Establishment of an ELISA Based on a Recombinant Antigenic Protein Containing Multiple Prominent Epitopes for Detection of African Swine Fever Virus Antibodies.

African swine fever virus (ASFV) poses a significant threat to the global pig industry, necessitating accurate and efficient diagnostic methods for its infection. Previous studies have often focused on a limited number of epitopes from a few proteins for detecting antibodies against ASFV. Therefore, the current study aimed to use multiple B-cell epitopes in developing an indirect Enzyme-Linked Immunosorbent Assay (ELISA) for enhanced detection of ASFV antibodies. For the expression of recombinant protein, k3 derived from 27 multiple peptides of 11 ASFV proteins, such as p72, pA104R, pB602L, p12, p14.5, p49, pE248R, p30, p54, pp62, and pp220, was used. To confirm the expression of the recombinant protein, we used the Western blotting analysis. The purified recombinant K3 protein served as the antigen in our study, and we employed the indirect ELISA technique to detect anti-ASFV antibodies. The present finding showed that there was no cross-reactivity with antibodies targeting Foot-and-mouth disease virus (FMDV), Porcine circovirus type 2 (PCV2), Pseudorabies virus (PRV), Porcine reproductive and respiratory syndrome virus (PRRSV), and Classical swine fever virus (CSFV). Moreover, the current finding was sensitive enough to find anti-ASFV in serum samples that had been diluted up to 32 times. The test (k3-iELISA) showed diagnostic specificity and sensitivity of 98.41% and 97.40%, respectively. Moreover, during the present investigation, we compared the Ingenasa kit and the k3-iELISA to test clinical pig serum, and the results revealed that there was 99.00% agreement between the two tests, showing good detection capability of the k3-iELISA method. Hence, the current finding showed that the ELISA kit we developed can be used for the rapid detection of ASFV antibodies and used as an alternative during serological investigation of ASF in endemic areas.

Mast cell activation triggered by SARS-CoV-2 causes inflammation in brain microvascular endothelial cells and microglia.

SARS-CoV-2-induced excessive inflammation in brain leads to damage of blood-brain barrier, hypoxic-ischemic injury, and neuron degeneration. The production of inflammatory cytokines by brain microvascular endothelial cells and microglia is reported to be critically associated with the brain pathology of COVID-19 patients. However, the cellular mechanisms for SARS-CoV-2-inducing activation of brain cells and the subsequent neuroinflammation remain to be fully delineated. Our research, along with others', has recently demonstrated that SARS-CoV-2-induced accumulation and activation of mast cells (MCs) in mouse lung could further induce inflammatory cytokines and consequent lung damages. Intracerebral MCs activation and their cross talk with other brain cells could induce neuroinflammation that play important roles in neurodegenerative diseases including virus-induced neuro-pathophysiology. In this study, we investigated the role of MC activation in SARS-CoV-2-induced neuroinflammation. We found that (1) SARS-CoV-2 infection triggered MC accumulation in the cerebrovascular region of mice; (2) spike/RBD (receptor-binding domain) protein-triggered MC activation induced inflammatory factors in human brain microvascular endothelial cells and microglia; (3) MC activation and degranulation destroyed the tight junction proteins in brain microvascular endothelial cells and induced the activation and proliferation of microglia. These findings reveal a cellular mechanism of SARS-CoV-2-induced neuroinflammation.

Mast cell degranulation-triggered by SARS-CoV-2 induces tracheal-bronchial epithelial inflammation and injury.

SARS-CoV-2 infection-induced hyper-inflammation is a key pathogenic factor of COVID-19. Our research, along with others', has demonstrated that mast cells (MCs) play a vital role in the initiation of hyper-inflammation caused by SARS-CoV-2. In previous study, we observed that SARS-CoV-2 infection induced the accumulation of MCs in the peri-bronchus and bronchioalveolar-duct junction in humanized mice. Additionally, we found that MC degranulation triggered by the spike protein resulted in inflammation in alveolar epithelial cells and capillary endothelial cells, leading to subsequent lung injury. The trachea and bronchus are the routes for SARS-CoV-2 transmission after virus inhalation, and inflammation in these regions could promote viral spread. MCs are widely distributed throughout the respiratory tract. Thus, in this study, we investigated the role of MCs and their degranulation in the development of inflammation in tracheal-bronchial epithelium. Histological analyses showed the accumulation and degranulation of MCs in the peri-trachea of humanized mice infected with SARS-CoV-2. MC degranulation caused lesions in trachea, and the formation of papillary hyperplasia was observed. Through transcriptome analysis in bronchial epithelial cells, we found that MC degranulation significantly altered multiple cellular signaling, particularly, leading to upregulated immune responses and inflammation. The administration of ebastine or loratadine effectively suppressed the induction of inflammatory factors in bronchial epithelial cells and alleviated tracheal injury in mice. Taken together, our findings confirm the essential role of MC degranulation in SARS-CoV-2-induced hyper-inflammation and the subsequent tissue lesions. Furthermore, our results support the use of ebastine or loratadine to inhibit SARS-CoV-2-triggered degranulation, thereby preventing tissue damage caused by hyper-inflammation.

OAS1 suppresses African swine fever virus replication by recruiting TRIM21 to degrade viral major capsid protein.

IMPORTANCE: African swine fever virus (ASFV) completes the replication process by resisting host antiviral response via inhibiting interferon (IFN) secretion and interferon-stimulated genes (ISGs) function. 2', 5'-Oligoadenylate synthetase gene 1 (OAS1) has been reported to inhibit the replication of various RNA and some DNA viruses. However, the regulatory mechanisms involved in the ASFV-induced IFN-related pathway still need to be fully elucidated. Here, we found that OAS1, as a critical host factor, inhibits ASFV replication in an RNaseL-dependent manner. Furthermore, overexpression of OAS1 can promote the activation of the JAK-STAT pathway promoting innate immune responses. In addition, OAS1 plays a new function, which could interact with ASFV P72 protein to suppress ASFV infection. Mechanistically, OAS1 enhances the proteasomal degradation of P72 by promoting TRIM21-mediated ubiquitination. Meanwhile, P72 inhibits the production of avSG and affects the interaction between OAS1 and DDX6. Our findings demonstrated OAS1 as an important target against ASFV replication and revealed the mechanisms and intrinsic regulatory relationships during ASFV infection.

Development of an Effective Neutralizing Antibody Assay for SARS-CoV-2 Diagnosis.

Introduction: Neutralizing antibodies (NAbs) are essential for preventing reinfection with SARS-CoV-2 and the recurrence of COVID-19; nonetheless, the formation of NAbs following vaccination and infection remains enigmatic due to the lack of a practical and effective NAb assay in routine laboratory settings. In this study, we developed a convenient lateral flow assay for the rapid and precise measurement of serum NAb levels within 20 minutes. Methods: Receptor-binding domain-fragment crystallizable (RBD-Fc) and angiotensin-converting enzyme 2-histidine tag (ACE2-His) were expressed by the eukaryotic expression systems of Spodoptera frugiperda clone 9 and human embryonic kidney 293T, respectively. Then, colloidal gold was synthesized and conjugated with ACE2. After optimizing various operating parameters, an NAb lateral flow assay was constructed. Subsequently, its detection limit, specificity, and stability were systematically evaluated, and clinical samples were analyzed to validate its clinical feasibility. Results: RBD-Fc and ACE2-His were obtained with 94.01% and 90.05% purity, respectively. The synthesized colloidal gold had a uniform distribution with an average diameter of 24.15 ± 2.56 nm. With a detection limit of 2 µg/mL, the proposed assay demonstrated a sensitivity of 97.80% and a specificity of 100% in 684 uninfected clinical samples. By evaluating 356 specimens from infected individuals, we observed that the overall concordance rate between the proposed assay and conventional enzyme-linked immunosorbent assay was 95.22%, and we noticed that 16.57% (59/356) of individuals still did not produce NAbs after infection (both by ELISA and the proposed assay). All the above tests by this assay can obtain results within 20 minutes by the naked eye without any additional instruments or equipment. Conclusion: The proposed assay can expediently and reliably detect anti-SARS-CoV-2 NAbs after infection, and the results provide valuable data to facilitate effective prevention and control of SARS-CoV-2. Clinical trial registration: Serum and blood samples were used under approval from the Biomedical Research Ethics Subcommittee of Henan University, and the clinical trial registration number was HUSOM-2022-052. We confirm that this study complies with the Declaration of Helsinki.

Analgesic Effect of Perineural Injection of BoNT/A on Neuropathic Pain Induced by Chronic Constriction Injury of Sciatic Nerve in Rats.

This study was designed to investigate the analgesic effect of perineural injection of BoNT/A on neuropathic pain induced by sciatic nerve chronic constriction injury (CCI) and possible mechanisms. SD rats were randomly divided into Sham group, CCI group and BoNT/A group. Paw mechanical withdrawal threshold (pMWT) and paw thermal withdrawal latency (pTWL) of each group were detected at different time points after surgery. The expression of myelin markers, autophagy markers and NLRP3 inflammasome-related molecules in injured sciatic nerves were examined at 12 days after surgery. Moreover, C-fiber evoked potential in spinal dorsal horn was recorded. The expression of SNAP-25, neuroinflammation and synaptic plasticity in spinal dorsal horn of each group were examined. Then rats treated with BoNT/A were randomly divided into DMSO group and Wnt agonist group to further explore the regulatory effect of BoNT/A on Wnt pathway. We found that pMWT and pTWL of ipsilateral paw were significantly decreased in CCI group compared with Sham group, which could be improved by perineural injection of BoNT/A at days 7, 9 and 12 after surgery. The peripheral analgesic mechanisms of perineural injection of BoNT/A might be related to the protective effect on myelin sheath by inhibiting NLRP3 inflammasome and promoting autophagy flow, while the central analgesic mechanisms might be associated with inhibition of neuroinflammation and synaptic plasticity in spinal dorsal horn due to inhibiting SNAP-25 and Wnt pathway. As a new route of administration, perineural injection of BoNT/A can relieve CCI induced neuropathic pain probably via both peripheral and central analgesic mechanisms.

TRIM72 Alleviates Muscle Inflammation in mdx Mice via Promoting Mitophagy-Mediated NLRP3 Inflammasome Inactivation.

Chronic muscle inflammation exacerbates the pathogenesis of Duchenne muscular dystrophy (DMD), which is characterized by progressive muscle degeneration and weakness. NLRP3 (nucleotide-binding domain and leucine-rich repeat pyrin domain containing 3) inflammasome plays a key role in the inflammatory process, and its abnormal activation leads to a variety of inflammatory or immune diseases. TRIM72 (MG53) is a protective myokine for tissue repair and regeneration. However, little is known about the potential impact of TRIM72 in the crosstalk between mitophagy and inflammatory process of DMD. Here, 10-week-old male mdx mice were injected intramuscularly with adeno-associated virus (AAV-TRIM72) to overexpress TRIM72 protein for 6 weeks. Then, skeletal muscle samples were collected, and relevant parameters were measured by histopathological analysis and molecular biology techniques. C2C12 cell line was transfected with lentivirus (LV-TRIM72) to overexpress or siRNA (si-TRIM72) to suppress the TRIM72 expression for the following experiment. Our data firstly showed that the TRIM72 expression was decreased in skeletal muscles of mdx mice. Then, we observed the increased NLRP3 inflammasome and impaired mitophagy in mdx mice compared with wild type mice. In mdx mice, administration of AAV-TRIM72 alleviated the accumulation of NLRP3 inflammasome and the consequent IL-18 and IL1ß maturation by inducing autophagy, while this protective effect was reversed by chloroquine. Mitochondrial reactive oxygen species (mtROS), as a recognized activator for NLRP3 inflammasome, was attenuated by TRIM72 through the induction of mitophagy in C2C12 cells. Additionally, we proposed that the TRIM72 overexpression might promote mitophagy through both the early stage by PI3K-AKT pathway and the late stage by autolysosome fusion. In conclusion, the current study suggests that TRIM72 prevents DMD inflammation via decreasing NLRP3 inflammasomes and enhancing mitophagy. Collectively, our study provides insight into TRIM72 as a promising target for therapeutic intervention for DMD.

Coping efficacy is associated with the domain specificity in risk-taking behaviors during the COVID-19 pandemic.

The current study is to explore the associations between the threat to life and risk-taking behaviors across different domains during the coronavirus disease (COVID-19), and the role of the perceived threat and coping efficacy in these associations based on protection motivation theory. This study conducted an online survey on 2983 participants from 30 provinces in China. It found that people's risk-taking behaviors in the wake of the COVID-19 pandemic could be divided into stimulating risk-taking (SRT) behaviors and instrumental risk-taking (IRT) behaviors. The exposure level to the COVID-19 pandemic was negatively related to SRT behaviors in natural/physical, gambling, safety, moral, and reproductive domains, but not related to IRT behaviors in financial and corporation/competition domains. Two parallel routes were found in domain-specific risk-taking behaviors when people were faced with a life-threatening epidemic. Specifically, perceived threat consistently mediated the positive relationship between exposure level and risk-taking behaviors across domains. In contrast, coping efficacy mediated the negative relationship between exposure level and SRT behaviors but positive associations with IRT behaviors. These findings indicated that coping efficacy, rather than perceived threat is the factor that explains the people's domain-specific risk-taking behaviors in the context of the epidemic. The study holds implications for emergency policy-making that targets disaster risk reduction by increasing the public coping efficacy, which could prevent unnecessary SRT behaviors and improve necessary IRT behaviors in business and investment for economic recoveries.

Combinational benefit of antihistamines and remdesivir for reducing SARS-CoV-2 replication and alleviating inflammation-induced lung injury in mice.

COVID-19 is an immune-mediated inflammatory disease caused by SARS-CoV-2 infection, the combination of anti-inflammatory and antiviral therapy is predicted to provide clinical benefits. We recently demonstrated that mast cells (MCs) are an essential mediator of SARS-CoV-2-initiated hyperinflammation. We also showed that spike protein-induced MC degranulation initiates alveolar epithelial inflammation for barrier disruption and suggested an off-label use of antihistamines as MC stabilizers to block degranulation and consequently suppress inflammation and prevent lung injury. In this study, we emphasized the essential role of MCs in SARS-CoV-2-induced lung lesions in vivo, and demonstrated the benefits of co-administration of antihistamines and antiviral drug remdesivir in SARS-CoV-2-infected mice. Specifically, SARS-CoV-2 spike protein-induced MC degranulation resulted in alveolar-capillary injury, while pretreatment of pulmonary microvascular endothelial cells with antihistamines prevented adhesion junction disruption; predictably, the combination of antiviral drug remdesivir with the antihistamine loratadine, a histamine receptor 1 (HR1) antagonist, dampened viral replication and inflammation, thereby greatly reducing lung injury. Our findings emphasize the crucial role of MCs in SARS-CoV-2-induced inflammation and lung injury and provide a feasible combination antiviral and anti-inflammatory therapy for COVID-19 treatment.

Development of a Point-of-Care Test Based on Selenium Nanoparticles for Heart-Type Fatty Acid-Binding Proteins in Human Plasma and Blood.

Purpose: A rapid, convenient, cost-effective in-home test method for identifying heart-type fatty acid-binding protein (H-FABP) in plasma and blood by a lateral-flow immunoassay (LFIA) based on selenium nanoparticles (SeNPs) was developed. Methods: SeNPs were synthesized by using L-ascorbic acid to reduce seleninic acid at room temperature and conjugated with an anti-H-FABP monoclonal antibody. The limit of detection, specificity, and stability were measured, and clinical samples were analyzed. Results: The SeNPs were spherical with a diameter of 39.48 ± 3.72 nm and were conjugated successfully with an anti-H-FABP antibody, resulting in a total diameter of 46.52 ± 2.95 nm. The kit was designed for the determination of H-FABP in plasma specimens and whole blood specimens. The limit of detection was 1 ng/mL in plasma and blood, and the results could be determined within 10 min. No cross-reaction occurred with cardiac troponin I, creatine kinase-MB or myoglobin. The kits were stored at 40 °C for up to 30 days without significant loss of activity. The sensitivity was determined to be 100%, the specificity 96.67%, and the overall coincidence rate 97.83%. Conclusion: This SeNP assay kit can conveniently, rapidly, and sensitively detect H-FABP in plasma or blood with a readout of a simple color change visible to the naked eye with no special device, and can be used as an auxiliary means for the early screening of AMI. Clinical Trial Registration: Plasma and blood samples were used under approval from the Experimental Animal Ethics committee of the Joint National Laboratory for Antibody Drug Engineering, Henan University. The clinical trial registration number was HUSOM-2019-047.

Mast Cell Activation Triggered by Retrovirus Promotes Acute Viral Infection.

Mast cells (MCs) are strategically located at the host-environment interface and their non-allergic roles in the immune-surveillance of pathogens have recently gained more attention. However, MC-caused detrimental regulation of immune inflammations can promote viral invasion. Currently, the role of MCs in retroviral infection remains elusive. We have recently proved that human gut MCs could capture and transfer HIV-1 to CD4+ T cells for promoting viral spread; MC-released histamine augments HIV-1-induced functional polarization of dendritic cells to cause immunosuppression via stimulating the differentiation of regulatory T cells. In this study, we used a murine model of MuLV/Friend virus infection to address MC role in acute retroviral infection in vivo. The acute infection of MuLV/Friend virus could be established in C57BL/6 wild type mice, but viral acquisition showed low efficiency in C57BL/6-Kit W - sh/W - sh (Sash) mice which lack MCs. In mechanism, we found that MuLV/Friend virus triggered MC activation for degranulation; MC degranulation subsequently activated the granulocyte-like myeloid derived suppressive cells (G-MDSCs) to inhibit CD8+ T cells- and NK cells-mediated antiviral immune responses. The reconstruction of MCs in Sash mice promoted acute retroviral infection by regulating G-MDSCs functions and antiviral immune responses. Importantly, the administration of MC stabilizers to block cell degranulation elevated antiviral immune response and consequently suppressed retrovirus infection. This study uncovers a specific role of MCs in acute retroviral infection and elucidates the underlying immune-mechanisms. Targeting MCs may provide a novel approach for controlling acute infection by retroviruses.

Pharmacological suppression of glycogen synthase kinase-3 reactivates HIV-1 from latency via activating Wnt/ß-catenin/TCF1 axis in CD4+ T cells.

ABSTRACTHIV-1 latency posts a major obstacle for HIV-1 eradication. Currently, no desirable latency reversing agents (LRAs) have been implicated in the "Shock and Kill" strategy to mobilize the latently infected cells to be susceptible for clearance by immune responses. Identification of key cellular pathways that modulate HIV-1 latency helps to develop efficient LRAs. In this study, we demonstrate that the Wnt downstream ß-catenin/TCF1 pathway is a crucial modulator for HIV-1 latency. The pharmacological activation of the ß-catenin/TCF1 pathway with glycogen synthase kinase-3 (GSK3) inhibitors promoted transcription of HIV-1 proviral DNA and reactivated latency in CD4+ T cells; the GSK3 kinase inhibitor 6-bromoindirubin-3'-oxime (6-BIO)-induced HIV-1 reactivation was subsequently confirmed in resting CD4+ T cells from cART-suppressed patients and SIV-infected rhesus macaques. These findings advance our understanding of the mechanisms responsible for viral latency, and provide the potent LRA that can be further used in conjunction of immunotherapies to eradicate viral reservoirs.

SARS-CoV-2-triggered mast cell rapid degranulation induces alveolar epithelial inflammation and lung injury.

SARS-CoV-2 infection-induced hyper-inflammation links to the acute lung injury and COVID-19 severity. Identifying the primary mediators that initiate the uncontrolled hypercytokinemia is essential for treatments. Mast cells (MCs) are strategically located at the mucosa and beneficially or detrimentally regulate immune inflammations. In this study, we showed that SARS-CoV-2-triggered MC degranulation initiated alveolar epithelial inflammation and lung injury. SARS-CoV-2 challenge induced MC degranulation in ACE-2 humanized mice and rhesus macaques, and a rapid MC degranulation could be recapitulated with Spike-RBD binding to ACE2 in cells; MC degranulation altered various signaling pathways in alveolar epithelial cells, particularly, the induction of pro-inflammatory factors and consequential disruption of tight junctions. Importantly, the administration of clinical MC stabilizers for blocking degranulation dampened SARS-CoV-2-induced production of pro-inflammatory factors and prevented lung injury. These findings uncover a novel mechanism for SARS-CoV-2 initiating lung inflammation, and suggest an off-label use of MC stabilizer as immunomodulators for COVID-19 treatments.

Efficacy and Safety of Immunotherapies in Refractory Myasthenia Gravis: A Systematic Review and Meta-Analysis.

Introduction: Approximately 10-20% of patients WITH myasthenia gravis (MG) are refractory to conventional immunotherapies. The purpose of this study was to conduct a systematic review and meta-analysis to explore the optimal therapies for refractory MG. Method: Correlative studies were performed through a search in PubMed, Cochrane Library, and Embase databases. The primary outcome was defined by changes in the quantitative myasthenia gravis score (QMG). Secondary outcomes were defined by the Myasthenia Gravis Activities of Daily Living Scale (MG-ADL), Myasthenia Gravis Foundation of America (MGFA) post intervention status, adverse events, and disease exacerbation after treatment. Result: A total of 16 studies were included with 403 patients with refractory MG on therapies with rituximab, eculizumab, tacrolimus, and cladribine. Therapeutic efficacy of rituximab and eculizumab was identified with an estimated reduction in QMG score (4.158 vs. 6.928) and MG-ADL (4.400 vs. 4.344), respectively. No significant changes were revealed in efficacy or exacerbation density between the two independent therapeutic cohorts. The estimated adverse event density of eculizumab was more significant than that in the rituximab group (1.195 vs. 0.134 per patient-year), while the estimated serious event density was similar. Conclusion: The efficacy and safety of rituximab and eculizumab have been approved in patients with refractory MG. Rituximab had a superior safety profile than eculizumab with a lower incidence of adverse events. Systematic Review Registration: https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42021236818, identifier CRD42021236818.

Mechanistic Insight into Antiretroviral Potency of 2'-Deoxy-2'-ß-fluoro-4'-azidocytidine (FNC) with a Long-Lasting Effect on HIV-1 Prevention.

In preclinical and phase I and II clinical studies, 2'-deoxy-2'-ß-fluoro-4'-azidocytidine (FNC) displays a potent and long-lasting inhibition of HIV-1 infection. To investigate its mechanism of action, we compared it with the well-documented lamivudine (3TC). Pharmacokinetic studies revealed that the intracellular retention of FNC triphosphate in peripheral blood mononuclear cells was markedly longer than that of the 3TC triphosphate. FNC selectively enters and is retained in HIV target cells, where it exerts long-lasting prevention of HIV-1 infection. In addition to inhibition of HIV-1 reverse transcription, FNC also restores A3G expression in CD4+ T cells in FNC-treated HIV-1 patients. FNC binds to the Vif-E3 ubiquitin ligase complex, enabling A3G to avoid Vif-induced ubiquitination and degradation. These data reveal the mechanisms underlying the superior anti-HIV potency and long-lasting action of FNC. Our results also suggest a potential clinical application of FNC as a long-lasting pre-exposure prophylactic agent capable of preventing HIV infection.

Data relating to sequencing statistics and the reads and genomic coverage aligning to Taenia solium in the cerebrospinal fluid.

This data can serve as a reference for other next-generation sequencing (NGS) of the cerebrospinal fluid (CSF). In the related research article, entitled "Next-Generation Sequencing of Cerebrospinal Fluid for the Diagnosis of Neurocysticercosis", we reported NGS of the CSF might be an auxiliary method for neurocysticercosis (NCC) patients who have complicated manifestations and courses to receive early diagnosis and treatment. In this article, we retrieved the available data about the sequencing statistics of the CSF samples and the number of unique reads and genomic coverage aligning to microorganic sequences. The data were generated by the Illumina MiniSeq system for sequencing and computational subtraction of the human host sequences was performed. Finally, the remaining sequencing data were aligned to the Microbial Genome Databases. This data can serve as a reference for other NGS of the CSF.

Quercetin Attenuates d-GaLN-Induced L02 Cell Damage by Suppressing Oxidative Stress and Mitochondrial Apoptosis via Inhibition of HMGB1.

High mobility group box-1 (HMGB1) plays an important role in various liver injuries. In the case of acute liver injury, it leads to aseptic inflammation and other reactions, and also regulates specific cell death responses in chronic liver injury. HMGB1 has been demonstrated to be a good therapeutic target for treating liver failure. Quercetin (Que), as an antioxidant, is a potential phytochemical with hepatocyte protection and is also considered to be an inhibitor of HMGB1. However, the mechanism of its hepatoprotective effects remains to be characterized. The present study explored whether the hepatoprotective effect of Que antagonizes HMGB1, and subsequent molecular signaling events. Our results indicated that Que protects L02 cells from d-galactosamine (d-GaLN)-induced cellular damage by reducing intracellular reactive oxygen species (ROS) production and apoptotic responses in the mitochondrial pathway. Immunofluorescence and Western blot assays showed that HMGB1 was involved in d-GaLN-induced L02 cell damage. Further research showed that after transfection with HMGB1 short hairpin RNA (shRNA), cell viability was improved, and intracellular ROS production and apoptosis were suppressed. When co-treated with Que, the expression of HMGB1 was decreased significantly, the expression of proteins in the corresponding signal pathway were further reduced, and the production of ROS and apoptosis were further suppressed. Molecular docking also indicated the binding of Que and HMGB1. Taken together, these results indicate that Que significantly improves d-GaLN-induced cellular damage by inhibiting oxidative stress and mitochondrial apoptosis via inhibiting HMGB1.

Next-generation sequencing of cerebrospinal fluid for the diagnosis of neurocysticercosis.

OBJECTIVE: Neurocysticercosis (NCC) is the most common parasitic disease of the human central nervous system (CNS). However, a diagnosis of NCC may be hard to make if the specific clinical and routine neuroimaging manifestations are lacking, which hinders physicians from considering further immunodiagnostic tests. PATIENTS AND METHODS: Seven patients presented with fever, headache, nausea, cognitive decline, confusion, or progressive leg weakness. There were no pathogens found in the cerebrospinal fluid (CSF); patients were clinically suspected of meningoencephalitis or cerebrovascular disease. To clearly determine the etiology, next generation sequencing (NGS) of the CSF was used to detect pathogens in these seven patients. RESULTS: Taenia solium DNA sequences were detected in the seven patients, but not in the non-template controls (NTCs) or the other patients with clinically suspected CNS infections. Based on the patients' medical data and the diagnostic criteria for NCC, seven patients were diagnosed with probable NCC. The unique reads aligning to Taenia solium ranged from 6 to 261064, with genomic coverage ranging from 0.0003% to 14.8079%. The number of unique reads and genomic coverage dropped in three of the seven patients after antiparasitic treatment, consistent with the relief of symptoms. CONCLUSION: This study showed that NGS of the CSF might be an auxiliary diagnostic method for NCC patients. Larger studies are required.

Reduction of Human DNA Contamination in Clinical Cerebrospinal Fluid Specimens Improves the Sensitivity of Metagenomic Next-Generation Sequencing.

Metagenomics next-generation sequencing (mNGS) is increasingly available for the detection of obscure infectious diseases of the central nervous system. However, human DNA contamination from elevated white cells, one of the characteristic cerebrospinal fluid (CSF) features in meningitis patients, greatly reduces the sensitivity of mNGS in the pathogen detection. Currently, effective approaches to selectively reduce host DNA contamination from clinical CSF samples are still lacking. In this study, a total of 20 meningitis patients were enrolled, including 10 definitively diagnosed tuberculous meningitis (TBM) and 10 definite cryptococcal meningitis (CM) cases. To evaluate the effect of reduced human DNA in the sensitivity of mNGS detection, three specimen-processing protocols were performed: (i) To remove human DNA, saponin, a nonionic surfactant, was used to selectively lyse white cells in CSF followed by DNase treatment prior to the extraction of DNA; (ii) to reduce host DNA, CSF was centrifuged to remove human cells, and the supernatant was collected for DNA extraction; and (iii) DNA extraction from the unprocessed specimens was set as the control. We found that saponin processing significantly elevated the NGS unique reads for Cryptococcus (P < 0.01) compared with the control but had no effects for Mycobacterium tuberculosis (P > 0.05). However, detection of centrifuged supernatants improved the NGS unique reads for both TBM and CM compared with controls (P < 0.01). Our results demonstrate that the use of mNGS of centrifuged supernatants from clinical CSF samples in patients with TBM and CM is a simple and effective method to improve the sensitivity of pathogen detection.