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BACKGROUND: Recent advances in sequencing have facilitated large-scale analyses of the metagenomic composition of different samples, including the environmental microbiome of air, water, and soil, as well as the microbiome of living humans and other animals. Analyses of the microbiome of ancient human samples may provide insights into human health and disease, as well as pathogen evolution, but the field is still in its very early stages and considered highly challenging. - RESULTS: The metagenomic and pathogen content of Egyptian mummified individuals from different time periods was investigated via genetic analysis of the microbial composition of various tissues. The analysis of the dental calculus’ microbiome identified Red Complex bacteria, which are correlated with periodontal diseases. From bone and soft tissue, genomes of two ancient pathogens, a 2200-year-old Mycobacterium leprae strain and a 2000-year-old human hepatitis B virus, were successfully reconstructed. - CONCLUSIONS: The results show the reliability of metagenomic studies on Egyptian mummified individuals and the potential to use them as a source for the extraction of ancient pathogen DNA. Background Results - Sample information and dating - General metagenomic assessment - Mycobacterium leprae (individual Abusir1630) - Hepatitis B virus (individual Abusir1543) - Oral microbiome assessment Discussion Conclusions Methods - Sample extraction and radiocarbon dating - Sample extraction and library preparation - Metagenomic screening - Authentication of ancient DNA - Content of endogenous DNA (SourceTracker2) - Data processing of sample Abusir1630b (M. leprae) -- Read processing, mapping, and variant calling -- SNP typing -- Anthropological analysis -- Phylogeny -- Beast analysis -- Temporal signal - Data processing individual Abusir1543 (hepatitis B virus) -- Read processing, mapping, and variant calling -- Phylogeny -- Recombination analysis -- Beast analysis -- Temporal signal

Alzheimer's disease (AD) involves changes in both lipid and RNA metabolism, but it remained unknown if these differences associate with AD's cognition and/or post-mortem neuropathology indices. Here, we report RNA-sequencing evidence of inter-related associations between lipid processing, cognition level, and AD neuropathology. In two unrelated cohorts, we identified pathway-enriched facilitation of lipid processing and alternative splicing genes, including the neuronal-enriched NOVA1 and hnRNPA1. Specifically, this association emerged in temporal lobe tissue samples from donors where postmortem evidence demonstrated AD neuropathology, but who presented normal cognition proximate to death. The observed changes further associated with modified ATP synthesis and mitochondrial transcripts, indicating metabolic relevance; accordingly, mass-spectrometry-derived lipidomic profiles distinguished between individuals with and without cognitive impairment prior to death. In spite of the limited group sizes, tissues from persons with both cognitive impairment and AD pathology showed elevation in several drug-targeted genes of other brain, vascular and autoimmune disorders, accompanied by pathology-related increases in distinct lipid processing transcripts, and in the RNA metabolism genes hnRNPH2, TARDBP, CLP1 and EWSR1. To further detect 3′-polyadenylation variants, we employed multiple cDNA primer pairs. This identified variants that showed limited differences in scope and length between the tested cohorts, yet enabled superior clustering of demented and non-demented AD brains versus controls compared to total mRNA expression values. Our findings indicate inter-related cognition-associated differences in AD's lipid processing, alternative splicing and 3′-polyadenylation, calling for pursuing the underlying psychological and therapeutics implications., Highlights • RNA transcripts differ between brains from Alzheimer's patients with or without dementia. • In non-demented patients with pathology, brain neurons show facilitated RNA and lipid processing. • Cognition-related differences included brain, vascular and autoimmune disease-related genes. • Polyadenylation changes classify patients better than transcript expression levels.

International audience; AbstractThe intestinal microbiota of honey bees consists of only few bacterial species and may have effects on health and pathogen resilience. Honey is usually harvested and replaced by sugar syrup. We hypothesized that replacing honey may change the composition of the intestinal microbiota, and therefore compromise pathogen resilience. Fifteen colonies were fed with wheat starch syrup, sucrose syrup, or blossom honey. 16S-based bacterial community analysis was performed on three individuals per hive in summer and winter, and Nosema ceranae and Crithidia/Lotmaria levels were assessed by qPCR. Seasonal differences in the intestinal microbiota and N. ceranae were found; however, microbiota and parasite levels were very similar between the feed types. Rhizobiales and Bifidobacteria were found to be increased in the bees that had received honey or wheat starch syrup, as compared to sucrose syrup. In conclusion, intestinal microbiota and parasites were found to be largely unaffected by the winter feed type.

Studying ancient DNA allows us to retrace the evolutionary history of human pathogens, such as Mycobacterium leprae, the main causative agent of leprosy. Leprosy is one of the oldest recorded and most stigmatizing diseases in human history. The disease was prevalent in Europe until the 16th century and is still endemic in many countries with over 200,000 new cases reported annually. Previous worldwide studies on modern and European medieval M. leprae genomes revealed that they cluster into several distinct branches of which two were present in medieval Northwestern Europe. In this study, we analyzed 10 new medieval M. leprae genomes including the so far oldest M. leprae genome from one of the earliest known cases of leprosy in the United Kingdom—a skeleton from the Great Chesterford cemetery with a calibrated age of 415–545 C.E. This dataset provides a genetic time transect of M. leprae diversity in Europe over the past 1500 years. We find M. leprae strains from four distinct branches to be present in the Early Medieval Period, and strains from three different branches were detected within a single cemetery from the High Medieval Period. Altogether these findings suggest a higher genetic diversity of M. leprae strains in medieval Europe at various time points than previously assumed. The resulting more complex picture of the past phylogeography of leprosy in Europe impacts current phylogeographical models of M. leprae dissemination. It suggests alternative models for the past spread of leprosy such as a wide spread prevalence of strains from different branches in Eurasia already in Antiquity or maybe even an origin in Western Eurasia. Furthermore, these results highlight how studying ancient M. leprae strains improves understanding the history of leprosy worldwide., Author summary Many controversies surround leprosy, which is one of the oldest recorded diseases of humankind. The origin and past spread of its main causative agent, Mycobacterium leprae, remain unknown although many attempts have been made to reconstruct its past from historical and archeological sources. Analysis of ancient M. leprae genomes reconstructed from archaeological remains can contribute greatly to reconstructing the origin and evolution of this pathogen. With a new set of ancient M. leprae genomes from Europe, we traced back a so far unrecognized past diversity, which places Europe as a key region for the early spread and worldwide dissemination of leprosy. Our results hint to the potential dynamic changes in the prevalence of different M. leprae strains in Europe during Antiquity, and highlight the need to study ancient pathogen genomes in order to better understand our past.

The abrupt onslaught of the syphilis pandemic that started in the late fifteenth century established this devastating infectious disease as one of the most feared in human history1 . Surprisingly, despite the availability of effective antibiotic treatment since the mid-twentieth century, this bacterial infection, which is caused by Treponema pallidum subsp. pallidum (TPA), has been re-emerging globally in the last few decades with an estimated 10.6 million cases in 2008 (ref. 2). Although resistance to penicillin has not yet been identified, an increasing number of strains fail to respond to the secondline antibiotic azithromycin3. Little is known about the genetic patterns in current infections or the evolutionary origins of the disease due to the low quantities of treponemal DNA in clinical samples and difficulties in cultivating the pathogen4. Here, we used DNA capture and whole-genome sequencing to successfully interrogate genome-wide variation from syphilis patient specimens, combined with laboratory samples of TPA and two other subspecies. Phylogenetic comparisons based on the sequenced genomes indicate that the TPA strains examined share a common ancestor after the fifteenth century, within the early modern era. Moreover, most contemporary strains are azithromycin-resistant and are members of a globally dominant cluster, named here as SS14-Ω. The cluster diversified from a common ancestor in the mid-twentieth century subsequent to the discovery of antibiotics. Its recent phylogenetic divergence and global presence point to the emergence of a pandemic strain cluster. Fil: Arora, Natasha. Universitat Zurich; Suiza Fil: Schuenemann, Verena J.. Eberhard Karls Universität Tübingen. Institute For Archaeological Sciences.; Alemania Fil: Jäger, Hünter. Eberhard Karls Universität Tübingen.; Alemania Fil: Peltzer, Alexander. Eberhard Karls Universität Tübingen.; Alemania Fil: Seitz, Alexander. Eberhard Karls Universität Tübingen.; Alemania Fil: Herbig, Alexander. Eberhard Karls Universität Tübingen.; Alemania Fil: Strouhal, Michal. Masaryk University; República Checa Fil: Grillová, Linda. Masaryk University; República Checa Fil: Sánchez Busó, Leonor. Universidad de Valencia; España. University of Cambridge; Reino Unido Fil: Kühnert, Denise. Universitat Zurich; Suiza Fil: Bos, Kirsten I.. Eberhard Karls Universität Tübingen. Institute For Archaeological Sciences.; Alemania Fil: Davis Rivero, Leyla. Universitat Zurich; Suiza Fil: Mikalová, Lenka. Masaryk University; República Checa Fil: Bruisten, Sylvia. Public Health Laboratory. Department of Infectious Diseases; Países Bajos Fil: Komericki, Peter. Medical University of Graz; Austria Fil: French, Patrick. The Mortimer Market Centre ; Reino Unido Fil: Grant, Paul R.. University College London; Estados Unidos Fil: Pando, María de los Ángeles. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; Argentina. Universidad de Buenos Aires; Argentina Fil: Gallo Vaulet, Maria Lucia. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Bioquímica Clínica; Argentina Fil: Rodríguez Fermepin, Marcelo. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Bioquímica Clínica; Argentina Fil: Martinez, Antonio. Hospital General Universitario de Valencia; España Fil: Lara, Arturo Centurión. University of Washington; Estados Unidos Fil: Giacani, Lorenzo. University of Washington; Estados Unidos Fil: Norris, Steven J.. UTHealth McGovern Medical School. Department of Pathology and Laboratory Medicine; Estados Unidos Fil: Smajs, David. Masaryk University; República Checa Fil: Bosshard, Philipp P.. Universitat Zurich; Suiza Fil: González Candelas, Fernando. Universidad de Valencia; España Fil: Nieselt, Kay. Eberhard Karls Universität Tübingen.; Alemania Fil: Krause, Johannes. Eberhard Karls Universität Tübingen.; Alemania Fil: Bagheri, Homayoun C.. Universitat Zurich; Suiza

Syphilis swept across the world in the 16th century as one of most prominent documented pandemics and is re-emerging worldwide despite the availability of effective antibiotics. Little is known about the genetic patterns in current infections or the evolutionary origins of the disease due to the non-cultivable and clonal nature of the causative bacterium Treponema pallidum subsp. pallidum. In this study, we used DNA capture and next generation sequencing to obtain whole genome data from syphilis patient specimens and from treponemes propagated in laboratory settings. Phylogenetic analyses indicate that the syphilis strains examined here share a common ancestor after the 15th century. Moreover, most contemporary strains are azithromycin resistant and members of a globally dominant cluster named here as SS14-Ω. This cluster diversified from a common ancestor in the mid-20th century and has the population genetic and epidemiological features indicative of the emergence of a pandemic strain cluster.

QuantSeq provides an easy protocol to generate highly strand-specific next-generation sequencing (NGS) libraries close to the 3′ end of polyadenylated RNAs within 4.5 h. Only one fragment per transcript is generated, directly linking the number of reads mapping to a gene to its expression. QuantSeq reduces data analysis time and enables a higher level of multiplexing per run. QuantSeq is the RNA sample preparation method for accurate and affordable gene expression measurement.

The apolipoprotein E (ApoE) gene has been linked to maladies such as hypercholesterolemia, CNS injury and disease. In this study, we present evidence that, in addition to the known transcript (ApoE S1) that translates into ApoE, there are three additional transcripts in mice. Two of these transcripts, ApoE S2 and ApoE S3, which are predicted to be transmembrane proteins, are transcribed from the sense strand. ApoE AS1 is transcribed from the antisense strand and is complementary to exon 4 of ApoE S1. The open reading frame of ApoE AS1 is conserved between human and mouse. The antisense transcript falls within the region of the human epsilon 4 allele that has been linked to the familial onset form of Alzheimer's disease. We also demonstrate the expression of ApoE S3 and ApoE AS1 in ApoE knockout mice, and ApoE S1 and ApoE S2 do not get transcribed. We had previously identified ApoE S1 as being upregulated in mice after spinal cord injury. In this study, we show that in spinal cord-injured C57BL/6 mice, both ApoE S1 and ApoE S3 transcripts are 10-fold upregulated and the antisense ApoE AS1 is 100-fold upregulated compared with normal levels. Such data suggest that these alternate transcripts are involved in the molecular pathogenesis of CNS disease and perhaps in ApoE expression in general, as we show that ApoE S2 and AS1 are also transcribed in human.

The MRL/MpJ mouse has a greatly enhanced healing response and an absence of scarring compared with other mouse strains. Following lesions to the CNS mammals show a scarring response known as reactive gliosis, and this CNS scar tissue blocks regeneration of cut axons. We have therefore compared reactive gliosis in the MRL/MpJ mouse and the Swiss Webster mouse, which exhibits normal scarring in the periphery. The lesion model was a stab lesion to the cortex, in which reactive gliosis has previously been quantified. Axon regeneration was examined following a cut lesion to the dopaminergic projection from the substantia nigra to the striatum used in previous regeneration experiments. In the MRL/MpJ following the lesion compared with Swiss Webster mice there was greater cell loss around the lesion followed by greater and more widespread and more prolonged cellular proliferation. Early after the lesion there was a greater loss of glial fibrillary acidic protein (GFAP)-positive astrocytes around the injury site in the MRL/MpJ, and an enhancement and prolongation of the microglial inflammatory response. This was accompanied by greater and more widespread blood-brain barrier leakage following injury. RNA levels for the matrix metalloproteinases (MMP)-2 and MMP-9 as well as for the thrombin receptors PAR-1 and PAR-4 were also greater at the MRL/MpJ injury site. All of these differences were transient and by 14 days post-injury there were no differences observed between MRL/MpJ and control mice. No axonal regeneration was observed following axotomy to the nigrostriatal pathway of the MRL/MpJ or the Swiss Webster mice at any time point.

Apolipoprotein E (apo-E), a protein involved in lipid metabolism and cholesterol transport, has been found to be up-regulated in CNS injury and is associated with Alzheimer's disease in humans. In this study, we show that apo-E is also up-regulated after complete spinal cord transection in the C57BL/6 mouse. In the uninjured cord, the cellular localization of apo-E protein is in astrocytes, in individual neurons throughout the laminae except for the dorsal horn, and in endothelial cells of capillaries in the immediate vicinity of those neurons. After injury, RNA levels are elevated as early as 4 days and reach a maximal level between 1 and 2 weeks. Protein levels follow closely but remain up-regulated beyond 3 weeks. Early on, the protein can be found in neutrophils and macrophages at the injury site and only at later times in astrocytes during the remodeling of white matter tracts, most prominently in degenerating parts of the fasciculus gracilis. © 2002 Wiley-Liss, Inc.

Preadipocyte factor-1 (Pref-1), a delta-like protein containing epidermal growth factor-repeats, is expressed in proliferating cells in a variety of tissues and is believed to be involved in maintaining the undifferentiated state of these cells. Using microarray analysis, reverse transcriptase-polymerase chain reaction, in-situ hybridization, and immunohistochemistry, we have identified Pref-1 expression in the healing ears of two strains of mice, MRL and C57BL/6. MRL is unusual in that ear punches completely regenerate the ear tissue along with new cartilage with no scarring. Pref-1 is more highly expressed in the MRL wounds, is uniquely found in a condensation of cells within the regenerating tissue of the blastema, and may contribute to the regenerative capacity of the MRL ear wound.

In personalized medicine, SNPs are used to identify specific diseases of a patient. However, for many SNPs, no information about the pathogenicity is available. Current programs try to predict the effect of a SNP on the function of a protein, but give no possibility for visual interpretation. We have developed SNPViz, a program that first finds 3D structures of affected proteins and then highlights the affected amino acid in the 3D structure. This can give researchers and doctors more information about the probable pathogenicity of the SNP. In the future, we plan to add also further information, such as whether the position of the SNP is in a binding domain, or is involved in a protein-protein interaction.

Understanding disease-related metabolite interactions is a key issue in computational biology. We apply a modified Bayesian Optimization Algorithm to targeted metabolomics data from plasma samples of insulin-sensitive and -resistant subjects both suffering from non-alcoholic fatty liver disease. In addition to improving the classification accuracy by selecting relevant features, we extract the information that led to their selection and reconstruct networks from detected feature dependencies. We compare the influence of a variety of classifiers and different scoring metrics and examine whether the reconstructed networks represent physiological metabolite interconnections. We find that the presented method is capable of significantly improving the classification accuracy of otherwise hardly classifiable metabolomics data and that the detected metabolite dependencies can be mapped to physiological pathways, which in turn were affirmed by literature from the domain.

The MRL mouse was proposed as a model of mammalian regeneration because it can close ear holes completely with the restoration of normal tissue. This regeneration process involves the formation of a blastema during healing, the re-appearance of cartilage and hair follicles, and healing without scarring. Such a process requires extensive tissue remodeling. To characterize differences in ear wounding responses between regenerating and nonregenerating mice, we examined and compared the extracellular matrix remodeling and the matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) response in the MRL and C57BL/6 mouse strains after injury. We found a correlation between the MRL's ability to break down the basement membrane, form a blastema, and close ear hole wounds and an inflammatory response with neutrophils and macrophages seen in the ear after injury. These cells were positive for MMP-2 and MMP-9 as well as TIMP-2 and TIMP-3. Clear differences between the MRL and B6 response to injury were seen that could explain the differences in healing and blastema formation in the MRL and lack of it in the B6 mice. This finding was further supported by enzyme activity as determined by gelatin zymography.

The SENSE mRNA-seq library preparation kit generates ready-to-sequence libraries from low amounts of total RNA within 4 h. SENSE libraries are significantly more strand specific (>99.9%) than leading alternatives and are virtually free of rRNA contamination. The protocol does not rely on RNA or cDNA fragmentation or additional kits for rRNA depletion, library amplification or size selection.