Pulmonary hypoplasia, characterized by incomplete alveolar development, remains a major cause of mortality and morbidity in congenital diaphragmatic hernia. Recently demonstrated to differentiate from a common bipotent progenitor during development, the two cell types that line the alveoli type 1 and type 2 alveolar cells have shown to alter their relative ratio in congenital diaphragmatic hernia lungs.

Nanoparticle interactions with cellular membranes and the kinetics of their transport and localization are important determinants of their functionality and their biological consequences. Understanding these phenomena is fundamental for the translation of such NPs from in vitro to in vivo systems for bioimaging and medical applications. Two CdSe/ZnS quantum dots (QD) with differing surface functionality (NH2 or COOH moieties) were used here for investigating the intracellular uptake and transport kinetics of these QDs.

Contractile myofiber units are mainly composed of thick myosin and thin actin (F-actin) filaments. F-Actin interacts with Microtubule Associated Monooxygenase, Calponin And LIM Domain Containing 2 (MICAL2). Indeed, MICAL2 modifies actin subunits and promotes actin filament turnover by severing them and preventing repolymerization. In this study, we found that MICAL2 increases during myogenic differentiation of adult and pluripotent stem cells (PSCs) towards skeletal, smooth and cardiac muscle cells and localizes in the nucleus of acute and chronic regenerating muscle fibers. In vivo delivery of Cas9-Mical2 guide RNA complexes results in muscle actin defects and demonstrates that MICAL2 is essential for skeletal muscle homeostasis and functionality. Conversely, MICAL2 upregulation shows a positive impact on skeletal and cardiac muscle commitments. Taken together these data demonstrate that modulations of MICAL2 have an impact on muscle filament dynamics and its fine-tuned balance is essential for the regeneration of muscle tissues.

Signal transducer and activator of transcription 1 (STAT1) gain-of-function (GOF) mutations result in a primary immunodeficiency (PID) characterized typically by chronic mucocutaneous candidiasis (CMC), but a wider phenotypic range is reported and remains unexplained from a pathophysiological point-of-view. We hypothesized that different STAT1 GOF mutations may result in distinct molecular mechanisms, possibly explaining the variable phenotypes observed in patients. We selected STAT1 GOF mutants (R274W, R321S, T419R, and N574I) that are spread over the protein and studied their dynamic behavior in vitro in U3A and HeLa cell lines. All GOF mutants showed increased STAT1 phosphorylation compared to STAT1 WT. Real-time imaging demonstrated three underlying mechanisms for STAT1 GOF: (i) R274W showed a faster nuclear accumulation, (ii) both R321S and N574I showed a reduced nuclear mobility and slower dephosphorylation, whereas (iii) T419R was near-immobile in the nucleus, potentially due to enhanced binding to chromatin.

A compound's intratumoural distribution is an important determinant for the effectiveness of molecular therapy or imaging. Antibodies (Abs), though often used in the design of targeted compounds, struggle to achieve a homogenous distribution due to their large size and bivalent binding mechanism. In contrast, smaller compounds like nanobodies (Nbs) are expected to distribute more homogenously, though this has yet to be demonstrated in vivo at the microscopic level. We propose an intravital approach to evaluate the intratumoural distribution of different fluorescently labeled monomeric and dimeric Nb tracers and compare this with a monoclonal antibody (mAb). Monomeric and dimeric formats of the anti-HER2 (2Rb17c and 2Rb17c-2Rb17c) and control (R3B23 and R3B23-R3B23) Nb, as well as the dimeric monovalent Nb 2Rb17c-R3B23 were generated and fluorescently labeled with a Cy5 fluorophore. The mAb trastuzumab-Cy5 was also prepared. Whole-body biodistribution of all constructs was investigated in mice bearing subcutaneous xenografts (HER2+ SKOV3) using in vivo epi-fluorescence imaging. Next, for intravital experiments, GFP-expressing SKOV3 cells were grown under dorsal window chambers on athymic nude mice (n = 3/group), and imaged under a fluorescence stereo microscope immediately after intravenous injection of the tracers. Consecutive fluorescence images within the tumour were acquired over the initial 20 min after injection and later, single images were taken at 1, 3 and 24 h post-injection. Additionally, two-photon microscopy was used to investigate the colocalization of GFP (tumour cells) and Cy5 fluorescence (tracers) at higher resolution. Whole-body images showed rapid renal clearance of all Nbs, and fast tumour targeting for the specific Nbs. Specific tumour uptake of the mAb could only be clearly distinguished from background after several hours. Intravital imaging revealed that monomeric Nb tracers accumulated rapidly and distributed homogenously in the tumour mere minutes after intravenous injection. The dimeric compounds initially achieved lower fluorescence intensities than the monomeric. Furthermore, whereas the HER2-specific dimeric bivalent compound remained closely associated to the blood vessels over 24 h, the HER2-specific dimeric monovalent tracer achieved a more homogenous tumour distribution from 1 h post-injection onwards. Non-specific tracers were not retained in the tumour. Trastuzumab had the most heterogenous intratumoural distribution of all evaluated compounds, while -due to the long blood retention- achieving the highest overall tumour uptake at 24 h post-injection. In conclusion, monomeric Nbs very quickly and homogenously distribute through tumour tissue, at a rate significantly greater than dimeric Nbs and mAbs. This underlines the potential of monomeric Nb tracers and therapeutics in molecular imaging and targeted therapies.

Hematopoietic stem cell gene therapy is emerging as a promising therapeutic strategy for many diseases of the blood and immune system. However, several individuals who underwent gene therapy in different trials developed hematological malignancies caused by insertional mutagenesis. Preclinical assessment of vector safety remains challenging because there are few reliable assays to screen for potential insertional mutagenesis effects in vitro. Here we demonstrate that genotoxic vectors induce a unique gene expression signature linked to stemness and oncogenesis in transduced murine hematopoietic stem and progenitor cells. Based on this finding, we developed the surrogate assay for genotoxicity assessment (SAGA). SAGA classifies integrating retroviral vectors using machine learning to detect this gene expression signature during the course of in vitro immortalization. On a set of benchmark vectors with known genotoxic potential, SAGA achieved an accuracy of 90.9%. SAGA is more robust and sensitive and faster than previous assays and reliably predicts a mutagenic risk for vectors that led to leukemic severe adverse events in clinical trials. Our work provides a fast and robust tool for preclinical risk assessment of gene therapy vectors, potentially paving the way for safer gene therapy trials.

Stem cell tracking in cellular therapy and regenerative medicine is an urgent need, superparamagnetic iron oxide nanoparticles (IONPs) could be used as contrast agents in magnetic resonance imaging (MRI) that allows visualization of the implanted cells ensuring they reach the desired sites in vivo. Herein, we report the study of the interaction of 3,4-dihydroxyhydrocinnamic acid (DHCA) functionalized IONPs that have desirable properties for T2 - weighted MRI, with bone marrow-derived primary human mesenchymal stem cells (hMSCs). Using the multiparametric high-content imaging method, we evaluate cell viability, formation of reactive oxygen species, mitochondrial health, as well as cell morphology and determine that the hMSCs are minimally affected after labelling with IONPs. Their cellular uptake is visualized by transmission electron microscopy (TEM) and Prussian Blue staining, and quantified using an iron specific colourimetric method. In vitro and in vivo studies demonstrate that these IONPs are biocompatible and can produce significant contrast enhancement in T2-weighted MRI. Iron oxide nanoparticles are detected in vivo as hypointense regions in the liver up to two weeks post injection using 9.4 T MRI. These DHCA functionalized IONPs are promising contrast agents for stem cell tracking by T2-weighted MRI as they are biocompatible and show no evidence of cytotoxic effects on hMSCs.

Prompt appropriate treatment reduces mortality of severe febrile illness in sub-Saharan Africa. We studied the health itinerary of children under-five admitted to the hospital with severe febrile illness in a setting endemic for Plasmodium falciparum (Pf) malaria and invasive non-typhoidal Salmonella infections, identified delaying factors and assessed their associations with in-hospital death.

Although recruiting newborns is ethically challenging, clinical trials remain essential to improve neonatal care. There is a lack of empirical data on the parental perspectives following participation of their neonate in a clinical trial, especially at long term. The objective of this study is to assess experiences and emotions of parents, long term after trial participation in an interventional drug trial.

Skeletal muscle holds an intrinsic capability of growth and regeneration both in physiological conditions and in case of injury. Chronic muscle illnesses, generally caused by genetic and acquired factors, lead to deconditioning of the skeletal muscle structure and function, and are associated with a significant loss in muscle mass. At the same time, progressive muscle wasting is a hallmark of aging. Given the paracrine properties of myogenic stem cells, extracellular vesicle-derived signals have been studied for their potential implication in both the pathogenesis of degenerative neuromuscular diseases and as a possible therapeutic target. In this study, we screened the content of extracellular vesicles from animal models of muscle hypertrophy and muscle wasting associated with chronic disease and aging. Analysis of the transcriptome, protein cargo, and microRNAs (miRNAs) allowed us to identify a hypertrophic miRNA signature amenable for targeting muscle wasting, consisting of miR-1 and miR-208a. We tested this signature among others in vitro on mesoangioblasts (MABs), vessel-associated adult stem cells, and we observed an increase in the efficiency of myogenic differentiation. Furthermore, injections of miRNA-treated MABs in aged mice resulted in an improvement in skeletal muscle features, such as muscle weight, strength, cross-sectional area, and fibrosis compared to controls. Overall, we provide evidence that the extracellular vesicle-derived miRNA signature we identified enhances the myogenic potential of myogenic stem cells.

Background Arteriovenous fistulae (AVFs) are the gold standard for vascular access in those requiring hemodialysis but may put an extra hemodynamic stress on the cardiovascular system. The complex interactions between the heart, kidney, and AVFs remain incompletely understood. Methods and Results We characterized a novel rat model of five-sixths partial nephrectomy (NX) and AVFs. NX induced increases in urea, creatinine, and hippuric acid. The addition of an AVF (AVF+NX) further increased urea and a number of uremic toxins such as trimethylamine N-oxide and led to increases in cardiac index, left and right ventricular volumes, and right ventricular mass. Plasma levels of uremic toxins correlated well with ventricular morphology and function. Heart transcriptomes identified altered expression of 8 genes following NX and 894 genes following AVF+NX, whereas 290 and 1431 genes were altered in the kidney transcriptomes, respectively. Gene ontology and Kyoto Encyclopedia of Genes and Genomes analysis revealed gene expression changes related to cell division and immune activation in both organs, suppression of ribosomes and transcriptional activity in the heart, and altered renin-angiotensin signaling as well as chronodisruption in the kidney. All except the latter were worsened in AVF+NX compared with NX. Conclusions Inflammation and organ dysfunction in chronic kidney disease are exacerbated following AVF creation. Furthermore, our study provides important information for the discovery of novel biomarkers and therapeutic targets in the management of cardiorenal syndrome.

To infect nondividing cells, HIV-1 needs to cross the nuclear membrane. The importin transportin-SR2 (TRN-SR2 or transportin-3) has been proposed to mediate HIV-1 nuclear import, but the detailed mechanism remains unresolved. The direct interaction of TRN-SR2 with HIV-1 integrase (IN) has been proposed to drive HIV-1 nuclear import. Alternatively, TRN-SR2 may play an indirect role by mediating nuclear import of cleavage and polyadenylation specificity factor 6 (CPSF6). To unravel the role of TRN-SR2, we designed CRISPR/Cas9 guide RNAs targeting different exons of TNPO3. Although this approach failed to generate full knockouts, monoallelic knockout clones were generated with indel mutations. HIV-1 replication was hampered in those clones at the level of HIV-1 nuclear import without an effect on the cellular distribution of the TRN-SR2 cargoes CPSF6 or alternative splicing factor1/pre-mRNA splicing factor SF2 (ASF/SF2). Recombinant ΔV105 TRN-SR2 expressed in clone 15.15 was 2-fold impaired for interaction with HIV-1 IN and classified as an interaction mutant. Our data support a model whereby TRN-SR2 acts as a cofactor of HIV-1 nuclear import without compromising the nuclear import of cellular cargoes. CRISPR/Cas9-induced mutagenesis can be used as a method to generate interface mutants to characterize host factors of human pathogens. IMPORTANCE Combination antiretroviral therapy (cART) effectively controls HIV-1 by reducing viral loads, but it does not cure the infection. Lifelong treatment with cART is a prerequisite for sustained viral suppression. The rapid emergence of drug-resistant viral strains drives the necessity to discover new therapeutic targets. The nuclear import of HIV-1 is crucial in the HIV-1 replication cycle, but the detailed mechanism remains incompletely understood. This study provides evidence that TRN-SR2 directly mediates HIV-1 nuclear import via the interaction with HIV-1 integrase. The interaction between those proteins is therefore a promising target toward a rational drug design which could lead to new therapeutic strategies due to the bottleneck nature of HIV-1 nuclear import.

Magnetoliposomes (MLs) were synthesized and tested for longitudinal monitoring of transplanted pancreatic islets using magnetic resonance imaging (MRI) in rat models. The rat insulinoma cell line INS-1E and isolated pancreatic islets from outbred and inbred rats were used to optimize labeling conditions in vitro. Strong MRI contrast was generated by islets exposed to 50 µg Fe/ml for 24 hours without any increased cell death, loss of function or other signs of toxicity. In vivo experiments showed that pancreatic islets (50-1000 units) labeled with MLs were detectable for up to 6 weeks post-transplantation in the kidney subcapsular space. Islets were also monitored for two weeks following transplantation through the portal vein of the liver. Hereby, islets labeled with MLs and transplanted under the left kidney capsule were able to correct hyperglycemia and had stable MRI signals until nephrectomy. Interestingly, in vivo MRI of streptozotocin induced diabetic rats transplanted with allogeneic islets demonstrated loss of MRI contrast between 7-16 days, indicative of loss of islet structure. MLs used in this study were not only beneficial for monitoring the location of transplanted islets in vivo with high sensitivity but also reported on islet integrity and hereby indirectly on islet function and rejection.

Aging is associated with gradual alterations in the neurochemical characteristics of the brain, which can be assessed in-vivo with proton-magnetic resonance spectroscopy (1H-MRS). However, the impact of these age-related neurochemical changes on functional motor behavior is still poorly understood. Here, we address this knowledge gap and specifically focus on the neurochemical integrity of the left sensorimotor cortex (SM1) and the occipital lobe (OCC), as both regions are main nodes of the visuomotor network underlying bimanual control. 1H-MRS data and performance on a set of bimanual tasks were collected from a lifespan (20-75 years) sample of 86 healthy adults. Results indicated that aging was accompanied by decreased levels of N-acetylaspartate (NAA), glutamate-glutamine (Glx), creatine ​+ ​phosphocreatine (Cr) and myo-inositol (mI) in both regions, and decreased Choline (Cho) in the OCC region. Lower NAA and Glx levels in the SM1 and lower NAA levels in the OCC were related to poorer performance on a visuomotor bimanual coordination task, suggesting that NAA could serve as a potential biomarker for the integrity of the motor system supporting bimanual control. In addition, lower NAA, Glx, and mI levels in the SM1 were found to be correlates of poorer dexterous performance on a bimanual dexterity task. These findings highlight the role for 1H-MRS to study neurochemical correlates of motor performance across the adult lifespan.

Mendelian susceptibility to mycobacterial disease (MSMD) is caused by inborn errors of IFN-γ immunity. The most frequent genetic defects are found in IL12 or a subunit of its receptor. IL23R deficiency in MSMD has only been reported once, in two pediatric patients from the same kindred with isolated disseminated Bacille Calmette-Guérin disease. We evaluated the impact of a homozygous stop mutation in IL23R (R381X), identified by whole exome sequencing, in an adult patient with disseminated non-tuberculous mycobacterial disease.

Bronchopulmonary dysplasia (BPD) is a developmental disorder of infants born prematurely, characterized by disrupted alveolarization and microvascular maturation. However, the sequence of alveolar and vascular alterations is currently not fully understood. Therefore, we used a rabbit model to evaluate alveolar and vascular development under preterm birth and hyperoxia, respectively. Pups were born by cesarean section 3 days before term and exposed for 7 days to hyperoxia (95% O2) or normoxia (21% O2). In addition, term-born rabbits were exposed to normoxia for 4 days. Rabbit lungs were fixed by vascular perfusion and prepared for stereological analysis. Normoxic preterm rabbits had a significantly lower number of alveoli than term rabbits. The number of septal capillaries was lower in preterm rabbits but less pronounced than the alveolar reduction. In hyperoxic preterm rabbits, the number of alveoli was similar to that in normoxic preterm animals; however, hyperoxia had a severe additional negative effect on the capillary number. In conclusion, preterm birth had a strong effect on alveolar development, and hyperoxia had a more pronounced effect on capillary development. The data provide a complex picture of the vascular hypothesis of BPD which rather seems to reflect the ambient oxygen concentration than the effect of premature birth.

Preterm birth is the most significant problem in contemporary obstetrics accounting for 5-18% of worldwide deliveries. Encephalopathy of prematurity encompasses the multifaceted diffuse brain injury resulting from preterm birth. Current animal models exploring the underlying pathophysiology of encephalopathy of prematurity employ significant insults to generate gross central nervous system abnormalities. To date the exclusive effect of prematurity was only studied in a non-human primate model. Therefore, we aimed to develop a representative encephalopathy of prematurity small animal model only dependent on preterm birth. Time mated New-Zealand white rabbit does were either delivered on 28 (pre-term) or 31 (term) postconceptional days by caesarean section. Neonatal rabbits underwent neurobehavioral evaluation on 32 days post conception and then were transcardially perfuse fixed. Neuropathological assessments for neuron and oligodendrocyte quantification, astrogliosis, apoptosis and cellular proliferation were performed. Lastly, ex-vivo high-resolution Magnetic Resonance Imaging was used to calculate T1 volumetric and Diffusion Tensor Imaging derived fractional anisotropy and mean diffusivity. Preterm birth was associated with a motoric (posture instability, abnormal gait and decreased locomotion) and partial sensory (less pain responsiveness and failing righting reflex) deficits that coincided with global lower neuron densities, less oligodendrocyte precursors, increased apoptosis and less proliferation. These region-specific histological changes corresponded with Magnetic Resonance Diffusion Tensor Imaging differences. The most significant differences were seen in the hippocampus, caudate nucleus and thalamus of the preterm rabbits. In conclusion this model of preterm birth, in the absence of any other contributory events, resulted in measurable neurobehavioral deficits with associated brain structural and Magnetic Resonance Diffusion Tensor Imaging findings.

Stable integration in the host genome renders murine leukemia virus (MLV)-derived vectors attractive tools for gene therapy. Adverse events in otherwise successful clinical trials caused by proto-oncogene activation due to vector integration hamper their application. MLV and MLV-based vectors integrate near strong enhancers, active promoters, and transcription start sites (TSS) through specific interaction of MLV integrase (IN) with the bromodomain and extra-terminal (BET) family of proteins, accounting for insertional mutagenesis. We identified a BET-interaction motif in the C-terminal tail of MLV IN conserved among gammaretroviruses. By deletion of this motif or a single point mutation (INW390A), BET-independent MLV (BinMLV) were engineered. BinMLV vectors carrying INW390A integrate at wild-type efficiency, with an integration profile that no longer correlates with BET chromatin distribution nor with the traditional markers of MLV integration. In particular, BinMLV vector integration associated less with oncogene TSS compared to the MLV vectors currently used in clinical trials. Together, these findings open perspectives to increase the biosafety of gammaretroviral vectors for gene therapy.

Distinct integration patterns of different retroviruses, including HIV-1, have puzzled virologists for over 20 years. A tetramer of the viral integrase (IN) assembles on the two viral cDNA ends, docks onto the target DNA (tDNA), and catalyzes viral genome insertion into the host chromatin. We identified the amino acids in HIV-1 IN that directly contact tDNA bases and affect local integration site sequence selection. These residues also determine the propensity of the virus to integrate into flexible tDNA sequences. Remarkably, natural polymorphisms INS119G and INR231G retarget viral integration away from gene-dense regions. Precisely these variants were associated with rapid disease progression in a chronic HIV-1 subtype C infection cohort. These findings link integration site selection to virulence and viral evolution, but also to the host immune response and antiretroviral therapy, since HIV-1 IN119 is under selection by HLA alleles and integrase inhibitors.

Nuclear entry is a selective, dynamic process granting the HIV-1 pre-integration complex (PIC) access to the chromatin. Classical analysis of nuclear entry of heterogeneous viral particles only yields averaged information. We now have employed single-virus fluorescence methods to follow the fate of single viral pre-integration complexes (PICs) during infection by visualizing HIV-1 integrase (IN). Nuclear entry is associated with a reduction in the number of IN molecules in the complexes while the interaction with LEDGF/p75 enhances IN oligomerization in the nucleus. Addition of LEDGINs, small molecule inhibitors of the IN-LEDGF/p75 interaction, during virus production, prematurely stabilizes a higher-order IN multimeric state, resulting in stable IN multimers resistant to a reduction in IN content and defective for nuclear entry. This suggests that a stringent size restriction determines nuclear pore entry. Taken together, this work demonstrates the power of single-virus imaging providing crucial insights in HIV replication and enabling mechanism-of-action studies.