Augmenting CAR T Cell Functions with LIGHT.

Chimeric antigen receptor (CAR) T-cell therapy has resulted in remarkable clinical success in the treatment of B-cell malignancies. However, its clinical efficacy in solid tumors is limited, primarily by target antigen heterogeneity. To overcome antigen heterogeneity, we developed CAR T cells that overexpress LIGHT, a ligand of both LTßR on cancer cells and HVEM on immune cells. LIGHT-expressing CAR T cells displayed both antigen-directed cytotoxicity mediated by the CAR and antigen-independent killing mediated through the interaction of LIGHT with LTßR on cancer cells. Moreover, CAR T cells expressing LIGHT had immunostimulatory properties that improved the cells' proliferation and cytolytic profile. These data indicate that LIGHT-expressing CAR T cells may provide a way to eliminate antigen-negative tumor cells to prevent antigen-negative disease relapse.

Development of a water-soluble fluorescent Al3+ probe based on phenylsulfonyl-2-pyrone in biological systems.

BACKGROUND: Al exists naturally in the environment and is an important component in acidic soils, which harm almost all plants. Furthermore, Al is widely used in food additives, cosmetics, and medicines, resulting in living organisms ingesting traces of Al orally or dermally every day. Accordingly, Al accumulates in the body, which can cause negative bioeffects and diseases, and this concern is gaining increasing attention. Therefore, to detect and track Al in the environment and in living organisms, the development of novel Al-selective probes that are water-soluble and exhibit fluorescence at long wavelengths is necessary. RESULTS: In this study, an Al3+-selective fluorescent probe PSP based on a novel pyrone molecule was synthesized and characterized to detect and track Al in biological systems. PSP exhibited fluorescence enhancement at 580 nm in the presence of Al3+ in aqueous media. Binding analysis using Job's plot and structural analysis using 1H NMR showed that PSP formed a 1:1 complex with Al3+ at the two carbonyl groups of the dimethyl malonate of the pyrone ring. Upon testing in biological systems, PSP showed good cell membrane permeability, detected intracellular Al3+ in human breast cancer cells (MDA-MB-231), and successfully imaged accumulated Al3+ in Microcystis aeruginosa and the larvae of Rheocricotopus species. SIGNIFICANCE: The novel Al3+-selective fluorescent probe PSP is highly effective and is expected to aid in elucidating the role of Al3+ in the environment and living organisms.

Cytogenotoxicity of raw and treated dairy manure slurry by two-stage chemical and electrocoagulation: An application of the Allium cepa bioassay.

Livestock farming is an essential agricultural practice. However, the improper management of livestock wastes and discharge of untreated or partially treated livestock manure slurry poses significant environmental problems. In this study, we aimed to compare the cytogenotoxic potential of untreated and treated dairy manure slurry treated with a two-stage chemical and electrocoagulation (EC) using the Allium cepa bioassay. The A. cepa bioassay is a well-established standard tool for assessing the cytogenotoxic effects of environmental contaminants, especially those that are occurred as complex contaminant mixtures. The dairy manure slurry was subjected to chemical treatment utilizing polyaluminum chloride (PAC) and cationic polyacrylamide (CPAM) at optimized conditions, followed by EC utilizing either aluminum (Al) or steel anodes. The treated and untreated samples were then evaluated for their potential cytogenotoxicty using the A. cepa bioassay, by measuring the nuclear abnormalities (NAs) and chromosomal aberrations (CAs), along with the mitotic indices (MIs). Our findings revealed a significant reduction in cytogenotoxic indicators in the treated liquid fraction compared to the untreated dairy manure slurry. Specifically, the frequency of total NAs showed a significant reduction from 154 ‰ to 37 ‰ when the dairy manure slurry was treated with chemical coagulation followed by EC utilizing an Al anode. Moreover, the MI exhibited a significant improvement from 7 ‰ to 123 ‰, suggesting the mitigation of toxic effects. These results collectively demonstrate the effectiveness of the two-stage chemical and EC treatment under optimal conditions in treating diary manure slurry while reducing its cytogenotoxicity for living systems. The A. cepa bioassay proved to be a sensitive and reliable method for assessing the toxicity of the treated samples. The efficient solid-liquid separation and the reduction of toxicity in the liquid fraction for biological systems achieved through this treatment process highlight its potential for sustainable management of livestock waste and the preservation of water quality. Nevertheless, further studies are required to assess the toxicity of solid fraction.

Toxicity of triclosan, an antimicrobial agent, to a nontarget freshwater zooplankton species, Moina macrocopa.

The toxicity of triclosan (TCS) on the freshwater cladoceran Moina macrocopa was investigated by acute and chronic toxicity assessments followed by genotoxicity and oxidative stress response analyses. The 48-h LC50 of TCS for ≤24-h-old M. macrocopa was determined as 539 µg L-1 . Chronic exposure to TCS at concentrations ranging from 5 to 100 µg L-1 showed a stimulatory effect at low concentrations (≤10 µg L-1 ) and an inhibitory effect at high concentrations (≥50 µg L-1 ) on growth, reproduction, and population-growth-related parameters of M. macrocopa. The genotoxicity test results indicated that TCS concentrations ranging from 50 to 100 µg L-1 can alter individuals' DNA. Analysis of the antioxidant enzymes catalase (CAT) and glutathione s-transferase (GST) demonstrated increased levels of these enzymes at high TCS concentrations. Our results indicated that TCS concentrations found in the natural environment have minimal acute toxicity to M. macrocopa. However, TCS at even low concentrations can significantly affect its growth, reproduction, and population-growth-related characteristics. The observed responses suggest a hormetic dose-response pattern and imply a potential endocrine-disrupting effect of TCS. Our molecular and biochemical findings indicated that high concentrations of TCS have the potential to induce oxidative stress that may lead to DNA alterations in M. macrocopa.

Efficient selection of a biallelic and nonchimeric gene-edited tree using Oxford Nanopore Technologies sequencing.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 nuclease system is a versatile and essential biotechnological tool in the life sciences that allows efficient genome editing. When generating gene-edited trees, T0-generation plants are often used for subsequent analysis because of the time that is required to obtain the desired mutants via crossing. However, T0-generation plants exhibit various unexpected mutations, which emphasizes the need to identify mutants with expected mutation patterns. The two critical checkpoints in this process are to confirm the expected mutation patterns in both alleles and to exclude somatic chimeric plants. In this study, we generated gene-edited Cryptomeria japonica plants and established a method to determine chimerism and mutation patterns using fragment analysis and Oxford Nanopore Technologies (ONT)-based amplicon sequencing. In the first screening, fragment analysis, i.e., indel detection via amplicon analysis, was used to predict indel mutation patterns in both alleles and to discriminate somatic chimeric plants in 188 candidate mutants. In the second screening, we precisely determined the mutation patterns and chimerism in the mutants using ONT-based amplicon sequencing, where confirmation of both alleles can be achieved using allele-specific markers flanking the single guide RNA target site. In the present study, a bioinformatic analysis procedure was developed and provided for the rapid and accurate determination of DNA mutation patterns using ONT-based amplicon sequencing. As ONT amplicon sequencing has a low running cost compared with other long-read analysis methods, such as PacBio, it is a powerful tool in plant genetics and biotechnology to select gene-edited plants with expected indel patterns in the T0-generation.

Egeria densa organic extracts: an eco-friendly approach to suppress Microcystis aeruginosa growth through allelopathy.

Macrophytes play a significant role in shaping plankton communities by shading, controlling water turbulence, and nutrient availability, while also producing allelochemicals causing varying effects on different organisms. Many researchers have shown that when live macrophytes are present, they inhibit cyanobacteria. However, their widespread use is often limited due to numerous concerns, including invasive characteristics. This study focused on the applicability of Egeria densa extracts to suppress Microcystis aeruginosa. We employed pure water and dimethyl sulfoxide, to obtain compounds from E. densa. The results revealed that E. densa aqueous extracts stimulated M. aeruginosa growth, whereas organic extracts exhibited suppression. Specifically, at low concentrations of organics extracts (0.5 and 1 g/L), after day 4, the growth inhibition was confirmed by significantly higher (p < 0.05) stress levels shown in cells treated with low concentrations. The highest inhibition rate of 32% was observed at 0.5 g/L. However, high concentrations of organic extracts (3 and 6 g/L), showed increased growth compared with control. These results suggest that high concentrations of organic extracts from E. densa potentially suppress allelochemical-induced M. aeruginosa inhibition due to high nutrient availability. In comparison with an aqueous solvent, the use of organic solvent seems to be more effective in efficiently extracting allelochemicals from E. densa.

A single-nucleotide substitution of CjTKPR1 determines pollen production in the gymnosperm plant Cryptomeria japonica.

Pollinosis, also known as pollen allergy or hay fever, is a global problem caused by pollen produced by various plant species. The wind-pollinated Japanese cedar (Cryptomeria japonica) is the largest contributor to severe pollinosis in Japan, where increasing proportions of people have been affected in recent decades. The MALE STERILITY 4 (MS4) locus of Japanese cedar controls pollen production, and its homozygous mutants (ms4/ms4) show abnormal pollen development after the tetrad stage and produce no mature pollen. In this study, we narrowed down the MS4 locus by fine mapping in Japanese cedar and found TETRAKETIDE α-PYRONE REDUCTASE 1 (TKPR1) gene in this region. Transformation experiments using Arabidopsis thaliana showed that single-nucleotide substitution ("T" to "C" at 244-nt position) of CjTKPR1 determines pollen production. Broad conservation of TKPR1 beyond plant division could lead to the creation of pollen-free plants not only for Japanese cedar but also for broader plant species.

Backdoor Attack on Deep Neural Networks Triggered by Fault Injection Attack on Image Sensor Interface.

A backdoor attack is a type of attack method that induces deep neural network (DNN) misclassification. The adversary who aims to trigger the backdoor attack inputs the image with a specific pattern (the adversarial mark) into the DNN model (backdoor model). In general, the adversary mark is created on the physical object input to an image by capturing a photo. With this conventional method, the success of the backdoor attack is not stable because the size and position change depending on the shooting environment. So far, we have proposed a method of creating an adversarial mark for triggering backdoor attacks by means of a fault injection attack on the mobile industry processor interface (MIPI), which is the image sensor interface. We propose the image tampering model, with which the adversarial mark can be generated in the actual fault injection to create the adversarial mark pattern. Then, the backdoor model was trained with poison data images, which the proposed simulation model created. We conducted a backdoor attack experiment using a backdoor model trained on a dataset containing 5% poison data. The clean data accuracy in normal operation was 91%; nevertheless, the attack success rate with fault injection was 83%.

Response surface optimization of chemical coagulation for solid-liquid separation of dairy manure slurry through Box-Behnken design with desirability function.

Discharging livestock manure slurry without proper treatment causes various environmental and sociological problems. Chemical coagulation is a widely used and easily applicable method for treating such wastewater. However, the technique requires optimization to enhance coagulation efficiency while minimizing chemical usage. In this study, we propose an efficient, low-cost, and environmentally safe chemical coagulation method for solid-liquid separation of dairy manure slurry. Experiments were conducted in laboratory jar tests using dairy manure slurry to investigate the impact of coagulants, specifically polyaluminum chloride (PAC) and cationic polyacrylamide (CPAM), as well as pH, on the process of solid-liquid separation. Preliminary ranges of PAC, CPAM, and pH were estimated through single-factor experiments. Coagulation optimization and modeling were performed using the response surface methodology (RSM) with the Box-Behnken design (BBD), wherein the desired goal of each parameter was set to maximize solid-liquid separation efficiency while reducing chemical dosage to maintain residual aluminum (Al) concentrations below water quality standards. Numerical optimization predicted that the optimal dosages were 75 mg/L of PAC and 35 mg/L of CPAM at pH 7. Under these conditions, removal efficiencies of 99% for turbidity and 97% for chemical oxygen demand (COD) were achieved, with a minimal residual Al concentration of 0.045 mg/L. Positive zeta potential values in the treated water confirmed complete separation of negatively charged solids in the dairy manure slurry. The response values predicted by BBD aligned with the experimental results, and the analysis of variance (ANOVA) demonstrated the predictability and accuracy of the response models. Consequently, this study highlights the practical application of RSM with BBD in optimizing chemical coagulation using PAC and CPAM to achieve efficient solid-liquid separation in livestock wastewater while maintaining low residual Al concentrations.

Modulation of phytotoxic and cytogenetic effects of cadmium by humic acid: Findings from a short-term plant-based bioassay.

The study of the modulation of the toxicity of heavy metals by coexisting chemicals in the environment is vital for realistic ecological risk assessment. Our study was aimed at determining possible toxicity modulations of Cd by humic acid (HA) using the Allium cepa test system. A. cepa bulbs were exposed to Cd (1 and 5 mg/L) and HA (10 mg/L) individually or in mixtures. The root lengths of the bulbs and cytogenetic endpoints in root meristematic cells, including the mitotic index (MI), nuclear abnormalities (NAs), and chromosomal abnormalities (CAs), were determined. The results revealed that the MIs of A. cepa co-exposed to HA and Cd were significantly recovered by >15% compared with those of A. cepa subjected to Cd-only treatments, and this response was more sensitive than the phytotoxic response (root length). Furthermore, the burden of NAs was significantly decreased in the co-exposed bulbs by >20% compared with bulbs with Cd-only treatments. The frequencies of CAs were also reduced in the bulbs co-exposed to HA and 1 and 5 mg/L Cd by >15 and >25%, respectively, compared with bulbs receiving Cd-only treatments. Therefore, our findings indicated that HA plays a significant protective role in Cd toxicity in A. cepa.

New readers and writers of RNA modifications unique to leukemia stem cells.

Chemical modifications of RNA are regulated by a series of readers, writers, and erasers that dictate gene expression. Two new studies in Cell Stem Cell1,2identify roles for the N6-methyladenosine (m6A) methyltransferase METTL16 and the m6A reader IGF2BP2 in leukemia-initiating cells, illuminating exciting new therapeutic targets for leukemia.

IMPDH inhibition activates TLR-VCAM1 pathway and suppresses the development of MLL-fusion leukemia.

Inosine monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme in de novo guanine nucleotide synthesis pathway. Although IMPDH inhibitors are widely used as effective immunosuppressants, their antitumor effects have not been proven in the clinical setting. Here, we found that acute myeloid leukemias (AMLs) with MLL-fusions are susceptible to IMPDH inhibitors in vitro. We also showed that alternate-day administration of IMPDH inhibitors suppressed the development of MLL-AF9-driven AML in vivo without having a devastating effect on immune function. Mechanistically, IMPDH inhibition induced overactivation of Toll-like receptor (TLR)-TRAF6-NF-κB signaling and upregulation of an adhesion molecule VCAM1, which contribute to the antileukemia effect of IMPDH inhibitors. Consequently, combined treatment with IMPDH inhibitors and the TLR1/2 agonist effectively inhibited the development of MLL-fusion AML. These findings provide a rational basis for clinical testing of IMPDH inhibitors against MLL-fusion AMLs and potentially other aggressive tumors with active TLR signaling.

Mechanisms involved in hematopoietic stem cell aging.

Hematopoietic stem cells (HSCs) undergo progressive functional decline over time due to both internal and external stressors, leading to aging of the hematopoietic system. A comprehensive understanding of the molecular mechanisms underlying HSC aging will be valuable in developing novel therapies for HSC rejuvenation and to prevent the onset of several age-associated diseases and hematological malignancies. This review considers the general causes of HSC aging that range from cell-intrinsic factors to cell-extrinsic factors. In particular, epigenetics and inflammation have been implicated in the linkage of HSC aging, clonality, and oncogenesis. The challenges in clarifying mechanisms of HSC aging have accelerated the development of therapeutic interventions to rejuvenate HSCs, the major goal of aging research; these details are also discussed in this review.

[Pathophysiology of hematological malignancies associated with ASXL1 mutations].

Somatic mutations in the epigenetic regulator ASXL1 are considered a poor prognostic factor in myeloid malignancies, including myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). ASXL1 mutations coexist with other mutations in majority of patients, suggesting that its mutation alone is insufficient to cause cancer. ASXL1 mutations have been detected in age-related clonal hematopoiesis (CH), which has been linked to an increased risk of hematological malignancies. Therefore, ASXL1 mutations are likely to be one of the first events in the tumorigenesis process. With our most recent findings, we summarize the mechanisms by which ASXL1 mutations cause CH and hematological malignancies in this review.

High-Affinity Ratiometric Fluorescence Probe Based on 6-Amino-2,2'-Bipyridine Scaffold for Endogenous Zn2+ and Its Application to Living Cells.

Zinc is an essential trace element involved in many biological activities; however, its functions are not fully understood. To elucidate the role of endogenous labile Zn2+, we developed a novel ratiometric fluorescence probe, 5-(4-methoxyphenyl)-4-(methylsulfanyl)-[2,2'-bipyridin]-6-amine (6 (rBpyZ)) based on the 6-amino-2,2'-bipyridine scaffold, which acts as both the chelating agent for Zn2+ and the fluorescent moiety. The methoxy group acted as an electron donor, enabling the intramolecular charge transfer state of 6 (rBpyZ), and a ratiometric fluorescence response consisting of a decrease at the emission wavelength of 438 nm and a corresponding increase at the emission wavelength of 465 nm was observed. The ratiometric probe 6 (rBpyZ) exhibited a nanomolar-level dissociation constant (Kd = 0.77 nM), a large Stokes shift (139 nm), and an excellent detection limit (0.10 nM) under physiological conditions. Moreover, fluorescence imaging using A549 human lung adenocarcinoma cells revealed that 6 (rBpyZ) had good cell membrane permeability and could clearly visualize endogenous labile Zn2+. These results suggest that the ratiometric fluorescence probe 6 (rBpyZ) has considerable potential as a valuable tool for understanding the role of Zn2+ in living systems.

CHIP-associated mutant ASXL1 in blood cells promotes solid tumor progression.

Clonal hematopoiesis of indeterminate potential (CHIP) is an age-associated phenomenon characterized by clonal expansion of blood cells harboring somatic mutations in hematopoietic genes, including DNMT3A, TET2, and ASXL1. Clinical evidence suggests that CHIP is highly prevalent and associated with poor prognosis in solid-tumor patients. However, whether blood cells with CHIP mutations play a causal role in promoting the development of solid tumors remained unclear. Using conditional knock-in mice that express CHIP-associated mutant Asxl1 (Asxl1-MT), we showed that expression of Asxl1-MT in T cells, but not in myeloid cells, promoted solid-tumor progression in syngeneic transplantation models. We also demonstrated that Asxl1-MT-expressing blood cells accelerated the development of spontaneous mammary tumors induced by MMTV-PyMT. Intratumor analysis of the mammary tumors revealed the reduced T-cell infiltration at tumor sites and programmed death receptor-1 (PD-1) upregulation in CD8+ T cells in MMTV-PyMT/Asxl1-MT mice. In addition, we found that Asxl1-MT induced T-cell dysregulation, including aberrant intrathymic T-cell development, decreased CD4/CD8 ratio, and naïve-memory imbalance in peripheral T cells. These results indicate that Asxl1-MT perturbs T-cell development and function, which contributes to creating a protumor microenvironment for solid tumors. Thus, our findings raise the possibility that ASXL1-mutated blood cells exacerbate solid-tumor progression in ASXL1-CHIP carriers.

Short-duration exposure of 3-µm polystyrene microplastics affected morphology and physiology of watermilfoil (sp. roraima).

Microplastics are one of the most widely discussed environmental issues worldwide. Several studies have shown the effect of microplastic exposure on the marine environment; however, studies on freshwater systems are lacking. This study was conducted to investigate the effect of microplastics on hydroponically growing emergent freshwater macrophytes, watermilfoil (sp. roraima) under controlled environmental conditions. Plants were exposed to 0 mg L-1 (control), 0.05 mg L-1, 0.25 mg L-1, 1.25 mg L-1, and 6 mg L-1 of 3-µm polystyrene microspheres for 7 days. The oxidative stress, antioxidant response, pigmentations, Fv/Fm, and growth parameters in the above-water and below-water parts were analyzed separately. Microscopic observations were performed to confirm the tissue absorbance of the microplastics. Exposure to microplastics altered some parameters; however, growth was not affected. The effect of microplastics was not linear with the exposure concentration for most of the parameters and between 1.25 and 6 mg L-1 concentrations. The response trends mostly followed the second-order polynomial distributions. Under the 1.25 mg L-1 exposure, there were significant changes in root length, H2O2 content, catalase activity, anthocyanin content, and Fv/Fm. There were differences in parameters between the above-water and below-water parts, and the responses of the microplastics followed different trends. Microscopic observations confirmed the attachment of microplastic particles onto newly formed roots, except for older roots or shoot tissues.

Area-Efficient Post-Processing Circuits for Physically Unclonable Function with 2-Mpixel CMOS Image Sensor.

In order to realize image information security starting from the data source, challenge-response (CR) device authentication, based on a Physically Unclonable Function (PUF) with a 2 Mpixel CMOS image sensor (CIS), is studied, in which variation of the transistor in the pixel array is utilized. As each CR pair can be used only once to make the CIS PUF resistant to the modeling attack, CR authentication with CIS can be carried out 4050 times, with basic post-processing to generate the PUF ID. If a larger number of authentications is required, advanced post-processing using Lehmer encoding can be utilized to carry out authentication 14,858 times. According to the PUF performance evaluation, the authentication error rate is less than 0.001 ppm. Furthermore, the area overhead of the CIS chip for the basic and advanced post-processing is only 1% and 2%, respectively, based on a Verilog HDL model circuit design.

A histone modifier, ASXL1, interacts with NONO and is involved in paraspeckle formation in hematopoietic cells.

Paraspeckles are membraneless organelles formed through liquid-liquid phase separation and consist of multiple proteins and RNAs, including NONO, SFPQ, and NEAT1. The role of paraspeckles and the component NONO in hematopoiesis remains unknown. In this study, we show histone modifier ASXL1 is involved in paraspeckle formation. ASXL1 forms phase-separated droplets, upregulates NEAT1 expression, and increases NONO-NEAT1 interactions through the C-terminal intrinsically disordered region (IDR). In contrast, a pathogenic ASXL mutant (ASXL1-MT) lacking IDR does not support the interaction of paraspeckle components. Furthermore, paraspeckles are disrupted and Nono localization is abnormal in the cytoplasm of hematopoietic stem and progenitor cells (HSPCs) derived from ASXL1-MT knockin mice. Nono depletion and the forced expression of cytoplasmic NONO impair the repopulating potential of HSPCs, as does ASXL1-MT. Our study indicates a link between ASXL1 and paraspeckle components in the maintenance of normal hematopoiesis.