AAV mediated carboxyl terminus of Hsp70 interacting protein overexpression mitigates the cognitive and pathological phenotypes of APP/PS1 mice.

JOURNAL/nrgr/04.03/01300535-202501000-00033/figure1/v/2024-05-14T021156Z/r/image-tiff The E3 ubiquitin ligase, carboxyl terminus of heat shock protein 70 (Hsp70) interacting protein (CHIP), also functions as a co-chaperone and plays a crucial role in the protein quality control system. In this study, we aimed to investigate the neuroprotective effect of overexpressed CHIP on Alzheimer's disease. We used an adeno-associated virus vector that can cross the blood-brain barrier to mediate CHIP overexpression in APP/PS1 mouse brain. CHIP overexpression significantly ameliorated the performance of APP/PS1 mice in the Morris water maze and nest building tests, reduced amyloid-ß plaques, and decreased the expression of both amyloid-ß and phosphorylated tau. CHIP also alleviated the concentration of microglia and astrocytes around plaques. In APP/PS1 mice of a younger age, CHIP overexpression promoted an increase in ADAM10 expression and inhibited ß-site APP cleaving enzyme 1, insulin degrading enzyme, and neprilysin expression. Levels of HSP70 and HSP40, which have functional relevance to CHIP, were also increased. Single nuclei transcriptome sequencing in the hippocampus of CHIP overexpressed mice showed that the lysosomal pathway and oligodendrocyte-related biological processes were up-regulated, which may also reflect a potential mechanism for the neuroprotective effect of CHIP. Our research shows that CHIP effectively reduces the behavior and pathological manifestations of APP/PS1 mice. Indeed, overexpression of CHIP could be a beneficial approach for the treatment of Alzheimer's disease.

Transient inhibition of neutrophil functions enhances the antitumor effect of intravenously delivered oncolytic vaccinia virus.

Oncolytic viruses (OVs) possess the unique ability to selectively replicate within tumor cells, leading to their destruction, while also reversing the immunosuppression within the tumor microenvironment and triggering an antitumor immune response. As a result, OVs have emerged as one of the most promising approaches in cancer therapy. However, the effective delivery of intravenously administered OVs faces significant challenges imposed by various immune cells within the peripheral blood, hindering their access to tumor sites. Notably, neutrophils, the predominant white blood cell population comprising approximately 50%-70% of circulating white cells in humans, show phagocytic properties. Our investigation revealed that the majority of oncolytic vaccinia viruses (VV) are engulfed and degraded by neutrophils in the bloodstream. The depletion of neutrophils using the anti-LY6G Ab (1-A8) resulted in an increased accumulation of circulating oncolytic VV in the peripheral blood and enhanced deposition at the tumor site, consequently amplifying the antitumor effect. Neutrophils heavily rely on PI3K signaling to sustain their phagocytic process. Additionally, our study determined that the inhibition of the PI3Kinase delta isoform by idelalisib (CAL-101) suppressed the uptake of oncolytic VV by neutrophils. This inhibition led to a greater presence of oncolytic VV in both the peripheral blood and at the tumor site, resulting in improved efficacy against the tumor. In conclusion, our study showed that inhibiting neutrophil functions can significantly enhance the antitumor efficacy of intravenous oncolytic VV.

Rapid identification of full-length genome and tracing variations of monkeypox virus in clinical specimens based on mNGS and amplicon sequencing.

The monkeypox virus (MPXV) has triggered a current outbreak globally. Genome sequencing of MPXV and rapid tracing of genetic variants will benefit disease diagnosis and control. It is a significant challenge but necessary to optimize the strategy and application of rapid full-length genome identification and to track variations of MPXV in clinical specimens with low viral loads, as it is one of the DNA viruses with the largest genome and the most AT-biased, and has a significant number of tandem repeats. Here we evaluated the performance of metagenomic and amplicon sequencing techniques, and three sequencing platforms in MPXV genome sequencing based on multiple clinical specimens of five mpox cases in Chinese mainland. We rapidly identified the full-length genome of MPXV with the assembly of accurate tandem repeats in multiple clinical specimens. Amplicon sequencing enables cost-effective and rapid sequencing of clinical specimens to obtain high-quality MPXV genomes. Third-generation sequencing facilitates the assembly of the terminal tandem repeat regions in the monkeypox virus genome and corrects a common misassembly in published sequences. Besides, several intra-host single nucleotide variations were identified in the first imported mpox case. This study offers an evaluation of various strategies aimed at identifying the complete genome of MPXV in clinical specimens. The findings of this study will significantly enhance the surveillance of MPXV.

Hypoxia-regulated secretion of IL-12 enhances antitumor activity and safety of CD19 CAR-T cells in the treatment of DLBCL.

CD19-targeted chimeric antigen receptor-modified T (CD19 CAR-T) cell therapy has been demonstrated as one of the most promising therapeutic strategies for treating B cell malignancies. However, it has shown limited treatment efficacy for diffuse large B cell lymphoma (DLBCL). This is, in part, due to the tumor heterogeneity and the hostile tumor microenvironment. Human interleukin-12 (IL-12), as a potent antitumor cytokine, has delivered encouraging outcomes in preclinical studies of DLBCL. However, potentially lethal toxicity associated with systemic administration precludes its clinical application. Here, an armed CD19 CAR expressing hypoxia-regulated IL-12 was developed (CAR19/hIL12ODD). In this vector, IL-12 secretion was restricted to hypoxic microenvironments within the tumor site by fusion of IL-12 with the oxygen degradation domain (ODD) of HIF1α. In vitro, CAR19/hIL12ODD-T cells could only secrete bioactive IL-12 under hypoxic conditions, accompanied by enhanced proliferation, robust IFN-γ secretion, increased abundance of CD4+, and central memory T cell phenotype. In vivo, adoptive transfer of CAR19/hIL12ODD-T cells significantly enhanced regression of large, established DLBCL xenografts in a novel immunodeficient Syrian hamster model. Notably, this targeted and controlled IL-12 treatment was without toxicity in this model. Taken together, our results suggest that armed CD19 CARs with hypoxia-controlled IL-12 (CAR19/hIL12ODD) might be a promising and safer approach for treating DLBCL.

Distinct assembly processes and environmental adaptation of abundant and rare archaea in Arctic marine sediments.

Revealing the ecological processes and environmental adaptation of abundant and rare archaea is a central, but poorly understood, topic in ecology. Here, abundant and rare archaeal diversity, community assembly processes and co-occurrence patterns were comparatively analyzed in Arctic marine sediments. Our findings revealed that the rare taxa exhibited significantly higher diversity compared to the abundant taxa. Additionally, the abundant taxa displayed stronger environmental adaptation than the rare taxa. The co-occurrence network analysis demonstrated that the rare taxa developed more interspecies interactions and modules in response to environmental disturbance. Furthermore, the community assembly of abundant and rare taxa in sediments was primarily controlled by stochastic and deterministic processes, respectively. These findings provide valuable insights into the archaeal community assembly processes and significantly contribute to a deeper understanding of the environmental adaptability of abundant and rare taxa in Arctic marine sediments.

Genomic Surveillance for SARS-CoV-2 - China, September 26, 2022 to January 29, 2023.

Introduction: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has generated 2,431 variants over the course of its global transmission over the past 3 years. To better evaluate the genomic variation of SARS-CoV-2 before and after the optimization of coronavirus disease 2019 (COVID-19) prevention and control strategies, we analyzed the genetic evolution branch composition and genomic variation of SARS-CoV-2 in both domestic and imported cases in China (the data from Hong Kong and Macau Special Administrative Regions and Taiwan, China were not included) from September 26, 2022 to January 29, 2023. Methods: Analysis of the number of genome sequences, sampling time, dynamic changes of evolutionary branches, origin, and clinical typing of SARS-CoV-2 variants submitted by 31 provincial-level administrative divisions (PLADs) and Xinjiang Production and Construction Corps (XPCC) was conducted to assess the accuracy and timeliness of SARS-CoV-2 variant surveillance. Results: From September 26, 2022 to January 29, 2023, 20,013 valid genome sequences of domestic cases were reported in China, with 72 evolutionary branches. Additionally, 1,978 valid genome sequences of imported cases were reported, with 169 evolutionary branches. The prevalence of the Omicron variants of SARS-CoV-2 in both domestic and imported cases was consistent with that of international epidemic variants. Conclusions: This study provides an overview of the prevalence of Omicron variants of SARS-CoV-2 in China. After optimizing COVID-19 prevention and control strategies, no novel Omicron variants of SARS-CoV-2 with altered biological characteristics or public health significance have been identified since December 1, 2022.

CHCHD2 p.Thr61Ile knock-in mice exhibit motor defects and neuropathological features of Parkinson's disease.

The p.Thr61Ile (p.T61I) mutation in coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2) was deemed a causative factor in Parkinson's disease (PD). However, the pathomechanism of the CHCHD2 p.T61I mutation in PD remains unclear. Few existing mouse models of CHCHD2-related PD completely reproduce the features of PD, and no transgenic or knock-in (KI) mouse models of CHCHD2 mutations have been reported. In the present study, we generated a novel CHCHD2 p.T61I KI mouse model, which exhibited accelerated mortality, progressive motor deficits, and dopaminergic (DA) neurons loss with age, accompanied by the accumulation and aggregation of α-synuclein and p-α-synuclein in the brains of the mutant mice. The mitochondria of mouse brains and induced pluripotent stem cells (iPSCs)-derived DA neurons carrying the CHCHD2 p.T61I mutation exhibited aberrant morphology and impaired function. Mechanistically, proteomic and RNA sequencing analysis revealed that p.T61I mutation induced mitochondrial dysfunction in aged mice likely through repressed insulin-degrading enzyme (IDE) expression, resulting in the degeneration of the nervous system. Overall, this CHCHD2 p.T61I KI mouse model recapitulated the crucial clinical and neuropathological aspects of patients with PD and provided a novel tool for understanding the pathogenic mechanism and therapeutic interventions of CHCHD2-related PD.

Development of two multiplex real-time PCR assays for simultaneous detection and differentiation of monkeypox virus IIa, IIb, and I clades and the B.1 lineage.

An ongoing multi-country outbreak of monkeypox was reported in May 2022 with several deaths, affecting 107 countries of all six World Health Organization (WHO) regions. The WHO has declared the current monkeypox outbreak to be a Public Health Emergency of International Concern. It is, thus, necessary to rapidly and accurately detect and distinguish different monkeypox virus (MPXV) clades. We designed primers and probes based on the alignment of 138 complete genomes of poxviruses. In Panel 1, we mixed one pair of primers and three probes to detect and differentiate the MPXV Western Africa (IIa, IIb clade) and Congo Basin (I clade) and other orthopoxviruses. In Panel 2, we mixed one pair of primers and two probes to detect the 2022 MPXV (B.1 lineage and its descendant lineages). In addition, we tested the specificity and sensitivity of the assay using real-time PCR. In Panel 1, the assay reproducibly identified various concentrations of two plasmids of the monkeypox virus, whereas other orthopoxviruses did not cross-react. In Panel 2, the probe annealed well to MPXV B.1 and showed the expected linearity. These two multiple real-time assays are inclusive and highly specific for identifying different clades of MPXV.

Deoxythymidylate Kinase as a Promising Marker for Predicting Prognosis and Immune Cell Infiltration of Pan-cancer.

Background: Deoxythymidylate kinase (DTYMK) serves as a pyrimidine metabolic rate-limiting enzyme that catalyzes deoxythymidine monophosphate (dTMP) to generate deoxythymidine diphosphate (dTDP). It remains unclear whether DTYMK expression has the potential to predict outcome and immune cell infiltration in cancers. Methods: DTYMK expression profile was analyzed using Oncomine, TIMER, GEPIA and UALCAN databases. The influence of DTYMK on immune infiltration was examined using TIMER and TISIDB databases. DTYMK interactive gene hub and co-expressing genes were obtained and analyzed by STRING and Linkedomics, respectively. The relationship between DTYMK expression and patient prognosis was validated using GEPIA, Kaplan-Meier plotter, and PrognoScan databases. The functions of DTYMK in cancer cells were also biologically validated in vitro. Results: DTYMK expression was elevated in tumor tissues compared with their control counterparts. DTYMK expression varied in different stages and discriminatorily distributed in different immune and molecular subtypes. Higher expression of DTYMK predicted worse outcome in several cancer types such as liver hepatocellular carcinoma (LIHC) and lung adenocarcinoma (LUAD). High DTYMK expression was positively or negatively correlated with immune cell infiltration, including B cell, CD8+ cell, CD4+ T cell, macrophage, neutrophil and dendritic cell, depending on the type of cancers. Additionally, DTYMK co-expressing genes participated in pyrimidine metabolism as well as in T helper cell differentiation in LIHC and LUAD. In vitro, knockdown of DTYMK suppressed cell migration of liver and lung cancer cells. Conclusion: DTYMK might be taken as an useful prognostic and immunological marker in cancers and further investigation is warrented.

Implementation of a pathological diagnosis and treatment pathway may improve the molecular detection of lung cancer.

Background: The 2018 Guideline from the College of American Pathologists (CAP), the International Association for the Study of Lung Cancer (IASLC), and the Association for Molecular Pathology (AMP) established a benchmark turnaround time (TAT), according to which the results should be available to the treating oncologist within 10 working days. This article focused on the application process of a new protocol for pathological diagnosis and gene testing pathway and a sample collector. We want to solve the problem that there are not enough puncture samples for gene testing, and the benchmark turnaround time of gene detection was long in clinic. Methods: In this study, we established and validated a new protocol for a pathological diagnosis and treatment pathway that was tested in the Henan Cancer Hospital, China. The "Biology collector (BIOCO)" tool was designed by our team, was made of polyvinyl chloride (PVC) material (patent application number is 201820902335.6). It consisted of two round magnets on the collector that can be adsorbed on the microtome blade holder, thus making it to move arbitrarily. It collected specimens that were discarded when the wax block was trimmed. We analyzed the TAT, testing accuracy and anti-pollution of new protocol based on BIOCO, compared with the conventional process based on the Routine Collection (ROUCO). Results: The new pathway adopts a parallel approach to conventional pathology and molecular pathology, which significantly shortens the TAT to 4-6 days. The use of the BIOCO tool can effectively save pathological samples, avoid cross-contamination, and reduce the time delay caused by re-sampling. Most importantly, its accuracy and effectiveness are consistent with conventional collection methods. Conclusions: The new diagnosis and treatment pathway based on the BIOCO collector can be used as a practical approach for the molecular diagnostic platform of the hospital pathology department.

Analysis of 12 GWAS-Linked Loci With Parkinson's Disease in the Chinese Han Population.

A recent large-scale European-originated genome-wide association study identified 38 novel independent risk signals in 37 loci for Parkinson's disease (PD). However, whether these new loci are associated with PD in Asian populations remains elusive. The present study aimed to explore the relationship between the 12 most relevant loci with larger absolute values for these new risk loci and PD in the Chinese Han population. We performed a case-control study including 527 PD patients and 435 healthy controls. In the allele model, it was found that rs10748818/GBF1 was associated with PD in the Chinese Han population [p = 0.035, odds ratio (OR) 1.221, 95% confidence interval (CI) 1.014-1.472.

Chaperone-mediated autophagy affects tumor cell proliferation and cisplatin resistance in esophageal squamous cell carcinoma.

BACKGROUND: Chaperone-mediated autophagy (CMA) is a lysosomal degradation pathway of selective soluble proteins. Lysosomal membrane associated protein 2a (LAMP2a) is the lysosomal membrane receptor of CMA and influences CMA activity. Although it has been suggested that higher expression of LAMP2a is associated with more advanced tumor node metastasis (TNM) stages and shorter survival time in patients with esophageal squamous cell carcinoma (ESCC), the underlying mechanism has not been known yet. METHODS: In this study, we modulated the activity of CMA through LAMP2a or small molecular compounds in human ESCC cells to investigate its role in ESCC. RESULTS: We found that down-regulating the activity of CMA could inhibit the proliferation and colony formation of ESCC cells as well as increase their sensitivity to cisplatin. CONCLUSIONS: Our results promote better understanding of how CMA affects human ESCC and provide a new therapeutic target against ESCC through down-regulating LAMP2a.

A systemically deliverable Vaccinia virus with increased capacity for intertumoral and intratumoral spread effectively treats pancreatic cancer.

BACKGROUND: Pancreatic cancer remains one of the most lethal cancers and is refractory to immunotherapeutic interventions. Oncolytic viruses are a promising new treatment option, but current platforms demonstrate limited efficacy, especially for inaccessible and metastatic cancers that require systemically deliverable therapies. We recently described an oncolytic vaccinia virus (VV), VVLΔTKΔN1L, which has potent antitumor activity, and a regime to enhance intravenous delivery of VV by pharmacological inhibition of pharmacological inhibition of PI3 Kinase δ (PI3Kδ) to prevent virus uptake by macrophages. While these platforms improve the clinical prospects of VV, antitumor efficacy must be improved. METHODS: VVLΔTKΔN1L was modified to improve viral spread within and between tumors via viral B5R protein modification, which enhanced production of the extracellular enveloped virus form of VV. Antitumor immunity evoked by viral treatment was improved by arming the virus with interleukin-21, creating VVL-21. Efficacy, functional activity and synergy with α-programmed cell death protein 1 (α-PD1) were assessed after systemic delivery to murine and Syrian hamster models of pancreatic cancer. RESULTS: VVL-21 could reach tumors after systemic delivery and demonstrated antitumor efficacy in subcutaneous, orthotopic and disseminated models of pancreatic cancer. The incorporation of modified B5R improved intratumoural accumulation of VV. VVL-21 treatment increased the numbers of effector CD8+ T cells within the tumor, increased circulating natural killer cells and was able to polarize macrophages to an M1 phenotype in vivo and in vitro. Importantly, treatment with VVL-21 sensitized tumors to the immune checkpoint inhibitor α-PD1. CONCLUSIONS: Intravenously administered VVL-21 successfully remodeled the suppressive tumor-microenvironment to promote antitumor immune responses and improve long-term survival in animal models of pancreatic cancer. Importantly, treatment with VVL-21 sensitized tumors to the immune checkpoint inhibitor α-PD1. Combination of PI3Kδ inhibition, VVL-21 and α-PD1 creates an effective platform for treatment of pancreatic cancer.

CHIP protects against MPP+/MPTP-induced damage by regulating Drp1 in two models of Parkinson's disease.

Mitochondrial dysfunction has been implicated in the pathogenesis of Parkinson's disease (PD). Carboxyl terminus of Hsp70-interacting protein (CHIP) is a key regulator of mitochondrial dynamics, and mutations in CHIP or deficits in its expression have been associated with various neurological diseases. This study explores the protective role of CHIP in cells and murine PD models. In SH-SY5Y cell line, overexpression of CHIP improved the cell viability and increased the ATP levels upon treatment with 1-methyl-4-phenylpyridinium (MPP+). To achieve CHIP overexpression in animal models, we intravenously injected mice with AAV/BBB, a new serotype of adeno-associated virus that features an enhanced capacity to cross the blood-brain barrier. We also generated gene knock-in mice that overexpressed CHIP in neural tissue. Our results demonstrated that CHIP overexpression in mice suppressed 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced damage, including movement impairments, motor coordination, and spontaneous locomotor activity, as well as loss of dopaminergic neurons. In vitro and in vivo experiments showed that overexpression of CHIP inhibited the pathological increase in Drp1 observed in the PD models, suggesting that CHIP regulates Drp1 degradation to attenuate MPP+/MPTP-induced injury. We conclude that CHIP plays a protective role in MPP+/MPTP-induced PD models. Our experiments further revealed that CHIP maintains the integrity of mitochondria.

Impaired meningeal lymphatic drainage in patients with idiopathic Parkinson's disease.

Animal studies implicate meningeal lymphatic dysfunction in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease (PD). However, there is no direct evidence in humans to support this role1-5. In this study, we used dynamic contrast-enhanced magnetic resonance imaging to assess meningeal lymphatic flow in cognitively normal controls and patients with idiopathic PD (iPD) or atypical Parkinsonian (AP) disorders. We found that patients with iPD exhibited significantly reduced flow through the meningeal lymphatic vessels (mLVs) along the superior sagittal sinus and sigmoid sinus, as well as a notable delay in deep cervical lymph node perfusion, compared to patients with AP. There was no significant difference in the size (cross-sectional area) of mLVs in patients with iPD or AP versus controls. In mice injected with α-synuclein (α-syn) preformed fibrils, we showed that the emergence of α-syn pathology was followed by delayed meningeal lymphatic drainage, loss of tight junctions among meningeal lymphatic endothelial cells and increased inflammation of the meninges. Finally, blocking flow through the mLVs in mice treated with α-syn preformed fibrils increased α-syn pathology and exacerbated motor and memory deficits. These results suggest that meningeal lymphatic drainage dysfunction aggravates α-syn pathology and contributes to the progression of PD.

α-Synuclein in Parkinson's Disease: Does a Prion-Like Mechanism of Propagation from Periphery to the Brain Play a Role?

Parkinson's disease (PD) is one of the most common neurodegenerative diseases, defined as motor and non-motor symptoms associated with the loss of dopaminergic neurons and a decreased release of dopamine (DA). Currently, PD patients are believed to have a neuropathological basis denoted by the presence of Lewy bodies (LBs) or Lewy neurites (LNs), which mostly comprise α-synuclein (α-syn) inclusions. Remarkably, there is a growing body of evidence indicating that the inclusions undergo template-directed aggregation and propagation via template-directed among the brain and peripheral organs, mainly in a prion-like manner. Interestingly, some studies reported that an integral loop was reminiscent of the mechanism of Parkinson's disease, denoting that α-syn as prionoid was transmitted from the periphery to the brain via specific pathways. Also the systematic life cycle of α-syn in the cellular level is illustrated. In this review, we critically assess landmark evidence in the field of Parkinson's disease with a focus on the genesis and prion-like propagation of the α-syn pathology. The anatomical and cell-to-cell evidences are discussed to depict the theory behind the propagation and transferred pathways. Furthermore, we highlight effective therapeutic perspectives and clinical trials targeting prion-like mechanisms. Major controversies surrounding this topic are also discussed.

Treatment and Prevention of Lung Cancer Using a Virus-Infected Reprogrammed Somatic Cell-Derived Tumor Cell Vaccination (VIReST) Regime.

Lung cancer is one of the most commonly diagnosed cancer and despite therapeutic advances, mortality remains high. The long period of clinical latency associated with lung cancer provides an ideal window of opportunity to administer vaccines to at-risk individuals that can prevent tumor progression and initiate long-term anti-tumor immune surveillance. Here we describe a personalized vaccination regime that could be applied for both therapeutic and prophylactic prevention of lung cancer, based on the derivation of lung cancer cells from induced pluripotent stem cells. Stem cells from healthy mice were modified to express Cre-dependent KRASG12D and Trp53R172H prior to differentiation to lung progenitor cells. Subsequent viral delivery of Cre caused activation of exogenous driver mutations, resulting in transformation and development of lung cancer cells. iPSC-derived lung cancer cells were highly antigenically related to lung cancer cells induced in LSL-KRASG12D/+; Trp53R172H/+ transgenic mice and were antigenically unrelated to original pluripotent stem cells or pancreatic cancer cells derived using the same technological platform. For vaccination, induced lung cancer cells were infected with oncolytic Adenovirus or Vaccinia virus, to act as vaccine adjuvants, prior to delivery of vaccines sequentially to a murine inducible transgenic model of lung cancer. Application of this Virus-Infected, Reprogrammed Somatic cell-derived Tumor cell (VIReST) regime primed tumor-specific T cell responses that significantly prolonged survival in both subcutaneous post-vaccine challenge models and induced transgenic models of lung cancer, demonstrating that stem cell-derived prophylactic vaccines may be a feasible intervention for treatment or prevention of lung cancer development in at-risk individuals.