A 2-million-year-old ecosystem in Greenland uncovered by environmental DNA.

Late Pliocene and Early Pleistocene epochs 3.6 to 0.8 million years ago1 had climates resembling those forecasted under future warming2. Palaeoclimatic records show strong polar amplification with mean annual temperatures of 11-19 °C above contemporary values3,4. The biological communities inhabiting the Arctic during this time remain poorly known because fossils are rare5. Here we report an ancient environmental DNA6 (eDNA) record describing the rich plant and animal assemblages of the Kap København Formation in North Greenland, dated to around two million years ago. The record shows an open boreal forest ecosystem with mixed vegetation of poplar, birch and thuja trees, as well as a variety of Arctic and boreal shrubs and herbs, many of which had not previously been detected at the site from macrofossil and pollen records. The DNA record confirms the presence of hare and mitochondrial DNA from animals including mastodons, reindeer, rodents and geese, all ancestral to their present-day and late Pleistocene relatives. The presence of marine species including horseshoe crab and green algae support a warmer climate than today. The reconstructed ecosystem has no modern analogue. The survival of such ancient eDNA probably relates to its binding to mineral surfaces. Our findings open new areas of genetic research, demonstrating that it is possible to track the ecology and evolution of biological communities from two million years ago using ancient eDNA.

Human-Based New Approach Methodologies in Developmental Toxicity Testing: A Step Ahead from the State of the Art with a Feto-Placental Organ-on-Chip Platform.

Developmental toxicity testing urgently requires the implementation of human-relevant new approach methodologies (NAMs) that better recapitulate the peculiar nature of human physiology during pregnancy, especially the placenta and the maternal/fetal interface, which represent a key stage for human lifelong health. Fit-for-purpose NAMs for the placental-fetal interface are desirable to improve the biological knowledge of environmental exposure at the molecular level and to reduce the high cost, time and ethical impact of animal studies. This article reviews the state of the art on the available in vitro (placental, fetal and amniotic cell-based systems) and in silico NAMs of human relevance for developmental toxicity testing purposes; in addition, we considered available Adverse Outcome Pathways related to developmental toxicity. The OECD TG 414 for the identification and assessment of deleterious effects of prenatal exposure to chemicals on developing organisms will be discussed to delineate the regulatory context and to better debate what is missing and needed in the context of the Developmental Origins of Health and Disease hypothesis to significantly improve this sector. Starting from this analysis, the development of a novel human feto-placental organ-on-chip platform will be introduced as an innovative future alternative tool for developmental toxicity testing, considering possible implementation and validation strategies to overcome the limitation of the current animal studies and NAMs available in regulatory toxicology and in the biomedical field.

Phase 1/1b Studies of UCB0599, an Oral Inhibitor of α-Synuclein Misfolding, Including a Randomized Study in Parkinson's Disease.

BACKGROUND: Parkinson's disease (PD) and its progression are thought to be caused and driven by misfolding of α-synuclein (ASYN). UCB0599 is an oral, small-molecule inhibitor of ASYN misfolding, aimed at slowing disease progression. OBJECTIVE: The aim was to investigate safety/tolerability and pharmacokinetics (PK) of single and multiple doses of UCB0599. METHODS: Safety/tolerability and PK of single and multiple doses of UCB0599 and its metabolites were investigated in two phase 1 studies in healthy participants (HPs), where food effect and possible interaction with itraconazole (ITZ) were assessed (UP0030 [randomized, placebo-controlled, dose-escalation, crossover study, N = 65] and UP0078 [open-label study, N = 22]). Safety/tolerability and multi-dose PK of UCB0599 were subsequently investigated in a phase 1b randomized, double-blind, placebo-controlled study of participants with PD (UP0077 [NCT04875962], N = 31). RESULTS: Across all studies, UCB0599 displayed rapid absorption with linear, time-independent PK properties; PK of multiple doses of UCB0599 were predictable from single-dose exposures. No notable food-effect was observed; co-administration with ITZ affected UCB0599 disposition (maximum plasma concentration and area under the curve increased ~1.3- and ~2 to 3-fold, respectively) however, this did not impact the safety profile. Hypersensitivity reactions were reported in UP0030 (n = 2) and UP0077 (n = 2). Treatment-related adverse events occurred in 43% (UCB0599), and 30% (placebo) of participants with PD were predominantly mild-to-moderate in intensity and were not dose related. CONCLUSIONS: Seventy-three HPs and 21 participants with PD received UCB0599 doses; an acceptable safety/tolerability profile and predictable PK support continued development of UCB0599 for the slowing of PD progression. A phase 2 study in early-stage PD is underway (NCT04658186). © 2022 UCB Pharma. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Addressing microchimerism in pregnancy by ex vivo human placenta perfusion.

The physical connection of mother and offspring during pregnancy allows the bi-directional exchange of a small number of cells through the placenta. These cells, which can persist long-term in the recipient individual are genetically foreign to it and therefore fulfill the principle of microchimerism. Over the last years, pioneer research on microchimeric cells revealed their role in immune adaptation during pregnancy and priming of tolerogenic responses in the progeny. However, the mechanisms involved in cell transfer across the placenta barrier remain poorly investigated. In this review, we summarize the evidence of fetomaternal microchimerism, propose a mechanism for cell trafficking through the placenta and discuss the different models and techniques available for its analysis. Likewise, we aim to generate interest in the use of ex vivo placenta perfusion to investigate microchimerism in physiological and pathological settings.

The road (not) taken - Placental transfer and interspecies differences.

Species differences are among the main reasons for the high failure rate of preclinical studies. A better awareness and understanding of these differences might help to improve the outcome of preclinical research. In reproduction, the placenta is the central organ regulating fetal exposure to a substance circulating in the maternal organism. Exact information about placental transfer can help to better estimate the toxic potential of a substance. From an evolutionary point of view, the chorioallantoic placenta is the organ with the highest anatomical diversity among species. Moreover, frequently used animal models in reproduction belong to rodents and lagomorphs, two groups that are characterized by the generation of an additional type of placenta, which is crucial for fetal development, but absent from humans: the inverted yolk sac placenta. Taken together, the translatability of placental transfer studies from laboratory animals to humans is challenging, which is supported by the fact that numerous species-dependent toxic effects are described in literature. Thus, reliable human-relevant data are frequently lacking and the toxic potential of chemicals and pharmaceuticals for humans can hardly be estimated, often resulting in recommendations that medical treatments or exposure to chemicals should be avoided for safety reasons. Although species differences of placental anatomy have been described frequently and the need for human-relevant research models has been emphasized, analyses of substances with species-dependent placental transfer have been performed only sporadically. Here, we present examples for species-specific placental transfer, including that of nanoparticles and pharmaceuticals, and discuss potential underlying mechanisms. With respect to the COVID 19-pandemic it might be of interest that some antiviral drugs are reported to feature species-specific placental transfer. Further, differences in placental structure and antibody transfer may affect placental transfer of ZIKA virus.

Pregnancy and pandemics: Interaction of viral surface proteins and placenta cells.

Throughout history, pandemics of infectious diseases caused by emerging viruses have spread worldwide. Evidence from previous outbreaks demonstrated that pregnant women are at high risk of contracting the diseases and suffering from adverse outcomes. However, while some viruses can cause major health complications for the mother and her fetus, others do not appear to affect pregnancy. Viral surface proteins bind to specific receptors on the cellular membrane of host cells and begin therewith the infection process. During pregnancy, the molecular features of these proteins may determine specific target cells in the placenta, which may explain the different outcomes. In this review, we display information on Variola, Influenza, Zika and Corona viruses focused on their surface proteins, effects on pregnancy, and possible target placental cells. This will contribute to understanding viral entry during pregnancy, as well as to develop strategies to decrease the incidence of obstetrical problems in current and future infections.

An Arabidopsis FANCJ helicase homologue is required for DNA crosslink repair and rDNA repeat stability.

Proteins of the Fanconi Anemia (FA) complementation group are required for crosslink (CL) repair in humans and their loss leads to severe pathological phenotypes. Here we characterize a homolog of the Fe-S cluster helicase FANCJ in the model plant Arabidopsis, AtFANCJB, and show that it is involved in interstrand CL repair. It acts at a presumably early step in concert with the nuclease FAN1 but independently of the nuclease AtMUS81, and is epistatic to both error-prone and error-free post-replicative repair in Arabidopsis. The simultaneous knock out of FANCJB and the Fe-S cluster helicase RTEL1 leads to induced cell death in root meristems, indicating an important role of the enzymes in replicative DNA repair. Surprisingly, we found that AtFANCJB is involved in safeguarding rDNA stability in plants. In the absence of AtRTEL1 and AtFANCJB, we detected a synergetic reduction to about one third of the original number of 45S rDNA copies. It is tempting to speculate that the detected rDNA instability might be due to deficiencies in G-quadruplex structure resolution and might thus contribute to pathological phenotypes of certain human genetic diseases.

Combined Metabolic Targeting With Metformin and the NSAIDs Diflunisal and Diclofenac Induces Apoptosis in Acute Myeloid Leukemia Cells.

The accelerated metabolism of tumor cells, inevitable for maintaining high proliferation rates, is an emerging target for tumor therapy. Increased glucose and lipid metabolism as well as mitochondrial activity have been shown in solid tumors but also in leukemic cells. As tumor cells are able to escape the blockade of one metabolic pathway by a compensatory increase in other pathways, treatment strategies simultaneously targeting metabolism at different sites are currently developed. However, the number of clinically applicable anti-metabolic drugs is still limited. Here, we analyzed the impact of the anti-diabetic drug metformin alone or in combination with two non-steroidal anti-inflammatory drugs (NSAIDs) diclofenac and diflunisal on acute myeloid leukemia (AML) cell lines and primary patient blasts. Diclofenac but not diflunisal reduced lactate secretion in different AML cell lines (THP-1, U937, and KG-1) and both drugs increased respiration at low concentrations. Despite these metabolic effects, both NSAIDs showed a limited effect on tumor cell proliferation and viability up to a concentration of 0.2 mM. In higher concentrations of 0.4-0.8 mM diflunisal alone exerted a clear effect on proliferation of AML cell lines and blocked respiration. Single treatment with the anti-diabetic drug metformin blocked mitochondrial respiration, but proliferation and viability were not affected. However, combining all three drugs exerted a strong cytostatic and cytotoxic effect on THP-1 cells. Comparable to the results obtained with THP-1 cells, the combination of all three drugs significantly reduced proliferation of primary leukemic blasts and induced apoptosis. Furthermore, NSAIDs supported the effect of low dose chemotherapy with cytarabine and reduced proliferation of primary AML blasts. Taken together we show that low concentrations of metformin and the two NSAIDs diclofenac and diflunisal exert a synergistic inhibitory effect on AML proliferation and induce apoptosis most likely by blocking tumor cell metabolism. Our results underline the feasibility of applying anti-metabolic drugs for AML therapy.

Human serum alters cell culture behavior and improves spheroid formation in comparison to fetal bovine serum.

BACKGROUND: The use of fetal bovine serum (FBS) as growth supplement for human cell and tissue culture is widely spread in basic research as well as in clinical approaches, although several limitations must be considered, such as unstable composition and availability, biosafety and ethical aspects. Regarding interspecies differences, xenogeneic growth factors may evoke incompatibilities and non-desired interactions with human cells resulting in imprecise outcome of human-relevant data. METHODS: In this study the functionality of human serum (HS) has been investigated in comparison to FBS by assessing proliferation, migration and invasion of the human cervical cancer cell lines SiHa and HeLa. The effects of both sera on spheroid formation were analyzed microscopically. RESULTS: Both, FBS and HS, stimulate cell proliferation and migration similarly, whereas HS significantly enhanced cell invasion. The spheroid formation assay revealed remarkable differences between both sera, especially for SiHa cells. While in FBS supplemented medium cells only formed loose aggregates, HS induced regularly shaped spheroids under all tested conditions. CONCLUSION: We were able to demonstrate that HS and FBS differently influence behavior of cells in culture which may have an impact on experimental results, especially in 3D cultures.

Calculation of injection forces for highly concentrated protein solutions.

Protein solutions often manifest a high viscosity at high solution concentrations, thus impairing injectability. Accordingly, accurate prediction of the injection force based on solution viscosity can greatly support protein formulation and device development. In this study, the shear-dependent viscosity of three concentrated protein solutions is reported, and calculated injection forces obtained by two different mathematical models are compared against measured values. The results show that accurate determination of the needle dimensions and the shear-thinning behavior of the protein solutions is vital for injection force prediction. Additionally, one model delivered more accurate results, particularly for solutions with prominent shear-thinning behavior.

Ancient and modern environmental DNA.

DNA obtained from environmental samples such as sediments, ice or water (environmental DNA, eDNA), represents an important source of information on past and present biodiversity. It has revealed an ancient forest in Greenland, extended by several thousand years the survival dates for mainland woolly mammoth in Alaska, and pushed back the dates for spruce survival in Scandinavian ice-free refugia during the last glaciation. More recently, eDNA was used to uncover the past 50 000 years of vegetation history in the Arctic, revealing massive vegetation turnover at the Pleistocene/Holocene transition, with implications for the extinction of megafauna. Furthermore, eDNA can reflect the biodiversity of extant flora and fauna, both qualitatively and quantitatively, allowing detection of rare species. As such, trace studies of plant and vertebrate DNA in the environment have revolutionized our knowledge of biogeography. However, the approach remains marred by biases related to DNA behaviour in environmental settings, incomplete reference databases and false positive results due to contamination. We provide a review of the field.

GC content-independent amino acid patterns in bacteria and archaea.

Every organism can be characterized by the amino acid composition of its proteome. So far it was assumed that these compositions are determined by the GC content of the DNA or, in some cases, by extreme lifestyles, like thermophily or halophily. Here, we focussed our analysis on eight amino acids, each of which is encoded by both, GC and AT rich codons, to identify finer amino acid patterns beyond the GC dominance. We investigated the conceptually translated proteomes of 1029 bacterial and archaeal strains with sequenced genomes for amino acid composition. Using correspondence analysis, we found that phylogenetic groups within bacteria and archaea generally can be discriminated from other groups due to their amino acid composition. In some cases, single organisms, e.g. Treponema pallidum strains or Mycoplasma penetrans, are characterized by extreme amino acid compositions. We assume that our data could provide a basis for a new approach to analyze evolution of bacterial and archaeal groups. Furthermore, for single organisms, the detailed knowledge of the amino acid composition of the entire proteome encoded in the genome could lead to a better understanding, important for pharmaceutical or biotechnological applications. We recommend that information about amino acid compositions should be provided in databases, comparable to the GC content of genomes.

BRCC36A is epistatic to BRCA1 in DNA crosslink repair and homologous recombination in Arabidopsis thaliana.

BRCA1 is a well-known tumor suppressor protein in mammals, involved in multiple cellular processes such as DNA repair, chromosome segregation and chromatin remodeling. Interestingly, homologs of BRCA1 and several of its complex partners are also found in plants. As the respective mutants are viable, in contrast to mammalian mutants, detailed analyses of their biological role is possible. Here we demonstrate that the model plant Arabidopsis thaliana harbors two homologs of the mammalian BRCA1 interaction partner BRCC36, AtBRCC36A and AtBRCC36B. Mutants of both genes as well as the double mutants are fully fertile and show no defects in development. We were able to show that mutation of one of the homologs, AtBRCC36A, leads to a severe defect in intra- and interchromosomal homologous recombination (HR). A HR defect is also apparent in Atbrca1 mutants. As the Atbrcc36a/Atbrca1 double mutant behaves like the single mutants of AtBRCA1 and AtBRCC36A both proteins seem to be involved in a common pathway in the regulation of HR. AtBRCC36 is also epistatic to AtBRCA1 in DNA crosslink repair. Upon genotoxic stress, AtBRCC36A is transferred into the nucleus.

In silico prediction of potential metallothioneins and metallohistins in actinobacteria.

Metallothioneins and metallohistins are short peptides with a high cysteine and/or histidine content able to coordinate metals intracellularly, thereby increasing the tolerance against elevated concentrations of metals. Because of their features, they can be detected by in silico prediction from proteomes annotated from sequenced genomes. Here, we analyzed 73 sequenced actinobacterial genomes for peptides (≤ 100 amino acids) with a high content of cysteine and histidine (≥ 15%) and identified 103 putative metallothioneins and metallohistins. For 45 of these peptides, we found similarities to metal binding protein domains, including zinc fingers, heavy metal transporters or eukaryotic metallothioneins, which can serve as proof-of-principle in underscoring a potential function as metal binding peptides. An evolutionary origin from metal containing domains of enzymes is discussed and metallohistins not containing cysteine are described for the first time for bacteria.

In silico analysis of nickel containing superoxide dismutase evolution and regulation.

Superoxide dismutases are essential enzymes involved in detoxification of reactive oxygen by dismutation of the superoxide radical anion. A class of nickel containing superoxide dismutases has been described for streptomycetes and cyanobacteria. In silico analysis was used to study the distribution of genes coding for NiSOD in other taxa and to elucidate signals linked to nickel incorporation and maturation of NiSOD. Data mining revealed homologous proteins from actinobacteria, proteobacteria, chlamydiae, and eukarya (green algae) thus allowing a comparison of protein structural elements. Nickel ligands and maturation signals for N-terminal proteolysis were highly conserved. Genomic sequences surrounding genes encoding NiSOD homologs were compared in order to detect putative accessory enzymes involved in maturation. An endopeptidase gene linked to sodN coding for NiSOD was found in actinobacteria and cyanobacteria, but not in other taxa. The distribution of NiSOD encoding sequences showed four clusters which are not consistent with the phylogeny of the species. In addition, the different genomic context argues for heterologous gene transfer, most likely from actinobacteria to other taxa. In order to address regulation by nickel availability and incorporation into the mature protein, we present first evidence for putative regulatory nucleotide sequences which will be useful in future studies on nickel uptake and incorporation.

Superoxide dismutases of heavy metal resistant streptomycetes.

Heavy metal tolerant and resistant strains of streptomycetes isolated from a former uranium mining site were screened for their superoxide dismutase expression. From the strains tolerating high concentrations of different heavy metals, one was selected for its tolerance of concentrations of heavy metals (Ni, Cu, Cd, Cr, Mn, Zn, Fe). This strain, Streptomyces acidiscabies E13, was chosen for the purpose of superoxide dismutase analysis. Gel electrophoresis and activity staining revealed only one each of a nickel (NiSOD) and an iron (FeZnSOD) containing superoxide dismutase as shown by differential enzymatic repression studies. The gene for nickel containing superoxide dismutase, sodN, was cloned and sequenced from this strain. The genomic sequence shows 92.7% nucleotide identity and 96.1% amino acid identity to sodN of S. coelicolor. Expression can be activated by nickel as well as other heavy metals and active enzyme is produced in media lacking nickel but containing copper, iron or zinc. Thus, the selected strain is well suited for further characterization of the enzyme encoded by sodN.