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.

Portable Multi-Channel Electrochemical Device with Good Interaction and Wireless Connection for On-Site Testing.

It is very important to rapidly test the key indicators of water in the field to fully evaluate the quality of the regional water environment. However, a high-resolution measuring device that can generate small currents for low-concentration analytes in water samples is often bulky, complex to operate, and difficult for data sharing. This work introduces a portable multi-channel electrochemical device with a small volume, good interaction, and data-sharing capabilities called PMCED. The PMCED provides an easy-to-operate graphical interactive interface to conveniently set the parameters for cyclic voltammetry or a differential pulse method performed by the four electrode channels. At the same time, the device, with a current sensitivity of 100 nA V-1, was applied to the detection of water samples with high background current and achieved a high-resolution measurement at low current levels. The PMCED uses the Narrow Band Internet of Things (NB-IoT) to meet the needs for uploading data to the cloud in remote areas. The electrochemical signal preprocessing and chemometrics models run in the cloud, and the final results are visualized on a web page, providing a remote access channel for on-site testing results.

Evaluation of factors contributing to variability of qualitative and quantitative proficiency testing for SARS-CoV-2 nucleic acid detection.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to unprecedented social and economic disruption. Many nucleic acid testing (NAT) laboratories in China have been established to control the epidemic better. This proficiency testing (PT) aims to evaluate the participants' performance in qualitative and quantitative SARS-CoV-2 NAT and to explore the factors that contribute to differences in detection capabilities. Two different concentrations of RNA samples (A, B) were used for quantitative PT. Pseudovirus samples D, E (different concentrations) and negative sample (F) were used for qualitative PT. 50 data sets were reported for qualitative PT, of which 74.00% were entirely correct for all samples. Forty-two laboratories participated in the quantitative PT. 37 submitted all gene results, of which only 56.76% were satisfactory. For qualitative detection, it is suggested that laboratories should strengthen personnel training, select qualified detection kits, and reduce cross-contamination to improve detection accuracy. For quantitative detection, the results of the reverse transcription digital PCR (RT-dPCR) method were more comparable and reliable than those of reverse transcription quantitative PCR (RT-qPCR). The copy number concentration of ORF1ab and N in samples A and B scattered in 85, 223, 50, and 106 folds, respectively. The differences in the quantitative result of RT-qPCR was mainly caused by the non-standard use of reference materials and the lack of personnel operating skills. Comparing the satisfaction of participants participating in both quantitative and qualitative proficiency testing, 95.65% of the laboratories with satisfactory quantitative results also judged the qualitative results correctly, while 85.71% of the laboratories with unsatisfactory quantitative results were also unsatisfied with their qualitative judgments. Therefore, the quantitative ability is the basis of qualitative judgment. Overall, participants from hospitals reported more satisfactory results than those from enterprises and universities. Therefore, surveillance, daily qualitiy control and standardized operating procedures should be strengthened to improve the capability of SARS-CoV-2 NAT.

Accurate quantification of SARS-CoV-2 RNA by isotope dilution mass spectrometry and providing a correction of reverse transcription efficiency in droplet digital PCR.

The novel coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected more than 505 million confirmed cases, including over 6 million deaths. Reference materials (RMs) of SARS-CoV-2 RNA played a crucial role in performance evaluation and quality control of testing laboratories. As the potential primary characterization method of RMs, reverse transcription digital PCR (RT-dPCR) measures the copy number of RNA, but the accuracy of reverse transcription (RT) efficiency has yet to be confirmed. This study established a method of enzymatic digestion followed by isotope dilution mass spectrometry (IDMS), which does not require an RT reaction, to quantify in vitro-transcribed SARS-CoV-2 RNA. RNA was digested to nucleotide monophosphate (NMP) within 15 min and analyzed by IDMS within 5 min. The consistency among the results of four different NMPs demonstrated the reliability of the proposed method. Compared to IDMS, the quantitative result of RT-dPCR turned out to be about 10% lower, possibly attributed to the incompleteness of the reverse transcription process. Therefore, the proposed approach could be valuable and reliable for quantifying RNA molecules and evaluating the RT efficiency of RT-based methods.

Interlaboratory assessment of quantification of SARS-CoV-2 RNA by reverse transcription digital PCR.

The pandemic of the novel coronavirus disease 2019 (COVID-19) has caused severe harm to the health of people all around the world. Molecular detection of the pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), played a crucial role in the control of the disease. Reverse transcription digital PCR (RT-dPCR) has been developed and used in the detection of SARS-CoV-2 RNA as an absolute quantification method. Here, an interlaboratory assessment of quantification of SARS-CoV-2 RNA was organized by the National Institute of Metrology, China (NIMC), using in vitro transcribed RNA samples, among ten laboratories on six different dPCR platforms. Copy number concentrations of three genes of SARS-CoV-2 were measured by all participants. Consistent results were obtained with dispersion within 2.2-fold and CV% below 23% among different dPCR platforms and laboratories, and Z' scores of all the reported results being satisfactory. Possible reasons for the dispersion included PCR assays, partition volume, and reverse transcription conditions. This study demonstrated the comparability and applicability of RT-dPCR method for quantification of SARS-CoV-2 RNA and showed the capability of the participating laboratories at SARS-CoV-2 test by RT-dPCR platform.

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.

Establishment of primary reference measurement procedures and reference materials for EGFR variant detection in non-small cell lung cancer.

Circulating tumor DNA (ctDNA)-based mutation detection is promising to change the clinical practice of genotype-directed therapy for cancer. A growing number of non-invasive tests for cancer screening and monitoring that involve the detection of ctDNA have been commercialized. Primary reference measurement procedures (PRMPs) and reference materials (RMs) are urgently needed to assess the non-invasive tests. In this study, a PRMP based on digital PCR (dPCR) and ctDNA RMs for quantification of the frequently occurring variant in epidermal growth factor receptor (EGFR L858R, T790M, and 19Del) in non-small cell lung cancer (NSCLC) were established. The candidate dPCR PRMP showed high specificity (false positive rate 0-0.003%), good repeatability (coefficient of variance (CV), 2-3% for 104 copies/reaction), and high interlaboratory reproducibility (3-10%). A good linearity (0.97 < slope < 1.03, R2 ≥ 0.9999) between the measured mutant (MU) value and prepared value was observed for all assays over the fractional abundance (FA) range, between 25% and 0.05%. The limit of quantification (LoQ) was determined to be 34 L858R, 23 T790M, and 34 19Del copies/reaction, corresponding to a FA of 0.2%. An inter-laboratory study of using the EGFR ctDNA RMs and dPCR assays demonstrated that the participating laboratories produced consistent concentrations of MU and wild-type (WT), as well as FA. This study demonstrates that dPCR can act as a potential PRMP for EGFR mutation for validation of NSCLC genotyping tests and ctDNA quantitative tests. The PRMP and RMs established here could improve interlaboratory repeatability and reproducibility, which supports rapid translation and application of non-invasive tests into clinical practice.

Quantitative analysis of RNA by HPLC and evaluation of RT-dPCR for coronavirus RNA quantification.

Nucleic acid detection and quantification have been known to be important at various fields, from genetically modified organisms and gene expression to virus detection. For DNA molecules, digital PCR has been developed as an absolute quantification method which is not dependent on external calibrators. While when it comes to RNA molecules, reverse transcription (RT) step must be taken before PCR amplification to obtain cDNA. With different kinds of reverse transcriptase (RTase) and RT reaction conditions being used in laboratory assays, the efficiency of RT process differs a lot which led variety in quantification results of RNA molecules. In this study, we developed HPLC method combined with enzymatic digestion of RNA to nucleotides for quantification of RNA without RT process. This method was metrologically traceable to four nuceloside monophosphate (NMP) Certification Reference Materials of National Institute of Metrology, China (NIMC) for insurance of accuracy. The established method was used to evaluate the reverse transcription digital polymerase chain reaction (RT-dPCR) of three target genes of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) RNA, including open reading frame 1ab (ORF1ab), nucleocapsid protein (N) and envelope protein (E) gene. Three available RT kits had been evaluated and disparities were observed for the RT efficiency varied from 9% to 182%. It is thus demonstrated that HPLC combined with enzymatic digestion could be a useful method to quantify RNA molecules and evaluate RT efficiency. It is suggested that RT process should be optimized and identified in RNA quantification assays.

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.

Highly accurate and sensitive diagnostic detection of SARS-CoV-2 by digital PCR.

The outbreak of COVID-19 caused by a novel Coronavirus (termed SARS-CoV-2) has spread to over 210 countries around the world. Currently, reverse transcription quantitative qPCR (RT-qPCR) is used as the gold standard for diagnosis of SARS-CoV-2. However, the sensitivity of RT-qPCR assays of pharyngeal swab samples are reported to vary from 30% to 60%. More accurate and sensitive methods are urgently needed to support the quality assurance of the RT-qPCR or as an alternative diagnostic approach. A reverse transcription digital PCR (RT-dPCR) method was established and evaluated. To explore the feasibility of RT-dPCR in diagnostic of SARS-CoV-2, a total of 196 clinical pharyngeal swab samples from 103 suspected patients, 77 close contacts and 16 supposed convalescents were analyzed by RT-qPCR and then measured by the proposed RT-dPCR. For the 103 fever suspected patients, 19 (19/25) negative and 42 (42/49) equivocal tested by RT-qPCR were positive according to RT-dPCR. The sensitivity of SARS-CoV-2 detection was significantly improved from 28.2% by RT-qPCR to 87.4% by RT-dPCR. For 29 close contacts (confirmed by additional sample and clinical follow up), 16 (16/17) equivocal and 1 negative tested by RT-qPCR were positive according to RT-dPCR, which is implying that the RT-qPCR is missing a lot of asymptomatic patients. The overall sensitivity, specificity and diagnostic accuracy of RT-dPCR were 91%, 100% and 93%, respectively. RT-dPCR is highly accurate method and suitable for detection of pharyngeal swab samples from COVID-19 suspected patients and patients under isolation and observation who may not be exhibiting clinical symptoms.

Histone Deacetylase HDT1 is Involved in Stem Vascular Development in Arabidopsis.

Histone acetylation and deacetylation play essential roles in eukaryotic gene regulation. HD2 (HD-tuins) proteins were previously identified as plant-specific histone deacetylases. In this study, we investigated the function of the HDT1 gene in the formation of stem vascular tissue in Arabidopsis thaliana. The height and thickness of the inflorescence stems in the hdt1 mutant was lower than that of wild-type plants. Paraffin sections showed that the cell number increased compared to the wild type, while transmission electron microscopy showed that the size of individual tracheary elements and fiber cells significantly decreased in the hdt1 mutant. In addition, the cell wall thickness of tracheary elements and fiber cells increased. We also found that the lignin content in the stem of the hdt1 mutants increased compared to that of the wild type. Transcriptomic data revealed that the expression levels of many biosynthetic genes related to secondary wall components, including cellulose, lignin biosynthesis, and hormone-related genes, were altered, which may lead to the altered phenotype in vascular tissue of the hdt1 mutant. These results suggested that HDT1 is involved in development of the vascular tissue of the stem by affecting cell proliferation and differentiation.

The papain-like cysteine protease CEP1 is involved in programmed cell death and secondary wall thickening during xylem development in Arabidopsis.

Both tracheary elements and fiber cells undergo programmed cell death (PCD) during xylem development. In this study we investigated the role of papain-like cysteine protease CEP1 in PCD in the xylem of Arabidopsis. CEP1 was located in the cell wall of xylem cells, and CEP1 expression levels in inflorescence stems increased during stem maturation. cep1 mutant plants exhibited delayed stem growth and reduced xylem cell number compared to wild-type plants. Transmission electron microscopy demonstrated that organelle degradation was delayed during PCD, and thicker secondary walls were present in fiber cells and tracheary elements of the cep1 mutant. Transcriptional analyses of the maturation stage of the inflorescence stem revealed that genes involved in the biosynthesis of secondary wall components, including cellulose, hemicellulose, and lignin, as well as wood-associated transcriptional factors, were up-regulated in the cep1 mutant. These results suggest that CEP1 is directly involved in the clearing of cellular content during PCD and regulates secondary wall thickening during xylem development.

[Preliminary study on human mature placenta tissue-derived hematopoietic stem/progenitor cells].

Clinical transplantation has indicated that cord blood (CB) can be used in the hematopoietic reconstitution in the children, but not well used in the adult patients because of the low cell amount. The present study aimed to explore the capability of proliferation and differentiation of the hematopoietic stem/progenitor cells derived from human mature placenta tissue (PT) in vitro, and to find a new source of hematopoietic/progenitor cells for clinical transplantation. CD34(+) cells in human mature placenta tissue were isolated and characterized by using enzyme-digestion method and flow cytometry. A long culture system without cytokines was established with human mature placenta tissue-derived mononucleated cells and cord blood mononuclear cells. The number of nucleated cells was weekly counted in culture for 14 weeks. The number of CFC was counted in culture for 2 weeks. The results showed that the CFC yields (CFU-GM, 186.90 +/- 24.52; BFU-E, 101.40 +/- 13.35) and the percentage of CD34(+) cells (2.74 +/- 0.61%) and CD34(+)/CD38(-) cells (2.46 +/- 0.42%) in placenta tissue (PT) were higher than CFC (CFU-GM, 136.90 +/- 25.15; BFU-E, 49.20 +/- 8.13), CD34(+) cells (1.73 +/- 0.32%) and CD34(+)/CD38(-) cells (0.80 +/- 0.25%) in cord blood (CB). The MNCs from PT have shown more survival ability than the cells from CB in the long-term cell culture condition; and the cells from PT increased by 2 times. It is concluded that the placenta may be another hematopoietic organ in ontogeny. The cells from placenta were more juvenile, and may be favorable source for clinical stem cell transplantation.