Phenotypic switching of vascular smooth muscle cells is a central process in abdominal aortic aneurysm (AAA) pathology. We found that knockdown TCF7L1 (transcription factor 7-like 1), a member of the TCF/LEF (T cell factor/lymphoid enhancer factor) family of transcription factors, inhibits vascular smooth muscle cell differentiation. This study hints at potential interventions to maintain a normal, differentiated smooth muscle cell state, thereby eliminating the pathogenesis of AAA. In addition, our study provides insights into the potential use of TCF7L1 as a biomarker for AAA.

UNASSIGNED: Induction of potent, HBV-specific immune responses is crucial to control and finally cure HBV. The therapeutic hepatitis B vaccine TherVacB combines protein priming with a Modified Vaccinia virus Ankara (MVA)-vector boost to break immune tolerance in chronic HBV infection. Particulate protein and vector vaccine components, however, require a constant cooling chain for storage and transport, posing logistic and financial challenges to vaccine applications. We aimed to identify an optimal formulation to maintain stability and immunogenicity of the protein and vector components of the vaccine using a systematic approach.
UNASSIGNED: We used stabilizing amino acid (SAA)-based formulations to stabilize HBsAg and HBV core particles (HBcAg), and the MVA-vector. We then investigated the effect of lyophilization and short- and long-term high-temperature storage on their integrity. Immunogenicity and safety of the formulated vaccine was validated in HBV-naïve and adeno-associated virus (AAV)-HBV-infected mice.
UNASSIGNED: In vitro analysis proved the vaccine\'s stability against thermal stress during lyophilization and the long-term stability of SAA-formulated HBsAg, HBcAg and MVA during thermal stress at 40 °C for 3 months and at 25 °C for 12 months. Vaccination of HBV-naïve and AAV-HBV-infected mice demonstrated that the stabilized vaccine was well tolerated and able to brake immune tolerance established in AAV-HBV mice as efficiently as vaccine components constantly stored at 4 °C/-80 °C. Even after long-term exposure to elevated temperatures, stabilized TherVacB induced high titre HBV-specific antibodies and strong CD8+ T-cell responses, resulting in anti-HBs seroconversion and strong suppression of the virus in HBV-replicating mice.
UNASSIGNED: SAA-formulation resulted in highly functional and thermostable HBsAg, HBcAg and MVA vaccine components. This will facilitate global vaccine application without the need for cooling chains and is important for the development of prophylactic as well as therapeutic vaccines supporting vaccination campaigns worldwide.
UNASSIGNED: Therapeutic vaccination is a promising therapeutic option for chronic hepatitis B that may enable its cure. However, its application requires functional cooling chains during transport and storage that can hardly be guaranteed in many countries with high demand. In this study, the authors developed thermostable vaccine components that are well tolerated and that induce immune responses and control the virus in preclinical mouse models, even after long-term exposure to high surrounding temperatures. This will lower costs and ease application of a therapeutic vaccine and thus be beneficial for the many people affected by hepatitis B around the world.

In our quest to solve biomolecular structures to higher resolutions in cryoEM, care must be taken to deal with all aspects of image formation in the electron microscope. One of these is the Ewald sphere/focus gradient that derives from the scattering geometry in the microscope and its implications for recovering high resolution and handedness information. While several methods to deal with it has been proposed and implemented, there are still questions as to the correct approach. At the high acceleration voltages used for cryoEM, the traditional projection approximation that ignores the Ewald sphere breaks down around 2-3 Å and with large particles. This is likely not crucial for most biologically interesting molecules, but is required to understand detail about catalytic events, molecular orbitals, orientation of bound water molecules, etc. Through simulation I show that integration along the Ewald spheres in frequency space during reconstruction, the \"simple insertion method\" is adequate to reach resolutions to the Nyquist frequency. Both theory and simulations indicate that the handedness information encoded in such phases is irretrievably lost in the formation of real space images. The conclusion is that correct reconstruction along the Ewald spheres avoids the limitations of the projection approximation.

Alzheimer\'s disease (AD) is a progressively neurodegenerative disease without effective treatment. Here, we reported that the levels of expression and enzymatic activity of phosphatase magnesium-dependent 1A (PPM1A) were both repressed in brains of AD patient postmortems and 3 × Tg-AD mice, and treatment of adeno-associated virus (AAV)-ePHP-overexpression (OE)-PPM1A for brain-specific PPM1A overexpression or the new discovered PPM1A activator Miltefosine (MF, FDA approved oral anti-leishmanial drug) for PPM1A enzymatic activation improved the AD-like pathology in 3 × Tg-AD mice. The mechanism was intensively investigated by assay against the 3 × Tg-AD mice with brain-specific PPM1A knockdown (KD) through AAV-ePHP-KD-PPM1A injection. MF alleviated neuronal tauopathy involving microglia/neurons crosstalk by both promoting microglial phagocytosis of tau oligomers via PPM1A/Nuclear factor-κb (NF-κB)/C-X3-C Motif Chemokine Receptor 1 (CX3CR1) signaling and inhibiting neuronal tau hyperphosphorylation via PPM1A/NLR Family Pyrin Domain Containing 3 (NLRP3)/tau axis. MF suppressed microglial NLRP3 inflammasome activation by both inhibiting NLRP3 transcription via PPM1A/NF-κB/NLRP3 pathway in priming step and promoting PPM1A binding to NLRP3 to interfere NLRP3 inflammasome assembly in assembly step. Our results have highly addressed that PPM1A activation shows promise as a therapeutic strategy for AD and highlighted the potential of MF in treating this disease.

UNASSIGNED: Mesenchymal stromal cells (MSCs) hold the potential for application as cellular therapy products; however, there are many problems that need to be addressed before the use in clinical settings, these include the heterogeneity of MSCs, scalability in MSC production, timing and techniques for MSC administration, and engraftment efficiency and persistency of administered MSCs. In this study, problems regarding immune rejection caused by human leukocyte antigen (HLA) mismatches were addressed.
UNASSIGNED: Umbilical cord-derived MSCs (UC-MSCs) were gene-edited to avoid allogeneic immunity. The HLA class I expression was abrogated by the knock-out of the beta-2-microglobulin (B2M) gene; instead, the B2M-HLA-G fusion gene was knocked-in using the CRISPR/Cas9 system in combination with adeno-associated virus (AAV).
UNASSIGNED: Cell surface markers on gene-edited UC-MSCs were not different from those on primary UC-MSCs. The gene-edited UC-MSCs also retained the potential to differentiate into adipocytes, osteoblasts, and chondrocytes. B2M gene knock-out alone protected cells from allogeneic T cell immune responses but were vulnerable to NK cells. B2M gene knock-out in combination with B2M-HLA-G knock-in protected cells from both T cells and NK cells. The B2M-HLA-G knock-in MSCs retained a good immunosuppressive ability and the addition of these cells into the mixing lymphocyte reaction showed a significant inhibition of T cell proliferation.
UNASSIGNED: The results of this study demonstrated the possibility that the CRISPR/Cas9 system combined with AAV can be used to effectively disrupt/introduce any gene into UC-MSCs. Our findings suggest that the gene-edited cell line produced here using this method may have a higher ability to escape the cytotoxic activity of immune cells than primary cells, thereby being more advantageous for long-term graft survival.

UNASSIGNED: Subretinal injections (SRis) are commonly used in retinal gene therapy procedures to deliver adeno-associated virus (AAV) to photoreceptors and retinal pigment epithelial cells. We present an optimized surgical protocol to minimize off-target application of AAV in the vitreous, which in turn reduces the risk of extensive biodistribution and inflammation, ultimately leading to enhanced safety of the therapy.
UNASSIGNED: Experimental animal research study.
UNASSIGNED: Eight cynomolgus monkeys (Macaca fascicularis).
UNASSIGNED: Subretinal injections with an AAV2/8 vector were performed. The animals were allocated to 2 different vector dose groups (1×10ˆ 11 and 5×10ˆ 11 viral genomes [vg]). Samples of intravitreal fluid were taken at the end of the SRi procedure and again after a 3-minute lavage (wash-out) with balanced salt solution (BSS).
UNASSIGNED: Intravitreal vector genome copies were analyzed with quantitative polymerase chain reaction and compared between groups.
UNASSIGNED: Even uneventful SRi leads to dissemination of millions of AAV particles (0.1-0.7% of viral vector loading dose) into the vitreous cavity. Three minutes of lavage led to a substantial decrease (on average 96%) of intravitreal vector load.
UNASSIGNED: Multiple studies have shown that the intravitreal space is not as immune privileged as the subretinal space. Intravitreal AAV particles disseminate into the bloodstream, lead to increased biodistribution into lymphatic tissue, and help to stage an immune response with implications for both safety and efficacy. Therefore, minimizing off-target vector application after reflux of vector from the subretinal space is of significant interest. We show that a simple lavage of intravitreal fluid efficiently decreases the intravitreal vector load. Such a step should be considered when performing subretinal gene therapy.

UNASSIGNED: HBV exhibits wide genetic diversity with at least 9 genotypes (GTs), which differ in terms of prevalence, geographic distribution, natural history, disease progression, and treatment outcome. However, differences in HBV replicative capacity, gene expression, and infective capability across different GTs remain incompletely understood. Herein, we aimed to study these crucial aspects using newly constructed infectious clones covering the major HBV GTs.
UNASSIGNED: The replicative capacity of infectious clones covering HBV GTs A-E was analyzed in cell lines, primary hepatocytes and humanized mice. Host responses and histopathology induced by the different HBV GTs were characterized in hydrodynamically injected mice. Differences in treatment responses to entecavir and various HBV capsid inhibitors were also quantified across the different genetically defined GTs.
UNASSIGNED: Patient-derived HBV infectious clones replicated robustly both in vitro and in vivo. GTs A and D induce more pronounced intrahepatic and proinflammatory cytokine responses which correlated with faster viral clearance. Notably, all 5 HBV clones robustly produced viral particles following transfection into HepG2 cells, and these particles were infectious in HepG2-NTCP cells, primary human hepatocytes and human chimeric mice. Notably, GT D virus exhibited higher infectivity than GTs A, B, C and E in vitro, although it was comparable to GT A and B in the human liver chimeric mice in vivo. HBV capsid inhibitors were more readily capable of suppressing HBV GTs A, B, D and E than C.
UNASSIGNED: The infectious clones described here have broad utility as genetic tools that can mechanistically dissect intergenotypic differences in antiviral immunity and pathogenesis and aid in HBV drug development and screening.
UNASSIGNED: The hepatitis B virus (HBV) is a major contributor to human morbidity and mortality. HBV can be categorized into a number of genotypes, based on their specific genetic make-up, of which 9 are well known. We isolated and cloned the genomes of 5 of these genotypes and used them to create valuable tools for future research on this clinically important virus.

Respiratory depression (RD) is the primary cause of death due to opioids. Opioids bind to mu (µ)-opioid receptors (MORs) encoded by the MOR gene Oprm1, widely expressed in the central and peripheral nervous systems including centers that modulate breathing. Respiratory centers are located throughout the brainstem. Experiments with Oprm1-deleted knockout (KO) mice undertaken to determine which sites are necessary for the induction of opioid-induced respiratory depression (OIRD) showed that the pre-Bötzinger complex (preBötC) and the pontine Kölliker-Fuse nucleus (KF) contribute equally to OIRD but RD was not totally eliminated. Morphine showed a differential influence on preBötC and KF neurons - low doses attenuated RD following deletion of MORs from preBötC neurons and an increase in apneas after high doses whereas deletion of MORs from KF neurons but not the preBötC attenuated RD at both high and low doses. In other KO mice studies, morphine administration after deletion of Oprm1 from both the preBötC and the KF/PBN neurons, led to the conclusion that both respiratory centres contribute to OIRD but the preBötC predominates. MOR-mediated post-synaptic activation of GIRK potassium channels has been implicated as a cause of OIRD. A complementary mechanism in the preBötC involving KCNQ potassium channels independent of MOR signaling has been described. Recent experiments in rats showing that morphine depresses normal, but not gasping breathing, cast doubt on the belief that eupnea, sighs, and gasps, are under the control of preBötC neurons. Methadone, administered to alleviate symptoms of neonatal opioid withdrawal syndrome (NOWES), desensitized rats to OIRD. Protection lost between postnatal days 1 and 2 coincides with the preBötC becoming the dominant generator of respiratory rhythm. Neonatal antidepressant exposure syndrome (NADES) and serotonin toxicity (ST) show similarities including RD. Enzyme CYP2D6 involved in opioid detoxification is polymorphic. Individuals of different CYP2D6 genotype may show increased, decreased, or no enzyme activity, contributing to the variability of patient responses to different opioids and OIRD.

CRISPR-Cas9 is the most straightforward genome-editing tool to date. However, its implementation across disciplines is hampered by variable genome-editing efficiencies, reduced cell viability, and low success rates in obtaining clonal cell lines. This review aims to recognize all CRISPR-Cas9‒related work within the experimental dermatology field to identify key factors for successful strategies in the different keratinocyte (KC) cell sources available. On the basis of these findings, we conclude that most groups use immortalized KCs for generating knockout KCs. Our critical considerations for future studies using CRISPR-Cas9, both for fundamental and clinical applications, may guide implementation strategies of CRISPR-Cas9 technologies in the (experimental) dermatology field.

The primary etiology of a diverse range of cardiomyopathies is now understood to be genetic, creating a new paradigm for targeting treatments on the basis of the underlying molecular cause. This review provides a genetic and etiologic context for the traditional clinical classifications of cardiomyopathy, including molecular subtypes that may exhibit differential responses to existing or emerging treatments. The authors describe several emerging cardiomyopathy treatments, including gene therapy, direct targeting of myofilament function, protein quality control, metabolism, and others. The authors discuss advantages and disadvantages of these approaches and indicate areas of high potential for short- and longer term efficacy.