Rapid and Accurate Quantification of Viable Lactobacillus Cells in Infant Formula by Flow Cytometry Combined with Propidium Monoazide and Signal-Enhanced Fluorescence In Situ Hybridization.

Lactobacillus is an important member of the probiotic bacterial family for regulating human intestinal microflora and preserving its normalcy, and it has been widely used in infant formula. An appropriate and feasible method to quantify viable Lactobacilli cells is urgently required to evaluate the quality of probiotic-fortified infant formula. This study presents a rapid and accurate method to count viable Lactobacilli cells in infant formula using flow cytometry (FCM). First, Lactobacillus cells were specifically and rapidly stained by oligonucleotide probes based on a signal-enhanced fluorescence in situ hybridization (SEFISH) technique. A DNA-binding fluorescent probe, propidium monoazide (PMA), was then used to accurately recognize viable Lactobacillus cells. The entire process of this newly developed PMA-SEFISH-FCM method was accomplished within 2.5 h, which included pretreatment, dual staining, and FCM analysis; thus, this method showed considerably shorter time-to-results than other rapid methods. This method also demonstrated a good linear correlation (R2 = 0.9994) with the traditional plate-based method with a bacterial recovery rate of 91.24%. To the best of our knowledge, the present study is the first report of FCM combined with PMA and FISH for the specific detection of viable bacterial cells.

Reliable biological and multi-omics research through biometrology.

Metrology is the science of measurement and its applications, whereas biometrology is the science of biological measurement and its applications. Biometrology aims to achieve accuracy and consistency of biological measurements by focusing on the development of metrological traceability, biological reference measurement procedures, and reference materials. Irreproducibility of biological and multi-omics research results from different laboratories, platforms, and analysis methods is hampering the translation of research into clinical uses and can often be attributed to the lack of biologists' attention to the general principles of metrology. In this paper, the progresses of biometrology including metrology on nucleic acid, protein, and cell measurements and its impacts on the improvement of reliability and comparability in biological research are reviewed. Challenges in obtaining more reliable biological and multi-omics measurements due to the lack of primary reference measurement procedures and new standards for biological reference materials faced by biometrology are discussed. In the future, in addition to establishing reliable reference measurement procedures, developing reference materials from single or multiple parameters to multi-omics scale should be emphasized. Thinking in way of biometrology is warranted for facilitating the translation of high-throughput omics research into clinical practices.

Rapid and accurate quantification of viable Bifidobacterium cells in milk powder with a propidium monoazide - antibiotic fluorescence in situ hybridization - flow cytometry method.

Due to its beneficial effects on human health, Bifidobacterium is commonly added to milk powder. Accurate quantification of viable Bifidobacterium is essential for assessing the therapeutic efficacy of milk powder. In this study, we introduced a novel propidium monoazide (PMA) - antibiotic fluorescence in situ hybridization (AFISH) - flow cytometry (FCM) method to rapidly and accurately quantify viable Bifidobacterium cells in milk powder. Briefly, Bifidobacterium cells were treated with chloramphenicol (CM) to increase their rRNA content, followed by staining with RNA-binding oligonucleotide probes, based on the AFISH technique. Then, the DNA-binding dye PMA was used to differentiate between viable and non-viable cells. The PMA-AFISH-FCM method, including sample pretreatment, CM treatment, dual staining, and FCM analysis, required around 2 h and was found to be better than the current methods. This is the first study to implement FCM combined with PMA and oligonucleotide probe for detecting Bifidobacterium.

Rapid and Multiplexed Detection of Single Cells of Salmonella, Escherichia coli O157, and Shigella flexneri in Ground Beef by Flow Cytometry.

Salmonella, Escherichia coli O157, and Shigella flexneri are typical foodborne pathogens in ground beef, which can cause severe infection even when present as a single cell. Flow cytometry (FCM) methods are widely applied in the rapid detection of pathogens in food products. In this study, we report an FCM-based method for detecting single cells of Salmonella, E. coli O157, and S. flexneri in 25 g ground beef samples. We fluorescently labeled specific antibodies that could effectively identify bacterial cells, prepared single-cell samples by serial dilution, and optimized the pre-enrichment time. The results showed that 7 h of pre-enrichment is appropriate for sensitive single-cell detection by FCM. Finally, we evaluated this method in artificially contaminated and retail beef samples. This study outlines a novel highly sensitive FCM-based method to detect Salmonella, E. coli O157, and S. flexneri in beef samples within 8 h that can be applied to the rapid and multiplexed detection of foodborne pathogens.

Rapid Detection of Single Viable Escherichia coli O157 Cells in Fresh Lettuce and Strawberry by Immunomagnetic Flow Cytometry in Combination with Pre-Enrichment.

Enterohemorrhagic Escherichia coli are an important pathogen causing food poisoning. The rapid detection of viable E. coli O157 in vegetables and fruits at single-cell level is critical because of the low infective dose of this pathogen. In this study, an immunomagnetic flow cytometry (IMFC)-based method was developed to detect E. coli O157 in lettuce and strawberries inoculated with 1 CFU/25 g. This method developed immunomagnetic (IM)-beads to capture E. coli O157 cells. The pre-enrichment of E. coli O157 and IM-bead separation rapidly increased the concentration of cells to a detectable range for flow cytometry. Compared with the plate-based method, the diagnostic sensitivity and specificity of the IMFC-based method were 100% in 166 samples, including 100 artificially contaminated samples, 60 retail samples, and six O157-positive samples for proficiency testing. The developed IMFC-based method was found to be effective in detecting E. coli O157 at single-cell level in 25 g of lettuce or strawberry with relatively shorter associated time to results of 5.7 h. Therefore, the IMFC-based method could improve detection efficiency and also make early warnings in a short time.

Point-of-care test system for detection of immunoglobulin-G and -M against nucleocapsid protein and spike glycoprotein of SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) epidemic continues to ravage the world. In epidemic control, dealing with a large number of samples is a huge challenge. In this study, a point-of-care test (POCT) system was successfully developed and applied for rapid and accurate detection of immunoglobulin-G and -M against nucleocapsid protein (anti-N IgG/IgM) and receptor-binding domain in spike glycoprotein (anti-S-RBD IgG/IgM) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Any one of the IgG/IgM found in a sample was identified as positive. The POCT system contains colloidal gold-based lateral flow immunoassay test strips, homemade portable reader, and certified reference materials, which detected anti-N and anti-S-RBD IgG/IgM objectively in serum within 15 min. Receiver operating characteristic curve analysis was used to determine the optimal cutoff values, sensitivity, and specificity. It exhibited equal to or better performances than four approved commercial kits. Results of the system and chemiluminescence immunoassay kit detecting 108 suspicious samples had high consistency with kappa coefficient at 0.804 (P < 0.001). Besides, the levels and alterations of the IgG/IgM in an inpatient were primarily investigated by the POCT system. Those results suggested the POCT system possess the potential to contribute to rapid and accurate serological diagnosis and epidemiological survey of COVID-19.

Faster Detection of Staphylococcus aureus in Milk and Milk Powder by Flow Cytometry.

A flow cytometry (FCM)-based method was developed for the faster detection of Staphylococcus aureus in milk and milk powder. Viable S. aureus cells were recognized by highly selective, fluorescently labeled antibodies and Propidium Iodide, and then analyzed by FCM. Using a 5-h pre-enrichment period, the method could detect low numbers of S. aureus cells in 6 h, with a limit of detection of 7.50 cells/mL in milk and 8.30 cells/g in milk powder. The established method was compared with the plate-based method using 75 ultra-high-temperature-treated milk samples, 25 pasteurized milk samples, 66 raw milk samples, and 123 milk powder samples. The two methods yielded similar results for the detection of the pathogen in all sample types. The FCM-based method allows effective and faster monitoring of S. aureus contamination and can be applied to the rapid detection of microorganisms in milk and dairy products.

Rapid Flow Cytometric Detection of Single Viable Salmonella Cells in Milk Powder.

Salmonella, a highly virulent food-borne pathogen transmitted through food, can cause severe infectious diseases in a large number of people through a single outbreak, due to its low infective doses. In this study, a flow cytometry (FCM)-based method was developed for the rapid detection of single viable Salmonella cells with dual staining of fluorescein isothiocyanate (FITC)-labeled anti-Salmonella antibody and propidium iodide (PI) dyes. The FCM-based method includes 6 h of pre-enrichment, 40 min of target cell isolation, and 20 min of dual staining and FCM analysis. The developed method demonstrated high specificity for the detection of 23 Salmonella strains and 22 food-borne pathogenic non-Salmonella strains. Furthermore, the analyses of 30 samples of milk powder artificially contaminated with single Salmonella cells, 123 samples of retail milk powder, and 6 samples of Salmonella-positive milk powder were performed by the FCM-based as well as traditional plate-based methods for testing the efficiency of the methods. The two methods yielded similar results for the detection of pathogens in all milk powder samples. In conclusion, the developed FCM-based method was found to be efficient in detecting single viable Salmonella cells in milk powder within 7 h. The proposed dual-color FITC assay combined with pre-enrichment offers a great potential for the rapid and sensitive detection of other pathogens in dairy products.

International Comparison of Enumeration-Based Quantification of DNA Copy-Concentration Using Flow Cytometric Counting and Digital Polymerase Chain Reaction.

Enumeration-based determination of DNA copy-concentration was assessed through an international comparison among national metrology institutes (NMIs) and designated institutes (DIs). Enumeration-based quantification does not require a calibration standard thereby providing a route to "absolute quantification", which offers the potential for reliable value assignments of DNA reference materials, and International System of Units (SI) traceability to copy number 1 through accurate counting. In this study, 2 enumeration-based methods, flow cytometric (FCM) counting and the digital polymerase chain reaction (dPCR), were compared to quantify a solution of the pBR322 plasmid at a concentration of several thousand copies per microliter. In addition, 2 orthogonal chemical-analysis methods based on nucleotide quantification, isotope-dilution mass spectrometry (IDMS) and capillary electrophoresis (CE) were applied to quantify a more concentrated solution of the plasmid. Although 9 dPCR results from 8 laboratories showed some dispersion (relative standard deviation [RSD] = 11.8%), their means were closely aligned with those of the FCM-based counting method and the orthogonal chemical-analysis methods, corrected for gravimetric dilution factors. Using the means of dPCR results, the RSD of all 4 methods was 1.8%, which strongly supported the validity of the recent enumeration approaches. Despite a good overall agreement, the individual dPCR results were not sufficiently covered by the reported measurement uncertainties. These findings suggest that some laboratories may not have considered all factors contributing to the measurement uncertainty of dPCR, and further investigation of this possibility is warranted.

Evaluation of volume-based flow cytometry as a potential primary method for quantification of bacterial reference material.

Bacterial reference materials (RMs) play a crucial role in many analytical processes of microbiological detection. Currently, bacteria are typically counted using the traditional plate-based approach, which results in a higher uncertainty of bacterial RMs unfortunately. Therefore, novel methods are urgently required for the value assignment of RMs in the field of microbiology to derive measurement traceability and accuracy. A potential primary method for microbiological quantification based on flow cytometry (FCM) is described in this study using Escherichia coli O157 (E. coli O157) as an example. The proposed method was applied to determine the number of viable E. coli O157 cells in the RMs with a result of (5.48 ± 0.27) × 108 cells mL-1, which was in good agreement with the result obtained using the plate-based method (En = 0.47). Additionally, this method could be entirely described and understood by equations, and provides formal traceability to the SI for counts of viable bacterial cells, while the associated relative expanded uncertainty (4.93%, k = 2) was significantly lower in comparison to the plate-based method. Therefore, the FCM-based method might be a potential primary method for characterizing bacterial RMs. To our knowledge, this is the first description of FCM as a potential primary method for accurate and traceable quantification of viable bacterial cells with a comprehensive uncertainty statement in microbiological metrology.

Accurate and online quantification of viable Rhodobacter sphaeroides cells using a flow cytometry-dielectric spectroscopy (FCM-DS) method.

Accurate and online quantification of viable cells is one of the necessary requirements during the microbial fermentation process for high productivity. The flow cytometry (FCM)-based method accurately quantifies viable cells, but this offline method cannot reflect the counts constantly. The dielectric spectroscopy (DS) sensor is widely utilized to monitor viable cells online; however, accurately converting the capacitance value of the DS sensor to the viable bacterial cell counts has barely been tried. We have developed a method by coupling the principles and techniques of FCM and the DS sensor to quantify viable Rhodobacter sphaeroides cells. Using specific fluorescent antibodies and propidium iodide (PI), viable R. sphaeroides cells were accurately quantified within 30 min by FCM. The DS sensor was combined with the FCM to create a direct capacitance-viable cell count quantification system. The LOD (limit of detection) of the FCM-DS method was 8 × 108 CFU/mL, RSD (relative standard deviation) < 5%, along with good reproducibility of the results. Finally, the viable cell count, obtained from the FCM-DS method, was applied to regulate the specific oxygen uptake rate (QO2) that increased the production of coenzyme Q10 by 8.1%. Together, our results strongly suggest that viable cells can be accurately quantified online by the integrated FCM-DS method, which would help to devise precise fermentation control strategies.

Rapid and multiplexed quantification of Salmonella, Escherichia coli O157:H7, and Shigella flexneri in ground beef using flow cytometry.

Salmonella, Escherichia coli O157:H7 (E. coli O157:H7) and Shigella flexneri (S. flexneri) might contaminate similar types of meat products and cause deadly diseases in humans. In reality, ground beef samples may carry more than one pathogen and a rapid and accurate detection method for the simultaneous identification of multiple specific pathogenic strains in ground beef is crucial. In this study, a sample pretreatment protocol and a flow cytometry method were developed for rapid and multiplexed quantification of the three pathogens without cultural enrichment in ground beef. The whole process of sample pretreatment, staining, and instrument analysis can be accomplished within 1 h. The three bacteria upon sample pretreatment were demonstrated good recoveries (93.8%-101.2%). The quantitative detection range of the mothed was 103 to 108 cells/g for all three pathogens, and the detection limit for Salmonella, E. coli O157:H7 and S. flexneri in ground beef were 3.1 × 103 cells/g, 2.1 × 103 cells/g and 2.3 × 103 cells/g, respectively. Therefore, the as-developed approach is a rapid and quantitative method for multiplexed detection of Salmonella, E. coli O157:H7, and S. flexneri in ground beef.

The regulation of DOCK family proteins on T and B cells.

The dedicator of cytokinesis (DOCK) family proteins consist of 11 members, each of which contains 2 domains, DOCK homology region (DHR)-1 and DHR-2, and as guanine nucleotide exchange factors, they mediate activation of small GTPases. Both DOCK2 and DOCK8 deficiencies in humans can cause severe combined immunodeficiency, but they have different characteristics. DOCK8 defect mainly causes high IgE, allergic disease, refractory skin virus infection, and increased incidence of malignant tumor, whereas DOCK2 defect mainly causes early-onset, invasive infection with less atopy and increased IgE. However, the underlying molecular mechanisms causing the disease remain unclear. This paper discusses the role of DOCK family proteins in regulating B and T cells, including development, survival, migration, activation, immune tolerance, and immune functions. Moreover, related signal pathways or molecule mechanisms are also described in this review. A greater understanding of DOCK family proteins and their regulation of lymphocyte functions may facilitate the development of new therapeutics for immunodeficient patients and improve their prognosis.

Synthesis of TEMPO radical decorated hollow porous aromatic frameworks for selective oxidation of alcohols.

A bottom-up approach was developed to prepare TEMPO radical decorated hollow aromatic frameworks (HPAF-TEMPO) by using TEMPO radical functionalized monomers and SiO2 nanospheres as templates. The accessible inner layer, high density of TEMPO sites, and hybrid micro-/mesopores of the HPAF-TEMPO enable the aerobic oxidation of a broad range of alcohols with high efficiency and excellent selectivity.

SARS-CoV-2 infection causes immunodeficiency in recovered patients by downregulating CD19 expression in B cells via enhancing B-cell metabolism.

The SARS-CoV-2 infection causes severe immune disruption. However, it is unclear if disrupted immune regulation still exists and pertains in recovered COVID-19 patients. In our study, we have characterized the immune phenotype of B cells from 15 recovered COVID-19 patients, and found that healthy controls and recovered patients had similar B-cell populations before and after BCR stimulation, but the frequencies of PBC in patients were significantly increased when compared to healthy controls before stimulation. However, the percentage of unswitched memory B cells was decreased in recovered patients but not changed in healthy controls upon BCR stimulation. Interestingly, we found that CD19 expression was significantly reduced in almost all the B-cell subsets in recovered patients. Moreover, the BCR signaling and early B-cell response were disrupted upon BCR stimulation. Mechanistically, we found that the reduced CD19 expression was caused by the dysregulation of cell metabolism. In conclusion, we found that SARS-CoV-2 infection causes immunodeficiency in recovered patients by downregulating CD19 expression in B cells via enhancing B-cell metabolism, which may provide a new intervention target to cure COVID-19.

Viral dynamics and antibody responses in people with asymptomatic SARS-CoV-2 infection.

Over 40% of the coronavirus disease 2019 (COVID-19) COVID-19 patients were asymptomatically infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the immune responses of these asymptomatic individuals is a critical factor for developing the strategy to contain the COVID-19 pandemic. Here, we determined the viral dynamics and antibody responses among 143 asymptomatic individuals identified in a massive screening of more than 5 million people in eight districts of Wuhan in May 2020. Asymptomatic individuals were admitted to the government-designated centralized sites in accordance with policy. The incidence rate of asymptomatic infection is ~2.92/100,000. These individuals had low viral copy numbers (peaked at 315 copies/mL) and short-lived antibody responses with the estimated diminish time of 69 days. The antibody responses in individuals with persistent SARS-CoV-2 infection is much longer with the estimated diminish time of 257 days. These results imply that the immune responses in the asymptomatic individuals are not potent enough for preventing SARS-CoV-2 re-infection, which has recently been reported in recovered COVID-19 patients. This casts doubt on the efficacy of forming "herd-immunity" through natural SARS-CoV-2 infection and urges for the development of safe and effective vaccines.

Cross-protection against influenza virus infection by intranasal administration of M2-based vaccine with chitosan as an adjuvant.

Influenza vaccines based on conserved antigens could provide cross-protection against infections by multiple subtypes of influenza A virus. Influenza matrix protein 2 (M2) is highly conserved in all influenza A strains. In this study, we deleted the transmembrane domain of the M2 of the avian influenza virus (AIV) A/Chicken/Jiangsu/7/2002 (H9N2) strain to create an M2 without a transmembrane domain, named sM2, which was efficiently expressed in Escherichia coli. The sM2 protein was administered intranasally to mice in combination with chitosan adjuvant three times at an interval of 3 weeks. Three weeks after the last immunization, the mice were challenged with a lethal dose (5 x LD(50)) of A/Chicken/Jiangsu/7/2002 (H9N2) virus, PR8 (H1N1) virus and A/Chicken/Henan/12/2004 (H5N1) virus. The protective immunity of the vaccine was evaluated by determining the survival rates, residual lung virus titers, body weight, and the serum antibody titers of the mice. Nasal administration of 15 microg sM2 in combination with chitosan completely protected mice against the homologous virus and protected 90 and 30% of the mice against the heterologous H1N1 and H5N1 viruses, respectively. The study indicated that the sM2 protein was a candidate antigen for a broad-spectrum influenza virus vaccine and that the adjuvant chitosan improved the efficacy of the sM2 vaccine.

Protection against multiple influenza A virus subtypes by intranasal administration of recombinant nucleoprotein.

Vaccination is a cost-effective way to control the influenza epidemic. Vaccines based on highly conserved antigens can provide protection against different influenza A strains and subtypes. In this study, the recombinant nucleoprotein (rNP) of the A/PR/8/34 (H1N1) influenza virus strain was effectively expressed using a prokaryotic expression system and then purified with a nickel-charged Sepharose affinity column as a candidate component for an influenza vaccine. The rNP was administered intranasally three times at 3-week intervals to female BALB/c mice in combination with an adjuvant (cholera toxin B subunit containing 0.2% of the whole toxin). Twenty-one days after the last immunization, the mice were challenged with homologous or heterologous influenza viruses at a lethal dose. The results showed that intranasal immunization of 10 µg rNP with adjuvant completely protected the immunized mice against the homologous influenza virus, and immunization with 100 µg rNP in combination with adjuvant provided good cross-protection against heterologous H5N1 and H9N2 avian influenza viruses. The results indicate that such a vaccine administered intranasally can induce mucosal and cell-mediated immunity, thus having the potential to control epidemics caused by new emerging influenza viruses.

Calibration of an Upconverting Phosphor-Based Quantitative Immunochromatographic Assay for Detecting Yersinia pestis, Brucella spp., and Bacillus anthracis Spores.

Yersinia pestis, Brucella spp., and Bacillus anthracis are pathogens that can cause infectious zoonotic diseases with high mortality rates. An upconverting phosphor-based quantitative immunochromatographic (UPT-LF) assay, a point-of-care testing method suitable for resource-limited areas, was calibrated to quantitatively detect pathogenic bacteria. The bacterial purity or activity were ensured via staining methods and growth curves, respectively. Growth assays showed that the classic plate-counting method underestimated bacterial numbers compared with the bacterial counting method recommended by the reference material of the National Institutes for Food and Drug Control, China. The detection results of the UPT-LF assay differed significantly between the bacterial cultures in liquid and solid media and between different strains. Accelerated stability assessments and freeze-thaw experiments showed that the stability of the corresponding antigens played an important role in calibrating the UPT-LF assay. In this study, a new calibration system was developed for quantitative immunochromatography for detecting pathogenic bacteria. The results demonstrated the necessity of calibration for standardizing point-of-care testing methods.

Generation and evaluation of an H9N1 influenza vaccine derived by reverse genetics that allows utilization of a DIVA strategy for control of H9N2 avian influenza.

H9N2 avian influenza viruses have circulated widely in domestic poultry around the world, and their outbreaks have resulted in heavy morbidity and mortality. In addition, H9N2 avian influenza viruses were transmitted directly from birds to humans in Hong Kong and mainland China during 1998 and 2003, which prompted the public health authorities to seek protective strategies to control H9N2 influenza viruses. In this study, we attempted to develop a DIVA (differentiating infected and vaccinated animals) strategy for H9N2 avian influenza viruses. This strategy does not interfere with serological monitoring and allows effective control of H9N2 avian influenza. We generated a reassortant H9N1 influenza vaccine strain by reverse genetics and employed an enzyme-linked immunosorbent assay (ELISA) with a truncated N1 antigen expressed in E. coli to differentiate between vaccinated and naturally infected animals. Immunization of BALB/c mice with the inactivated reassortant H9N1 vaccine conferred protection against lethal challenge with H9N2 viruses. Meanwhile, the ELISA can be used to distinguish between vaccination and natural infection quickly and easily. Therefore, this study has opened up a new avenue for the control of H9N2 avian influenza.