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Volume overload (VO) induced hypertrophy is one of the hallmarks to the development of heart diseases. Understanding the compensatory mechanisms involved in this process might help preventing the disease progression.Therefore, the present study used 2 months old Wistar rats, which underwent an aortocaval fistula to develop VO-induced hypertrophy. The animals were subdivided into four different groups, two sham operated animals served as age-matched controls and two groups with aortocaval fistula. Echocardiography was performed prior termination after 4- and 8-month. Functional and molecular changes of several sarcomeric proteins and their signalling pathways involved in the regulation and modulation of cardiomyocyte function were investigated.The model was characterized with preserved ejection fraction in all groups and with elevated heart/body weight ratio, left/right ventricular and atrial weight at 4- and 8-month, which indicates VO-induced hypertrophy. In addition, 8-months groups showed increased left ventricular internal diameter during diastole, RV internal diameter, stroke volume and velocity-time index compared with their age-matched controls. These changes were accompanied by increased CaOur findings showed VO-induced cardiomyocyte dysfunction via deranged phosphorylation of myofilament proteins and signalling pathways due to increased oxidative state of CaMKII and PKG and this might contribute to the development of hypertrophy.

Multiple sclerosis (MS) is a progressive inflammatory-demyelinating disease of the central nervous system. Increasing evidence suggests that vulnerable neurons in MS exhibit fatal metabolic exhaustion over time, a phenomenon hypothesized to be caused by chronic hyperexcitability. Axonal Kv7 (outward rectifying) and oligodendroglial Kir4.1 (inward rectifying) potassium channels have important roles in regulating neuronal excitability at and around nodes of Ranvier. Here, we studied the spatial and functional relationship between neuronal Kv7 and oligodendroglial Kir4.1 channels and assessed the transcriptional and functional signatures of cortical and retinal projection neurons under physiological and inflammatory-demyelinating conditions. We found that both channels became dysregulated in MS and experimental autoimmune encephalomyelitis (EAE) with Kir4.1 channels being chronically downregulated and Kv7 channel subunits being transiently upregulated during inflammatory demyelination. Further, we observed that pharmacological Kv7 channel opening with retigabine reduced neuronal hyperexcitability in human and EAE neurons, improved clinical EAE signs and rescued neuronal pathology in oligodendrocyte-Kir4.1-deficient mice. In summary, our findings indicate that neuron-oligodendrocyte compensatory interactions promote resilience through Kv7 and Kir4.1 channels and suggest pharmacological activation of nodal Kv7 channels as a neuroprotective strategy against inflammatory demyelination.

It remains largely unclear how thymocytes translate relative differences in T cell receptor (TCR) signal strength into distinct developmental programs that drive the cell fate decisions towards conventional (Tconv) or regulatory T cells (Treg). Following TCR activation, intracellular calcium (Ca2+) is the most important second messenger, for which the potassium channel K2P18.1 is a relevant regulator. Here, we identify K2P18.1 as a central translator of the TCR signal into the thymus-derived Treg (tTreg) selection process. TCR signal was coupled to NF-κB-mediated K2P18.1 upregulation in tTreg progenitors. K2P18.1 provided the driving force for sustained Ca2+ influx that facilitated NF-κB- and NFAT-dependent expression of FoxP3, the master transcription factor for Treg development and function. Loss of K2P18.1 ion-current function induced a mild lymphoproliferative phenotype in mice, with reduced Treg numbers that led to aggravated experimental autoimmune encephalomyelitis, while a gain-of-function mutation in K2P18.1 resulted in increased Treg numbers in mice. Our findings in human thymus, recent thymic emigrants and multiple sclerosis patients with a dominant-negative missense K2P18.1 variant that is associated with poor clinical outcomes indicate that K2P18.1 also plays a role in human Treg development. Pharmacological modulation of K2P18.1 specifically modulated Treg numbers in vitro and in vivo. Finally, we identified nitroxoline as a K2P18.1 activator that led to rapid and reversible Treg increase in patients with urinary tract infections. Conclusively, our findings reveal how K2P18.1 translates TCR signals into thymic T cell fate decisions and Treg development, and provide a basis for the therapeutic utilization of Treg in several human disorders.

Numerous epidemiological studies have found co-morbidity between non-severe traumatic brain injury (TBI) and substance misuse in both civilian and military populations. Preclinical studies have also identified this relationship for some misused substances. We have previously demonstrated that repeated blast traumatic brain injury (rbTBI) increased oxycodone seeking without increasing oxycodone self-administration, suggesting that the neurological sequelae of traumatic brain injury can elevate opioid misuse liability. Here, we determined the chronicity of this effect by testing different durations of time between injury and oxycodone self-administration and durations of abstinence. We found that the subchronic (four weeks), but not the acute (three days) or chronic (four months) duration between injury and oxycodone self-administration was associated with increased drug seeking and re-acquisition of self-administration following a 10-day abstinence. Examination of other abstinence durations (two days, four weeks, or four months) revealed no effect of rbTBI on drug seeking at any of the abstinence durations tested. Together, these data indicate that there is a window of vulnerability after TBI when oxycodone self-administration is associated with elevated drug seeking and relapse-related behaviors.

The calcium-activated potassium channel 3.1 (K

Secondary damage obstructs functional recovery for individuals who have sustained a spinal cord injury (SCI). Two processes significantly contributing to tissue damage after trauma are spinal cord hemorrhage and inflammation: more specifically, the recruitment and activation of immune cells, frequently driven by pro-inflammatory factors. Cytokines are inflammatory mediators capable of modulating the immune response. While cytokines are necessary to elicit inflammation for proper healing, excessive inflammation can result in destructive processes. The pro-inflammatory cytokines IL-12 and IL-23 are pathogenic in multiple autoimmune diseases. The cytokine subunit IL-12p40 is necessary to form bioactive IL-12 and IL-23. In this study, we examined the relationship between spinal cord hemorrhage and IL-12-related factors, as well as the impact of IL-12p40 (IL-12/IL-23) on secondary damage and functional recovery after SCI. Using in vivo magnetic resonance imaging and protein tissue analyses, we demonstrated a positive correlation between IL-12 and tissue hemorrhage. Receptor and ligand subunits of IL-12 were significantly upregulated after injury and colocalized with astrocytes, demonstrating a myriad of opportunities for IL-12 to induce an inflammatory response. IL-12p40

Purpose Diffusion MRI provides unique contrast important for the detection and examination of pathophysiology after acute neurologic insults, including spinal cord injury. Diffusion weighted imaging of the rodent spinal cord has typically been evaluated with axial EPI readout. However, Diffusion weighted imaging is prone to motion artifacts, whereas EPI is prone to susceptibility artifacts. In the context of acute spinal cord injury, diffusion filtering has previously been shown to improve detection of injury by minimizing the confounding effects of edema. We propose a diffusion-preparation module combined with a rapid acquisition with relaxation enhancement readout to minimize artifacts for sagittal imaging. Methods Sprague-Dawley rats with cervical contusion spinal cord injury were scanned at 9.4 Tesla. The sequence optimization included the evaluation of motion-compensated encoding diffusion gradients, gating strategy, and different spinal cord-specific diffusion-weighting schemes. Results A diffusion-prepared rapid acquisition with relaxation enhancement achieved high-quality images free from susceptibility artifacts with both second-order motion-compensated encoding and gating necessary for reduction of motion artifacts. Axial diffusivity obtained from the filtered diffusion-encoding scheme had greater lesion-to-healthy tissue contrast (52%) compared to the similar metric from DTI (25%). Conclusion This work demonstrated the feasibility of high-quality diffusion sagittal imaging in the rodent cervical cord with diffusion-prepared relaxation enhancement. The sequence and results are expected to improve injury detection and evaluation in acute spinal cord injury.

The medical uses of cannabidiol (CBD), a constituent of the Cannabis sativa, have accelerated the legal and social acceptance for CBD-based medications but has also given the momentum for questioning whether the long-term use of CBD during the early years of life may induce adverse neurobiological effects in adulthood, including sleep disturbances. Given the critical window for neuroplasticity and neuro-functional changes that occur during stages of adolescence, we hypothesized that CBD might influence the sleep-wake cycle in adult rats after their exposure to CBD during the adolescence. Here, we investigated the effects upon behavior and neural activity in adulthood after long-term administrations of CBD in juvenile rats. We pre-treated juvenile rats with CBD (5 or 30 mg/Kg, daily) from post-natal day (PND) 30 and during 2 weeks. Following the treatments, the sleep-wake cycle and NeuN expression was analyzed at PND 80. We found that systemic injections of CBD (5 or 30 mg/Kg, i.p.) given to adolescent rats (post-natal day 30) for 14 days increased in adulthood the wakefulness and decreased rapid eye movement sleep during the lights-on period whereas across the lights-off period, wakefulness was diminished and slow wave sleep was enhanced. In addition, we found that adult animals that received CBD during the adolescence displayed disruptions in sleep rebound period after total sleep deprivation. Finally, we determined how the chronic administrations of CBD during the adolescence affected in the adulthood the NeuN expression in the suprachiasmatic nucleus, a sleep-related brain region. Our findings are relevant for interpreting results of adult rats that were chronically exposed to CBD during the adolescence and provide new insights into how CBD may impact the sleep-wake cycle and neuronal activity during developmental stages.

Objective To test the hypothesis that death with physiological parallels to human cases of sudden unexpected death in epilepsy (SUDEP) can be induced in seizing rats by ictal activation of oxygen-conserving reflexes (OCRs). Methods Urethane-anesthetized female Long-Evans rats were implanted with electrodes for electrocardiography (ECG), electrocorticography (ECoG), and respiratory thermocouple; venous and arterial cannulas; and a laryngoscope guide and cannula or nasal cannula for activation of the laryngeal chemoreflex (LCR) or mammalian diving reflex (MDR), respectively. Kainic acid injection, either systemic or into the ventral hippocampus, induced prolonged acute seizures. Results Reflex challenges during seizures caused sudden death in 18 of 20 rats-all MDR rats (10) and all but two LCR rats (8) failed to recover from ictal activation of OCRs and died within minutes of the reflexes. By comparison, 4 of 4 control (ie, nonseizing) rats recovered from 64 induced diving reflexes (16 per rat), and 4 of 4 controls recovered from 64 induced chemoreflexes (16 per rat). Multiple measures were consistent with reports of human SUDEP. Terminal central apnea preceded terminal asystole in all cases. Heart and respiratory rate fluctuations that paralleled those seen in human SUDEP occurred during OCR-induced sudden death, and mean arterial pressure (MAP) was predictive of death, showing a 17 or 15 mm Hg drop (MDR and LCR, respectively) in the 20 s window centered on the time of brain death. OCR activation was never fatal in nonseizing rats. Significance These results present a method of inducing sudden death in two seizure models that show pathophysiology consistent with that observed in human cases of SUDEP. This proposed mechanism directly informs previous findings by our group and others in the field; provides a repeatable, inducible animal model for the study of sudden death; and offers a potential explanation for observations made in cases of human SUDEP.

We have previously shown that immunization of C57BL/6 mice, prone to spontaneous development of experimental autoimmune encephalomyelitis (EAE), with three antigens (MOG35‐55, DNA‐histone complex or DNA‐methylated BSA complex), alters the differentiation profiles of bone marrow haematopoietic stem cells (HSCs). These are associated with the production of autoantibodies (auto‐Abs) against these antigens and the formation of abzymes hydrolysing DNA, MOG, myelin basic protein (MBP) and histones. Immunization of mice with antigens accelerates the development of EAE. This work is the first to analyse the ratio of auto‐Abs without and with catalytic activities at different stages of EAE development (onset, acute and remission phases) after immunization of mice with the three specific antigens. Prior to immunization and during spontaneous in‐time development of EAE, the concentration of auto‐Abs against MBP, MOG, histones and DNA and activities of IgG antibodies in the hydrolysis of substrates increased in parallel; correlation coefficients = +0.69‐0.94. After immunization with MOG, DNA‐histone complex or DNA‐met‐BSA complex, both positive (from +0.13 to +0.98) and negative correlations (from −0.09 to −0.69) were found between these values. Our study is the first showing that depending on the antigen, the relative amount of harmful auto‐Abs without and abzymes with low or high catalytic activities may be produced only at onset and in acute or remission phases of EAE. The antigen governs the EAE development rate, whereby the ratio of auto‐Abs without catalytic activity and with enzymatic activities of harmful abzymes hydrolysing MBP, MOG, histones and DNA varies strongly between different disease phases.

Exact mechanisms of autoimmune disease development are still yet unknown. However, it is known that the development of autoimmune diseases is associated with defects in the immune system, namely, the violation of the bone marrow hematopoietic stem cells (HSCs) differentiation profiles. Different characteristics of autoimmune reaction development in experimental autoimmune encephalomyelitis (EAE) prone Th mice characterizing T-lymphocytes response were analyzed using standard approaches. Profiles of several HSCs differentiation of bone marrow (BFU-E, CFU-E, CFU-GM, CFU-GEMM, T- and B-lymphocytes) of Th male and female mice during spontaneous development of EAE were noticeably different. Patterns of total lymphocytes, B- and T-cells proliferation in several different organs (bone marrow, blood, spleen, thymus, and lymph nodes) were also remarkably different. In addition, there were in time noticeable differences in their changes for some organs of male and female mice. Characters of changes in the profiles of CD4 and CD8 cells proliferation in some organs not always coincide with those for total T lymphocytes. The changes in the differentiation profiles of HSCs and the level of lymphocytes proliferation in the bone marrow and other organs were associated with the increase in the concentration of antibodies against DNA, myelin basic protein, and myelin oligodendrocyte glycoprotein, and catalytic antibodies hydrolyzing these substrates. Despite some differences in changes in the analyzed parameters, in general, the spontaneous development of EAE in male and female mice occurs to some extent in a comparable way.

Lymphomas with central nervous system (CNS) involvement confer a worse prognosis than those without CNS involvement, and patients currently have limited treatment options. T cells genetically engineered with CD19-targeted chimeric antigen receptors (CAR) are effective against B-cell malignancies and show tremendous potential in the treatment of systemic lymphoma. We aimed to leverage this strategy toward a more effective therapy for patients with lymphoma with CNS disease. NOD-scid IL2Rgammanull (NSG) mice with CNS and/or systemic lymphoma were treated with CD19-CAR T cells via intracerebroventricular (ICV) or intravenous (IV) injection. CAR T cells isolated after treatment were rigorously examined for phenotype, gene expression, and function. We observed that CAR T cells infused ICV, but not IV, completely and durably eradicated both CNS and systemic lymphoma. CAR T cells delivered ICV migrated efficiently to the periphery, homed to systemic tumors, and expanded in vivo, leading to complete elimination of disease and resistance to tumor rechallenge. Mechanistic studies indicated that ICV-delivered CAR T cells are conditioned by exposure to cerebrospinal fluid in the ICV environment for superior antilymphoma activity and memory function compared with IV-delivered CAR T cells. Further analysis suggested that manipulating cellular metabolism or preactivating therapeutic CAR T cells with antigen ex vivo may improve the efficacy of CAR T cells in vivo. Our demonstration that ICV-delivered CD19-CAR T cells had activity against CNS and systemic lymphoma could offer a valuable new strategy for treatment of B-cell malignancies with CNS involvement.

Cardiac MRI of small animal models of cancer radiation therapy (RT) is a valuable tool for studying the effect of RT on the heart. However, standard cardiac MRI exams require long scanning times, which is challenging for sick animals that may not survive extended periods of imaging under anesthesia. The purpose of this study is to develop an optimized, fast MRI exam for comprehensive cardiac functional imaging of small-animal models of cancer RT. Ten adult female rats (2 non-irradiated and 8 irradiated) were scanned using the developed exam. Optimal imaging parameters were determined, which minimized scanning time while ensuring measurement accuracy and avoiding imaging artifacts. This optimized, fast MRI exam lasted for 30 min, which was tolerated by all animals. EF was normal in all imaged rats, although it was significantly increased in the irradiated rats, which also showed ventricular hypertrophy. However, myocardial strain was significantly reduced in the irradiated rats. In conclusion, a fast MRI exam has been developed for comprehensive cardiac functional imaging of rats in 30 min, with optimized imaging parameters to ensure accurate measurements and tolerance by irradiated rats. The generated strain measurements provide an early marker of regional cardiac dysfunction before global function is affected.

The Ca2+ activated potassium channel 3.1 (KCa3.1) is involved in critical steps of the metastatic cascade, such as proliferation, migration, invasion and extravasation. Therefore, a fast and efficient protocol for imaging of KCa3.1 channels was envisaged. The novel fluorescently labeled small molecule imaging probes 1 and 2 were synthesized by connecting a dimethylpyrrole‐based BODIPY dye with a derivative of the KCa3.1 channel inhibitor senicapoc via linkers of different length. Patch‐clamp experiments revealed the inhibition of KCa3.1 channels by the probes confirming interaction with the channel. Both probes 1 and 2 were able to stain KCa3.1 channels in non‐small‐cell lung cancer (NSCLC) cells following a simple, fast and efficient protocol. Pre‐incubation with unlabeled senicapoc removed the punctate staining pattern showing the specificity of the new probes 1 and 2. Staining of the channel with the fluorescently labeled senicapoc derivatives 1 or 2 or with antibody‐based indirect immunofluorescence yielded identical or very similar densities of stained KCa3.1 channels. However, co‐staining using both methods did not lead to the expected overlapping punctate staining pattern. This observation was explained by docking studies showing that the antibody used for indirect immunofluorescence and the probes 1 and 2 label different channel populations. Whereas the antibody binds at the closed channel conformation, the probes 1 and 2 bind within the open channel., Open vs. closed: The KCa3.1 channel is involved in critical steps of the metastatic cascade. Analyzing KCa3.1 channel expression has predictive power with respect to patient survival. Novel fluorescently labeled small‐molecule imaging probes were able to stain KCa3.1 channels in NSCLC cells selectively and efficiently. Co‐staining using antibody‐based indirect immunofluorescence did not lead to the expected overlapping punctate staining pattern. This observation was explained by docking studies showing that the antibody and the novel imaging probes label different channel populations, closed and open channels, respectively.

The inhibiting effect of the secondary phosphite antioxidant degradation product bis(2,4-di-tert-butylphenyl)phosphate (bDtBPP) on cell growth is well-known. The present study describes structurally related compounds which are likely to be formed from similar widely used phosphite antioxidants used in materials for the manufacturing of single-use (SU) equipment. Two potential candidates of such compounds-3,3',5,5'-tetra-tert-butyl-2,2'-dihydroxybiphenylphosphate (TtBBP) and bis(p-nonylphenyl)phosphate (bNPP)-were identified by chromatography and mass spectrometry followed by synthesis and X-ray structure elucidation. Additionally, the formation of TtBBP was confirmed in an analytical degradation study and its migration from SU bioprocessing material was estimated. The cytotoxicity evaluation by means of cell culture spiking experiments and flow cytometry analysis revealed that' even if cell growth was inhibited by all the compounds to some extent, bDtBPP showed the most severe effect and stoods out from the other two degradants investigated. Graphical abstract.

Background Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS), characterized by inflammatory and neurodegenerative processes. Despite demyelination being a hallmark of the disease, how it relates to neurodegeneration has still not been completely unraveled, and research is still ongoing into how these processes can be tracked non-invasively. Magnetic resonance imaging (MRI) derived brain network characteristics, which closely mirror disease processes and relate to functional impairment, recently became important variables for characterizing immune-mediated neurodegeneration; however, their histopathological basis remains unclear. Methods In order to determine the MRI-derived correlates of myelin dynamics and to test if brain network characteristics derived from diffusion tensor imaging reflect microstructural tissue reorganization, we took advantage of the cuprizone model of general demyelination in mice and performed longitudinal histological and imaging analyses with behavioral tests. By introducing cuprizone into the diet, we induced targeted and consistent demyelination of oligodendrocytes, over a period of 5 weeks. Subsequent myelin synthesis was enabled by reintroduction of normal food. Results Using specific immune-histological markers, we demonstrated that 2 weeks of cuprizone diet induced a 52% reduction of myelin content in the corpus callosum (CC) and a 35% reduction in the neocortex. An extended cuprizone diet increased myelin loss in the CC, while remyelination commenced in the neocortex. These histologically determined dynamics were reflected by MRI measurements from diffusion tensor imaging. Demyelination was associated with decreased fractional anisotropy (FA) values and increased modularity and clustering at the network level. MRI-derived modularization of the brain network and FA reduction in key anatomical regions, including the hippocampus, thalamus, and analyzed cortical areas, were closely related to impaired memory function and anxiety-like behavior. Conclusion Network-specific remyelination, shown by histology and MRI metrics, determined amelioration of functional performance and neuropsychiatric symptoms. Taken together, we illustrate the histological basis for the MRI-driven network responses to demyelination, where increased modularity leads to evolving damage and abnormal behavior in MS. Quantitative information about in vivo myelination processes is mirrored by diffusion-based imaging of microstructural integrity and network characteristics.

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system that is characterized by the progressive loss of oligodendrocytes and myelin and is associated with thalamic dysfunction. Cuprizone (CPZ)-induced general demyelination in rodents is a valuable model for studying different aspects of MS pathology. CPZ feeding is associated with the altered distribution and expression of different ion channels along neuronal somata and axons. However, it is largely unknown whether the copper chelator CPZ directly influences ion channels. Therefore, we assessed the effects of different divalent cations (copper; zinc) and trace metal chelators (EDTA; Tricine; the water-soluble derivative of CPZ, BiMPi) on hyperpolarization-activated cyclic nucleotide-gated (HCN) channels that are major mediators of thalamic function and pathology. In addition, alterations of HCN channels induced by CPZ treatment and MS-related proinflammatory cytokines (IL-1β; IL-6; INF-α; INF-β) were characterized in C57Bl/6J mice. Thus, the hyperpolarization-activated inward current (I

Traumatic brain injury (TBI) and drug addiction are common comorbidities, but it is unknown if the neurological sequelae of TBI contribute to this relationship. We have previously reported elevated oxycodone seeking after drug self-administration in rats that received repeated blast TBI (rbTBI). TBI and exposure to drugs of abuse can each change structural and functional neuroimaging outcomes, but it is unknown if there are interactive effects of injury and drug exposure. To determine the effects of TBI and oxycodone exposure, we subjected rats to rbTBI and oxycodone self-administration and measured drug seeking and several neuroimaging measures. We found interactive effects of rbTBI and oxycodone on fractional anisotropy (FA) in the nucleus accumbens (NAc), and that FA in the medial prefrontal cortex (mPFC) was correlated with drug seeking. We also found an interactive effect of injury and drug on widespread functional connectivity and regional homogeneity of the blood oxygen level dependent (BOLD) response, and that intra-hemispheric functional connectivity in the infralimbic medial prefrontal cortex positively correlated with drug seeking. In conclusion, rbTBI and oxycodone self-administration had interactive effects on structural and functional magnetic resonance imaging (MRI) measures, and correlational effects were found between some of these measures and drug seeking. These data support the hypothesis that TBI and opioid exposure produce neuroadaptations that contribute to addiction liability.

Interstitial fibrosis and tubular atrophy, a major cause of kidney allograft dysfunction, has been linked to premature cellular senescence. The mTOR inhibitor Rapamycin protects from senescence in experimental models, but its antiproliferative properties have raised concern early after transplantation particularly at higher doses. Its effect on senescence has not been studied in kidney transplantation, yet. Rapamycin was applied to a rat kidney transplantation model (3 mg/kg bodyweight loading dose, 1.5 mg/kg bodyweight daily dose) for 7 days. Low Rapamycin trough levels (2.1-6.8 ng/mL) prevented the accumulation of p16(INK4a) positive cells in tubules, interstitium, and glomerula. Expression of the cytokines MCP-1, IL-1 beta, and TNF-alpha, defining the proinflammatory senescence-associated secretory phenotype, was abrogated. Infiltration with monocytes/macrophages and CD8(+) T-lymphocytes was reduced and tubular function was preserved by Rapamycin. Inhibition of mTOR was not associated with impaired structural recovery, higher glucose levels, or weight loss. mTOR inhibition with low-dose Rapamycin in the immediate posttransplant period protected from premature cellular senescence without negative effects on structural and functional recovery from preservation/reperfusion damage, glucose homeostasis, and growth in a rat kidney transplantation model. Reduced senescence might maintain the renal regenerative capacity rendering resilience to future injuries resulting in protection from interstitial fibrosis and tubular atrophy.

BackgroundMood disorders (including major depressive disorder and bipolar disorder) affect 10-20% of the population. They range from brief, mild episodes to severe, incapacitating conditions that markedly impact lives. Despite their diagnostic distinction, multiple approaches have shown considerable sharing of risk factors across the mood disorders.MethodsTo clarify their shared molecular genetic basis, and to highlight disorder-specific associations, we meta-analysed data from the latest Psychiatric Genomics Consortium (PGC) genome-wide association studies of major depression (including data from 23andMe) and bipolar disorder, and an additional major depressive disorder cohort from UK Biobank (total: 185,285 cases, 439,741 controls; non-overlapping N = 609,424).ResultsSeventy-three loci reached genome-wide significance in the meta-analysis, including 15 that are novel for mood disorders. More genome-wide significant loci from the PGC analysis of major depression than bipolar disorder reached genome-wide significance. Genetic correlations revealed that type 2 bipolar disorder correlates strongly with recurrent and single episode major depressive disorder. Systems biology analyses highlight both similarities and differences between the mood disorders, particularly in the mouse brain cell types implicated by the expression patterns of associated genes. The mood disorders also differ in their genetic correlation with educational attainment – positive in bipolar disorder but negative in major depressive disorder.ConclusionsThe mood disorders share several genetic associations, and can be combined effectively to increase variant discovery. However, we demonstrate several differences between these disorders. Analysing subtypes of major depressive disorder and bipolar disorder provides evidence for a genetic mood disorders spectrum.

Urinary extracellular vesicles (uEVs) are emerging as non-invasive biomarkers for various kidney diseases, but it is unknown how differences in nephron mass impact uEV excretion. To address this, uEV excretion was measured before and after human kidney donor nephrectomy and rat nephrectomy. In male and female donors, uEVs were quantified in cell-free spot and 24-h urine samples using nanoparticle tracking analysis (NTA), EVQuant, and CD9-time-resolved fluorescence immunoassay. Female donors had significantly lower total kidney volume (TKV) and excreted 49% fewer uEVs than male donors. uEV excretion correlated positively with estimated glomerular filtration rate (eGFR), creatinine clearance, and TKV (R's between 0.6 and 0.7). uEV excretion rate could also be predicted from spot urines after multiplying spot uEV/creatinine by 24-h urine creatinine. Donor nephrectomy reduced eGFR by 36% ± 10%, but the excretion of uEVs by only 16% (CD9+ uEVs -37%, CD9- uEVs no decrease). Donor nephrectomy increased the podocyte marker WT-1 and the proximal tubule markers NHE3, NaPi-IIa, and cubilin in uEVs two- to four-fold when correcting for the nephrectomy. In rats, the changes in GFR and kidney weight correlated with the changes in uEV excretion rate (R = 0.46 and 0.60, P th nephrectomy). Taken together, our data show that uEV excretion depends on nephron mass, and that nephrectomy reduces uEV excretion less than expected based on nephron loss due to compensatory hypertrophy. The major implication of our findings is that a measure for nephron mass or uEV excretion rate should be included when comparing uEV biomarkers between individuals.

Background and purpose Microstructure imaging with advanced diffusion MRI (dMRI) techniques have shown increased sensitivity and specificity to microstructural changes in various disease and injury models. Oscillating gradient spin echo (OGSE) dMRI, implemented by varying the oscillating gradient frequency, and microscopic anisotropy (μA) dMRI, implemented via tensor valued diffusion encoding, may provide additional insight by increasing sensitivity to smaller spatial scales and disentangling fiber orientation dispersion from true microstructural changes, respectively. The aims of this study were to characterize the test-retest reproducibility of in vivo OGSE and μA dMRI metrics in the mouse brain at 9.4 Tesla and provide estimates of required sample sizes for future investigations. Methods Twelve adult C57Bl/6 mice were scanned twice (5 days apart). Each imaging session consisted of multifrequency OGSE and μA dMRI protocols. Metrics investigated included μA, linear diffusion kurtosis, isotropic diffusion kurtosis, and the diffusion dispersion rate (Λ), which explores the power-law frequency dependence of mean diffusivity. The dMRI metric maps were analyzed with mean region-of-interest (ROI) and whole brain voxel-wise analysis. Bland-Altman plots and coefficients of variation (CV) were used to assess the reproducibility of OGSE and μA metrics. Furthermore, we estimated sample sizes required to detect a variety of effect sizes. Results Bland-Altman plots showed negligible biases between test and retest sessions. ROI-based CVs revealed high reproducibility for most metrics (CVs < 15%). Voxel-wise CV maps revealed high reproducibility for μA (CVs ~ 10%), but low reproducibility for OGSE metrics (CVs ~ 50%). Conclusion Most of the μA dMRI metrics are reproducible in both ROI-based and voxel-wise analysis, while the OGSE dMRI metrics are only reproducible in ROI-based analysis. Given feasible sample sizes (10–15), μA metrics and OGSE metrics may provide sensitivity to subtle microstructural changes (4–8%) and moderate changes (> 6%), respectively.

Inherited cardiomyopathies form a heterogenous group of disorders that affect the structure and function of the heart. Defects in the genes encoding sarcomeric proteins are associated with various perturbations that induce contractile dysfunction and promote disease development. In this review we aimed to outline the functional consequences of the major inherited cardiomyopathies in terms of myocardial contraction and kinetics, and to highlight the structural and functional alterations in some sarcomeric variants that have been demonstrated to be involved in the pathogenesis of the inherited cardiomyopathies. A particular focus was made on mutation-induced alterations in cardiomyocyte mechanics. Since no disease-specific treatments for familial cardiomyopathies exist, several novel agents have been developed to modulate sarcomere contractility. Understanding the molecular basis of the disease opens new avenues for the development of new therapies. Furthermore, the earlier the awareness of the genetic defect, the better the clinical prognostication would be for patients and the better the prevention of development of the disease.

Clinical methods for determining the severity of traumatic spinal cord injury (SCI) and long-term functional outcome in the acute setting are limited in their prognostic accuracy because of the heterogeneity of injury and dynamic injury progression. The aim of this study was to evaluate the time course and sensitivity of advanced magnetic resonance imaging (MRI) methods to neurological function after SCI in a rat contusion model. Rats received a graded contusion injury at T10 using a weight-drop apparatus. MRI consisted of morphological measures from T(2)-weighted imaging, quantitative T(2) imaging, and diffusion-weighted imaging (DWI) at 1, 30, and 90 days post-injury (dpi). The derived metrics were compared with neurological function assessed using weekly Basso, Beattie, and Bresnahan (BBB) locomotor scoring and return of reflexive micturition function. At the acute time point (1 dpi), diffusion metrics sensitive to axonal injury at the injury epicenter had the strongest correlation with time-matched BBB scores and best predicted 90-dpi BBB scores. At 30 dpi, axonal water fraction derived from DWI and T(2) values were both correlated with time-matched locomotor scores. At the chronic time point (90 dpi), cross-sectional area was most closely correlated to BBB. Overall, the results demonstrate differential sensitivity of MRI metrics at different time points after injury, but the metrics follow the expected pathology of acute axonal injury followed by continued degeneration and finally a terminal level of atrophy. Specificity of DWI in the acute setting may make it impactful as a prognostic tool while T(2) imaging provided the most information about injury severity in chronic injury.

Continuous processing is the future production method for monoclonal antibodies (mAbs). A fully continuous, fully automated downstream process based on disposable equipment was developed and implemented inside the MoBiDiK pilot plant. However, a study evaluating the comparability between batch and continuous processing based on product quality attributes was not conducted before. The work presented fills this gap comparing both process modes experimentally by purifying the same harvest material (side-by-side comparability). Samples were drawn at different time points and positions in the process for batch and continuous mode. Product quality attributes, product-related impurities, as well as process-related impurities were determined. The resulting polished material was processed to drug substance and further evaluated regarding storage stability and degradation behavior. The in-process control data from the continuous process showed the high degree of accuracy in providing relevant process parameters such as pH, conductivity, and protein concentration during the entire process duration. Minor differences between batch and continuous samples are expected as different processing conditions are unavoidable due to the different nature of batch and continuous processing. All tests revealed no significant differences in the intermediates and comparability in the drug substance between the samples of both process modes. The stability study of the final product also showed no differences in the stability profile during storage and forced degradation. Finally, online data analysis is presented as a powerful tool for online-monitoring of chromatography columns during continuous processing.

Molecular barcoding technologies that uniquely identify single cells are hampered by limitations in barcode measurement. Readout by sequencing does not preserve the spatial organization of cells in tissues, whereas imaging methods preserve spatial structure but are less sensitive to barcode sequence. Here we introduce a system for image-based readout of short (20bp) DNA barcodes. In this system, called Zombie, phage RNA polymerases transcribe engineered barcodes in fixed cells. The resulting RNA is subsequently detected by fluorescent in situ hybridization. Using competing match and mismatch probes, Zombie can accurately discriminate single-nucleotide differences in the barcodes. This method allows in situ readout of dense combinatorial barcode libraries and single-base mutations produced by CRISPR base editors without requiring barcode expression in live cells. Zombie functions across diverse contexts, including cell culture, chick embryos, and adult mouse brain tissue. The ability to sensitively read out compact and diverse DNA barcodes by imaging will facilitate a broad range of barcoding and genomic recording strategies., Editorial summary: The spatial location and sequence of DNA barcodes are detected with high sensitivity in fixed tissues.

OBJECTIVE: To investigate carotid body (CB) mechanisms related to sudden death during seizure. Ictal activation of oxygen-conserving reflexes (OCRs) can trigger fatal cardiorespiratory collapse in seizing rats, which presents like human sudden unexpected death in epilepsy (SUDEP). The CB is strongly implicated in OCR pathways; we hypothesize that modulating CB activity will provide insight into these mechanisms of death. METHODS: Long-Evans rats were anesthetized with urethane. Recordings included: electrocorticography, electrocardiography, respiration via nasal thermocouple, and blood pressure (BP). The mammalian diving reflex (MDR) was activated by cold water delivered through a nasal cannula. Reflex and stimulation trials were repeated up to 16 times (4 pre-intervention, 12 post-intervention) or until death. In some animals, one or both carotid bodies were denervated. In some animals, the CB was electrically stimulated, both with and without MDR. Seizures were induced with kainic acid (KA). RESULTS: Animals without seizure and with no CB modulation survived all reflexes. Non-seizing animals with CB denervation survived 7.1 ± 5.4 reflexes before death, and only 1 of 7 survived past the 12-trial threshold. Electrical CB stimulation without seizure and without reflex caused significant tachypnea and hypotension. Electrical CB stimulation with seizure and without reflex required higher amplitudes to replicate the physiological responses seen outside seizure. Seizing animals without CB intervention survived 3.2 ± 3.6 trials (per-reflex survival rate 42.0% ± 44.4%), and 0 of 7 survived past the 12-trial threshold. Seizing animals with electrical CB stimulation survived 10.5 ± 4.7 ictal trials (per-reflex survival rate 86.3% ± 35.0%), and 6 of 8 survived past the 12-trial threshold. SIGNIFICANCE: These results suggest that, during seizure, the ability of the CB to stimulate a restart of respiration is impaired. The CB and its afferents may be relevant to fatal ictal apnea and SUDEP in humans, and CB stimulation may be a relevant intervention technique in these deaths.

Background Cladribine is a synthetic purine analogue that interferes with DNA synthesis and repair next to disrupting cellular proliferation in actively dividing lymphocytes. The compound is approved for the treatment of multiple sclerosis (MS). Cladribine can cross the blood–brain barrier, suggesting a potential effect on central nervous system (CNS) resident cells. Here, we explored compartment-specific immunosuppressive as well as potential direct neuroprotective effects of oral cladribine treatment in experimental autoimmune encephalomyelitis (EAE) mice. Methods In the current study, we compare immune cell frequencies and phenotypes in the periphery and CNS of EAE mice with distinct grey and white matter lesions (combined active and focal EAE) either orally treated with cladribine or vehicle, using flow cytometry. To evaluate potential direct neuroprotective effects, we assessed the integrity of the primary auditory cortex neuronal network by studying neuronal activity and spontaneous synaptic activity with electrophysiological techniques ex vivo. Results Oral cladribine treatment significantly attenuated clinical deficits in EAE mice. Ex vivo flow cytometry showed that cladribine administration led to peripheral immune cell depletion in a compartment-specific manner and reduced immune cell infiltration into the CNS. Histological evaluations revealed no significant differences for inflammatory lesion load following cladribine treatment compared to vehicle control. Single cell electrophysiology in acute brain slices was performed and showed an impact of cladribine treatment on intrinsic cellular firing patterns and spontaneous synaptic transmission in neurons of the primary auditory cortex. Here, cladribine administration in vivo partially restored cortical neuronal network function, reducing action potential firing. Both, the effect on immune cells and neuronal activity were transient. Conclusions Our results indicate that cladribine exerts a neuroprotective effect after crossing the blood–brain barrier independently of its peripheral immunosuppressant action.

A consensus is yet to be reached regarding the exact prevalence of epileptic seizures or epilepsy in multiple sclerosis (MS). In addition, the underlying pathophysiological basis of the reciprocal interaction among neuroinflammation, demyelination, and epilepsy remains unclear. Therefore, a better understanding of cellular and network mechanisms linking these pathologies is needed. Cuprizone-induced general demyelination in rodents is a valuable model for studying MS pathologies. Here, we studied the relationship among epileptic activity, loss of myelin, and pro-inflammatory cytokines by inducing acute, generalized demyelination in a genetic mouse model of human absence epilepsy, C3H/HeJ mice. Both cellular and network mechanisms were studied using in vivo and in vitro electrophysiological techniques. We found that acute, generalized demyelination in C3H/HeJ mice resulted in a lower number of spike–wave discharges, increased cortical theta oscillations, and reduction of slow rhythmic intrathalamic burst activity. In addition, generalized demyelination resulted in a significant reduction in the amplitude of the hyperpolarization-activated inward current (Ih) in thalamic relay cells, which was accompanied by lower surface expression of hyperpolarization-activated, cyclic nucleotide-gated channels, and the phosphorylated form of TRIP8b (pS237-TRIP8b). We suggest that demyelination-related changes in thalamic Ih may be one of the factors defining the prevalence of seizures in MS.

For many biological systems, a variety of simulation models exist. A new simulation model is rarely developed from scratch, but rather revises and extends an existing one. A key challenge, however, is to decide which model might be an appropriate starting point for a particular problem and why. To answer this question, we need to identify entities and activities that contributed to the development of a simulation model. Therefore, we exploit the provenance data model, PROV-DM, of the World Wide Web Consortium and, building on previous work, continue developing a PROV ontology for simulation studies. Based on a case study of 19 Wnt/β-catenin signaling models, we identify crucial entities and activities as well as useful metadata to both capture the provenance information from individual simulation studies and relate these forming a family of models. The approach is implemented in WebProv, a web application for inserting and querying provenance information. Our specialization of PROV-DM contains the entities Research Question, Assumption, Requirement, Qualitative Model, Simulation Model, Simulation Experiment, Simulation Data, and Wet-lab Data as well as activities referring to building, calibrating, validating, and analyzing a simulation model. We show that most Wnt simulation models are connected to other Wnt models by using (parts of) these models. However, the overlap, especially regarding the Wet-lab Data used for calibration or validation of the models is small. Making these aspects of developing a model explicit and queryable is an important step for assessing and reusing simulation models more effectively. Exposing this information helps to integrate a new simulation model within a family of existing ones and may lead to the development of more robust and valid simulation models. We hope that our approach becomes part of a standardization effort and that modelers adopt the benefits of provenance when considering or creating simulation models., Author summary We revise a provenance ontology for simulation studies of cellular biochemical models. Provenance information is useful for understanding the creation of a simulation model because it not only contains information about the entities and activities that have led to a simulation model but also their relations, all of which can be visualized. It provides additional structure by explicitly recording research questions, assumptions, and requirements and relating them along with data, qualitative models, simulation models, and simulation experiments through a small set of predefined but extensible activities. We have applied our concept to a family of 19 Wnt signaling models and implemented a web-based tool (WebProv) to store the provenance information from these studies. The resulting provenance graph visualizes the story line of simulation studies and demonstrates the creation and calibration of simulation models, the successive attempts of validation and extension, and shows, beyond an individual simulation study, how the Wnt models are related. Thereby, the steps and sources that contributed to a simulation model are made explicit. Our approach complements other approaches aimed at facilitating the reuse and assessment of simulation products in systems biology such as model repositories as well as annotation and documentation guidelines.

Capsule polymers are crucial virulence factors of pathogenic bacteria and are used as antigens in glycoconjugate vaccine formulations. Some Gram-negative pathogens express poly(glycosylglycerol phosphate) capsule polymers that resemble Gram-positive wall teichoic acids and are synthesized by TagF-like capsule polymerases. So far, the biotechnological use of these enzymes for vaccine developmental studies was restricted by the unavailability of enantiopure CDP-glycerol, one of the donor substrates required for polymer assembly. Here, we use CTP:glycerol-phosphate cytidylyltransferases (GCTs) and TagF-like polymerases to synthesize the poly(glycosylglycerol phosphate) capsule polymer backbones of the porcine pathogen Actinobacillus pleuropneumoniae, serotypes 3 and 7 (App3 and App7). GCT activity was confirmed by high-performance liquid chromatography, and polymers were analyzed using comprehensive nuclear magnetic resonance studies. Solid-phase synthesis protocols were established to allow potential scale-up of polymer production. In addition, one-pot reactions exploiting glycerol-kinase allowed us to start the reaction from inexpensive, widely available substrates. Finally, this study highlights that multidomain TagF-like polymerases can be transformed by mutagenesis of active site residues into single-action transferases, which in turn can act in trans to build-up structurally new polymers. Overall, our protocols provide enantiopure, nature-identical capsule polymer backbones from App2, App3, App7, App9, and App11, Neisseria meningitidis serogroup H, and Bibersteinia trehalosi serotypes T3 and T15.

With the rapidly emerging field of autologous therapies, Single-Use (SU) technologies are increasingly used in personalized medicine due to their manifold advantages. Although qualification of the starting material of autologous therapies such as the CAR-T process has been highlighted, little attention has been paid to the effect of leachables on cell-based therapies, even if recent studies indicate interactions of leachables with cells. To close this gap, this study presents a risk-analysis of SU-material on a CAR-T process and identifies hazards imposed by tubing materials and leachables thereof. In order to represent a CAR-T process in its entirety, two test systems, namely a lentivirus production process and primary T-cells, were used. While the effects on lentivirus production are comparable to those reported for antibody production processes in CHO cells, we found that PVC material and corresponding leachables, i.e. plasticizer, inhibit cell growth of primary T-cells to a great extent. Additionally, our results indicate that critical quality attributes are affected by the PVC material.

To evaluate if routine screening for aortic calcification using unenhanced CT lowers the risk of stroke and alters the surgical approach in patients undergoing general cardiac surgery compared with standard of care (SoC).In this prospective, multicenter, randomized controlled trial, adult patients scheduled for cardiac surgery from September 2014 to October 2019 were randomized 1:1 into two groups: SoC alone, including chest radiography, vs. SoC plus preoperative noncontrast CT. The primary endpoint was in-hospital perioperative stroke. Secondary endpoints were preoperative change of the surgical approach, in-hospital mortality, and postoperative delirium. The trial was halted halfway for expected futility, as the conditional power analysis showed a chance 1% of finding the hypothesized effect.A total of 862 patients were evaluated (SoC-group: 433 patients (66 ± 11 years; 74.1% male) vs. SoC + CT-group: 429 patients (66 ± 10 years; 69.9% male)). The perioperative stroke rate (SoC + CT: 2.1%, 9/429 vs. SoC: 1.2%, 5/433, p = 0.27) and rate of changed surgical approach (SoC + CT: 4.0% (17/429) vs. SoC: 2.8% (12/433, p = 0.35) did not differ between groups. In-hospital mortality and postoperative delirium were comparable between groups. In the SoC + CT group, aortic calcification was observed on CT in the ascending aorta in 28% (108/380) and in the aortic arch in 70% (265/379).Preoperative noncontrast CT in cardiac surgery candidates did not influence the surgical approach nor the incidence of perioperative stroke compared with standard of care. Aortic calcification is a frequent finding on the CT scan in these patients but results in major surgical alterations to prevent stroke in only few patients.• Aortic calcification is a frequent finding on noncontrast computed tomography prior to cardiac surgery. • Routine use of noncontrast computed tomography does not often lead to a change of the surgical approach, when compared to standard of care. • No effect was observed on perioperative stroke after cardiac surgery when using routine noncontrast computed tomography screening on top of standard of care.

The 2-pore potassium channel subfamily K member 9 (KCNK9) regulates intracellular calcium concentration and thus modulates cell survival and inflammatory signaling pathways. It also was recognized as a risk allele for inflammatory bowel disease. However, it remains unclear whether KCNK9 modulates inflammatory bowel disease via its impact on immune cell function or whether its influence on calcium homeostasis also is relevant in intestinal epithelial cells.Kcnk9Compared with controls, KCNK9 deficiency or its pharmacologic blockade were associated with aggravated DSS-induced colitis compared with wild-type animals. In the absence of KCNK9, intestinal epithelial cells showed increased intracellular calcium levels and were more prone to mitochondrial damage and caspase-9-dependent apoptosis. We found that expression of KCNK3 was increased in Kcnk9KCNK9 enhances mitochondrial stability, reduces apoptosis, und thus supports epithelial cell survival after DSS exposure in vivo and in vitro. Conversely, its increased expression in Kcnk3

Multicellular development depends on the differentiation of cells into specific fates with precise spatial organization. Lineage history plays a pivotal role in cell fate decisions, but is inaccessible in most contexts. Engineering cells to actively record lineage information in a format readablein situwould provide a spatially resolved view of lineage in diverse developmental processes. Here, we introduce a serine integrase-based recording system that allowsin situreadout, and demonstrate its ability to reconstruct lineage relationships in cultured stem cells and flies. The system, termed intMEMOIR, employs an array of independent three-state genetic memory elements that can recombine stochastically and irreversibly, allowing up to 59,049 distinct digital states. intMEMOIR accurately reconstructed lineage trees in stem cells and enabled simultaneous analysis of single cell clonal history, spatial position, and gene expression inDrosophilabrain sections. These results establish a foundation for microscopy-readable clonal analysis and recording in diverse systems.One sentence summaryA new genetic editing system termed intMEMOIR reveals the lineage histories of individual cells directly within their native tissue context.

Sudden death in epilepsy, or SUDEP, is a fatal condition that accounts for more than 4000 deaths each year. Limited clinical and preclinical data on sudden death suggests critical contributions from autonomic, cardiac, and respiratory pathways. Recent animal (rat) studies on kainic acid induced seizure models explored a potential mechanism for such sudden and severe cardiorespiratory dysregulation being linked to acid reflux induced laryngospasm. Here, we expand on those previous investigations and utilize a multimodal approach to provide visual evidence of acid reflux-initiated laryngospasm and subsequent fatal cardiorespiratory distress in seizing rats.We used systemic kainic acid to acutely induce seizure activity in Long Evans rats, under urethane anesthesia. We recorded electroencephalography (EEG), electrocardiography (ECG), chest plethysmography and esophageal pH signals during simultaneous fast MRI scans of the rat stomach and esophagus. MRI images, in conjunction with electrophysiology data were used to identify seizure progression, stomach acid movement up the esophagus, cardiorespiratory changes, and sudden death.In all cases of sudden death, esophageal pH recordings alongside MRI images visualized stomach acid movement up the esophagus. Severe cardiac (ST segment elevation), respiratory (intermittent apnea) and brain activity (EEG narrowing due to hypoxia) changes were observed only after acid reached the larynx, which strongly suggests onset of laryngospasm following acid reflux. Additionally, absence of stomach acid in the esophagus of animals that survived acute seizure, provided evidence of a causal relationship between acid reflux and sudden death. The complimentary information coming from electrophysiology and fast MRI scans provided insight into the mechanism of esophageal reflux, laryngospasm, obstructive apnea, and subsequent sudden death in seizing animals. The results carry clinical significance as they outline a potential mechanism that may be relevant to SUDEP in humans.

Despite the potential to guide clinical management of spinal cord injury and disease, noninvasive methods of monitoring perfusion status of the spinal cord clinically remain an unmet need. In this study, we optimized pseudo-continuous arterial spin labeling (pCASL) for the rodent cervical spinal cord and demonstrate its utility in identifying perfusion deficits in an acute contusion injury model. High-resolution perfusion sagittal images with reduced imaging artifacts were obtained with optimized background suppression and imaging readout. Following moderate contusion injury, perfusion was clearly and reliably decreased at the site of injury. Implementation of time-encoded pCASL confirmed injury site perfusion deficits with blood flow measurements corrected for variability in arterial transit times. The noninvasive protocol of pCASL in the spinal cord can be utilized in future applications to examine perfusion changes after therapeutic interventions in the rat and translation to patients may offer critical implications for patient management.

The presence of leachables in biopharmaceutical processes using single-use technologies (SUT) is well known. For the detection and quantification of the latter, extractable studies of SUT are very common nowadays. Although a mixture of compounds is regularly found in extractable studies, research has only been carried out regarding the effect of individual compounds on cell culture and the cumulative effect of a mix of leachables has not been investigated yet. In this study, a set of leachable model compounds (LMCs) was chosen and the effect of the LMCs on a Chinese hamster ovary DG44 cell line producing an IgG antibody was investigated concerning cell growth, cell cycle distribution and productivity. It was shown that even if worst-case concentrations were used, the LMCs solely impact cell growth. Additionally, interaction studies revealed that the inhibiting effect of the mix is lower than the expected cumulative effect. A strong antagonism between the antioxidant butylated hydroxytoluene and the plasticizer Tris(2-ethylhexyl)trimellitate was found using an isobologram analysis. © 2021 The Authors. Biotechnology Progress published by Wiley Periodicals LLC on behalf of American Institute of Chemical Engineers.

mTOR inhibitors offer advantages after kidney transplantation including antiviral and antitumor activity besides facilitating low calcineurin inhibitor exposure to reduce nephrotoxicity. Concerns about adverse effects due to antiproliferative and antiangiogenic properties have limited their clinical use particularly early after transplantation. Interference with vascular endothelial growth factor (VEGF)-A, important for physiologic functioning of renal endothelial cells and tubular epithelium, has been implicated in detrimental renal effects of mTOR inhibitors. Low doses of Rapamycin (loading dose 3 mg/kg bodyweight, daily doses 1.5 mg/kg bodyweight) were administered in an allogenic rat kidney transplantation model resulting in a mean through concentration of 4.30 ng/mL. Glomerular and peritubular capillaries, tubular cell proliferation, or functional recovery from preservation/reperfusion injury were not compromised in comparison to vehicle treated animals. VEGF-A, VEGF receptor 2, and the co-receptor Neuropilin-1 were upregulated by Rapamycin within 7 days. Rat proximal tubular cells (RPTC) responded in vitro to hypoxia with increased VEGF-A and VEGF-R1 expression that was not suppressed by Rapamycin at therapeutic concentrations. Rapamycin did not impair proliferation of RPTC under hypoxic conditions. Low-dose Rapamycin early posttransplant does not negatively influence the VEGF network crucial for recovery from preservation/reperfusion injury. Enhancement of VEGF signaling peritransplant holds potential to further improve outcomes.

More than 500 molecules have been identified as components of Cannabis sativa (C. sativa), of which the most studied is Δ

Maternal exposures during pregnancy affect the onset and progression of adult diseases in the offspring. A prior mouse study indicated that maternal tobacco smoke exposure affects hepatic fibrosis in adult offspring. Gutkha, a broadly used smokeless tobacco (ST) product, is widely used by pregnant woman in many countries. The objective of this murine study was to evaluate whether oral maternal exposure to gutkha during pregnancy alters non-alcoholic fatty liver disease (NAFLD) in adult offspring: risk factors for the progression of NAFLD to cirrhosis in adults remain elusive. Buccal cavity &lsquo, painting&rsquo, of pregnant mice with gutkha began on gestational days (GD) 2&ndash, 4 and continued until parturition. Beginning at 12 weeks of age, a subset of offspring were transitioned to a high-fat diet (HFD). Results demonstrated that prenatal exposure to gutkha followed by an HFD in adulthood significantly increased the histologic evidence of fatty liver disease only in adult male offspring. Changes in hepatic fibrosis-related cytokines (interleukin (IL)-1b and IL-6) and in hepatic collagen mRNA expression were observed when comparing adult male offspring exposed to gutkha in utero to those not exposed. These findings indicate that maternal use of gutkha during pregnancy affects NAFLD in adult offspring in a sex-dependent manner.

For in situ tissue engineering (TE) applications it is important that implant degradation proceeds in concord with neo-tissue formation to avoid graft failure. It will therefore be valuable to have an imaging contrast agent (CA) available that can report on the degrading implant. For this purpose, a biodegradable radiopaque biomaterial is presented, modularly composed of a bisurea chain-extended polycaprolactone (PCL2000-U4U) elastomer and a novel iodinated bisurea-modified CA additive (I-U4U). Supramolecular hydrogen bonding interactions between the components ensure their intimate mixing. Porous implant TE-grafts are prepared by simply electrospinning a solution containing PCL2000-U4U and I-U4U. Rats receive an aortic interposition graft, either composed of only PCL2000-U4U (control) or of PCL2000-U4U and I-U4U (test). The grafts are explanted for analysis at three time points over a 1-month period. Computed tomography imaging of the test group implants prior to explantation shows a decrease in iodide volume and density over time. Explant analysis also indicates scaffold degradation. (Immuno)histochemistry shows comparable cellular contents and a similar neo-tissue formation process for test and control group, demonstrating that the CA does not have apparent adverse effects. A supramolecular approach to create solid radiopaque biomaterials can therefore be used to noninvasively monitor the biodegradation of synthetic implants.

Hyperpolarization-activated cation channels are involved, among other functions, in learning and memory, control of synaptic transmission and epileptogenesis. The importance of the HCN1 and HCN2 isoforms for brain function has been demonstrated, while the role of HCN4, the third major neuronal HCN subunit, is not known. Here we show that HCN4 is essential for oscillatory activity in the thalamocortical (TC) network. HCN4 is selectively expressed in various thalamic nuclei, excluding the thalamic reticular nucleus. HCN4-deficient TC neurons revealed a massive reduction of Ih and strongly reduced intrinsic burst firing, whereas the current was normal in cortical pyramidal neurons. In addition, evoked bursting in a thalamic slice preparation was strongly reduced in the mutant mice probes. HCN4-deficiency also significantly slowed down thalamic and cortical oscillations during active wakefulness. Taken together, these results establish that thalamic HCN4 channels are essential for the production of rhythmic intrathalamic oscillations and determine regular TC oscillatory activity during alert states.

In the traumatically injured spinal cord, decreased perfusion is believed to contribute to secondary tissue damage beyond the primary mechanical impact, and restoration of perfusion is believed to be a promising therapeutic target. However, methods to monitor spinal cord perfusion non-invasively are limited. Perfusion magnetic resonance imaging (MRI) techniques established for the brain have not been routinely adopted to the spinal cord. The purpose of this study was to examine the relationship between spinal cord blood flow (SCBF) and injury severity in a rat thoracic spinal cord contusion injury (SCI) model using flow-sensitive alternating inversion recovery (FAIR) with two variants of the label position. SCBF as a marker of severity was compared to T1 mapping and to spinal cord-optimized diffusion weighted imaging (DWI) with filtered parallel apparent diffusion coefficient. Thirty-eight rats underwent a T10 contusion injury with varying severities (8 sham; 10 mild; 10 moderate; 10 severe) with MRI performed at 1 day post injury at the lesion site and follow-up neurological assessments using the Basso, Beattie, Bresnahan (BBB) locomotor scoring up to 28 days post injury. Using whole-cord regions of interest at the lesion epicenter, SCBF was decreased with injury severity and had a significant correlation with BBB scores at 28 days post injury. Importantly, estimates of arterial transit times (ATT) in the injured spinal cord were not altered after injury, which suggests that FAIR protocols optimized to measure SCBF provide more value in the context of acute traumatic injury to the cord. T1-relaxation time constants were strongly related to injury severity and had a larger extent of changes than either SCBF or DWI measures. These findings suggest that perfusion decreases in the spinal cord can be monitored non-invasively after injury, and multi-parametric MRI assessments of perfusion, diffusion, and relaxation capture unique features of the pathophysiology of preclinical injury.

Contains fulltext : 202738.pdf (Publisher’s version ) (Open Access) Sensory information processing and higher cognitive functions rely on the interactions between thalamus and cortex. Many types of neurological and psychiatric disorders are accompanied or driven by alterations in the brain connectivity. In the present study putative changes in functional and effective cortico-cortical (CC), thalamo-cortical (TC) and cortico-thalamic (CT) connectivity during wakefulness and slow-wave sleep in a model of thalamo-cortical dysrhythmia (TCD), TRIP8b-/- mice and in control (WT) mice are described. Coherence and nonlinear Granger Causality (GC) were calculated for twenty 10 s length epochs of slow-wave sleep and active-wakefulness of each animal. Coherence was reduced between 4 and ca 20 Hz in the cortex and between cortex and thalamus during slow-wave sleep compared to active-wakefulness in WT, but not in TRIP8b-/- mice. Moreover, TRIP8b-/- mice showed lower CT coherence during active-wakefulness compared to WT mice; these differences were no longer present during slow-wave sleep. Unconditional GC analysis also showed sleep related reductions in TC and CT couplings in WT mice, while TRIP8b-/- mice showed diminished wake and enhanced sleep CC coupling and rather strong CT directed coupling during wake and sleep, albeit smaller during sleep. Conditional GC coupling analysis confirmed the diminished CC and enhanced CT coupling in TRIP8b-/- mice. Our findings indicate that altered properties of HCN channels, characterizing TRIP8b-/- mice, have clear effects on CC, TC and CT networks. A more complete understanding of the function of the altered communication within these networks awaits detailed phenotyping of TRIP8b-/- mice aimed at specifics of sensory and attentional processes. 12 p.

Diffusion tensor distribution (DTD) imaging builds on principles from diffusion, solid‐state and low‐field NMR spectroscopies, to quantify the contents of heterogeneous voxels as nonparametric distributions, with tensor “size”, “shape” and orientation having direct relations to corresponding microstructural properties of biological tissues. The approach requires the acquisition of multiple images as a function of the magnitude, shape and direction of the diffusion‐encoding gradients, leading to long acquisition times unless fast image read‐out techniques like EPI are employed. While in previous in vivo human brain studies performed at 3 T this proved a viable option, porting these measurements to very high magnetic fields and/or to heterogeneous organs induces B 0‐ and B 1‐inhomogeneity artifacts that challenge the limits of EPI. To overcome such challenges, we demonstrate here that high spatial resolution DTD of mouse brain can be carried out at 15.2 T with a surface‐cryoprobe, by relying on SPatiotemporal ENcoding (SPEN) imaging sequences. These new acquisition and data‐processing protocols are demonstrated with measurements on in vivo mouse brain, and validated with synthetic phantoms designed to mimic the diffusion properties of white matter, gray matter and cerebrospinal fluid. While still in need of full extensions to 3D mappings and of scanning additional animals to extract more general physiological conclusions, this work represents another step towards the model‐free, noninvasive in vivo characterization of tissue microstructure and heterogeneity in animal models, at ≈0.1 mm resolutions., Diffusion tensor distribution (DTD) imaging allows to quantify the heterogeneity of tissues in terms of local “sizes”, “shapes” and orientations, which are in turn directly related to the microstructural properties of biological tissues. Here, a high‐definition DTD analysis of a mouse brain is presented, carried out at 15.2 T with a surface‐cryoprobe and relying on a SPatiotemporal ENcoding (SPEN) imaging sequence. These results enabled the model‐free, noninvasive characterization of tissue microstructure and heterogeneity in live animals, at ≈0.1 mm resolutions.