Development of an SI-traceable N-terminal pro-B-type natriuretic peptide certified reference material using structure-based impurity-corrected isotope dilution mass spectrometry approaches.

N-Terminal pro-B-type natriuretic peptide (NT-proBNP) is a pivotal biomarker for the diagnosis and prognosis of heart failure (HF). However, no SI-traceable certified reference material (CRM) or reference measurement procedure (RMP) is available for this biomarker, and so clinical testing results obtained in different laboratories cannot be traced to a higher-order standard, leading to incomparable measurements. Protein hydrolysis and protein cleavage isotope dilution mass spectrometry (AAA-IDMS and PepA-IDMS) were used to develop a CRM. Structurally related impurities were identified by high-resolution mass spectrometry. The quantitative AAA-IDMS results were corrected according to the amino acid compositions of the impurities. Using PepA-IDMS, two peptides from the proteolyzed product were confirmed as signature peptides. To obtain traceable and accurate results, the signature peptides were quantified using impurity-corrected AAA-IDMS. The candidate NT-proBNP solution was denatured and enzymatically digested using the Glu-C endoproteinase. The released signature peptides were measured using an isotopic dilution approach. The homogeneity and stability of the candidate CRM were characterized, and their uncertainties were combined with the value assignment process. The developed CRM can be considered a unique SI-traceable NT-proBNP reference material and is expected to be used as a primary calibrator for matrix NT-proBNP CRM development.

The Possible Role of Electrical Stimulation in Osteoporosis: A Narrative Review.

Osteoporosis is mainly a geriatric disease with a high incidence, and the resulting spinal fractures and hip fractures cause great harm to patients. Anti-osteoporosis drugs are the main treatment for osteoporosis currently, but these drugs have potential clinical limitations and side effects, so the development of new therapies is of great significance to patients with osteoporosis. Electrical stimulation therapy mainly includes pulsed electromagnetic fields (PEMF), direct current (DC), and capacitive coupling (CC). Meanwhile, electrical stimulation therapy is clinically convenient without side effects. In recent years, many researchers have explored the use of electrical stimulation therapy for osteoporosis. Based on this, the role of electrical stimulation therapy in osteoporosis was summarized. In the future, electrical stimulation might become a new treatment for osteoporosis.

A Specific Mass-Tag Approach for Detection of Foodborne Pathogens Using MALDI-TOF Mass Spectrometry.

Pathogen infections present a considerable threat to global health owing to the high morbidity and mortality, and usually multiple pathogens coexist in food and the environment. Consequently, it is in urgent need to develop some multiplexed and sensitive approaches for pathogen detection. Here, we presented a novel strategy using mass tag-mediated surface engineering for simultaneous detection of multiple bacteria by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Following aptamer binding, primer amplification, and DNA hybridization, bacteria were specifically labeled by their corresponding mass tags, which could be released and ionized after laser irradiation. This strategy converted the detection of bacteria to the analysis of mass tags, allowing simultaneous detection of multiple bacteria and avoiding the dependence of microbial mass spectra databases. In addition, this approach applied two rolling circle amplification (RCA) reactions to improve both the capture efficiency and detection sensitivity of the target bacteria. The specificity and the real sample detection were evaluated, and the results demonstrated a potential application of this approach in milk safety monitoring.

Inkjet-Patterned Microdroplets as Individual Microenvironments for Adherent Single Cell Culture.

Adhesion of single cells is the foundation of manifold cellular behaviors and life processes. However, investigating the function of a specific cell is still challenging due to deficiency of adhesion or interference from surrounding cells. Herein, an open microfluidic system is reported for culturing adherent single cells, implemented by a micrometer-scale droplet matrix on an inkjet-printed polylysine template. The target cells are isolated from any cell from other droplets, and their adhesion strength is determined to be comparable to conventional petri dishes via an in-situ investigation with a microfluidic extractor. On this proposed platform, isolated single cells are observed to display an entirely distinct spreading behavior featuring total absence of elongation, indicating drastic cell behavior change from their "singleness." This system has high versatility and compatibility for various assaying methods, assuring a promising potential in detailed single cell behavior and cell heterogeneity studies.

Advances in the research of osteosarcoma stem cells and its related genes.

Osteosarcoma is a malignant tumor that often occurs in adolescents. There is an urgent need for new treatment options for osteosarcoma due to its poor prognosis after metastasis. Cancer stem cell (CSC) theory states that CSCs represent a small proportion of cancer cells. These CSC have self-renewal ability and are closely associated with cancer growth and metastasis as well as chemotherapy resistance. Similarly, osteosarcoma stem cells (OSCs) play an important role in the growth, metastasis, and chemotherapy resistance of osteosarcoma cells. Targeting OSCs may represent a future treatment of osteosarcoma. Furthermore, some genes have been shown to regulate the growth, metastasis, and chemotherapy resistance of osteosarcoma cells by altering the stemness of OSCs. Targeting these genes may help in the treatment of osteosarcoma. This review mainly discusses recent advances in the research of OSCs and their related genes.

Metabolism-Based Capture and Analysis of Circulating Tumor Cells in an Open Space.

The level of circulating tumor cells (CTCs) in blood is a predictor of metastatic cancer progress, serving as an important biomarker for cancer diagnosis, prognosis, and therapy. Currently, there are mainly two conventional strategies to distinguish CTCs, including biological property-based affinity capture and physical property-based label-free isolation. Although great progress has been made in this field, the ability to distinguish CTCs still needs to be improved further due to the cell heterogeneity. Herein, a metabolism-based isolation approach was applied to identify tumor cells according to the "Warburg effect", and a bifunctional open-space platform with fluid walls was developed for real-time monitoring and in situ capture/analysis of tumor cells. A drop-on-demand inkjet printing technique was introduced to create a single cell-containing droplet array with high throughput and high encapsulation rate, and the homogeneous crystalline matrix spots ejected from the inkjet also provided high-quality and reproducible lipid profiling. This platform could combine both microscopic image and mass data, and it has been proven to be capable of isolating and identifying CTCs in complex blood samples, making it a promising tool for evaluating the efficacy of therapy and monitoring the disease progression.

Concentrating Single Cells in Picoliter Droplets for Phospholipid Profiling on a Microfluidic System.

Cellular membranes are composed of a variety of lipids in different amounts and proportions, and alterations of them are usually closely related to various diseases. To reveal the intercellular heterogeneity of the lipid variation, an integrated microfluidic system is designed, which consists of droplet-based inkjet printing, dielectrophoretic electrodes, and de-emulsification interface to achieve on-line single-cell encapsulation, manipulation, and mass spectrometry (MS) detection. This integrated system effectively improves the single-cell encapsulation rate, and meanwhile reduces the matrix interference and continuous oil phase interference to the MS detection. Using this system, the heterogeneities between the normal and cancer cells are compared, and the heterogeneity of the same cells before and after the drug treatment changed obviously, indicating that this system can be used as a promising tool for studying the link between the alterations of lipid homeostasis and various diseases.

Nongenetically Encoded and Erasable Imaging Strategy for Receptor-Specific Glycans on Live Cells.

Glycans on specific receptors play a crucial role in regulating receptor functions and indicating cell pathological states. For a detailed glycosylation regulatory mechanism, live cell imaging of receptor-specific glycans becomes significantly important. In this work, we present a nongenetically encoded labeling strategy to specifically install a fluorescence resonance energy transfer (FRET) pair onto the receptor of interest by ligand-receptor binding and metabolic glycan engineering. This method breaks the limitation that the receptors have to possess an extracellular terminus which can be used to attach a fluorescent tag, avoiding the undesired effects introduced by inserting amino acids into proteins. Furthermore, the donor-equipped ligand can be flexibly competed with an unlabeled compound, leading to an efficient erasure of donor fluorescence signal. We envision that this strategy will have the potential to sequentially identify and characterize multiple receptor-specific glycans on the live cell surface through multiple cycles including labeled ligand binding, FRET-induced fluorescence imaging, and the unlabeled compound competing for fluorescence erasure. Besides, this in situ glycan profiling strategy will have wide applications in molecular diagnosis and cellular targeted therapies.

Inkjet Printing Based Droplet Generation for Integrated Online Digital Polymerase Chain Reaction.

We report on the development of a novel and flexible online digital polymerase chain reaction (dPCR) system. The system was composed of three parts: an inkjet for generating the droplets, a coiled fused-silica capillary for thermal cycling, and a laser-induced fluorescence detector (LIFD) for positive droplet counting. Upon inkjet printing, monodisperse droplets were continuously generated in the oil phase and then introduced into the capillary in the form of a stable dispersion. The droplets containing one or zero molecules of target DNA passed through the helical capillary that was attached to a cylindrical thermal cycler for PCR amplification, resulting in the generation of fluorescence for the DNA-positive droplet. After 36 PCR cycles, the fluorescence signal intensity was detected by laser-induced fluorescence located at the downstream of the capillary, followed by a positive/negative counting. The present system was successfully applied to the absolute quantification of the HPV sequence in Caski cells with dynamic ranges spanning 4 orders of magnitude.

Inkjet Printing Based Separation of Mammalian Cells by Capillary Electrophoresis.

This study describes a method to investigate the separation of cells by capillary electrophoresis (CE) coupled with inkjet printing system. The results validated the feasibility of inkjet printing for mammalian cells to achieve the drop-on-demand and convenient sampling into capillary then zone electrophoresis was applied to separate different cells according to their electrophoretic mobility, finally the peak signal were measured by UV detector. Linear relationship between the peak area and the droplet number was obtained within the range of 25-400 drops (R2 = 0.996) at a fixed cell concentration 106/mL, indicating that this system could be used for rapid and accurate quantification of cells.

Convection-Diffusion Layer in an "Open Space" for Local Surface Treatment and Microfabrication using a Four-Aperture Microchemical Pen.

A four-aperture microchemical pen was used to produce a stable convection-diffusion layer in an "open space" for microreactions and microfabrication. The process represents a new method for microreactions and microfabrication in a convection-diffusion layer. To prove the concept of a convection-diffusion layer in an "open space", bovine serum albumin was labeled with 4-fluoro-7-nitro-2,1,3-benzoxadiazole to confirm that the small convection-diffusion layer was effective for local surface treatment. To demonstrate the potential for microfabrication, silver patterns were fabricated on a glass surface with a convection-diffusion layer by using the silver-mirror reaction. The widths of each silver pattern could be easily controlled from 10 to 60 µm. Patterned silver lines with uniform widths or gradient widths were prepared. This is the first proof of concept study of a convection-diffusion layer in an "open space" used in local surface treatment and microfabrication on a surface. The microchemical pen represents a potential method for the region-selective microtreatment of tissues, cells, and other biological interfaces.

A DNA-directed covalent conjugation fluorescence probe for in vitro detection of functional matrix metalloproteinases.

Matrix metalloproteinases (MMPs) have been considered to contribute to the progression of tumorigenesis and tumor invasion; MMP-9 in particular, has been regarded as a priority target in cancer treatment due to its massive up-regulation in malignant tissues and its ability to degrade type IV collagen. In this work, we employed a DNA-directed covalent conjugation method to design a fluorescence probe for in vitro detection of functional matrix metalloproteinases, by which a nitrilotriacetic acid (NTA)-modified DNA probe can combine with the Zn2+ in the active site of MMPs, and then a molecule beacon (MB) modified FITC and BHQ1 can open to bond with their complementary base, NTA-modified DNA. We can evaluate the amount of MMPs in the medium according to the fluorescence intensity. The detection procedure can be finished in 30 min with good selectivity, cheap reagents and easy preparation. All the results and the amount of secreted MMPs under three different cell culture conditions are in accordance with previous reports. Satisfactory results are obtained. Furthermore, owing to the importance of MMP-9, we designed an approach to achieve the desired selectivity and specificity of our work, using dual amplification for improving fluorescence intensity based on RCA to detect the amount of MMP-9.

Emerging N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD) and 6PPD quinone in paired human plasma and urine from Tianjin, China: Preliminary assessment with demographic factors.

With increasing concerns about N-(1,3-Dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD) and 6PPD-quinone (6PPD-Q), relevant environmental investigations and toxicological research have sprung up in recent years. However, limited information could be found for human body burden assessment. This work collected and analyzed 200 samples consisting of paired urine and plasma samples from participants (50 male and 50 female) in Tianjin, China. Low detection frequencies (DF, <15 %) were found except for urinary 6PPD-Q (86 %), which suggested the poor residue tendency of 6PPD and 6PPD-Q in blood. The low DFs also lead to no substantial association between two chemicals. Data analysis based on urinary 6PPD-Q showed a significant difference between males and females (p < 0.05). No significant correlation was found for other demographic factors (Body Mass Index (BMI), age, drinking, and smoking). The mean values of daily excretion (ng/kg bw/day) calculated using urinary 6PPD-Q for females and males were 7.381 ng/kg bw/day (female) and 3.360 ng/kg bw/day (male), and apparently female suffered higher daily exposure. Further analysis with daily excretion and ALT (alanine aminotransferase)/TSH (thyroid stimulating hormone)/ blood cell analysis indicators found a potential correlation with 6PPD-Q daily excretion and liver/immune functions. Considering this preliminary assessment, systematic research targeting the potential organs at relevant concentrations is required.

Metabolic markers and microecological characteristics of tongue coating in patients with chronic gastritis.

BACKGROUND: In Traditional Chinese Medicine (TCM), tongue diagnosis has been an important diagnostic method for the last 3000 years. Tongue diagnosis is a non-invasive, simple and valuable diagnostic tool. TCM treats the tongue coating on a very sensitive scale that reflects physiological and pathological changes in the organs, especially the spleen and stomach. Tongue coating can diagnose disease severity and determine the TCM syndrome ("Zheng" in Chinese). The biological bases of different tongue coating appearances are still poorly understood and lack systematic investigation at the molecular level. METHODS: Tongue coating samples were collected from 70 chronic gastritis patients and 20 normal controls. 16S rRNA denatured gradient gel electrophoresis (16S rRNA-DGGE) and liquid chromatography and mass spectrometry (LC-MS) were designed to profile tongue coatings. The statistical techniques used were principal component analysis and partial least squares-discriminate analysis. RESULTS: Ten potential metabolites or markers were found in chronic gastritis patients, including UDP-D-galactose, 3-ketolactose, and vitamin D2, based on LC-MS. Eight significantly different strips were observed in samples from chronic gastritis patients based on 16S rRNA-DGGE. Two strips, Strips 8 and 10, were selected for gene sequencing. Strip 10 sequencing showed a 100% similarity to Rothia mucilaginosa. Strip 8 sequencing showed a 96.2% similarity to Moraxella catarrhalis. CONCLUSIONS: Changes in glucose metabolism could possibly form the basis of tongue coating conformation in chronic gastritis patients. The study revealed important connections between metabolic components, microecological components and tongue coating in chronic gastritis patients. Compared with other diagnostic regimens, such as blood tests or tissue biopsies, tongue coating is more amenable to, and more convenient for, both patients and doctors.

MOFs-Modified Electrochemical Sensors and the Application in the Detection of Opioids.

Opioids are widely used in clinical practice, but drug overdoses can lead to many adverse reactions, and even endanger life. Therefore, it is essential to implement real-time measurement of drug concentrations to adjust the dosage given during treatment, keeping drug levels within therapeutic levels. Metal-Organic frameworks (MOFs) and their composite materials modified bare electrode electrochemical sensors have the advantages of fast production, low cost, high sensitivity, and low detection limit in the detection of opioids. In this review, MOFs and MOFs composites, electrochemical sensors modified with MOFs for the detection of opioids, as well as the application of microfluidic chips in combination with electrochemical methods are all reviewed, and the potential for the development of microfluidic chips electrochemical methods with MOFs surface modifications for the detection of opioids is also prospected. We hope that this review will provide contributions to the study of electrochemical sensors modified with MOFs for the detection of opioids.

Advances in droplet digital polymerase chain reaction on microfluidic chips.

The PCR technique has been known to the general public since the pandemic outbreak of COVID-19. This technique has progressed through three stages: from simple PCR to real-time fluorescence PCR to digital PCR. Among them, the microfluidic-based droplet digital PCR technique has attracted much attention and has been widely applied due to its advantages of high throughput, high sensitivity, low reagent consumption, low cross-contamination, and absolute quantification ability. In this review, we introduce various designs of microfluidic-based ddPCR developed within the last decade. The microfluidic-based droplet generation methods, thermal cycle strategies, and signal counting approaches are described, and the applications in the fields of single-cell analysis, disease diagnosis, and pathogen detection are introduced. Further, the challenges and prospects of microfluidic-based ddPCR are discussed. We hope that this review can contribute to the further development of the microfluidic-based ddPCR technique.

Identification and validation of a nomogram predicting cancer-specific survival for elderly patients with adult fibrosarcoma: a multicenter retrospective study.

Background: Due to the low incidence of adult fibrosarcoma (AFS), it is difficult for clinicians to assess cancer-specific survival (CSS) in elderly patients based on this study. The study aimed to develop nomograms capable of accurately predicting 3-, 5-, and 8-year CSS in patients over 40 years of age with AFS. Methods: Data were collected from The Surveillance, Epidemiology, and End Results (SEER) registry. 586 patients were included in this study. Univariate as well as multivariate Cox regression analyses were applied to identify independent risk factors. A nomogram was constructed and validated to predict the 3-, 5-, and 8-year CSS of patients. Results: Five variables including age, sex, stage, grade, and chemotherapy status were considered independent risk factors and were used to construct the nomogram. The nomogram was well validated. The C-indexes of the training cohort and the validation cohort are 0.766 and 0.780, respectively. In addition, the area under the curves for 3-, 5- and 8-year CSS are 0.824, 0.846 and 0.840 in the training cohort, 0.835, 0.806 and 0.829 in the validation cohort. Calibration curves were also plotted to show that predicted endings have a well fit for the true endings. Finally, decision curve analysis demonstrates that the nomogram can bring a high benefit to patients. Conclusion: We successfully constructed a highly accurate nomogram to predict the CSS of AFS patients at 3-, 5-, and 8 years. The nomogram can greatly help clinicians and patients with AFS.

LncRNA XLOC_015548 affects the proliferation and differentiation of myoblasts via the MAPK signaling pathway.

In recent years, an increasing number of studies have reported that long non-coding RNAs (lncRNAs) play essential regulatory roles in myogenic differentiation. In this study, a specific LncRNA XLOC_015548 (Lnc000280) was identified. However, little research has explored its mechanism of action by constructing XLOC_015548 gene editing cell models. In this study, relevant sequences were obtained according to the RNA-seq results. Subsequently, XLOC_015548 knockdown and over-expression lentiviral vectors were constructed, and the C2C12 myoblast cell line was transfected to prepare the XLOC_015548 gene-edited myoblast model. The in vitro analysis revealed that over-expression of XLOC_015548 significantly promoted the proliferation and differentiation of myoblasts and the formation of myotubes, whereas the opposite result was obtained in the knockdown group. XLOC_015548 regulated myogenic differentiation and affected the expression of myogenic differentiation regulators such as Myod, myogenin, and MyHC. Regarding the signaling pathway, we found that XLOC_015548 correlated with the phosphorylation level of MAPK/MEK/ERK pathway proteins. And the degree of phosphorylation was positively correlated with the protein expression of myogenic differentiation regulators. In conclusion, a new gene-edited myoblast model was constructed based on the lncRNA regulator XLOC_015548. The in vitro cell experiments verified that XLOC_015548 had regulatory effects on muscle growth and myoblast differentiation. These findings provide a laboratory foundation for the clinical application of lncRNAs as regulatory factors in the treatment of disuse muscle atrophy.

A Simple-to-Use Nomogram for Predicting Postoperative Early Death Risk in Elderly Patients with Spinal Tumors: A Population-Based Study.

Background: For elderly patients with primary spinal tumors, surgery is the best option for many elderly patients, in addition to palliative care. However, due to the unique physical function of elderly patients, the short-term prognosis is often unpredictable. It is therefore essential to develop a novel nomogram as a clinical aid to predict the risk of early death for elderly patients with primary spinal tumors who undergo surgery. Materials and Methods: In this study, clinical data were obtained from 651 patients through the SEER database, and they were retrospectively analyzed. Logistic regression analyses were used for risk-factor screening. Predictive modeling was performed through the R language. The prediction models were calibrated as well as evaluated for accuracy in the validation cohort. The receiver operating characteristic (ROC) curve and the decision curve analysis (DCA) were used to evaluate the functionality of the nomogram. Results: We identified four separate risk factors for constructing nomograms. The area under the receiver operating characteristic curve (training set 0.815, validation set 0.815) shows that the nomogram has good discrimination ability. The decision curve analysis demonstrates the clinical use of this nomogram. The calibration curve indicates that this nomogram has high accuracy. At the same time, we have also developed a web version of the online nomogram for clinical practitioners to apply. Conclusions: We have successfully developed a nomogram that can accurately predict the risk of early death of elderly patients with primary spinal tumors undergoing surgery, which can provide a reference for clinicians.

The Role of m6A in Osteoporosis and the Differentiation of Mesenchymal Stem Cells into Osteoblasts and Adipocytes.

Osteoporosis is a systemic disease in which bone mass decreases, leading to an increased risk of bone fragility and fracture. The occurrence of osteoporosis is believed to be related to the disruption of the differentiation of mesenchymal stem cells into osteoblasts and adipocytes. N6-adenylate methylation (m6A) modification is the most common type of chemical RNA modification and refers to a methylation modification formed by the nitrogen atom at position 6 of adenine (A), which is catalyzed by a methyltransferase. The main roles of m6A are the post-transcriptional level regulation of the stability, localization, transportation, splicing, and translation of RNA; these are key elements of various biological activities, including osteoporosis and the differentiation of mesenchymal stem cells into osteoblasts and adipocytes. The main focus of this review is the role of m6A in these two biological processes.