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.

[Discussion of Application Status and Key Attentions on Registration for Ossicular Replacement Prosthesis].

The ossicular replacement prosthesis should have good biocompatibility, stability, easy to install, and excellent sound transmission capacity. In this study, the characteristics of ideal materials for the ossicular replacement prosthesis were analyzed by searching the types of materials used in clinical practice and comparing the advantages and disadvantages of various materials and structures. At the same time, in combination with the current evaluation requirements and evaluation experience, the focus of the performance research project of ossicular replacement prosthesis in the process of registration is discussed to clarify the performance evaluation requirements of these products, so as to provide reference for the future work of manufacturers and regulators. The performance evaluation of ossicular replacement prosthesis focuses on its mechanical properties, fixation stability, sound transmission characteristics, biological characteristics, and magnetic resonance compatibility.

Chronic Unpredictable Mild Stress in Rats based on the Mongolian medicine.

Depression is a prevalent affective disorder and constitutes a leading cause of global disability. The limitations of current pharmacological interventions contribute to the substantial health burden attributed to this condition. There is a pressing need for a deeper understanding of the underlying mechanisms of depression, making pre-clinical models with translational potential highly valuable. Mongolian medicine, a subset of traditional medicine, posits that disease occurrence is closely tied to the equilibrium of wind, bile, and Phlegm. In this study, we introduce a protocol for the chronic unpredictable mild stress (CUMS) method in rats. Within this framework, rats are subjected to a series of fluctuating, mild stressors to induce a depression-like phenotype, mimicking the pathogenesis of human depression. Behavioral assays employed in this protocol include the sucrose preference test (SPT), indicative of anhedonia-a core symptom of depression; the open field test (OFT), which measures anxiety levels; and the Morris water maze test (MWM), which evaluates spatial memory and learning abilities. The CUMS method demonstrates the capability to induce anhedonia and to cause long-term behavioral deficits. Furthermore, this protocol is more aligned with Mongolian medical theory than other animal models designed to elicit depression-like behavior. The development of this animal model and subsequent research provide a robust foundation for future innovative studies in the realm of Mongolian medicine.

[Discussion and Concern on Animal Study for Orthopaedic Implantable Devices].

In recent years, new orthopaedic implantable devices continue to emerge, which require higher requirements for technical evaluation. Animal study is an important part of the research and development process for the new orthopedic implantable devices, which provides relevant evidence for product design and stereotyping. By introducing the purpose of animal study, and the application of 3R principle (replacement, reduction, refinement) in this field, we summarize the concern on the animal study, in order to provide reference for the development and research of new orthopedic implantable devices and biomaterials. At the same time, the application of evidence-based research methods such as systematic review in the field is introduced, which provides new tools and approaches for the technical review and regulatory science.

Increased Gal-3 Mediates Microglia Activation and Neuroinflammation via the TREM2 Signaling Pathway in Prion Infection.

Galectin 3 (Gal-3) is one of the major elements for activating microglia and mediating neuroinflammation in some types of neurodegenerative diseases. However, its role in the pathogenesis of prion disease is seldom addressed. In this study, markedly increased brain Gal-3 was identified in three scrapie-infected rodent models at the terminal stage. The increased Gal-3 was mainly colocalized with the activated microglia. Coincidental with the increased brain Gal-3 in prion-infected animals, the expression of brain trigger receptor expressed in myeloid cell 2 (TREM2), one of the Gal-3 receptors, and some components in the downstream pathway also significantly increased, whereas Toll-like receptor 4 (TLR4), another Gal-3 receptor, and the main components in its downstream signaling were less changed. The increased Gal-3 signals were distributed at the areas with PrPSc deposit but looked not to colocalize directly with PrPSc/PrP signals. Similar changing profiles of Gal-3, the receptors TREM2 and TLR4, as well as the proteins in the downstream pathways were also observed in prion-infected cell line SMB-S15. Removal of PrPSc replication in SMB-S15 cells reversed the upregulation of cellular Gal-3, TREM2, and the relevant proteins. Moreover, we presented data for interactions of Gal-3 with TREM2 and with TLR4 morphologically and molecularly in the cultured cells. Stimulation of prion-infected cells or their normal partner cells with recombinant mouse Gal-3 in vitro induced obvious responses for activation of TREM2 signaling and TLR4 signaling. Our data here strongly indicate that prion infection or PrPSc deposit induces remarkably upregulated brain Gal-3, which is actively involved in the microglia activation and neuroinflammation mainly via TREM2 signaling.

Accumulation of Prion Triggers the Enhanced Glycolysis via Activation of AMKP Pathway in Prion-Infected Rodent and Cell Models.

Mitochondrial dysfunction is one of the hallmarks in the pathophysiology of prion disease and other neurodegenerative diseases. Various metabolic dysfunctions are identified and considered to contribute to the progression of some types of neurodegenerative diseases. In this study, we evaluated the status of glycolysis pathway in prion-infected rodent and cell models. The levels of the key enzymes, hexokinase (HK), phosphofructokinase (PFK), and pyruvate kinase (PK) were significantly increased, accompanying with markedly downregulated mitochondrial complexes. Double-stained IFAs revealed that the increased HK2 and PFK distributed widely in GFAP-, Iba1-, and NeuN-positive cells. We also identified increased levels of AMP-activated protein kinase (AMPK) and the downstream signaling. Changes of AMPK activity in prion-infected cells by the AMPK-specific inhibitor or activator induced the corresponding alterations not only in the downstream signaling, but also the expressions of three key kinases in glycolysis pathway and the mitochondrial complexes. Transient removal or complete clearance of prion propagation in the prion-infected cells partially but significantly reversed the increases of the key enzymes in glycolysis, the upregulation of AMPK signaling pathway, and the decreases of the mitochondrial complexes. Measurements of the cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) showed lower OCR and higher ECAR in prion-infected cell line, which were sufficiently reversed by clearance of prion propagation. Those data indicate a metabolic reprogramming from oxidative phosphorylation to glycolysis in the brains during the progression of prion disease. Accumulation of PrPSc is critical for the switch to glycolysis, largely via activating AMPK pathway.

[Concerns of Technical Evaluation on Registration of All-inside Meniscus Suture System].

OBJECTIVE: To summarize the product registration declaration ideas and registration technical review of the all-inside meniscal suture system, and to systematically think about of the technical review concerns of the all-inside meniscal suture system products to provide technical guidance for improving the quality of registration and application and regulatory efficiency. METHODS: Consult the public information of such products at home and abroad, and summarize the experience of registration review of such products. RESULTS: The technical review of the all-inside meniscus suture system registration mainly focuses on product basic information, pre-clinical research, clinical evaluation and product technical requirements. CONCLUSIONS: The difficulty of product registration and declaration of the all-inside meniscus suture system lies in the provision of pre-clinical research data of the product, and the applicant needs to strengthen the basic research ability, formulate scientific technical indicators and test methods to ensure the safety and effectiveness of the product, and also provide sufficient supporting data for the registration declaration.

Abnormal Changes of IL3/IL3R and Its Downstream Signaling Pathways in the Prion-Infected Cell Line and in the Brains of Scrapie-Infected Rodents.

Interleukin 3 (IL-3) plays an important role in hematopoiesis and immune regulation, brain IL-3/IL-3R signaling has been shown to involve in the physiological and pathological processes of a variety of neurodegenerative diseases, but its role in prion diseases is rarely described. Here, the changes of IL-3/IL-3R and its downstream signaling pathways in a scrapie-infected cell line and in the brains of several scrapie-infected rodent models were evaluated by various methods. Markedly decreased IL-3Rα were observed in the brains of scrapie-infected rodents at terminal stage and in the prion-infected cell model, which showed increased in the brain samples collected at early and middle stage of infection. The IL-3 levels were almost unchanged in the brains of scrapie-infected mice and in the prion-infected cell line. Morphological assays identified close co-localization of the increased IL-3Rα signals with NeuN- and Iba1-positive cells, whereas co-localization of IL-3 signals with NeuN- and GFAP-positive cells in the scrapie-infected brain tissues. Some downstream components of IL-3/IL-3R pathways, including JAK2-STAT5 and PI3K/AKT/mTOR pathways, were downregulated in the brains of scrapie-infected rodents at terminal stage and in the prion-infected cells. Stimulation of recombinant IL-3 on the cultured cells showed prion that the prion-infected cells displayed markedly more reluctant responses of JAK2-STAT5 and PI3K/AKT/mTOR pathways than the normal partner cells. These data suggest that although prion infection or PrPSc accumulation in brain tissues does not affect IL-3 expression, it significantly downregulates IL-3R levels, thereby inhibiting the downstream pathways of IL-3/IL-3R and blocking the neuroregulatory and neuroprotective activities of IL-3.

Detecting SARS-CoV-2 BA.2, BA.4, and BA.5 Variants Utilizing a Robust RT-RPA-CRISPR/Cas12a-Based Method - China, 2023.

Introduction: Since 2019, numerous variants of concern for severe acute respiratory syndrome virus 2 (SARS-CoV-2) have emerged, leading to significant outbreaks. The development of novel, highly accurate, and rapid detection techniques for these new SARS-CoV-2 variants remains a primary focus in the ongoing efforts to control and prevent the coronavirus disease 2019 (COVID-19) pandemic. Methods: Reverse transcription-recombinase polymerase amplification combined with the clustered regularly interspaced short palindromic repeats-associated protein 12a (CRISPR/Cas12a) system was used to validate the detection of the Omicron BA.2, BA.4, and BA.5 variants of SARS-CoV-2. Results: Our results demonstrate that the CRISPR/Cas12a assay is capable of effectively detecting the SARS-CoV-2 BA.2, BA.4, and BA.5 variants with a limit of detection of 10, 1, and 10 copies/µL, respectively. Importantly, our assay successfully differentiated the three SARS-CoV-2 Omicron strains from one another. Additionally, we evaluated 46 SARS-CoV-2 positive clinical samples consisting of BA.2 (n=20), BA.4 (n=6), and BA.5 (n=20) variants, and the sensitivity of our assay ranged from 90% to 100%, while the specificity was 100%. Discussion: This research presents a swift and reliable CRISPR-based method that may be employed to track the emergence of novel SARS-CoV-2 variants.

A recombinant adenovirus vector containing the synNotch receptor gene for the treatment of triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is known as the most difficult molecular subtype of breast cancer to treat. Recent studies revealed that cancer stem cells (CSCs) play a critical role in TNBC recurrence and metastasis. In this study, we developed a recombinant replication-deficient adenoviral vector (Ad-CD44-N-HIF-3α4), which contains a gene encoding a synthetic Notch (synNotch) receptor composed of the extracellular domain of CD44 (CD44-ECD) and the hypoxia-inducible factor (HIF)-3α4 connected by the Notch core regulatory region. CD44 is a transmembrane glycoprotein and known as a CSC marker in breast cancer and other malignancies. HIF-3α4 is a dominant-negative regulator of HIF-1α and HIF-2α and inhibits hypoxia-inducing effect. Both CD44 and HIF signals contribute cancer stemness and maintaining CSCs in breast cancer. The CD44-ECD in the synNotch receptor acts as the CD44 decoy receptor, and after a ligand such as a hyaluronic acid binds to the CD44-ECD, HIF-3α4 is released from the Notch core domain. We performed an in vivo study using a mouse xenograft model of MDA-MB-231, a highly invasive TNBC cell, and confirmed the significant antitumor activity of the intratumoral injections of Ad-CD44-N-HIF3α4. Our findings in this study warrant the further development of Ad-CD44-N-HIF3α4 for the treatment of patients with TNBC.

Re-interpretation of the mechanism of type 2 diabetes mellitus based on a framework of psychosomatic medicine: a real-world study.

OBJECTIVE: Using bipolar disorder (BD) as a control, we explored the possible developmental process of impaired glucose metabolism rhythm. METHODS: In total, 441 subjects (77, 162, 134, 54, and 14 in the pre-diabetes [pre-DM], DM, BD, BD + pre-DM, and BD + DM groups, respectively) and 160 controls were included. All subjects were assessed using the Neuroticism Extraversion Openness Five-Factor Inventory (NEO-FFI). The hypothalamic-pituitary-adrenal (HPA) and hypothalamic-pituitary-thyroid (HPT) axes were measured. RESULTS: Cluster analysis showed that the BD, BD + DM, and DM groups were classified as the 'disease group, the BD + pre-DM group as the 'mixed period group', and the pre-DM group as the 'pre-disease group'. The conscientiousness factor scores of the NEO-FFI in the 'disease group' were higher than the norm but lower than the norm in the 'pre-disease group'. The scores of neurotic factors in the 'pre-disease' and 'mixed period' groups were both significantly higher than that in the 'disease group' (corrected p < 0.001). The incidences of the abnormal HPA axis decreased gradually from the 'pre-disease group' to the 'mixed period group' then to the 'disease group', while those of the HPT axis slightly increased at first and then significantly decreased. The overall prediction rate of the multiple logistic regression model was 92.7%. CONCLUSION: This study suggests that progression of pre-diabetes to DM is a continuous process from local abnormalities to rhythm disorder of glucose metabolism. This understanding can be applied to the whole course management and early intervention of DM and to the future development of optimised treatment based on rhythm regulation. TRIAL REGISTRATION: Clinical trial registration number: ChiCTR1800019064. Name of trial registration: Identify and the optimization of treatment for non-infectious chronic diseases under the "stress-dysrhythmia" theory hypothesis (Registration date: 24/10/2018). The full trial protocol can be accessed at the Chinese Clinical Trial Registry ( http://www.chictr.org.cn/ ).

Genomic Surveillance for SARS-CoV-2 Variants of Concern from Imported COVID-19 Cases - the Mainland of China, 2021.

Introduction: After the epidemic in Wuhan City was brought under control in 2020, local outbreaks of coronavirus disease 2019 (COVID-19) in the mainland of China were mainly due to imported COVID-19 cases. The ongoing evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has continued to generate new variants. Some have been designated as variants of concern (VOCs) by the World Health Organization (WHO). To better assess the role of imported SARS-CoV-2 surveillance and the prevalence of VOCs in 2021, the genomic surveillance data of SARS-CoV-2 from imported COVID-19 cases of 2021 in the mainland of China were analyzed. Methods: The analyses included the number of sequence submissions, time of sequence deposition, and time of detection of the VOCs in order to determine the timeliness and sensitivity of the surveillance. The proportions of VOCs were analyzed and compared with data from the Global Initiative of Sharing All Influenza Data (GISAID). Results: A total of 3,355 sequences of imported cases were submitted from 29 provincial-level administrative divisions, with differences in the number of sequence submissions and median time of sequence deposition. A total of 2,388 sequences with more than 90% genomic coverage were used for lineage analysis. The epidemic trend from Alpha to Delta to Omicron in imported cases was consistent with that in the GISAID. In addition, VOCs from imported cases were usually identified after WHO designation and before causing local outbreaks. Conclusions: The global distribution of SARS-CoV-2 VOCs changed rapidly in 2021. Robust genomic surveillance of the imported SARS-CoV-2 in the mainland of China is of great significance.

IOT based portable heart rate and SpO2 pulse oximeter.

Ever since the COVID-19 pandemic, the necessity of new,innovative medical devices to analyse and monitor the situation has become profound. With this in mind, we have developed an IOT based portable and cost-efficient heart rate and SpO2/level sensor device with efficient performance. The proposed system is equipped with an onboard OLED with simple internet connectivity, which publishes data on the OLED and to a HTML webpage. The salient features include its portability, user compatibility and is unique in its way of transmitting collected data online, making it achieve better results than the current available solutions in various ways. The system has on board a MAX30100 sensor(Pulse Oximeter sensor) and along with the IOT based microcontroller(WeMos D1 mini) enables the facility to monitor the real time health data of patients in real-time through a HTML page.

A tandem-repeat dimeric RBD protein-based covid-19 vaccine zf2001 protects mice and nonhuman primates.

Safe, efficacious, and deployable vaccines are urgently needed to control COVID-19 in the large-scale vaccination campaigns. We report here the preclinical studies of an approved protein subunit vaccine against COVID-19, ZF2001, which contains tandem-repeat dimeric receptor-binding domain (RBD) protein with alum-based adjuvant. We assessed vaccine immunogenicity and efficacy in both mice and non-human primates (NHPs). ZF2001 induced high levels of RBD-binding and SARS-CoV-2 neutralizing antibody in both mice and non-human primates, and elicited balanced TH1/TH2 cellular responses in NHPs. Two doses of ZF2001 protected Ad-hACE2-transduced mice against SARS-CoV-2 infection, as detected by reduced viral RNA and relieved lung injuries. In NHPs, vaccination of either 25 µg or 50 µg ZF2001 prevented infection with SARS-CoV-2 in lung, trachea, and bronchi, with milder lung lesions. No evidence of disease enhancement was observed in both animal models. ZF2001 has been approved for emergency use in China, Uzbekistan, Indonesia, and Columbia. The high safety, immunogenicity, and protection efficacy in both mice and NHPs found in this preclinical study was consistent with the results in human clinical trials.

[Discussion on Key Attention in Technical Review for Additive Manufacturing Acetabular Reconstruction Implantable Device].

With the development of additive manufacturing, the advantages of this type implant devices in the treatment of acetabular defects and reconstruction are becoming more and more prominent. The number of registration and declaration of such products is increasing day by day. According to the relevant requirements of the National Medical Products Administration for registration and application documents, combined with the characteristics of acetabular reconstruction implant products made of additive manufacturing, this study analyzes and summarizes the relevant requirements on raw material control, product performance research, product manufacturing, clinical evaluation, et al. We should pay more attention to in the registration and application materials submitted by the applicant. Provide opinions and suggestions for the next registration applicant to standardize product R&D and registration application documents, in order to help them optimize product R&D process, improve product quality and registration application efficiency.

Surveillance and Analysis of SARS-CoV-2 Variant Importation - China, January-June 2022.

Introduction: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant is the dominant circulating strain worldwide. To assess the importation of SARS-CoV-2 variants in the mainland of China during the Omicron epidemic, the genomic surveillance data of SARS-CoV-2 from imported coronavirus disease 2019 (COVID-19) cases in the mainland of China during the first half of 2022 were analyzed. Methods: Sequences submitted from January to July 2022, with a collection date before June 30, 2022, were incorporated. The proportions of SARS-CoV-2 variants as well as the relationships between the origin and destination of each Omicron imported case were analyzed. Results: 4,946 sequences of imported cases were submitted from 27 provincial-level administrative divisions (PLADs), and the median submission interval was within 1 month after collection. In 3,851 Omicron sequences with good quality, 1 recombinant (XU) and 4 subvariants under monitoring (BA.4, BA.5, BA.2.12.1, and BA.2.13) were recorded, and 3 of them (BA.4, BA.5, and BA.2.12.1) caused local transmissions in the mainland of China later than that recorded in the surveillance. Omicron subvariants dominated in the first half of 2022 and shifted from BA.1 to BA.2 then to BA.4 and BA.5. The percentage of BA.2 in the imported SARS-CoV-2 surveillance data was far higher than that in the Global Initiative on Sharing All Influenza Data (GISAID). The imported cases from Hong Kong Special Administrative Region, China, accounted for 32.30% of Omicron cases sampled, and 98.71% of them were BA.2. Conclusions: The Omicron variant showed the intra-Omicron evolution in the first half of 2022, and all of the Omicron subvariants were introduced into the mainland of China multiple times from multiple different locations.