Microscopic morphomolecular evaluation of transgenic humanized ACE2 murine models of SARS-CoV2

INTRODUCTION: SARS-CoV-2 is the causative agent for the ongoing pandemic that was first declared in 2020, taking the lives of almost six million people and disrupting communities worldwide. Although an impressive global effort from the scientific community has yielded multiple vaccines and therapeutics, more research is crucial for continued progress against SARS-CoV-2 and future emerging infectious diseases. Animal models have played a significant role in the development of many advancements throughout the pandemic, and better models are needed for more effective research. OBJECTIVE: Although many animals have been utilized for SARS-CoV-2 research, a model to recapitulate severe pulmonary disease is still lacking. Routinely utilized models have consisted of non-human primates, Syrian hamsters, and mice. Excluding ethical concerns, non-human primates are expensive and limited in supply, limiting the ability to execute more statistically powerful studies. Pneumonia caused by SARS-CoV-2 in non-human primates is also very mild, with nearly all animals surviving, creating substantial skepticism surrounding the frequency of acute respiratory distress syndrome (ARDS) and diffuse alveolar damage (DAD) occurrence in these animal models. Syrian hamsters are also naturally permissive to SARS-CoV-2 and consistently display the most severe lung pathology of any existing animal model, but the lack of availability of species specific reagents and research tools makes studying this model difficult. Utilization of mouse model does not require development of new research tools, as mice have been classically utilized for preclinical research for decades. This work seeks to characterize and evaluate two human ACE2-expressing transgenic mouse models to provide the scientific community with knowledge on their translational relevance. METHODS: K18-hACE2 (K18) and Rosa26-hACE2 (Rosa26) mice were infected with SARS-CoV-2 and checked daily for temperature and weight. Plaque assays and qPCR were utilized to determine viral load. Tissues were stained with H&E for histopathological scoring and qualitative analysis. K18-HACE2and Rosa26-hACE2tissues were fluorescently labeled using two different multiplex immunohistochemistry panels. Slides were digitized by a Vectra Polaris™ fluorescent whole slide scanner, unmixed using inFORMTM vxxx and digital analysis was completed using HALO™ vxxx. Statistical analysis was conducted using GraphPad Prism™ 9.0.1. RESULTS: Both transgenic models succumbed to SARS-CoV-2 infection, with neurodissemination and death/euthanasia corresponding with peak viral loads in both models. hACE2 mRNA and ACE2 protein anatomical distribution and expression levels was similar in both models as determined by RNAscope® ISH and IHC respectively. In brains, hACE2 was expressed sporadically in neurons, but consistently in blood vessels and choroidal epithelium. In the lungs, viral load peaked on day 2 and 4 while lung infiltrate steadily increased throughout the course of infection, peaking on day 7- 8. However, severe lung pathology was not observed in any animals and many of the hallmarks of diffuse alveolar damage were absent, namely the formation of a hyaline membrane, hemorrhage, edema, alveolar fibrin polymerization and neutrophil influx. K18-HACE2 mice showed less lung infiltrate when compared to Rosa26-hACE2mice, which had more T-cell rich infiltrate. No significant difference exists between the two strains in terms of IBA1+ cells and CD11b+ cells in the lungs, though both cell populations increased throughout the course of infection. Both models demonstrated neuroinvasion as early as day 4, but neurodissemination in the Rosa26-hACE2infection was limited to ventral portions of the brain, while the K18-hACE2 showed severe and near global dissemination within the brain aside from cerebellar sparing which was observed in both models. K18-HACE2mice showed a significant decrease in neuronal density and an increase in microglial reactive processes consistent with SARS-CoV-2 induced neuronal loss and microglial reactivity. Together, these findings show that neither hACE2 transgenic mouse model represents a model of severe lung disease for SARS-CoV-2 and that the main determinant of lethality is viral neuroinvasion and neurodissemination. Although hACE2 was under the control of the keratin 18 promoter in both models, the distinct insertion location resulted in distinct clinicopathological outcomes that are not easily explained but bring appreciation to the complexity of the central dogma of biology. CONCLUSION: Using digital image analysis of immunohistochemistry paired with histopathological scoring and traditional molecular and virological techniques, this study demonstrates that although the transgenic hACE2 mouse models available to researchers result in lethal disease following SARS-CoV-2 infection, death/euthanasia is invariably resulting of neurodissemination with mild pneumonia limiting their translational relevance of mirroring severe COVID-19 in humans.

Resistance to cytotoxicity and sustained release of interleukin-6 and interleukin-8 in the presence of decreased interferon-γ after differentiation of glioblastoma by human natural killer cells.

Natural killer (NK) cells are functionally suppressed in the glioblastoma multiforme (GBM) tumor microenvironment. We have recently shown that survival and differentiation of cancer stem-like cells (CSCs)/poorly differentiated tumors are controlled through two distinct phenotypes of cytotoxic and non-cytotoxic/split anergized NK cells, respectively. In this paper, we studied the function of NK cells against brain CSCs/poorly differentiated GBM and their NK cell-differentiated counterparts. Brain CSCs/poorly differentiated GBM, differentiated by split anergized NK supernatants (supernatants from NK cells treated with IL-2 + anti-CD16mAb) expressed higher levels of CD54, B7H1 and MHC-I and were killed less by the NK cells, whereas their CSCs/poorly differentiated counterparts were highly susceptible to NK cell lysis. Resistance to NK cells and differentiation of brain CSCs/poorly differentiated GBM by split anergized NK cells were mediated by interferon (IFN)-γ and tumor necrosis factor (TNF)-α. Brain CSCs/poorly differentiated GBM expressed low levels of TNFRs and IFN-γRs, and when differentiated and cultured with IL-2-treated NK cells, they induced increased secretion of pro-inflammatory cytokine interleukin (IL)-6 and chemokine IL-8 in the presence of decreased IFN-γ secretion. NK-induced differentiation of brain CSCs/poorly differentiated GBM cells was independent of the function of IL-6 and/or IL-8. The inability of NK cells to lyse GBM tumors and the presence of a sustained release of pro-inflammatory cytokines IL-6 and chemokine IL-8 in the presence of a decreased IFN-γ secretion may lead to the inadequacy of NK cells to differentiate GBM CSCs/poorly differentiated tumors, thus failing to control tumor growth

Direct correlation of crystal structure and optical properties in wurtzite/zinc-blende GaAs nanowire heterostructures

A novel method for the direct correlation at the nanoscale of structural and optical properties of single GaAs nanowires is reported. Nanowires consisting of 100% wurtzite and nanowires presenting zinc-blende/wurtzite polytypism are investigated by photoluminescence spectroscopy and transmission electron microscopy. The photoluminescence of wurtzite GaAs is consistent with a band gap of 1.5 eV. In the polytypic nanowires, it is shown that the regions that are predominantly composed of either zinc-blende or wurtzite phase show photoluminescence emission close to the bulk GaAs band gap, while regions composed of a nonperiodic superlattice of wurtzite and zinc-blende phases exhibit a redshift of the photoluminescence spectra as low as 1.455 eV. The dimensions of the quantum heterostructures are correlated with the light emission, allowing us to determine the band alignment between these two crystalline phases. Our first-principles electronic structure calculations within density functional theory, employing a hybrid-exchange functional, predict band offsets and effective masses in good agreement with experimental results

Timing of births and oral contraceptive use influences ovarian cancer risk

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/139076/1/ijc30910_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/139076/2/ijc30910.pd

Single-Molecule Real-Time (SMRT) Full-Length RNA-Sequencing Reveals Novel and Distinct mRNA Isoforms in Human Bone Marrow Cell Subpopulations.

Hematopoietic cells are continuously replenished from progenitor cells that reside in the bone marrow. To evaluate molecular changes during this process, we analyzed the transcriptomes of freshly harvested human bone marrow progenitor (lineage-negative) and differentiated (lineage-positive) cells by single-molecule real-time (SMRT) full-length RNA-sequencing. This analysis revealed a ~5-fold higher number of transcript isoforms than previously detected and showed a distinct composition of individual transcript isoforms characteristic for bone marrow subpopulations. A detailed analysis of messenger RNA (mRNA) isoforms transcribed from the ANXA1 and EEF1A1 loci confirmed their distinct composition. The expression of proteins predicted from the transcriptome analysis was evaluated by mass spectrometry and validated previously unknown protein isoforms predicted e.g., for EEF1A1. These protein isoforms distinguished the lineage negative cell population from the lineage positive cell population. Finally, transcript isoforms expressed from paralogous gene loci (e.g., CFD, GATA2, HLA-A, B, and C) also distinguished cell subpopulations but were only detectable by full-length RNA sequencing. Thus, qualitatively distinct transcript isoforms from individual genomic loci separate bone marrow cell subpopulations indicating complex transcriptional regulation and protein isoform generation during hematopoiesis

Irradiation Damage Independent Deuterium Retention in WMoTaNbV

High entropy alloys are a promising new class of metal alloys with outstanding radiation resistance and thermal stability. The interaction with hydrogen might, however, have desired (H storage) or undesired effects, such as hydrogen-induced embrittlement or tritium retention in the fusion reactor wall. High entropy alloy WMoTaNbV and bulk W samples were used to study the quantity of irradiation-induced trapping sites and properties of D retention by employing thermal desorption spectrometry, secondary ion mass spectrometry, and elastic recoil detection analysis. The D implantation was not found to create additional hydrogen traps in WMoTaNbV as it does in W, while 90 at% of implanted D is retained in WMoTaNbV, in contrast to 35 at% in W. Implantation created damage predicted by SRIM is 0.24 dpa in WMoTaNbV, calculated with a density of 6.044×1022 atoms/cm3. The depth of the maximum damage was 90 nm. An effective trapping energy for D in WMoTaNbV was found to be about 1.7 eV, and the D emission temperature was close to 700 °C

Hydrogen isotope exchange experiments in high entropy alloy WMoTaNbV

Plasma–facing components in future fusion reactors must endure high temperatures as well as high fluxes and fluences of high energy particles. Currently tungsten has been chosen as the primary plasma-facing material due to its good thermal conductivity, low erosion rate and low fuel retention. Materials with even better properties are still being investigated to be used in reactor regions with demanding plasma conditions. High entropy alloys (HEA) are a new class of metallic alloys and their exploitation in fusion applications has not been widely studied. In this work, the hydrogen isotope exchange effect in an equiatomic HEA containing W, Mo, Ta, Nb, and V was studied. Deuterium was implanted into HEA samples with 30 keV/D energy and the HEA and reference samples were annealed in H2 atmosphere and in vacuum at various temperatures up to 400 °C, respectively. The near-surface D concentration profiles were measured with ERDA and the isotope exchange was observed to remove over 90 % of the trapped deuterium from the implantation region at temperatures above 200 °C. TDS was used to measure retention deeper in the bulk in which the reduction of trapped deuterium was significantly lower. High total retention of H was found in the bulk after H2 atmosphere annealing which indicates permeation and deep trapping of H in the material.Plasma-facing components in future fusion reactors must endure high temperatures as well as high fluxes and fluences of high energy particles. Currently tungsten has been chosen as the primary plasma-facing material due to its good thermal conductivity, low erosion rate and low fuel retention. Materials with even better properties are still being investigated to be used in reactor regions with demanding plasma conditions. High entropy alloys (HEA) are a new class of metallic alloys and their exploitation in fusion applications has not been widely studied. In this work, the hydrogen isotope exchange effect in an equiatomic HEA containing W, Mo, Ta, Nb, and V was studied. Deuterium was implanted into HEA samples with 30 keV/D energy and the HEA and reference samples were annealed in H2 atmosphere and in vacuum at various temperatures up to 400 °C, respectively. The near-surface D concentration profiles were measured with ERDA and the isotope exchange was observed to remove over 90 % of the trapped deuterium from the implantation region at temperatures above 200 °C. TDS was used to measure retention deeper in the bulk in which the reduction of trapped deuterium was significantly lower. High total retention of H was found in the bulk after H2 atmosphere annealing which indicates permeation and deep trapping of H in the material.Peer reviewe

Augmenting hematoma-scavenging capacity of innate immune cells by CDNF reduces brain injury and promotes functional recovery after intracerebral hemorrhage

During intracerebral hemorrhage (ICH), hematoma formation at the site of blood vessel damage results in local mechanical injury. Subsequently, erythrocytes lyse to release hemoglobin and heme, which act as neurotoxins and induce inflammation and secondary brain injury, resulting in severe neurological deficits. Accelerating hematoma resorption and mitigating hematoma-induced brain edema by modulating immune cells has potential as a novel therapeutic strategy for functional recovery after ICH. Here, we show that intracerebroventricular administration of recombinant human cerebral dopamine neurotrophic factor (rhCDNF) accelerates hemorrhagic lesion resolution, reduces peri-focal edema, and improves neurological outcomes in an animal model of collagenase-induced ICH. We demonstrate that CDNF acts on microglia/macrophages in the hemorrhagic striatum by promoting scavenger receptor expression, enhancing erythrophagocytosis and increasing anti-inflammatory mediators while suppressing the production of pro-inflammatory cytokines. Administration of rhCDNF results in upregulation of the Nrf2-HO-1 pathway, but alleviation of oxidative stress and unfolded protein responses in the perihematomal area. Finally, we demonstrate that intravenous delivery of rhCDNF has beneficial effects in an animal model of ICH and that systemic application promotes scavenging by the brain's myeloid cells for the treatment of ICH.Peer reviewe

Strain-engineering of the charge and spin-orbital interactions in Sr2IrO4

In the high spin-orbit coupled Sr2IrO4, the high sensitivity of the ground state to the details of the local lattice structure shows a large potential for the manipulation of the functional properties by inducing local lattice distortions. We use epitaxial strain to modify the Ir-O bond geometry in Sr2IrO4 and perform momentum-dependent Resonant Inelastic X-ray Scattering (RIXS) at the metal and at the ligand sites to unveil the response of the low energy elementary excitations. We observe that the pseudospin-wave dispersion for tensile-strained Sr2IrO4 films displays large softening along the [h,0] direction, while along the [h,h] direction it shows hardening. This evolution reveals a renormalization of the magnetic interactions caused by a strain-driven crossover from anisotropic to isotropic interactions between the magnetic moments. Moreover, we detect dispersive electron-hole pair excitations which shift to lower (higher) energies upon compressive (tensile) strain, manifesting a reduction (increase) in the size of the charge gap. This behavior shows an intimate coupling between charge excitations and lattice distortions in Sr2IrO4, originating from the modified hopping elements between the t2g orbitals. Our work highlights the central role played by the lattice degrees of freedom in determining both the pseudospin and charge excitations of Sr2IrO4 and provides valuable information towards the control of the ground state of complex oxides in the presence of high spin-orbit coupling.Comment: Published in Proceedings of the National Academy of Sciences, September 202