A comprehensive assessment of four whole blood stabilizers for flow-cytometric analysis of leukocyte populations.

Though cryopreservation of cell fractions is widely used in flow cytometry studies, whole blood cryopreservation is more challenging due to the presence of erythrocytes and effects of fixatives commonly used for preservation. Here, we evaluated and compared head-to-head the performance of four commercial whole blood cryopreservation kits; (1) Cytodelics, (2) Stable-Lyse V2 and Stable-Store V2 (SLSS-V2), (3) Proteomic stabilizer (PROT-1), and (4) Transfix. We found that PROT-1, Transfix, and Cytodelics maintained the distribution of major leukocyte subsets-granulocytes, T cells, natural killer cells, and B cells, on a comparable level to unpreserved samples, despite the attenuation of fluorescence intensities in flow cytometric assays. Moreover, these three stabilizers also maintained the activated phenotypes of neutrophils upon stimulation with N-formylmethionyl-leucyl-phenylalanine and lipopolysaccharides. The upregulation of adhesion molecules (CD11b), Fc receptors (CD16), and granule proteins (CD66b), as well as the shedding of surface L-selectin (CD62L), was conserved most efficiently in PROT-1 and Cytodelics when compared to samples only treated with erythrocyte lysing. However, none of the stabilizers provided a reliable detection of CCR7 for accurate quantification of T cell maturation stages. We also evaluated the performance of Cytodelics in longitudinal clinical samples obtained from acute COVID-19 patients, where it allowed reliable detection of lymphopenia and granulocyte expansion. These results support the feasibility of whole blood cryopreservation for immunophenotyping by flow cytometry, particularly in longitudinal studies. In conclusion, the performance of different stabilizers is variable and therefore the choice of stabilizers should depend on cell type of interest, as well as antibody clones and experimental design of each study.

Production of a major metabolite of niclosamide using bacterial cytochrome P450 enzymes.

Niclosamide has been proposed as a possible candidate for a Covid-19 drug. However, the metabolites of niclosamide are difficult to investigate because they are usually not available commercially or they are quite expensive in the commercial market. In this study, the major metabolite of niclosamide in human liver microsomes (HLMs) was confirmed to be 3-OH niclosamide. Because the production of 3-OH niclosamide using HLMs has a slow turnover rate, a new method of producing niclosamide metabolite with an easier and highly cost-efficient method was thus conducted. Bacterial CYP102A1 (BM3) is one of the bacterial cytochrome P450s (CYPs) from Bacillus megaterium that structurally show similar activities to human CYPs. Here, the BM3 mutants were used to produce niclosamide metabolites and the metabolites were analyzed using high-performance liquid chromatography and LC-mass spectrometry. Among a set of mutants tested here, BM3 M14 mutant was the most active in producing 3-OH niclosamide, the major metabolite of niclosamide. Comparing BM3 M14 and HLMs, BM3 M14 production of 3-OH niclosamide was 34-fold higher than that of HLMs. Hence, the engineering of BM3 can be a cost-efficient method to produce 3-OH niclosamide.

The Impact of COVID-19 on Ethnic Business Households Involved in Tourism in Ninh Thuan, Vietnam

Since the COVID-19 outbreak at the end of 2019, there have been many studies on its impact on the tourism industry. However, research on the effects of this pandemic on ethnic tourism business households is minimal. This study explores how COVID-19 has affected ethnic minority tourism businesses and how they have responded to the crisis. Two ancient craft villages of the Cham people, which are popular destinations in Ninh Thuan province, were selected as case studies. Data were collected from late 2021 to early 2022 through fieldwork and in-depth interviews with 20 subjects who ran Cham-owned business households. A mini-survey of 52 Cham-owned tourism business households was also conducted as a qualitative method to supplement the statistical data. The findings of this study are: the Cham tourism business households have been affected by the COVID-19 pandemic, but not significantly;and that the Cham business households have utilized their own advantages (such as a small business scale, utilization of available advantages, reasonable gender division of labor in the family, changing business strategies, and taking advantage of social media) to weather the crisis. This article contributes to the literature on the impact of disasters on tourism by focusing on how ethnic minorities use the business household model to overcome a crisis and by presenting evidence that ethnic tourism combined with business households is a sustainable model.

Semi-nested RT-PCR enables sensitive and high-throughput detection of SARS-CoV-2 based on melting analysis.

BACKGROUND: Asymptomatic transmission was found to be the Achilles' heel of the symptom-based screening strategy, necessitating the implementation of mass testing to efficiently contain the transmission of COVID-19 pandemic. However, the global shortage of molecular reagents and the low throughput of available realtime PCR facilities were major limiting factors. METHODS: A novel semi-nested and heptaplex (7-plex) RT-PCR assay with melting analysis for detection of SARS-CoV-2 RNA has been established for either individual testing or 96-sample pooled testing. The complex melting spectrum collected from the heptaplex RT-PCR amplicons was interpreted with the support of an artificial intelligence algorithm for the detection of SARS-CoV-2 RNA. The analytical and clinical performance of the semi-nested RT-PCR assay was evaluated using RNAs synthesized in-vitro and those isolated from nasopharyngeal samples. RESULTS: The LOD of the assay for individual testing was estimated to be 7.2 copies/reaction. Clinical performance evaluation indicated a sensitivity of 100% (95% CI: 97.83-100) and a specificity of 99.87% (95% CI: 99.55-99.98). More importantly, the assay supports a breakthrough sample pooling method, which makes possible parallel screening of up to 96 samples in one real-time PCR well without loss of sensitivity. As a result, up to 8,820 individual pre-amplified samples could be screened for SARS-CoV-2 within each 96-well plate of realtime PCR using the pooled testing procedure. CONCLUSION: The novel semi-nested RT-PCR assay provides a solution for highly multiplex (7-plex) detection of SARS-CoV-2 and enables 96-sample pooled detection for increase of testing capacity. .

Characterization of low-density granulocytes in COVID-19.

Severe COVID-19 is characterized by extensive pulmonary complications, to which host immune responses are believed to play a role. As the major arm of innate immunity, neutrophils are one of the first cells recruited to the site of infection where their excessive activation can contribute to lung pathology. Low-density granulocytes (LDGs) are circulating neutrophils, whose numbers increase in some autoimmune diseases and cancer, but are poorly characterized in acute viral infections. Using flow cytometry, we detected a significant increase of LDGs in the blood of acute COVID-19 patients, compared to healthy controls. Based on their surface marker expression, COVID-19-related LDGs exhibit four different populations, which display distinctive stages of granulocytic development and most likely reflect emergency myelopoiesis. Moreover, COVID-19 LDGs show a link with an elevated recruitment and activation of neutrophils. Functional assays demonstrated the immunosuppressive capacities of these cells, which might contribute to impaired lymphocyte responses during acute disease. Taken together, our data confirms a significant granulocyte activation during COVID-19 and suggests that granulocytes of lower density play a role in disease progression.

A novel sample pooling strategy and its application for mass screening of SARS-CoV-2 in an outbreak of COVID-19 in Vietnam (preprint)

We present a sample pooling approach and the results of its application for mass screening of SARS-CoV-2 in >96,000 asymptomatic individuals. Our approach did not compromise the sensitivity of PCR, while increasing the throughput and reducing 77% of the costs. 22/32 asymptomatic cases would have been missed without mass screening.

Systems-Level Immunomonitoring from Acute to Recovery Phase of Severe COVID-19.

Severe disease of SARS-CoV-2 is characterized by vigorous inflammatory responses in the lung, often with a sudden onset after 5-7 days of stable disease. Efforts to modulate this hyperinflammation and the associated acute respiratory distress syndrome rely on the unraveling of the immune cell interactions and cytokines that drive such responses. Given that every patient is captured at different stages of infection, longitudinal monitoring of the immune response is critical and systems-level analyses are required to capture cellular interactions. Here, we report on a systems-level blood immunomonitoring study of 37 adult patients diagnosed with COVID-19 and followed with up to 14 blood samples from acute to recovery phases of the disease. We describe an IFNγ-eosinophil axis activated before lung hyperinflammation and changes in cell-cell co-regulation during different stages of the disease. We also map an immune trajectory during recovery that is shared among patients with severe COVID-19.