Protocol for isolating and identifying small extracellular vesicles derived from human umbilical cord mesenchymal stem cells.

Small extracellular vesicles (sEVs) are lipid bilayer-enclosed particles secreted by living cells. Here, we present a protocol for the collection and isolation of sEVs derived from human umbilical cord mesenchymal stem cells (hucMSCs). We describe steps for characterizing their morphology and integrity by transmission electron microscopy (TEM) and size distribution using nanoparticle tracking analysis (NTA) and an atomic force microscope (AFM). We then detail procedures for assessing nanoparticle size analysis and molecular markers by western blotting and Flow NanoAnalyzer.

CD8+ T cell-based cancer immunotherapy.

The immune system in humans is a defense department against both exogenous and endogenous hazards, where CD8+ T cells play a crucial role in opposing pathological threats. Various immunotherapies based on CD8+ T cells have emerged in recent decades, showing their promising results in treating intractable diseases. However, in the fight against the constantly changing and evolving cancers, the formation and function of CD8+ T cells can be challenged by tumors that might train a group of accomplices to resist the T cell killing. As cancer therapy stepped into the era of immunotherapy, understanding the physiological role of CD8+ T cells, studying the machinery of tumor immune escape, and thereby formulating different therapeutic strategies become the imperative missions for clinical and translational researchers to fulfill. After brief basics of CD8+ T cell-based biology is covered, this review delineates the mechanisms of tumor immune escape and discusses different cancer immunotherapy regimens with their own advantages and setbacks, embracing challenges and perspectives in near future.

Translational medicine for acute lung injury.

Acute lung injury (ALI) is a complex disease with numerous causes. This review begins with a discussion of disease development from direct or indirect pulmonary insults, as well as varied pathogenesis. The heterogeneous nature of ALI is then elaborated upon, including its epidemiology, clinical manifestations, potential biomarkers, and genetic contributions. Although no medication is currently approved for this devastating illness, supportive care and pharmacological intervention for ALI treatment are summarized, followed by an assessment of the pathophysiological gap between human ALI and animal models. Lastly, current research progress on advanced nanomedicines for ALI therapeutics in preclinical and clinical settings is reviewed, demonstrating new opportunities towards developing an effective treatment for ALI.

Visualization of microRNA therapy in cancers delivered by small extracellular vesicles.

MicroRNA (miRNA) delivery by extracellular vesicles (EVs) has recently inspired tremendous developments in cancer treatments. However, hybridization between miRNA and its target mRNA is still difficult to be imaged in vivo to assess the therapeutic effects in time. Herein we design a nano-scale fluorescent "off-on" complex encapsulated by small extracellular vesicles (sEVs) for real-time visualization and evaluation of gene therapy efficiency in human gastric cancer cells and murine xenograft tumor models. The complex is formed by π-π stacking between graphene quantum dots (GQDs) and tumor suppressor miR-193a-3p conjugated fluorescent tag whose signals remain off when binding to GQDs. Loaded into sEVs using tunable sonication techniques, the GQDs/Cy5-miR particles enter the tumor cells and promote miR-193a-3p escape from endosomes. The miR-193a-3p in GQDs/Cy5-miR is unleashed to pair the specific target oncogene cyclin D1 (CCND1), therefore turning on the fluorescence of miRNA tags. We find out that GQDs/Cy5-miR@sEVs can activate the "turn-on" fluorescent signal and exhibit the longest retention time in vivo, which suggests a minimized degradation of miR-193a-3p in dynamic processes of miRNA-mRNA binding. More importantly, GQDs/Cy5-miR@sEVs significantly promote cancer apoptosis in vitro and in vivo via the enhanced cellular uptake. Our study demonstrates that GQDs/Cy5-miR@sEVs represent an efficient and refined theranostic platform for gene therapy in cancers.

Fluorescence Tracking of Small Extracellular Vesicles In Vivo.

In this study, we employed organic and inorganic dyes that have fluorescence under visible or near-infrared light region to stain human umbilical cord (Huc) mesenchymal stem cell (MSC)-, HEK293T cell- and HGC cell-derived small extracellular vesicles (sEVs), and then tracked their fluorescence signals in human gastric cancer xenografted murine models. Several biological characteristics were examined and compared when different dye-stained sEVs in the same tumor model or the same dye-stained sEVs between different tumor models were applied, including sEVs circulation in the blood, biodistribution of sEVs in major organs, and time-dependent tumor accumulation of sEVs. The results demonstrated that distinct tumor accumulation features were presented by sEVs if labeled by different fluorescent dyes, while sEVs derived from different cell lines showed homologous blood circulation and tumor accumulation. To conclude, although fluorescence imaging remains a reliable way to trace sEVs, single staining of sEVs membrane should be obviated in future work when examining the biological fate of sEVs.

Construction of a novel nomogram based on competing endogenous RNAs and tumor-infiltrating immune cells for prognosis prediction in elderly patients with colorectal cancer.

Competitive endogenous RNAs (ceRNAs) and tumor-infiltrating immune cells play essential roles in colorectal cancer (CRC) tumorigenesis. However, their prognostic role in elderly patients with CRC is unclear. Gene expression profiles and clinical information for elderly patients with CRC were downloaded from The Cancer Genome Atlas. Univariate, LASSO, and multivariate Cox regression analyses were utilized for screening key ceRNAs and prevent overfitting. A total of 265 elderly patients with CRC were included. We constructed a novel ceRNA network consisting of 17 lncRNAs, 35 miRNAs, and 5 mRNAs. We established three prognosis predictive nomograms based on four key ceRNAs (ceRNA nomogram), five key immune cells (immune cell nomogram), and their combination (ceRNA-immune cell nomogram). Among them, the ceRNA-immune cell nomogram had the best accuracy. Furthermore, the areas under the curve of the ceRNA-immune cell nomogram were also significantly greater than the TNM stage at 1 (0.818 vs. 0.693), 3 (0.865 vs. 0.674), and 5 (0.832 vs. 0.627) years. Co-expression analysis revealed that CBX6 was positively correlated with activated dendritic cells (R = 0.45, p < 0.01), whereas negatively correlated with activated mast cells (R =- 0.43, p < 0.01). In conclusion, our study constructed three nomograms to predict prognosis in elderly patients with CRC, among which the ceRNA-immune cell nomogram had the best prediction accuracy. We inferred that the mechanism underlying the regulation of activated dendritic cells and mast cells by CBX6 might play a crucial role in tumor development and prognosis of elderly patients with CRC.

A handheld contactless conductivity detector for monitoring the desalting of low-volume virus and cell samples.

Desalting of biosamples is crucial for analytical techniques intolerant to abundant salts. However, there is no simple tool to monitor the desalting of low-volume biosamples so far. Here we developed a handheld capacitively coupled contactless conductivity detector (hC4D) as a miniaturized device to measure the conductivity of 75 µL biosamples. Polyether-ether-ketone (PEEK) tubing was selected as the sample reservoir for sample loading via a pipette. Another pipetting of air pushed the sample solution out of the tubing to recollect the sample. Owing to the low sample consumption and easy sample recollection, hC4D is advantageous for testing expensive biosamples, such as viruses and cells. In addition, the whole process of sample injection, conductivity measurement, recollection, and calibration of conductivity can be completed within 1 min. To verify the feasibility of hC4D, we monitored the desalting progress of gel filtration (GF) of 200 µL blood samples, ultrafiltration (UF) of 300 µL virus samples, and dialysis of 7 mL cell samples. Three rounds of GF and UF completely removed the salts but led to poor sample recovery. In contrast, low concentrations of residual salts remained and better recovery was achieved after two rounds of GF and UF. We further utilized the hC4D to monitor the dialysis and tuned the salt concentration in the cell sample, such that we maintained the viability of cells in a low conductivity environment. These results indicated that hC4D is a promising tool for optimizing the desalting procedure of low-volume biosamples.

Stalk-derived carbon dots as nanosensors for Fe3+ ions detection and biological cell imaging.

Introduction: Iron is one of the most important needed elements for the growth and reproduction of living organisms. The detection of iron levels is important and developing fluorescent probes with excellent sensitivity for Fe3+ ions is of great significance. Carbon dot (CDs) is a new type of fluorescent nanomaterial based on abundant and low-cost carbon elements. The use of widely distributed renewable agricultural waste straw as a carbon precursor to prepare CDs sensor can not only reduce the pollution caused by burning straw to the atmospheric environment, but also achieve the transformation of resources from waste to treasure. Methods: In this study, CDs were obtained from corn stalk powder by pyrolysis and microwave process. The sensitivity and linear response range of CDs sensor was studied through analyzing the effect of different Fe3+ ions concentrations on the fluorescence quenching. The application of CDs in biological cell imaging was investigated using HGC-27 cells. Results: The fluorescence quenching showed a good linear relationship with the Fe3+ concentration in the range from 0 to 128 µM, and a low detection limit of 63 nM. In addition, the CDs have high recognition for Fe3+ ions. Meanwhile, the CDs have a low cytotoxicity and desirable biocompatibility, allowing the multicolor living cell imaging. Conclusion: The prepared CDs can be used as fluorescent sensors for the selective detection of Fe3+ ions and biological cell imaging. Our results supported that the conversion of agricultural waste into carbon nanomaterials has great potential to be developed.

The early warning research on nursing care of stroke patients with intelligent wearable devices under COVID-19.

Stroke patients under the background of the new crown epidemic need to be home-based care. However, traditional nursing methods cannot take care of the patients' lives in all aspects. Based on this, based on machine learning algorithms, our work combines regression models and SVM to build a smart wearable device system and builds a system prediction module to predict patient care needs. The node is used to collect human body motion and physiological parameter information and transmit data wirelessly. The software is used to quickly process and analyze the various motion and physiological parameters of the patient and save the analysis and processing structure in the database. By comparing the results of nursing intervention experiments, we can see that the smart wearable device designed in this paper has a certain effect in stroke care.

Correction to: The early warning research on nursing care of stroke patients with intelligent wearable devices under COVID-19.

[This corrects the article DOI: 10.1007/s00779-021-01520-9.].

Risk factors for hospitalization and pneumonia development of pediatric patients with seasonal influenza during February-April 2023.

Objectives: In China influenza remains a low activity for continuous 3 years due to COVID-19 controls. We here sought to study the clinical characteristics and risk factors of the influenza infection among children after the mandatory COVID-19 restrictions were lifted. Methods: We included 1,006 pediatric patients with influenza A virus (IAV) infection, enrolled in one tertiary hospital in Zhenjiang, Jiangsu Province, China, during February to April 2023. Patients were divided into the outpatient (n = 798) and inpatient (n = 208) groups, and their baseline characteristics were compared between two groups to conclude the risk factors for pediatric hospitalization. Separately, pediatric inpatients (n = 208) were further divided into the pneumonia and non-pneumonia groups with comparison of their clinical characteristics, including their laboratory test results and representative radiological features, to derive the key determinants for pneumonia development after hospitalization. Results: Compared to outpatients, IAV-infected pediatric inpatients exhibited younger age, higher female: male ratio, more co-infection of influenza B virus (IBV) and hematological abnormality. Multivariate regression analysis determined the independent risk factors of hospitalization to be the clinical symptom of abdominal pain (OR = 2.63, [95% CI, 1.05-6.57], p = 0.039), co-infection of IBV (OR = 44.33, [95% CI, 25.10-78.30], p = 0.001), elevated levels of lymphocytes (OR = 2.24, [95% CI,1.65-3.05], p = 0.001) and c-reactive proteins (CRPs) (OR = 1.06, [95% CI, 1.03-1.08], p = 0.001) upon hospital admission. Furthermore, the cough symptom (OR = 17.39, [95% CI, 3.51-86.13], p = 0.001) and hospitalization length (OR = 1.36, [95% CI, 1.12-1.67], p = 0.002) were determined to be risk factors of pneumonia acquirement for pediatric inpatients. Conclusion: While the abdominal pain, viral co-infection and some hematological abnormality mainly contribute to hospitalization of pediatric patients with IAV infection, the length of hospital stay and clinical sign of coughing upon hospital admission constitute the key determinants for nosocomial pneumonia development.

Hepatic dysfunctions in COVID-19 patients infected by the omicron variant of SARS-CoV-2.

Background: Presently, the omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) dominates amid the coronavirus disease 2019 (COVID-19) pandemic, but its clinical characteristics with intrinsic severity and organ tropism remain understudied. Methods: We reported 1,001 mild COVID-19 patients that were infected with the omicron variant of SARS-CoV-2 and hospitalized in China from February to June 2022, including their demographic information, medical/immunization history, clinical symptom, and hematological profile. Patients with one-, two- and three-dose vaccination were compared to assess the vaccine effectiveness. Importantly, liver damage caused by the omicron variant infection was evaluated, in comparison to that caused by the wild-type or the delta variant SARS-CoV-2 infection. Results: For the reported COVID-19 patients infected by the omicron variant of SARS-CoV-2, their median age was 36.0 [interquartile range (IQR): 26.0-50.0] and 49.7% were female. Hypertension, diabetes, and bronchitis were the leading comorbidities, and asymptomatic patients took up a major portion (61.2%). While most hematological parameters revealed the alleviated pathogenicity, full vaccination or booster shot showed effective protection against clinical severity. Furthermore, liver damages caused by viral infection of the omicron variant were largely attenuated when compared to those by infection of the wild-type or the delta variant SARS-CoV-2. Conclusions: Our results supported that the viremic effect of the omicron variant tended to be modest, while the liver damage caused by this strain became milder than the previous circulating variants.

Correlation of clinical characteristics between patients with seasonal influenza and patients infected by the wild type or delta variant of SARS-CoV-2.

Background: We compared the clinical characteristics of the patients with COVID-19, infected by the wild type or delta variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in connection with those of patients with seasonal influenza, all in mild cases. Methods: We retrospectively studied 245 and 115 patients with mild COVID-19 infected by the wild type and the delta variant of SARS-CoV-2, respectively, with their demographic information, medical history, and laboratory data from hospital records, individually compared to 377 patients with mild seasonal influenza, before and after individual treatment. Results: Compared to the influenza cohort, the COVID-19 cohort or the COVID-19 delta variant cohort demonstrated younger median age, lower male ratio, and shorter duration from disease onset to hospitalization. Hypertension remained the top comorbidity among all cohorts. Based on patients' data upon hospitalization, the correlation of clinical characteristics between patients with influenza and those with the wild-type COVID-19 is greater than that between patients with influenza and those with the delta variant COVID-19. Individual treatment in each viral disease alleviated most hematological parameters, but some compromised biomarkers at the time of hospital discharge revealed persistent renal or myocardial impairment among patients with COVID-19 and influenza in recovery. Conclusion: Timely and proper treatment using broad-spectrum antibiotics and antiviral drugs could moderately alleviate the acute viremia and possible bacterial co-infection in patients with mild COVID-19 and influenza, followed by compromised recovery. To prepare for the flu season amid the COVID-19 pandemic, preventive and adequate immunizations of both flu and COVID-19 vaccines, as well as specific therapeutics to effectively reverse viral impairments, are in urgent need.

Polymalic acid for translational nanomedicine.

With rich carboxyl groups in the side chain, biodegradable polymalic acid (PMLA) is an ideal delivery platform for multifunctional purposes, including imaging diagnosis and targeting therapy. This polymeric material can be obtained via chemical synthesis, or biological production where L-malic acids are polymerized in the presence of PMLA synthetase inside a variety of microorganisms. Fermentative methods have been employed to produce PMLAs from biological sources, and analytical assessments have been established to characterize this natural biopolymer. Further functionalized, PMLA serves as a versatile carrier of pharmaceutically active molecules at nano scale. In this review, we first delineate biosynthesis of PMLA in different microorganisms and compare with its chemical synthesis. We then introduce the biodegradation mechanism PMLA, its upscaled bioproduction together with characterization. After discussing advantages and disadvantages of PMLA as a suitable delivery carrier, and strategies used to functionalize PMLA for disease diagnosis and therapy, we finally summarize the current challenges in the biomedical applications of PMLA and envisage the future role of PMLA in clinical nanomedicine.

Effect of Comorbid Diabetes on Clinical Characteristics of COVID-19 Patients Infected by the Wild-Type or Delta Variant of SARS-CoV-2.

Background: Diabetes is one of the most common comorbidities in COVID-19 patients that pertains to disease severity, but the causal mechanism regarding its negative impact on COVID-19 outcome has yet been uncovered. Methods: We retrospectively analyzed 459 COVID-19 patients admitted in early 2020 and 336 COVID-19 patients admitted in August 2021, with their demographic information, medical history, vaccination status (if applied), and laboratory data reported. Results: Among COVID-19 patients, compared to the non-diabetic group, the diabetic group exhibited elder age, higher proportion of patients with other major comorbidities, more severe dysfunction of innate immune cells, more refractory blood coagulopathy and more detrimental organ damage. For the wild-type SARS-CoV-2 infection, diabetic comorbidity was associated with COVID-19 severity but not mortality, and the glycemic levels in the non-diabetic group upon infection experienced high and analogous to those in the diabetic group. Besides, infected by the delta variant of SARS-CoV-2, the non-diabetic patients did not demonstrate hyperglycemia, and despite different vaccination statuses, the diabetic patients exhibited comparable antibody responses to non-diabetic, showing the robustness of acquired immunity. Conclusions: SARS-CoV-2 infection may superimpose the deterioration of innate immune systems in diabetic patients, which contributes to their worsened disease outcome, but timely COVID-19 immunization could provide adequate protection in diabetic population that leads to favored prognosis.

Clinical Characteristics of COVID-19 Patients Infected by the Omicron Variant of SARS-CoV-2.

Background: Currently, as the omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) surges amid the coronavirus disease 2019 (COVID-19) pandemic, its clinical characteristics with intrinsic severity and the protection from vaccination have been understudied. Methods: We reported 169 COVID-19 patients that were infected with the omicron variant of SARS-CoV-2 and hospitalized in Suzhou, China, from February to March 2022, with their demographic information, medical/immunization history, clinical symptom, and hematological profile. At the same time, patients with none/partial (one-dose), full (two-dose) and three-dose vaccination were also compared to assess the vaccine effectiveness. Findings: For the omicron COVID-19 patients included in this study, their median age was 33.0 [interquartile range (IQR): 24.0-45.5], 53.3% were male and the median duration from illness onset to hospitalization was 2 days. Hypertension, bronchitis, and diabetes were the leading comorbidities among patients. While the common clinical symptoms included cough, fever, expectoration, and fatigue, etc., asymptomatic patients took up a significant portion (46.7%). For hematological parameters, most values revealed the alleviated pathogenicity induced by the omicron variant infection. No critically ill or deceased patients due to COVID-19 infection were reported in this study. Interpretation: Our results supported that the viremic effect of the omicron variant became milder than the previous circulating variants, while full vaccination or booster shot was greatly desired for an effective protection against clinical severity.

Association Between Red Blood Cell Distribution Width and COVID-19 Severity in Delta Variant SARS-CoV-2 Infection.

Studies have discovered that wild-type SARS-CoV-2 infections are commonly linked to abnormalities in the hematological profiles of COVID-19 patients, one such abnormality being characterized by elevations in red blood cell distribution width (RDW). Whether this linkage reoccurs in delta variant SARS-CoV-2 infection remains unexamined. Here we compared baseline blood parameters in COVID-19 patients infected by wild type and its delta variant, respectively. Our results here point to that although the delta variant has shown increased virulence, transmissibility, and vaccine escape, it has a minimally negative impact on RDW values that were previously found prognostic for COVID-19 severity.

Comparative Study of Acute Lung Injury in COVID-19 and Non-COVID-19 Patients.

Background: Amid the coronavirus disease 2019 (COVID-19) pandemic, we analyzed clinical characteristics of acute lung injury (ALI) in COVID-19 patients and reported their similarity and dissimilarity to those of non-COVID-19 patients in the intensive care unit (ICU). Methods: We reported on 90 COVID-19 and 130 non-COVID-19 ALI patients in the ICUs of multiple centers. Demographic data, medical histories, laboratory findings, and radiological images were analyzed and compared between the two cohorts and within each cohort between survivors and non-survivors. For ALI survivors, clinical characteristics before and after treatment were also compared. Findings: Aberrations in blood parameters, such as leukocytosis, neutrophilia, and thrombocytopenia, were observed in both cohorts. More characteristic abnormalities, including significantly higher red cell distribution width (RDW), C-reactive proteins, and lactic dehydrogenase (LDH) but lower troponin (TnT) and procalcitonin, were observed in the COVID-19 cohort than in the non-COVID-19 cohort, whereas D-dimer levels showed a similar elevation in both cohorts. The COVID-19 cohort also showed more diversified CT patterns where severe features such as consolidations and crazy paving patterns were more frequently observed. Multivariate analysis indicated that age, fever symptom, prothrombin time, procalcitonin, partial pressure of carbon dioxide, oxygenated hemoglobin, and crazy paving patterns in CT scans were independent risk factors associated with COVID-19. Interpretation: Comparison of ALI characteristics between COVID-19 and non-COVID-19 patients in the ICU setting provided insight into the pathogenesis of ALI induced by different risk factors, suggesting distinct treatment plans.

Extracellular vesicles as delivery systems at nano-/micro-scale.

Extracellular vesicles (EVs) have shown significant promises as nano-/micro-size carriers in drug delivery and bioimaging. With more characteristics of EVs explored through tremendous research efforts, their unmatched physicochemical properties, biological features, and mechanical aspects make them unique vehicles, owning exceptional pharmacokinetics, circulatory metabolism and biodistribution pattern when delivering theranostic cargoes. In this review we firstly analyzed pros and cons of the EVs as a delivery platform. Secondly, compared to engineered nanoparticle delivery systems, such as biocompatible di-block co-polymers, rational design to improve EVs (exosomes in particular) were elaborated. Lastly, different pharmaceutical loading approaches into EVs were compared, reaching a conclusion on how to construct a clinically available and effective nano-/micro-carrier for a satisfactory medical mission.

Differently PEGylated Polymer Nanoparticles for Pancreatic Cancer Delivery: Using a Novel Near-Infrared Emissive and Biodegradable Polymer as the Fluorescence Tracer.

In this study, a chemically synthetic polymer, benzo[1,2-b:4,5-b']difuran(BDF)-based donor-acceptor copolymer PBDFDTBO, was individually coated by amphiphilic poly(ethylene oxide)-block-poly(ε-caprolactone) (PEO-PCL) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy(polyethylene glycol) (DSPE-PEG or PEG-DSPE), to form stably fluorescent nanoparticles in the near-infrared (NIR) window. The physicochemical properties of the synthesized nanoparticles were characterized and compared, including their size, surface charge, and morphology. In addition, in vitro studies were also performed using two pancreatic cancer cell lines, assessing the cell viability of the PBDFDTBO-included PEGylated nanoparticles formulations. Moreover, in vivo studies were also conducted, using subcutaneous murine cancer models to investigate the polymeric nanoparticles' circulation time, tumor accumulation, and preferred organ biodistribution. The overall results demonstrated that even with the same PEGylated surface, the hydrophobic composition anchored on the encapsulated PBDFDTBO core strongly affected the biodistribution and tumor accumulation of the nanoparticles, to a degree possibly determined by the hydrophobic interactions between the hydrophobic segment of amphiphilic polymers (DSPE or PCL moiety) and the enwrapped PBDFDTBO. Both PEGylated nanoparticles were compared to obtain an optimized coating strategy for a desired biological feature in pancreatic cancer delivery.