CTCF binds to sites in the major histocompatibility complex that are rapidly reconfigured in response to interferon-gamma

Activation of the major histocompatibility complex (MHC) by interferon-gamma (IFN−γ) is a fundamental step in the adaptive immune response to pathogens. Here, we show that reorganization of chromatin loop domains in the MHC is evident within the first 30 min of IFN−γ treatment of fibroblasts, and that further dynamic alterations occur up to 6 h. These very rapid changes occur at genomic sites which are occupied by CTCF and are close to IFN−γ-inducible MHC genes. Early responses to IFN−γ are thus initiated independently of CIITA, the master regulator of MHC class II genes and prepare the MHC for subsequent induction of transcription

Widespread Expression of BORIS/CTCFL in Normal and Cancer Cells

BORIS (CTCFL) is the paralog of CTCF (CCCTC-binding factor; NM_006565), a ubiquitously expressed DNA-binding protein with diverse roles in gene expression and chromatin organisation. BORIS and CTCF have virtually identical zinc finger domains, yet display major differences in their respective C- and N-terminal regions. Unlike CTCF, BORIS expression has been reported only in the testis and certain malignancies, leading to its classification as a “cancer-testis” antigen. However, the expression pattern of BORIS is both a significant and unresolved question in the field of DNA binding proteins. Here, we identify BORIS in the cytoplasm and nucleus of a wide range of normal and cancer cells. We compare the localization of CTCF and BORIS in the nucleus and demonstrate enrichment of BORIS within the nucleolus, inside the nucleolin core structure and adjacent to fibrillarin in the dense fibrillar component. In contrast, CTCF is not enriched in the nucleolus. Live imaging of cells transiently transfected with GFP tagged BORIS confirmed the nucleolar accumulation of BORIS. While BORIS transcript levels are low compared to CTCF, its protein levels are readily detectable. These findings show that BORIS expression is more widespread than previously believed, and suggest a role for BORIS in nucleolar function

Interactions between mouse CNS cells : Microglia and neural precursor cells

The mammalian central nervous system (CNS) contains a variety of cells, all specialized to perform different functions. Most numerous are the three types of glia cells, whose basic role is to support the signaling units of the CNS, the neurons. The perspective of this thesis is from the glia cells; particularly the microglia cells, a non-neural population of cells that are spread throughout the CNS. In the healthy CNS these cells are resting, but as a consequence of various CNS disturbances they rapidly become activated, frequently together with astrocytes, the second type of glia cells (oligodendrocytes being the third). This can happen slowly, as in neurodegenerative diseases, or quickly as in acute CNS lesions such as stroke. Both processes involve microglia cells, and sometimes macrophages from the circulation. These latter cells are difficult to distinguish from the microglia cells. In an effort to generate microglia specific markers we used phage display technology to select microglia specific peptides from a random peptide library displayed on the phage surface. Two sets of selection strategies were compared with regard to phage-clone enrichment. The first strategy was based on phage binding to monolayers of primary microglia fixed to a solid surface, while the second strategy was based on fluorescence activated cell sorting (FACS) of microglia cells with bound phages. The latter protocol was found to be superior. Five phage-clones that preferentially bound to microglia cells were isolated. One of the selected clones was shown to be microglia specific by free peptide inhibition and selective in binding to microglia cells, as compared to blood-derived monocytes. Much of our current knowledge in neurobiology derives from studies of cells in culture, a less complex substitute for in vivo studies. As a bridge between monolayer CNS cell cultures and in vivo animal models we set up a three-dimensional culture system, so called aggregate cultures from mouse CNS cells. These aggregates were characterized in detail regarding cellular composition and dynamics, as well as the expression of several neuropeptides and neurotransmitters. All the principle brain cells were present in the aggregates and their numbers changed over time, neurons being the most numerous. The cells appeared to mature as judged by their morphology and, in the case of neurons, the increased expression of synapse specific proteins. Among the investigated neuropeptides, enkephalin and dynorphin were the most abundant followed by galanin, approximating their expression in CNS development. We also found that neural precursor cells, capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes, were maintained in the aggregates, even after more than two months of culturing. Treating the aggregates with EGF led to the formation of an outer layer of nestin-positive precursor cells. Using the aggregate culture in part, we found that factor(s) secreted from microglia cells attracts neural precursor cells in a chemotactic manner. This finding may explain the preferred migration of precursor cells to sites of CNS injury. Furthermore, microglia derived factors could affect the differentiation of neural precursor cells, such that more neurons were formed. Together these results suggest important functions of microglia cells in CNS development and pathology. It is reasonable to believe that the migration of neural precursor cells is directed both by attractant and repellant cues. Reactive astrocytes are well known to inhibit growing axons and recently also suggested to inhibit the migration of neural precursor cells. We show that astrocytes in culture repel neural precursor cells and that this effect is mediated by secreted Slit proteins. This conclusion is based on several observations; astrocytes produce Slit and the astrocyte-repellant effect was blocked by the ectodomain of the Slit receptor, and finally, recombinant Slit could substitute for astrocyte derived Slit. Knowledge about the interplay between attractive and repulsive cues may be important for the manipulation of neural precursor cells for medical purposes

Use of Weight Elements to Improve the Dynamic Performance of Umbilicals

The development strategy in the North Sea has over the last years been towards tie in of smaller fields to existing infrastructure. Detailed engineering is required to obtain an optimal design for each application. This can be challenging for systems with multiple design constraints e.g. interference with adjacent structures combined with strict floater interface design requirements. Flexible risers and umbilicals are usually arranged in compliant configurations, where floater motions are absorbed by large changes of the configuration geometry in the 3D space. A vital design issue for such systems is to avoid overbending and fulfill fatigue life design criteria at floater supports. Another design challenge often encountered in design of such systems is to avoid/limit compressive effective tension in extreme load cases. In this paper it is proposed to introduce weight elements along parts of the riser/umbilical to improve the overall dynamic behavior and thereby address these design challenges. This principle was applied when designing the configuration for Morvin DEH riser. Morvin DEH riser is connected to A˚sgard B semi-submersible platform in the North Sea at 300m water depth. For this particular problem the design challenge was to limit the angle response in extreme load cases and fulfill fatigue design requirements in the bellmouth interface. Another constraint was at the same time to avoid interference between adjacent mooring lines and umbilicals. A feasible solution was found by introducing weight elements in the upper part of the umbilical. This had a minor impact on the cost schedule of the project. The Morvin DEH riser was installed successfully in the summer of 2009.</jats:p

First case of mpox with monkeypox virus clade Ib outside Africa in a returning traveller, Sweden, August 2024 [Elektronisk resurs] : public health measures

An unprecedented upsurge in mpox, caused by clade I of the monkeypox virus (MPXV) was noted in Central Africa in 2024. The first mpox case with MPXV clade Ib outside Africa was reported in Sweden in mid-August. The case experienced a mild disease course after travelling to an affected country. No additional cases of this clade have been detected in Sweden. Strengthened public health measures and surveillance including whole genome sequencing are crucial to prevent establishment of novel MPXV clades

Analysis of circulating protein aggregates as a route of investigation into neurodegenerative disorders

Abstract Plasma proteome composition reflects the inflammatory and metabolic state of the organism and can be predictive of system-level and organ-specific pathologies. Circulating protein aggregates are enriched with neurofilament heavy chain—axonal proteins involved in brain aggregate formation and recently identified as biomarkers of the fatal neuromuscular disorder amyotrophic lateral sclerosis. Using unbiased proteomic methods, we have fully characterized the content in neuronal proteins of circulating protein aggregates from amyotrophic lateral sclerosis patients and healthy controls, with reference to brain protein aggregate composition. We also investigated circulating protein aggregate protein aggregation propensity, stability to proteolytic digestion and toxicity for neuronal and endothelial cell lines. Circulating protein aggregates separated by ultracentrifugation are visible as electron-dense macromolecular particles appearing as either large globular or as small filamentous formations. Analysis by mass spectrometry revealed that circulating protein aggregates obtained from patients are enriched with proteins involved in the proteasome system, possibly reflecting the underlying basis of dysregulated proteostasis seen in the disease, while those from healthy controls show enrichment of proteins involved in metabolism. Compared to the whole human proteome, proteins within circulating protein aggregates and brain aggregates show distinct chemical features of aggregation propensity, which appear dependent on the tissue or fluid of origin and not on the health status. Neurofilaments’ two high-mass isoforms (460 and 268 kDa) showed a strong differential expression in amyotrophic lateral sclerosis compared to healthy control circulating protein aggregates, while aggregated neurofilament heavy chain was also partially resistant to enterokinase proteolysis in patients, demonstrated by immunoreactive bands at 171 and 31 kDa fragments not seen in digested healthy controls samples. Unbiased proteomics revealed that a total of 4973 proteins were commonly detected in circulating protein aggregates and brain, including 24 expressed from genes associated with amyotrophic lateral sclerosis. Interestingly, 285 circulating protein aggregate proteins (5.7%) were regulated (P &amp;lt; 0.05) and are present in biochemical pathways linked to disease pathogenesis and protein aggregation. Biologically, circulating protein aggregates from both patients and healthy controls had a more pronounced effect on the viability of hCMEC/D3 endothelial and PC12 neuronal cells compared to immunoglobulins extracted from the same plasma samples. Furthermore, circulating protein aggregates from patients exerted a more toxic effect than healthy control circulating protein aggregates on both cell lines at lower concentrations (P: 0.03, in both cases). This study demonstrates that circulating protein aggregates are significantly enriched with brain proteins which are representative of amyotrophic lateral sclerosis pathology and a potential source of biomarkers and therapeutic targets for this incurable disorder.</jats:p

Enzymatic degradation of RNA causes widespread protein aggregation in cell and tissue lysates

ABSTRACTMost proteins in cell and tissue lysates are soluble. Here, we show that many of these proteins, including several that are implicated in neurodegenerative diseases, are maintained in a soluble and functional state by association with endogenous RNA, as degradation of RNA invariably leads to protein aggregation. We identify the importance of nucleic acid structure, with single-stranded pyrimidine-rich bulges or loops surrounded by double-stranded regions being particularly efficient in this role, revealing an apparent one-to-one protein-nucleic acid stoichiometry. The relationship of these findings to pathological protein aggregation is suggested by our discovery that protein aggregates isolated from brain tissue from Amyotrophic Lateral Sclerosis patients can be rendered soluble after refolding by both RNA and synthetic oligonucleotides. Together, these findings open new avenues for understanding the mechanism behind protein aggregation and shed light on how certain proteins remain soluble.</jats:p

Analysis of circulating protein aggregates reveals pathological hallmarks of amyotrophic lateral sclerosis

AbstractBlood-based biomarkers can be informative of brain disorders where protein aggregation play a major role. The proteome of plasma and circulating protein aggregates (CPA) reflect the inflammatory and metabolic state of the organism and can be predictive of system-level and/or organ-specific pathologies. CPA are enriched with heavy chain neurofilaments (NfH), key axonal constituents involved in brain aggregates formation and biomarkers of the fatal neurodegenerative disorder amyotrophic lateral sclerosis (ALS). Here we show that CPA and brain protein aggregates (BPA) from ALS differ in protein composition and appear as a combination of electron-dense large globular and small filamentous formations on transmission electron microscopy. CPA are highly enriched with proteins involved in the proteasome and energy metabolism. Compared to the human proteome, proteins within aggregates show distinct and tissue-dependent chemical features of aggregation propensity. The use of a TMTcalibrator™ proteomics workflow with ALS brain as calibrant reveals 4973 brain-derived low-abundance proteins in CPA, including the products of translation of 24 ALS risk genes. 285 of these (5.7%) are regulated in ALS CPA including FUS (p&lt;0.05). CPA from both ALS and healthy controls affect cell viability when testing endothelial and PC12 neuronal cell lines, while CPA from ALS exert a more toxic effect at lower concentrations. The analysis of resistance to protease enzymes hydrolysis indicates an ALS-specific digestion pattern for NfH using enterokinase. This study reveals how peripheral protein aggregates are significantly enriched with brain proteins which are highly representative of ALS pathology and a potential alternative source of biomarkers and therapeutic targets for this incurable disorder.Significance StatementMolecular mechanism of neurodegeneration like protein aggregation are important brain-specific alterations which need to be addressed therapeutically. Recently described fluid biomarkers of neurodegenerative disorders provide means for stratification and monitoring of disease progression. Here we show that circulating protein aggregates are easily accessible in blood and reproduce important features of brain pathology for an incurable disorder like amyotrophic lateral sclerosis. They represent a source of biomarkers and of novel therapeutics for ALS.</jats:sec

Analysis of Circulating Protein Aggregates Reveals Pathological Hallmarks of Amyotrophic Lateral Sclerosis

Abstract Background: plasma proteins composition reflects the inflammatory and metabolic state of an organism and can be predictive of system-level and organ-specific pathologies. Circulating protein aggregates (CPA) are enriched with heavy chain neurofilaments (NfH), axonal proteins involved in brain protein aggregates (BPA) formation and recently identified as biomarkers of the fatal neuromuscular disorder amyotrophic lateral sclerosis (ALS). Methods: here we use mass spectrometry and brain-enhanced TMTcalibrator™-based proteomics to evaluate the composition and the brain-derived protein component of CPA extracted from ALS and healthy controls (HC) plasma samples using high-performance ultracentrifugation. We also test CPA and BPA proteins aggregation propensity and the resistance to proteases digestion by trypsin, chymotrypsin, calpain and enterokinase of NFH within aggregates. Finally, we study CPA biological effects on neuronal and endothelial cell lines. Results: electron microscopy confirms the presence in CPA of electron-dense macromolecular particles appearing as either large globular or as small filamentous formations. CPA from ALS are enriched with proteasome system proteins while HC CPA show a prominent expression of proteins involved in metabolism. CPA enterokinase digestion in ALS generates 171 and 31 KDa NfH fragments not seen in HC samples. Compared to the whole human proteome, proteins within CPA and BPA show distinct chemical features of aggregation propensity, which appear dependent on the tissue or fluid of origin and not on the healthy or pathological source of plasma. The use of a TMTcalibrator™ proteomics workflow reveals 4973 brain-derived low-abundance proteins in CPA, including products of translation of 24 ALS risk genes. 285 (5.7%) are regulated in ALS (p &lt; 0.05) and belong to biochemical pathways previously linked to ALS pathogenesis and aggregates formation. CPA from both ALS and HC have a higher effect on hCMEC/D3 endothelial and PC12 neuronal cells viability than immunoglobulins extracted from the same plasma samples. Compared to HC, CPA from ALS plasma samples exert a higher toxic effect on both cell lines at lower concentrations.Conclusions: this study demonstrates that CPA are significantly enriched with brain proteins which are representative of ALS pathology and a potential source of biomarkers and therapeutic targets for this incurable disorder.</jats:p

Massive and rapid COVID-19 testing is feasible by extraction-free SARS-CoV-2 RT-PCR

AbstractCoronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is commonly diagnosed by reverse transcription polymerase chain reaction (RT-PCR) to detect viral RNA in patient samples, but RNA extraction constitutes a major bottleneck in current testing. Methodological simplification could increase diagnostic availability and efficiency, benefitting patient care and infection control. Here, we describe methods circumventing RNA extraction in COVID-19 testing by performing RT-PCR directly on heat-inactivated or lysed samples. Our data, including benchmarking using 597 clinical patient samples and a standardised diagnostic system, demonstrate that direct RT-PCR is viable option to extraction-based tests. Using controlled amounts of active SARS-CoV-2, we confirm effectiveness of heat inactivation by plaque assay and evaluate various generic buffers as transport medium for direct RT-PCR. Significant savings in time and cost are achieved through RNA-extraction-free protocols that are directly compatible with established PCR-based testing pipelines. This could aid expansion of COVID-19 testing.</jats:p