NPAS2, transcriptionally activated by ARRB1, promotes the malignant behaviours of lung adenocarcinoma cells and regulates the reprogramming of glucose metabolism.

Lung adenocarcinoma (LUAD) is a serious threat to public health and is accompanied by increased morbidity and mortality worldwide. Neuronal PAS domain protein2 (NPAS2) has been confirmed as an oncogene in LUAD; however, little is known about its molecular mechanism. Here, the expression level of NPAS2 was detected in LUAD cell lines and 16HBE cells. Gain- and loss-of-function experiments were performed. Cell Counting Kit-8, colony formation, flow cytometry, wound-healing and Transwell assays were conducted to assess cell proliferation, apoptosis, migration and invasion, respectively. Reprogramming of glucose metabolism was evaluated via oxygen consumption rate (OCR), complexes activities, lactic production and glucose consumption. The expression of critical proteins was examined by western blot. We demonstrated aberrant upregulation of NPAS2 and ß-arrestin-1 (ARRB1) in LUAD cell lines. ARRB1 was found to be a critical transcription factor of NPAS2 with binding sites within the promoter region of NPAS2, thereby causing its transcriptional activation. Functional experiments revealed that NPAS2 depletion significantly inhibited the malignant behaviours of A549 cells by suppressing cell proliferation, migration, invasion and epithelial-mesenchymal transition and promoting cell apoptosis. Meanwhile, NPAS2 depletion increased OCR and activities of complexes (I, II, III and V), and reduced lactic acid production and glucose uptake in A549 cells, indicating that NPAS2 depletion inhibited aerobic glycolysis, accompanied by reduced expression of glycolytic enzymes. However, the changes caused by NPAS2 knockdown were partly restored by ARRB1 overexpression. In conclusion, our study suggests that ARRB1 could transcriptionally activate NPAS2, facilitating malignant activities and glycolysis, and ultimately promoting the progression of LUAD, proving a novel therapeutic strategy for the treatment of LUAD.

Determination of the cycle threshold value of the Xpert Xpress SARS-CoV-2/Flu/RSV test that corresponds to the presence of infectious SARS-CoV-2 in anterior nasal swabs.

Despite having high analytical sensitivities and specificities, qualitative SARS-CoV-2 nucleic acid amplification tests (NAATs) cannot distinguish infectious from non-infectious virus in clinical samples. In this study, we determined the highest cycle threshold (Ct) value of the SARS-CoV-2 targets in the Xpert Xpress SARS-CoV-2/Flu/RSV (Xpert 4plex) test that corresponded to the presence of detectable infectious SARS-CoV-2 in anterior nasal swab samples. A total of 111 individuals with nasopharyngeal swab specimens that were initially tested by the Xpert Xpress SARS-CoV-2 test were enrolled. A healthcare worker subsequently collected anterior nasal swabs from all SARS-CoV-2-positive individuals, and those specimens were tested by the Xpert 4plex test, viral culture, and laboratory-developed assays for SARS-CoV-2 replication intermediates. SARS-CoV-2 Ct values from the Xpert 4plex test were correlated with data from culture and replication intermediate testing to determine the Xpert 4plex assay Ct value that corresponded to the presence of infectious virus. Ninety-eight of the 111 (88.3%) individuals initially tested positive by the Xpert Xpress SARS-CoV-2 test. An anterior nasal swab specimen collected from positive individuals a median of 2 days later (range, 0-9 days) tested positive for SARS-CoV-2 by the Xpert 4plex test in 39.8% (39/98) of cases. Of these samples, 13 (33.3%) were considered to contain infectious virus based on the presence of cultivable virus and replication intermediates, and the highest Ct value observed for the Xpert 4plex test in these instances was 26.3. Specimens that yielded Ct values of ≤26.3 when tested by the Xpert 4plex test had a likelihood of containing infectious SARS-CoV-2; however, no infectious virus was detected in specimens with higher Ct values.IMPORTANCEUnderstanding the correlation between real-time PCR test results and the presence of infectious SARS-CoV-2 may be useful for informing patient management and workforce return-to-work or -duty. Further studies in different patient populations are needed to correlate Ct values or other biomarkers of viral replication along with the presence of infectious virus in clinical samples.

Varespladib mitigates acute liver injury via suppression of excessive mitophagy on Naja atra envenomed mice by inhibiting PLA2.

Snakebite envenomation often leads to severe visceral injuries, including acute liver injury (ALI). However, the toxicity mechanism remains unclear. Moreover, varespladib can directly inhibit phospholipase A2 (PLA2) in snake venom, but its protective effect on snakebite-induced ALI and the mechanism have not been clarified. Previous studies have shown that snake venom PLA2 leads to neuron cell death via reactive oxygen species (ROS), one of the initial factors related to the mitophagy pathway. The present study group also found that ROS accumulation occurred after Naja atra envenoming. Hematoxylin and eosin (H/E) staining and immunohistochemistry (IHC) were performed to identify the expression of inflammatory factors in the liver tissue, and flow cytometry and immunofluorescence were used to detect ROS levels and mitochondrial function. Immunofluorescence and western blotting were also used for detecting mitophagy pathway-related proteins. The results showed that N. atra bite induced ALI by activating mitophagy and inducing inflammation and that varespladib had a protective effect. Collectively, these results showed the pathological mechanism of ALI caused by N. atra bite and revealed the protective effect of varespladib.

Development of an integrated and project-based laboratory course in upper-level biochemistry and molecular biology.

An integrated and projected-based laboratory course was described, integrating interconnected knowledge points and biochemistry and molecular biology techniques on a research project-based system. The program, which served as an essential extension of theoretical courses to practice, was conducted with a sophomore of basic medical science who had completed the course in medical biochemistry and molecular biology. This course engaged students in learning "genetic manipulation" and "recombinant DNA technology" to understand the target gene's role in disease mechanics, thus altering evaluation and treatment for clinical disease. Students could master applied and advanced techniques, such as cell culture, transfection, inducing exogenous fusion protein expression, purifying protein and its concentration assay, quantitative polymerase chain reaction, and western bot analysis. This laboratory exercise links laboratory practices with the methods of current basic research. Students need to complete the experimental design report and laboratory report, which could be advantageous for improving their ability to write lab summaries and scientific papers in the future. The reliability and validity analyses were conducted on the questionnaire, and we examined students' satisfaction with the course and their gains from the course. The student feedback was generally positive, indicating that the exercise helped consolidate theoretical knowledge, increase scientific research enthusiasm, and provide a powerful tool to be a better person and make informed decisions.

Elevated CD169 expressing monocyte/macrophage promotes systemic inflammation and disease progression in cirrhosis.

Systemic inflammation is related to disease progression and prognosis in patients with advanced cirrhosis. However, the mechanisms underlying the initiation of inflammation are still not fully understood. The role of CD169+ monocyte/macrophage in cirrhotic systemic inflammation was undetected. Flow cytometry analysis was used to detect the percentage and phenotypes of CD169+ monocytes as well as their proinflammatory function in patient-derived cirrhotic tissue and blood. Transcriptome differences between CD169+ and CD169- monocytes were also compared. Additionally, a mouse model with specific depletion of CD169+ monocytes/macrophages was utilized to define their role in liver injury and fibrosis. We observed increased CD169 expression in monocytes from cirrhotic patients, which was correlated with inflammatory cytokine production and disease progression. CD169+ monocytes simultaneously highly expressed M1- and M2-like markers and presented immune-activated profiles. We also proved that CD169+ monocytes robustly prevented neutrophil apoptosis. Depletion of CD169+ monocytes/macrophages significantly inhibited inflammation and liver necrosis in acute liver injury, but the spontaneous fibrin resolution after repeated liver injury was impaired. Our results indicate that CD169 defines a subset of inflammation-associated monocyte that correlates with disease development in patients with cirrhosis. This provides a possible therapeutic target for alleviating inflammation and improving survival in cirrhosis.

Assessment of an automated Cytomegalovirus nucleic acid amplification test using clinical plasma, bronchoalveolar lavage, and tissue specimens.

BACKGROUND: Cytomegalovirus (CMV) causes significant morbidity and mortality in immunocompromised patients, particularly transplant recipients. Quantitation of CMV DNA in peripheral blood is used to monitor prophylactic and pre-emptive approaches to prevent CMV disease, whereas CMV DNA testing of non-plasma specimens may aid in the diagnosis of end-organ disease. METHODS: The analytical performance of the FDA-approved Aptima CMV Quant Assay was evaluated using reference CMV (SeraCare) diluted in defibrinated human plasma, as well as negative bronchoalveolar lavage fluid and tissue. Agreement was determined using 100 clinical acid-citrate-dextrose (ACD) plasma specimens, 77 bronchoalveolar lavage (BAL) fluids, and 101 tissues previously tested using artus CMV qPCR. RESULTS: Aptima CMV lower limit of detection (LLOD) was 169 IU/mL for ACD plasma, 100 IU/mL for BAL, and 50 IU/mL for tissue. Positive percent agreement (PPA) was 100.0% (50/50; 95% CI: 92.9% - 100.0%) and negative percent agreement (NPA) was 94.0% (47/50; 95% CI: 83.5% - 98.8%) for ACD plasma. Bland-Altman analysis revealed a bias of 0.20 log10 IU/mL (Aptima - artus) with 95% limits of agreement of -0.53 to 0.93. For BAL fluids, PPA was 70.0% (14/20; 95% CI: 45.7% - 88.1%) and NPA was 82.4% (43/51; 95% CI: 69.1% - 91.6%). For tissues, PPA was 90.0% (45/50; 95% CI: 78.2% - 96.7%) and NPA was 94.0% (47/50; 95% CI: 83.5% - 98.8%). CONCLUSIONS: The Aptima CMV Quant Assay demonstrates high analytical sensitivity and good overall agreement using clinical plasma and tissue specimens.

The anti-myotoxic effects and mechanisms of Sinonatrix annularis serum and a novel plasma metalloproteinase inhibitor towards Deinagkistrodon acutus envenomation.

Non-venomous snakes commonly evolve natural resistance to venom to escape predators. Sinonatrix annularis serum has been shown to inhibit Deinagkistrodon acutus venom-induced hemorrhage and upregulation of serum CK, CK-MB, LDH, AST and ALT levels. Using TMT-labeled proteomics analysis, 168 proteins were found to be altered significantly in the envenomed gastrocnemius muscle and categorized into pathways such as complement and coagulation cascades, leukocyte transendothelial migration, and JAK/STAT signaling. These alterations were mitigated by S. annularis serum. Subsequently, a novel metalloproteinase inhibitor, SaMPI, was isolated from S. annularis serum by two-step chromatography. It showed strong antidotal effects against D. acutus envenomation, including inhibition of subcutaneous bleeding caused by crude venom and DaMP (a metalloproteinase derived from D. acutus) activity in a 1:1 ratio. Histology and immunoblotting analyses demonstrated that SaMPI mitigated myonecrosis, reduced neutrophil infiltration and local inflammatory factor release, and retarded JAK/STAT and MAPK signaling activation. Analysis of the SaMPI gene cloned by 5'-RACE revealed a shared sequence identity of 58-79% with other SVMP inhibitors. These findings demonstrate the protective effects of SaMPI and indicate its potential value as a candidate for viper bite adjuvant therapy.

Vanadium-doped graphitic carbon nitride for high performance lithium-sulfur batteries.

To promote polysulfide conversion in lithium sulfur batteries (LSB) and alleviate the shuttle effect, we designed and fabricated a novel catalyst of vanadium-doped graphite phase carbon nitride with nitrogen defects (V@gC3N4-ND) and high vanadium loading (3.46 at%) by defect engineering and two-step pyrolysis. Employing a V@gC3N4-ND modified separator, the LSB yielded capacities of 934 mA h g-1 at 1C and 404 mA h g-1 at 4C; the former was retained by 61% and 45% after 500 and 1000 cycles, respectively. In particular, the initial capacity of the battery reached 969 mA h g-1 at a sulfur loading of 10.0 mg cm-2. This work provides a facile route to the preparation of high-loading vanadium active site catalysts with nitrogen defects in the support, which are promising for high performance LSB applications.

Phosphorylated Hsp27 promotes adriamycin resistance in breast cancer cells through regulating dual phosphorylation of c-Myc.

Chemotherapy resistance of breast cancer cells is one of the major factors affecting patient survival rate. Heat shock protein 27 (Hsp27) is a member of the small heat shock protein family that has been reported to be associated with chemotherapy resistance in tumor cells, but the exact mechanism is not fully understood. Here, we explored the regulation of Hsp27 in adriamycin-resistant pathological conditions of breast cancer in vitro and in vivo. We found that overexpression of Hsp27 in MCF-7 breast cancer cells reversed DNA damage induced by adriamycin, and thereby reduced subsequent cell apoptosis. Non-phosphorylated Hsp27 accelerated ubiquitin-mediated degradation of c-Myc under normal physiological conditions. After stimulation with adriamycin, Hsp27 was phosphorylated and translocated from the cytoplasm into the nucleus, where phosphorylated Hsp27 upregulated c-Myc and Nijmegen breakage syndrome 1 (NBS1) protein levels thus leading to ATM activation. We further showed that phosphorylated Hsp27 promoted c-Myc nuclear import and stabilization by regulating T58/S62 phosphorylation of c-Myc through a protein phosphatase 2A (PP2A)-dependent mechanism. Collectively, the data presented in this study demonstrate that Hsp27, in its phosphorylation state, plays a critical role in adriamycin-resistant pathological conditions of breast cancer cells.

Affected inflammation-related signaling pathways in snake envenomation: A recent insight.

Snake envenomation is well known to cause grievous pathological signs, including haemorrhagic discharge, necrosis, and respiratory distress. However, inflammatory reactions are also common envenoming manifestations that lead to successive damage, such as oedema, ulceration, lymphadenectasis, systemic inflammatory response syndrome (SIRS) and even multiple organ dysfunction syndrome (MODS). Interference with the inflammatory burst is hence important in the clinical treatment of snake envenomation. Here, we summarize the typical snake toxins (or venoms) that cause inflammatory reactions and the underlying signaling pathways. In brief, inflammatory reactions are usually triggered by snake venom phospholipase A2 (svPLA2), snake venom metalloprotease (SVMP), snake venom serine protease (SVSP) and C-type lectin/snaclec (CTL) as well as disintegrin (DIS) via multiple signaling pathways. They are nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing 3 (NLRP3), nuclear factor kappa-B (NF-κB), mitogen-activated protein kinase (MAPK), janus kinase/signal transducer and activator of transcription (JAK-STAT) and phosphoinositide 3-Kinase/protein kinase B (PI3K/PKB also called PI3K-AKT) signaling pathways. Activation of these pathways promotes the expression of pro-inflammatory molecules such as cytokines, especially interleukin-1ß (IL-1ß) which causes further inflammatory cascades and manifestations, such as swelling, fever, pain, and severe complications. Remarkably, almost half of introduced snake toxins (or venoms) have anti-inflammatory effects through blocking these pathways and suppressing the expression of pro-inflammatory molecules. Investigation of affected inflammation-related signaling pathways is meaningful to achieve better clinical treatment.

Two cases of MPXV infection during pregnancy in heterosexual cisgender women without classic cutaneous lesions, Northern California, 2022.

As part of an epidemiologic survey, we screened remnant samples collected for STI testing for mpox virus. We identified two cases of presumed MPXV infection in pregnant, heterosexual cisgender women. Here, we describe their pregnancy and birth outcomes. Both patients required induction of labor and experienced labor complicated by chorioamnionitis.

Rapid emergence and transmission of virulence-associated mutations in the oral poliovirus vaccine following vaccination campaigns.

There is an increasing burden of circulating vaccine-derived polioviruses (cVDPVs) due to the continued use of oral poliovirus vaccine (OPV). However, the informativeness of routine OPV VP1 sequencing for the early identification of viruses carrying virulence-associated reversion mutations has not been directly evaluated in a controlled setting. We prospectively collected 15,331 stool samples to track OPV shedding from children receiving OPV and their contacts for ten weeks following an immunization campaign in Veracruz State, Mexico and sequenced VP1 genes from 358 samples. We found that OPV was genetically unstable and evolves at an approximately clocklike rate that varies across serotypes and by vaccination status. Overall, 61% (11/18) of OPV-1, 71% (34/48) OPV-2, and 96% (54/56) OPV-3 samples with available data had evidence of a reversion at the key 5' UTR attenuating position and 28% (13/47) of OPV-1, 12% (14/117) OPV-2, and 91% (157/173) OPV-3 of Sabin-like viruses had ≥1 known reversion mutations in the VP1 gene. Our results are consistent with previous work documenting rapid reversion to virulence of OPV and underscores the need for intensive surveillance following OPV use.

Evaluation of an automated system for the quantitation of human Herpesvirus-6 DNA from clinical specimens.

Background: Quantitation of human herpesvirus-6 (HHV-6) DNA in clinical specimens is important for the diagnosis and management of HHV-6-associated infection and reactivation in immunocompromised patients, particularly transplant recipients. Methods: The analytical performance of the Altona RealStar ASR HHV-6 qPCR on the semi-automated AltoStar AM16 system was assessed using HHV-6 reference material in plasma and cerebral spinal fluid (CSF). Qualitative and quantitative agreement was determined using 123 clinical EDTA plasma specimens tested using a laboratory-developed HHV-6 qPCR. Results: The 95% Lower Limit of Detection was 20 IU/mL [95% confidence interval (CI): 10 to 29] in plasma and 78 IU/mL (95% CI: 55 to 146) in CSF. The assay was linear from 7.0 to 2.0 log10 IU/mL in both matrices. Overall agreement of the RealStar ASR HHV-6 qPCR on the AltoStar AM16 with a laboratory-developed test was 95.9% (95% CI: 90.8 to 98.7). Passing-Bablok analysis of specimens quantifiable by both methods and at levels >1000 copies/mL revealed a regression line of Y = 1.00*X-0.20, with neither systematic (95% CI Y-intercept: -0.66 to 0.26) nor proportional (95% CI slope: 0.89 to 1.10) bias compared to the reference. Conclusions: The RealStar ASR HHV-6 qPCR on the AltoStar AM16 provides accurate quantitation for clinical monitoring of HHV-6 in immunocompromised hosts.

Mott-Schottky MXene@WS2 Heterostructure: Structural and Thermodynamic Insights and Application in Ultra Stable Lithium-Sulfur Batteries.

Due to the "shuttle effect" and low conversion kinetics of polysulfides, the cycle stability of lithium sulfur (Li-S) battery is unsatisfactory, which hinders its practical application. The Mott-Schottky heterostructures for Li-S batteries not only provide more catalytic/adsorption active sites, but also facilitate electrons transport by a built-in electric field, which are both beneficial for polysulfides conversion and long-term cycle stability. Here, MXene@WS2 heterostructure was constructed by in-situ hydrothermal growth for separator modification. In-depth ultraviolet photoelectron spectroscopy and ultraviolet visible diffuse reflectance spectroscopy analysis reveals that there is an energy band difference between MXene and WS2 , confirming the heterostructure nature of MXene@WS2 . DFT calculations indicate that the Mott-Schottky MXene@WS2 heterostructure can effectively promote electron transfer, improve the multi-step cathodic reaction kinetics, and further enhance polysulfides conversion. The built-in electric field of the heterostructure plays an important role in reducing the energy barrier of polysulfides conversion. Thermodynamic studies reveal the best stability of MXene@WS2 during polysulfides adsorption. As a result, the Li-S battery with MXene@WS2 modified separator exhibits high specific capacity (1613.7 mAh g-1 at 0.1 C) and excellent cycling stability (2000 cycles with 0.0286 % decay per cycle at 2 C). Even at a high sulfur loading of 6.3 mg cm-2 , the specific capacity could be retained by 60.0 % after 240 cycles at 0.3 C. This work provides deep structural and thermodynamic insights into MXene@WS2 heterostructure and its promising prospect of application in high performance Li-S batteries.

Hepatitis E virus seropositivity in an ethnically diverse community blood donor population.

BACKGROUND AND OBJECTIVES: Hepatitis E virus (HEV) is an underrecognized and emerging infectious disease that may threaten the safety of donor blood supply in many parts of the world. We sought to elucidate whether our local community blood supply is at increased susceptibility for transmission of transfusion-associated HEV infections. MATERIALS AND METHODS: We screened 10,002 randomly selected donations over an 8-month period between 2017 and 2018 at the Stanford Blood Center for markers of HEV infection using commercial IgM/IgG serological tests and reverse transcriptase quantitative polymerase chain reaction assays (RT-qPCR). Donor demographic information, including gender, age, self-identified ethnicity, location of residence and recent travel, were obtained from the donor database and used to generate multivariate binary logistic regressions for risk factors of IgG seropositivity. RESULTS: A total of 10,002 blood donations from 7507 unique donors were screened, and there was no detectable HEV RNA by RT-qPCR. The overall seropositivity rate was 12.1% for IgG and 0.56% for IgM. Multivariate analysis of unique donors revealed a significantly higher risk of IgG seropositivity with increasing age, White/Asian ethnicities and residence in certain local counties. CONCLUSION: Although HEV IgG seroprevalence in the San Francisco Bay Area is consistent with ongoing infection, the screening of a large donor population did not identify any viraemic blood donors. While HEV is an underrecognized and emerging infection in other regions, there is no evidence to support routine blood screening for HEV in our local blood supply currently; however, periodic monitoring may still be required to assess the ongoing risk.

Prevalence of Mpox (Monkeypox) in patients undergoing STI screening in northern California, April-September 2022.

BACKGROUND: Despite the sharp increase in mpox (formerly monkeypox) incidence and the wide geographic spread of mpox during the 2022 outbreak, the community prevalence of infection remains poorly characterized. This study is a retrospective epidemiologic survey to estimate mpox prevalence. METHODS: Samples obtained for sexually transmitted infection (STI) testing from April to September 2022 in the public hospital and clinic system of San Mateo County, California were screened for mpox virus (MPXV) using polymerase chain reaction. RESULTS: 16/1,848 samples from 11/1,645 individuals were positive for MPXV by qPCR. 4/11 individuals with positive MPXV testing were cisgender women, 2 of whom were pregnant at the time of sample collection. Both deliveries were complicated by chorioamnionitis. Anorectal and oropharyngeal samples were the most likely to be positive for MPXV (4/60 anorectal samples and 4/66 oropharyngeal samples compared with 5/1,264 urine samples and 3/445 vaginal samples). CONCLUSIONS: Our study is one of the first epidemiologic surveys for MPXV infection outside of sexual health/STI clinic settings. Relatively high rates of MPXV from oropharyngeal and anorectal samples reinforces the importance of MPXV testing at various anatomic sites, particularly if patients are presenting with non-lesional symptoms (pharyngitis, proctitis). However, the United States Food and Drug Administration (FDA) has not yet authorized non-lesional MPXV testing. The identification of MPXV in women in our cohort suggests that the rates of mpox in women may have previously been underestimated and highlights the risk of pregnancy complications associated with mpox.

Comparison of nucleocapsid antigen with strand-specific reverse-transcription PCR for monitoring SARS-CoV-2 infection.

BACKGROUND: Tests that sensitively detect the presence of actively replicating SARS-CoV-2 may improve patient care by allowing the safe and timely discontinuation of isolation. Correlates of active replication include nucleocapsid antigen and virus minus-strand RNA. METHODS: Qualitative agreement of the DiaSorin LIAISON SARS-CoV-2 nucleocapsid antigen chemiluminescent immunoassay (CLIA) with minus-strand RNA was determined using 402 upper respiratory specimens from 323 patients previously tested using a laboratory-developed SARS-CoV-2 strand-specific RT-qPCR. Nucleocapsid antigen levels, minus-strand and plus-strand cycle threshold values, as well as virus culture, were used to evaluate discordant specimens. Receiver operating characteristic curves were also used to identify virus RNA thresholds for active replication, including values harmonized to the World Health Organization International Standard. RESULTS: Overall agreement was 92.0% [95% confidence interval (CI): 89.0 - 94.5], positive percent agreement was 90.6% (95% CI: 84.4 - 95.0), and negative percent agreement was 92.8% (95% CI: 89.0 - 95.6). The kappa coefficient was 0.83 (95% CI: 0.77 - 0.88). Discordant specimens contained low levels of nucleocapsid antigen and minus-strand RNA. 84.8% (28/33) were negative by culture. Sensitivity-optimized plus-strand RNA thresholds for active replication were 31.6 cycles or 3.64 log10 IU/mL; resulting in 100.0% sensitivity (95% CI: 97.6 to 100.0) and 55.9 specificity (95% CI: 49.7 to 62.0). CONCLUSIONS: Detection of nucleocapsid antigen by CLIA performs equivalently to minus-strand detection via strand-specific RT-qPCR, though these methods may overestimate replication-competent virus compared to culture. Careful implementation of biomarkers for actively replicating SARS-CoV-2 has the potential to inform infection control decision-making and patient management.

Evaluation of a multiplexed oligonucleotide ligation assay for SARS-CoV-2 variant identification.

BACKGROUND: SARS-CoV-2 variant surveillance informs vaccine composition and decisions to de-authorize antibody therapies. Though detailed genetic characterization requires whole-genome sequencing, targeted mutation analysis may complement pandemic surveillance efforts. METHODS: This study investigated the qualitative performance of a multiplex oligonucleotide ligation assay targeting 19 spike mutations using 192 whole genome sequenced upper respiratory samples representing SARS-CoV-2 variants of concern. RESULTS: Initial valid results were obtained from 95.8% [95% confidence interval (CI): 92.0 - 98.2; 184/192] of samples. All eight invalid samples were valid on repeat testing. When comparing SARS-CoV-2 oligonucleotide ligase assay SARS-CoV-2 variant calls with whole genome sequencing, overall positive percent agreement was 100% (95% CI: 98.1 - 100.0; 192/192), as was the positive and negative percent agreement for each of the tested variants; Gamma, Delta, Omicron BA.1, BA.2, and BA.4/BA.5. CONCLUSIONS: This multiplexed oligonucleotide ligation assays demonstrated accurate SARS-CoV-2 variant typing compared to whole genome sequencing. Such an approach has the potential to provide improved turnaround compared to sequencing and more detailed mutation coverage than RT-qPCR.

Comparison of Real-Time PCR and Digital PCR for Detection of Plasma Epstein-Barr Virus DNA in Nasopharyngeal Carcinoma.

Plasma Epstein-Barr virus (EBV) DNA is an established biomarker for endemic nasopharyngeal carcinoma. However, existing real-time quantitative PCR (qPCR) assays are limited by poor interlaboratory reproducibility. This is a barrier to biomarker integration into staging systems and management. It was hypothesized that EBV digital PCR (dPCR) would have similar sensitivity but improved precision relative to qPCR. Using the World Health Organization EBV standard and patient specimens, the NRG-HN001 BamHI-W qPCR, two commercial EBNA-1 qPCR assays, and two laboratory-developed dPCR assays amplifying the BamHI-W, EBNA-1, and EBER targets were compared. Testing was conducted in the North American reference laboratory for the NRG-HN001 randomized trial. The EBV dPCR assays achieved similar performance compared with qPCR. Although dPCR does not require quantitation standards, different dPCR thresholding algorithms yielded significant qualitative and quantitative variation. This was most evident with low levels of EBV DNA. No-template control-informed thresholding (ddpcRquant) mitigated false-positive/false-negative findings. The NRG-HN001 BamHI-W qPCR and laboratory-developed BamHI-W droplet dPCR offered higher sensitivity, lower limit of blank, higher precision at low plasma EBV DNA levels (≤1500 IU/mL), and higher overall agreement with clinical specimens versus single-copy qPCR/dPCR targets (EBNA-1/EBER). These data confirm the rationale for using the BamHI-W target to define prognostic thresholds and indicate that both qPCR and dPCR methods harmonized to the World Health Organization standard can provide the necessary analytical performance.

A20 affects macrophage polarization through the NLRP3 inflammasome signaling pathway and promotes breast cancer progression.

Breast cancer is the most common malignant tumor in females, and the majority of patients succumb to metastasis. The present study aimed to investigate the association between tumor necrosis factor alpha-induced protein 3 (A20), NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) and tumor-associated macrophage polarization, and their effects on the proliferation and metastasis of breast cancer cells. The expression of A20 in breast cancer cells was analyzed by reverse transcription-quantitative PCR (RT-qPCR) and western blotting. RT-qPCR and western blotting were also used to confirm the transfection efficiency. The viability, clone formation, migration, invasion and angiogenesis of transfected breast cancer cells were detected by Cell Counting Kit-8, colony formation, wound healing, Transwell and tube formation assays, respectively. Activated macrophages, namely M1 and M2 type macrophages, were observed by double staining immunofluorescence. The levels of M1 and M2 macrophage markers were analyzed by qPCR. The expression of angiogenesis-related proteins and NLRP3 inflammasome activation-associated proteins was detected by western blotting. The results revealed that A20 was highly expressed in breast cancer cells. Interference with A20 inhibited the proliferation, invasion, migration and angiogenesis of breast cancer cells, and inhibited the M2-like polarization of macrophages. Interference with A20 promoted the activation of the NLRP3 inflammasome. The NLRP3 inhibitor MCC950 alleviated the effect of interference with A20 to promote macrophage proliferation and recruitment, as well as M2-like polarization. In conclusion, interference with A20 inhibited macrophage proliferation and M2-like polarization through the NLRP3 inflammasome signaling pathway to inhibit breast cancer progression.