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Keyser, C.

Publications and source records attributed to Keyser, C..

5 recordsLinked to original sources

Quantification of Human DNA from Century-Old Archived FFPE Samples for Retrospective Genomic Studies

BackgroundFormalin-fixed, paraffin-embedded (FFPE) tissue archives are an invaluable resource for genomic research, offering the potential to link genomic data to long-term clinical outcomes. Their utility has however been limited by the degradation caused by fixation and long-term storage, particularly for samples archived for many decades. MethodsThis study evaluates a cohort of 79 FFPE tissue blocks collected and archived in 1973 from the Strasbourg Pathological Tissue Archive (SPTA), and a cohort of 51 FFPE tissue blocks from the Geneva Brain Bank (GBB), collected between 1928 and 1971. DNA was quantified using a forensic-grade quantitative PCR (qPCR) assay (QIAGEN Investigator Quantiplex Pro Kit on a Rotor-Gene Q) to precisely measure human DNA concentration, assess degradation, and detect PCR inhibition. ResultsA high proportion of the samples yielded human DNA fragments of 80bp and 95bp and very few fragments of 205bp. All organ samples treated with Bouin liquid (n=12) gave poor results, but among samples fixed in formalin (n=117), 58.1% showed over 0.25ng/{micro}L of 80bp human DNA fragments and 37.6% over 1ng/{micro}L. We propose a model to describe the decay of these samples and estimate the proportion of samples that should yield at least a 0.25ng/{micro}L concentration of fragments over 100bp to 45.1%. ConclusionsThis work demonstrates that automated extraction methods optimized for FFPE allow for the recovery of usable material even in century-old archived samples with inconsistent conditions of conservation. Our data suggests that the time spent in storage is much less influential on DNA quality than initial fixation time. Crucially, given the fragmented nature of the material recovered (an expected result), future analyses of this material will have to be conducted using next-generation sequencing (NGS) technologies and approaches that rely on short fragments.

genetics↗

Combining MicroED and native mass spectrometry for structural discovery of enzyme-biosynthetic inhibitor complexes

With the goal of accelerating the discovery of small molecule-protein complexes, we leverage fast, low-dose, event based electron counting microcrystal electron diffraction (MicroED) data collection and native mass spectrometry. This approach resolves structures of the epoxide-based cysteine protease inhibitor, and natural product, E-64, and its biosynthetic analogs bound to the model cysteine protease, papain. The combined structural power of MicroED and the analytical capabilities of native mass spectrometry (ED-MS) allows assignment of papain structures bound to E-64-like ligands with data obtained from crystal slurries soaked with mixtures of known inhibitors, and crude biosynthetic reactions. ED-MS further discriminates the highest-affinity ligand soaked into microcrystals from a broad inhibitor cocktail, and identifies multiple similarly high-affinity ligands soaked into microcrystals simultaneously. This extends to libraries of printed ligands dispensed directly onto TEM grids and later soaked with papain microcrystal slurries. ED-MS identifies papain binding to its preferred natural products, by showing that two analogues of E-64 outcompete others in binding to papain crystals, and by detecting papain bound to E-64 and an analogue from crude biosynthetic reactions, without purification. This illustrates the utility of ED-MS for natural product ligand discovery and for structure-based screening of small molecule binders to macromolecular targets.

biophysics↗

Splenic clearance of rigid erythrocytes as an inherited mechanism for splenomegaly and natural resistance to malaria

In malaria-endemic areas, subjects from specific groups like Fulani have a peculiar protection against malaria, with high levels of IgM but also frequent anemia and splenomegaly. The mechanisms underlying this phenotype remain elusive. In Benin, West Africa, we measured the deformability of circulating erythrocytes in genetically distinct groups (including Fulani) living in sympatry, using ektacytometry and microsphiltration, a mimic of how the spleen clears rigid erythrocytes. Compared to non-Fulani, Fulani displayed a higher deformability of circulating erythrocytes, pointing to an enhanced clearance of rigid erythrocytes by the spleen. This phenotype was observed in individuals displaying markers of Plasmodium falciparum infection. The heritability of this new trait was high, with a strong multigenic component. Five of the top 10 genes selected by a population structure-adjusted GWAS, expressed in the spleen, are potentially involved in splenic clearance of erythrocytes (CHERP, MB, PALLD, SPARC, PDE10A), through control of vascular tone, collagen synthesis and macrophage activity. In specific ethnic groups, genetically-controlled processes likely enhance the innate retention of infected and uninfected erythrocytes in the spleen, explaining splenomegaly, anemia, cryptic intrasplenic parasite loads, hyper-IgM, and partial protection against malaria. Beyond malaria-related phenotypes, inherited splenic hyper-filtration of erythrocytes may impact the pathogenesis of other hematologic diseases. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSThe genetic background of individuals influences their susceptibility to infectious diseases. Specific human groups, like the Fulani in Africa, react to malaria parasites (named Plasmodium) in a specific way. Upon infection, Fulani develop a grossly enlarged spleen, and high levels of anti-Plasmodium antibodies in their blood. They also carry smaller numbers of parasites in their blood, and thus are considered partially protected against malaria. The mechanisms underlying this natural protection, different from other natural protective mechanisms such as the sickle cell trait, are not well understood. Malaria impairs the deformability of red blood cells and the spleen is a key organ to controlling red blood cell quality. We have recently demonstrated that red blood cells containing live malaria parasites accumulate intensely in the spleen of subjects with long term exposure to these parasites. Enhanced retention of infected and uninfected red blood cells in the spleen would explain why the spleen is larger and why lower numbers of parasites are left in circulation. We thus explored whether the retention of infected and uninfected red blood cells could explain why Fulani are partially protected against malaria. Because it is unethical to perform spleen puncture or biopsies for research purposes, our explorations were indirect by carefully analyzing the properties of circulating red blood cells in a large number of subjects and by assessing whether observations could be explained by their genetic make-up. Added value of this studyIn more than 500 subjects, we confirmed the high frequency of large spleens in Fulani and, through 2 different methods, we demonstrated an enhanced deformability of their circulating red blood cells, that likely stems from the more efficient removal of the less deformable ones. This enhanced deformability was found to be inheritable based on carefully collected family links and refined analysis of genetic markers. Implications of all the available evidenceOur findings indicate that genes potentially driving the filtration of red blood cells by the spleen likely influence how subjects in specific groups in Africa and elsewhere react to malaria. While most previous hypotheses pointed to conventional immunological mechanisms as the trigger, we propose that a simple physiological mechanism that controls the quality of red blood cells may drive natural protection from malaria even before the intervention of immunological cells. A better understanding of these processes is of great importance in the context of malaria elimination efforts. These findings may also have an impact on the understanding of other red blood cell-related disorders, such as inherited red cell diseases, in which splenic filtration of abnormal red blood cells may precipitate splenic complications.

physiology↗

Surveying the sweetpotato rhizosphere, endophyte, and surrounding soil microbiomes at two North Carolina farms reveals underpinnings of sweetpotato microbiome community assembly

Farmers grow sweetpotatoes worldwide and some sub-Saharan African and Asian diets include sweetpotato as a staple, yet the sweetpotato microbiome is conspicuously less studied relative to crops such as maize, soybean, and wheat. Studying sweetpotato microbiome ecology may reveal paths to engineer the microbiome to improve sweetpotato yield, and/or combat sweetpotato pests and diseases. We sampled sweetpotatoes and surrounding soil from two North Carolina farms. We took samples from sweetpotato fields under two different land management regimes, conventional and organic, and collected two sweetpotato cultivars, Beauregard and Covington. By comparing SSU rRNA gene amplicon sequence profiles from sweetpotato storage root skin, rhizosphere, and surrounding soil we found the skin microbiome possessed the least composition heterogeneity among samples and lowest alpha-diversity and was significantly nested by the rhizosphere in amplicon sequence variant (ASV) membership. Many ASVs were specific to a single field and/or only found in either the skin, rhizosphere, or surrounding soil. Notably, sweetpotato skin enriched for Planctomycetaceae in relative abundance at both farms. This study elucidates underpinnings of sweetpotato microbiome community assembly, quantifies microbiome composition variance within a single farm, and reveals microorganisms associated with sweetpotato skin that belong to common but uncultured soil phylotypes.

microbiology↗

Relationships between clans and genetic kin explain cultural similarities over vast distances: the case of Yakutia

Archaeological studies sample ancient human populations one site at a time, often limited to a fraction of the regions and periods occupied by a given group. While this bias is known and discussed in the literature, few model populations span areas as large and unforgiving as the Yakuts of Eastern Siberia. We systematically surveyed 31,000 square kilometres in the Sakha Republic (Yakutia) and completed the archaeological study of 174 frozen graves, assembled between the 15th and the 19th century. We analysed genetic data (autosomal genotypes, Y-chromosome haplotypes and mitochondrial haplotypes) for all ancient subjects and confronted these to data on 190 modern subjects from the same area and the same population. Ancient familial links were identified between graves up to 1500 km apart, as well as paternal clans. We provide new insights on the origins of the contemporary Yakut population and demonstrate that cultural similarities in the past were linked to (i) the expansion of specific paternal clans, (ii) preferential marriage among the elites and (iii) funeral choices that could constitute a bias in any ancient population study.

genetics↗