Aging‐related cell type‐specific pathophysiologic immune responses that exacerbate disease severity in aged COVID‐19 patients

Coronavirus disease 2019 (COVID‐19) is especially severe in aged patients, defined as 65 years or older, for reasons that are currently unknown. To investigate the underlying basis for this vulnerability, we performed multimodal data analyses on immunity, inflammation, and COVID‐19 incidence and severity as a function of age. Our analysis leveraged age‐specific COVID‐19 mortality and laboratory testing from a large COVID‐19 registry, along with epidemiological data of ~3.4 million individuals, large‐scale deep immune cell profiling data, and single‐cell RNA‐sequencing data from aged COVID‐19 patients across diverse populations. We found that decreased lymphocyte count and elevated inflammatory markers (C‐reactive protein, D‐dimer, and neutrophil–lymphocyte ratio) are significantly associated with age‐specific COVID‐19 severities. We identified the reduced abundance of naïve CD8 T cells with decreased expression of antiviral defense genes (i.e., IFITM3 and TRIM22) in aged severe COVID‐19 patients. Older individuals with severe COVID‐19 displayed type I and II interferon deficiencies, which is correlated with SARS‐CoV‐2 viral load. Elevated expression of SARS‐CoV‐2 entry factors and reduced expression of antiviral defense genes (LY6E and IFNAR1) in the secretory cells are associated with critical COVID‐19 in aged individuals. Mechanistically, we identified strong TGF‐beta‐mediated immune–epithelial cell interactions (i.e., secretory‐non‐resident macrophages) in aged individuals with critical COVID‐19. Taken together, our findings point to immuno‐inflammatory factors that could be targeted therapeutically to reduce morbidity and mortality in aged COVID‐19 patients.     

Bone marrow‐derived mesenchymal stem cells promote Helicobacter pylori‐associated gastric cancer progression by secreting thrombospondin‐2

ObjectivesBone marrow‐derived cells (BMDCs), especially mesenchymal stem cells (MSCs), may be involved in the development of Helicobacter pylori‐associated gastric cancer (GC) in mice, but the specific mechanism remains unclear, and evidence from human studies is lacking.Materials and MethodsTo verify the role of BM‐MSCs in H pylori‐associated GC, green fluorescent protein (GFP)‐labelled BM‐MSCs were transplanted into the subserosal layers of the stomach in a mouse model of chronic H pylori infection. Three months post‐transplantation, the mice were sacrificed, and the gastric tissues were subjected to histopathological and immunofluorescence analyses. In addition, we performed fluorescence in situ hybridization (FISH) and immunofluorescence analyses of gastric tissue from a female patient with H pylori infection and a history of acute myeloid leukaemia who received a BM transplant from a male donor.ResultsIn mice with chronic H pylori infection, GFP‐labelled BM‐MSCs migrated from the serous layer to the mucosal layer and promoted GC progression. The BM‐MSCs differentiated into pan‐cytokeratin‐positive epithelial cells and α‐smooth muscle actin‐positive cancer‐associated fibroblasts (CAFs) by secreting the protein thrombospondin‐2. FISH analysis of gastric tissue from the female patient revealed Y‐chromosome‐positive cells. Immunofluorescence analyses further confirmed that Y‐chromosome‐positive cells showed positive BM‐MSCs marker. These results suggested that allogeneic BMDCs, including BM‐MSCs, can migrate to the stomach under chronic H pylori infection.ConclusionsTaken together, these findings imply that BM‐MSCs participate in the development of chronic H pylori‐associated GC by differentiating into both gastric epithelial cells and CAFs.     

Study on the development and integration of 3D‐printed optics in small‐scale productions of single‐use cultivation vessels

Integrating optical sensors and 3D‐printed optics into single‐use (SU) cultivation vessels for customized, tailor‐made equipment could be a next big step in the bioreactor and screening platform development enabling online bioprocess monitoring. Many different parameters such as pH, oxygen, carbon dioxide and optical density (OD) can be monitored more easily using online measuring instruments compared to offline sampling. Space‐saving integrated sensors in combination with adapted optics such as prisms open up vastly new possibilities to precisely guide light through SU vessels. This study examines how optical prisms can be 3D‐printed with a 3D‐inkjet printer, modified and then evaluated in a custom made optical bench. The prisms are coated or bonded with thin cover glasses. For the examination of reflectance performance and conformity prisms are compared on the basis of measured characteristics of a conventional glass prism. In addition, the most efficient and reproducible prism geometry and modification technique is applied to a customized 3D‐printed cultivation vessel. The vessel is evaluated on a commercial sensor‐platform, a shake flask reader (SFR) vario, to investigate its sensing‐characteristics while monitoring scattered light with the turbidity standard formazine and a cell suspension of Saccharomyces cerevisiae as model organism. It is demonstrated that 3D‐printed prisms can be used in combination with commercial scattered light sensor‐platforms to determine OD of a microbial culture and that a 3D‐printed unibody design with integrated optics in a cultivation vessel is feasible. In the range of OD₆₀₀ 0–1.16 rel.AU a linear correlation between sensor amplitude and offline determined OD can be achieved. Thus, enabling for the first time a measurement of low cell densities with the SFR vario platform. Moreover, sensitivity is also at least three times higher compared to the commonly used method.     

Crystal structure of bacterial cytotoxic necrotizing factor CNFY reveals molecular building blocks for intoxication

Cytotoxic necrotizing factors (CNFs) are bacterial single‐chain exotoxins that modulate cytokinetic/oncogenic and inflammatory processes through activation of host cell Rho GTPases. To achieve this, they are secreted, bind surface receptors to induce endocytosis and translocate a catalytic unit into the cytosol to intoxicate host cells. A three‐dimensional structure that provides insight into the underlying mechanisms is still lacking. Here, we determined the crystal structure of full‐length Yersinia pseudotuberculosis CNF_Y. CNF_Y consists of five domains (D1–D5), and by integrating structural and functional data, we demonstrate that D1–3 act as export and translocation module for the catalytic unit (D4–5) and for a fused β‐lactamase reporter protein. We further found that D4, which possesses structural similarity to ADP‐ribosyl transferases, but had no equivalent catalytic activity, changed its position to interact extensively with D5 in the crystal structure of the free D4–5 fragment. This liberates D5 from a semi‐blocked conformation in full‐length CNF_Y, leading to higher deamidation activity. Finally, we identify CNF translocation modules in several uncharacterized fusion proteins, which suggests their usability as a broad‐specificity protein delivery tool.     

Nonproductive exposure of PBMCs to SARS‐CoV‐2 induces cell‐intrinsic innate immune responses

Cell‐intrinsic responses mounted in PBMCs during mild and severe COVID‐19 differ quantitatively and qualitatively. Whether they are triggered by signals emitted by productively infected cells of the respiratory tract or result from physical interaction with virus particles remains unclear. Here, we analyzed susceptibility and expression profiles of PBMCs from healthy donors upon ex vivo exposure to SARS‐CoV and SARS‐CoV‐2. In line with the absence of detectable ACE2 receptor expression, human PBMCs were refractory to productive infection. RT–PCR experiments and single‐cell RNA sequencing revealed JAK/STAT‐dependent induction of interferon‐stimulated genes (ISGs) but not proinflammatory cytokines. This SARS‐CoV‐2‐specific response was most pronounced in monocytes. SARS‐CoV‐2‐RNA‐positive monocytes displayed a lower ISG signature as compared to bystander cells of the identical culture. This suggests a preferential invasion of cells with a low ISG baseline profile or delivery of a SARS‐CoV‐2‐specific sensing antagonist upon efficient particle internalization. Together, nonproductive physical interaction of PBMCs with SARS‐CoV‐2‐ and, to a much lesser extent, SARS‐CoV particles stimulate JAK/STAT‐dependent, monocyte‐accentuated innate immune responses that resemble those detected in vivo in patients with mild COVID‐19.     

A novel multikinase inhibitor SKLB‐YTH‐60 ameliorates inflammation and fibrosis in bleomycin‐induced lung fibrosis mouse models

ObjectivesIdiopathic pulmonary fibrosis (IPF) is marked by the excessive accumulation of extracellular matrix, which participates in a variety of chronic diseases or injuries and seriously threatens human health. Due to the side effects of clinical drugs, there is still a need to develop novel and less toxic drugs to treat pulmonary fibrosis.Materials and MethodsSKLB‐YTH‐60 was developed through computer‐aided drug design, de novo synthesis and high‐throughput screening. We employed the bleomycin (BLM)‐induced lung fibrosis animal models and used TGF‐β₁ to induce the epithelial‐mesenchymal transition (EMT) of A549 cells in vitro. Meanwhile, the protein expression of collagen I and the α‐smooth muscle actin (α‐SMA), E‐cadherin, p‐FGFR1, p‐PLCγ, p‐Smad2/3 and p‐Erk1/2 was detected by western blot.ResultsYTH‐60 has obvious anti‐proliferative activity on fibroblasts and A549 cells. Moreover, YTH‐60 could impair the EMT of A549 cells and suppressed fibrosis by inhibiting FGFR and TGF‐β/Smad‐dependent pathways. Intraperitoneal administration of preventive YTH‐60 could significantly reduce the degree of fibrosis in mice and regulate the imbalance of the immune microenvironment. In addition, we observed that therapeutic YTH‐60 treatment attenuated fibrotic changes in mice during the period of fibrosis. Importantly, YTH‐60 has shown an acceptable oral bioavailability (F = 17.86%) and appropriate eliminated half‐life time (T _1/2 = 8.03 hours).ConclusionsTaken together, these preclinical evaluations suggested that YTH‐60 could be a promising drug candidate for treating IPF.     

Purification and characterization of the receptor‐binding domain of SARS‐CoV‐2 spike protein from Escherichia coli

SARS‐CoV‐2 is responsible for a disruptive worldwide viral pandemic, and renders a severe respiratory disease known as COVID‐19. Spike protein of SARS‐CoV‐2 mediates viral entry into host cells by binding ACE2 through the receptor‐binding domain (RBD). RBD is an important target for development of virus inhibitors, neutralizing antibodies, and vaccines. RBD expressed in mammalian cells suffers from low expression yield and high cost. E. coli is a popular host for protein expression, which has the advantage of easy scalability with low cost. However, RBD expressed by E. coli (RBD‐1) lacks the glycosylation, and its antigenic epitopes may not be sufficiently exposed. In the present study, RBD‐1 was expressed by E. coli and purified by a Ni Sepharose Fast Flow column. RBD‐1 was structurally characterized and compared with RBD expressed by the HEK293 cells (RBD‐2). The secondary structure and tertiary structure of RBD‐1 were largely maintained without glycosylation. In particular, the major β‐sheet content of RBD‐1 was almost unaltered. RBD‐1 could strongly bind ACE2 with a dissociation constant (K_D) of 2.98 × 10^–8 M. Thus, RBD‐1 was expected to apply in the vaccine development, screening drugs and virus test kit.     

Using fractal self‐similarity to increase precision of shrub biomass estimates

We show that aerial tips are self‐similar fractals of whole shrubs and present a field method that applies this fact to improves accuracy and precision of biomass estimates of tall‐shrubs, defined here as those with diameter at root collar (DRC) ≥ 2.5 cm. Power function allometry of biomass to stem diameter generates a disproportionate prediction error that increases rapidly with diameter. Thus, biomass should be modeled as a single measure of stem diameter only if stem diameter is less than a threshold D_max. When stem diameter exceeds D_max, then the stem internode should be treated as a conic frustrum requiring two additional measures: a second, node‐adjacent diameter and a length. If the second diameter is less than D_max, then the power function allometry can be applied to the aerial tip; otherwise an additional internode is measured. This “two‐component” allometry—internodes as frustra and aerial tips as shrubs—can reduce estimated biomass error propagated to the plot‐level by as much as 50% or more where very large shrubs are present D_max is any diameter such that the ratio of single‐component to two‐component uncertainty exceeds the ratio of two‐component to single‐component measurement time. Guidelines for estimating D_max based on pilot field data are provided. Tall shrubs are increasing in abundance and distribution across Arctic, alpine, boreal, and dryland ecosystems. Estimating their biomass is important for both ecological studies and carbon accounting. Reducing field‐sample prediction error increases precision in multi‐stage modeling because additional measures efficiently improve plot‐level biomass precision, reducing uncertainty for shrub biomass estimates.     

Cytochrome P450 26A1 regulates the clusters and killing activity of NK cells during the peri‐implantation period

Cytochrome P450 26A1 (CYP26A1) plays a vital role in early pregnancy in mice. Our previous studies have found that CYP26A1 affects embryo implantation by modulating natural killer (NK) cells, and that there is a novel population of CYP26A1⁺ NK cells in the uteri of pregnant mice. The aim of this study was to investigate the effects of CYP26A1 on the subsets and killing activity of NK cells. Through single‐cell RNA sequencing (scRNA‐seq), we identified four NK cell subsets in the uterus, namely, conventional NK (cNK), tissue‐resident NK (trNK) 1 and 2, and proliferating trNK (trNKp). The two most variable subpopulations after uterine knockdown of CYP26A1 were trNKp and trNK2 cells. CYP26A1 knockdown significantly downregulated the expression of the NK cell function‐related genes Cd44, Cd160, Vegfc, and Slamf6 in trNK2 cells, and Klra17 and Ogn in trNKp cells. Both RNA‐seq and cytotoxicity assays confirmed that CYP26A1⁺ NK cells had low cytotoxicity. These results indicate that CYP26A1 may affect the immune microenvironment at the maternal‐foetal interface by regulating the activity of NK cells.     

Mesenchymal stem cell‐conditioned medium attenuates the retinal pathology in amyloid‐β‐induced rat model of Alzheimer's disease: Underlying mechanisms

Amyloid‐beta (Aβ) oligomer is known to contribute to the pathophysiology of age‐related macular degeneration. Herein, we aimed to elucidate the in vivo and in vitro effects of Aβ_1‐42 application on retinal morphology in rats. Our in vivo studies revealed that intracerebroventricular administration of Aβ_1‐42 oligomer caused dysmorphological changes in both retinal ganglion cells and retinal pigment epithelium. In addition, in vitro studies revealed that ARPE‐19 cells following Aβ_1‐42 oligomer application had decreased viability along with apoptosis and decreased expression of the tight junction proteins, increased expression of both phosphor‐AKT and phosphor‐GSK3β and decreased expression of both SIRT1 and β‐catenin. Application of conditioned medium (CM) obtained from mesenchymal stem cells (MSC) protected against Aβ_1‐42 oligomer‐induced retinal pathology in both rats and ARPE‐19 cells. In order to explore the potential role of peptides secreted from the MSCs, we applied mass spectrometry to compare the peptidomics profiles of the MSC‐CM. Gene ontology enrichment analysis and String analysis were performed to explore the differentially expressed peptides by predicting the functions of their precursor proteins. Bioinformatics analysis showed that 3‐8 out of 155–163 proteins in the MSC‐CM maybe associated with SIRT1/pAKT/pGSK3β/β‐catenin, tight junction proteins, and apoptosis pathway. In particular, the secretomes information on the MSC‐CM may be helpful for the prevention and treatment of retinal pathology in age‐related macular degeneration.     

Elucidation and control of low and high active populations of alkaline phosphatase molecules for quantitative digital bioassay

Alkaline phosphatase (ALP), a homo‐dimeric enzyme has been widely used in various bioassays as disease markers and enzyme probes. Recent advancements of digital bioassay revolutionized ALP‐based diagnostic assays as seen in rapid growth of digital ELISA and the emerging multiplex profiling of single‐molecule ALP isomers. However, the intrinsic heterogeneity found among ALP molecules hampers the ALP‐based quantitative digital bioassays. This study aims quantitative analysis of single‐molecule activities of ALP from Escherichia coli and reveals the static heterogeneity in catalytic activity of ALP with two distinct populations: half‐active and fully‐active portions. Digital assays with serial buffer exchange uncovered single‐molecule Michaelis–Menten kinetics of ALP; half‐active molecules have halved values of the catalytic turnover rate, k _cat, and the rate constant of productive binding, k _on, of the fully active molecules. These findings suggest that half‐active ALP molecules are heterogenic dimers composed of inactive and active monomer units, while fully active ALP molecules comprise two active units. Static heterogeneity was also observed for ALP with other origins: calf intestine or shrimp, showing how the findings can be generalized across species. Cell‐free expression of ALP with disulfide bond enhancer and spiked zinc ion resulted in homogenous population of ALP of full activity, implying that inactive monomer units of ALP are deficient in correct disulfide bond formation and zinc ion coordination. These findings provide basis for further study on molecular mechanism and biogenesis of ALP, and also offer the way to prepare homogenous and active populations of ALP for highly quantitative and sensitive bioassays with ALP.     

Understanding of mouse and human bladder at single‐cell resolution: integrated analysis of trajectory and cell‐cell interactive networks based on multiple scRNA‐seq datasets

ObjectivesTo elaborately decipher the mouse and human bladders at single‐cell levels.Materials and MethodsWe collected more than 50,000 cells from multiple datasets and created, up to date, the largest integrated bladder datasets. Pseudotime trajectory of urothelium and interstitial cells, as well as dynamic cell‐cell interactions, was investigated. Biological activity scores and different roles of signaling pathways between certain cell clusters were also identified.ResultsThe glucose score was significantly high in most urothelial cells, while the score of H3 acetylation was roughly equally distributed across all cell types. Several genes via a pseudotime pattern in mouse (Car3, Dkk2, Tnc, etc.) and human (FBLN1, S100A10, etc.) were discovered. S100A6, TMSB4X, and typical uroplakin genes seemed as shared pseudotime genes for urothelial cells in both human and mouse datasets. In combinational mouse (n = 16,688) and human (n = 22,080) bladders, we verified 1,330 and 1,449 interactive ligand‐receptor pairs, respectively. The distinct incoming and outgoing signaling was significantly associated with specific cell types. Collagen was the strongest signal from fibroblasts to urothelial basal cells in mouse, while laminin pathway for urothelial basal cells to smooth muscle cells (SMCs) in human. Fibronectin 1 pathway was intensely sent by myofibroblasts, received by urothelial cells, and almost exclusively mediated by SMCs in mouse bladder. Interestingly, the cell cluster of SMCs 2 was the dominant sender and mediator for Notch signaling in the human bladder, while SMCs 1 was not. The expression of integrin superfamily (the most common communicative pairs) was depicted, and their co‐expression patterns were located in certain cell types (eg, Itgb1 and Itgb4 in mouse and human basal cells).ConclusionsThis study provides a complete interpretation of the normal bladder at single‐cell levels, offering an in‐depth resource and foundation for future research.     

ORP2 couples LDL‐cholesterol transport to FAK activation by endosomal cholesterol/PI(4,5)P2 exchange

Low‐density lipoprotein (LDL)‐cholesterol delivery from late endosomes to the plasma membrane regulates focal adhesion dynamics and cell migration, but the mechanisms controlling it are poorly characterized. Here, we employed auxin‐inducible rapid degradation of oxysterol‐binding protein‐related protein 2 (ORP2/OSBPL2) to show that endogenous ORP2 mediates the transfer of LDL‐derived cholesterol from late endosomes to focal adhesion kinase (FAK)‐/integrin‐positive recycling endosomes in human cells. In vitro, cholesterol enhances membrane association of FAK to PI(4,5)P₂‐containing lipid bilayers. In cells, ORP2 stimulates FAK activation and PI(4,5)P₂ generation in endomembranes, enhancing cell adhesion. Moreover, ORP2 increases PI(4,5)P₂ in NPC1‐containing late endosomes in a FAK‐dependent manner, controlling their tubulovesicular trafficking. Together, these results provide evidence that ORP2 controls FAK activation and LDL‐cholesterol plasma membrane delivery by promoting bidirectional cholesterol/PI(4,5)P₂ exchange between late and recycling endosomes.