Reactive astrocytes evolve after brain injury, inflammatory and degenerative diseases, whereby they undergo transcriptomic re-programming. In malignant brain tumors, their function and crosstalk to ...other components of the environment is poorly understood. Here we report a distinct transcriptional phenotype of reactive astrocytes from glioblastoma linked to JAK/STAT pathway activation. Subsequently, we investigate the origin of astrocytic transformation by a microglia loss-of-function model in a human organotypic slice model with injected tumor cells. RNA-seq based gene expression analysis of astrocytes reveals a distinct astrocytic phenotype caused by the coexistence of microglia and astrocytes in the tumor environment, which leads to a large release of anti-inflammatory cytokines such as TGFβ, IL10 and G-CSF. Inhibition of the JAK/STAT pathway shifts the balance of pro- and anti-inflammatory cytokines towards a pro-inflammatory environment. The complex interaction of astrocytes and microglia cells promotes an immunosuppressive environment, suggesting that tumor-associated astrocytes contribute to anti-inflammatory responses.
Significance Chronic hepatitis B virus (HBV) infection puts >250 million humans at risk for developing liver cirrhosis and liver cancer. Current therapies are not curative because they do not target ...HBV́s persistence reservoir, the plasmid-like covalently closed circular DNA (cccDNA). RNA production from cccDNA initiates the generation of progeny virus via protein-primed reverse transcription, yielding viral polymerase-linked relaxed-circular DNA (RC-DNA). Its conversion, upon infection, into cccDNA requires multiple poorly understood steps, including polymerase removal. We found that the host enzyme tyrosyl-DNA-phosphodiesterase 2 (TDP2), important for repair of cellular protein–DNA adducts, performed this step in vitro and that TDP2 depletion impaired the conversion of RC-DNA to cccDNA in cells. These data establish a functional link between HBV and cellular DNA repair and pave the way for targeting HBV persistence.
Hepatitis B virus (HBV), the causative agent of chronic hepatitis B and prototypic hepadnavirus, is a small DNA virus that replicates by protein-primed reverse transcription. The product is a 3-kb relaxed circular DNA (RC-DNA) in which one strand is linked to the viral polymerase (P protein) through a tyrosyl–DNA phosphodiester bond. Upon infection, the incoming RC-DNA is converted into covalently closed circular (ccc) DNA, which serves as a viral persistence reservoir that is refractory to current anti-HBV treatments. The mechanism of cccDNA formation is unknown, but the release of P protein is one mandatory step. Structural similarities between RC-DNA and cellular topoisomerase–DNA adducts and their known repair by tyrosyl-DNA-phosphodiesterase (TDP) 1 or TDP2 suggested that HBV may usurp these enzymes for its own purpose. Here we demonstrate that human and chicken TDP2, but only the yeast ortholog of TDP1, can specifically cleave the Tyr–DNA bond in virus-adapted model substrates and release P protein from authentic HBV and duck HBV (DHBV) RC-DNA in vitro, without prior proteolysis of the large P proteins. Consistent with TPD2’s having a physiological role in cccDNA formation, RNAi-mediated TDP2 depletion in human cells significantly slowed the conversion of RC-DNA to cccDNA. Ectopic TDP2 expression in the same cells restored faster conversion kinetics. These data strongly suggest that TDP2 is a first, although likely not the only, host DNA-repair factor involved in HBV cccDNA biogenesis. In addition to establishing a functional link between hepadnaviruses and DNA repair, our results open new prospects for directly targeting HBV persistence.
BACKGROUND AND PURPOSE—Whether maximal treatment should be offered to elderly patients suffering from poor-grade aneurysmal subarachnoid hemorrhage (aSAH) is controversial. The survival of patients ...in this subgroup beyond the usual outcome measurements 6 to 12 months after aSAH is unclear. The purpose of this study is to provide survival and outcome data to support clinicians making decisions on treatment for this subgroup of patients.
METHODS—We performed a retrospective analysis of the Bernese SAH database for poor-grade (World Federation of Neurosurgical Societies grade IV and V) elderly patients (age ≥60 years) suffering from aSAH admitted to our institution from 2005 to 2017. Patients were divided into 3 age groups (60–69, 70–79, and 80–90 years). Survival analysis was performed to estimate mean survival and hazard ratios for death. Binary logarithmic regression was used to estimate the odds ratio for favorable (modified Rankin Scale score of 0–3) and unfavorable (modified Rankin Scale score of 4–6) outcome.
RESULTS—Increasing age was associated with an increasing risk of death after aSAH. The hazard ratio increased by 6% per year of age (P<0.001; hazard ratio, 1.06; 95% CI, 1.03–1.09) and 76% per decade (P<0.001; hazard ratio, 1.76; 95% CI, 1.35–2.29). Mean survival was 56.3±8 months (patients aged 60–69 years), 31.6±7.6 months (70–79 years), and 7.6±5.8 months (80–90 years). Unfavorable outcomes 6 to 12 months after aSAH were strongly related to older age. The odds ratio increased by 11% per year of age (P<0.001; odds ratio, 1.11; 95% CI, 1.05–1.18) and 192% per decade (P<0.001; odds ratio, 2.92; 95% CI, 1.63–5.26).
CONCLUSIONS—Risk for death and unfavorable outcome increases markedly with older age in elderly patients with poor-grade aSAH. Despite a high initial mortality, treatment resulted in a reasonable proportion of favorable outcomes up to 79 years of age and only a small number of patients who were moderately or severely disabled 6 to 12 months after aSAH. Mean survival and proportion of favorable outcomes decreased markedly in patients older than 80 years.
Screening improves outcomes related to colorectal cancer (CRC); however, suboptimal participation for available screening tests limits the full benefits of screening. Non-invasive screening using a ...blood based assay may potentially help reach the unscreened population.
To compare the performance of a new Septin9 DNA methylation based blood test with a fecal immunochemical test (FIT) for CRC screening.
In this trial, fecal and blood samples were obtained from enrolled patients. To compare test sensitivity for CRC, patients with screening identified colorectal cancer (n = 102) were enrolled and provided samples prior to surgery. To compare test specificity patients were enrolled prospectively (n = 199) and provided samples prior to bowel preparation for screening colonoscopy.
Plasma and fecal samples were analyzed using the Epi proColon and OC Fit-Check tests respectively.
For all samples, sensitivity for CRC detection was 73.3% (95% CI 63.9-80.9%) and 68.0% (95% CI 58.2-76.5%) for Septin9 and FIT, respectively. Specificity of the Epi proColon test was 81.5% (95% CI 75.5-86.3%) compared with 97.4% (95% CI 94.1-98.9%) for FIT. For paired samples, the sensitivity of the Epi proColon test (72.2% -95% CI 62.5-80.1%) was shown to be statistically non-inferior to FIT (68.0%-95% CI 58.2-76.5%). When test results for Epi proColon and FIT were combined, CRC detection was 88.7% at a specificity of 78.8%.
At a sensitivity of 72%, the Epi proColon test is non- inferior to FIT for CRC detection, although at a lower specificity. With negative predictive values of 99.8%, both methods are identical in confirming the absence of CRC.
ClinicalTrials.gov NCT01580540.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
OBJECTIVE Frontal ventriculostomy is one of the most frequent and standardized procedures in neurosurgery. However, many first and subsequent punctures miss the target, and suboptimal placement or ...misplacement of the catheter is common. The authors therefore reexamined the landmarks and rules to determine the entry point and trajectory with the best hit rate (HtR). METHODS The authors randomly selected CT scans from their institution's DICOM pool that had been obtained in 50 patients with normal ventricular and skull anatomy and without ventricular puncture. Using a 5 × 5-cm frontal grid with 25 entry points referenced to the bregma, the authors examined trajectories 1) perpendicular to the skull, 2) toward classic facial landmarks in the coronal and sagittal planes, and 3) toward an idealized target in the middle of the ipsilateral anterior horn (ILAH). Three-dimensional virtual reality ventriculostomies were simulated for these entry points; trajectories and the HtRs were recorded, resulting in an investigation of 8000 different virtual procedures. RESULTS The best HtR for the ILAH was 86% for an ideal trajectory, 84% for a landmark trajectory, and 83% for a 90° trajectory, but only at specific entry points. The highest HtRs were found for entry points 3 or 4 cm lateral to the midline, but only in combination with a trajectory toward the contralateral canthus; and 1 or 2 cm lateral to the midline, but only paired with a trajectory toward the nasion. The same "pairing" exists for entry points and trajectories in the sagittal plane. For perpendicular (90°) trajectories, the best entry points were at 3-5 cm lateral to the midline and 3 cm anterior to the bregma, or 4 cm lateral to the midline and 2 cm anterior to the bregma. CONCLUSIONS Only a few entry points offer a chance of a greater than 80% rate of hitting the ILAH, and then only in combination with a specific trajectory. This "pairing" between entry point and trajectory was found both for landmark targeting and for perpendicular trajectories, with very limited variability. Surprisingly, the ipsilateral medial canthus, a commonly reported landmark, had low HtRs, and should not be recommended as a trajectory target.
A defining feature of the brain is the ability of its synaptic contacts to adapt structurally and functionally in an experience-dependent manner. In the human cortex, however, direct experimental ...evidence for coordinated structural and functional synaptic adaptation is currently lacking. Here, we probed synaptic plasticity in human cortical slices using the vitamin A derivative all-trans retinoic acid (atRA), a putative treatment for neuropsychiatric disorders such as Alzheimer's disease. Our experiments demonstrated that the excitatory synapses of superficial (layer 2/3) pyramidal neurons underwent coordinated structural and functional changes in the presence of atRA. These synaptic adaptations were accompanied by ultrastructural remodeling of the calcium-storing spine apparatus organelle and required mRNA translation. It was not observed in synaptopodin-deficient mice, which lack spine apparatus organelles. We conclude that atRA is a potent mediator of synaptic plasticity in the adult human cortex.
Reproducible segmentation of brain tumors on magnetic resonance images is an important clinical need. This study was designed to evaluate the reliability of a novel fully automated segmentation tool ...for brain tumor image analysis in comparison to manually defined tumor segmentations.
We prospectively evaluated preoperative MR Images from 25 glioblastoma patients. Two independent expert raters performed manual segmentations. Automatic segmentations were performed using the Brain Tumor Image Analysis software (BraTumIA). In order to study the different tumor compartments, the complete tumor volume TV (enhancing part plus non-enhancing part plus necrotic core of the tumor), the TV+ (TV plus edema) and the contrast enhancing tumor volume CETV were identified. We quantified the overlap between manual and automated segmentation by calculation of diameter measurements as well as the Dice coefficients, the positive predictive values, sensitivity, relative volume error and absolute volume error.
Comparison of automated versus manual extraction of 2-dimensional diameter measurements showed no significant difference (p = 0.29). Comparison of automated versus manual segmentation of volumetric segmentations showed significant differences for TV+ and TV (p<0.05) but no significant differences for CETV (p>0.05) with regard to the Dice overlap coefficients. Spearman's rank correlation coefficients (ρ) of TV+, TV and CETV showed highly significant correlations between automatic and manual segmentations. Tumor localization did not influence the accuracy of segmentation.
In summary, we demonstrated that BraTumIA supports radiologists and clinicians by providing accurate measures of cross-sectional diameter-based tumor extensions. The automated volume measurements were comparable to manual tumor delineation for CETV tumor volumes, and outperformed inter-rater variability for overlap and sensitivity.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Objective
Spinal cerebrospinal fluid (CSF) leaks cause spontaneous intracranial hypotension (SIH). Microsurgery can sufficiently seal spinal CSF leaks. Yet, some patients suffer from residual ...symptoms. Aim of the study was to assess predictors for favorable outcome after surgical treatment of SIH.
Methods
We included consecutive patients with SIH treated surgically from January 2013 to May 2020. Subjects were surveyed by a questionnaire. Primary outcome was resolution of symptoms as rated by the patient. Secondary outcome was postoperative headache intensity on the numeric rating scale (NRS). Association between variables and outcome was assessed using univariate and multivariate regression. A cut-off value for continuous variables was calculated by a ROC analysis.
Results
Sixty-nine out of 86 patients (80.2%) returned the questionnaire and were analyzed. Mean age was 46.7 years and 68.1% were female. A significant association with the primary and secondary outcome was found only for preoperative symptom duration (
p
= 0.001 and
p
< 0.001), whereby a shorter symptom duration was associated with a better outcome. Symptom duration remained a significant predictor in a multivariate model (
p
= 0.013). Neither sex, age, type of pathology, lumbar opening pressure, nor initial presentation were associated with the primary outcome. ROC analysis yielded treatment within 12 weeks as a cut-off for better outcome.
Conclusion
Shorter duration of preoperative symptoms is the most powerful predictor of favorable outcome after surgical treatment of SIH. While an initial attempt of conservative treatment is justified, we advocate early definitive treatment within 12 weeks in case of persisting symptoms.
Dendritic spines are highly dynamic neuronal compartments that control the synaptic transmission between neurons. Spines form ultrastructural units, coupling synaptic contact sites to the dendritic ...shaft and often harbor a spine apparatus organelle, composed of smooth endoplasmic reticulum, which is responsible for calcium sequestration and release into the spine head and neck. The spine apparatus has recently been linked to synaptic plasticity in adult human cortical neurons. While the morphological heterogeneity of spines and their intracellular organization has been extensively demonstrated in animal models, the influence of spine apparatus organelles on critical signaling pathways, such as calcium-mediated dynamics, is less well known in human dendritic spines. In this study we used serial transmission electron microscopy to anatomically reconstruct nine human cortical spines in detail as a basis for modeling and simulation of the calcium dynamics between spine and dendrite. The anatomical study of reconstructed human dendritic spines revealed that the size of the postsynaptic density correlates with spine head volume and that the spine apparatus volume is proportional to the spine volume. Using a newly developed simulation pipeline, we have linked these findings to spine-to-dendrite calcium communication. While the absence of a spine apparatus, or the presence of a purely passive spine apparatus did not enable any of the reconstructed spines to relay a calcium signal to the dendritic shaft, the calcium-induced calcium release from this intracellular organelle allowed for finely tuned "all-or-nothing" spine-to-dendrite calcium coupling; controlled by spine morphology, neck plasticity, and ryanodine receptors. Our results suggest that spine apparatus organelles are strategically positioned in the neck of human dendritic spines and demonstrate their potential relevance to the maintenance and regulation of spine-to-dendrite calcium communication.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Hepadnaviruses, including hepatitis B virus (HBV) as a major human pathogen, replicate their tiny 3 kb DNA genomes by capsid-internal protein-primed reverse transcription of a pregenomic (pg) RNA. ...Initiation requires productive binding of the viral polymerase, P protein, to a 5´ proximal bipartite stem-loop, the RNA encapsidation signal ε. Then a residue in the central ε bulge directs the covalent linkage of a complementary dNMP to a Tyr sidechain in P protein´s Terminal Protein (TP) domain. After elongation by two or three nucleotides (nt) the TP-linked DNA oligo is transferred to a 3´ proximal acceptor, enabling full-length minus-strand DNA synthesis. No direct structural data are available on hepadnaviral initiation complexes but their cell-free reconstitution with P protein and ε RNA (Dε) from duck HBV (DHBV) provided crucial mechanistic insights, including on a major conformational rearrangement in the apical Dε part. Analogous cell-free systems for human HBV led at most to P-ε binding but no detectable priming. Here we demonstrate that local relaxation of the highly basepaired ε upper stem, by mutation or via synthetic split RNAs, enables ε-dependent in vitro priming with full-length P protein from eukaryotic translation extract yet also, and without additional macromolecules, with truncated HBV miniP proteins expressed in bacteria. Using selective 2-hydroxyl acylation analyzed by primer extension (SHAPE) we confirm that upper stem destabilization correlates with in vitro priming competence and show that the supposed bulge-closing basepairs are largely unpaired even in wild-type ε. We define the two 3´ proximal nt of this extended bulge as main initiation sites and provide evidence for a Dε-like opening of the apical ε part upon P protein binding. Beyond new HBV-specific basic aspects our novel in vitro priming systems should facilitate the development of high-throughput screens for priming inhibitors targeting this highly virus-specific process.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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