Developmental effect of RASopathy mutations on neuronal network activity on a chip.

RASopathies are a group of genetic disorders caused by mutations in genes encoding components and regulators of the RAS/MAPK signaling pathway, resulting in overactivation of signaling. RASopathy patients exhibit distinctive facial features, cardiopathies, growth and skeletal abnormalities, and varying degrees of neurocognitive impairments including neurodevelopmental delay, intellectual disabilities, or attention deficits. At present, it is unclear how RASopathy mutations cause neurocognitive impairment and what their neuron-specific cellular and network phenotypes are. Here, we investigated the effect of RASopathy mutations on the establishment and functional maturation of neuronal networks. We isolated cortical neurons from RASopathy mouse models, cultured them on multielectrode arrays and performed longitudinal recordings of spontaneous activity in developing networks as well as recordings of evoked responses in mature neurons. To facilitate the analysis of large and complex data sets resulting from long-term multielectrode recordings, we developed MATLAB-based tools for data processing, analysis, and statistical evaluation. Longitudinal analysis of spontaneous network activity revealed a convergent developmental phenotype in neurons carrying the gain-of-function Noonan syndrome-related mutations Ptpn11 D61Y and Kras V14l. The phenotype was more pronounced at the earlier time points and faded out over time, suggesting the emergence of compensatory mechanisms during network maturation. Nevertheless, persistent differences in excitatory/inhibitory balance and network excitability were observed in mature networks. This study improves the understanding of the complex relationship between genetic mutations and clinical manifestations in RASopathies by adding insights into functional network processes as an additional piece of the puzzle.

The dynamics of the female microbiome: unveiling abrupt changes of microbial domains across body sites from prepartum to postpartum phases.

The microbial ecosystem of women undergoes enormous changes during pregnancy and the perinatal period. Little is known about the extent of changes in the maternal microbiome beyond the vaginal cavity and its recovery after birth. In this study, we followed pregnant women [maternal prepartum (mpre), n = 30] into the postpartum period [1 month postpartum, maternal postpartum (mpost), n = 30]. We profiled their oral, urinary, and vaginal microbiome; archaeome; mycobiome; and urinary metabolome and compared them with those of nonpregnant (np) women (n = 29). Overall, pregnancy status (np, mpre, and mpost) had a smaller effect on the microbiomes than body site, but massive transitions were observed for the oral and urogenital (vaginal and urinary) microbiomes. While the oral microbiome fluctuates during pregnancy but stabilizes rapidly within the first month postpartum, the urogenital microbiome is characterized by a major remodeling caused by a massive loss of Lactobacillus and thus a shift from Vaginal Community State Type (CST) I (40% of women) to CST IV (85% of women). The urinary metabolome rapidly reached an np-like composition after delivery, apart from lactose and oxaloacetic acid, which were elevated during active lactation. Fungal and archaeal profiles were indicative of pregnancy status. Methanobacterium signatures were found mainly in np women, and Methanobrevibacter showed an opposite behavior in the oral cavity (increased) and vagina (decreased) during pregnancy. Our findings suggest that the massive remodeling of the maternal microbiome and metabolome needs more attention and that potential interventions could be envisioned to optimize recovery and avoid long-term effects on maternal health and subsequent pregnancies. IMPORTANCE: The perinatal microbiome is of specific interest for the health of the mother and infant. We therefore investigate the dynamics of the female microbiome from nonpregnant over prepartum to the postpartum period in urine and the oral and vaginal cavities. A specific focus of this study is put not only on the bacterial part of the microbiome but also on the underinvestigated contribution of fungi and archaea. To our knowledge, we present the first study highlighting those aspects. Our findings suggest that the massive remodeling of the maternal microbiome and metabolome needs more attention and that potential interventions could be envisioned to optimize recovery and avoid long-term effects on maternal health and subsequent pregnancies.

Hydroxynorketamine, but not ketamine, acts via α7 nicotinic acetylcholine receptor to control presynaptic function and gene expression.

Ketamine is clinically used fast-acting antidepressant. Its metabolite hydroxynorketamine (HNK) shows a robust antidepressant effect in animal studies. It is unclear, how these chemically distinct compounds converge on similar neuronal effects. While KET acts mostly as N-methyl-d-aspartate receptor (NMDAR) antagonist, the molecular target of HNK remains enigmatic. Here, we show that KET and HNK converge on rapid inhibition of glutamate release by reducing the release competence of synaptic vesicles and induce nuclear translocation of pCREB that controls expression of neuroplasticity genes connected to KET- and HNK-mediated antidepressant action. Ro25-6981, a selective antagonist of GluN2B, mimics effect of KET indicating that GluN2B-containing NMDAR might mediate the presynaptic effect of KET. Selective antagonist of α7 nicotinic acetylcholine receptors (α7nAChRs) or genetic deletion of Chrna7, its pore-forming subunit, fully abolishes HNK-induced synaptic and nuclear regulations, but leaves KET-dependent cellular effects unaffected. Thus, KET or HNK-induced modulation of synaptic transmission and nuclear translocation of pCREB can be mediated by selective signaling via NMDAR or α7nAChRs, respectively. Due to the rapid metabolism of KET to HNK, it is conceivable that subsequent modulation of glutamatergic and cholinergic neurotransmission affects circuits in a cell-type-specific manner and contributes to the therapeutic potency of KET. This finding promotes further exploration of new combined medications for mood disorders.

Macrophages influence Schwann cell myelin autophagy after nerve injury and in a model of Charcot-Marie-Tooth disease.

BACKGROUND AND AIMS: The complex cellular and molecular interactions between Schwann cells (SCs) and macrophages during Wallerian degeneration are a prerequisite to allow rapid uptake and degradation of myelin debris and axonal regeneration after peripheral nerve injury. In contrast, in non-injured nerves of Charcot-Marie-Tooth 1 neuropathies, aberrant macrophage activation by SCs carrying myelin gene defects is a disease amplifier that drives nerve damage and subsequent functional decline. Consequently, targeting nerve macrophages might be a translatable treatment strategy to mitigate disease outcome in CMT1 patients. Indeed, in previous approaches, macrophage targeting alleviated the axonopathy and promoted sprouting of damaged fibers. Surprisingly, this was still accompanied by robust myelinopathy in a model for CMT1X, suggesting additional cellular mechanisms of myelin degradation in mutant peripheral nerves. We here investigated the possibility of an increased SC-related myelin autophagy upon macrophage targeting in Cx32def mice. METHODS: Combining ex vivo and in vivo approaches, macrophages were targeted by PLX5622 treatment. SC autophagy was investigated by immunohistochemical and electron microscopical techniques. RESULTS: We demonstrate a robust upregulation of markers for SC autophagy after injury and in genetically-mediated neuropathy when nerve macrophages are pharmacologically depleted. Corroborating these findings, we provide ultrastructural evidence for increased SC myelin autophagy upon treatment in vivo. INTERPRETATION: These findings reveal a novel communication and interaction between SCs and macrophages. This identification of alternative pathways of myelin degradation may have important implications for a better understanding of therapeutic mechanisms of pharmacological macrophage targeting in diseased peripheral nerves.

Enhanced recovery after cesarean section (ERAC): Where are we in Austria?

OBJECTIVE: Enhanced recovery after surgery (ERAS) recommendations for cesarean section (ERAC), likely the most common reason for laparotomy in women, were issued in 2018-19. We examined how current perioperative management at cesarean section in Austrian hospitals aligns with ERAS recommendations. STUDY DESIGN: We surveyed the 21 largest public obstetric units in Austria for alignment with 20 of the 31 strong ERAS recommendations regarding perioperative maternal care at cesarean section. We also looked at how the German-language clinical guideline for cesarean section (AWMF Guideline Sectio caesarea) aligns with ERAS recommendations. RESULTS: The 21 obstetric units cared for about 51% of all births in Austria in 2019. Cesarean section rates ranged from 17.7% to 50.4%. All 21 units implemented the five strong recommendations regarding patient information and counselling, regional anesthesia, euvolemia and multimodal analgesia. The least implemented strong recommendation was the one for the use of pneumatic compression stockings to prevent thromboembolic disease (0/21 units). Overall, all 21 units implemented ≥11 and 13 (62%) implemented ≥15 (≥75%) of the 20 strong recommendations; no unit implemented all 20 strong recommendations. There were no differences in the implementation of strong recommendations according to hospital volume. CONCLUSIONS: Even in the absence of formal adoption of ERAS program for cesarean section many perioperative ERAS recommendations are already implemented in Austria. The least implemented recommendations were the use of pneumatic compression stockings (0 of 21 units) and immediate catheter removal (4 of 21 units). Only 10 of the 20 ERAS recommendations we looked at are included in the current German-language clinical guideline for cesarean section.

Development and Application of Automatized Routines for Optical Analysis of Synaptic Activity Evoked by Chemical and Electrical Stimulation.

The recent development of cellular imaging techniques and the application of genetically encoded sensors of neuronal activity led to significant methodological progress in neurobiological studies. These methods often result in complex and large data sets consisting of image stacks or sets of multichannel fluorescent images. The detection of synapses, visualized by fluorescence labeling, is one major challenge in the analysis of these datasets, due to variations in synapse shape, size, and fluorescence intensity across the images. For their detection, most labs use manual or semi-manual techniques that are time-consuming and error-prone. We developed SynEdgeWs, a MATLAB-based segmentation algorithm that combines the application of an edge filter, morphological operators, and marker-controlled watershed segmentation. SynEdgeWs does not need training data and works with low user intervention. It was superior to methods based on cutoff thresholds and local maximum guided approaches in a realistic set of data. We implemented SynEdgeWs in two automatized routines that allow accurate, direct, and unbiased identification of fluorescently labeled synaptic puncta and their consecutive analysis. SynEval routine enables the analysis of three-channel images, and ImgSegRout routine processes image stacks. We tested the feasibility of ImgSegRout on a realistic live-cell imaging data set from experiments designed to monitor neurotransmitter release using synaptic phluorins. Finally, we applied SynEval to compare synaptic vesicle recycling evoked by electrical field stimulation and chemical depolarization in dissociated cortical cultures. Our data indicate that while the proportion of active synapses does not differ between stimulation modes, significantly more vesicles are mobilized upon chemical depolarization.

The Antiplatelet Action of S-Nitroso Human Serum Albumin in Whole Blood.

Nitric oxide donors (NO-donors) have been shown to have therapeutic potential (e.g., ischemia/reperfusion injury). However, due to their release rate/antiplatelet properties, they may cause bleeding in patients. We therefore studied the antiplatelet effects of the two different NO-donors, i.e., S-NO-Human Serum Albumin (S-NO-HSA) and Diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA-NONOate) in whole blood (WB) samples. WB samples were spiked with S-NO-HSA or DEA-NONOate (100 µmol/L or 200 µmol/L), and the NO release rate (nitrite/nitrate levels via HPLC) and antiplatelet efficacy (impedance aggregometry, platelet function analyzer, Cone-and-platelet analyzer, thrombelastometry) were assessed. S-NO-HSA had a significantly lower NO release compared to equimolar concentrations of DEA-NONOate. Virtually no antiplatelet action of S-NO-HSA was observed in WB samples, whereas DEA-NONOate significantly attenuated platelet function in WB. Impedance aggregometry measurements revealed that Amplitudes (slope: -0.04022 ± 0.01045 ohm/µmol/L, p = 0.008) and Lag times (slope: 0.6389 ± 0.2075 s/µmol/L, p = 0.0051) were dose-dependently decreased and prolonged by DEA-NONOate. Closure times (Cone-and-platelet analyzer) were dose-dependently prolonged (slope: 0.3738 ± 0.1403 s/µmol/L, p = 0.0174 with collagen/ADP coating; slope: -0.5340 ± 0.1473 s/µmol/L, p = 0.0019 with collagen/epinephrine coating) by DEA-NONOate. These results in WB further support the pharmacological potential of S-NO-HSA as an NO-donor due to its ability to presumably prevent bleeding events even at high concentrations up to 200 µmol/L.

In vitro functional analysis of gRNA sites regulating assembly of hepatitis B virus.

The roles of RNA sequence/structure motifs, Packaging Signals (PSs), for regulating assembly of an HBV genome transcript have been investigated in an efficient in vitro assay containing only core protein (Cp) and RNA. Variants of three conserved PSs, within the genome of a strain not used previously, preventing correct presentation of a Cp-recognition loop motif are differentially deleterious for assembly of nucleocapsid-like particles (NCPs). Cryo-electron microscopy reconstruction of the T = 4 NCPs formed with the wild-type gRNA transcript, reveal that the interior of the Cp shell is in contact with lower resolution density, potentially encompassing the arginine-rich protein domains and gRNA. Symmetry relaxation followed by asymmetric reconstruction reveal that such contacts are made at every symmetry axis. We infer from their regulation of assembly that some of these contacts would involve gRNA PSs, and confirmed this by X-ray RNA footprinting. Mutation of the ε stem-loop in the gRNA, where polymerase binds in vivo, produces a poor RNA assembly substrate with Cp alone, largely due to alterations in its conformation. The results show that RNA PSs regulate assembly of HBV genomic transcripts in vitro, and therefore may play similar roles in vivo, in concert with other molecular factors.

Physical exercise mitigates neuropathic changes in an animal model for Charcot-Marie-Tooth disease 1X.

Inherited neuropathies of the Charcot-Marie-Tooth (CMT) type 1 are still untreatable diseases of the peripheral nervous system. We have previously shown that macrophages substantially amplify neuropathic changes in various mouse models of CMT1 subforms and that targeting innate immune cells substantially ameliorates disease outcome. However, up to date, specific approaches targeting macrophages pharmacologically might entail side effects. Here, we investigate whether physical exercise dampens peripheral nerve inflammation in a model for an X-linked dominant form of CMT1 (CMT1X) and whether this improves neuropathological and clinical outcome subsequently. We found a moderate, but significant decline in the number of macrophages and an altered macrophage activation upon voluntary wheel running. These observations were accompanied by an improved clinical outcome and axonal preservation. Most interestingly, exercise restriction by ~40% accelerated amelioration of clinical outcome and further improved nerve structure by increasing myelin thickness compared to the unrestricted running group. This myelin-preserving effect of limited exercise was accompanied by an elevated expression of brain-derived neurotrophic factor (BDNF) in peripheral nerves, while the expression of other trophic factors like neuregulin-1, glial cell line-derived neurotrophic factor (GDNF) or insulin-like growth factor 1 (IGF-1) were not influenced by any mode of exercise. We demonstrate for the first time that exercise dampens inflammation and improves nerve structure in a mouse model for CMT1, likely leading to improved clinical outcome. Reducing the amount of exercise does not automatically decrease treatment efficacy, reflecting the need of optimally designed exercise studies to achieve safe and effective treatment options for CMT1 patients.

Aß1-16 controls synaptic vesicle pools at excitatory synapses via cholinergic modulation of synapsin phosphorylation.

Amyloid beta (Aß) is linked to the pathology of Alzheimer's disease (AD). At physiological concentrations, Aß was proposed to enhance neuroplasticity and memory formation by increasing the neurotransmitter release from presynapse. However, the exact mechanisms underlying this presynaptic effect as well as specific contribution of endogenously occurring Aß isoforms remain unclear. Here, we demonstrate that Aß1-42 and Aß1-16, but not Aß17-42, increased size of the recycling pool of synaptic vesicles (SV). This presynaptic effect was driven by enhancement of endogenous cholinergic signalling via α7 nicotinic acetylcholine receptors, which led to activation of calcineurin, dephosphorylation of synapsin 1 and consequently resulted in reorganization of functional pools of SV increasing their availability for sustained neurotransmission. Our results identify synapsin 1 as a molecular target of Aß and reveal an effect of physiological concentrations of Aß on cholinergic modulation of glutamatergic neurotransmission. These findings provide new mechanistic insights in cholinergic dysfunction observed in AD.

Presynaptic vesicular accumulation is required for antipsychotic efficacy in psychotic-like rats.

BACKGROUND: The therapeutic effects of antipsychotic drugs (APDs) are mainly attributed to their postsynaptic inhibitory functions on the dopamine D2 receptor, which, however, cannot explain the delayed onset of full therapeutic efficacy. It was previously shown that APDs accumulate in presynaptic vesicles during chronic treatment and are released like neurotransmitters in an activity-dependent manner triggering an auto-inhibitory feedback mechanism. Although closely mirroring therapeutic action onset, the functional consequence of the APD accumulation process remained unclear. AIMS: Here we tested whether the accumulation of the APD haloperidol (HAL) is required for full therapeutic action in psychotic-like rats. METHODS: We designed a HAL analog compound (HAL-F), which lacks the accumulation property of HAL, but retains its postsynaptic inhibitory action on dopamine D2 receptors. RESULTS/OUTCOMES: By perfusing LysoTracker fluorophore-stained cultured hippocampal neurons, we confirmed the accumulation of HAL and the non-accumulation of HAL-F. In an amphetamine hypersensitization psychosis-like model in rats, we found that subchronic intracerebroventricularly delivered HAL (0.1 mg/kg/day), but not HAL-F (0.3-1.5 mg/kg/day), attenuates psychotic-like behavior in rats. CONCLUSIONS/INTERPRETATION: These findings suggest the presynaptic accumulation of HAL may serve as an essential prerequisite for its full antipsychotic action and may explain the time course of APD action. Targeting accumulation properties of APDs may, thus, become a new strategy to improve APD action.

Human Milk Oligosaccharides Modulate the Risk for Preterm Birth in a Microbiome-Dependent and -Independent Manner.

Preterm birth (PTB) is one of the leading causes of neonatal mortality. The causes for spontaneous PTB are multifactorial and often remain unknown. In this study, we tested the hypothesis that human milk oligosaccharides (HMOs) in blood and urine modulate the maternal urinary and vaginal microbiome and influence the risk for PTB. We analyzed the vaginal and urinary microbiome of a cross-sectional cohort of women with or without preterm labor and correlated our findings with measurements of metabolites and HMOs in urine and blood. We identified several microbial signatures, such as Lactobacillus jensenii, L. gasseri, Ureaplasma sp., and Gardnerella sp., associated with a short cervix, PTB, and/or preterm contractions. In addition, we observed associations between sialylated HMOs, in particular 3'-sialyllactose, with PTB, short cervix, and increased inflammation and confirmed an influence of HMOs on the microbiome profile. Since they identify serum and urinary HMOs and several key microorganisms associated with PTB, our findings point at two distinct processes modulating the risk for PTB. One process seems to be driven by sterile inflammation, characterized by increased concentrations of sialylated HMOs in serum. Another process might be microbiome mediated and potentially associated with specific HMO signatures in urine. Our results support current efforts to improve diagnostics and therapeutic strategies in PTB.IMPORTANCE The causes for preterm birth (PTB) often remain elusive. We investigated whether circulating human milk oligosaccharides (HMOs) might be involved in modulating urinary and vaginal microbiome promoting or preventing PTB. We identified here HMOs and key microbial taxa associated with indicators of PTB. Based on our results, we propose two models for how HMOs might modulate risk for PTB: (i) by changes in HMOs associated with sterile inflammation (microbiome-independent) and (ii) by HMO-driven shifts in microbiome (microbiome-dependent). Our findings will guide current efforts to better predict the risk for PTB in seemingly healthy pregnant women and also provide appropriate preventive strategies.

Tumor Cell-Based Vaccine Generated With High Hydrostatic Pressure Synergizes With Radiotherapy by Generating a Favorable Anti-tumor Immune Microenvironment.

Dendritic cell (DC)-based vaccines pulsed with high hydrostatic pressure (HHP)-inactivated tumor cells have been demonstrated to be a promising immunotherapy for solid tumors. We focused on sole injection of tumor cells that were inactivated by HHP and their combination with local radiotherapy (RTx) for in vivo induction of anti-tumor immune responses. HHP-treatment of tumor cells resulted in pre-dominantly necrotic cells with degraded DNA. We confirmed that treatments at 200 MPa or higher completely inhibited the formation of tumor cell colonies in vitro. No tumor growth was seen in vivo after injection of HHP-treated tumor cells. Single vaccination with HHP-killed tumor cells combined with local RTx significantly retarded tumor growth and improved the survival as shown in B16-F10 and CT26 tumor models. In B16-F10 tumors that were irradiated with 2 × 5Gy and vaccinated once with HHP-killed tumor cells, the amount of natural killer (NK) cells, monocytes/macrophages, CD4+ T cells and NKT cells was significantly increased, while the amount of B cells was significantly decreased. In both models, a trend of increased CD8+ T cell infiltration was observed. Generally, in irradiated tumors high amounts of CD4+ and CD8+ T cells expressing PD-1 were found. We conclude that HHP generates inactivated tumor cells that can be used as a tumor vaccine. Moreover, we show for the first time that tumor cell-based vaccine acts synergistically with RTx to significantly retard tumor growth by generating a favorable anti-tumor immune microenvironment.

New insights into neonatal coagulation: normal clot formation despite lower intra-clot thrombin levels.

BACKGROUND: Healthy neonates exhibit no bleeding tendencies, but exhibit longer partial thromboplastin times than adults. Lower clotting factor levels may be balanced by lower inhibitor levels, which is not reflected in routine coagulation assays, but could result in normal clot formation in vivo. The novel thrombodynamics assay simulates a damaged vessel with tissue factor immobilized to a surface. We hypothesized that intra-clot thrombin levels and spatial fibrin clot formation with this assay are comparable in neonates and adults. METHODS: Coagulation was tested in plasma from venous neonatal blood (N = 12), cord blood (N = 30), and adult blood (N = 20) using thrombodynamics and calibrated automated thrombography. RESULTS: Neonates exhibited a higher initial rate of clot formation than adults (adult: 60.7 ± 3.9 µm/min; neonatal: 66.8 ± 3.9 µm/min; cord: 68.1 ± 3.3 µm/min; P < 0.001) and a comparable stationary rate of clot formation (adult: 35.8 ± 8.5 µm/min; neonatal: 37.0 ± 4.6 µm/min; cord: 36.0 ± 5.2 µm/min; P = 0.834). Intra-clot thrombin levels were lower in neonates (adult: 41.9 ± 11.2 AU/l; neonatal: 22.6 ± 10.2 AU/l; cord: 23.6 ± 9.7 AU/l; P < 0.001), but the longitudinal rate of thrombin propagation was comparable (adult: 27.2 ± 4.2 µm/min neonatal; 27.9 ± 2.9 µm/min; cord: 27.6 ± 3.4 µm/min; P = 0.862). CONCLUSIONS: Despite lower intra-clot thrombin levels, neonates exhibit normal spatial fibrin clot growth, which concurs with clinically well-functioning hemostasis in healthy neonates.

RNA processing bodies are disassembled during Old World alphavirus infection.

RNA processing bodies (P-bodies) are non-membranous cytoplasmic aggregates of mRNA and proteins involved in mRNA decay and translation repression. P-bodies actively respond to environmental stresses, associated with another type of RNA granules, known as stress granules (SGs). Alphaviruses were previously shown to block SG induction at late stages of infection, which is important for efficient viral growth. In this study, we found that P-bodies were disassembled or reduced in number very early in infection with Semliki Forest virus (SFV) or chikungunya virus (CHIKV) in a panel of cell lines. Similar to SGs, reinduction of P-bodies by a second stress (sodium arsenite) was also blocked in infected cells. The disassembly of P-bodies still occurred in non-phosphorylatable eIF2α mouse embryonal fibroblasts (MEFs) that are impaired in SG assembly. Studies of translation status by ribopuromycylation showed that P-body disassembly is independent of host translation shutoff, which requires the phosphorylation of eIF2α in the SFV- or CHIKV-infected cells. Labelling of newly synthesized RNA with bromo-UTP showed that host transcription shutoff correlated with P-body disassembly at the same early stage (3-4 h) after infection. However, inhibition of global transcription with actinomycin D (ActD) failed to disassemble P-bodies as effectively as the viruses did. Interestingly, blocking nuclear import with importazole led to an efficient P-bodies loss. Our data reveal that P-bodies are disassembled independently from SG formation at early stages of Old World alphavirus infection and that nuclear import is involved in the dynamic of P-bodies.

Phytate degradation, intestinal microbiota, microbial metabolites and immune values are changed in growing pigs fed diets with varying calcium-phosphorus concentration and fermentable substrates.

The present study assessed effects of diets containing varying calcium-phosphorus (CaP) concentration and fermentable substrates on digestibility of diets, intestinal microbiota and immune system using 32 crossbred pigs (initial BW 54.7 kg). In a 2 × 2 factorial arrangement, pigs were fed either a corn-soybean meal (CSB) or corn-field pea (CFP) diet with either low [-] (4.4 g Ca/kg; 4.2 g total P/kg) or high [+] (8.3 g Ca/kg; 7.5 g total P/kg; supplemented with monocalcium phosphate) CaP content for a period of 9 weeks. In week 8, blood samples were taken, and at the end of the trial, all pigs were euthanized to collect digesta and mesenteric lymphoid tissue. Apparent total tract digestibility (ATTD) of P was greater (p < 0.05) for pigs fed the CaP+ and CFP diets than CaP- and CSB diets. The myo-inositol 1,2,3,4,5,6-hexakis (dihydrogen phosphate) (InsP6 ) concentration in jejunal digesta was higher (p < 0.05) for CaP+ than in CaP- fed pigs. In addition, caecal and faecal InsP5 isomer concentration were greater (p < 0.05) for CSB than CFP diets. In the caecum, gene copy numbers of saccharolytic bacteria, such as Eubacterium rectale and Roseburia spp., as well as SCFA concentration were higher (p < 0.05) for CaP+ than CaP- diets. In particular, innate immune cell numbers, such as natural killer cells, dendritic cells, monocytes and neutrophils, were greater (p < 0.05) for CaP+ than CaP- fed pigs. Diets high in CaP resulted in higher abundance of potential beneficial bacteria and might promote the first line of defence enhancing the activation of the cellular adaptive immune response, thereby possibly decreasing the risk for intestinal disturbances. These results strongly suggest that both, CaP supply and dietary ingredients differing in fermentability, may beneficially affect gut health through increase in SCFA-producing bacteria and/or bacteria with anti-inflammatory properties.

The conundrum of postpartum thrombotic Microangiopathy: case report and considerations for management.

BACKGROUND: Microangiopathic hemolytic anemias and thrombocytopenias in pregnant or postpartum women constitute an interdisciplinary diagnostic and therapeutic challenge in the evaluation of thrombotic microangiopathies (TMA), where urgent care must be considered. CASE PRESENTATION: We here report the case of a 21-year-old Somali woman, who was delivered by emergency caesarean section at 35 weeks of gestational age with acute dyspnea, placental abruption and gross edema due to severe preeclampsia/HELLP syndrome. After delivery, she developed acute kidney failure and thrombotic microangiopathy as revealed by kidney biopsy. The lack of early response to plasma exchange prompted extensive laboratory workup. Ultimately, the patient completely recovered with negative fluid balance and control of severe hypertension. CONCLUSIONS: This case report emphasizes the importance to differentiate between primary TMA syndromes and microangiopathic hemolytic anemias due to systemic disorders. Delayed recovery from preeclampsia/HELLP syndrome and malignant hypertension can clinically mimic primary TMA syndromes in the postpartum period.

Polyphosphate in Neonates: Less Shedding from Platelets and Divergent Prothrombotic Capacity Due to Lower TFPI Levels.

Background: The neonatal hemostatic system exhibits a fragile balance featuring lower levels of clotting factors as well as inhibitors. Neonatal platelets show in-vitro hypoaggregability, but neonates exhibit well-functioning primary and secondary hemostasis despite this impairment. Recently, polyphosphate shed by activated platelets has been shown to induce a prothrombotic shift on the plasmatic coagulation system of adults. The impact of platelet derived polyphosphate might differ in neonates due to aforementioned peculiarities. Aims: We aimed to comparatively determine polyphosphate content and release from adult and neonatal platelets and to determine its impact on thrombin generation in plasma from adult and cord blood. Methods: Polyphosphate was extracted from adult and neonatal platelet lysates and releasates using silica spin-columns and quantified with a DAPI based fluorescence assay. The impact of exogenous polyphosphate in various concentrations (208-0.026 µg/ml) on thrombin generation was evaluated in plasma from adult and cord blood as well as in adult plasma with reduced tissue factor pathway inhibitor (TFPI) levels using calibrated automated thrombography. Results: Polyphosphate content was comparable in both groups, but the fraction of released polyphosphate upon stimulation with thrombin receptor activating peptide was lower in neonatal samples (adult: 84.1 ± 12.9%; cord: 58.8 ± 11.2%). Relative impact of polyphosphate on lag time of thrombin generation was higher in adult samples compared to samples from cord blood (adult: 41.0% [IQR: 35.2-71.8%] of vehicle; cord: 73.4% [IQR: 70.2-91.4%] of vehicle). However, in samples from cord blood, lower concentrations of polyphosphate were required to obtain maximal impact on thrombin generation (adult: 26 µg/ml; cord: 0.814 µg/ml). PolyP affected thrombin generation in adult plasma similarly to cord plasma, when the TFPI concentration was reduced to neonatal levels. Conclusion: Differences in the impact of polyphosphate on adult and neonatal coagulation are largely caused by differences in TFPI levels. Lower polyphosphate release from neonatal platelets, but lower optimum concentration to drive neonatal plasmatic hemostasis emphasizes the well-matched, but fragile interplay between platelets and coagulation in newborns. A potential developmental mismatch should be considered when transfusing adult platelets into neonates.

HBV RNA pre-genome encodes specific motifs that mediate interactions with the viral core protein that promote nucleocapsid assembly.

Formation of the hepatitis B virus nucleocapsid is an essential step in the viral lifecycle, but its assembly is not fully understood. We report the discovery of sequence-specific interactions between the viral pre-genome and the hepatitis B core protein that play roles in defining the nucleocapsid assembly pathway. Using RNA SELEX and bioinformatics, we identified multiple regions in the pre-genomic RNA with high affinity for core protein dimers. These RNAs form stem-loops with a conserved loop motif that trigger sequence-specific assembly of virus-like particles (VLPs) at much higher fidelity and yield than in the absence of RNA. The RNA oligos do not interact with preformed RNA-free VLPs, so their effects must occur during particle assembly. Asymmetric cryo-electron microscopy reconstruction of the T = 4 VLPs assembled in the presence of one of the RNAs reveals a unique internal feature connected to the main core protein shell via lobes of density. Biophysical assays suggest that this is a complex involving several RNA oligos interacting with the C-terminal arginine-rich domains of core protein. These core protein-RNA contacts may play one or more roles in regulating the organization of the pre-genome during nucleocapsid assembly, facilitating subsequent reverse transcription and acting as a nucleation complex for nucleocapsid assembly.

Short- and long-term perinatal outcome in twin pregnancies affected by weight discordance.

INTRODUCTION: The objective was to investigate the association between chorionicity-specific intertwin birthweight discordance and adverse outcomes including long-term follow up at 6, 18, and 48-60 months after term via Ages and Stages Questionnaire. MATERIAL AND METHODS: In this secondary analysis of a cohort study (Oldenburg et al., n = 1688) and a randomized controlled trial (PREDICT study, n = 1045) twin pairs were divided into three groups according to chorionicity-specific birthweight discordance: <75th percentile, 75th-90th percentile and >90th percentile. Information on infant mortality, admittance to neonatal intensive care units, and gestational age at delivery was available for all pairs. Detailed neonatal outcomes were available for 656 pairs from PREDICT, of which 567 pairs had at least one Ages and Stages Questionnair follow-up. Logistic regression models were used for dichotomous outcomes. Ages and Stages Questionnair scores were compared using the method of generalized estimating equation to account for the correlation within twins. RESULTS: The 75th and 90th percentiles for birthweight discordance were 14.8 and 21.4% for monochorionic and 16.0 and 23.8% for dichorionic twins. After adjustment for small for gestational age and gender, birthweight discordance >75th and >90th percentile was associated with induced delivery <34 weeks [odds ratio 1.71 (95% confidence interval 1.11-2.65) and odds ratio 2.83 (95% confidence interval 1.73-4.64), respectively]. Discordance >75th-percentile was associated with an increased risk of infant mortality after 28 days [odds ratio 4.69 (95% confidence interval 1.07-20.45)] but not with major neonatal complications or with low mean Ages and Stages Questionnair scores at 6, 18, and 48-60 months after term. CONCLUSION: Chorionicity-specific intertwin birthweight discordance is a risk factor for induced preterm delivery and infant mortality, but not for lower scores for neurophysiological development at 6, 18, and 48-60 months.