Engineering of glycoside hydrolase family 7 cellobiohydrolases directed by natural diversity screening.

Protein engineering and screening of processive fungal cellobiohydrolases (CBHs) remain challenging due to limited expression hosts, synergy-dependency, and recalcitrant substrates. In particular, glycoside hydrolase family 7 (GH7) CBHs are critically important for the bioeconomy and typically difficult to engineer. Here, we target the discovery of highly active natural GH7 CBHs and engineering of variants with improved activity. Using experimentally assayed activities of genome mined CBHs, we applied sequence and structural alignments to top performers to identify key point mutations linked to improved activity. From ∼1500 known GH7 sequences, an evolutionarily diverse subset of 57 GH7 CBH genes was expressed in Trichoderma reesei and screened using a multiplexed activity screening assay. Ten catalytically enhanced natural variants were identified, produced, purified, and tested for efficacy using industrially relevant conditions and substrates. Three key amino acids in CBHs with performance comparable or superior to Penicillium funiculosum Cel7A were identified and combinatorially engineered into P. funiculosum cel7a, expressed in T. reesei, and assayed on lignocellulosic biomass. The top performer generated using this combined approach of natural diversity genome mining, experimental assays, and computational modeling produced a 41% increase in conversion extent over native P. funiculosum Cel7A, a 55% increase over the current industrial standard T. reesei Cel7A, and 10% improvement over Aspergillus oryzae Cel7C, the best natural GH7 CBH previously identified in our laboratory.

Single-Digit Nanobubble Sensing via Nanopore Technology.

Nanobubbles play an important role in diverse fields, including engineering, medicine, and agriculture. Understanding the characteristics of individual nanobubbles is essential for comprehending fluid dynamics behaviors and advancing nanoscale science across various fields. Here, we report a strategy based on nanopore sensors for characterizing single-digit nanobubbles. We investigated the sizes and diffusion coefficients of nanobubbles at different voltages. Additionally, the finite element simulation and molecular dynamics simulation were introduced to account for counterion concentration variation around nanobubbles in the nanopore. In particular, the differences in stability and surface charge density of nanobubbles under various solution environments have been studied by the ion-stabilized model and the DLVO theory. Additionally, a straightforward method to mitigate nanobubble generation in the bulk for reducing current noise in nanopore sensing was suggested. The results hold significant implications for enhancing the understanding of individual nanobubble characterizations, especially in the nanofluid field.

Identification the Role of Grain Boundaries in Polycrystalline Photovoltaics via Advanced Atomic Force Microscope.

Atomicforce microscopy (AFM)-based scanning probing techniques, including Kelvinprobe force microscopy (KPFM) and conductive atomic force microscopy (C-AFM), have been widely applied to investigate thelocal electromagnetic, physical, or molecular characteristics of functional materials on a microscopic scale. The microscopic inhomogeneities of the electronic properties of polycrystalline photovoltaic materials can be examined by these advanced AFM techniques, which bridge the local properties of materials to overall device performance and guide the optimization of the photovoltaic devices. In this review, the critical roles of local optoelectronic heterogeneities, especially at grain interiors (GIs) and grain boundaries (GBs) of polycrystalline photovoltaic materials, including versatile polycrystalline silicon, inorganic compound materials, and emerging halide perovskites, studied by KPFM and C-AFM, are systematically identified. How the band alignment and electrical properties of GIs and GBs affect the carrier transport behavior are discussed from the respective of photovoltaic research. Further exploiting the potential of such AFM-based techniques upon a summary of their up-to-date applications in polycrystalline photovoltaic materials is beneficial to acomprehensive understanding of the design and manipulation principles of thenovel solar cells and facilitating the development of the next-generation photovoltaics and optoelectronics.

Unsupervised OCT image despeckling with ground-truth- and repeated-scanning-free features.

Optical coherence tomography (OCT) can resolve biological three-dimensional tissue structures, but it is inevitably plagued by speckle noise that degrades image quality and obscures biological structure. Recently unsupervised deep learning methods are becoming more popular in OCT despeckling but they still have to use unpaired noisy-clean images or paired noisy-noisy images. To address the above problem, we propose what we believe to be a novel unsupervised deep learning method for OCT despeckling, termed Double-free Net, which eliminates the need for ground truth data and repeated scanning by sub-sampling noisy images and synthesizing noisier images. In comparison to existing unsupervised methods, Double-free Net obtains superior denoising performance when trained on datasets comprising retinal and human tissue images without clean images. The efficacy of Double-free Net in denoising holds significant promise for diagnostic applications in retinal pathologies and enhances the accuracy of retinal layer segmentation. Results demonstrate that Double-free Net outperforms state-of-the-art methods and exhibits strong convenience and adaptability across different OCT images.

Identifying the active compounds and mechanism of action of TongFu XieXia Decoction for treating intestinal obstruction using network pharmacology combined with ultra-high performance liquid chromatography-quadrupole-orbitrap mass spectrometry.

RATIONALE: TongFu XieXia Decoction (TFXXD), a formulation rooted in traditional Chinese medicine and optimized through clinical practice, serves as an advanced version of the classic Da Cheng Qi decoction used for treating intestinal obstruction (IO), demonstrating significant therapeutic efficacy. However, due to the intricate nature of herbal compositions, the principal constituents and potential mechanisms of TFXXD have yet to be clarified. Accordingly, this study seeks to identify the active compounds and molecular targets of TFXXD, as well as to elucidate its anti-IO mechanisms. METHODS: Qualitative identification of the principal constituents of TFXXD was accomplished using ultra-high preformance liquid chromatography-quadrupole-orbitrap mass spectrometry (UPLC-Q-Orbitrap-MS/MS) analysis. PharmMapper facilitated the prediction of potential molecular targets, whereas protein-protein interaction analysis was conducted using STRING 11.0. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed using the Metascape database. A "compounds-target-pathway" network was meticulously constructed within Cytoscape 3.8.2. Finally, molecular docking studies were performed to investigate the interactions between the core target and the crucial compound. RESULTS: UPLC-Q-Orbitrap-MS/MS analysis identified 65 components with high precision and sensitivity. Furthermore, 64 potential targets were identified as integral to TFXXD bioactivity in IO treatment. Gene Ontology enrichment analysis revealed 995 distinct biological functions, while the Kyoto Encyclopedia of Genes and Genomes enrichment analysis identified 143 intricate signaling pathways. CONCLUSION: Molecular docking studies substantiated the substantial affinity between the TFXXD bioactive constituents and their corresponding targets in the context of IO. TFXXD exerts its therapeutic efficacy in IO through a multifaceted interplay between multiple compounds, targets, and pathways. The integration of network pharmacology with UPLC-Q-Orbitrap-MS/MS has emerged as a promising strategy to unravel the intricate web of molecular interactions underlying herbal medicine. However, it is imperative to emphasize the necessity for further in vivo and in vitro experiments.

Single-incision plus one-port laparoscopy surgery versus conventional multi-port laparoscopy surgery for colorectal cancer: a systematic review and meta-analysis.

OBJECTIVE: The efficacy of single-incision plus one-port laparoscopic surgery (SILS + 1) versus conventional laparoscopic surgery (CLS) for colorectal cancer treatment remains unclear. This study compares the short-term and long-term outcomes of SILS + 1 and CLS using a high-quality systematic review and meta-analysis. METHOD: Literature search followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, drawing from PubMed, Embase, Web of Science, and the Cochrane Library until December 10, 2023. Statistical analysis was conducted using RevMan and Stata. RESULT: The review and meta-analysis included seven studies with 1740 colorectal cancer patients. Compared to CLS, SILS + 1 showed significant improvements in operation time (WMD = - 18.33, P < 0.00001), blood loss (WMD = - 21.31, P < 0.00001), incision length (WMD = - 2.07, P < 0.00001), time to first defecation (WMD = - 14.91, P = 0.009), time to oral intake (WMD = - 11.46, P = 0.04), and time to ambulation (WMD = - 11.52, P = 0.01). There were no significant differences in lymph node harvest, resection margins, complications, anastomotic leakage, hospital stay, disease-free survival, overall survival, and postoperative recurrence. CONCLUSIONS: Compared to CLS, SILS + 1 demonstrates superiority in shortening the surgical incision and promoting postoperative recovery. SILS + 1 can provide a safe and feasible alternative to CLS.

Multiparameter MRI-based radiomics nomogram for preoperative prediction of brain invasion in atypical meningioma:a multicentre study.

OBJECTIVE: To develop a nomogram based on tumor and peritumoral edema (PE) radiomics features extracted from preoperative multiparameter MRI for predicting brain invasion (BI) in atypical meningioma (AM). METHODS: In this retrospective study, according to the 2021 WHO classification criteria, a total of 469 patients with pathologically confirmed AM from three medical centres were enrolled and divided into training (n = 273), internal validation (n = 117) and external validation (n = 79) cohorts. BI was diagnosed based on the histopathological examination. Preoperative contrast-enhanced T1-weighted MR images (T1C) and T2-weighted MR images (T2) for extracting meningioma features and T2-fluid attenuated inversion recovery (FLAIR) sequences for extracting meningioma and PE features were obtained. The multiple logistic regression was applied to develop separate multiparameter radiomics models for comparison. A nomogram was developed by combining radiomics features and clinical risk factors, and the clinical usefulness of the nomogram was verified using decision curve analysis. RESULTS: Among the clinical factors, PE volume and PE/tumor volume ratio are the risk of BI in AM. The combined nomogram based on multiparameter MRI radiomics features of meningioma and PE and clinical indicators achieved the best performance in predicting BI in AM, with area under the curve values of 0.862 (95% CI, 0.819-0.905) in the training cohort, 0.834 (95% CI, 0.780-0.908) in the internal validation cohort and 0.867 (95% CI, 0.785-0.950) in the external validation cohort, respectively. CONCLUSIONS: The nomogram based on tumor and PE radiomics features extracted from preoperative multiparameter MRI and clinical factors can predict the risk of BI in patients with AM.

Guided Lamb Wave Array Time-Delay-Based MUSIC Algorithm for Impact Imaging.

Composite materials, valued in aerospace for their stiffness, strength and lightness, require impact monitoring for structural health, especially against low-velocity impacts. The MUSIC algorithm, known for efficient directional scanning and easy sensor deployment, is gaining prominence in this area. However, in practical engineering applications, the broadband characteristics of impact response signals and the time delay errors in array elements' signal reception lead to inconsistencies between the steering vector and the actual signal subspace, affecting the precision of the MUSIC impact localization method. Furthermore, the anisotropy of composite materials results in time delay differences between array elements in different directions. If the MUSIC algorithm uses a fixed velocity value, this also introduces time delay errors, further reducing the accuracy of localization. Addressing these challenges, this paper proposes an innovative MUSIC algorithm for impact imaging using a guided Lamb wave array, with an emphasis on time delay management. This approach focuses on the extraction of high-energy, single-frequency components from impact response signals, ensuring accurate time delay measurement across array elements and enhancing noise resistance. It also calculates the average velocity of single-frequency components in varying directions for an initial impact angle estimation. This estimated angle then guides the selection of a specific single-frequency velocity, culminating in precise impact position localization. The experimental evaluation, employing equidistantly spaced array elements to capture impact response signals, assessed the effectiveness of the proposed method in accurately determining array time delays. Furthermore, impact localization tests on reinforced composite structures were conducted, with the results indicating high precision in pinpointing impact locations.

Research on Tire Surface Damage Detection Method Based on Image Processing.

The performance of the tire has a very important impact on the safe driving of the car, and in the actual use of the tire, due to complex road conditions or use conditions, it will inevitably cause immeasurable wear, scratches and other damage. In order to effectively detect the damage existing in the key parts of the tire, a tire surface damage detection method based on image processing was proposed. In this method, the image of tire side is captured by camera first. Then, the collected images are preprocessed by optimizing the multi-scale bilateral filtering algorithm to enhance the detailed information of the damaged area, and the optimization effect is obvious. Thirdly, the image segmentation based on clustering algorithm is carried out. Finally, the Harris corner detection method is used to capture the "salt and pepper" corner of the target region, and the segmsegmed binary image is screened and matched based on histogram correlation, and the target region is finally obtained. The experimental results show that the similarity detection is accurate, and the damage area can meet the requirements of accurate identification.

DNA Carrier-Assisted Molecular Ping-Pong in an Asymmetric Nanopore.

Nanopore analysis relies on ensemble averaging of translocation signals obtained from numerous molecules, requiring a relatively high sample concentration and a long turnaround time from the sample to results. The recapture and subsequent re-reading of the same molecule is a promising alternative that enriches the signal information from a single molecule. Here, we describe how an asymmetric nanopore improves molecular ping-pong by promoting the recapture of the molecule in the trans reservoir. We also demonstrate that the molecular recapture could be improved by linking the target molecule to a long DNA carrier to reduce the diffusion, thereby achieving over 100 recapture events. Using this ping-pong methodology, we demonstrate its use in accurately resolving nanostructure motifs along a DNA scaffold through repeated detection. Our method offers novel insights into the control of DNA polymer dynamics within nanopore confinement and opens avenues for the development of a high-fidelity DNA detection platform.

Design and construction of chemical-biological module clusters for degradation and assimilation of poly(ethylene terephthalate) waste.

The rising accumulation of poly(ethylene terephthalate) (PET) waste presents an urgent ecological challenge, necessitating an efficient and economical treatment technology. Here, we developed chemical-biological module clusters that perform chemical pretreatment, enzymatic degradation, and microbial assimilation for the large-scale treatment of PET waste. This module cluster included (i) a chemical pretreatment that involves incorporating polycaprolactone (PCL) at a weight ratio of 2% (PET:PCL = 98:2) into PET via mechanical blending, which effectively reduces the crystallinity and enhances degradation; (ii) enzymatic degradation using Thermobifida fusca cutinase variant (4Mz), that achieves complete degradation of pretreated PET at 300 g/L PET, with an enzymatic loading of 1 mg protein per gram of PET; and (iii) microbial assimilation, where Rhodococcus jostii RHA1 metabolizes the degradation products, assimilating each monomer at a rate above 90%. A comparative life cycle assessment demonstrated that the carbon emissions from our module clusters (0.25 kg CO2-eq/kg PET) are lower than those from other established approaches. This study pioneers a closed-loop system that seamlessly incorporates pretreatment, degradation, and assimilation processes, thus mitigating the environmental impacts of PET waste and propelling the development of a circular PET economy.

Photo-Induced Disproportionation-Mediated Photodynamic Therapy: Simultaneous Oxidation of Tetrahydrobiopterin and Generation of Superoxide Radicals.

We herein present an approach of photo-induced disproportionation for preparation of Type-I photodynamic agents. As a proof of concept, BODIPY-based photosensitizers were rationally designed and prepared. The photo-induced intermolecular electron transfer between homotypic chromophores leads to the disproportionation reaction, resulting in the formation of charged intermediates, cationic and anionic radicals. The cationic radicals efficiently oxidize the cellularimportant coenzyme, tetrahydrobiopterin (BH4 ), and the anionic radicals transfer electrons to oxygen to produce superoxide radicals (O2 - ⋅). One of our Type-I photodynamic agents not only self-assembles in water but also effectively targets the endoplasmic reticulum. It displayed excellent photocytotoxicity even in highly hypoxic environments (2 % O2 ), with a half-maximal inhibitory concentration (IC50 ) of 0.96 µM, and demonstrated outstanding antitumor efficacy in murine models bearing HeLa tumors.

Spontaneous Symmetry Breaking of Achiral Molecules Leading to the Formation of Homochiral Superstructures that Exhibit Mechanoluminescence.

Chirality, with its intrinsic symmetry-breaking feature, is frequently utilized in the creation of acentric crystalline functional materials that exhibit intriguing optoelectronic properties. On the other hand, the development of chiral crystals from achiral molecules offers a solution that bypasses the need for enantiopure motifs, presenting a promising alternative and thereby expanding the possibilities of the self-assembly toolkit. Nevertheless, the rational design of achiral molecules that prefer spontaneous symmetry breaking during crystallization has so far been obscure. In this study, we present a series of six achiral molecules, demonstrating that when these conformationally flexible molecules adopt a cis-conformation and engage in multiple non-covalent interactions along a helical path, they collectively self-assemble into chiral superstructures consisting of single-handed supramolecular columns. When these homochiral supramolecular columns align in parallel, they form polar crystals that exhibit intense luminescence upon grinding or scraping. We therefore demonstrate our molecular design strategy could significantly increase the likelihood of symmetry breaking in achiral molecular synthons during self-assembly, offering a facile access to novel chiral crystalline materials with unique optoelectronic properties.

Development of a cGMP-compliant process to manufacture donor-derived, CD45RA-depleted memory CD19-CAR T cells.

Autologous chimeric antigen receptor (CAR) T cells targeting the CD19 antigen have demonstrated a high complete response rate in relapsed/refractory B-cell malignancies. However, autologous CAR T cell therapy is not an option for all patients. Here we optimized conditions for clinical-grade manufacturing of allogeneic CD19-CAR T cells using CD45RA-depleted donor memory T cells (Tm) for a planned clinical trial. Tm were activated using the MACS GMP T Cell TransAct reagent and transduced in the presence of LentiBOOST with a clinical-grade lentiviral vector that encodes a 2nd generation CD19-CAR with a 41BB.zeta endodomain. Transduced T cells were transferred to a G-Rex cell culture device for expansion and harvested on day 7 or 8 for cryopreservation. The resulting CD19-CAR(Mem) T cells expanded on average 34.2-fold, and mean CAR expression was 45.5%. The majority of T cells were CD4+ and had a central memory or effector memory phenotype, and retained viral specificity. CD19-CAR(Mem) T cells recognized and killed CD19-positive target cells in vitro and had potent antitumor activity in an ALL xenograft model. Thus we have successfully developed a current good manufacturing practice-compliant process to manufacture donor-derived CD19-CAR(Mem) T cells. Our manufacturing process could be readily adapted for CAR(Mem) T cells targeting other antigens.

Establishment of pseudorabies virus latency and reactivation model in mice dorsal root ganglia culture.

Pseudorabies virus (PRV) belongs to the alpha herpesvirus family and is responsible for Aujeszky's disease in pigs. Similar to other alpha herpesviruses, PRV establishes a lifelong latent infection in trigeminal ganglion. These latently infected pigs serve as a reservoir for recurrent infections when reactivation is triggered, making the eradication of PRV a challenging task. However, the molecular mechanism underlying PRV latency and reactivation in neurons is still poorly understood due to limitations in the in vitro model. To establish a pseudorabies virus latency and reactivation model in primary neuron cultures, we isolated dorsal root ganglion (DRG) from newborn Kunming mice using a method named epineurium-pulling for DRG collection (EPDC) and cultured primary neurons in vitro. A dual-colour recombinant PRV BAC mRuby-VP16 was constructed and 0.5 multiplicity of infection (MOI) was found as an appropriate dose in the presence of aciclovir to establish latency. Reactivation was induced using UV-inactivated herpesviruses or a series of chemical inhibitors. Interestingly, we found that not only UV-PRV, but also UV-HSV-1 and UV-BHoV-5 were able to induce rapid PRV reactivation. The efficiency of reactivation for LY294002, forskolin, etoposide, dexamethasone, and acetylcholine was found to be dependent on their concentration. In conclusion, we developed a valuable model of PRV latency and reactivation, which provides a basis for future mechanism research.

Bile from Patients with Primary Sclerosing Cholangitis Contains Mucosal-Associated Invariant T-Cell Antigens.

Primary sclerosing cholangitis (PSC) is associated with altered microbiota of the gut and bile. Mucosal-associated invariant T (MAIT) cells, enriched in human liver, uniquely recognize microbial-derived metabolites. This study aimed to determine whether bile from patients with PSC contains antigens activating MAIT cells. Bile was collected at the time of liver transplantation from patients with PSC (n = 28). The bile samples were either directly incubated with peripheral blood mononuclear cells from healthy donors or with antigen-presenting cells followed by co-culture with peripheral blood mononuclear cells. MAIT cell activation was assessed by flow cytometry. An anti-MR1 antibody was used to determine whether the activation was major histocompatibility complex class I-related protein (MR1) restricted. Biliary microbiota profiles were generated using 16S rRNA amplicon sequencing, and the abundance of the bacterial gene ribD was predicted. Eight of 28 bile samples could activate MAIT cells. This activation was partly MR1-dependent in five of eight bile samples. Microbial DNA was detected in 15 of 28 bile samples, including the five bile samples leading to MR1-dependent activation. A higher abundance of the ribD gene expression in the group of bile samples that could activate MAIT cells was predicted on the basis of the 16S sequencing. In co-culture experiments, cholangiocytes could take up and present biliary antigens to MAIT cells. These findings suggest a pathophysiological pathway in PSC connecting the immune system and the microbiome.

Decreased ubiquitin modifying enzyme A20 associated with hyper-responsiveness to ovalbumin challenge following intrauterine growth restriction.

BACKGROUND: Intrauterine growth restriction (IUGR) is strongly correlated with an increased risk of asthma later in life. Farm dust protects mice from developing house dust mite-induced asthma, and loss of ubiquitin modifying enzyme A20 in lung epithelium would abolish this protective effect. However, the mechanisms of A20 in the development of asthma following IUGR remains unknown. METHODS: An IUGR rat model induced by maternal nutrient restriction was used for investigating the role of A20 in the response characteristics of IUGR rats to ovalbumin (OVA) challenge. The ubiquitination of proteins and N6-methyladenosine (m6A) modifications were used to further assess the potential mechanism of A20. RESULTS: IUGR can reduce the expression of A20 protein in lung tissue of newborn rats and continue until 10 weeks after birth. OVA challenging can increase the expression of A20 protein in lung tissue of IUGR rats, but its level was still significantly lower than the control OVA group. The differentially ubiquitinated proteins in lung tissues were also observed in IUGR and normal newborn rats. Furthermore, this ubiquitination phenomenon continued from the newborn to adulthood. In the detected RNA methylations, m6A abundance of the motif GGACA was the highest. The higher abundances of m6A modification of A20 mRNA from IUGR were negatively correlated with the trend of A20 protein levels. CONCLUSION: These findings indicate A20 as a key regulator during the development of asthma following IUGR, providing further insight into the prevention of asthma induced by environmental factors.

Clinical nomogram assisting in discrimination of juvenile dermatomyositis-associated interstitial lung disease.

OBJECTIVE: To establish a prediction model using non-invasive clinical features for early discrimination of DM-ILD in clinical practice. METHOD: Clinical data of pediatric patients with JDM were retrospectively analyzed using machine learning techniques. The early discrimination model for JDM-ILD was established within a patient cohort diagnosed with JDM at a children's hospital between June 2015 and October 2022. RESULTS: A total of 93 children were included in the study, with the cohort divided into a discovery cohort (n = 58) and a validation cohort (n = 35). Univariate and multivariate analyses identified factors associated with JDM-ILD, including higher ESR (OR, 3.58; 95% CI 1.21-11.19, P = 0.023), higher IL-10 levels (OR, 1.19; 95% CI, 1.02-1.41, P = 0.038), positivity for MDA-5 antibodies (OR, 5.47; 95% CI, 1.11-33.43, P = 0.045). A nomogram was developed for risk prediction, demonstrating favorable discrimination in both the discovery cohort (AUC, 0.736; 95% CI, 0.582-0.868) and the validation cohort (AUC, 0.792; 95% CI, 0.585-0.930). Higher nomogram scores were significantly associated with an elevated risk of disease progression in both the discovery cohort (P = 0.045) and the validation cohort (P = 0.017). CONCLUSION: The nomogram based on the ESIM predictive model provides valuable guidance for the clinical evaluation and long-term prognosis prediction of JDM-ILD.

Preparation of cryopreserved chimeric antigen receptor T cells for the locoreogional delivery to the neural axis.

BACKGROUND AIMS: Intracranial (IC) locoregional delivery of chimeric antigen receptor (CAR) T cells presents an attractive delivery method to central nervous system tumors. Although IC delivery is actively being employed in early-phase clinical studies, no thaw/wash methods have been published to remove the neurotoxic cryoprotectant dimethyl sulfoxide (DMSO) from CAR T-cell products before IC administration. Thus, the aim of this study was to develop and validate a simple thaw/wash procedure. METHODS: We developed a thaw/wash procedure that consist of product thaw at 37°C, equilibration for 5 min in 1 volume of preservative-free normal saline (PFNS), dilution with an additional 8 volumes of PFNS, removal of DMSO through a washing step, resuspension in 2.0 mL of PFNS and storage in a syringe at 20-25°C. Final formulated products (FPs) were assessed for quality and safety attributes and stability over 3 h from the completion of the thaw. Stability parameters included CAR T-cell viability, transgene surface expression and cytolytic activity. RESULTS: The developed procedure reduced the calculated % of DMSO to less than 0.025%. FP cell viability and recovery (versus pre-cryopreservation) were within acceptable specifications (mean viability: 85.3%, range: 83%-88%; total nucleated cell recovery mean: 76.5%, range: 65.4%-82.5%). Other prespecified quality assurance/quality control parameters including appearance/ integrity, sterility and endotoxin level (≤1.0 EU/mL), were also met by all FPs (n = 3). Three hours' post thaw/wash stability was confirmed. All products maintained cell viability greater than 70% (mean, 80.0%; range, 79%-81%), with no significant change in transgene expression or cytolytic activity of B7-H3-CAR T cells compared with thawed not diluted/washed control CAR T cells. CONCLUSIONS: We have developed a simple thaw/wash procedure to prepare B7-H3-CAR T cells for their locoregional delivery to the neural axis. While we focus here on CAR T cells, the methods could be readily adapted to other cryopreserved immune effector cell products.

Prognostic value of Hematoxylin and eosin staining tumor-infiltrating lymphocytes (H&E-TILs) in patients with esophageal squamous cell carcinoma treated with chemoradiotherapy.

BACKGROUND: Tumor-infiltrating lymphocytes (TILs) by routine hematoxylin and eosin staining (H&E-TILs) are a robust prognostic biomarker in various cancers. However, the role of H&E-TILs in esophageal squamous cell carcinoma (ESCC) treated with concurrent chemoradiotherapy (CCRT) has not been reported. The purpose of this study was to assess the prognostic value of H&E-TILs in ESCC treated with CCRT. METHODS: The clinical data of 160 patients with ESCC treated with CCRT in our center between Jan. 2014 and Dec. 2021 were collected and retrospectively reviewed, and propensity score matching (PSM) analyses were performed. The H&E-TILs sections before CCRT were reassessed by two experienced pathologists independently. The H&E-TILs sections were classified into a positive group (+, > 10%) and a negative group (-, ≤ 10%) using 10% as the cutoff. The effects of H&E-TILs on overall survival (OS), progression-free survival (PFS), distant metastasis-free survival (DMFS), and locoregional recurrence-free survival (LRFS) were explored using the Kaplan‒Meier method, and the log-rank test was used to test the differences. Multivariable analysis was performed using the Cox proportion hazards model. RESULTS: The short-term response to CCRT and the OS (P < 0.001), DMFS (P = 0.001), and LRFS (P < 0.001) rates were significantly different between the H&E-TILs (+) and H&E-TILs (-) groups. Subgroup analysis showed that H&E-TILs(+) with CR + PR group had a longer survival than H&E-TILs(-) with CR + PR, H&E-TILs(+) with SD + PD and H&E-TILs(-) with SD + PD group, respectively(P < 0.001). Furthermore, based on TCGA data, patients in the high TILs group had a better prognosis than those in the low TILs group. Multivariate analyses indicated that H&E-TILs and the short-term response to CCRT were the only two independent factors affecting OS, PFS, DMFS, and LRFS simultaneously, and H&E-TILs expression was associated with an even better prognosis for those patients with CR + PR. CONCLUSIONS: H&E-TILs may be an effective and beneficial prognostic biomarker for ESCC patients treated with CCRT. Patients with H&E-TILs (+) with PR + CR would achieve excellent survival. Further prospective studies are required to validate the conclusions.