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Abstract Epilepsy is defined by the abrupt emergence of harmful seizures, but the nature of these regime shifts remains enigmatic. From the perspective of dynamical systems theory, such critical transitions occur upon inconspicuous perturbations in highly interconnected systems and can be modeled as mathematical bifurcations between alternative regimes. The predictability of critical transitions represents a major challenge, but the theory predicts the appearance of subtle dynamical signatures on the verge of instability. Whether such dynamical signatures can be measured before impending seizures remains uncertain. Here, we verified that predictions on bifurcations applied to the onset of hippocampal seizures, providing concordant results from in silico modeling, optogenetics experiments in male mice and intracranial EEG recordings in human patients with epilepsy. Leveraging pharmacological control over neural excitability, we showed that the boundary between physiological excitability and seizures can be inferred from dynamical signatures passively recorded or actively probed in hippocampal circuits. Of importance for the design of future neurotechnologies, active probing surpassed passive recording to decode underlying levels of neural excitability, notably when assessed from a network of propagating neural responses. Our findings provide a promising approach for predicting and preventing seizures, based on a sound understanding of their dynamics.

Abstract This study provides a detailed understanding of the preclinical pharmacokinetics and metabolism of ELP‐004, an osteoclast inhibitor in development for the treatment of bone erosion. Current treatments for arthritis, including biological disease‐modifying antirheumatic drugs, are not well‐tolerated in a substantial subset of arthritis patients and are expensive; therefore, new treatments are needed. Pharmacokinetic parameters of ELP‐004 were tested with intravenous, oral, and subcutaneous administration and found to be rapidly absorbed and distributed. We found that ELP‐004 was non‐mutagenic, did not induce chromosome aberrations, non‐cardiotoxic, and had minimal off‐target effects. Using in vitro hepatic systems, we found that ELP‐004 is primarily metabolized by CYP1A2 and CYP2B6 and predicted metabolic pathways were identified. Finally, we show that ELP‐004 inhibits osteoclast differentiation without suppressing overall T‐cell function. These preclinical data will inform future development of an oral compound as well as in vivo efficacy studies in mice.

Abstract Background Herpes simplex virus (HSV) encephalitis (HSE) is a serious and potentially life-threatening disease, affecting both adults and newborns. Progress in understanding the virus and host factors involved in neonatal HSE has been hampered by the limitations of current brain models that do not fully recapitulate the tissue structure and cell composition of the developing human brain in health and disease. Here, we developed a human fetal organotypic brain slice culture (hfOBSC) model and determined its value in mimicking the HSE neuropathology in vitro. Methods Cell viability and tissues integrity were determined by lactate dehydrogenase release in supernatant and immunohistological (IHC) analyses. Brain slices were infected with green fluorescent protein (GFP-) expressing HSV-1 and HSV-2. Virus replication and spread were determined by confocal microscopy, PCR and virus culture. Expression of pro-inflammatory cytokines and chemokines were detected by PCR. Cell tropism and HSV-induced neuropathology were determined by IHC analysis. Finally, the in situ data of HSV-infected hfOBSC were compared to the neuropathology detected in human HSE brain sections. Results Slicing and serum-free culture conditions were optimized to maintain the viability and tissue architecture of ex vivo human fetal brain slices for at least 14 days at 37 °C in a CO2 incubator. The hfOBSC supported productive HSV-1 and HSV-2 infection, involving predominantly infection of neurons and astrocytes, leading to expression of pro-inflammatory cytokines and chemokines. Both viruses induced programmed cell death—especially necroptosis—in infected brain slices at later time points after infection. The virus spread, cell tropism and role of programmed cell death in HSV-induced cell death resembled the neuropathology of HSE. Conclusions We developed a novel human brain culture model in which the viability of the major brain-resident cells—including neurons, microglia, astrocytes and oligodendrocytes—and the tissue architecture is maintained for at least 2 weeks in vitro under serum-free culture conditions. The close resemblance of cell tropism, spread and neurovirulence of HSV-1 and HSV-2 in the hfOBSC model with the neuropathological features of human HSE cases underscores its potential to detail the pathophysiology of other neurotropic viruses and as preclinical model to test novel therapeutic interventions.

Abstract Acetalated dextran (Ac-Dex) nanoparticles are currently of immense interest due to their sharp pH-responsive nature and high biodegradability. Ac-Dex nanoparticles are often formulated through single- or double-emulsion methods utilizing polyvinyl alcohol as the stabilizer. The emulsion methods utilize toxic organic solvents such as dichloromethane or chloroform and require multi-step processing to form stable Ac-Dex nanoparticles. Here, we introduce a simple flash nanoprecipitation (FNP) approach that utilizes a confined impinging jet mixer and a non-toxic solvent, ethanol, to form Ac-Dex nanoparticles rapidly. Ac-Dex nanoparticles were stabilized using nonionic PEGylated surfactants, D-α-Tocopherol polyethylene glycol succinate (TPGS), or Pluronic (F-127). Ac-Dex nanoparticles formed using FNP were highly monodisperse and stably encapsulated a wide range of payloads, including hydrophobic, hydrophilic, and macromolecules. When lyophilized, Ac-Dex TPGS nanoparticles remained stable for at least one year with greater than 80% payload retention. Ac-Dex nanoparticles were non-toxic to cells and achieved intracellular release of payloads into the cytoplasm. In vivo studies demonstrated a predominant biodistribution of Ac-Dex TPGS nanoparticles in the liver, lungs, and spleen after intravenous administration. Taken together, the FNP technique allows easy fabrication and loading of Ac-Dex nanoparticles that can precisely release payloads into intracellular environments for diverse therapeutic applications. Graphical abstract pH-responsive Acetalateddextran can be formulated using nonionic surfactants, such as TPGS or F-127, for intracellular release of payloads. Highly monodisperse and stable nanoparticles can be created through the simple, scalable flash nanoprecipitation technique, which utilizes a confined impingement jet mixer.

The aetiology of chronic aseptic meningitis is difficult to establish. Candida meningitis in particular is often diagnosed late, as cerebrospinal fluid (CSF) work-up and imaging findings are nonspecific. A 35-year-old patient with chronic aseptic meningitis, for which repeated microbiological testing of CSF was unrevealing, was finally diagnosed with Candida albicans (C. albicans) meningitis with cauda equina involvement using metagenomic next-generation sequencing (mNGS). This report highlights the diagnostic challenges and the difficulties of treating shunt-associated fungal meningitis.

The race towards decarbonization is driving major oil and gas companies to explore means to use renewable heat and power for their plants as part of their commitment to reduce their carbon intensity by 80% to 100% by 2050 [1]. In terms of greenhouse gas (GHG) emissions, natural gas is considered the cleanest fossil fuel option available, and decarbonization of new liquefied natural gas (LNG) projects is on the radar of many LNG projects developers. In addition, LNG cargos will have to be certified in the future by accredited authorities to meet defined GHG emission levels [2]. This research study investigates a concept of providing both heat and power from a photovoltaic (PV) and concentrated solar power (CSP) solar tower hybrid plant to meet the energy demand of LNG export terminals. Two locations have been investigated for potential future LNG projects: Karratha in Australia and Ras Laffan in Qatar. Both locations have direct normal irradiance (DNI) values higher than 2000 kWh/m2/year, which is the minimum level required for CSP technology [3]. A techno-economic assessment was carried-out taking into consideration the electricity price, the grid carbon intensity and the carbon dioxide (CO2) tax in the region. The results indicate that the CSP/PV hybrid plant significantly accelerates the decarbonization of energy supply to the “All electric” LNG Plant. The quantity of CO2 emitted between 2025 and 2050 is reduced by 81% for Karratha (Australia) and by 88% for Ras Laffan (Qatar) compared to a grid connected LNG plant.

Abstract Background Trigeminal ganglia (TG) neurons are an important site of lifelong latent varicella-zoster virus (VZV) infection. Although VZV-specific T-cells are considered pivotal to control virus reactivation, their protective role at the site of latency remains uncharacterized. Methods Paired blood and TG specimens were obtained from ten latent VZV-infected adults, of which nine were co-infected with herpes simplex virus type 1 (HSV-1). Short-term TG-derived T-cell lines (TG-TCL), generated by mitogenic stimulation of TG-derived T-cells, were probed for HSV-1- and VZV-specific T-cells using flow cytometry. We also performed VZV proteome-wide screening of TG-TCL to determine the fine antigenic specificity of VZV reactive T-cells. Finally, the relationship between T-cells and latent HSV-1 and VZV infections in TG was analyzed by reverse transcription quantitative PCR (RT-qPCR) and in situ analysis for T-cell proteins and latent viral transcripts. Results VZV proteome-wide analysis of ten TG-TCL identified two VZV antigens recognized by CD8 T-cells in two separate subjects. The first was an HSV-1/VZV cross-reactive CD8 T-cell epitope, whereas the second TG harbored CD8 T-cells reactive with VZV specifically and not the homologous peptide in HSV-1. In silico analysis showed that HSV-1/VZV cross reactivity of TG-derived CD8 T-cells reactive with ten previously identified HSV-1 epitopes was unlikely, suggesting that HSV-1/VZV cross-reactive T-cells are not a common feature in dually infected TG. Finally, no association was detected between T-cell infiltration and VZV latency transcript abundance in TG by RT-qPCR or in situ analyses. Conclusions The low presence of VZV- compared to HSV-1-specific CD8 T-cells in human TG suggests that VZV reactive CD8 T-cells play a limited role in maintaining VZV latency.

T cells are considered important for controlling skin infections. Here, the authors report that varicella-zoster virus-specific T cells preferentially persist as tissue-resident-memory T cells in rash-involved skin after recovery from zoster.

Abstract Background Trigeminal ganglia (TG) neurons are the main site of lifelong latent herpes simplex virus type 1 (HSV-1) infection. T-cells in ganglia contribute to long-term control of latent HSV-1 infection, but it is unclear whether these cells are bona fide tissue-resident memory T-cells (TRM). We optimized the processing of human post-mortem nervous tissue to accurately phenotype T-cells in human TG ex vivo and in situ. Methods Peripheral blood mononuclear cells (PBMC; 5 blood donors) were incubated with several commercial tissue digestion enzyme preparations to determine off-target effect on simultaneous detection of 15 specific T-cell subset markers by flow cytometry. Next, optimized enzymatic digestion was applied to ex vivo phenotype T-cells in paired PBMC, normal appearing white matter (NAWM) and TG of 8 deceased brain donors obtained

Abstract The interphotoreceptor matrix (IPM) is a specialized extracellular mesh of molecules surrounding the inner and outer segments of photoreceptor neurons. Interphotoreceptor matrix proteoglycan 1 and 2 (IMPG1 and IMPG2) are major components of the IPM. Both proteoglycans possess SEA (sperm protein, enterokinase and agrin) domains, which may support proteolysis. Interestingly, mutations in the SEA domains of IMPG1 and IMPG2 are associated with vision disease in humans. However, if SEA domains in IMPG molecules undergo proteolysis, and how this contributes to vision pathology is unknown. Therefore, we investigated SEA-mediated proteolysis of IMPG1 and IMPG2 and its significance to IPM physiology. Immunoblot analysis confirmed proteolysis of IMPG1 and IMPG2 in the retinas of wildtype mice. Point mutations mimicking human mutations in the SEA domain of IMPG1 that are associated with vision disease inhibited proteolysis. These findings demonstrate that proteolysis is part of the maturation of IMPG1 and IMPG2, in which deficits are associated with vision diseases. Further, immunohistochemical assays showed that proteolysis of IMPG2 generated two subunits, a membrane-attached peptide and an extracellular peptide. Notably, the extracellular portion of IMPG2 trafficked from the IPM around the inner segment toward the outer segment IPM by an IMPG1-dependent mechanism. This result provides the first evidence of a trafficking system that shuttles IMPG1 and IMPG2 from the inner to outer IPM in a co-dependent manner. In addition, these results suggest an interaction between IMPG1–IMPG2 and propose that mutations affecting one IMPG could affect the localization of the normal IMPG partner, contributing to the disease mechanism of vision diseases associated with defective IMPG molecules.

Abstract Background Fatigue is common in patents with breast cancer (BC), and can occur in patients with early stage disease and in the absence of muscle wasting (i.e. cachexia). We have reported transcriptional and proteomic alterations in muscles from BC patients, which are associated with fatigue. Mice implanted with human BC xenografts recapitulate the muscle molecular composition changes seen in patients, coupled with a greater rate of contraction‐induced fatigue. Multiple bioinformatics platforms in both human and mouse muscles have identified peroxisome proliferator activated receptor gamma (PPARG) as central to this phenotype, with several PPARG target genes downregulated in muscle in response to tumour growth. The current study tested the hypothesis that the PPARG agonist pioglitazone (pio), a commonly prescribed diabetes drug, would rescue the transcriptional alterations observed in muscles of tumour‐bearing mice. Methods Sixteen female NSG mice were implanted with breast cancer patient‐derived orthotopic xenografts (BC‐PDOX) via transplantation of Her2/neu+ human tumour fragments. BC‐PDOX mice were randomly assigned to a treatment group that received daily oral pio at 30 mg/kg (n = 8), or a control group that received a similar volume of vehicle (n = 8). Treatment was initiated when tumours reached a volume of 600 mm3, and lasted for 2 weeks. Hindlimb muscles were isolated from BC‐PDOX and non‐tumour bearing mice for RNA‐sequencing, gene expression validation, and ATP quantification. Differentially expressed genes (DEGs) in muscles from BC‐PDOX mice relative to non‐tumour bearing controls were identified using DESeq2, and multiple bioinformatics platforms were employed to contextualize the DEGs. Results We found that the administration of pio restored the muscle gene expression patterns of BC‐PDOX mice to a profile resembling muscles of non‐tumour bearing NSG control mice. Validation of skeletal muscle gene expression by qPCR confirmed pio increased the expression of PPARG target genes (P

Xanthine oxidase (XO) catalyzes the catabolism of hypoxanthine to xanthine and xanthine to uric acid, generating oxidants as a byproduct. Importantly, XO activity is elevated in numerous hemolytic conditions including sickle cell disease (SCD); however, the role of XO in this context has not been elucidated. Whereas long-standing dogma suggests elevated levels of XO in the vascular compartment contribute to vascular pathology via increased oxidant production, herein, we demonstrate, for the first time, that XO has an unexpected protective role during hemolysis. Using an established hemolysis model, we found that intravascular hemin challenge (40 μmol/kg) resulted in a significant increase in hemolysis and an immense (20-fold) elevation in plasma XO activity in Townes sickle cell phenotype (SS) sickle mice compared to controls. Repeating the hemin challenge model in hepatocyte-specific XO knockout mice transplanted with SS bone marrow confirmed the liver as the source of enhanced circulating XO as these mice demonstrated 100% lethality compared to 40% survival in controls. In addition, studies in murine hepatocytes (AML12) revealed hemin mediates upregulation and release of XO to the medium in a toll like receptor 4 (TLR4)-dependent manner. Furthermore, we demonstrate that XO degrades oxyhemoglobin and releases free hemin and iron in a hydrogen peroxide-dependent manner. Additional biochemical studies revealed purified XO binds free hemin to diminish the potential for deleterious hemin-related redox reactions as well as prevents platelet aggregation. In the aggregate, data herein reveals that intravascular hemin challenge induces XO release by hepatocytes through hemin-TLR4 signaling, resulting in an immense elevation of circulating XO. This increased XO activity in the vascular compartment mediates protection from intravascular hemin crisis by binding and potentially degrading hemin at the apical surface of the endothelium where XO is known to be bound and sequestered by endothelial glycosaminoglycans (GAGs).

Abstract Background The COVID‐19 pandemic has greatly increased the incidence and clinical importance of critical illness myopathy (CIM), because it is one of the most common complications of modern intensive care medicine. Current diagnostic criteria only allow diagnosis of CIM at an advanced stage, so that patients are at risk of being overlooked, especially in early stages. To determine the frequency of CIM and to assess a recently proposed tool for early diagnosis, we have followed a cohort of COVID‐19 patients with acute respiratory distress syndrome and compared the time course of muscle excitability measurements with the definite diagnosis of CIM. Methods Adult COVID‐19 patients admitted to the Intensive Care Unit of the University Hospital Bern, Switzerland requiring mechanical ventilation were recruited and examined on Days 1, 2, 5, and 10 post‐intubation. Clinical examination, muscle excitability measurements, medication record, and laboratory analyses were performed on all study visits, and additionally nerve conduction studies, electromyography and muscle biopsy on Day 10. Muscle excitability data were compared with a cohort of 31 age‐matched healthy subjects. Diagnosis of definite CIM was made according to the current guidelines and was based on patient history, results of clinical and electrophysiological examinations as well as muscle biopsy. Results Complete data were available in 31 out of 44 recruited patients (mean [SD] age, 62.4 [9.8] years). Of these, 17 (55%) developed CIM. Muscle excitability measurements on Day 10 discriminated between patients who developed CIM and those who did not, with a diagnostic precision of 90% (AUC 0.908; 95% CI 0.799–1.000; sensitivity 1.000; specificity 0.714). On Days 1 and 2, muscle excitability parameters also discriminated between the two groups with 73% (AUC 0.734; 95% CI 0.550–0.919; sensitivity 0.562; specificity 0.857) and 82% (AUC 0.820; CI 0.652–0.903; sensitivity 0.750; specificity 0.923) diagnostic precision, respectively. All critically ill COVID‐19 patients showed signs of muscle membrane depolarization compared with healthy subjects, but in patients who developed CIM muscle membrane depolarization on Days 1, 2 and 10 was more pronounced than in patients who did not develop CIM. Conclusions This study reports a 55% prevalence of definite CIM in critically ill COVID‐19 patients. Furthermore, the results confirm that muscle excitability measurements may serve as an alternative method for CIM diagnosis and support its use as a tool for early diagnosis and monitoring the development of CIM.

The race towards decarbonization is driving major oil and gas companies to explore means to use renewable heat and power for their plants as part of their commitment to reduce their carbon intensity by 80% to 100% by 2050 (Holbrook, 2023). In terms of GHG (Greenhouse Gas) emissions, natural gas is considered the cleanest fossil fuel option available, and decarbonization of new LNG (Liquefied Natural Gas) projects is on the radar of many LNG projects developers. In addition, LNG cargos will have to be certified in the future by accredited authorities to meet defined GHG emission levels (Stern, 2019).This research study investigates a new concept of providing both heat and power from a PV (Photovoltaic) and CSP (Concentrated Solar Power) hybrid plant to meet the energy demand of an LNG export terminal. Two locations have been investigated for potential future LNG projects: Karratha in Australia and Ras Laffan in Qatar. Both locations have DNI values higher than 2000 kWh/m2/year, which is the minimum level required for CSP technology (Lovegrove and Stein, 2021).The LCCA (Levelized Cost of CO2 abatement) was used to compare the various solar configurations to select the option that will bring the greatest amount of CO2 reduction for the same investment cost (CGEP, 2020). The solar configurations technical parameters were defined based on the surface land available, natural gas feed composition and LNG plant design characteristics. The techno-economic assessment was carried-out taking into consideration the electricity price, the grid carbon intensity, and the CO2 tax in the region.Whilst in Australia a collocated single plant is favourable in terms of land availability and DNI (Direct Normal Irradiation) level, in Qatar, heat and power production have been separated into two distinct locations. Next to the LNG plant, a CSP plant provides heat only, whereas 130 km away, the hybrid PV/CSP plant located at Qurain EL Bawl provides electric power.The results indicate that the PV/CSP hybrid plant significantly accelerates the decarbonization of energy supply to the “All electric” LNG Plant. Depending on the solar field size, the quantity of CO2 emitted between 2025 and 2050 is reduced by 78–81% for Karratha (Australia) and by 82–88% for Ras Laffan (Qatar) compared to a grid connected plant.In addition, the economic assessment indicated that buying electricity from the grid during the period 2025–2050 would be more expensive for Australia then investing in a PV/CSP plant. In Qatar, the LCOE range of 93–110 US$/MWh is not competitive with the price of electricity in the region Qatar, mainly because of energy subsidies, however both the carbon tax and electricity price will need to be raised to the same level as in Australia to diversify and expand the power sector in the region.

Introduction: Decompressive hemicraniectomy (DCE) is routinely performed for intracranial pressure control after malignant middle cerebral artery (MCA) infarction. Decompressed patients are at risk of traumatic brain injury and the syndrome of the trephined until cranioplasty. Cranioplasty after DCE is itself associated with high complication rates. Single-stage surgical strategies may eliminate the need for follow-up surgery while allowing for safe brain expansion and protection from environmental factors. Research question: Assess the volume needed for safe expansion of the brain to enable single-stage surgery. Materials and methods: We performed a retrospective radiological and volumetric analysis of all patients that had DCE in our clinic between January 2009 and December 2018 and met inclusion criteria. We investigated prognostic parameters in perioperative imaging and assessed clinical outcome. Results: Of 86 patients with DCE, 44 fulfilled the inclusion criteria. Median brain swelling was 75.35 mL (8.7–151.2 mL). Median bone flap volume was 113.3 mL (73.34–146.1 mL). Median brain swelling was 1.62 mm below the previous outer rim of the skull (5.3 mm to –2.19 mm). In 79.6% of the patients, the volume of removed bone alone was equivalent to or larger than the additional intracranial volume needed for brain swelling. Discussion and conclusion: The space provided by removal of the bone alone was sufficient to match the expansion of the injured brain after malignant MCA infarction in the vast majority of our patientsA subgaleal space-expanding flap with a minimal offset can provide protection from trauma and atmospheric pressure without compromising brain expansion.

CISD-1/mitoNEET is an evolutionarily conserved outer mitochondrial membrane [2Fe-2S] protein that regulates mitochondrial function and morphology. The [2Fe-2S] clusters are redox reactive and shown to mediate oxidative stress in vitro and in vivo. However, there is limited research studying CISD-1/mitoNEET mediation of oxidative stress in response to environmental stressors. In this study, we have determined the X-ray crystal structure of Caenorhabditis elegans CISD-1/mitoNEET homologue and evaluated the mechanisms of oxidative stress resistance to the pro-oxidant paraquat in age-synchronized populations by generating C. elegans gain and loss of function CISD-1 models. The structure of the C. elegans CISD-1/mitoNEET soluble domain refined at 1.70-Å resolution uniquely shows a reversible disulfide linkage at the homo-dimeric interface and also represents the N-terminal tail domain for dimerization of the cognate kinesin motor protein KLP-17 involved in chromosome segregation dynamics and germline development of the nematode. Moreover, overexpression of CISD-1/mitoNEET in C. elegans has revealed beneficial effects on oxidative stress resistance against paraquat-induced reactive oxygen species generation, corroborated by increased activation of the p38 mitogen-activated protein kinase (MAPK) signaling cascade.

This overview of reviews (i.e., an umbrella review) is designed to reappraise the validity of systematic reviews (SRs) and meta-analyses related to the performance of Aspergillus PCR tests for the diagnosis of invasive aspergillosis in immunocompromised patients. The methodological quality of the SRs was assessed using the AMSTAR-2 checklist; the quality of the evidence (QOE) within each SR was appraised following the GRADE approach. Eight out of 12 SRs were evaluated for qualitative and quantitative assessment. Five SRs evaluated Aspergillus PCR on bronchoalveolar lavage fluid (BAL) and three on blood specimens. The eight SRs included 167 overlapping reports (59 evaluating PCR in blood specimens, and 108 in BAL), based on 107 individual primary studies (98 trials with a cohort design, and 19 with a case−control design). In BAL specimens, the mean sensitivity and specificity ranged from 0.57 to 0.91, and from 0.92 to 0.97, respectively (QOE: very low to low). In blood specimens (whole blood or serum), the mean sensitivity ranged from 0.57 to 0.84, and the mean specificity from 0.58 to 0.95 (QOE: low to moderate). Across studies, only a low proportion of AMSTAR-2 critical domains were unmet (1.8%), demonstrating a high quality of methodological assessment. Conclusions. Based on the overall methodological assessment of the reviews included, on average we can have high confidence in the quality of results generated by the SRs.

Abstract We measured plasma and cerebrospinal fluid (CSF) metabolite concentrations in a 5-day porcine sepsis model of fecal peritonitis. The objectives were: (i) to verify whether the expected pathways that had emerged in previous studies pertain only to the early inflammatory response or persist for the subsequent days; (ii) to identify metabolic derangements that arise later; (iii) to verify whether CSF metabolite concentrations were altered and if these alterations were similar to those in the blood or delayed. We observed an early response to inflammation and cytokine storms with alterations in lipid and glucose metabolism. The arginine/asymmetric dimethylarginine (ADMA) and phenylalanine/tyrosine balances changed 24 h after resuscitation in plasma, and later in CSF. There was a rise in ammonia concentration, with altered concentrations of metabolites in the urea cycle. Whether persistent derangement of these pathways have a role not only on short-term outcomes but also on longer-term comorbidities, such as septic encephalopathy, should be addressed in further studies.

Abstract B-cell acute lymphoblastic leukemia (ALL) is characterized by accumulation of immature hematopoietic cells in the bone marrow, a well-established sanctuary site for leukemic cell survival during treatment. While standard of care treatment results in remission in most patients, a small population of patients will relapse, due to the presence of minimal residual disease (MRD) consisting of dormant, chemotherapy-resistant tumor cells. To interrogate this clinically relevant population of treatment refractory cells, we developed an in vitro cell model in which human ALL cells are grown in co-culture with human derived bone marrow stromal cells or osteoblasts. Within this co-culture, tumor cells are found in suspension, lightly attached to the top of the adherent cells, or buried under the adherent cells in a population that is phase dim (PD) by light microscopy. PD cells are dormant and chemotherapy-resistant, consistent with the population of cells that underlies MRD. In the current study, we characterized the transcriptional signature of PD cells by RNA-Seq, and these data were compared to a published expression data set derived from human MRD B-cell ALL patients. Our comparative analyses revealed that the PD cell population is markedly similar to the MRD expression patterns from the primary cells isolated from patients. We further identified genes and key signaling pathways that are common between the PD tumor cells from co-culture and patient derived MRD cells as potential therapeutic targets for future studies.

BackgroundBrain fog is a common and highly disturbing symptom for patients with neuropathic postural tachycardia syndrome (POTS). Cognitive deficits have been measured exclusively in the upright body position and mainly comprised impairments of higher cognitive functions. The cause of brain fog is still unclear today. This study aimed to investigate whether increased autonomic activation might be an underlying mechanism for the occurrence of brain fog in neuropathic POTS. We therefore investigated cognitive function in patients with neuropathic POTS and a healthy control group depending on body position and in relation to catecholamine release as a sensitive indicator of acute stress. The second aim was to test the effect of water intake on cardiovascular regulation, orthostatic symptoms, cognitive function and catecholamine release.MethodsThirteen patients with neuropathic POTS and 15 healthy control subjects were included. All participants completed a total of four rounds of cognitive testing: two before and two after the intake of 500 ml still water, each first in the supine position and then during head-up tilt. At the end of each cognitive test, a blood sample was collected for determination of plasma catecholamines. After each head-up tilt phase participants were asked to rate their current symptoms on a visual analogue scale.ResultsWorking memory performance in the upright body position was impaired in patients, which was associated with self-reported symptom severity. Patients had elevated plasma norepinephrine independent of body position and water intake that increased excessively in the upright body position. The excessive increase of plasma norepinephrine was related to heart rate and symptom severity. Water intake in patients decreased norepinephrine concentrations and heart rate, and improved symptoms as well as cognitive performance.ConclusionBrain fog and symptom severity in neuropathic POTS are paralleled by an excessive norepinephrine secretion. Bolus water drinking down-regulates norepinephrine secretion and improves general symptom severity including brain fog.

Abstract Background Early prognostication in patients with acute consciousness impairment is a challenging but essential task. Current prognostic guidelines vary with the underlying etiology. In particular, electroencephalography (EEG) is the most important paraclinical examination tool in patients with hypoxic ischemic encephalopathy (HIE), whereas it is not routinely used for outcome prediction in patients with traumatic brain injury (TBI). Method Data from 364 critically ill patients with acute consciousness impairment (GCS ≤ 11 or FOUR ≤ 12) of various etiologies and without recent signs of seizures from a prospective randomized trial were retrospectively analyzed. Random forest classifiers were trained using 8 visual EEG features—first alone, then in combination with clinical features—to predict survival at 6 months or favorable functional outcome (defined as cerebral performance category 1–2). Results The area under the ROC curve was 0.812 for predicting survival and 0.790 for predicting favorable outcome using EEG features. Adding clinical features did not improve the overall performance of the classifier (for survival: AUC = 0.806, p = 0.926; for favorable outcome: AUC = 0.777, p = 0.844). Survival could be predicted in all etiology groups: the AUC was 0.958 for patients with HIE, 0.955 for patients with TBI and other neurosurgical diagnoses, 0.697 for patients with metabolic, inflammatory or infectious causes for consciousness impairment and 0.695 for patients with stroke. Training the classifier separately on subgroups of patients with a given etiology (and thus using less training data) leads to poorer classification performance. Conclusions While prognostication was best for patients with HIE and TBI, our study demonstrates that similar EEG criteria can be used in patients with various causes of consciousness impairment, and that the size of the training set is more important than homogeneity of ACI etiology.

Varicella-zoster virus (VZV) establishes lifelong neuronal latency in humans. Here, Ouwendijk and Depledge et al. identify a fusion transcript, VLT-ORF63, which is expressed during lytic and latent infection, and demonstrate a role for the translated fusion protein in induction of lytic gene expression from latent VZV genomes.

Background: Postural tachycardia syndrome (POTS) is a form of autonomic dysregulation. There is increasing evidence that the etiology may be immune-mediated in a subgroup of patients. Patients with POTS often experience an exacerbation of their symptoms associated with (viral) infections and often fear the same symptom aggravation after vaccination. In this report we describe the tolerability of messenger ribonucleic acid (mRNA) vaccines against coronavirus disease 19 (COVID-19) and the consequences of a COVID-19 infection on POTS symptoms in our cohort of patients with neuropathic POTS. Methods: We conducted a standardized, checklist-based interview with 23 patients and recorded the acute side effects of mRNA vaccination, acute symptoms of COVID-19 infection as well as the effects of vaccination and COVID-19 infection on POTS symptoms. Results: Of all included patients, 20 patients received two mRNA vaccines without having had a previous COVID-19 infection, and five patients in total had suffered a COVID-19 infection. Of these, three had COVID-19 without and two after being vaccinated. No increased frequency of side effects after both doses of mRNA vaccines was observed. Six patients reported a mild and short-term aggravation of their POTS symptoms beyond the duration of acute vaccine side effects. All five patients who suffered a COVID-19 infection subsequently reported a pronounced and persistent exacerbation of POTS symptoms. Conclusions: Our observations suggest that mRNA vaccines are not associated with a higher frequency of acute side effects in patients with POTS. Symptom exacerbation as a consequence of mRNA vaccination seems to be less frequent and of shorter duration compared to patients who suffered a COVID-19 infection.

BackgroundWe demonstrated experimentally that inhaled nitric oxide (iNO) dilates hypoperfused arterioles, increases tissue perfusion, and improves neurological outcome following subarachnoid hemorrhage (SAH) in mice. We performed a prospective pilot study to evaluate iNO in patients with delayed cerebral ischemia after SAH.MethodsSAH patients with delayed cerebral ischemia and hypoperfusion despite conservative treatment were included. iNO was administered at a maximum dose of 40 ppm. The response to iNO was considered positive if: cerebral artery diameter increased by 10% in digital subtraction angiography (DSA), or tissue oxygen partial pressure (PtiO2) increased by > 5 mmHg, or transcranial doppler (TCD) values decreased more than 30 cm/sec, or mean transit time (MTT) decreased below 6.5 secs in CT perfusion (CTP). Patient outcome was assessed at 6 months with the modified Rankin Scale (mRS).ResultsSeven patients were enrolled between February 2013 and September 2016. Median duration of iNO administration was 23 h. The primary endpoint was reached in all patients (five out of 17 DSA examinations, 19 out of 29 PtiO2 time points, nine out of 26 TCD examinations, three out of five CTP examinations). No adverse events necessitating the cessation of iNO were observed. At 6 months, three patients presented with a mRS score of 0, one patient each with an mRS score of 2 and 3, and two patients had died.ConclusionAdministration of iNO in SAH patients is safe. These results call for a larger prospective evaluation.

Drug hypersensitivity (DH) reactions are clinically unusual because the underlying immune stimulations are not antigen-driven, but due to non-covalent drug-protein binding. The drugs may bind to immune receptors like HLA or TCR which elicits a strong T cell reaction (p-i concept), the binding may enhance the affinity of antibodies (enhanced affinity model), or drug binding may occur on soluble proteins which imitate a true antigen (fake antigen model). These novel models of DH could have a major impact on how to perform risk assessments in drug development. Herein, we discuss the difficulties of detecting such non-covalent, labile and reversible, but immunologically relevant drug-protein interactions early on in drug development. The enormous diversity of the immune system, varying interactions, and heterogeneous functional consequences make it to a challenging task. We propose that a realistic approach to detect clinically relevant non-covalent drug interactions for a new drug could be based on a combination of in vitro cell culture assays (using a panel of HLA typed donor cells) and functional analyses, supplemented by structural analysis (computational data) of the reactive cells/molecules. When drug-reactive cells/molecules with functional impact are detected in these risk assessments, a close clinical monitoring of the drug may reveal the true incidence of DH, as suppressing but also enhancing factors occurring in vivo can influence the clinical manifestation of a DH.

The evacuation of a chronic subdural hematoma (cSDH) is one of the most common procedures in neurosurgery. The aim of this study was to assess the influence of drainage suction in the surgical treatment of cSDH on the recurrence rate. Post hoc analysis was conducted on two randomized controlled trials (cSDH-Drain-Trial and TOSCAN trial) stratifying a total of 581 patients into active or passive drain type. Of the 581 patients, 359 (61.8%) and 220 (37.9%) were stratified into the active and passive drainage groups, respectively. The reoperation rate following postoperative recurrence was 23.1% and 14.1% in the active and passive drainage groups, respectively (p < 0.011). After propensity score matching, the differences in recurrence rate remained significant (26.6% versus 15.6%, p = 0.012). However, the functional outcome (mRS) at 6–12 months did not differ significantly (median [IQR]) between the 2 groups (passive drainage group 0.00 [0.00, 2.00], active drainage group 1.00 [0.00, 2.00], p = 0.431). Mortality was comparable between the groups (passive drainage group 12 (5.5%), active drainage group 20 (5.6%), p = 0.968). In the univariate analysis, active drainage, short (

The proteins glutamate dehydrogenase (GDH) and mitoNEET are both targets of drug development efforts to treat metabolic disorders, cancer, and neurodegenerative diseases. However, these two proteins differ starkly in the current knowledge about ligand binding sites. MitoNEET is a [2Fe-2S]-containing protein with no obvious binding site for small ligands observed in its crystal structures. In contrast, GDH is known to have a variety of ligands at multiple allosteric sites thereby leading to complex regulation in activity. In fact, while GDH can utilize either NAD(H) or NADP(H) for catalysis at the active site, only NAD(H) binds at a regulatory site to inhibit GDH activity. Previously, we found that mitoNEET forms a covalent bond with GDH in vitro and increases the catalytic activity of the enzyme. In this study we evaluated the effects of mitoNEET binding on the allosteric control of GDH conferred by inhibitors. We examined all effectors using NAD or NADP as the coenzyme to determine allosteric linkage by the NAD-binding regulatory site. We found that GDH activity, in the presence of the inhibitory palmitoyl-CoA and EGCG, can be rescued by mitoNEET, regardless of the coenzyme used. This suggests that mitoNEET rescues GDH by stabilizing the open conformation.

The SARS-CoV-2 pandemic has claimed many lives and disrupted the quality of life of most individuals. Diagnostic tests not only serve to confirm past exposure but can provide information crucial for guiding healthcare options for patients. Current diagnostic tests for the presence of the SARS-CoV-2 virus or anti-spike protein antibodies do not address the question whether longer lasting cellular immunity is mounted in most individuals. Using an activation marker flow cytometric immunoassay (SARS-CoV-2 lymphocytes analysis), we showed that both CD4+/CD8+ T cell and B cell activation differ between naïve and infected individuals up to 11 months after infection. On the basis of the specificity of this diagnostic tool for detecting both SARS-CoV-2-experienced T and B cells, we propose that this assay could benefit immunocompromised individuals who are unable to mount sustained antibody responses, by determining cellular immunity as possible partial protection, and for studying immune correlates of protection – thereby increasing knowledge of COVID-19 in a wider range of patient groups.

Background: Delayed cerebral ischemia and cerebral vasospasm remain the leading causes of poor outcome in survivors of aneurysmal subarachnoid hemorrhage. Refractory cerebral vasospasms can be treated with endovascular vasodilator therapy, which can either be performed in conscious sedation or general anesthesia. The aim of this study is to compare the effect of the anesthesia modality on long-term clinical outcomes in patients undergoing endovascular vasodilator therapy due to cerebral vasospasm and hypoperfusion. Methods: Modified Rankin Scale (mRS) scores were retrospectively analyzed at time of discharge from the hospital and six months after aneurysmal subarachnoid hemorrhage. Additionally, National Institutes of Health Stroke Scale (NIHSS) was assessed 24 hours before, immediately before, immediately after, and 24 hours after endovascular vasodilator therapy, and at discharge and six months. Interventional parameters such as duration of intervention, choice and dosage of vasodilator and number of arteries treated were also recorded. Results: A total of 98 patients were included in this analysis and separated into patients who had interventions in conscious sedation, general anesthesia and a mix of both. Neither mRS at discharge nor at six months showed a significant difference for functionally independent outcomes (mRS 0-2) between groups. NIHSS before endovascular vasodilator therapy was significantly higher in patients receiving interventions in general anesthesia but did not differ anymore between groups six months after the initial bleed. Conclusion: This study did not observe a difference in outcome whether patients underwent endovascular vasodilator therapy in general anesthesia or conscious sedation for refractory cerebral vasospasms. Hence, the choice should be made for each patient individually.

Tellurium has two-dimensional phases and some Weyl nodes around its Femi level that make it a suitable candidate for the study and application in nonlinear optics. Here the authors show the synthesis and use of bio-grown optical Te(0) nanoparticles for optical modulation and thermo-optic switching.

Objectives: Novel formulations (gastro-resistant tablet and intravenous solution) of posaconazole (POS) have been approved in prophylaxis and therapy of invasive fungal diseases (IFDs). Study aim was to analyze treatment strategies and clinical effectiveness. Methods: We set up a web-based registry on www.ClinicalSurveys.net for documentation of comprehensive data of patients who received novel POS formulations. Data analysis was split into two groups of patients who received novel POS formulations for antifungal prophylaxis (posaconazole prophylaxis group) and antifungal therapy (posaconazole therapy group), respectively. Results: Overall, 180 patients (151 in the posaconazole prophylaxis group and 29 in the posaconazole therapy group) from six German tertiary care centers and hospitalized between 05/2014 – 03/2016 were observed. Median age was 58 years (range: 19 – 77 years) and the most common risk factor for IFD was chemotherapy (n = 136; 76%). In the posaconazole prophylaxis group and posaconazole therapy group, median POS serum levels at steady-state were 1,068 μg/L (IQR 573–1,498 μg/L) and 904 μg/L (IQR 728–1,550 μg/L), respectively (P = 0.776). During antifungal prophylaxis with POS, nine (6%) probable/proven fungal breakthroughs were reported and overall survival rate of hospitalization was 86%. The median overall duration of POS therapy was 18 days (IQR: 7 – 23 days). Fourteen patients (48%) had progressive IFD under POS therapy, of these five patients (36%) died related to or likely related to IFD. Conclusions: Our study demonstrates clinical effectiveness of antifungal prophylaxis with novel POS formulations. In patients treated for possible/probable/proven IFD, we observed considerable mortality in patients receiving salvage treatment and with infections due to rare fungal species. Keywords: Invasive fungal infection, Neutropenia, Posaconazole serum level, Clinical effectiveness, High-risk patient

Endometriosis is a common gynecological disorder seen in women and is characterized by chronic pelvic pain and infertility. This disorder is becoming more prevalent with increased morbidity. The etiology of endometriosis remains to be fully elucidated, which will lead to improved therapeutic options. In this review, we will evaluate the biochemical mechanisms leading to oxidative stress and their implication in the pathophysiology of endometriosis, as well as potential treatments that target these processes. A comprehensive exploration of previous research revealed that endometriosis is associated with elevated reactive oxygen species and oxidation products, decreased antioxidants and detoxification enzymes, and dysregulated iron metabolism. High levels of oxidative stress contributed to inflammation, extracellular matrix degradation, angiogenesis, and cell proliferation, which may explain its role in endometriosis. Endometriosis-associated pain was attributed to neurogenic inflammation and a feed-forward mechanism involving macrophages, pro-inflammatory cytokines, and pain-inducing prostaglandins. N-acetylcysteine, curcumin, melatonin, and combined vitamin C and E supplementation displayed promising results for the treatment of endometriosis, but further research is needed for their use in this population.

Cholesterol biosynthesis is a multi-step process involving several subcellular compartments, including peroxisomes. Cells adjust their sterol content by both transcriptional and post-transcriptional feedback regulation, for which sterol regulatory element-binding proteins (SREBPs) are essential; such homeostasis is dysregulated in peroxisome-deficient Pex2 knockout mice. Here, we compared the regulation of cholesterol biosynthesis in Chinese hamster ovary (CHO-K1) cells and in three isogenic peroxisome-deficient CHO cell lines harboring Pex2 gene mutations. Peroxisome deficiency activated expression of cholesterogenic genes, however, cholesterol levels were unchanged. 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) protein levels were increased in mutant cells, whereas HMGCR activity was significantly decreased, resulting in reduced cholesterol synthesis. U18666A, an inhibitor of lysosomal cholesterol export, induced cholesterol biosynthetic enzymes; yet, cholesterol synthesis was still reduced. Interestingly, peroxisome deficiency promoted ER-to-Golgi SREBP cleavage-activating protein (SCAP) trafficking even when cells were cholesterol-loaded. Restoration of functional peroxisomes normalized regulation of cholesterol synthesis and SCAP trafficking. These results highlight the importance of functional peroxisomes for maintaining cholesterol homeostasis and efficient cholesterol synthesis.

ABSTRACT Varicella-zoster virus (VZV), a double-stranded DNA virus, causes varicella, establishes lifelong latency in ganglionic neurons, and reactivates later in life to cause herpes zoster, commonly associated with chronic pain. The VZV genome is densely packed and produces multitudes of overlapping transcripts deriving from both strands. While 71 distinct open reading frames (ORFs) have thus far been experimentally defined, the full coding potential of VZV remains unknown. Here, we integrated multiple short-read RNA sequencing approaches with long-read direct RNA sequencing on RNA isolated from VZV-infected cells to provide a comprehensive reannotation of the lytic VZV transcriptome architecture. Through precise mapping of transcription start sites, splice junctions, and polyadenylation sites, we identified 136 distinct polyadenylated VZV RNAs that encode canonical ORFs, noncanonical ORFs, and ORF fusions, as well as putative noncoding RNAs (ncRNAs). Furthermore, we determined the kinetic class of all VZV transcripts and observed, unexpectedly, that transcripts encoding the ORF62 protein, previously designated Immediate-Early, were expressed with Late kinetics. Our work showcases the complexity of the VZV transcriptome and provides a comprehensive resource that will facilitate future functional studies of coding RNAs, ncRNAs, and the biological mechanisms underlying the regulation of viral transcription and translation during lytic VZV infection. IMPORTANCE Transcription from herpesviral genomes, executed by the host RNA polymerase II and regulated by viral proteins, results in coordinated viral gene expression to efficiently produce infectious progeny. However, the complete coding potential and regulation of viral gene expression remain ill-defined for the human alphaherpesvirus varicella-zoster virus (VZV), causative agent of both varicella and herpes zoster. Here, we present a comprehensive overview of the VZV transcriptome and the kinetic class of all identified viral transcripts, using two virus strains and two biologically relevant cell types. Additionally, our data provide an overview of how VZV diversifies its transcription from one of the smallest herpesviral genomes. Unexpectedly, the transcript encoding the major viral transactivator protein (pORF62) was expressed with Late kinetics, whereas orthologous transcripts in other alphaherpesviruses are typically expressed during the immediate early phase. Therefore, our work both establishes the architecture of the VZV transcriptome and provides insight into regulation of alphaherpesvirus gene expression.

In mammals, peroxisomes perform crucial functions in cellular metabolism, signaling and viral defense which are essential to the viability of the organism. Molecular cues triggered by changes in the cellular environment induce a dynamic response in peroxisomes, which manifests itself as a change in peroxisome number, altered enzyme levels and adaptations to the peroxisomal morphology. How the regulation of this process is integrated into the cell’s response to different stimuli, including the signaling pathways and factors involved, remains unclear. Here, a cell-based peroxisome proliferation assay has been applied to investigate the ability of different stimuli to induce peroxisome proliferation. We determined that serum stimulation, long-chain fatty acid supplementation and TGFβ application all increase peroxisome elongation, a prerequisite for proliferation. Time-resolved mRNA expression during the peroxisome proliferation cycle revealed a number of peroxins whose expression correlated with peroxisome elongation, including the β isoform of PEX11, but not the α or γ isoforms. An initial map of putative regulatory motif sites in the respective promoters showed a difference between binding sites in PEX11α and PEX11β, suggesting that these genes may be regulated by distinct pathways. A functional SMAD2/3 binding site in PEX11β points to the involvement of the TGFβ signaling pathway in expression of this gene and thus peroxisome proliferation/dynamics in humans.

Ketohexokinase (KHK) is the first and rate-limiting enzyme of fructose metabolism. Expression of the two alternatively spliced KHK isoforms, KHK-A and KHK-C, is tissue-specific and KHK-C is predominantly expressed in liver, kidney and intestine and responsible for the fructose-catabolizing function. While KHK isoform choice has been linked to the development of disorders such as obesity, diabetes, cardiovascular disease and cancer, little is known about the regulation of total KHK expression. In the present study, we investigated how hypoxic signaling influences fructose metabolism in the liver. Hypoxia or von Hippel-Lindau (VHL) tumor suppressor loss leads to the stabilization of hypoxia-inducible factors alpha (HIF-1α and HIF-2α) and the activation of their signaling to mediate adaptive responses. By studying liver-specific Vhl, Vhl/Hif1a, and Vhl/Epas1 knockout mice, we found that KHK expression is suppressed by HIF-2α (encoded by Epas1) but not by HIF-1α signaling on mRNA and protein levels. Reduced KHK levels were accompanied by downregulation of aldolase B (ALDOB) in the livers of Vhl and Vhl/Hif1a knockout mice, further indicating inhibited fructose metabolism. HIF-1α and HIF-2α have both overlapping and distinct target genes but are differentially regulated depending on the cell type and physiologic or pathologic conditions. HIF-2α activation augments peroxisome degradation in mammalian cells by pexophagy and thereby changes lipid composition reminiscent of peroxisomal disorders. We further demonstrated that fructose metabolism is negatively regulated by peroxisome-deficiency in a Pex2 knockout Zellweger mouse model, which lacks functional peroxisomes and is characterized by widespread metabolic dysfunction. Repression of fructolytic genes in Pex2 knockout mice appeared to be independent of PPARα signaling and nutritional status. Interestingly, our results demonstrate that both HIF-2α and peroxisome-deficiency result in downregulation of Khk independent of splicing as both isoforms, Khka as well as Khkc, are significantly downregulated. Hence, our study offers new and unexpected insights into the general regulation of KHK, and therefore fructolysis. We revealed a novel regulatory function of HIF-2α, suggesting that HIF-1α and HIF-2α have tissue-specific opposing roles in the regulation of Khk expression, isoform choice and fructolysis. In addition, we discovered a previously unknown function of peroxisomes in the regulation of fructose metabolism.

Herpes simplex virus 1 (HSV-1) and varicella-zoster virus (VZV) are two closely related human alphaherpesviruses that persistently infect most adults worldwide and cause a variety of clinically important diseases. Herpesviruses are extremely well adapted to their hosts and interact broadly with cellular proteins to regulate virus replication and spread. However, it is incompletely understood how HSV-1 and VZV interact with the host proteome during productive infection. This study determined the temporal changes in virus and host protein expression during productive HSV-1 and VZV infection in the same cell type. Results demonstrated the temporally coordinated expression of HSV-1 and VZV proteins in infected cells. Analysis of the host proteomes showed that both viruses affected extracellular matrix composition, transcription, RNA processing and cell division. Moreover, the prominent role of epidermal growth factor receptor (EGFR) signaling during productive HSV-1 and VZV infection was identified. Stimulation and inhibition of EGFR leads to increased and decreased virus replication, respectively. Collectively, the comparative temporal analysis of viral and host proteomes in productively HSV-1 and VZV-infected cells provides a valuable resource for future studies aimed to identify target(s) for antiviral therapy development.

The introduction of targeted agents has revolutionized the treatment of chronic lymphocytic leukemia but only few patients achieve complete remissions and minimal residual disease negativity with ibrutinib monotherapy. This multicenter, investigator-initiated phase-II study evaluates a sequential treatment with two cycles of bendamustine debulking for patients with a higher tumor load, followed by ofatumumab and ibrutinib induction and maintenance treatment. An all-comer population, irrespective of prior treatment, physical fitness and genetic factors was included. The primary endpoint was the investigator assessed overall response rate at the end of induction treatment. Of 66 patients enrolled, one patient with early treatment discontinuation was excluded from the efficacy analysis as predefined by the protocol. Thirty-nine patients (60%) were treatment-naive and 26 patients (40%) had relapsed/refractory CLL, 21 patients (32%) had a del(17p) and/or TP53 mutation and 45 patients (69%) had an unmutated IGHV status. At the end of the induction, 60 of 65 patients (92%) responded and 9 (14%) achieved minimal residual disease negativity (

Varicella-zoster virus (VZV) establishes lifelong infection in the majority of the population, but mechanisms underlying latency remain unclear. Here, the authors use ultra-deep RNA sequencing, enriched for viral RNAs, of latently infected human trigeminal ganglia and identify a spliced, latency-associated VZV mRNA.

Primary varicella-zoster virus (VZV) infection leads to varicella and the establishment of lifelong latency in sensory ganglion neurons. Reactivation of latent VZV causes herpes zoster, which is frequently associated with chronic pain. Latent viral gene expression is restricted to the VZV latency-associated transcript (VLT) and VLT-ORF63 (VLT63) fusion transcripts. Since VLT and VLT63 encode proteins that are expressed during lytic infection, we investigated whether pVLT and pVLT-ORF63 are essential for VZV replication by performing VZV genome mutagenesis using CRISPR/Cas9 and BAC technologies. We first established that CRISPR/Cas9 can efficiently mutate VZV genomes in lytically VZV-infected cells through targeting non-essential genes ORF8 and ORF11 and subsequently show recovery of viable mutant viruses. By contrast, the VLT region was markedly resistant to CRISPR/Cas9 editing. Whereas most mutants expressed wild-type or N-terminally altered versions of pVLT and pVLT-ORF63, only a minority of the resulting mutant viruses lacked pVLT and pVLT-ORF63 coding potential. Growth curve analysis showed that pVLT/pVLT-ORF63 negative viruses were viable, but impaired in growth in epithelial cells. We confirmed this phenotype independently using BAC-derived pVLT/pVLT-ORF63 negative and repaired viruses. Collectively, these data demonstrate that pVLT and/or pVLT-ORF63 are dispensable for lytic VZV replication but promote efficient VZV infection in epithelial cells.

Hippocampal neurogenesis is important for certain forms of cognition, and failing neurogenesis has been implicated in neuropsychiatric diseases. The neurogenic capacity of hippocampal neural stem/progenitor cells (NSPCs) depends on a balance between quiescent and proliferative states. Here, we show that the rate of fatty acid oxidation (FAO) regulates the activity of NSPCs. Quiescent NSPCs show high levels of carnitine palmitoyltransferase 1a (Cpt1a)-dependent FAO, which is downregulated in proliferating NSPCs. Pharmacological inhibition and conditional deletion of Cpt1a in vitro and in vivo leads to altered NSPC behavior, showing that Cpt1a-dependent FAO is required for stem cell maintenance and proper neurogenesis. Strikingly, manipulation of malonyl-CoA, the metabolite that regulates levels of FAO, is sufficient to induce exit from quiescence and to enhance NSPC proliferation. Thus, the data presented here identify a shift in FAO metabolism that governs NSPC behavior and suggest an instructive role for fatty acid metabolism in regulating NSPC activity.

Abstract Background Disorders of the oxidative phosphorylation (OXPHOS) system represent a large group among the inborn errors of metabolism. The most frequently observed biochemical defect is isolated deficiency of mitochondrial complex I (CI). No effective treatment strategies for CI deficiency are so far available. The purpose of this study was to investigate whether and how mesenchymal stem cells (MSCs) are able to modulate metabolic function in fibroblast cell models of CI deficiency. Methods We used human and murine fibroblasts with a defect in the nuclear DNA encoded NDUFS4 subunit of CI. Fibroblasts were co-cultured with MSCs under different stress conditions and intercellular mitochondrial transfer was assessed by flow cytometry and fluorescence microscopy. Reactive oxygen species (ROS) levels were measured using MitoSOX-Red. Protein levels of CI were analysed by blue native polyacrylamide gel electrophoresis (BN-PAGE). Results Direct cellular interactions and mitochondrial transfer between MSCs and human as well as mouse fibroblast cell lines were demonstrated. Mitochondrial transfer was visible in 13.2% and 6% of fibroblasts (e.g. fibroblasts containing MSC mitochondria) for human and mouse cell lines, respectively. The transfer rate could be further stimulated via treatment of cells with TNF-α. MSCs effectively lowered cellular ROS production in NDUFS4-deficient fibroblast cell lines (either directly via co-culture or indirectly via incubation of cell lines with cell-free MSC supernatant). However, CI protein expression and activity were not rescued by MSC treatment. Conclusion This study demonstrates the interplay between MSCs and fibroblast cell models of isolated CI deficiency including transfer of mitochondria as well as modulation of cellular ROS levels. Further exploration of these cellular interactions might help to develop MSC-based treatment strategies for human CI deficiency.

Objectives This study evaluated color differences (ΔEs) and translucency parameter changes (ΔTPs) of various computer-aided design/computer-aided manufacturing (CAD/CAM) blocks after immersion in coffee. Materials and Methods Eight CAD/CAM blocks and four restorative composite resins were evaluated. The CIE L*a*b* values of 2.0 mm thick disk-shaped specimens were measured using the spectrophotometer on white and black backgrounds (n = 6). The ΔEs and ΔTPs of one day, one week, and one month immersion in coffee or water were calculated. The values of each material were analyzed by two-way ANOVA and Tukey's multiple comparisons (α = 0.05). The ΔEs after prophylaxis paste polishing of 1 month coffee immersion specimens, water sorption and solubility were also evaluated. Results After one month in coffee, ΔEs of CAD/CAM composite resin blocks and restorative composites ranged from 1.6 to 3.7 and from 2.1 to 7.9, respectively, and ΔTPs decreased. The ANOVA of ΔEs and ΔTPs revealed significant differences in two main factors, immersion periods and media, and their interaction except for ΔEs of TEL (Telio CAD, Ivoclar Vivadent). The ΔEs significantly decreased after prophylaxis polishing except GRA (Gradia Block, GC). There was no significant correlation between ΔEs and water sorption or solubility in water. Conclusions The ΔEs of CAD/CAM blocks after immersion in coffee varied among products and were comparable to those of restorative composite resins. The discoloration of CAD/CAM composite resin blocks could be effectively removed with prophylaxis paste polishing, while that of some restorative composites could not be removed.