Immunogenicity of standard and extended dosing intervals of BNT162b2 mRNA vaccine.

Extension of the interval between vaccine doses for the BNT162b2 mRNA vaccine was introduced in the United Kingdom to accelerate population coverage with a single dose. At this time, trial data were lacking, and we addressed this in a study of United Kingdom healthcare workers. The first vaccine dose induced protection from infection from the circulating alpha (B.1.1.7) variant over several weeks. In a substudy of 589 individuals, we show that this single dose induces severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing antibody (NAb) responses and a sustained B and T cell response to the spike protein. NAb levels were higher after the extended dosing interval (6-14 weeks) compared with the conventional 3- to 4-week regimen, accompanied by enrichment of CD4+ T cells expressing interleukin-2 (IL-2). Prior SARS-CoV-2 infection amplified and accelerated the response. These data on dynamic cellular and humoral responses indicate that extension of the dosing interval is an effective immunogenic protocol.

IgG3 regulates tissue-like memory B cells in HIV-infected individuals.

Immunoglobulin G3 (IgG3) has an uncertain role in the response to infection with and vaccination against human immunodeficiency virus (HIV). Here we describe a regulatory role for IgG3 in dampening the immune system-activating effects of chronic HIV viremia on B cells. Secreted IgG3 was bound to IgM-expressing B cells in vivo in HIV-infected chronically viremic individuals but not in early-viremic or aviremic individuals. Tissue-like memory (TLM) B cells, a population expanded by persistent HIV viremia, bound large amounts of IgG3. IgG3 induced clustering of B cell antigen receptors (BCRs) on the IgM+ B cells, which was mediated by direct interactions between soluble IgG3 and membrane IgM of the BCR (IgM-BCR). The inhibitory IgG receptor CD32b (FcγRIIb), complement component C1q and inflammatory biomarker CRP contributed to the binding of secreted IgG3 onto IgM-expressing B cells of HIV-infected individuals. Notably, IgG3-bound TLM B cells were refractory to IgM-BCR stimulation, thus demonstrating that IgG3 can regulate B cells during chronic activation of the immune system.

Methane emission from a cool brown dwarf.

Beyond our Solar System, aurorae have been inferred from radio observations of isolated brown dwarfs1,2. Within our Solar System, giant planets have auroral emission with signatures across the electromagnetic spectrum including infrared emission of H3+ and methane. Isolated brown dwarfs with auroral signatures in the radio have been searched for corresponding infrared features, but only null detections have been reported3. CWISEP J193518.59-154620.3. (W1935 for short) is an isolated brown dwarf with a temperature of approximately 482 K. Here we report James Webb Space Telescope observations of strong methane emission from W1935 at 3.326 µm. Atmospheric modelling leads us to conclude that a temperature inversion of approximately 300 K centred at 1-10 mbar replicates the feature. This represents an atmospheric temperature inversion for a Jupiter-like atmosphere without irradiation from a host star. A plausible explanation for the strong inversion is heating by auroral processes, although other internal and external dynamical processes cannot be ruled out. The best-fitting model rules out the contribution of H3+ emission, which is prominent in Solar System gas giants. However, this is consistent with rapid destruction of H3+ at the higher pressure where the W1935 emission originates4.

The first assessment of the genetic diversity and structure of the endangered West Indian manatee in Cuba.

The coastal waters of Cuba are home to a small, endangered population of West Indian manatee, which would benefit from a comprehensive characterization of the population's genetic variation. We conducted the first genetic assessment of Cuban manatees to determine the extent of the population's genetic structure and characterize the neutral genetic diversity among regions within the archipelago. We genotyped 49 manatees at 18 microsatellite loci, a subset of 27 samples on 1703 single nucleotide polymorphisms (SNPs), and sequenced 59 manatees at the mitochondrial control region. The Cuba manatee population had low nuclear (microsatellites HE = 0.44, and SNP HE = 0.29) and mitochondrial genetic diversity (h = 0.068 and π = 0.00025), and displayed moderate departures from random mating (microsatellite FIS = 0.12, SNP FIS = 0.10). Our results suggest that the western portion of the archipelago undergoes periodic exchange of alleles based on the evidence of shared ancestry and low but significant differentiation. The southeast Guantanamo Bay region and the western portion of the archipelago were more differentiated than southwest and northwest manatees. The genetic distinctiveness observed in the southeast supports its recognition as a demographically independent unit for natural resource management regardless of whether it is due to historical isolation or isolation by distance. Estimates of the regional effective population sizes, with the microsatellite and SNP datasets, were small (all Ne < 60). Subsequent analyses using additional samples could better examine how the observed structure is masking simple isolation by distance patterns or whether ecological or biogeographic forces shape genetic patterns.

Expression of serum response factor in the lung mesenchyme is essential for development of pulmonary fibrosis.

Extracellular matrix deposition characterizes idiopathic pulmonary fibrosis (IPF) and is orchestrated by myofibroblasts. The lung mesenchyme is an essential source of myofibroblasts in pulmonary fibrosis. Although the transcription factor serum response factor (SRF) has shown to be critical in the process of myofibroblast differentiation, its role in development of pulmonary fibrosis has not been determined in vivo. In this study, we observed that SRF expression localized to mesenchymal compartments, areas of dense fibrosis, and fibroblastic foci in human (IPF and normal) and bleomycin-treated mouse lungs. To determine the role of mesenchymal SRF in pulmonary fibrosis, we utilized a doxycycline-inducible, Tbx4 lung enhancer (Tbx4LE)-driven Cre-recombinase to disrupt SRF expression in the lung mesenchyme in vivo. Doxycycline-treated Tbx4LE-rtTA/TetO-Cre/tdTom/SRFf,f (and controls) were treated with a single intratracheal dose of bleomycin to induce pulmonary fibrosis and examined for lung mesenchymal expansion, pulmonary fibrosis, and inflammatory response. Bleomycin-treated Tbx4LE-rtTA/TetO-Cre/tdTom/SRFf,f mice showed decreased numbers of Tbx4LE-positive lung mesenchymal cells (LMCs) and collagen accumulation (via hydroxyproline assay) compared with controls. This effect was associated with SRF-null LMCs losing their proliferative and myofibroblast differentiation potential compared with SRF-positive controls. Together, these data demonstrate that SRF plays a critical role in LMC myofibroblast expansion during bleomycin-induced pulmonary fibrosis. This sets the stage for pharmacological strategies that specifically target SRF in the lung mesenchyme as a potential means of treating pulmonary fibrosis.

Isolating the roles of movement and reproduction on effective connectivity alters conservation priorities for an endangered bird.

Movement is important for ecological and evolutionary theory as well as connectivity conservation, which is increasingly critical for species responding to environmental change. Key ecological and evolutionary outcomes of movement, such as population growth and gene flow, require effective dispersal: movement that is followed by successful reproduction. However, the relative roles of movement and postmovement reproduction for effective dispersal and connectivity remain unclear. Here we isolate the contributions of movement and immigrant reproduction to effective dispersal and connectivity across the entire breeding range of an endangered raptor, the snail kite (Rostrhamus sociabilis plumbeus). To do so, we unite mark-resight data on movement and reproduction across 9 years and 27 breeding patches with an integrated model that decomposes effective dispersal into its hierarchical levels of movement, postmovement breeding attempt, and postmovement reproductive success. We found that immigrant reproduction limits effective dispersal more than movement for this endangered species, demonstrating that even highly mobile species may have limited effective connectivity due to reduced immigrant reproduction. We found different environmental limitations for the reproductive component of effective dispersal compared with movement, indicating that different conservation strategies may be needed when promoting effective dispersal rather than movement alone. We also demonstrate that considering immigrant reproduction, rather than movement alone, alters which patches are the most essential for connectivity, thereby changing conservation priorities. These results challenge the assumption that understanding movement alone is sufficient to infer connectivity and highlight that connectivity conservation may require not only fostering movement but also successful reproduction of immigrants.

Chronic virus infection enforces demethylation of the locus that encodes PD-1 in antigen-specific CD8(+) T cells.

Functionally exhausted T cells have high expression of the PD-1 inhibitory receptor, and therapies that block PD-1 signaling show promise for resolving chronic viral infections and cancer. By using human and murine systems of acute and chronic viral infections, we analyzed epigenetic regulation of PD-1 expression during CD8(+) T cell differentiation. During acute infection, naive to effector CD8(+) T cell differentiation was accompanied by a transient loss of DNA methylation of the Pdcd1 locus that was directly coupled to the duration and strength of T cell receptor signaling. Further differentiation into functional memory cells coincided with Pdcd1 remethylation, providing an adapted program for regulation of PD-1 expression. In contrast, the Pdcd1 regulatory region was completely demethylated in exhausted CD8(+) T cells and remained unmethylated even when virus titers decreased. This lack of DNA remethylation leaves the Pdcd1 locus poised for rapid expression, potentially providing a signal for premature termination of antiviral functions.

Urbanization and Population Genetic Structure of the Panama City crayfish (Procambarus econfinae).

For species with geographically restricted distributions, the impacts of habitat loss and fragmentation on long-term persistence may be particularly pronounced. We examined the genetic structure of Panama City crayfish (PCC), Procambarus econfinae, whose historical distribution is limited to an area approximately 145 km2, largely within the limits of Panama City and eastern Bay County, FL. Currently, PCC occupy approximately 28% of its historical range, with suitable habitat composed of fragmented patches in the highly urbanized western portion of the range and managed plantations in the more contiguous eastern portion of the range. We used 1640 anonymous single-nucleotide polymorphisms to evaluate the effects of anthropogenic habitat modification on the genetic diversity and population structure of 161 PCC sampled from across its known distribution. First, we examined urban habitat patches in the west compared with less-developed habitat patches in the east. Second, we used approximate Bayesian computation to model inferences on the demographic history of eastern and western populations. We found anthropogenic habitat modifications explain the genetic structure of PCC range-wide. Clustering analyses revealed significant genetic structure between and within eastern and western regions. Estimates of divergence between east and west were consistent with urban growth in the mid-20th century. PCC have low genetic diversity and high levels of inbreeding and relatedness, indicating populations are small and isolated. Our results suggest that PCC have been strongly affected by habitat loss and fragmentation and management strategies, including legal protection, translocations, or reintroductions, may be necessary to ensure long-term persistence.

Cancerous inhibitor of protein phosphatase 2A (CIP2A) modifies energy metabolism via 5' AMP-activated protein kinase signalling in malignant cells.

Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an adverse biomarker across many malignancies. Using K562 cells engineered to have high or low CIP2A expression, we show that high CIP2A levels significantly bias cellular energy production towards oxidative phosphorylation (OXPHOS) rather than glycolysis. Mass spectrometric analysis of CIP2A interactors and isobaric tagging for relative and absolute protein quantitation (ITRAQ) experiments identified many associated proteins, several of which co-vary with CIP2A level. Many of these CIP2A associating and co-varying proteins are involved in energy metabolism including OXPHOS, or in 5' AMP-activated protein kinase (AMPK) signalling, and manipulating AMPK activity mimics the effects of low/high CIP2A on OXPHOS. These effects are dependent on the availability of nutrients, driven by metabolic changes caused by CIP2A. CIP2A level did not affect starvation-induced AMPK phosphorylation of Unc-51 autophagy activating kinase 1 (ULK-1) at Ser555, but autophagy activity correlated with an increase in AMPK activity, to suggest that some AMPK processes are uncoupled by CIP2A, likely via its inhibition of protein phosphatase 2A (PP2A). The data demonstrate that AMPK mediates this novel CIP2A effect on energy generation in malignant cells.

The Central Role and Possible Mechanisms of Bacterial DNAs in Sepsis Development.

The pathological roles of bacterial DNA have been documented many decades ago. Bacterial DNAs are different from mammalian DNAs; the latter are heavily methylated. Mammalian cells have sensors such as TLR-9 to sense the DNAs with nonmethylated CpGs and distinguish them from host DNAs with methylated CpGs. Further investigation has identified many other types of DNA sensors distributed in a variety of cellular compartments. These sensors not only sense foreign DNAs, including bacterial and viral DNAs, but also sense damaged DNAs from the host cells. The major downstream signalling pathways includeTLR-9-MyD88-IKKa-IRF-7/NF-κB pathways to increase IFN/proinflammatory cytokine production, STING-TBK1-IRF3 pathway to increase IFN-beta, and AIM2-ASC-caspas-1 pathway to release IL-1beta. The major outcome is to activate host immune response by inducing cytokine production. In this review, we focus on the roles and potential mechanisms of DNA sensors and downstream pathways in sepsis. Although bacterial DNAs play important roles in sepsis development, bacterial DNAs alone are unable to cause severe disease nor lead to death. Priming animals with bacterial DNAs facilitate other pathological factors, such as LPS and other virulent factors, to induce severe disease and lethality. We also discuss compartmental distribution of DNA sensors and pathological significance as well as the transport of extracellular DNAs into cells. Understanding the roles of DNA sensors and signal pathways will pave the way for novel therapeutic strategies in many diseases, particularly in sepsis.

The number of breeders explains genetic connectivity in an endangered bird.

Connectivity is central to ecology and evolution as it focuses on the movement of individuals or genes across landscapes. Genetic connectivity approaches aim to understand gene flow but often estimate it indirectly based on metrics of genetic differentiation, which can also be affected by other evolutionary forces such as genetic drift. Gene flow and genetic drift are driven by separate ecological mechanisms with potentially differing effects on genetic differentiation and interpretations of genetic connectivity. The ecological mechanisms contributing to gene flow and genetic drift are primarily effective dispersal, or movement followed by successful reproduction, and the number of breeders in a local population, Nb , respectively. Yet, rarely are these ecological mechanisms and genetic connectivity measured simultaneously across landscapes. We examine the roles of effective dispersal and Nb on genetic connectivity across the entire range of the endangered snail kite (Rostrhamus sociabilis plumbeus), between 2006-2015. We find that both Nb and effective dispersal are important predictors of genetic connectivity across this landscape, but that Nb has a 3 × stronger effect on genetic connectivity. Furthermore, Nb is positively correlated with heterozygosity and allelic richness within patches, suggesting a potentially important role of genetic drift, in addition to gene flow, on genetic connectivity. These results emphasize that conservation efforts should focus on not only between-patch processes of movement but also within-patch processes regarding habitat quality and local population size for increasing genetic connectivity.

Mixing rates in weakly differentiated stocks of greater amberjack (Seriola dumerili) in the Gulf of Mexico.

The greater amberjack (Seriola dumerili) is a commercially and recreationally important marine fish species in the southeastern United States, where it has been historically managed as two non-mixing stocks (Gulf of Mexico and Atlantic). Mark-recapture studies and analysis of mitochondrial DNA have suggested the two stocks are demographically independent; however, little is currently known about when and where spawning occurs in Gulf of Mexico amberjack, and whether stock mixture occurs on breeding grounds. The primary objective of this study was to quantify stock mixture among breeding populations of amberjack collected from the Atlantic and Gulf of Mexico. Genetic data based on 11 loci identified very low, though statistically significant differentiation among Gulf of Mexico samples (GST = 0.007, [Formula: see text] = 0.009; all P = 0.001) and between reproductive adults collected from two spawning areas (GST = 0.007, [Formula: see text] = 0.014; all P = 0.001). Naïve Bayesian mixture analysis supported a single genetic cluster [p(S|data) = 0.734] whereas trained clustering (using Atlantic and Gulf spawning fish) gave the highest support to a two-cluster model (p(S|data) = 1.0). Our results support the argument that the genetic structuring of greater amberjack is more complex than the previously assumed two, non-mixing stock model. Although our data provide evidence of limited population structure, we argue in favour of non-panmixia among reproductive fish collected from the Gulf of Mexico and Florida Keys.

Genetic and Epigenetic Regulation of PD-1 Expression.

The inhibitory immune receptor programmed cell death-1 (PD-1) is intricately regulated. In T cells, PD-1 is expressed in response to most immune challenges, but it is rapidly downregulated in acute settings, allowing for normal immune responses. On chronically stimulated Ag-specific T cells, PD-1 expression remains high, leading to an impaired response to stimuli. Ab blockade of PD-1 interactions during chronic Ag settings partially restores immune function and is now used clinically to treat a variety of devastating cancers. Understanding the regulation of PD-1 expression may be useful for developing novel immune-based therapies. In this review, the molecular mechanisms that drive dynamic PD-1 expression during acute and chronic antigenic stimuli are discussed. An array of cis-DNA elements, transcription factors, and epigenetic components, including DNA methylation and histone modifications, control PD-1 expression. The interplay between these regulators fine-tunes PD-1 expression in different inflammatory environments and across numerous cell types to modulate immune responses.

The molecular signature of murine T cell homeostatic proliferation reveals both inflammatory and immune inhibition patterns.

T lymphocyte homeostatic proliferation, driven by the engagement of T cell antigen receptor with self-peptide/major histocompatibility complexes, and signaling through the common γ-chain-containing cytokine receptors, is critical for the maintenance of the T cell compartment and is regulated by the Fas death receptor (Fas, CD95). In the absence of Fas, Fas-deficient lymphoproliferation spontaneous mutation (lpr) mice accumulate homeostatically expanded T cells. The functional consequences of sequential rounds of homeostatic expansion are not well defined. We thus examined the gene expression profiles of murine wild-type and Fas-deficient lpr CD8+ T cell subsets that have undergone different amounts of homeostatic proliferation as defined by their level of CD44 expression, and the CD4-CD8-TCRαß+ T cell subset that results from extensive homeostatic expansion of CD8+ T cells. Our studies show that recurrent T cell homeostatic proliferation results in global gene expression changes, including the progressive upregulation of both cytolytic proteins such as Fas-Ligand and granzyme B as well as inhibitory proteins such as programmed cell death protein 1 (PD-1) and lymphocyte activating 3 (Lag3). These findings provide an explanation for how augmented T cell homeostatic expansion could lead to the frequently observed clinical paradox of simultaneous autoinflammatory and immunodeficiency syndromes and provide further insight into the regulatory programs that control chronically stimulated T cells.

Did Late Pleistocene climate change result in parallel genetic structure and demographic bottlenecks in sympatric Central African crocodiles, Mecistops and Osteolaemus?

The mid-Holocene has had profound demographic impacts on wildlife on the African continent, although there is little known about the impacts on species from Central Africa. Understanding the impacts of climate change on codistributed species can enhance our understanding of ecosystem dynamics and for formulating restoration objectives. We took a multigenome comparative approach to examine the phylogeographic structure of two poorly known Central African crocodile species-Mecistops sp. aff. cataphractus and Osteolaemus tetraspis. In addition, we conducted coalescent-based demographic reconstructions to test the hypothesis that population decline was driven by climate change since the Last Glacial Maximum, vs. more recent anthropogenic pressures. Using a hierarchical Bayesian model to reconstruct demographic history, we show that both species had dramatic declines (>97%) in effective population size in the 'period following the Last Glacial Maximum 1,500-18,000 YBP. Identification of genetic structuring showed both species have similar regional structure corresponding to major geological features (i.e., hydrologic basin) and that small observed differences between them are best explained by the differences in their ecology and the likely impact that climate change had on their habitat needs. Our results support our hypothesis that climatic effects, presumably on forest and wetland habitat, had a congruent negative impact on both species.

The causes of dispersal and the cost of carry-over effects for an endangered bird in a dynamic wetland landscape.

The decision to disperse or remain philopatric between breeding seasons has important implications for both ecology and evolution, including the potential for carry-over effects, where an individual's previous history affects its current performance. Carry-over effects are increasingly documented although underlying mechanisms remain unclear. Here we test for potential carry-over effects and their mechanisms by uniting hypotheses for the causes and consequences of habitat selection and dispersal across space and time. We linked hypotheses regarding different types of factors and information (environmental conditions, personal and public information) predicted to impact reproductive success and dispersal for an endangered, wetland-dependent bird, the snail kite (Rostrhamus sociabilis plumbeus). To do so, we coupled structural equation modelling with 20 years of mark-recapture and nesting data across the breeding range of this species to isolate potential direct and indirect effects of these factors. We found that water depth at nest sites explained subsequent emigration rates via an indirect path through the use of personal, not public, information. Importantly, we found that these dispersers tended to initiate nests later the following breeding season. This pattern explained a phenological mismatch of nesting with hydrological conditions, whereby immigrants tended to nest later, late nesters tended to experience lower water depths, higher nest failure occurred at lower water depths and higher nest failure explained subsequent breeding dispersal. These results identified a novel potential mechanism for carry-over effects: a phenological mismatch with environmental conditions (water depth) that occurred potentially due to time costs of dispersal. Our results also highlighted a substantial benefit of philopatry - earlier initiation of reproduction - which allows philopatric individuals to better coincide with environmental conditions that are beneficial for successful reproduction. These results have implications for our mechanistic understanding and prediction of carryover effects, and emphasize that local conservation strategies, such as water management, can explain future demography at distant sites connected through dispersal.

NF-κB regulates PD-1 expression in macrophages.

Programmed cell death-1 (PD-1) is responsible for T cell exhaustion during chronic viral infections and is expressed on a variety of immune cells following activation. Despite its importance, the mechanisms that regulate PD-1 in cell types other than CD8 T cells are poorly defined. In this study, the molecular mechanisms for inducing PD-1 expression in CD4 T cells, macrophages, and B cells were explored. In CD4 T cells, PD-1 induction following TCR stimulation required NFAT, as the calcineurin/NFAT pathway inhibitor cyclosporin A was able to block PD-1 induction in a manner similar to that seen in CD8 T cells. In contrast, LPS but not PMA and ionomycin stimulation was able to induce PD-1 expression in macrophages in a manner insensitive to cyclosporin A-mediated inhibition. B cells could use both pathways, although the levels of PD-1 expression were highest with PMA and ionomycin. An NF-κB binding site located upstream of the gene in conserved region C was required for NF-κB-dependent PD-1 gene activation in macrophages. Chromatin immunoprecipitation showed NF-κB p65 binding to this region following stimulation of macrophages with LPS. PD-1 induction was associated with histone modifications characteristic of accessible chromatin; however, in contrast to CD8 T cells, conserved region B in macrophages did not lose CpG methylation upon stimulation and PD-1 expression. The linkage of TLR/NF-κB signaling to the induction of PD-1 suggests the possibility of an opportunistic advantage to microbial infections in manipulating immune inhibitory responses.

Microsatellite Mutation Rate in Atlantic Sturgeon (Acipenser oxyrinchus).

Understanding mutation rates can greatly extend the utility of population and conservation genetic analyses. Herein, we present an estimate of genome-wide microsatellite mutation rate in Atlantic sturgeon (Acipenser oxyrinchus) based on parent-offspring transmission patterns. We screened 307 individuals for parentage and mutation-rate analysis applying 43 variable markers. Out of 13228 allele transfers, 11 mutations were detected, producing a mutation rate of 8.3 × 10-4 per locus per generation (95% confidence interval: 1.48 × 10-3, 4.15 × 10-4). Single-step mutations predominated and there were trends toward mutations in loci with greater polymorphism and allele length. Two of the detected mutations were most probably cluster mutations, being identified in 12 and 28 sibs, respectively. Finally, we observed evidences of polyploidy based on the sporadic presence of 3 or 4 alleles per locus in the genotyped individuals, supporting previous reports of incomplete diploidization in Atlantic sturgeon.

Disease causing mutants of TDP-43 nucleic acid binding domains are resistant to aggregation and have increased stability and half-life.

Over the last two decades many secrets of the age-related human neural proteinopathies have been revealed. A common feature of these diseases is abnormal, and possibly pathogenic, aggregation of specific proteins in the effected tissue often resulting from inherent or decreased structural stability. An archetype example of this is superoxide dismutase-1, the first genetic factor to be linked with amyotrophic lateral sclerosis (ALS). Mutant or posttranslationally modified TAR DNA binding protein-32 (TDP-43) is also strongly associated with ALS and an increasingly large number of other neurodegenerative diseases, including frontotemporal lobar degeneration (FTLD). Cytoplasmic mislocalization and elevated half-life is a characteristic of mutant TDP-43. Furthermore, patient age at the onset of disease symptoms shows a good inverse correlation with mutant TDP-43 half-life. Here we show that ALS and FTLD-associated TDP-43 mutations in the central nucleic acid binding domains lead to elevated half-life and this is commensurate with increased thermal stability and inhibition of aggregation. It is achieved without impact on secondary, tertiary, or quaternary structure. We propose that tighter structural cohesion contributes to reduced protein turnover, increasingly abnormal proteostasis and, ultimately, faster onset of disease symptoms. These results contrast our perception of neurodegenerative diseases as misfolded proteinopathies and delineate a novel path from the molecular characteristics of mutant TDP-43 to aberrant cellular effects and patient phenotype.

Heparin Isomeric Oligosaccharide Separation Using Volatile Salt Strong Anion Exchange Chromatography.

The complexity of heparin and heparan sulfate saccharides makes their purification, including many isomeric structures, very challenging and is a bottleneck for structure-activity studies. High-resolution separations have been achieved by strong anion exchange (SAX) chromatography on Propac PA1 and cetyltrimethylammonium (CTA)-C18 silica columns; however, these entail subsequent desalting methodologies and consequent sample losses and are incompatible with orthogonal chromatography methodologies and, in particular, mass spectrometry. Here, we present the CTA-SAX purification of heparin oligosaccharides using volatile salt (VS) buffer. In VSCTA-SAX, the use of ammonium bicarbonate buffer for elution improves resolution through both weaker dissociation and conformational coordination of the ammonium across the sulfate groups. Using ion mobility mass spectrometry, we demonstrate that isomeric structures have different structural conformations, which makes chromatographic separation achievable. Resolution of such structures is improved compared to standard SAX methods, and in addition, VSCTA-SAX provides an orthogonal method to isolate saccharides with higher purity. Because ammonium bicarbonate is used, the samples can be evaporated rather than desalted, preventing substantial sample loss and allowing more effective subsequent analysis by electrospray mass spectrometry. We conclude that VSCTA-SAX is a powerful new tool that helps address the difficult challenge of heparin/heparan sulfate saccharide separation and will enhance structure-activity studies.