High-level production of nervonic acid in the oleaginous yeast Yarrowia lipolytica by systematic metabolic engineering.

Nervonic acid benefits the treatment of neurological diseases and the health of brain. In this study, we employed the oleaginous yeast Yarrowia lipolytica to overproduce nervonic acid oil by systematic metabolic engineering. First, the production of nervonic acid was dramatically improved by iterative expression of the genes ecoding ß-ketoacyl-CoA synthase CgKCS, fatty acid elongase gELOVL6 and desaturase MaOLE2. Second, the biosynthesis of both nervonic acid and lipids were further enhanced by expression of glycerol-3-phosphate acyltransferases and diacylglycerol acyltransferases from Malania oleifera in endoplasmic reticulum (ER). Third, overexpression of a newly identified ER structure regulator gene YlINO2 led to a 39.3% increase in lipid production. Fourth, disruption of the AMP-activated S/T protein kinase gene SNF1 increased the ratio of nervonic acid to lignoceric acid by 61.6%. Next, pilot-scale fermentation using the strain YLNA9 exhibited a lipid titer of 96.7 g/L and a nervonic acid titer of 17.3 g/L (17.9% of total fatty acids), the highest reported titer to date. Finally, a proof-of-concept purification and separation of nervonic acid were performed and the purity of it reached 98.7%. This study suggested that oleaginous yeasts are attractive hosts for the cost-efficient production of nervonic acid and possibly other very long-chain fatty acids (VLCFAs).

CD7 protein plays a crucial role in T cell infiltration in tumors.

CD7 protein as a target is being used to treat CD7+ lymphoma; however, the role of CD7 in the hematopoietic system remains largely unknown. Therefore, we evaluated the effects of CD7 KO in mice. The differentiation of the hematopoietic system in the bone marrow and the number of various cell types in the thymus and spleen did not differ between CD7 KO and WT mice. After subcutaneous inoculation of B16-F10 melanoma cells, tumors from CD7 KO mice grew more rapidly, and the proportion of CD8+ T cells in the spleen and tumors decreased. In vitro, the infiltration and adhesion of CD8+ T cells from the spleen of CD7 KO mice were weakened. Blocking CD7 in normal T cells did not alter the migration and infiltration, but in Jurkat, CCRF-CEM, and KG-1a tumor cell lines, migration and invasion were significantly reduced after blocking CD7. Therefore, CD7 does not affect hematopoietic system development but plays a crucial role in T cell infiltration into tumors.

B7-H3 chimeric antigen receptor-modified T cell shows potential for targeted treatment of acute myeloid leukaemia.

BACKGROUND AND AIMS: Chimeric antigen receptor (CAR)-T cell therapy is a novel type of immunotherapy. However, the use of CAR-T cells to treat acute myeloid leukaemia (AML) has limitations. B7-H3 is expressed in several malignancies, including some types of AML cells. However, its expression in normal tissues is low. Therefore, B7-H3 is ideal for targeted AML therapy. MATERIALS AND METHODS: First, we constructed B7-H3 CAR that can target B7-H3, and then constructed B7-H3-CAR-T cells in vitro, which were co-incubated with six AML cell lines expressing different levels of B7-H3, respectively. The toxicity and cytokines were detected by flow cytometry. In vivo, AML model was established in B-NSG mice to study the toxicity of B7-H3-CAR T on AML cells. RESULTS: In vitro functional tests showed that B7-H3-CAR-T cells were cytotoxic to B7-H3-positive AML tumor cells and had good scavenging effect on B7-H3-expressing AML cell lines, and the cytokine results were consistent. In vivo, B7-H3-CAR-T cells significantly inhibited tumor cell growth in a mouse model of AML, prolonging mouse survival compared with controls. CONCLUSION: B7-H3-CAR-T cells may serve as a novel therapeutic method for the targeted treatment of AML.

Corrosion Resistance of Amorphous-Nanocrystalline Composite Structure Materials.

The purpose of this paper is to investigate the corrosion resistance of different nanoscale microstructures in the same material system and propose a novel method to obtain high-performance materials. During the last 2 decades, microstructure refinement and microalloying have become the main methods to prepare high-performance materials. The tensile strength of nanocrystalline solid solutions can reach 2.3 gigapascal, which is more than 1 fold the strength of traditional steel. However, there are few studies about the corrosion resistance of different nanoscale microstructures. In this paper, coatings with different microstructures (nanocrystalline, amorphous, and amorphous-nanocrystalline composite) have been successfully prepared by electrodeposition in the same material system (nickel-phosphorus alloy). Electrochemical test and high-pressure corrosion immersion test were carried out. The results show that the material loss of amorphous-nanocrystalline coating (P = 9.2 wt %) is about 1/4 that of crystalline coating at 8 MPa. In the range of 0.1 and 8 MPa, the average acceleration effect of hydrostatic pressure on the corrosion rate was calculated to be 1.611 × 10-6 g·cm-2·d-1·MPa-1.

In situ fabrication of MIL-68(In)@ZnIn2S4 heterojunction for enhanced photocatalytic hydrogen production.

Metal-organic frameworks (MOFs), as a class of semiconductor-like materials, are widely used in photocatalysis. However, the limited visible light absorption and poor charge separation efficiency are the main challenges restricting their photocatalytic performance. Herein, the type II heterojunction MIL-68(In)@ZIS was successfully fabricated by in situ growth of ZnIn2S4 (ZIS) on the surface of a representative MOF, i.e. MIL-68(In). After composition optimization, MIL-68(In)-20@ZIS shows an extraordinary photocatalytic hydrogen production efficiency of 9.09 mmol g-1 h-1 and good photochemical stability, which far exceeds those of most photocatalysts. The hierarchical loose structure of MIL-68(In)-20@ZIS is conducive to the adsorption of reactants and mass transfer. Meanwhile, a large number of tight 2D contact interfaces significantly reduce the obstruction of charge transfer, paving the way for high-perform photocatalytic hydrogen evolution. The experimental results demonstrate that the MIL-68(In)@ZIS heterojunction achieves intensive photoresponse and effective charge separation and transfer benefiting from unique charge transport paths of a type II heterojunction. This study opens an avenue toward MOF-based heterojunctions for solar energy conversion.

Unexpected High-Performance Photocatalytic Hydrogen Evolution in Co@NCNT@ZnIn2 S4 Triggered by Directional Charge Separation and Transfer.

The structural design of photocatalysts is highly related to the separation and transfer of photogenerated carriers, which is essential for the improvement of photocatalytic hydrogen evolution performance. Here, the hybrid photocatalyst M@NCNT@ZIS (M: Fe, Co, Ni; NCNT: nitrogen-doped carbon nanotube; ZIS: ZnIn2 S4 ) with a hierarchical structure is rationally designed and precisely synthesized. The unique hollow structure with a large specific surface area offers abundant reactive sites, thus increasing the adsorption of reactants. Importantly, the properly positioned metal nanoparticles realize the directional charge migration from ZIS to M@NCNT, which significantly improves the efficiency of charge separation. Furthermore, the intimate interface between M@NCNT and ZIS effectively facilitates charge migration by shortening the transfer distance and providing numerous transport channels. As a result, the optimized Co@NCNT@ZIS exhibits a remarkable photocatalytic hydrogen evolution efficiency (43.73 mmol g-1 h-1 ) without Pt as cocatalyst. Experimental characterizations and density functional theory calculations demonstrate that the synergistic effect between hydrogen adsorption and interfacial charge transport is of great significance for improving photocatalytic hydrogen production performance.

Model development to predict central lymph node metastasis in cN0 papillary thyroid microcarcinoma by machine learning.

Background: Whether prophylactic central lymph node dissection is necessary for cN0 papillary thyroid microcarcinoma (PTMC) patients remains highly debatable. Surgeons desperately need a way to help with surgical decision-making. While traditional predictive models can better explain changes in variables, machine learning (ML) models may have better predictive performance. This study aims to develop models for predicting the risk of central lymph node metastasis (CLNM) by utilizing ML algorithms. Methods: The clinical records of 1,121 patients with cN0 PTMC who underwent initial thyroid resection at our hospital between January 2014 and December 2018 were retrospectively retrieved. Univariate and multivariate analyses were performed to examine risk factors associated with CLNM. Six ML algorithms for predicting CLNM were established and internally validated. Indices including the area under the receiver operating characteristic (AUROC), sensitivity, specificity, accuracy, positive predictive value (PPV), and negative predictive value (NPV) were calculated to test the performance of the model. Results: The results showed 33.5% (376 out of 1,121) of patients had CLNM. In multivariate logistic regression (LR) analyses, gender, age, tumor size, multifocal lesions, and extrathyroidal extension (ETE) were all independent predictors of CLNM. The AUROC predictive values of the six ML algorithms were between 0.664 and 0.794, with the random forest (RF) model performing the best with an AUROC of 0.794. Therefore, we used the RF model and uploaded the results to a web-based risk calculator to predict an individual's probability of CLNM (https://xijing-thyroid.shinyapps.io/ptmc_clnm). Conclusions: Developing predictive models of CLNM in cN0 PTMC patients using the ML algorithm is a feasible method. Our online risk calculator based on the RF model may be a useful tool for surgical decisions.

CD7-directed CAR T-cell therapy: a potential immunotherapy strategy for relapsed/refractory acute myeloid leukemia.

Relapsed/refractory acute myeloid leukemia (AML) patients generally have a dismal prognosis and the treatment remains challenging. Due to the expression of CD7 on 30% AML and not on normal myeloid and erythroid cells, CD7 is an attractive target for immunotherapy of AML. CD7-targeted CAR T-cells had demonstrated encouraging efficacy in xenograft models of AML. We report here on the use of autologous CD7 CAR T-cells in the treatment of a relapsed/refractory AML patient with complex karyotype, TP53 deletion, FLT3-ITD mutation, and SKAP2-RUNX1 fusion gene. Before the CAR T-cell therapy, the patient achieved partial remission with IA regimen and attained complete remission after reinduction therapy (decitabine and venentoclax). Relapse occurred after consolidation (CLAG regimen). Then she failed CLIA regimen combined with venetoclax and exhibited resistance to FLT3 inhibitors. Bone marrow showed 20% blasts (CD7+ 95.6%). A total dose of 5 × 106/kg CD7 CAR T-cells was administered after the decitabine +FC regimen. Seventeen days after CAR T-cells infusion, she achieved morphologic leukemia-free state. The patient developed grade 3 cytokine release syndrome. No severe organ toxicity or immune effector cell-associated neurotoxicity syndrome was observed. In summary, the autologous CD7 CAR T-cell therapy could be considered a potential approach for AML with CD7 expression (NCT04762485).Trial registration Clinical Trials.gov, NCT04762485. Registered on February 21, 2021, prospectively registered.

Preventing crossinfection during reflectance confocal microscopy, using a transparent film dressing.

Contamination and crossinfection are always a significant concern when using reflectance confocal microscopy in the clinic because the glass window and metal ring at the front of the probe must contact the skin and mucosal surfaces, and sterilization of the imaging probes is usually impossible. We describe use of a transparent, single-use film dressing to solve this problem.

Autologous Nanobody-Derived Fratricide-Resistant CD7-CAR T-cell Therapy for Patients with Relapsed and Refractory T-cell Acute Lymphoblastic Leukemia/Lymphoma.

PURPOSE: Since CD7 may represent a potent target for T-lymphoblastic leukemia/lymphoma (T-ALL/LBL) immunotherapy, this study aimed to investigate safety and efficacy of autologous CD7-chimeric antigen receptor (CAR) T cells in patients with relapsed and refractory (R/R) T-ALL/LBL, as well as its manufacturing feasibility. PATIENTS AND METHODS: Preclinical phase was conducted in NPG mice injected with Luc+ GFP+CCRF-CEM cells. Open-label phase I clinical trial (NCT04004637) enrolled patients with R/R CD7-positive T-ALL/LBL who received autologous CD7-CAR T-cell infusion. Primary endpoint was safety; secondary endpoints included efficacy and pharmacokinetic and pharmacodynamic parameters. RESULTS: CD7 blockade strategy was developed using tandem CD7 nanobody VHH6 coupled with an endoplasmic reticulum/Golgi-retention motif peptide to intracellularly fasten CD7 molecules. In preclinical phase CD7 blockade CAR T cells prevented fratricide and exerted potent cytolytic activity, significantly relieving leukemia progression and prolonged the median survival of mice. In clinical phase, the complete remission (CR) rate was 87.5% (7/8) 3 months after CAR T-cell infusion; 1 patient with leukemia achieved minimal residual disease-negative CR and 1 patient with lymphoma achieved CR for more than 12 months. Majority of patients (87.5%) only had grade 1 or 2 cytokine release syndrome with no T-cell hypoplasia or any neurologic toxicities observed. The median maximum concentration of CAR T cells was 857.2 cells/µL at approximately 12 days and remained detectable up to 270 days. CONCLUSIONS: Autologous nanobody-derived fratricide-resistant CD7-CAR T cells demonstrated a promising and durable antitumor response in R/R T-ALL/LBL with tolerable toxicity, warranting further studies in highly aggressive CD7-positive malignancies.

Haploidentical CD7 CAR T-cells induced remission in a patient with TP53 mutated relapsed and refractory early T-cell precursor lymphoblastic leukemia/lymphoma.

Patients with relapsed/refractory early T-cell precursor lymphoblastic leukemia/lymphoma (ETP-ALL/LBL) respond poorly to traditional therapy and have dismal prognosis. CD7 is a promising therapeutic targets for chimeric antigen receptor modified T cell therapy (CART) due to its widely expression in almost all T-cell malignancies. Here we present the anti-CD7 CART therapy in a 11-year-old male with TP53 mutated relapsed/refractory ETP-ALL/LBL. The patient suffered second relapse after haploidentical hematopoietic stem cell transplantation, showing resistance to 4 lines salvage therapies including venetoclax. Nanobody derived CD7-CART cells were manufactured by co-transducing CAR-T cells with a CD7 protein expression blocker. 70.5% of blasts (CD7 expression: 92.6%) and extensive extramedullary disease (mediastinal mass, enlarged lymph nodes and spleen) were observed prior to CD7-CART-cell therapy. A total of 5 × 106/kg donor-derived CD7-CART-cells were infused. Hematological and extramedullary remission were both achieved, with persistence of CD7-CART-cells be detected until the last followup at 96th days after the infusion. Reversible adverse effects including grade 3 cytokine release syndrome and macrophage activation syndrome were observed. This case demonstrated that CD7-CART was a potent and safe salvage therapy in relapsed/refractory ETP-ALL/LBL patient with high tumor burden.Trial registration: ClinicalTrials. gov, NCT04785833 , Registered on March 8, 2021, prospectively registered.

Nanobody-based anti-CD22-chimeric antigen receptor T cell immunotherapy exhibits improved remission against B-cell acute lymphoblastic leukemia.

Chimeric antigen receptor (CAR) T-cell immunotherapies targeting CD19 can achieve impressive clinical remission rates in the treatment of B-cell non-Hodgkin lymphoma and B-cell acute lymphoblastic leukemia. However, relapse after CD19-CAR T treatment remains a major issue, with CD19 antigen-negative relapse being one of the main reasons. CD22, another antigen expressed in a B-cell lineage-specific pattern, is retained following CD19 loss. Accordingly, we hypothesized that CD22 could represent an alternative target to alleviate or compensate for the ineffectiveness of CD19-CAR T therapy. To this end, we generated camelid-derived CD22 nanobodies, whose smaller size, greater stability, and lower immunogenicity offer better quality than classical antibodies, and we used them to construct third-generation CD22-CARs containing 4-1BB and ICOS co-stimulatory domains. The novel CD22-CAR T cells exhibited impressive cytotoxicity both in vitro and in vivo and significantly prolonged the overall survival of tumor-bearing NSG mice. These findings provide the basis for further translational studies employing CD22-CARs.

Zkscan3 affects erythroblast development by regulating the transcriptional activity of GATA1 and KLF1 in mice.

ZKSCAN3 encodes a zinc-finger transcription factor that regulates the expression of important genes and plays a significant role in tumor development, pathogenesis, and metastasis. However, its biological functions under normal physiological conditions remain largely unknown. In our previous studies, using flow cytometry, we found that the deletion of Zkscan3 may cause abnormal erythropoiesis. In this study, we found that, in a Zkscan3 knockout mice model, the number of splenic early-stage (basophilic-erythroblasts) and late-stage (chromatophilic-erythroblasts to polychromatophilic-erythroblasts through orthochromatophilic-erythroblasts) erythroblasts increased, whereas the number of late erythroblasts in the bone marrow decreased. Moreover, the phenotype was exacerbated after treating mice with phenylhydrazine (PHZ), which causes severe hemolytic anemia. In the knockout mice treated with PHZ, the percentage of reticulocyte in the peripheral blood conspicuously increased, whereas MCHC and red blood cells decreased. Then, we performed RNA-seq and quantitative-polymerase chain reaction assay and found that the expression of GATA1 and Tiam1 in erythroblasts were upregulated, whereas KLF1 was downregulated. Luciferase assays showed that Zkscan3 inhibited the transcription of GATA1 and Tiam1 and promoted the expression of KLF1. Additionally, ChIP and CO-IP results confirmed that Zkscan3 directly interacts with GATA1 and inhibits its transcriptional activity in MEL cells. Our results demonstrate, for the first time, the significant role of Zkscan3 in physiological erythropoiesis through the interaction with GATA1, both at the DNA and protein level, and with Tiam1 and KLF1 at the DNA level.

Chimeric antigen receptor T cells derived from CD7 nanobody exhibit robust antitumor potential against CD7-positive malignancies.

The great success of chimeric antigen receptor T (CAR-T)-cell therapy in B-cell malignancies has significantly promoted its rapid expansion to other targets and indications, including T-cell malignancies and acute myeloid leukemia. However, owing to the life-threatening T-cell hypoplasia caused by CD7-CAR-T cells specific cytotoxic against normal T cells, as well as CAR-T cell-fratricide caused by the shared CD7 antigen on the T-cell surface, the clinical application of CD7 as a potential target for CD7+ malignancies is lagging. Here, we generated CD7ΔT cells using an anti-CD7 nanobody fragment coupled with an endoplasmic reticulum/Golgi retention domain and demonstrated that these cells transduced with CD7-CAR could prevent fratricide and achieve expansion. Additionally, CD7ΔCD7-CAR-T cells exhibited robust antitumor potiential against CD7+ tumors in vitro as well as in cell-line and patient-derived xenograft models of CD7-positive malignancies. Furthermore, we confirmed that the antitumor activity of CD7-CAR-T cells was positively correlated with the antigen density of tumor cells. This strategy adapts well with current clinical-grade CAR-T-cell manufacturing processes and can be rapidly applied for the therapy of patients with CD7+ malignancies.