Zika virus (ZIKV) is an arbovirus belonging to the genus Flavivirus (family Flaviviridae) and was first described in 1947 in Uganda following blood analyses of sentinel Rhesus monkeys. Until the ...twentieth century, the African and Asian lineages of the virus did not cause meaningful infections in humans. However, in 2007, vectored by Aedes aegypti mosquitoes, ZIKV caused the first noteworthy epidemic on the Yap Island in Micronesia. Patients experienced fever, skin rash, arthralgia and conjunctivitis. From 2013 to 2015, the Asian lineage of the virus caused further massive outbreaks in New Caledonia and French Polynesia. In 2013, ZIKV reached Brazil, later spreading to other countries in South and Central America. In Brazil, the virus has been linked to congenital malformations, including microcephaly and other severe neurological diseases, such as Guillain-Barré syndrome. Despite clinical evidence, direct experimental proof showing that the Brazilian ZIKV (ZIKV(BR)) strain causes birth defects remains absent. Here we demonstrate that ZIKV(BR) infects fetuses, causing intrauterine growth restriction, including signs of microcephaly, in mice. Moreover, the virus infects human cortical progenitor cells, leading to an increase in cell death. We also report that the infection of human brain organoids results in a reduction of proliferative zones and disrupted cortical layers. These results indicate that ZIKV(BR) crosses the placenta and causes microcephaly by targeting cortical progenitor cells, inducing cell death by apoptosis and autophagy, and impairing neurodevelopment. Our data reinforce the growing body of evidence linking the ZIKV(BR) outbreak to the alarming number of cases of congenital brain malformations. Our model can be used to determine the efficiency of therapeutic approaches to counteracting the harmful impact of ZIKV(BR) in human neurodevelopment.
This work analyses the distribution of living benthic foraminiferal assemblages of surface sediments in different intertidal areas of Ria de Aveiro (Portugal), a polihaline and anthropized coastal ...lagoon. The relationships among foraminiferal assemblages in association with environmental parameters (temperature, salinity, Eh and pH), grain size, the quantity and quality of organic matter (enrichment in carbohydrates, proteins and lipids), pollution caused by metals, and mineralogical data are studied in an attempt to identify indicators of adaptability to environmental stress. In particular, concentrations of selected metals in the surficial sediment are investigated to assess environmental pollution levels that are further synthetically parameterised by the Pollution Load Index (PLI). The PLI variations allowed the identification of five main polluted areas. Concentrations of metals were also analysed in three extracted phases to evaluate their possible mobility, bioavailability and toxicity in the surficial sediment. Polluted sediment in the form of both organic matter and metals can be found in the most confined zones. Whereas enrichment in organic matter and related biopolymers causes an increase in foraminifera density, pollution by metals leads to a decline in foraminiferal abundance and diversity in those zones. The first situation may be justified by the existence of opportunistic species (with high reproduction rate) that can live in low oxic conditions. The second is explained by the sensitivity of some species to pressure caused by metals. The quality of the organic matter found in these places and the option of a different food source should also explain the tolerance of several species to pollution caused by metals, despite their low reproductive rate in the most polluted areas. In this study, species that are sensitive and tolerant to organic matter and metal enrichment are identified, as is the differential sensitivity/tolerance of some species to metals enrichment.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
N
-methyladenosine (m
A) is an abundant internal RNA modification
that is catalysed predominantly by the METTL3-METTL14 methyltransferase complex
. The m
A methyltransferase METTL3 has been linked to ...the initiation and maintenance of acute myeloid leukaemia (AML), but the potential of therapeutic applications targeting this enzyme remains unknown
. Here we present the identification and characterization of STM2457, a highly potent and selective first-in-class catalytic inhibitor of METTL3, and a crystal structure of STM2457 in complex with METTL3-METTL14. Treatment of tumours with STM2457 leads to reduced AML growth and an increase in differentiation and apoptosis. These cellular effects are accompanied by selective reduction of m
A levels on known leukaemogenic mRNAs and a decrease in their expression consistent with a translational defect. We demonstrate that pharmacological inhibition of METTL3 in vivo leads to impaired engraftment and prolonged survival in various mouse models of AML, specifically targeting key stem cell subpopulations of AML. Collectively, these results reveal the inhibition of METTL3 as a potential therapeutic strategy against AML, and provide proof of concept that the targeting of RNA-modifying enzymes represents a promising avenue for anticancer therapy.
Despite technological advances, two-dimensional electrophoresis (2DE) of biological fluids, such as vitreous, remains a major challenge. In this study, artificial neural network was applied to ...optimize the recovery of vitreous proteins and its detection by 2DE analysis through the combination of several solubilizing agents (CHAPS, Genapol, DTT, IPG buffer), temperature, and total voltage. The highest protein recovery (94.9% ± 4.5) was achieved using 4% (
w
/
v
) CHAPS, 0.1% (
v
/v) Genapol, 20 mM DTT, and 2% (
v
/
v
) IPG buffer. Two iterations were required to achieve an optimized response (580 spots) using 4% (
w
/
v
) CHAPS, 0.2% (
v
/
v
) Genapol, 60 mM DTT, and 0.5% (
v
/
v
) IPG buffer at 35 kVh and 25 °C, representing a 2.4-fold improvement over the standard initial conditions of the experimental design. The analysis of depleted vitreous using the optimized protocol resulted in an additional 1.3-fold increment in protein detection over the optimal output, with an average of 761 spots detected in vitreous from different vitreoretinopathies. Our results clearly indicate the importance of combining the appropriate amount of solubilizing agents with a suitable control of the temperature and voltage to obtain high-quality gels. The high-throughput of this model provides an effective starting point for the optimization of 2DE protocols. This experimental design can be adapted to other types of matrices.
Graphical abstract
Whole-genome sequencing (WGS) permits comprehensive cancer genome analyses, revealing mutational signatures, imprints of DNA damage and repair processes that have arisen in each patient's cancer. We ...performed mutational signature analyses on 12,222 WGS tumor-normal matched pairs, from patients recruited via the UK National Health Service. We contrasted our results to two independent cancer WGS datasets, the International Cancer Genome Consortium (ICGC) and Hartwig Foundation, involving 18,640 WGS cancers in total. Our analyses add 40 single and 18 double substitution signatures to the current mutational signature tally. Critically, we show for each organ, that cancers have a limited number of 'common' signatures and a long tail of 'rare' signatures. We provide a practical solution for utilizing this concept of common versus rare signatures in future analyses.
The main objective of the present study was to investigate the use of in situ 2D fluorometry for monitoring key bioprocess variables in mammalian cell cultures, namely the concentration of viable ...cells and the concentration of recombinant proteins. All studies were conducted using a recombinant Baby Hamster Kidney (BHK) cell line expressing a fusion glycoprotein IgG1-IL2 cultured in batch and fed-batch modes. It was observed that the intensity of fluorescence signals in the excitation/emission wavelength range of amino acids, vitamins and NAD(P)H changed along culture time, although the dynamics of single fluorophors could not be correlated with the dynamics of the target state variables. Therefore, multivariate chemometric modeling was adopted as a calibration methodology. 2D fluorometry produced large volumes of redundant spectral data, which were first filtered by principal components analysis (PCA). Then, a partial least squares (PLS) regression was applied to correlate the reduced fluorescence maps with the target state variables. Two validation strategies were used to evaluate the predictive capacity of the developed PLS models. Accurate estimations of viable cells density (r² = 0.95; 99.2% of variance captured in the training set; r² = 0.91; 97.7% of variance captured in the validation set) and of glycoprotein concentration (r² = 0.99 and 99.7% of variance captured in the training set; r² = 0.99 and 99.3% of variance captured in the validation set) were obtained over a wide range of reactor operation conditions. The results presented herein confirm that 2D fluorometry constitutes a reliable methodology for on-line monitoring of viable cells and recombinant protein concentrations in mammalian cell cultures. Biotechnol. Bioeng. 2009;102: 1098-1106.
Numerous bacteria have been found to exhibit the capacity for intracellular polyhydroxyalkanoates (PHA) accumulation. Current methods for PHA production at the industrial scale are based on their ...synthesis from microbial isolates in either their wild form or by recombinant strains. High production costs are associated with these methods; thus, attempts have been made to develop more cost‐effective processes. Reducing the cost of the carbon substrates (e.g., through feeding renewable wastes) and increasing the efficiency of production technologies (including both fermentation and downstream extraction and recovery) are two such examples of these attempts. PHA production processes based on mixed microbial cultures are being investigated as a possible technology to decrease production costs, since no sterilization is required and bacteria can adapt quite well to the complex substrates that may be present in waste material. PHA accumulation by mixed cultures has been found under various operational conditions and configurations at both bench‐scale and full‐scale production. The process known as “feast and famine” or as “aerobic dynamic feeding” seems to have a high potential for PHA production by mixed cultures. Enriched cultures submitted to a transient carbon supply can synthesize PHA at levels comparable to those of pure cultures. Indeed, the intracellular PHA content can reach around 70% of the cell dry weight, suggesting that this process could be competitive with pure culture PHA production when fully developed. Basic and applied research of the PHA production process by mixed cultures has been carried out in the past decade, focusing on areas such as microbial characterization, process configuration, reactor operational strategies, process modeling and control, and polymer characterization. This paper presents a review of the PHA production process with mixed cultures, encompassing the findings reported in the literature as well as our own experimental results in relation to each of these areas.
Production of PHA by mixed cultures under feast and famine conditions.
Genomic instability is a hallmark of cancer and targeting DNA damage response (DDR) is emerging as a promising therapeutic strategy in different solid tumors. The effectiveness of targeting DDR in ...colorectal cancer has not been extensively explored.
We challenged 112 cell models recapitulating the genomic landscape of metastatic colorectal cancer with ATM, ATR, CHK1, WEE1, and DNA-PK inhibitors, in parallel with chemotherapeutic agents. We focused then on ATR inhibitors (ATRi) and, to identify putative biomarkers of response and resistance, we analyzed at multiple levels colorectal cancer models highly sensitive or resistant to these drugs.
We found that around 30% of colorectal cancers, including those carrying KRAS and BRAF mutations and unresponsive to targeted agents, are sensitive to at least one DDR inhibitor. By investigating potential biomarkers of response to ATRi, we found that ATRi-sensitive cells displayed reduced phospho-RPA32 foci at basal level, while ATRi-resistant cells showed increased RAD51 foci formation in response to replication stress. Lack of ATM and RAD51C expression was associated with ATRi sensitivity. Analysis of mutational signatures and HRDetect score identified a subgroup of ATRi-sensitive models. Organoids derived from patients with metastatic colorectal cancer recapitulated findings obtained in cell lines.
In conclusion, a subset of colorectal cancers refractory to current therapies could benefit from inhibitors of DDR pathways and replication stress. A composite biomarker involving phospho-RPA32 and RAD51 foci, lack of ATM and RAD51C expression, as well as analysis of mutational signatures could be used to identify colorectal cancers likely to respond to ATRi.
Despite the conventional view that a truly random V(D)J recombination process should generate a highly diverse immune repertoire, emerging reports suggest that there is a certain bias toward the ...generation of shared/public immune receptor chains. These studies were performed in viral diseases where public T cell receptors (TCR) appear to confer better protective responses. Selective pressures generating common TCR clonotypes are currently not well understood, but it is believed that they confer a growth advantage. As very little is known about public TCR clonotypes in cancer, here we set out to determine the extent of shared TCR clonotypes in the intra-tumor microenvironments of virus- and non-virus-driven head and neck cancers using TCR sequencing. We report that tumor-infiltrating T cell clonotypes were indeed shared across individuals with the same cancer type, where the majority of shared sequences were specific to the cancer type (i.e., viral versus non-viral). These shared clonotypes were not particularly enriched in EBV-associated nasopharynx cancer but, in both cancers, exhibited distinct characteristics, namely shorter CDR3 lengths, restricted V- and J-gene usages, and also demonstrated convergent V(D)J recombination. Many of these shared TCRs were expressed in patients with a shared HLA background. Pattern recognition of CDR3 amino acid sequences revealed strong convergence to specific pattern motifs, and these motifs were uniquely found to each cancer type. This suggests that they may be enriched for specificity to common antigens found in the tumor microenvironment of different cancers. The identification of shared TCRs in infiltrating tumor T cells not only adds to our understanding of the tumor-adaptive immune recognition but could also serve as disease-specific biomarkers and guide the development of future immunotherapies.
The diagnosis of hematologic malignancies relies on multidisciplinary workflows involving morphology, flow cytometry, cytogenetic, and molecular genetic analyses. Advances in cancer genomics have ...identified numerous recurrent mutations with clear prognostic and/or therapeutic significance to different cancers. In myeloid malignancies, there is a clinical imperative to test for such mutations in mainstream diagnosis; however, progress toward this has been slow and piecemeal. Here we describe Karyogene, an integrated targeted resequencing/analytical platform that detects nucleotide substitutions, insertions/deletions, chromosomal translocations, copy number abnormalities, and zygosity changes in a single assay. We validate the approach against 62 acute myeloid leukemia, 50 myelodysplastic syndrome, and 40 blood DNA samples from individuals without evidence of clonal blood disorders. We demonstrate robust detection of sequence changes in 49 genes, including difficult-to-detect mutations such as FLT3 internal-tandem and mixed-lineage leukemia (MLL) partial-tandem duplications, and clinically significant chromosomal rearrangements including MLL translocations to known and unknown partners, identifying the novel fusion gene MLL-DIAPH2 in the process. Additionally, we identify most significant chromosomal gains and losses, and several copy neutral loss-of-heterozygosity mutations at a genome-wide level, including previously unreported changes such as homozygosity for DNMT3A R882 mutations. Karyogene represents a dependable genomic diagnosis platform for translational research and for the clinical management of myeloid malignancies, which can be readily adapted for use in other cancers.
•We develop and validate Karyogene, a comprehensive one-stop diagnostic platform for the genomic analysis of myeloid malignancies.•Karyogene simultaneously detects substitutions, insertions/deletions, translocations, copy number and zygosity changes in a single assay.
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