Major geological and ecological events, such as the last glacial maximum (18,000 years ago) and the modern-day expansion of tick habitats, are likely causes of the observed "founder effects" for the two organisms in the Northeast. We therefore conclude that the genetic structure of B. burgdorferi has been intimately shaped by the natural history of its main vector, the northern lineage of I. scapularis ticks.     

An efficient haplotyping method with DNA pools

Determination of haplotype frequencies (the joint distribution of genetic markers) in large population samples is a powerful tool for association studies. This is due to their greater extent of polymorphism since any two bi-allelic single nucleotide polymorphisms (SNPs) generate a potential four-allele genetic marker. Therefore, a haplotype may capture a given functional polymorphism with higher statistical power than its SNP components. The statistical estimation of haplotype frequencies, usually employed in linkage disequilibrium studies, requires individual genotyping for each SNP in the haplotype, thus making it an expensive process. In this study, we describe a new method for direct measurement of haplotype frequencies in DNA pools by allele-specific, long-range haplotype amplification. The proposed method allows the efficient determination of haplotypes composed of two SNPs in close vicinity (up to 20 kb).     

In-depth qualitative and quantitative analysis of composite glycosylation profiles and other micro-heterogeneity on intact monoclonal antibodies by high-resolution native mass spectrometry using a modified Orbitrap

Here, we describe a fast, easy-to-use, and sensitive method to profile in-depth structural micro-heterogeneity, including intricate N-glycosylation profiles, of monoclonal antibodies at the native intact protein level by means of mass spectrometry using a recently introduced modified Orbitrap Exactive Plus mass spectrometer. We demonstrate the versatility of our method to probe structural micro-heterogeneity by describing the analysis of three types of molecules: (1) a non-covalently bound IgG4 hinge deleted full-antibody in equilibrium with its half-antibody, (2) IgG4 mutants exhibiting highly complex glycosylation profiles, and (3) antibody-drug conjugates. Using the modified instrument, we obtain baseline separation and accurate mass determination of all different proteoforms that may be induced, for example, by glycosylation, drug loading and partial peptide backbone-truncation. We show that our method can handle highly complex glycosylation profiles, identifying more than 20 different glycoforms per monoclonal antibody preparation and more than 30 proteoforms on a single highly purified antibody. In analyzing antibody-drug conjugates, our method also easily identifies and quantifies more than 15 structurally different proteoforms that may result from the collective differences in drug loading and glycosylation. The method presented here will aid in the comprehensive analytical and functional characterization of protein micro-heterogeneity, which is crucial for successful development and manufacturing of therapeutic antibodies     

Soil nutrients trump intraspecific effects on understory plant communities

Understanding the links between intraspecific genetic variation and patterns of diversity in associated communities has been the primary focus of community genetics or ‘genes-to-ecosystem’ research in ecology. While other ecological factors, such as the abiotic environment, have well-documented influences on communities, the relative contributions of genetic variation versus the environment to species interactions remains poorly explored. In this study, we use a common garden experiment to study a coastal dune plant community dominated by the shrub, Baccharis pilularis, which displays a morphological dimorphism in plant architecture. We found the differences in the understory plant community between erect and prostrate morphs of Baccharis to be statistically significant, but small relative to the impacts of nutrient additions (NPK and C additions), for the richness, cover, and biomass of the understory plant community. There were no significant interactions between Baccharis morphology and nutrient-addition treatments, suggesting the influence of nutrient addition was consistent between erect and prostrate morphs. Moreover, we found no difference in overall plant community composition between Baccharis morphs, while NPK additions led to shifts in understory community composition compared to unfertilized shrubs. In sum, our results indicate that nutrients are the more important factor governing understory plant community structure in a coastal dunes ecosystem followed by intraspecific variation in dominant shrub architecture. Our results address a growing call to understand the extended consequences of intraspecific variation across heterogeneous environments in terrestrial ecosystems.     

Population genetics,demographic and evolutionary history of the Dudley’s lousewort (Pedicularis dudleyi), a rare redwood forest specialist

Pedicularis dudleyi (Dudley’s Lousewort, Orobanchaceae) is an extremely rare plant endemic to the redwood forests of Central California. Until recently, the species was known only from three extant natural populations. However, in 2019, one of those populations was described as a novel species (P. rigginsiae D.J. Keil) based on morphological and ecological data leaving only two populations described as P. dudleyi. While little is known about the past distribution of the species, historical records have led to speculation that the species was once more widespread and may have suffered from habitat destruction as a result of widespread logging during the early twentieth century. We utilized a combination of ddRAD SNP and Sanger sequencing data to: (1) Test the morphological hypothesis that P. rigginsiae is distinct from P. dudleyi; (2) Describe the genetic diversity and population structure of P. dudleyi and; (3) Test the hypothesis that the species underwent a bottleneck corresponding with increased logging of redwood forests in the early twentieth century. Our results support the recognition of P. rigginsiae as distinct from P. dudleyi, increasing the conservation priority of both species. Genetic diversity statistics and analyses of genetic structure suggest that both populations of P. dudleyi are highly differentiated from each other with one population exhibiting unexpected substructure. Finally, demographic modeling supports a scenario where the contemporary rarity of the species is explained by a recent bottleneck.

     

A rapid serological assay for prediction of Salmonella infection status in slaughter pigs using surface plasmon resonance

We present a rapid surface plasmon resonance-based serological assay for the detection of Salmonella Typhimurium infection in pigs using the Plasmonic((R)) SPR device. Lipopolysaccharide (LPS, 10 microg mL(-1)) from Salmonella Typhimurium was immobilised by self-assembly on a hydrophobic SPR chip. Using this LPS-coated chip, it was possible to bind and detect the anti-Salmonella Typhimurium antibodies in serum of pigs infected with the bacteria. The developed SPR assay is able to differentiate between sera obtained from pigs having low, medium, and high levels of Salmonella infection. A commercial ELISA kit was used to classify the sera for levels of Salmonella infection on the basis of optical density (OD%). A strong positive correlation was observed between the SPR-based assay and the ELISA (n=38, r=0.90, p<0.01). The sensitivity and specificity of the assay are 0.93 and 0.87, respectively. The SPR-based assay is label-free and does not require any sample preparation or dilution steps. The total analysis time is 45 min for each serum sample. The assay was found to be specific for Salmonella Typhimurium and shows no cross-reactivity to Salmonella Choleraesuis or Escherichia coli antibodies. As no sample preparation is required the developed assay has the potential to be used as a reliable tool for Salmonella monitoring programmes in pork production.     

The cellular basis for immune interferon production in autoimmune MRL-Ipr/Ipr mice

Disturbances in immune interferon (IFN gamma) activity have been implicated in the development of human systemic lupus erythematosus (SLE) and the spontaneous disease sustained by autoimmune-prone mice. We therefore investigated the cellular basis for IFN gamma production in MRL-Ipr/Ipr mice and examined the relationship between synthesis of interleukin 2 (IL 2) and IFN gamma. In vitro IL 2 and IFN gamma production in 3 to 6-mo-old, autoimmune MRL-Ipr/Ipr and MRL-+/+ mice was compared with that seen in age- and sex-matched, immunologically normal CBA/J mice. 5 X 10(6) spleen cells were pulsed with 5 micrograms of concanavalin A (Con A), and the cellfree supernatant was assayed for IL 2 and IFN gamma activity at various times up to 72 hr. We found that peak levels of IL 2 in MRL mice were less than 10% of those in the CBA/J. Yet, production of IFN gamma by cells from the autoimmune and normal strains was quite comparable. The addition of murine IL 2 to optimally Con A-stimulated cells from the MRL-Ipr/Ipr or normal mice did not affect the subsequent peak production of IFN gamma. Although the primary producers of IFN gamma in cultures of normal mice bear the Lyt-2+ phenotype, the Lyt-1+2- T-cell subset was found to be the principal source of IFN gamma in the aged MRL-Ipr/Ipr. These data suggest that Lyt-1+ cells from MRL-Ipr/Ipr mice may be differentially responsive to the signal delivered by the same mitogenic lectin with respect to lymphokine production and may indicate a distorted commitment of such cells toward production of IFN gamma and repression of IL 2 synthesis. The relationship between hypoproduction of IL 2, this usual source of IFN gamma, and the autoimmune disease sustained by MRL-Ipr/Ipr mice remains unclear.