A new strategy for the treatment of Parkinson's disease: Discovery and bio-evaluation of the first central-targeting tyrosinase inhibitor.

The high level of tyrosinase leads to the generation of neuromelanin, further causing the abnormality of redox-related protein level and mediating the occurrence and development of Parkinson's disease (PD). However, the existing tyrosinase inhibitors are mostly natural product extracts or polyphenolic derivatives, which hindered them from penetrating the blood-brain barrier (BBB). Herein, we obtained a novel tyrosinase inhibitor, 2-06 (tyrosinase: monophenolase IC50 = 70.44 ± 22.69 µM, diphenolase IC50 = 1.89 ± 0.64 µM), through the structure-based screening method. The compound 2-06 presented good in vitro and in vivo safety, and can inhibit the tyrosinase and melanogenesis in B16F10. Moreover, this compound showed neuroprotective effects and Parkinsonism behavior improving function. 2-06 was proved to penetrate the BBB and enter the central nervous system (CNS). The exploration of the binding mode between 2-06 and tyrosinase provided the foundation for the subsequent structural optimization. This is the first research to develop a central-targeting tyrosinase inhibitor, which is crucial for in-depth study on the new strategy for utilizing tyrosinase inhibitors to treat PD.

Consensus statement on extracellular vesicles in liquid biopsy for advancing laboratory medicine.

Extracellular vesicles (EVs) represent a diverse class of nanoscale membrane vesicles actively released by cells. These EVs can be further subdivided into categories like exosomes and microvesicles, based on their origins, sizes, and physical attributes. Significantly, disease-derived EVs have been detected in virtually all types of body fluids, providing a comprehensive molecular profile of their cellular origins. As a result, EVs are emerging as a valuable addition to liquid biopsy techniques. In this collective statement, the authors share their current perspectives on EV-related research and product development, with a shared commitment to translating this newfound knowledge into clinical applications for cancer and other diseases, particularly as disease biomarkers. The consensus within this document revolves around the overarching recognition of the merits, unresolved questions, and existing challenges surrounding EVs. This consensus manuscript is a collaborative effort led by the Committee of Exosomes, Society of Tumor Markers, Chinese anti-Cancer Association, aimed at expediting the cultivation of robust scientific and clinically applicable breakthroughs and propelling the field forward with greater swiftness and efficacy.

Phospholipid-Anchored Ligand Conjugation on Extracellular Vesicles for Enhanced Cancer Targeting.

Extracellular vesicles (EVs) are recognized as potential candidates for next-generation drug delivery systems. However, the inherent cancer-targeting efficiency is unsatisfactory, necessitating surface modification to attach cell-binding ligands. By utilizing phospholipase D from Streptomyces in combination with maleimide-containing primary alcohol, the authors successfully anchored ligands onto milk-derived EVs (mEVs), overcoming the issues of ligand leakage or functional alteration seen in traditional methods. Quantitative nano-flow cytometry demonstrated that over 90% of mEVs are effectively modified with hundreds to thousands of ligands. The resulting mEV formulations exhibited remarkable long-term stability in conjugation proportion, ligand number, size distribution, and particle concentration, even after months of storage. It is further shown that conjugating transferrin onto mEVs significantly enhanced cellular uptake and induced pronounced cytotoxic effects when loaded with paclitaxel. Overall, this study presents a highly efficient, stable, cost-effective, and scalable ligand conjugation approach, offering a promising strategy for targeted drug delivery of EVs.

Nano-flow cytometry unveils mitochondrial permeability transition process and multi-pathway cell death induction for cancer therapy.

Mitochondrial permeability transition (mPT)-mediated mitochondrial dysfunction plays a pivotal role in various human diseases. However, the intricate details of its mechanisms and the sequence of events remain elusive, primarily due to the interference caused by Bax/Bak-induced mitochondrial outer membrane permeabilization (MOMP). To address these, we have developed a methodology that utilizes nano-flow cytometry (nFCM) to quantitatively analyze the opening of mitochondrial permeability transition pore (mPTP), dissipation of mitochondrial membrane potential ( Δ Ψm), release of cytochrome c (Cyt c), and other molecular alternations of isolated mitochondria in response to mPT induction at the single-mitochondrion level. It was identified that betulinic acid (BetA) and antimycin A can directly induce mitochondrial dysfunction through mPT-mediated mechanisms, while cisplatin and staurosporine cannot. In addition, the nFCM analysis also revealed that BetA primarily induces mPTP opening through a reduction in Bcl-2 and Bcl-xL protein levels, along with an elevation in ROS content. Employing dose and time-dependent strategies of BetA, for the first time, we experimentally verified the sequential occurrence of mPTP opening and Δ Ψm depolarization prior to the release of Cyt c during mPT-mediated mitochondrial dysfunction. Notably, our study uncovers a simultaneous release of cell-death-associated factors, including Cyt c, AIF, PNPT1, and mtDNA during mPT, implying the initiation of multiple cell death pathways. Intriguingly, BetA induces caspase-independent cell death, even in the absence of Bax/Bak, thereby overcoming drug resistance. The presented findings offer new insights into mPT-mediated mitochondrial dysfunction using nFCM, emphasizing the potential for targeting such dysfunction in innovative cancer therapies and interventions.

Novel Colorimetric and NIR Fluorescent Probe for Bisulfite/Sulfite Detection in Food and Water Samples and Living Cells Based on the PET Mechanism.

Despite their status of being widely used as food additives, bisulfite (HSO3-)/sulfite (SO32-) can pose serious health risks when they are excessively added. Therefore, it is vital to develop a new method for detecting HSO3-/SO32- in foodstuff. In this paper, a benzopyran-benzothiazole derivative (probe DCA-Btl) with near-infrared emission was designed and synthesized by constructing a "push-pull" electronic system. DCA-Btl can selectively recognize HSO3-/SO32- via a colorimetric and fluorescence dual channel in DMF/PBS (1:1, v/v, pH = 8.4), and the emission wavelength of DCA-Btl can reach 710 nm. The fluorescence quenching of DCA-Btl after recognition of HSO3- is attributed to the photoinduced electron transfer (PET) process of the adduct DCA-Btl-HSO3- as evaluated by the DFT/TD-DFT method. In addition, DCA-Btl has many advantages, including a large Stokes shift (95 nm), good anti-interference ability, and little cytotoxicity. What's more, DCA-Btl has been successfully applied for the detection of HSO3-/SO32- in actual water samples and food samples such as sugar, red wine, and biscuits with satisfying results, as well as for fluorescent imaging of HSO3- in living MCF-7 cells.

Mesenchymal stem cell-derived extracellular vesicles for immunomodulation and regeneration: a next generation therapeutic tool?

Mesenchymal stem cells (MSCs) can be widely isolated from various tissues including bone marrow, umbilical cord, and adipose tissue, with the potential for self-renewal and multipotent differentiation. There is compelling evidence that the therapeutic effect of MSCs mainly depends on their paracrine action. Extracellular vesicles (EVs) are fundamental paracrine effectors of MSCs and play a crucial role in intercellular communication, existing in various body fluids and cell supernatants. Since MSC-derived EVs retain the function of protocells and have lower immunogenicity, they have a wide range of prospective therapeutic applications with advantages over cell therapy. We describe some characteristics of MSC-EVs, and discuss their role in immune regulation and regeneration, with emphasis on the molecular mechanism and application of MSC-EVs in the treatment of fibrosis and support tissue repair. We also highlight current challenges in the clinical application of MSC-EVs and potential ways to overcome the problem of quality heterogeneity.

Excessive IL-10 and IL-18 trigger hemophagocytic lymphohistiocytosis-like hyperinflammation and enhanced myelopoiesis.

BACKGROUND: Hyperinflammation is a life-threatening condition associated with various clinical disorders characterized by excessive immune activation and tissue damage. Multiple cytokines promote the development of hyperinflammation; however, the contribution of IL-10 remains unclear despite emerging speculations for a pathological role. Clinical observations from hemophagocytic lymphohistiocytosis (HLH), a prototypical hyperinflammatory disease, suggest that IL-18 and IL-10 may collectively promote the onset of a hyperinflammatory state. OBJECTIVE: We aimed to investigate the collaborative roles of IL-10 and IL-18 in hyperinflammation. METHODS: A comprehensive plasma cytokine profile for 87 secondary HLH patients was first depicted and analyzed. We then investigated the systemic and cellular effects of coelevated IL-10 and IL-18 in a transgenic mouse model and cultured macrophages. Single-cell RNA sequencing was performed on the monocytes/macrophages isolated from secondary HLH patients to explore the clinical relevance of IL-10/IL-18-mediated cellular signatures. The therapeutic efficacy of IL-10 blockade was tested in HLH mouse models. RESULTS: Excessive circulating IL-10 and IL-18 triggered a lethal hyperinflammatory disease recapitulating HLH-like phenotypes in mice, driving peripheral lymphopenia and a striking shift toward enhanced myelopoiesis in the bone marrow. IL-10 and IL-18 polarized cultured macrophages to a distinct proinflammatory state with pronounced expression of myeloid cell-recruiting chemokines. Transcriptional characterization suggested the IL-10/IL-18-mediated cellular features were clinically relevant with HLH, showing enhanced granzyme expression and proteasome activation in macrophages. IL-10 blockade protected against the lethal disease in HLH mouse models. CONCLUSION: Coelevated IL-10 and IL-18 are sufficient to drive HLH-like hyperinflammatory syndrome, and blocking IL-10 is protective in HLH models.

Noninvasive Diagnosis of Nasopharyngeal Carcinoma Based on Phenotypic Profiling of Viral and Tumor Markers on Plasma Extracellular Vesicles.

Nasopharyngeal carcinoma (NPC) is a malignant tumor commonly associated with Epstein-Barr virus (EBV) infection, and its early diagnosis as well as its differentiation from nasopharyngitis (NPG) remains challenging due to the insufficient sensitivity of routine screening methods in clinical practice. To date, circulating extracellular vesicles (EVs, 40-1000 nm) have shown appealing potential in liquid biopsy for cancer diagnosis and prognosis. Herein, nanoflow cytometry (nFCM) capable of single EV analysis was applied to examine the expression of surface proteins with very low copy numbers on individual EVs as small as 40 nm. The particle concentrations of five EV subsets exposing EBV-encoded latent membrane proteins (LMP1 and LMP2A) and tumor markers (PD-L1, EGFR, and EpCAM) in plasma were determined rapidly via single-particle enumeration. We identified a five-marker panel named EVSUM5 (an unweighted sum of the concentration of the five individual EV subsets) that significantly surpassed the traditional VCA-IgA assay in discriminating NPC patients from both healthy donors and NPG patients with accuracies of 96.3 and 83.1%, respectively. Moreover, EVSUM2 (an unweighted sum of virus-specific LMP1- and LMP2A-positive EVs) could achieve the diagnosis of NPG with an accuracy of 82.6%. Collectively, the work presented a rapid, reliable, and noninvasive method as well as two diagnostic markers to help more accurately differentiate NPC from NPG patients and healthy donors in clinical practice.

Single-particle analysis of tear fluid reveals abundant presence of tissue factor-exposing extracellular vesicles with strong coagulation activity.

Extracellular vesicles (EVs) in easily accessible body fluids have emerged as a promising source for liquid biopsy. Although tear collection is fast, safe, and noninvasive, EVs of tear fluid are less studied and their involvement in physiological and pathological processes is largely unknown. The aim of present study was to analyze and characterize EVs in tear fluid at the single-particle level to reveal the population heterogeneity. A laboratory-built nano-flow cytometer (nFCM) was used to analyze the purity, size distribution, and particle concentration of EVs isolated from unstimulated tears (basal tears) upon double ultracentrifugation (17 min at 100,000×g, 4 °C) via side scattering detection. The expression of CD9, CD63, CD81, CD47, CD45, CD24, and EpCAM was assessed via immunofluorescent detection. The EV concentration in tear fluid was measured to be 1.1 ± 0.7 × 1011 particles/mL, which is approximately 100-fold higher than that of plasma EVs. In particular, it was identified for the first time that tears have strong coagulant activity owing to the abundant presence of tissue factor (TF) on tear EVs. The concentration of TF-exposing EVs (4.4 ± 3.1 × 1010 particles/mL) was found to be approximately 100-fold higher than their counterparts in saliva (4.5 ± 2.1 × 108 particles/mL). We postulate that TF-exposing vesicles in tears might play a role in host defense by promoting clot formation and thus reducing the risk of pathogen invasion. The coagulant activity of tears triggered by TF-exposing EVs could provide a new research perspective for ophthalmic research.

Active cargo loading into extracellular vesicles: Highlights the heterogeneous encapsulation behaviour.

Extracellular vesicles (EVs) have demonstrated unique advantages in serving as nanocarriers for drug delivery, yet the cargo encapsulation efficiency is far from expectation, especially for hydrophilic chemotherapeutic drugs. Besides, the intrinsic heterogeneity of EVs renders it difficult to evaluate drug encapsulation behaviour. Inspired by the active drug loading strategy of liposomal nanomedicines, here we report the development of a method, named "Sonication and Extrusion-assisted Active Loading" (SEAL), for effective and stable drug encapsulation of EVs. Using doxorubicin-loaded milk-derived EVs (Dox-mEVs) as the model system, sonication was applied to temporarily permeabilize the membrane, facilitating the influx of ammonium sulfate solution into the lumen to establish the transmembrane ion gradient essential for active loading. Along with extrusion to downsize large mEVs, homogenize particle size and reshape the nonspherical or multilamellar vesicles, SEAL showed around 10-fold enhancement of drug encapsulation efficiency compared with passive loading. Single-particle analysis by nano-flow cytometry was further employed to reveal the heterogeneous encapsulation behaviour of Dox-mEVs which would otherwise be overlooked by bulk-based approaches. Correlation analysis between doxorubicin auto-fluorescence and the fluorescence of a lipophilic dye DiD suggested that only the lipid-enclosed particles were actively loadable. Meanwhile, immunofluorescence analysis revealed that more than 85% of the casein positive particles was doxorubicin free. These findings further inspired the development of the lipid-probe- and immuno-mediated magnetic isolation techniques to selectively remove the contaminants of non-lipid enclosed particles and casein assemblies, respectively. Finally, the intracellular assessments confirmed the superior performance of SEAL-prepared mEV formulations, and demonstrated the impact of encapsulation heterogeneity on therapeutic outcome. The as-developed cargo-loading approach and nano-flow cytometry-based characterization method will provide an instructive insight in the development of EV-based delivery systems.

High-Throughput Human Telomere Length Analysis at the Single-Chromosome Level by FISH Coupled with Nano-Flow Cytometry.

Telomere length (TL) is a highly relevant biomarker for age-associated diseases and cancer, yet its clinical applications have been hindered by the inability of existing methods to rapidly measure the TL distribution and the percentage of chromosomes with critically short telomeres (CSTs, < 3 kb). Herein, we report the development of a high-throughput method to measure TL at the single-chromosome level. Metaphase chromosomes are isolated, hybridized with the Alexa Fluor 488-labeled telomeric peptide nucleic acid probe, and analyzed using a laboratory-built ultrasensitive nano-flow cytometer. The fluorescence intensity of individual chromosomes is converted to TL in kilobases upon external calibration. With an analysis rate of several thousand chromosomes per minute, a statistically robust TL distribution histogram is acquired in minutes, and the percentage of chromosomes with CSTs can be quickly assessed. By analyzing peripheral blood lymphocytes of 158 healthy donors, TL is found to shorten with age at a rate of 64 ± 3 bp/year and the percentage of chromosomes with CSTs increases with age at a rate of 0.32 ± 0.02%/year. Moreover, the data of 28 patients with chronic myeloid leukemia (CML) indicate that telomeres are significantly shorter at the time of diagnosis and the clinical phases of CML are closely associated with TL and the percentage of chromosomes with CSTs. This powerful tool could greatly deepen our understanding of telomere biology and improve the clinical utility of telomere biomarkers.

Tumor Microenvironment-Derived R-spondins Enhance Antitumor Immunity to Suppress Tumor Growth and Sensitize for Immune Checkpoint Blockade Therapy.

Natural killer (NK) cells and T cells are key effectors of antitumor immune responses and major targets of checkpoint inhibitors. In multiple cancer types, we find that the expression of Wnt signaling potentiator R-spondin genes (e.g., RSPO3) is associated with favorable prognosis and positively correlates with gene signatures of both NK cells and T cells. Although endothelial cells and cancer-associated fibroblasts comprise the R-spondin 3-producing cells, NK cells and T cells correspondingly express the R-spondin 3 receptor LGR6 within the tumor microenvironment (TME). Exogenous expression or intratumor injection of R-spondin 3 in tumors enhanced the infiltration and function of cytotoxic effector cells, which led to tumor regression. NK cells and CD8+ T cells independently and cooperatively contributed to R-spondin 3-induced control of distinct tumor types. The effect of R-spondin 3 was mediated in part through upregulation of MYC and ribosomal biogenesis. Importantly, R-spondin 3 expression enhanced tumor sensitivity to anti-PD-1 therapy, thereby highlighting new therapeutic avenues. SIGNIFICANCE: Our study identifies novel targets in enhancing antitumor immunity and sensitizing immune checkpoint inhibition, which provides a rationale for developing new immunotherapies against cancers. It also offers mechanistic insights on Wnt signaling-mediated modulation of anticancer immunity in the TME and implications for a putative R-spondin-LGR6 axis in regulating NK-cell biology. This article is highlighted in the In This Issue feature, p. 2945.

Geographic distribution of the cagA, vacA, iceA, oipA and dupA genes of Helicobacter pylori strains isolated in China.

BACKGROUND: There are geographic variations in the genotypes of Helicobacter pylori (H. pylori) cagA, vacA, iceA, oipA and dupA. The aim of the study was to investigate the distribution of these genotypes among H. pylori strains from five regions of China and their association with clinical outcomes. MATERIALS AND METHODS: Gastric biopsy specimens were obtained from 348 patients with different gastrointestinal diseases in the five regions of China. The regional distribution was 89 patients from Shandong, 91 from Guangxi, 57 from Hunan, 58 from Qinghai and 53 from Heilongjiang. The presence of cagA, vacA, iceA, oipA and dupA genotypes was determined by polymerase chain reaction (PCR) from H. pylori DNA. RESULTS: A total of 269 H. pylori isolates were obtained, of which 74 isolates were from Shandong, 78 from Guangxi, 46 from Hunan, 33 from Qinghai and 38 from Heilongjiang. The cagA-positive status was predominant in the five regions. The predominant vacA genotypes were s1c (73.4%), m2 (70.6%) and i1 (92.9%). In strains from Shandong, s1a and m1 were dominant. By contrast, s1c was dominant in Guangxi and i1 was dominant in Hunan and Heilongjiang. The prevalence of m2 subtype in Qinghai (78.8%) was significantly higher than that in other regions (P < 0.05). The predominant iceA genotype was iceA1 and the frequency of iceA1 was significantly more prevalent in Hunan than in other regions (P < 0.05). The oipA status "on" gene was more frequent in Shandong (91.9%) and Guangxi (91%) than in Heilongjiang (71.7%) (P < 0.05). Conversely, the dupA-positive status was less than half in Shandong (31.1%) and Guangxi (15.4%), whereas it was 73.9% in Hunan and 81.8% in Qinghai (P < 0.001). There were no significant associations between the cagA, vacA, iceA, oipA genotypes and clinical outcomes. The dupA-positive strains were more common in peptic ulcer disease (PUD) patients than in non-ulcer dyspepsia (NUD) patients in Shandong and Guangxi (P < 0.05), but the association was not observed in other geographic regions. CONCLUSIONS: There was significant geographic diversity of H. pylori genotypes in different regions of China and the presence of dupA gene can be considered as a marker for the development of gastroduodenal diseases. However, the cagA, iceA, vacA and oipA genes cannot be regarded for prediction of the clinical presentation of H. pylori infection in China.

Multiplexed detection of bacterial pathogens based on a cocktail of dual-modified phages.

Rapid, quantitative, and sensitive assays for the multiplexed detection of bacterial pathogens are urgently needed for public health. Here, we report the generation of dual-modified phage sensors for the simultaneous detection of multiple pathogenic bacteria. The M13KE phage was dual modified to display the targeting peptide on the minor coat protein pIII (∼5 copies) and the streptavidin-binding (StrB) peptide on the major coat protein pVIII (∼2700 copies). The targeting peptide specifically recognizes the target bacteria, and the StrB peptide acts as the efficient signal amplification and transduction unit upon binding with fluorescently tagged streptavidin. The bright fluorescence emitted from individual target bacteria can be clearly distinguished from the background via both the flow cytometry and fluorescence microscopy. Three different dual-modified phages targeting E. coli O157:H7, Salmonella Typhimurium, and Pseudomonas aeruginosa were constructed, and high specificity was verified via a large excess of other non-target bacteria. Using a 40 mL sample volume, the target bacteria detection limit was approximately 102 cells/mL via flow cytometry measurement in the presence of other non-target bacteria. By combining these three dual-modified phages into a cocktail, simultaneous detection and quantification of three target bacterial pathogens was demonstrated with good linearity. The strategy of constructing dual-modified phage represents a promising tool in the detection of bacterial pathogens.

Diversity of 3' variable region of cagA gene in Helicobacter pylori strains isolated from Chinese population.

BACKGROUND: The cytotoxin-associated gene A (cagA) is one of the most important virulence factors of Helicobacter pylori (H. pylori). There is a highly polymorphic Glu-Pro-Ile-Tyr-Ala (EPIYA) repeat region in the C-terminal of CagA protein. This repeat region is thought to play an important role in the pathogenesis of gastrointestinal diseases. The aim of this study was to investigate the diversity of cagA 3' variable region and the amino acid polymorphisms in the EPIYA segments of the CagA C-terminal region of H. pylori, and their association with gastroduodenal diseases. METHODS: A total of 515 H. pylori strains from patients in 14 different geographical regions of China were collected. The genomic DNA from each strain was extracted and the cagA 3' variable region was amplified by polymerase chain reaction (PCR). The PCR products were sequenced and analyzed using MEGA 7.0 software. RESULTS: A total of 503 (97.7%) H. pylori strains were cagA-positive and 1,587 EPIYA motifs were identified, including 12 types of EPIYA or EPIYA-like sequences. In addition to the four reported major segments, several rare segments (e.g., B', B″ and D') were defined and 20 different sequence types (e.g., ABD, ABC) were found in our study. A total of 481 (95.6%) strains carried the East Asian type CagA, and the ABD subtypes were most prevalent (82.1%). Only 22 strains carried the Western type CagA, which included AC, ABC, ABCC and ABCCCC subtypes. The CagA-ABD subtype had statistical difference in different geographical regions (P = 0.006). There were seven amino acid polymorphisms in the sequences surrounding the EPIYA motifs, among which amino acids 893 and 894 had a statistical difference with gastric cancer (P = 0.004). CONCLUSIONS: In this study, 503 CagA sequences were studied and analyzed in depth. In Chinese population, most H. pylori strains were of the CagA-ABD subtype and its presence was associated with gastroduodenal diseases. Amino acid polymorphisms at residues 893 and 894 flanking the EPIYA motifs had a statistically significant association with gastric cancer.

Arsenic Dispensing Powder Promotes Erythropoiesis in Myelodysplastic Syndromes via Downregulation of HIF1A and Upregulation of GATA Factors.

Traditional Chinese Medicine (TCM) is a practical medicine based on thousands of years of medical practice in China. Arsenic dispensing powder (ADP) has been used as a treatment for MDS patients with a superior efficacy on anemia at Xiyuan Hospital of China Academy of Chinese Medical Sciences. In this study, we retrospectively analyzed MDS patients that received ADP treatment in the past 9 years and confirmed that ADP improves patients' anemia and prolongs overall survival in intermediate-risk MDS patients. Then, we used the MDS transgenic mice model and cell line to explore the drug mechanism. In normal and MDS cells, ADP does not show cellular toxicity but promotes differentiation. In mouse MDS models, we observed that ADP showed significant efficacy on promoting erythropoiesis. In the BFU-E and CFU-E assays, ADP could promote erythropoiesis not only in normal clones but also in MDS clones. Mechanistically, we found that ADP could downregulate HIF1A in MDS clones through upregulation of VHL, P53 and MDM2, which is involved in two parallel pathways to downregulate HIF1A. We also confirmed that ADP upregulates GATA factors in normal clones. Thus, our clinical and experimental studies indicate that ADP is a promising drug to promote erythropoiesis in both MDS and normal clones with a superior outcome than current regular therapies. ADP promotes erythropoiesis in myelodysplastic syndromes via downregulation of HIF1A and upregulation of GATA factors.

Quantitative Assessment of the Physical Virus Titer and Purity by Ultrasensitive Flow Virometry.

Rapid quantification of viruses is vital for basic research on viral diseases as well as biomedical application of virus-based products. Here, we report the development of a high-throughput single-particle method to enumerate intact viral particles by ultrasensitive flow virometry, which detects single viruses as small as 27 nm in diameter. The nucleic acid dye SYTO 82 was used to stain the viral (or vector) genome, and a laboratory-built nano-flow cytometer (nFCM) was employed to simultaneously detect the side-scatter and fluorescence signals of individual viral particles. Using the bacteriophage T7 as a model system, intact virions were completely discriminated from empty capsids and naked viral genomes. Successful measurement of the physical virus titer and purity was demonstrated for recombinant adenoviruses, which could be used for gene delivery, therapeutic products derived from phage cocktails, and infected cell supernatants for veterinary vaccine production.

General and mild modification of food-derived extracellular vesicles for enhanced cell targeting.

Food-derived extracellular vesicles (FDEVs) have attracted increasing attention as potential delivery vehicles for therapeutic agents due to their desirable features such as excellent biocompatibility, easy accessibility and cost effectiveness. However, the intrinsic targeting capability of FDEVs is unsatisfactory compared to artificial nanoparticles or other source-derived EVs, which calls for efficient surface engineering strategies to equip them with specific ligands. Here we report a general and mild modification method via reduction of disulfide groups to maleimide reactive thiols. Taking milk-derived EVs (mEVs) as a model system, we demonstrated the feasibility for tethering various ligands on the surface without compromising the vesicular structures. Building an ultra-sensitive nano-flow cytometer (nFCM), the heterogeneous nature of the functionalized samples was revealed, and a magnetic separation approach was proposed accordingly to remove the as-observed non-EV particles. The cellular uptake and cytotoxicity experiments provided direct evidence showing an enhanced cell targeting and cargo delivery capability of the ligand conjugated mEVs. In addition, the in vivo imaging further proved the applicability of transferrin conjugation for increased tumor enrichment of mEVs. Collectively, this general and mild ligand conjugation method enables an efficient surface functionalization of FDEVs, which is of vital importance for enhanced targeting delivery.

Alterations of Fucosyltransferase Genes and Fucosylated Glycans in Gastric Epithelial Cells Infected with Helicobacter pylori.

Helicobacter pylori (H. pylori) adhesion to human gastric epithelial cells is closely linked with fucosylated glycans. Therefore, investigation of fucosylation in the interaction of gastric epithelial cells with H. pylori is critical. In this study we used lectin microarrays to detect the expression of fucosylated glycans in gastric epithelial cells (GES-1) infected with H. pylori strains isolated from patients with different diseases including chronic gastritis, duodenal ulcers, and gastric cancer (each containing two strains) at 4 h. In addition, we investigated the time-course expression of fucosyltransferase (FUT) 1-6 genes in GES-1 cells stimulated with H. pylori strains at 0.5-8 h. At 4 h post-infection, Lotus, AAA, BC2LCN, PA-IIL, CNL and ACG lectins had increased signals in H. pylori-infected GES-1 cells compared to uninfected cells. Higher expression of FUT1 and FUT2 was detected in all H. pylori-infected GES-1 cells within 2 h, regardless of the H. pylori strain. In particular, the expression of FUT2 was higher in H. pylori-infected GES-1 cells with a higher fold change in levels of BC2LCN lectin specific to α1-2 linked fucose (Fuc) at 4 h. The results suggest that the high levels of α1, 2-linked Fuc synthesized by FUT1/2, might play a role in the preliminary stage of H. pylori infection. This provides us with pivotal information to understand the adhesion of H. pylori to human gastric epithelial cells.

Activatable Mitochondria-Targeting Organoarsenic Prodrugs for Bioenergetic Cancer Therapy.

Despite widespread applications for cancer treatment, chemotherapy is restricted by several limitations, including low targeting specificity, acquired drug resistance, and concomitant adverse side effects. It remains challenging to overcome these drawbacks. Herein, we report a new bioenergetic approach for treating cancer efficiently. As a proof-of-concept, we construct activatable mitochondria-targeting organoarsenic prodrugs from organoarsenic compounds and traditional chemotherapeutics. These prodrugs could accomplish selective delivery and controlled release of both therapeutic agents to mitochondria, which synergistically promote mitochondrial ROS production and induce mitochondrial DNA damage, finally leading to mitochondria-mediated apoptosis of cancer cells. Our in vitro and in vivo experiments reveal the excellent anticancer efficacy of these prodrugs, underscoring the encouraging outlook of this strategy for effective cancer therapy.