DNA Framework Programmed Conformational Reconstruction of Antibody Complementary Determining Region.

The conformation of complementary determining region (CDR) is crucial in dictating its specificity and affinity for binding with an antigen, making it a focal point in artificial antibody engineering. Although desirable, programmable scaffolds that can regulate the conformation of individual CDRs with nanometer precision are still lacking. Here, we devise a strategy to program the CDR conformation by anchoring both ends of a free CDR loop to specific sites of a DNA framework structure. This method allows us to define the span of a single CDR loop with an ∼2 nm resolution. Using this approach, we create a series of DNA framework based artificial antibodies (DNFbodies) with varied CDR loop spans, leading to different antibody-antigen binding affinities. We find that an optimized single CDR loop (∼2.3 nm span) exhibits ∼3-fold improved affinity relative to natural antibodies, confirming the critical role of the CDR conformation. This study may inspire the rational design of artificial antibodies.

Refining chimeric antigen receptors via barcoded protein domain combination pooled screening.

Chimeric antigen receptor (CAR)-T cells represent a promising frontier in cancer immunotherapy. However, the current process for developing new CAR constructs is time consuming and inefficient. To address this challenge and expedite the evaluation and comparison of full-length CAR designs, we have devised a novel cloning strategy. This strategy involves the sequential assembly of individual CAR domains using blunt ligation, with each domain being assigned a unique DNA barcode. Applying this method, we successfully generated 360 CAR constructs that specifically target clinically validated tumor antigens CD19 and GD2. By quantifying changes in barcode frequencies through next-generation sequencing, we characterize CARs that best mediate proliferation and expansion of transduced T cells. The screening revealed a crucial role for the hinge domain in CAR functionality, with CD8a and IgG4 hinges having opposite effects in the surface expression, cytokine production, and antitumor activity in CD19- versus GD2-based CARs. Importantly, we discovered two novel CD19-CAR architectures containing the IgG4 hinge domain that mediate superior in vivo antitumor activity compared with the construct used in Kymriah, a U.S. Food and Drug Administration (FDA)-approved therapy. This novel screening approach represents a major advance in CAR engineering, enabling accelerated development of cell-based cancer immunotherapies.

Screening lactic acid bacteria and yeast strains for soybean paste fermentation in northeast of China.

Soybean paste was a traditional fermented product in northeast China, mainly fermented by molds, yeast, Bacillus, and lactic acid bacteria. In this study, the safety and fermentation ability of lactic acid bacteria and yeast strains isolated from traditional soybean paste in northeast China were evaluated, and the dynamic changes of biogenic amines, aflatoxin, total acids, amino acid nitrogen, and volatile compounds were investigated during the fermentation of the traditional soybean paste. Among the tested strains, Lactiplantibacillus plantarum DPUL-J8 could decompose putrescine by 100%, and no biogenic amine was produced by Pichia kudriavzevii DPUY-J8. Lactiplantibacillus plantarum DPUL-J8 and P. kudriavzevii DPUY-J8 with strong biogenic amine degrading capacities were inoculated into the soybean paste. After 30 days of fermentation, the content of biogenic amines and aflatoxin in the fermented soybean paste declined by more than 60% and 50%, respectively. At the same time, compared with the control group without inoculation, the contents of total acid (1.29 ± 0.05 g/100 g), amino acid nitrogen (0.82 ± 0.01 g/100 g), and volatile compounds in soybean paste fermented by L. plantarum DPUL-J8 and P. kudriavzevii DPUY-J8 were significantly increased, which had a good flavor. These results indicated that the use of L. plantarum DPUL-J8 and P. kudriavzevii DPUY-J8 as starter cultures for soybean paste might be a good strategy to improve the safety and flavor of traditional Chinese soybean paste.

Programming Aggregate States of DNA Nanorods with Sub-10 nm Hydrophobic Patterns for Tunable Cell Entry.

The intracellular application of DNA nanodevices is challenged by their inadequate cellular entry efficiency, which may be addressed by the development of amphiphilic DNA nanostructures. However, the impact of the spatial distribution of hydrophobicity in cell entry has not been fully explored. Here, we program a spectrum of amphiphilic DNA nanostructures displaying diverse sub-10 nm patterns of cholesterol, which result in distinct aggregate states in the aqueous solution and thus varied cell entry efficiencies. We find that the hydrophobic patterns can lead to discrete aggregate states, from monomers to low-number oligomers (n = 1-6). We demonstrate that the monomers or oligomers with moderate hydrophobic density are preferred for cell entry, with up to ∼174-fold improvement relative to unmodified ones. Our study provides a new clue for the rational design of amphiphilic DNA nanostructures for intracellular applications.

Anti-GD2 CAR-NKT cells in relapsed or refractory neuroblastoma: updated phase 1 trial interim results.

Vα24-invariant natural killer T cells (NKTs) have anti-tumor properties that can be enhanced by chimeric antigen receptors (CARs). Here we report updated interim results from the first-in-human phase 1 evaluation of autologous NKTs co-expressing a GD2-specific CAR with interleukin 15 (IL15) (GD2-CAR.15) in 12 children with neuroblastoma (NB). The primary objectives were safety and determination of maximum tolerated dose (MTD). The anti-tumor activity of GD2-CAR.15 NKTs was assessed as a secondary objective. Immune response evaluation was an additional objective. No dose-limiting toxicities occurred; one patient experienced grade 2 cytokine release syndrome that was resolved by tocilizumab. The MTD was not reached. The objective response rate was 25% (3/12), including two partial responses and one complete response. The frequency of CD62L+NKTs in products correlated with CAR-NKT expansion in patients and was higher in responders (n = 5; objective response or stable disease with reduction in tumor burden) than non-responders (n = 7). BTG1 (BTG anti-proliferation factor 1) expression was upregulated in peripheral GD2-CAR.15 NKTs and is a key driver of hyporesponsiveness in exhausted NKT and T cells. GD2-CAR.15 NKTs with BTG1 knockdown eliminated metastatic NB in a mouse model. We conclude that GD2-CAR.15 NKTs are safe and can mediate objective responses in patients with NB. Additionally, their anti-tumor activity may be enhanced by targeting BTG1. ClinicalTrials.gov registration: NCT03294954 .

Interaction between tumor cell TNFR2 and monocyte membrane-bound TNF-α triggers tumorigenic inflammation in neuroblastoma.

BACKGROUND: Tumor progression and resistance to therapy in children with neuroblastoma (NB), a common childhood cancer, are often associated with infiltration of monocytes and macrophages that produce inflammatory cytokines. However, the mechanism by which tumor-supportive inflammation is initiated and propagated remains unknown. Here, we describe a novel protumorigenic circuit between NB cells and monocytes that is triggered and sustained by tumor necrosis factor alpha (TNF-α). METHODS: We used NB knockouts (KOs) of TNF-α and TNFRSF1A mRNA (TNFR1)/TNFRSF1B mRNA (TNFR2) and TNF-α protease inbitor (TAPI), a drug that modulates TNF-α isoform expression, to assess the role of each component in monocyte-associated protumorigenic inflammation. Additionally, we employed NB-monocyte cocultures and treated these with clinical-grade etanercept, an Fc-TNFR2 fusion protein, to neutralize signaling by both membrane-bound (m) and soluble (s)TNF-α isoforms. Further, we treated NOD/SCID/IL2Rγ(null) mice carrying subcutaneous NB/human monocyte xenografts with etanercept and evaluated the impact on tumor growth and angiogenesis. Gene set enrichment analysis (GSEA) was used to determine whether TNF-α signaling correlates with clinical outcomes in patients with NB. RESULTS: We found that NB expression of TNFR2 and monocyte membrane-bound tumor necrosis factor alpha is required for monocyte activation and interleukin (IL)-6 production, while NB TNFR1 and monocyte soluble TNF-α are required for NB nuclear factor kappa B subunit 1 (NF-κB) activation. Treatment of NB-monocyte cocultures with clinical-grade etanercept completely abrogated release of IL-6, granulocyte colony-stimulating factor (G-CSF), IL-1α, and IL-1ß and eliminated monocyte-induced enhancement of NB cell proliferation in vitro. Furthermore, etanercept treatment inhibited tumor growth, ablated tumor angiogenesis, and suppressed oncogenic signaling in mice with subcutaneous NB/human monocyte xenografts. Finally, GSEA revealed significant enrichment for TNF-α signaling in patients with NB that relapsed. CONCLUSIONS: We have described a novel mechanism of tumor-promoting inflammation in NB that is strongly associated with patient outcome and could be targeted with therapy.

Self-assembly Induced Enhanced Electrochemiluminescence of Copper Nanoclusters Using DNA Nanoribbon Templates.

Copper nanoclusters (CuNCs) are attractive electrochemiluminescence (ECL) emitters as Cu is comparatively inexpensive, nontoxic, and highly abundant. However, their ECL yield is relatively low. Herein, we report that orderly self-assembly of CuNCs using DNA nanoribbon as the template (DNR/CuNCs) conferred the CuNCs with improved ECL properties compared with individual CuNCs in both annihilation and co-reactant processes. The DNR/CuNCs resulted in a high ECL yield of 46.8 % in K2 S2 O8 , which was ≈68 times higher than that of individual CuNCs. This strategy was successfully extended to other ECL emitters, such as gold nanoclusters and the Ru(bpy)3 2+ /TPrA system. Furthermore, as an application of DNR/CuNCs, a DNR/CuNC-based ECL biosensor with higher sensitivity was constructed for dopamine determination (two orders of magnitude lower than that previously reported), showing that DNR/CuNCs have a potential for application in ECL bioanalysis as a new type of superior luminophore candidate.

Safety evaluation and application of lactic acid bacteria and yeast strains isolated from Sichuan broad bean paste.

Broad bean paste is one of the most popular characteristic traditional fermented bean products in China, which is prepared by mixed fermentation of a variety of microorganisms, among which lactic acid bacteria and yeast played an important role in the improvement of the fermented broad bean paste quality. However, the traditional open-air fermentation of broad bean paste brought some risks of harmful microorganisms. In this study, the safety and fermentation ability of lactic acid bacteria and yeast strains isolated from traditional broad bean paste was evaluated. The results showed that the protease activity of the strain Lactobacillus plantarum DPUL-J5 (366.73 ± 9.00 U/L) and Pichia kudriavzevii DPUY-J5 (237.18 ± 10.93 U/L) were the highest. Both strains produced little biogenic amines, and did not exhibit α-hemolytic activity or antibiotic resistance for some of the antibiotics most used in human medicine. Furthermore, the broad bean paste fermentation involving DPUL-J5 and DPUY-J5 was beneficial for accumulating higher total acid (1.69 ± 0.01 g/100 g), amino-acid nitrogen (0.85 ± 0.03 g/100 g), and more volatile flavor compounds, meanwhile, reducing the levels of biogenic amines and aflatoxin B1. Therefore, this study provided a new strategy to improve the safety and quality of traditional broad bean paste.

Acute pancreatitis after nitrous oxide abuse: A case report.

The recreational use of nitrous oxide (N2O) is sharply increasing among young adults. N2O abuse can cause serious complications. However, the association between acute pancreatitis and N2O is rarely reported. Here we report a case of a young and previously healthy female with widespread cutaneous lesions, neurologic symptoms, and abdominal pain. Acute pancreatitis was the patient's primary diagnosis when she was admitted to the gastroenterology unit. We added the diagnosis of N2O intoxication after an additional inquiry into the patient's personal history revealed that she had abused N2O for 1 month. With the application of mecobalamin and other symptomatic treatments, the symptoms and laboratory indexes improved gradually. In this case, we highlight that acute pancreatitis may be a rare complication induced by N2O.

DNA framework carriers with asymmetric hydrophobic drug patterns for enhanced cellular cytotoxicity.

We devise a class of amphiphilic drug complexes by programming hydrophobic drug patterns (HDPs) on DNA frameworks. We investigate the effect of HDPs on cellular uptake efficiency and drug potency. We achieve enhanced cytotoxicity against tumor cells by using an asymmetric HDP.

LEF1 Drives a Central Memory Program and Supports Antitumor Activity of Natural Killer T Cells.

Vα24-invariant natural killer T cells (NKT) possess innate antitumor properties that can be exploited for cancer immunotherapy. We have shown previously that the CD62L+ central memory-like subset of these cells drives the in vivo antitumor activity of NKTs, but molecular mediators of NKT central memory differentiation remain unknown. Here, we demonstrate that relative to CD62L- cells, CD62L+ NKTs express a higher level of the gene encoding the Wnt/ß-catenin transcription factor lymphoid enhancer binding factor 1 (LEF1) and maintain active Wnt/ß-catenin signaling. CRISPR/Cas9-mediated LEF1 knockout reduced CD62L+ frequency after antigenic stimulation, whereas Wnt/ß-catenin activator Wnt3a ligand increased CD62L+ frequency. LEF1 overexpression promoted NKT expansion and limited exhaustion following serial tumor challenge and was sufficient to induce a central memory-like transcriptional program in NKTs. In mice, NKTs expressing a GD2-specific chimeric-antigen receptor (CAR) with LEF1 demonstrated superior control of neuroblastoma xenograft tumors compared with control CAR-NKTs. These results identify LEF1 as a transcriptional activator of the NKT central memory program and advance development of NKT cell-based immunotherapy. See related Spotlight by Van Kaer, p. 144.

Epidemiological investigation of a cluster of COVID-19 in badminton venues / 预防医学

Objective@#To perform an epidemiological survey of the first case with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in Pinghu City of Jiaxing City, Zhejiang Province on March 13, 2022, so as to provide insights into the management of coronavirus disease (COVID-19) epidemics. @*Methods@#According to the requirements of the Protocol on Prevention and Control of COVID-19 (8th Edition), epidemiological investigations were performed among 39 cases with SARS-CoV-2 infections in Pinghu City from March 13 to 20, 2022. Cases' demographics, clinical symptoms, history of immunization and exposure were collected, and close contacts were identified. Pharyngeal swabs were sampled from infected cases for detection of SARS-CoV-2 nucleic acid and whole-genome sequencing, and the source of infection and transmission route were investigated. @*Results@#The index case for this COVID-19 epidemic was an imported case from Shanghai Municipality, who infected 6 persons via aerosol transmission when playing in the badminton venue of Pinghu National Fitness Center on March 9; subsequently, one of these infected cases infected another 18 persons when playing in the badminton venue of Jiadian Village Resident's Fitness Center in Zhapu Township on March 12. Sixteen confirmed cases were reported, and all cases were mild; another 23 asymptomatic cases were diagnosed, with no death reported. This epidemic occurred from March 11 to 20, with 3 generations of spread and a median incubation period of 3 days. The SARS-CoV-2 infected cases had a median age of 33.5 (interquartile range, 12.0) years and included 36 cases with a history of COVID-19 vaccination. There were 16 cases with fever, cough, runny nose and sore throat, and 13 cases with imaging features of pneumonia. The effective reproductive number (Rt) of the COVID-19 epidemic was 7.73 at early stage, and was less than 1 since March 21. Whole-genome sequencing identified Omicron BA.2 variant among 33 cases, which had high homology with the index cases. @*Conclusion@#This epidemic was a cluster of COVID-19 caused by imported Omicron BA.2 variant infection from Shanghai Municipality, and the COVID-19 transmission was mainly caused by indoor aerosols.

ERK Inhibitor Ulixertinib Inhibits High-Risk Neuroblastoma Growth In Vitro and In Vivo.

Neuroblastoma (NB) is a pediatric tumor of the peripheral nervous system. Approximately 80% of relapsed NB show RAS-MAPK pathway mutations that activate ERK, resulting in the promotion of cell proliferation and drug resistance. Ulixertinib, a first-in-class ERK-specific inhibitor, has shown promising antitumor activity in phase 1 clinical trials for advanced solid tumors. Here, we show that ulixertinib significantly and dose-dependently inhibits cell proliferation and colony formation in different NB cell lines, including PDX cells. Transcriptomic analysis revealed that ulixertinib extensively inhibits different oncogenic and neuronal developmental pathways, including EGFR, VEGF, WNT, MAPK, NGF, and NTRK1. The proteomic analysis further revealed that ulixertinib inhibits the cell cycle and promotes apoptosis in NB cells. Additionally, ulixertinib treatment significantly sensitized NB cells to the conventional chemotherapeutic agent doxorubicin. Furthermore, ulixertinib potently inhibited NB tumor growth and prolonged the overall survival of the treated mice in two different NB mice models. Our preclinical study demonstrates that ulixertinib, either as a single agent or in combination with current therapies, is a novel and practical therapeutic approach for NB.

Identifying a confused cell identity for esophageal squamous cell carcinoma.

The cell identity of malignant cells and how they acquire it are fundamental for our understanding of cancer. Here, we report that esophageal squamous cell carcinoma (ESCC) cells display molecular features equally similar but distinct to all three types of normal esophageal epithelial cells, which we term as confused cell identity (CCI). CCI is an independent prognostic marker associated with poor prognosis in ESCC. Further, we identify tropomyosin 4 (TPM4) as a critical CCI gene that promotes the aggressiveness of ESCC in vitro and in vivo. And TPM4 creates CCI through activating the Jak/STAT-SOX2 pathway. Thus, our study suggests an unrecognized feature of ESCC cells, which might be of value for clinic prognosis and potential interference.

Artificial Intelligence for Detecting and Delineating Margins of Early ESCC Under WLI Endoscopy.

INTRODUCTION: Conventional white light imaging (WLI) endoscopy is the most common screening technique used for detecting early esophageal squamous cell carcinoma (ESCC). Nevertheless, it is difficult to detect and delineate margins of early ESCC using WLI endoscopy. This study aimed to develop an artificial intelligence (AI) model to detect and delineate margins of early ESCC under WLI endoscopy. METHODS: A total of 13,083 WLI images from 1,239 patients were used to train and test the AI model. To evaluate the detection performance of the model, 1,479 images and 563 images were used as internal and external validation data sets, respectively. For assessing the delineation performance of the model, 1,114 images and 211 images were used as internal and external validation data sets, respectively. In addition, 216 images were used to compare the delineation performance between the model and endoscopists. RESULTS: The model showed an accuracy of 85.7% and 84.5% in detecting lesions in internal and external validation, respectively. For delineating margins, the model achieved an accuracy of 93.4% and 95.7% in the internal and external validation, respectively, under an overlap ratio of 0.60. The accuracy of the model, senior endoscopists, and expert endoscopists in delineating margins were 98.1%, 78.6%, and 95.3%, respectively. The proposed model achieved similar delineating performance compared with that of expert endoscopists but superior to senior endoscopists. DISCUSSION: We successfully developed an AI model, which can be used to accurately detect early ESCC and delineate the margins of the lesions under WLI endoscopy.

Directing Multivalent Aptamer-Receptor Binding on the Cell Surface with Programmable Atom-Like Nanoparticles.

Multivalent interactions of biomolecules play pivotal roles in physiological and pathological settings. Whereas the directionality of the interactions is crucial, the state-of-the-art synthetic multivalent ligand-receptor systems generally lack programmable approaches for orthogonal directionality. Here, we report the design of programmable atom-like nanoparticles (aptPANs) to direct multivalent aptamer-receptor binding on the cell interface. The positions of the aptamer motifs can be prescribed on tetrahedral DNA frameworks to realize atom-like orthogonal valence and direction, enabling the construction of multivalent molecules with fixed aptamer copy numbers but different directionality. These directional-yet-flexible aptPAN molecules exhibit the adaptability to the receptor distribution on cell surfaces. We demonstrate the high-affinity tumor cell binding with a linear aptPAN oligomer (≈13-fold improved compared to free aptamers), which leads to ≈50 % suppression of cell growth.