Annals of the New York Academy of Sciences, Apr 1, 2012
Sialic acids are a diverse family of monosaccharides widely expressed on all cell surfaces of ver... more Sialic acids are a diverse family of monosaccharides widely expressed on all cell surfaces of vertebrates and so-called "higher" invertebrates, and on certain bacteria that interact with vertebrates. This overview surveys examples of biological roles of sialic acids in immunity, with emphasis on an evolutionary perspective. Given the breadth of the subject, the treatment of individual topics is brief. Subjects discussed include biophysical effects regulation of factor H; modulation of leukocyte trafficking via selectins; Siglecs in immune cell activation; sialic acids as ligands for microbes; impact of microbial and endogenous sialidases on immune cell responses; pathogen molecular mimicry of host sialic acids; Siglec recognition of sialylated pathogens; bacteriophage recognition of microbial sialic acids; polysialic acid modulation of immune cells; sialic acids as pathogen decoys or biological masks; modulation of immunity by sialic acid Oacetylation, sialic acids as antigens and xeno-autoantigens; anti-sialoglycan antibodies in reproductive incompatibility, and sialic-acid based blood groups.
bioRxiv (Cold Spring Harbor Laboratory), Apr 17, 2021
Influenza A viruses (IAVs) exploit host glycans in airway epithelial mucosa to gain entry and ini... more Influenza A viruses (IAVs) exploit host glycans in airway epithelial mucosa to gain entry and initiate infection. Rapid detection of changes in IAV specificity towards host glycan classes can provide early indication of virus transmissibility and infection potential. IAVs use hemagglutinins (HA) to bind sialic acids linked to larger glycan structures and a switch in HA specificity from a2,3to a2,6-linked sialoglycans is considered a prerequisite for viral transmission from birds to humans. While the changes in HA structure associated with the evolution of binding phenotype have been mapped, the influence of glycan receptor presentation on IAV specificity remains obscured. Here, we describe a glycan array platform which uses synthetic mimetics of mucin glycoproteins to model how receptor presentation in the mucinous glycocalyx, including glycan type and valency of the glycoconjugates and their surface density, impact IAV binding. We found that H1N1 virus produced in embryonated chicken eggs, which recognizes both receptor types, exclusively engaged mucin-mimetics carrying a2,3-linked sialic acids in their soluble form. The virus was able utilize both receptor structures when the probes were immobilized on the array; however, increasing density in the mucin-mimetic brush diminished viral adhesion. Propagation in mammalian cells produced a change in receptor pattern recognition by the virus, without altering its HA affinity, toward improved binding of a2,6-sialylated mucin mimetics and reduced sensitivity to surface crowding of the probes. Application of a support vector machine (SVM) learning algorithm as part of the glycan array binding analysis efficiently characterized this shift in binding preference and may prove useful to study the evolution of viral responses to a new host. .
Deep infiltrating endometriosis (DIE) is a debilitating subset of endometriosis with an ill-defin... more Deep infiltrating endometriosis (DIE) is a debilitating subset of endometriosis with an ill-defined set of symptoms and lacks specific diagnostic criteria to identify, leading to significant delays in treatment. Endometriosis itself is associated with chronic pain and infertility, leading to significant healthcare costs. A literature has shown that DIE is a non-progressive and curable disease. Currently, diagnosis is based purely on surgical diagnosis, which carriers a higher rate of morbidity, and fear of surgery or reluctance on behalf of provider, may delay diagnosis and thus treatment. Multiple attempts have been made to create an appropriate classification system mainly based on symptoms or imaging, however no one scheme has been successful to appropriately identify DIE. Research has now been geared towards the use of biomarkers, including blood, urine, peritoneal/follicular fluid, or endometrial tissue, to construct a diagnostic tool for DIE. This has the potential to create a less invasive mechanism for earlier identification and treatment of this curable disease.
Humans can incorporate the xenoglycan N-glycolylneuraminic acid (Neu5Gc) from the diet into repro... more Humans can incorporate the xenoglycan N-glycolylneuraminic acid (Neu5Gc) from the diet into reproductive tissues and secretions. Most humans also have circulating antibodies specific for this dietary xenoglycan. The potential for inflammation induced by incorporated Neu5Gc and circulating anti-Neu5Gc antibodies, termed xenosialitis, has been discussed as a factor influencing several human diseases. Potential effects of xenosialitis on human fertility remain unknown. Here, we investigate possible adverse effects of the presence of Neu5Gc on sperm or endometrium combined with anti-Neu5Gc antibodies in semen or uterine secretions in a mouse model. We use Cmah ؊/؊ mice, humanized for Neu5Gc deficiency. We find that the viability, migration, and capacitation of sperm with incorporated Neu5Gc are negatively affected when these are exposed to anti-Neu5Gc antibodies. In addition, we find that after copulation, activated uterine neutrophils and macrophages show increased phagocytosis of sperm in the presence of anti-Neu5Gc antibodies via the complement receptor 3 (C3R) and Fc␥ I/II/III (Fc receptor). Furthermore, Neu5Gc in endometrial cells combined with the presence of anti-Neu5Gc antibodies alters the receptivity and decidualization of endometrial explants. These studies provide mechanistic insights on how Neu5Gc on sperm and/or endometrium combined with anti-Neu5Gc antibodies in semen and uterine fluid might contribute to unexplained human infertility.
Establishment of adequate levels of sialylation is crucial for sperm survival and function after ... more Establishment of adequate levels of sialylation is crucial for sperm survival and function after insemination; however, the mechanism for the addition of the sperm sialome has not been identified. Here, we report evidence for several different mechanisms that contribute to the establishment of the mature sperm sialome. Directly quantifying the source of the nucleotide sugar CMP-beta-N-acetylneuraminic acid in epididymal fluid indicates that transsialylation occurs in the upper epididymis. Western blots for the low-molecular-mass sialoglycoprotein (around 20-50 kDa) in C57BL/6 mice epididymal fluid reflect that additional sialome could be obtained by glycosylphosphatidylinositol-anchored sialoglycopeptide incorporation during epididymal transit in the caput of the epididymis. Additionally, we found that in Cmah (CMP-N-acetylneuraminic acid hydroxylase)À/À transgenic mice, epididymal sperm obtained sialylated-CD52 from seminal vesicle fluid (SVF). Finally, we used Gfp (green fluorescent protein)+/+ mouse sperm to test the role of sialylation on sperm for protection from female leukocyte attack. There is very low phagocytosis of the epididymal sperm when compared to that of sperm coincubated with SVF. Treating sperm with Arthrobacter ureafaciens sialidase (AUS) increased phagocytosis even further. Our results highlight the different mechanisms of increasing sialylation, which lead to the formation of the mature sperm sialome, as well as reveal the sialome's function in sperm survival within the female genital tract.
Sugars are the most functionally and structurally diverse molecules in the biological world. Glyc... more Sugars are the most functionally and structurally diverse molecules in the biological world. Glycan structures range from tiny single monosaccharide units to giant chains thousands of units long. Some glycans are branched, their monosaccharides linked together in many different combinations and orientations. Some exist as solitary molecules; others are conjugated to proteins and lipids and alter their collective functional properties. In addition to structural and storage roles, glycan molecules participate in and actively regulate physiological and developmental processes. Glycans also mediate cellular interactions within and between individuals. Their roles in ecology and evolution are pivotal, but not well studied because glycan biochemistry requires different methods than standard molecular biology practice. The properties of glycans are in some ways convenient, and in others challenging. Glycans vary on organismal timescales, and in direct response to physiological and ecological conditions. Their mature structures are physical records of both genetic and environmental influences during maturation. We describe the scope of natural glycan variation and discuss how studying glycans will allow researchers to further integrate the fields of ecology and evolution.
Proceedings of the National Academy of Sciences of the United States of America, Apr 27, 1999
Phylogenetic trees for the four extant species of African hominoids are presented, based on mtDNA... more Phylogenetic trees for the four extant species of African hominoids are presented, based on mtDNA control region-1 sequences from 1,158 unique haplotypes. We include 83 new haplotypes of western chimpanzees and bonobos. Phylogenetic analysis of this enlarged database, which takes intraspecific geographic variability into account, reveals different patterns of evolution among species and great heterogeneity in species-level variation. Several chimpanzee and bonobo clades (and even single social groups) have retained substantially more mitochondrial variation than is seen in the entire human species. Among the 811 human haplotypes, those that branch off early are predominantly but not exclusively African. Neighbor joining trees provide strong evidence that eastern chimpanzee and human clades have experienced reduced effective population sizes, the latter apparently since the Homo sapiens-neanderthalensis split. Application of topiary pruning resolves ambiguities in the phylogenetic tree that are attributable to homoplasies in the data set. The diverse patterns of mtDNA sequence variation seen in today's hominoid taxa probably ref lect historical differences in ecological plasticity, female-biased dispersal, range fragmentation over differing periods of time, and competition among social groups. These results are relevant to the origen of zoonotic diseases, including HIV-1, and call into question some aspects of the current taxonomic treatment and conservation management of gorillas and chimpanzees.
In the context of a study of wild chimpanzees, Pan troglodytes vems, we found that genotypes base... more In the context of a study of wild chimpanzees, Pan troglodytes vems, we found that genotypes based on single PCR amplifications of microsatellite loci from single shed hair have a high error rate. We quantified error rates using the comparable results of 791 single shed hair PCR amplifications of 11 microsatellite loci of 18 known individuals. The most frequent error was the amplification of only one of the two alleles present at a heterozygous locus. This phenomenon, called allelic dropout, produced false homozygotes in 31% of single-hair amplifications. There was no difference in the probability of preferential amplification between longer and shorter alleles. The probability of scoring false homozygotes can be reduced to below 0.05 by three separate amplifications from single hairs of the same individual or by pooling hair samples from the same individual. In this study an additional 5.6% of the amplifications gave wrong genotypes because of contamination, labelling and loading errors, and possibly amplification artefacts. In contrast, amplifications from plucked hair taken from four dead individuals gave consistent results (error rate < 0.01%, n = 120). Allelic dropout becomes a problem when the DNA concentration falls below 0.05 ng/lO pL in the template as it can with shed hair, and extracts from faeces and masticated plant matter.
The more than two hundred species of primates live and reproduce in a large variety of ways. Most... more The more than two hundred species of primates live and reproduce in a large variety of ways. Most species are highly social. Recent studies of primates in nature are revealing new facets of their social and mating systems. Female primates follow their own reproductive strategies, which are less conspicuous than male strategies. The slow development of young primates makes them crucially dependent on intense and prolonged maternal care, exacerbating the imbalance in reproductive effort between the sexes. Primate mating systems seem more flexible than previously thought, as social changes, ecological conditions, and individual character all affect the ways in which primates mate and reproduce. Interactions between males and females range from the molecular, such as interactions between egg and sperm or harmonious expression of paternal and maternal genes in the offspring, to the behavioral, including mating patterns, social affiliation, and parental care. There is increasing evidence for the importance of multiple paternity for females. This evidence includes the need for diverse offspring, for good and compatible genes from the fathers, and for male assistance. Primate mating systems result from a delicate balance between male and female strategies and the constraints exerted by the physical and social environments.
ABSTRACT Efforts to control the global pandemic of human hepatitis B virus (hHBV) infection have ... more ABSTRACT Efforts to control the global pandemic of human hepatitis B virus (hHBV) infection have been hampered by incomplete understanding of viral-host interactions in this disease. This situation has been confounded by the fact that hHBV has a limited host range and cannot be propagated in simple cell culture (1). Reproducible experimental infection with determination of infectivity was demonstrated in chimpanzees (Pan troglodytes), but not other primates (2–4), long before other animal models such as the woodchuck were identified. After successful inoculation of chimpanzees was reported in 1972, multiple institutions, including a multigroup collaboration between the FDA, CDC, and NIH, initiated studies to evaluate them as a model for the study of HBV. For the majority of studies only chimpanzees “with no prior exposure” to virus were used because those with positive serology [either from exposure to hHBV or chimpanzee HBV (chHBV)], with an estimated prevalence of 3–6% in Africa (5), were not reproducibly susceptible to infection (3). It has been widely reported that the effects of HBV infection in chimpanzees are milder than in humans, that is, few have developed fulminant hepatitis, and inoculated chimpanzees exhibit few symptoms or signs of infection. Furthermore, the incidence of chronic infection with HBV (see Table 2) and horizontal and vertical transmission (from mother to offspring) in chimpanzees is lower than in humans (6,7). Chimpanzees have been the cornerstone of all research on infectivity of HBV and safety and efficacy of vaccines.
Molecular Reproduction and Development, Jun 9, 2015
Mammalian spermatozoa are coated with a thick glycocalyx that is assembled during sperm developme... more Mammalian spermatozoa are coated with a thick glycocalyx that is assembled during sperm development, maturation, and upon contact with seminal fluid. The sperm glycocalyx is critical for sperm survival in the female reproductive tract and is modified during capacitation. The complex interplay among the various glycoconjugates generates numerous signaling motifs that may regulate sperm function and, as a result, fertility. Nascent spermatozoa assemble their own glycans while the cells still possess a functional endoplasmic reticulum and Golgi in the seminiferous tubule, but once spermatogenesis is complete, they lose the capacity to produce glycoconjugates de novo. Sperm glycans continue to be modified, during epididymal transit by extracellular glycosidases and glycosyltransferases. Furthermore, epididymal cells secrete glycoconjugates (glycophosphatidylinositol-anchored glycoproteins and glycolipids) and glycan-rich microvesicles that can fuse with the maturing sperm membrane. The sperm glycocalyx mediates numerous functions in the female reproductive tract, including the following: inhibition of premature capacitation; passage through the cervical mucus; protection from innate and adaptive female immunity; formation of the sperm reservoir; and masking sperm proteins involved in fertilization. The immense diversity in sperm-associated glycans within and between species forms a remarkable challenge to our understanding of essential sperm glycan functions.
Background: Influenza A virus (IAV) neuraminidase (NA) cleaves sialic acids (Sias) from glycans. ... more Background: Influenza A virus (IAV) neuraminidase (NA) cleaves sialic acids (Sias) from glycans. Inhibiting NA with oseltamivir suppresses both viral infection, and viral release from cultured human airway epithelial cells. The role of NA in viral exit is well established: it releases budding virions by cleaving Sias from glycoconjugates on infected cells and progeny virions. The role of NA in viral entry remains unclear. Host respiratory epithelia secrete a mucus layer rich in heavily sialylated glycoproteins; these could inhibit viral entry by mimicking sialylated receptors on the cell surface. It has been suggested that NA allows influenza to penetrate the mucus by cleaving these sialylated decoys, but the exact mechanism is not yet established. Methods: We tested IAV interaction with secreted mucus using frozen human trachea/bronchus tissue sections, and bead-bound purified human salivary mucins (HSM) and purified porcine submaxillary mucins (PSM). The protective effect of mucus was analyzed using MDCK cells coated with purified HSM and PSM with known Sia content. Oseltamivir was used to inhibit NA activity, and the fluorescent reporter substrate, 4MU-Neu5Ac, was used to quantify NA activity. Results: IAV binds to the secreted mucus layer of frozen human trachea/bronchus tissues in a Sia dependent manner. HSM inhibition of IAV infection is Sia dose-dependent, but PSM cannot inhibit infection of underlying cells. HSM competitively inhibits NA cleavage of 4MU-Neu5Ac, reporter substrate. Human IAV effectively cleaves Sias from HSM but not from PSM, and binds to HSM but not to PSM. Conclusion: IAV interacts with human mucus on frozen tissue sections and mucus-coated beads. Inhibition of IAV infection by sialylated human mucus is dose-dependent, and enhanced when NA is inhibited with oseltamivir. Thus NA cleaves sialylated decoys during initial stages of infection. Understanding IAV interactions with host mucins is a promising new avenue for drug development.
Mucins are complex and heavily glycosylated O-linked glycoproteins, which contain more than 70% c... more Mucins are complex and heavily glycosylated O-linked glycoproteins, which contain more than 70% carbohydrate by weight 1-3. Secreted mucins, produced by goblet cells and the gastric mucosa, provide the scaffold for a micrometers-thick mucus layer that lines the epithelia of the gut and respiratory tract 3,4. In addition to mucins, mucus layers also contain antimicrobial peptides, cytokines, and immunoglobulins 5-9. The mucus layer is an important part of host innate immunity, and forms the first line of defense against invading microorganisms 8,10-12. As such, the mucus is subject to numerous interactions with microbes, both pathogens and symbionts, and secreted mucins form an important interface for these interactions. The study of such biological interactions usually involves histological methods for tissue collection and staining. The two most commonly used histological methods for tissue collection and preservation in the clinic and in research laboratories are: formalin fixation followed by paraffin embedding, and tissue freezing, followed by embedding in cryo-protectant media. Paraffin-embedded tissue samples produce sections with optimal qualities for histological visualization including clarity and well-defined morphology. However, during the paraffin embedding process a number of epitopes become altered and in order to study these epitopes, tissue sections have to be further processed with one of many epitope retrieval methods
Proceedings of the National Academy of Sciences of the United States of America, Sep 28, 2021
Membrane-associated mucins protect epithelial cell surfaces against pathogenic threats by serving... more Membrane-associated mucins protect epithelial cell surfaces against pathogenic threats by serving as nonproductive decoys that capture infectious agents and clear them from the cell surface and by erecting a physical barrier that restricts their access to target receptors on host cells. However, the mechanisms through which mucins function are still poorly defined because of a limited repertoire of tools available for tailoring their structure and composition in living cells with molecular precision. Using synthetic glycopolymer mimetics of mucins, we modeled the mucosal glycocalyx on red blood cells (RBCs) and evaluated its influence on lectin (SNA) and virus (H1N1) adhesion to endogenous sialic acid receptors. The glycocalyx inhibited the rate of SNA and H1N1 adhesion in a size- and density-dependent manner, consistent with the current view of mucins as providing a protective shield against pathogens. Counterintuitively, increasing the density of the mucin mimetics enhanced the retention of bound lectins and viruses. Careful characterization of SNA behavior at the RBC surface using a range of biophysical and imaging techniques revealed lectin-induced crowding and reorganization of the glycocalyx with concomitant enhancement in lectin clustering, presumably through the formation of a more extensive glycan receptor patch at the cell membrane. Our findings indicate that glycan-targeting pathogens may exploit the biophysical and biomechanical properties of mucins to overcome the mucosal glycocalyx barrier.
Anthropogeny is a classic term encompassing transdisciplinary investigations of the origens of th... more Anthropogeny is a classic term encompassing transdisciplinary investigations of the origens of the human species. Comparative anthropogeny is a systematic comparison of humans and other living nonhuman hominids (so-called “great apes”), aiming to identify distinctly human features in health and disease, with the overall goal of explaining human origens. We begin with a historical perspective, briefly describing how the field progressed from the earliest evolutionary insights to the current emphasis on in-depth molecular and genomic investigations of “human-specific” biology and an increased appreciation for cultural impacts on human biology. While many such genetic differences between humans and other hominids have been revealed over the last two decades, this information remains insufficient to explain the most distinctive phenotypic traits distinguishing humans from other living hominids. Here we undertake a complementary approach of “comparative physiological anthropogeny,” along the lines of the preclinical medical curriculum, i.e., beginning with anatomy and considering each physiological system and in each case considering genetic and molecular components that are relevant. What is ultimately needed is a systematic comparative approach at all levels from molecular to physiological to sociocultural, building networks of related information, drawing inferences, and generating testable hypotheses. The concluding section will touch on distinctive considerations in the study of human evolution, including the importance of gene-culture interactions.
Annals of the New York Academy of Sciences, Apr 1, 2012
Sialic acids are a diverse family of monosaccharides widely expressed on all cell surfaces of ver... more Sialic acids are a diverse family of monosaccharides widely expressed on all cell surfaces of vertebrates and so-called "higher" invertebrates, and on certain bacteria that interact with vertebrates. This overview surveys examples of biological roles of sialic acids in immunity, with emphasis on an evolutionary perspective. Given the breadth of the subject, the treatment of individual topics is brief. Subjects discussed include biophysical effects regulation of factor H; modulation of leukocyte trafficking via selectins; Siglecs in immune cell activation; sialic acids as ligands for microbes; impact of microbial and endogenous sialidases on immune cell responses; pathogen molecular mimicry of host sialic acids; Siglec recognition of sialylated pathogens; bacteriophage recognition of microbial sialic acids; polysialic acid modulation of immune cells; sialic acids as pathogen decoys or biological masks; modulation of immunity by sialic acid Oacetylation, sialic acids as antigens and xeno-autoantigens; anti-sialoglycan antibodies in reproductive incompatibility, and sialic-acid based blood groups.
bioRxiv (Cold Spring Harbor Laboratory), Apr 17, 2021
Influenza A viruses (IAVs) exploit host glycans in airway epithelial mucosa to gain entry and ini... more Influenza A viruses (IAVs) exploit host glycans in airway epithelial mucosa to gain entry and initiate infection. Rapid detection of changes in IAV specificity towards host glycan classes can provide early indication of virus transmissibility and infection potential. IAVs use hemagglutinins (HA) to bind sialic acids linked to larger glycan structures and a switch in HA specificity from a2,3to a2,6-linked sialoglycans is considered a prerequisite for viral transmission from birds to humans. While the changes in HA structure associated with the evolution of binding phenotype have been mapped, the influence of glycan receptor presentation on IAV specificity remains obscured. Here, we describe a glycan array platform which uses synthetic mimetics of mucin glycoproteins to model how receptor presentation in the mucinous glycocalyx, including glycan type and valency of the glycoconjugates and their surface density, impact IAV binding. We found that H1N1 virus produced in embryonated chicken eggs, which recognizes both receptor types, exclusively engaged mucin-mimetics carrying a2,3-linked sialic acids in their soluble form. The virus was able utilize both receptor structures when the probes were immobilized on the array; however, increasing density in the mucin-mimetic brush diminished viral adhesion. Propagation in mammalian cells produced a change in receptor pattern recognition by the virus, without altering its HA affinity, toward improved binding of a2,6-sialylated mucin mimetics and reduced sensitivity to surface crowding of the probes. Application of a support vector machine (SVM) learning algorithm as part of the glycan array binding analysis efficiently characterized this shift in binding preference and may prove useful to study the evolution of viral responses to a new host. .
Deep infiltrating endometriosis (DIE) is a debilitating subset of endometriosis with an ill-defin... more Deep infiltrating endometriosis (DIE) is a debilitating subset of endometriosis with an ill-defined set of symptoms and lacks specific diagnostic criteria to identify, leading to significant delays in treatment. Endometriosis itself is associated with chronic pain and infertility, leading to significant healthcare costs. A literature has shown that DIE is a non-progressive and curable disease. Currently, diagnosis is based purely on surgical diagnosis, which carriers a higher rate of morbidity, and fear of surgery or reluctance on behalf of provider, may delay diagnosis and thus treatment. Multiple attempts have been made to create an appropriate classification system mainly based on symptoms or imaging, however no one scheme has been successful to appropriately identify DIE. Research has now been geared towards the use of biomarkers, including blood, urine, peritoneal/follicular fluid, or endometrial tissue, to construct a diagnostic tool for DIE. This has the potential to create a less invasive mechanism for earlier identification and treatment of this curable disease.
Humans can incorporate the xenoglycan N-glycolylneuraminic acid (Neu5Gc) from the diet into repro... more Humans can incorporate the xenoglycan N-glycolylneuraminic acid (Neu5Gc) from the diet into reproductive tissues and secretions. Most humans also have circulating antibodies specific for this dietary xenoglycan. The potential for inflammation induced by incorporated Neu5Gc and circulating anti-Neu5Gc antibodies, termed xenosialitis, has been discussed as a factor influencing several human diseases. Potential effects of xenosialitis on human fertility remain unknown. Here, we investigate possible adverse effects of the presence of Neu5Gc on sperm or endometrium combined with anti-Neu5Gc antibodies in semen or uterine secretions in a mouse model. We use Cmah ؊/؊ mice, humanized for Neu5Gc deficiency. We find that the viability, migration, and capacitation of sperm with incorporated Neu5Gc are negatively affected when these are exposed to anti-Neu5Gc antibodies. In addition, we find that after copulation, activated uterine neutrophils and macrophages show increased phagocytosis of sperm in the presence of anti-Neu5Gc antibodies via the complement receptor 3 (C3R) and Fc␥ I/II/III (Fc receptor). Furthermore, Neu5Gc in endometrial cells combined with the presence of anti-Neu5Gc antibodies alters the receptivity and decidualization of endometrial explants. These studies provide mechanistic insights on how Neu5Gc on sperm and/or endometrium combined with anti-Neu5Gc antibodies in semen and uterine fluid might contribute to unexplained human infertility.
Establishment of adequate levels of sialylation is crucial for sperm survival and function after ... more Establishment of adequate levels of sialylation is crucial for sperm survival and function after insemination; however, the mechanism for the addition of the sperm sialome has not been identified. Here, we report evidence for several different mechanisms that contribute to the establishment of the mature sperm sialome. Directly quantifying the source of the nucleotide sugar CMP-beta-N-acetylneuraminic acid in epididymal fluid indicates that transsialylation occurs in the upper epididymis. Western blots for the low-molecular-mass sialoglycoprotein (around 20-50 kDa) in C57BL/6 mice epididymal fluid reflect that additional sialome could be obtained by glycosylphosphatidylinositol-anchored sialoglycopeptide incorporation during epididymal transit in the caput of the epididymis. Additionally, we found that in Cmah (CMP-N-acetylneuraminic acid hydroxylase)À/À transgenic mice, epididymal sperm obtained sialylated-CD52 from seminal vesicle fluid (SVF). Finally, we used Gfp (green fluorescent protein)+/+ mouse sperm to test the role of sialylation on sperm for protection from female leukocyte attack. There is very low phagocytosis of the epididymal sperm when compared to that of sperm coincubated with SVF. Treating sperm with Arthrobacter ureafaciens sialidase (AUS) increased phagocytosis even further. Our results highlight the different mechanisms of increasing sialylation, which lead to the formation of the mature sperm sialome, as well as reveal the sialome's function in sperm survival within the female genital tract.
Sugars are the most functionally and structurally diverse molecules in the biological world. Glyc... more Sugars are the most functionally and structurally diverse molecules in the biological world. Glycan structures range from tiny single monosaccharide units to giant chains thousands of units long. Some glycans are branched, their monosaccharides linked together in many different combinations and orientations. Some exist as solitary molecules; others are conjugated to proteins and lipids and alter their collective functional properties. In addition to structural and storage roles, glycan molecules participate in and actively regulate physiological and developmental processes. Glycans also mediate cellular interactions within and between individuals. Their roles in ecology and evolution are pivotal, but not well studied because glycan biochemistry requires different methods than standard molecular biology practice. The properties of glycans are in some ways convenient, and in others challenging. Glycans vary on organismal timescales, and in direct response to physiological and ecological conditions. Their mature structures are physical records of both genetic and environmental influences during maturation. We describe the scope of natural glycan variation and discuss how studying glycans will allow researchers to further integrate the fields of ecology and evolution.
Proceedings of the National Academy of Sciences of the United States of America, Apr 27, 1999
Phylogenetic trees for the four extant species of African hominoids are presented, based on mtDNA... more Phylogenetic trees for the four extant species of African hominoids are presented, based on mtDNA control region-1 sequences from 1,158 unique haplotypes. We include 83 new haplotypes of western chimpanzees and bonobos. Phylogenetic analysis of this enlarged database, which takes intraspecific geographic variability into account, reveals different patterns of evolution among species and great heterogeneity in species-level variation. Several chimpanzee and bonobo clades (and even single social groups) have retained substantially more mitochondrial variation than is seen in the entire human species. Among the 811 human haplotypes, those that branch off early are predominantly but not exclusively African. Neighbor joining trees provide strong evidence that eastern chimpanzee and human clades have experienced reduced effective population sizes, the latter apparently since the Homo sapiens-neanderthalensis split. Application of topiary pruning resolves ambiguities in the phylogenetic tree that are attributable to homoplasies in the data set. The diverse patterns of mtDNA sequence variation seen in today's hominoid taxa probably ref lect historical differences in ecological plasticity, female-biased dispersal, range fragmentation over differing periods of time, and competition among social groups. These results are relevant to the origen of zoonotic diseases, including HIV-1, and call into question some aspects of the current taxonomic treatment and conservation management of gorillas and chimpanzees.
In the context of a study of wild chimpanzees, Pan troglodytes vems, we found that genotypes base... more In the context of a study of wild chimpanzees, Pan troglodytes vems, we found that genotypes based on single PCR amplifications of microsatellite loci from single shed hair have a high error rate. We quantified error rates using the comparable results of 791 single shed hair PCR amplifications of 11 microsatellite loci of 18 known individuals. The most frequent error was the amplification of only one of the two alleles present at a heterozygous locus. This phenomenon, called allelic dropout, produced false homozygotes in 31% of single-hair amplifications. There was no difference in the probability of preferential amplification between longer and shorter alleles. The probability of scoring false homozygotes can be reduced to below 0.05 by three separate amplifications from single hairs of the same individual or by pooling hair samples from the same individual. In this study an additional 5.6% of the amplifications gave wrong genotypes because of contamination, labelling and loading errors, and possibly amplification artefacts. In contrast, amplifications from plucked hair taken from four dead individuals gave consistent results (error rate < 0.01%, n = 120). Allelic dropout becomes a problem when the DNA concentration falls below 0.05 ng/lO pL in the template as it can with shed hair, and extracts from faeces and masticated plant matter.
The more than two hundred species of primates live and reproduce in a large variety of ways. Most... more The more than two hundred species of primates live and reproduce in a large variety of ways. Most species are highly social. Recent studies of primates in nature are revealing new facets of their social and mating systems. Female primates follow their own reproductive strategies, which are less conspicuous than male strategies. The slow development of young primates makes them crucially dependent on intense and prolonged maternal care, exacerbating the imbalance in reproductive effort between the sexes. Primate mating systems seem more flexible than previously thought, as social changes, ecological conditions, and individual character all affect the ways in which primates mate and reproduce. Interactions between males and females range from the molecular, such as interactions between egg and sperm or harmonious expression of paternal and maternal genes in the offspring, to the behavioral, including mating patterns, social affiliation, and parental care. There is increasing evidence for the importance of multiple paternity for females. This evidence includes the need for diverse offspring, for good and compatible genes from the fathers, and for male assistance. Primate mating systems result from a delicate balance between male and female strategies and the constraints exerted by the physical and social environments.
ABSTRACT Efforts to control the global pandemic of human hepatitis B virus (hHBV) infection have ... more ABSTRACT Efforts to control the global pandemic of human hepatitis B virus (hHBV) infection have been hampered by incomplete understanding of viral-host interactions in this disease. This situation has been confounded by the fact that hHBV has a limited host range and cannot be propagated in simple cell culture (1). Reproducible experimental infection with determination of infectivity was demonstrated in chimpanzees (Pan troglodytes), but not other primates (2–4), long before other animal models such as the woodchuck were identified. After successful inoculation of chimpanzees was reported in 1972, multiple institutions, including a multigroup collaboration between the FDA, CDC, and NIH, initiated studies to evaluate them as a model for the study of HBV. For the majority of studies only chimpanzees “with no prior exposure” to virus were used because those with positive serology [either from exposure to hHBV or chimpanzee HBV (chHBV)], with an estimated prevalence of 3–6% in Africa (5), were not reproducibly susceptible to infection (3). It has been widely reported that the effects of HBV infection in chimpanzees are milder than in humans, that is, few have developed fulminant hepatitis, and inoculated chimpanzees exhibit few symptoms or signs of infection. Furthermore, the incidence of chronic infection with HBV (see Table 2) and horizontal and vertical transmission (from mother to offspring) in chimpanzees is lower than in humans (6,7). Chimpanzees have been the cornerstone of all research on infectivity of HBV and safety and efficacy of vaccines.
Molecular Reproduction and Development, Jun 9, 2015
Mammalian spermatozoa are coated with a thick glycocalyx that is assembled during sperm developme... more Mammalian spermatozoa are coated with a thick glycocalyx that is assembled during sperm development, maturation, and upon contact with seminal fluid. The sperm glycocalyx is critical for sperm survival in the female reproductive tract and is modified during capacitation. The complex interplay among the various glycoconjugates generates numerous signaling motifs that may regulate sperm function and, as a result, fertility. Nascent spermatozoa assemble their own glycans while the cells still possess a functional endoplasmic reticulum and Golgi in the seminiferous tubule, but once spermatogenesis is complete, they lose the capacity to produce glycoconjugates de novo. Sperm glycans continue to be modified, during epididymal transit by extracellular glycosidases and glycosyltransferases. Furthermore, epididymal cells secrete glycoconjugates (glycophosphatidylinositol-anchored glycoproteins and glycolipids) and glycan-rich microvesicles that can fuse with the maturing sperm membrane. The sperm glycocalyx mediates numerous functions in the female reproductive tract, including the following: inhibition of premature capacitation; passage through the cervical mucus; protection from innate and adaptive female immunity; formation of the sperm reservoir; and masking sperm proteins involved in fertilization. The immense diversity in sperm-associated glycans within and between species forms a remarkable challenge to our understanding of essential sperm glycan functions.
Background: Influenza A virus (IAV) neuraminidase (NA) cleaves sialic acids (Sias) from glycans. ... more Background: Influenza A virus (IAV) neuraminidase (NA) cleaves sialic acids (Sias) from glycans. Inhibiting NA with oseltamivir suppresses both viral infection, and viral release from cultured human airway epithelial cells. The role of NA in viral exit is well established: it releases budding virions by cleaving Sias from glycoconjugates on infected cells and progeny virions. The role of NA in viral entry remains unclear. Host respiratory epithelia secrete a mucus layer rich in heavily sialylated glycoproteins; these could inhibit viral entry by mimicking sialylated receptors on the cell surface. It has been suggested that NA allows influenza to penetrate the mucus by cleaving these sialylated decoys, but the exact mechanism is not yet established. Methods: We tested IAV interaction with secreted mucus using frozen human trachea/bronchus tissue sections, and bead-bound purified human salivary mucins (HSM) and purified porcine submaxillary mucins (PSM). The protective effect of mucus was analyzed using MDCK cells coated with purified HSM and PSM with known Sia content. Oseltamivir was used to inhibit NA activity, and the fluorescent reporter substrate, 4MU-Neu5Ac, was used to quantify NA activity. Results: IAV binds to the secreted mucus layer of frozen human trachea/bronchus tissues in a Sia dependent manner. HSM inhibition of IAV infection is Sia dose-dependent, but PSM cannot inhibit infection of underlying cells. HSM competitively inhibits NA cleavage of 4MU-Neu5Ac, reporter substrate. Human IAV effectively cleaves Sias from HSM but not from PSM, and binds to HSM but not to PSM. Conclusion: IAV interacts with human mucus on frozen tissue sections and mucus-coated beads. Inhibition of IAV infection by sialylated human mucus is dose-dependent, and enhanced when NA is inhibited with oseltamivir. Thus NA cleaves sialylated decoys during initial stages of infection. Understanding IAV interactions with host mucins is a promising new avenue for drug development.
Mucins are complex and heavily glycosylated O-linked glycoproteins, which contain more than 70% c... more Mucins are complex and heavily glycosylated O-linked glycoproteins, which contain more than 70% carbohydrate by weight 1-3. Secreted mucins, produced by goblet cells and the gastric mucosa, provide the scaffold for a micrometers-thick mucus layer that lines the epithelia of the gut and respiratory tract 3,4. In addition to mucins, mucus layers also contain antimicrobial peptides, cytokines, and immunoglobulins 5-9. The mucus layer is an important part of host innate immunity, and forms the first line of defense against invading microorganisms 8,10-12. As such, the mucus is subject to numerous interactions with microbes, both pathogens and symbionts, and secreted mucins form an important interface for these interactions. The study of such biological interactions usually involves histological methods for tissue collection and staining. The two most commonly used histological methods for tissue collection and preservation in the clinic and in research laboratories are: formalin fixation followed by paraffin embedding, and tissue freezing, followed by embedding in cryo-protectant media. Paraffin-embedded tissue samples produce sections with optimal qualities for histological visualization including clarity and well-defined morphology. However, during the paraffin embedding process a number of epitopes become altered and in order to study these epitopes, tissue sections have to be further processed with one of many epitope retrieval methods
Proceedings of the National Academy of Sciences of the United States of America, Sep 28, 2021
Membrane-associated mucins protect epithelial cell surfaces against pathogenic threats by serving... more Membrane-associated mucins protect epithelial cell surfaces against pathogenic threats by serving as nonproductive decoys that capture infectious agents and clear them from the cell surface and by erecting a physical barrier that restricts their access to target receptors on host cells. However, the mechanisms through which mucins function are still poorly defined because of a limited repertoire of tools available for tailoring their structure and composition in living cells with molecular precision. Using synthetic glycopolymer mimetics of mucins, we modeled the mucosal glycocalyx on red blood cells (RBCs) and evaluated its influence on lectin (SNA) and virus (H1N1) adhesion to endogenous sialic acid receptors. The glycocalyx inhibited the rate of SNA and H1N1 adhesion in a size- and density-dependent manner, consistent with the current view of mucins as providing a protective shield against pathogens. Counterintuitively, increasing the density of the mucin mimetics enhanced the retention of bound lectins and viruses. Careful characterization of SNA behavior at the RBC surface using a range of biophysical and imaging techniques revealed lectin-induced crowding and reorganization of the glycocalyx with concomitant enhancement in lectin clustering, presumably through the formation of a more extensive glycan receptor patch at the cell membrane. Our findings indicate that glycan-targeting pathogens may exploit the biophysical and biomechanical properties of mucins to overcome the mucosal glycocalyx barrier.
Anthropogeny is a classic term encompassing transdisciplinary investigations of the origens of th... more Anthropogeny is a classic term encompassing transdisciplinary investigations of the origens of the human species. Comparative anthropogeny is a systematic comparison of humans and other living nonhuman hominids (so-called “great apes”), aiming to identify distinctly human features in health and disease, with the overall goal of explaining human origens. We begin with a historical perspective, briefly describing how the field progressed from the earliest evolutionary insights to the current emphasis on in-depth molecular and genomic investigations of “human-specific” biology and an increased appreciation for cultural impacts on human biology. While many such genetic differences between humans and other hominids have been revealed over the last two decades, this information remains insufficient to explain the most distinctive phenotypic traits distinguishing humans from other living hominids. Here we undertake a complementary approach of “comparative physiological anthropogeny,” along the lines of the preclinical medical curriculum, i.e., beginning with anatomy and considering each physiological system and in each case considering genetic and molecular components that are relevant. What is ultimately needed is a systematic comparative approach at all levels from molecular to physiological to sociocultural, building networks of related information, drawing inferences, and generating testable hypotheses. The concluding section will touch on distinctive considerations in the study of human evolution, including the importance of gene-culture interactions.
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Papers by Pascal Gagneux