Single-cell transcriptomic sequencing identifies subcutaneous patient-derived xenograft recapitulated medulloblastoma.

BACKGROUND: Medulloblastoma (MB) is one of the most common malignant brain tumors that mainly affect children. Various approaches have been used to model MB to facilitate investigating tumorigenesis. This study aims to compare the recapitulation of MB between subcutaneous patient-derived xenograft (sPDX), intracranial patient-derived xenograft (iPDX), and genetically engineered mouse models (GEMM) at the single-cell level. METHODS: We obtained primary human sonic hedgehog (SHH) and group 3 (G3) MB samples from six patients. For each patient specimen, we developed two sPDX and iPDX models, respectively. Three Patch+/- GEMM models were also included for sequencing. Single-cell RNA sequencing was performed to compare gene expression profiles, cellular composition, and functional pathway enrichment. Bulk RNA-seq deconvolution was performed to compare cellular composition across models and human samples. RESULTS: Our results showed that the sPDX tumor model demonstrated the highest correlation to the overall transcriptomic profiles of primary human tumors at the single-cell level within the SHH and G3 subgroups, followed by the GEMM model and iPDX. The GEMM tumor model was able to recapitulate all subpopulations of tumor microenvironment (TME) cells that can be clustered in human SHH tumors, including a higher proportion of tumor-associated astrocytes and immune cells, and an additional cluster of vascular endothelia when compared to human SHH tumors. CONCLUSIONS: This study was the first to compare experimental models for MB at the single-cell level, providing value insights into model selection for different research purposes. sPDX and iPDX are suitable for drug testing and personalized therapy screenings, whereas GEMM models are valuable for investigating the interaction between tumor and TME cells.

A peptide derived from the N-terminus of charged multivesicular body protein 6 (CHMP6) promotes the secretion of gene editing proteins via small extracellular vesicle production.

Extracellular vesicles (EVs) are a promising new therapeutic platform. However, the low cargo-loading efficiency limits their clinical translation. In this study, we developed a high-yield EV cargo-loading device and explored its ability to encapsulate gene editing proteins. A series of fusion protein-based systems were constructed and their cargo loading efficiencies were compared by a NanoGlo luciferase assay. A myristoylated (Myr) peptide tag cloned from the N-terminal region of charged multivesicular body protein 6 (CHMP6), termed Myr(CHMP6), outcompeted CD9, ARRDC1, and other short polypeptides as an active packaging device. As determined by nanoparticle tracking analysis and transmission electron microscopy, the overexpression of Myr(CHMP6) increased small EV (sEV) production in Lenti-X 293T  cells without altering sEV morphology. The high passive packaging efficiency of Myr(CHMP6) was also elucidated for unmodified cargo loading. Western blotting revealed that Myr(CHMP6) facilitated the loading of Cre and Cas9 into sEVs without the generation of packaging device-cargo fusion proteins. Furthermore, Myr(CHMP6)-modified sEVs loaded with Cre or Cas9 promoted gene-editing in recipient cells, as observed using a fluorescence reporter system. Subsequent investigation demonstrated a dose-dependent effect of Myr(CHMP6) tag-induced cargo-loading. Mechanistically, N-myristoylation alone was necessary but not sufficient for the effective packaging of proteins into EVs. Thus, our results indicated that Myr(CHMP6) induces sEV production and may be effective in loading gene editing proteins into sEVs for therapeutic purposes.

Lipid Droplets Are Beneficial for Rabies Virus Replication by Facilitating Viral Budding.

Rabies is an old zoonotic disease caused by rabies virus (RABV), but the pathogenic mechanism of RABV is still not completely understood. Lipid droplets (LDs) have been reported to play a role in pathogenesis of several viruses. However, their role in RABV infection remains unclear. Here, we initially found that RABV infection upregulated LD production in multiple cells and mouse brains. After treatment with atorvastatin, a specific inhibitor of LDs, RABV replication in N2a cells decreased. Then we found that RABV infection could upregulate N-myc downstream regulated gene-1 (NDRG1), which in turn enhanced the expression of diacylglycerol acyltransferase 1/2 (DGAT1/2). DGAT1/2 could elevate cellular triglyceride synthesis and ultimately promote intracellular LD formation. Furthermore, we found that RABV-M and RABV-G, which were mainly involved in the viral budding process, could colocalize with LDs, indicating that RABV might utilize LDs as a carrier to facilitate viral budding and eventually increase virus production. Taken together, our study reveals that lipid droplets are beneficial for RABV replication, and their biogenesis is regulated via the NDRG1-DGAT1/2 pathway, which provides novel potential targets for developing anti-RABV drugs. IMPORTANCE Lipid droplets have been proven to play an important role in viral infections, but their role in RABV infection has not yet been elaborated. Here, we find that RABV infection upregulates the generation of LDs by enhancing the expression of N-myc downstream regulated gene-1 (NDRG1). Then NDRG1 elevated cellular triglycerides synthesis by increasing the activity of diacylglycerol acyltransferase 1/2 (DGAT1/2), which promotes the biogenesis of LDs. RABV-M and RABV-G, which are the major proteins involved in viral budding, could utilize LDs as a carrier for transport to cell membrane, resulting in enhanced virus budding. Our findings will extend the knowledge of lipid metabolism in RABV infection and help to explore potential therapeutic targets for RABV.

Establishment of Full-Length cDNA Clones and an Efficient Oral Infection Model for Feline Coronavirus in Cats.

Feline infectious peritonitis virus (FIPV) is the etiologic agent of feline infectious peritonitis (FIP) and causes fatal disease in cats of almost all ages. Currently, there are no clinically approved drugs or effective vaccines for FIP. Furthermore, the pathogenesis of FIP is still not fully understood. There is an urgent need for an effective infection model of feline infectious peritonitis induced by FIPV. Here, we constructed a field type I FIPV full-length cDNA clone, pBAC-QS, corresponding to the isolated FIPV QS. By replacing the FIPV QS spike gene with the commercially available type II FIPV 79-1146 (79-1146_CA) spike gene, we established and rescued a recombinant virus, designated rQS-79. Moreover, we constructed 79-1146_CA infectious full-length cDNA pBAC-79-1146_CA, corresponding to recombinant feline coronavirus (FCoV) 79-1146_CA (r79-1146_CA). In animal experiments with 1- to 2-year-old adult cats orally infected with the recombinant virus, rQS-79 induced typical FIP signs and 100% mortality. In contrast to cats infected with rQS-79, cats infected with 79-1146_CA did not show obvious signs. Furthermore, by rechallenging rQS-79 in surviving cats previously infected with 79-1146_CA, we found that there was no protection against rQS-79 with different titers of neutralizing antibodies. However, high titers of neutralizing antibodies may help prolong the cat survival time. Overall, we report the first reverse genetics of virulent recombinant FCoV (causing 100% mortality in adult cats) and attenuated FCoV (causing no mortality in adult cats), which will be powerful tools to study pathogenesis, antiviral drugs, and vaccines for FCoV. IMPORTANCE Tissue- or cell culture-adapted feline infectious peritonitis virus (FIPV) usually loses pathogenicity. To develop a highly virulent FIPV, we constructed a field isolate type I FIPV full-length clone with the spike gene replaced by the 79-1146 spike gene, corresponding to a virus named rQS-79, which induces high mortality in adult cats. rQS-79 represents the first described reverse genetics system for highly pathogenic FCoV. By further constructing the cell culture-adapted FCoV 79-1146_CA, we obtained infectious clones of virulent and attenuated FCoV. By in vitro and in vivo experiments, we established a model that can serve to study the pathogenic mechanisms of FIPV. Importantly, the wild-type FIPV replicase skeleton of serotype I will greatly facilitate the screening of antiviral drugs, both in vivo and in vitro.

Synaptic signalling in a network of dopamine neurons: what prevents proper intercellular crosstalk?

Human embryonic stem cell (hESC)-derived midbrain dopamine (DA) neurons stand out as a cell source for transplantation with their sustainability and consistency superior to the formerly used fetal tissues. However, multiple studies of DA neurons in culture failed to register action potential (AP) generation upon synaptic input. To test whether this is due to deficiency of NMDA receptor (NMDAR) coagonists released from astroglia, we studied the functional properties of neural receptors in hESC-derived DA neuronal cultures. We find that, apart from an insufficient amount of coagonists, lack of interneuronal crosstalk is caused by hypofunction of synaptic NMDARs due to their direct inhibition by synaptically released DA. This inhibitory tone is independent of DA receptors and affects the NMDAR coagonist binding site.

Cost-Effectiveness of Tofacitinib for Patients with Moderate-to-Severe Rheumatoid Arthritis in China.

BACKGROUND: Patients with moderate-to-severe rheumatoid arthritis have a heavy financial burden. The cost-effectiveness of introducing tofacitinib to the current treatment sequence for patients with moderate-to-severe rheumatoid arthritis who have inadequate response or intolerance to conventional synthetic disease-modifying antirheumatic drugs (csDMARDs-IR) in China remains unknown. OBJECTIVE: The objective of this study was to assess the cost-effectiveness of introducing tofacitinib into the current treatment sequence in China for patients with moderate-to-severe rheumatoid arthritis who have csDMARDs-IR. METHODS: A Markov model was constructed from the perspective of the Chinese healthcare system to compare treatment sequences with and without first-line tofacitinib for patients with rheumatoid arthritis with csDMARDs-IR. The treatment sequence without tofacitinib included adalimumab, etanercept, recombinant human tumor necrosis factor receptor-Fc fusion protein, infliximab, and tocilizumab. Costs were derived from publicly available sources. Clinical trials, network meta-analysis, and real-world data were used to generate quality-adjusted life-years (QALYs), transition probabilities, and the incidence of adverse events. Mortality probabilities were estimated from rheumatoid arthritis-based, Chinese all-cause mortality data. One-way and probabilistic sensitivity analyses were conducted to verify the robustness of the model. In addition, the cost-effectiveness of adding tofacitinib as second- and third-line treatment options was evaluated in our analyses. Costs and effects were discounted at 5% per anum. RESULTS: Compared to the current treatment sequence, adding tofacitinib as first-line treatment led to a cost-saving of $US880.11 (2018 values) and incremental QALYs of 1.34. Sensitivity analyses showed the results to be robust. Adding tofacitinib at second-line therapy was also a cost-saving option with a cost saving of $US653.65 and incremental QALYs of 1.34, while the incremental cost-effectiveness ratio of adding tofacitinib at third-line therapy was $US5588.14 per QALY gained. CONCLUSIONS: Using the WHO-recommended ICER acceptability threshold of ≤ 1-time per capita Gross Domestic Product (GDP), our analysis suggests that the introduction of tofacitinib into the current treatment sequence for moderate-to-severe RA patients with csDMARDs-IR in China was a cost saving option as first- and second-line treatment, and cost-effective as a third-line treatment option. Of note, use of tofacitinib as first- and second-line treatment post-csDMARDs-IR appeared to be cost saving.

Engineering synucleinopathy-resistant human dopaminergic neurons by CRISPR-mediated deletion of the SNCA gene.

An emerging treatment for Parkinson's disease (PD) is cell replacement therapy. Authentic midbrain dopaminergic (mDA) neuronal precursors can be differentiated from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs). These laboratory-generated mDA cells have been demonstrated to mature into functional dopaminergic neurons upon transplantation into preclinical models of PD. However, clinical trials with human fetal mesenchephalic cells have shown that cell replacement grafts in PD are susceptible to Lewy body formation suggesting host-to-graft transfer of α-synuclein pathology. Here, we have used CRISPR/Cas9n technology to delete the endogenous SNCA gene, encoding for α-synuclein, in a clinical-grade hESC line to generate SNCA+/- and SNCA-/- cell lines. These hESC lines were first differentiated into mDA neurons, and then challenged with recombinant α-synuclein preformed fibrils (PFFs) to seed the formation for Lewy-like pathology as measured by phosphorylation of serine-129 of α-synuclein (pS129-αSyn). Wild-type neurons were fully susceptible to the formation of protein aggregates positive for pS129-αSyn, while SNCA+/- and SNCA-/- neurons exhibited significant resistance to the formation of this pathological mark. This work demonstrates that reducing or completely removing SNCA alleles by CRISPR/Cas9n-mediated gene editing confers a measure of resistance to Lewy pathology.

Sex hormones affect acute and chronic stress responses in sexually dimorphic patterns: Consequences for depression models.

BACKGROUND: Alterations in peripheral sex hormones may play an important role in sex differences in terms of stress responses and mood disorders. It is not yet known whether and how stress-related brain systems and brain sex steroid levels fluctuate in relation to changes in peripheral sex hormone levels, or whether the different sexes show different patterns. We aimed to investigate systematically, in male and female rats, the effect of decreased circulating sex hormone levels following gonadectomy on acute and chronic stress responses, manifested as changes in plasma and hypothalamic sex steroids and hypothalamic stress-related molecules. METHOD: Experiment (Exp)-1: Rats (14 males, 14 females) were gonadectomized or sham-operated (intact); Exp-2: gonadectomized and intact rats (28 males, 28 females) were exposed to acute foot shock or no stressor; and Exp-3: gonadectomized and intact rats (32 males, 32 females) were exposed to chronic unpredictable mild stress (CUMS) or no stressor. For all rats, plasma and hypothalamic testosterone (T), estradiol (E2), and the expression of stress-related molecules were determined, including corticotropin-releasing hormone, vasopressin, oxytocin, aromatase, and the receptors for estrogens, androgens, glucocorticoids, and mineralocorticoids. RESULTS: Surprisingly, no significant correlation was observed in terms of plasma sex hormones, brain sex steroids, and hypothalamic stress-related molecule mRNAs (p > 0.113) in intact or gonadectomized, male or female, rats. Male and female rats, either intact or gonadectomized and exposed to acute or chronic stress, showed different patterns of stress-related molecule changes. CONCLUSION: Diminished peripheral sex hormone levels lead to different peripheral and central patterns of change in the stress response systems in male and female rats. This has implications for the choice of models for the study of the different types of mood disorders which also show sex differences.

Economic analysis of empiric versus diagnostic-driven strategies for immunocompromised patients with suspected fungal infections in the People's Republic of China.

Invasive fungal infections (IFIs) require rapid diagnosis and treatment. A decision-analytic model was used to estimate total costs and survival associated with a diagnostic-driven (DD) or an empiric treatment approach in neutropenic patients with hematological malignancies receiving chemotherapy or autologous/allogeneic stem cell transplants in Shanghai, Beijing, Chengdu, and Guangzhou, the People's Republic of China. Treatment initiation for the empiric approach occurred after clinical suspicion of an IFI; treatment initiation for the DD approach occurred after clinical suspicion and a positive IFI diagnostic test result. Model inputs were obtained from the literature; treatment patterns and resource use were based on clinical opinion. Total costs were lower for the DD versus the empiric approach in Shanghai (¥3,232 vs ¥4,331), Beijing (¥3,894 vs ¥4,864), Chengdu, (¥4,632 vs ¥5,795), and Guangzhou (¥8,489 vs ¥9,795). Antifungal administration was lower using the DD (5.7%) than empiric (9.8%) approach, with similar survival rates. Results from one-way and probabilistic sensitivity analyses were most sensitive to changes in diagnostic test sensitivity and IFI incidence; the DD approach dominated the empiric approach in 88% of scenarios. These results suggest that a DD compared to an empiric treatment approach in the People's Republic of China may be cost saving, with similar overall survival in immunocompromised patients with suspected IFIs.

Mouse precision-cut liver slices as an ex vivo model to study idiosyncratic drug-induced liver injury.

Idiosyncratic drug-induced liver injury (IDILI) has been the top reason for withdrawing drugs from the market or for black box warnings. IDILI may arise from the interaction of a drug's reactive metabolite with a mild inflammation that renders the liver more sensitive to injury resulting in increased toxicity (inflammatory stress hypothesis). Aiming to develop a robust ex vivo screening method to study inflammatory stress-related IDILI mechanisms and to find biomarkers that can detect or predict IDILI, mouse precision-cut liver slices (mPCLS) were coincubated for 24 h with IDILI-related drugs and lipopolysaccharide. Lipopolysaccharide exacerbated ketoconazole (15 µM) and clozapine (45 µM) toxicity but not their non-IDILI-related comparators, voriconazole (1500 µM) and olanzapine (45 µM). However, the other IDILI-related drugs tested [diclofenac (200 µM), carbamazepine (400 µM), and troglitazone (30 µM)] did not cause synergistic toxicity with lipopolysaccharide after 24 h of incubation. Lipopolysaccharide further decreased the reduced glutathione levels caused by ketoconazole or clozapine in mPCLS after 24 h of incubation, which was not the case for the other drugs. Lipopolysaccharide significantly increased nitric oxide (NO), cytokine, and chemokine release into the mPCLS media, while the treatment with the drugs alone did not cause any substantial change. All seven drugs drastically reduced lipopolysaccharide-induced NO production. Interestingly, only ketoconazole and clozapine increased the lipopolysaccharide-induced granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) release. Pilot experiments showed that diclofenac and troglitazone, but not carbamazepine, demonstrated synergistic toxicity with lipopolysaccharide after a longer incubation of 48 h in mPCLS. In conclusion, we have developed an ex vivo model to detect inflammatory stress-related liver toxicity and identified ketoconazole, clozapine, troglitazone, and diclofenac as drugs that showed synergistic toxicity with lipopolysaccharide. Reduced glutathione, G-CSF, and GM-CSF were identified to be potential biomarkers for IDILI-inducing drugs mediated by inflammatory stress, and mPCLS appear to be a promising screening tool to further unravel the mechanism of IDILI.