Whole-genome CRISPR screening identifies molecular mechanisms of PD-L1 expression in adult T-cell leukemia/lymphoma.

ABSTRACT: Adult T-cell leukemia/lymphoma (ATLL) is an aggressive T-cell malignancy with a poor prognosis and limited treatment options. Programmed cell death ligand 1(PD-L1) is recognized to be involved in the pathobiology of ATLL. However, what molecules control PD-L1 expression and whether genetic or pharmacological intervention might modify PD-L1 expression in ATLL cells are still unknown. To comprehend the regulatory mechanisms of PD-L1 expression in ATLL cells, we performed unbiased genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screening in this work. In ATLL cells, we discovered that the neddylation-associated genes NEDD8, NAE1, UBA3, and CUL3 negatively regulated PD-L1 expression, whereas STAT3 positively did so. We verified, in line with the genetic results, that treatment with the JAK1/2 inhibitor ruxolitinib or the neddylation pathway inhibitor pevonedistat resulted in a decrease in PD-L1 expression in ATLL cells or an increase in it, respectively. It is significant that these results held true regardless of whether ATLL cells had the PD-L1 3' structural variant, a known genetic anomaly that promotes PD-L1 overexpression in certain patients with primary ATLL. Pevonedistat alone showed cytotoxicity for ATLL cells, but compared with each single modality, pevonedistat improved the cytotoxic effects of the anti-PD-L1 monoclonal antibody avelumab and chimeric antigen receptor (CAR) T cells targeting PD-L1 in vitro. As a result, our work provided insight into a portion of the complex regulatory mechanisms governing PD-L1 expression in ATLL cells and demonstrated the in vitro preliminary preclinical efficacy of PD-L1-directed immunotherapies by using pevonedistat to upregulate PD-L1 in ATLL cells.

Analysis and therapeutic targeting of the IL-1R pathway in anaplastic large cell lymphoma.

Anaplastic large cell lymphoma (ALCL), a subgroup of mature T-cell neoplasms with an aggressive clinical course, is characterized by elevated expression of CD30 and anaplastic cytology. To achieve a comprehensive understanding of the molecular characteristics of ALCL pathology and to identify therapeutic vulnerabilities, we applied genome-wide CRISPR library screenings to both anaplastic lymphoma kinase positive (ALK+) and primary cutaneous (pC) ALK- ALCLs and identified an unexpected role of the interleukin-1R (IL-1R) inflammatory pathway in supporting the viability of pC ALK- ALCL. Importantly, this pathway is activated by IL-1α in an autocrine manner, which is essential for the induction and maintenance of protumorigenic inflammatory responses in pC-ALCL cell lines and primary cases. Hyperactivation of the IL-1R pathway is promoted by the A20 loss-of-function mutation in the pC-ALCL lines we analyze and is regulated by the nonproteolytic protein ubiquitination network. Furthermore, the IL-1R pathway promotes JAK-STAT3 signaling activation in ALCLs lacking STAT3 gain-of-function mutation or ALK translocation and enhances the sensitivity of JAK inhibitors in these tumors in vitro and in vivo. Finally, the JAK2/IRAK1 dual inhibitor, pacritinib, exhibited strong activities against pC ALK- ALCL, where the IL-1R pathway is hyperactivated in the cell line and xenograft mouse model. Thus, our studies revealed critical insights into the essential roles of the IL-1R pathway in pC-ALCL and provided opportunities for developing novel therapeutic strategies.

The RNA-binding motif protein 14 regulates telomere integrity at the interface of TERRA and telomeric R-loops.

Telomeric repeat-containing RNA (TERRA) and its formation of RNA:DNA hybrids (or TERRA R-loops), influence telomere maintenance, particularly in human cancer cells that use homologous recombination-mediated alternative lengthening of telomeres. Here, we report that the RNA-binding motif protein 14 (RBM14) is associated with telomeres in human cancer cells. RBM14 negatively regulates TERRA expression. It also binds to TERRA and inhibits it from forming TERRA R-loops at telomeres. RBM14 depletion has several effects, including elevated TERRA levels, telomeric R-loops, telomere dysfunction-induced DNA damage foci formation, particularly in the presence of DNA replication stress, pRPA32 accumulation at telomeres and telomere signal-free ends. Thus, RBM14 protects telomere integrity via modulating TERRA levels and its R-loop formation at telomeres.

Yersinia ruckeri Infection and Enteric Redmouth Disease among Endangered Chinese Sturgeons, China, 2022.

During October 2022, enteric redmouth disease (ERM) affected Chinese sturgeons at a farm in Hubei, China, causing mass mortality. Affected fish exhibited characteristic red mouth and intestinal inflammation. Investigation led to isolation of a prominent bacterial strain, zhx1, from the internal organs and intestines of affected fish. Artificial infection experiments confirmed the role of zhx1 as the pathogen responsible for the deaths. The primary pathologic manifestations consisted of degeneration, necrosis, and inflammatory reactions, resulting in multiple organ dysfunction and death. Whole-genome sequencing of the bacteria identified zhx1 as Yersinia ruckeri, which possesses 135 drug-resistance genes and 443 virulence factor-related genes. Drug-susceptibility testing of zhx1 demonstrated high sensitivity to chloramphenicol and florfenicol but varying degrees of resistance to 18 other antimicrobial drugs. Identifying the pathogenic bacteria associated with ERM in Chinese sturgeons establishes a theoretical foundation for the effective prevention and control of this disease.

Genome-wide CRISPR screens identify CD48 defining susceptibility to NK cytotoxicity in peripheral T-cell lymphomas.

Adult T-cell leukemia/lymphoma (ATLL) is one of the aggressive peripheral T-cell neoplasms with a poor prognosis. Accumulating evidence demonstrates that escape from adaptive immunity is a hallmark of ATLL pathogenesis. However, the mechanisms by which ATLL cells evade natural killer (NK)-cell-mediated immunity have been poorly understood. Here we show that CD48 expression in ATLL cells determines the sensitivity for NK-cell-mediated cytotoxicity against ATLL cells. We performed unbiased genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screening using 2 ATLL-derived cell lines and discovered CD48 as one of the best-enriched genes whose knockout conferred resistance to YT1-NK cell line-mediated cytotoxicity. The ability of CD48-knockout ATLL cells to evade NK-cell effector function was confirmed using human primary NK cells with reduced interferon-γ (IFNγ) induction and degranulation. We found that primary ATLL cells had reduced CD48 expression along with disease progression. Furthermore, other subgroups among aggressive peripheral T-cell lymphomas (PTCLs) also expressed lower concentrations of CD48 than normal T cells, suggesting that CD48 is a key molecule in malignant T-cell evasion of NK-cell surveillance. Thus, this study demonstrates that CD48 expression is likely critical for malignant T-cell lymphoma cell regulation of NK-cell-mediated immunity and provides a rationale for future evaluation of CD48 as a molecular biomarker in NK-cell-associated immunotherapies.

Genome-wide CRISPR screen identifies CDK6 as a therapeutic target in adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/lymphoma (ATLL) is an aggressive T-cell malignancy with a poor prognosis with current therapy. Here we report genome-wide CRISPR-Cas9 screening of ATLL models, which identified CDK6, CCND2, BATF3, JUNB, STAT3, and IL10RB as genes that are essential for the proliferation and/or survival of ATLL cells. As a single agent, the CDK6 inhibitor palbociclib induced cell cycle arrest and apoptosis in ATLL models with wild-type TP53. ATLL models that had inactivated TP53 genetically were relatively resistant to palbociclib owing to compensatory CDK2 activity, and this resistance could be reversed by APR-246, a small molecule activator of mutant TP53. The CRISPR-Cas9 screen further highlighted the dependence of ATLL cells on mTORC1 signaling. Treatment of ATLL cells with palbociclib in combination with mTORC1 inhibitors was synergistically toxic irrespective of the TP53 status. This work defines CDK6 as a novel therapeutic target for ATLL and supports the clinical evaluation of palbociclib in combination with mTORC1 inhibitors in this recalcitrant malignancy.

Effects of phoxim on antibacterial infection of silver carp.

The efficacy of phoxim in treating bacterial sepsis in silver carp is significant, yet its underlying mechanism remains elusive. This study aimed to establish a model of Aeromonas veronii infection in silver carp and subsequently treat the infected fish with 10 µg/L phoxim. Kidney and intestine samples from silver carp were collected for transcriptome analysis and assessment of intestinal microbial composition, with the aim of elucidating the mechanism underlying the efficacy of phoxim in treating bacterial sepsis in silver carp. The results of transcriptome and intestinal microbial composition analysis of silver carp kidney indicated that A. veronii infection could up-regulate the expression of il1ß, il6, nos2, ctsl, casp3 et al., which means, signifying that the kidney of silver carp would undergo inflammation, induce apoptosis, and alter the composition of intestinal microorganisms. Phoxim immersion might enhance the energy metabolism of silver carp and change its intestinal microbial composition, potentially elevating the antibacterial infection resistance of silver carp. These findings may contribute to an understanding of how phoxim can effectively treat bacterial sepsis in silver carp.

Targeting inflammatory macrophages rebuilds therapeutic efficacy of DOT1L inhibition in hepatocellular carcinoma.

Epigenetic reprogramming is a promising therapeutic strategy for aggressive cancers, but its limitations in vivo remain unclear. Here, we showed, in detailed studies of data regarding 410 patients with human hepatocellular carcinoma (HCC), that increased histone methyltransferase DOT1L triggered epithelial-mesenchymal transition-mediated metastasis and served as a therapeutic target for human HCC. Unexpectedly, although targeting DOT1L in vitro abrogated the invasive potential of hepatoma cells, abrogation of DOT1L signals hardly affected the metastasis of hepatoma in vivo. Macrophages, which constitute the major cellular component of the stroma, abrogated the anti-metastatic effect of DOT1L targeting. Mechanistically, NF-κB signal elicited by macrophage inflammatory response operated via a non-epigenetic machinery to eliminate the therapeutic efficacy of DOT1L targeting. Importantly, therapeutic strategy combining DOT1L-targeted therapy with macrophage depletion or NF-κB inhibition in vivo effectively and successfully elicited cancer regression. Moreover, we found that the densities of macrophages in HCC determined malignant cell DOT1L-associated clinical outcome of the patients. Our results provide insight into the crosstalk between epigenetic reprogramming and cancer microenvironments and suggest that strategies to influence the functional activities of inflammatory cells may benefit epigenetic reprogramming therapy.

Mechanism of sturgeon intestinal inflammation induced by Yersinia ruckeri and the effect of florfenicol intervention.

The mechanism by which Y. ruckeri infection induces enteritis in Chinese sturgeon remains unclear, and the efficacy of drug prevention and control measures is not only poor but also plagued with numerous issues. We conducted transcriptomic and 16 S rRNA sequencing analyses to examine the differences in the intestinal tract of hybrid sturgeon before and after Y. ruckeri infection and florfenicol intervention. Our findings revealed that Y. ruckeri induced the expression of multiple inflammatory factors, including il1ß, il6, and various chemokines, as well as casp3, casp8, and multiple tumor necrosis factor family members, resulting in pathological injury to the body. Additionally, at the phylum level, the relative abundance of Firmicutes and Bacteroidota increased, while the abundance of Plesiomonas and Cetobacterium decreased at the genus level, altering the composition of the intestinal flora. Following florfenicol intervention, the expression of multiple apoptosis and inflammation-related genes was down-regulated, promoting tissue repair. However, the flora became further dysregulated, increasing the risk of infection. In conclusion, our analysis of the transcriptome and intestinal microbial composition demonstrated that Y. ruckeri induces intestinal pathological damage by triggering apoptosis and altering the composition of the intestinal microbiota. Florfenicol intervention can repair pathological damage, but it also exacerbates flora imbalance, leading to a higher risk of infection. These findings help elucidate the molecular mechanism of Y. ruckeri-induced enteritis in sturgeon and evaluate the therapeutic effect of drugs on intestinal inflammation in sturgeon.

Efficacy and Safety of Ferric Citrate on Hyperphosphatemia among Chinese Patients with Chronic Kidney Disease Undergoing Hemodialysis: A Phase III Multicenter Randomized Open-Label Active-Drug-Controlled Study.

INTRODUCTION: Hyperphosphatemia in chronic kidney disease (CKD) patients is positively associated with mortality. Ferric citrate is a potent phosphorus binder that lowers serum phosphorus level and improves iron metabolism. We compared its efficacy and safety with active drugs in Chinese CKD patients with hemodialysis. METHODS: Chinese patients undergoing hemodialysis were randomized into two treatment groups in a 1:1 ratio, receiving either ferric citrate or sevelamer carbonate, respectively, for 12 weeks. Serum phosphorus levels, calcium concentration, and iron metabolism parameters were evaluated every 2 weeks. Frequency and severity of adverse events were recorded. RESULTS: 217 (90.4%) patients completed the study with balanced demographic and baseline characteristics between two groups. Ferric citrate decreased the serum phosphorus level to 0.59 ± 0.54 mmol/L, comparable to 0.56 ± 0.62 mmol/L by sevelamer carbonate. There was no significant difference between two groups (p > 0.05) in the proportion of patients with serum phosphorus levels reaching the target range, the response rate to the study drug, and the changes of corrected serum calcium concentrations, and intact-PTH levels at the end of treatment. The change of iron metabolism indicators in the ferric citrate group was significantly higher than those in the sevelamer carbonate group. There are 47 (40.5%) patients in the ferric citrate group, and 26 (21.3%) patients in the sevelamer carbonate group experienced drug-related treatment emergent adverse events (TEAEs); most were mild and tolerable. Common drug-related TEAEs were gastrointestinal disorders, including diarrhea (12.9 vs. 2.5%), fecal discoloration (14.7 vs. 0%), and constipation (1.7 vs. 7.4%) in ferric citrate and sevelamer carbonate group. CONCLUSION: Ferric citrate capsules have good efficacy and safety in the control of hyperphosphatemia in adult patients with CKD undergoing hemodialysis. Efficacy is not inferior to sevelamer carbonate. The TEAEs were mostly mild and tolerated by the patients.

Comparative analysis of the transcriptional responses of Acetilactobacillus jinshanensis BJ01 to organic acids.

As the important functional microorganism in the brewing process of Chinese Baijiu, lactic acid bacteria influences the microbial community and production of flavor substances in the Baijiu brewing process. In this study, we first isolated an Acetilactobacillus jinshanensis strain from baijiu fermented grains and named it A. jinshanensis BJ01. Its optimal growth conditions are 30 °C and pH 3.5. In particular, A. jinshanensis BJ01 cannot utilize inorganic acids and most organic acids, except for lactic acid (HL) and acetic acid (HAc). The observed phenotypes showed good growth with HL. When the mixed acid of HL-HAc (V:V = 1:1) was used, the growth rate of A. jinshanensis BJ01 greatly accelerated. Transcriptomic sequencing revealed the specific responses of the strain to the acidulants used. The number of upregulated genes in HL-HAc medium was more than that in single acid medium (HL or HAc). KEGG enrichment analyses indicated that the glycometabolism level of HAc regulation was relatively downregulated. The gene expression of quorum sensing and ABC transporter pathways were remarkably upregulated under HL-HAc regulation. Pyruvate metabolic pathway may be an important reason for the difference in A. jinshanensis BJ01 response to different organic acids. Our study reported a new organic acid-inducible growth type of bacteria mainly depending on the presence of HL and HAc, and was beneficial to the improvement of fermentation technology of Baijiu.

Effects of diets rich in Agaricus bisporus polysaccharides on the growth, antioxidant, immunity, and resistance to Yersinia ruckeri in channel catfish.

To promote the application of Agaricus bisporus polysaccharides (ABPs) in channel catfish (Ictalurus punctatus) culture, we evaluated the effects of ABPs on the growth, immunity, antioxidant, and antibacterial activity of channel catfish. When the amount of ABPs was 250 mg/kg, channel catfish's weight gain and specific growth rates increased significantly while the feed coefficient decreased. We also found that adding ABPs in the feed effectively increased the activities of ACP, MDA, T-SOD, AKP, T-AOC, GSH, and CAT enzymes and immune-related genes such as IL-1ß, Hsp70, and IgM in the head kidney of channel catfish. Besides, long-term addition will not cause pathological damage to the head kidney. When the amount of ABPs was over 125 mg/kg, the protection rate of channel catfish was more than 60%. According to the intestinal transcriptome analysis, the addition of ABPs promoted the expression of intestinal immunity genes and growth metabolism-related genes and enriched multiple related KEEG pathways. When challenged by Yersinia ruckeri infection, the immune response of channel catfish fed with ABPs was intenser and quicker. Additionally, the 16S rRNA gene sequencing analysis showed that the composition of the intestinal microbial community of channel catfish treated with ABPs significantly changed, and the abundance of microorganisms beneficial to channel catfish growth, such as Firmicutes and Bacteroidota increased. In conclusion, feeding channel catfish with ABPs promoted growth, enhanced immunity and antioxidant, and improved resistance to bacterial infections. Our current results might promote the use of ABPs in channel catfish and even other aquacultured fish species.

Effect of Agaricus bisporus Polysaccharides (ABPs) on anti-CCV immune response of channel catfish.

Herein, the effects of Agaricus bisporus Polysaccharides (ABPs) on anti-channel catfish virus (CCV) infections to promote their application in channel catfish culture were explored. Transcriptome and metabolome analyses were conducted on the spleen of a CCV-infected channel catfish model fed with or without ABPs. CCV infections upregulated many immune and apoptosis-related genes, such as IL-6, IFN-α3, IFN-γ1, IL-26, Casp3, Casp8, and IL-10, and activated specific immunity mediated by B cells. However, after adding ABPs, the expression of inflammation-related genes decreased in CCV-infected channel catfish, and the inflammatory inhibitors NLRC3 were upregulated. Meanwhile, the expression of apoptosis-related genes was reduced, indicating that ABPs can more rapidly and strongly enhance the immunity of channel catfish to resist viral infection. Moreover, the metabonomic analysis showed that channel catfish had a high energy requirement during CCV infection, and ABPs could enhance the immune function of channel catfish. In conclusion, ABPs can enhance the antiviral ability of channel catfish by enhancing immune response and regulating inflammation. Thus, these findings provided new insights into the antiviral response effects of ABPs, which might support their application in aquaculture.

Essential role of the linear ubiquitin chain assembly complex and TAK1 kinase in A20 mutant Hodgkin lymphoma.

More than 70% of Epstein-Barr virus (EBV)-negative Hodgkin lymphoma (HL) cases display inactivation of TNFAIP3 (A20), a ubiquitin-editing protein that regulates nonproteolytic protein ubiquitination, indicating the significance of protein ubiquitination in HL pathogenesis. However, the precise mechanistic roles of A20 and the ubiquitination system remain largely unknown in this disease. Here, we performed high-throughput CRISPR screening using a ubiquitin regulator-focused single-guide RNA library in HL lines carrying either wild-type or mutant A20. Our CRISPR screening highlights the essential oncogenic role of the linear ubiquitin chain assembly complex (LUBAC) in HL lines, which overlaps with A20 inactivation status. Mechanistically, LUBAC promotes IKK/NF-κB activity and NEMO linear ubiquitination in A20 mutant HL cells, which is required for prosurvival genes and immunosuppressive molecule expression. As a tumor suppressor, A20 directly inhibits IKK activation and HL cell survival via its C-terminal linear-ubiquitin binding ZF7. Clinically, LUBAC activity is consistently elevated in most primary HL cases, and this is correlated with high NF-κB activity and low A20 expression. To further understand the complete mechanism of NF-κB activation in A20 mutant HL, we performed a specifically designed CD83-based NF-κB CRISPR screen which led us to identify TAK1 kinase as a major mediator for NF-κB activation in cells dependent on LUBAC, where the LUBAC-A20 axis regulates TAK1 and IKK complex formation. Finally, TAK1 inhibitor Takinib shows promising activity against HL in vitro and in a xenograft mouse model. Altogether, these findings provide strong support that targeting LUBAC or TAK1 could be attractive therapeutic strategies in A20 mutant HL.

Dual RNA-seq of spleens extracted from channel catfish infected with Aeromonas veronii reveals novel insights into host-pathogen interactions.

Interactions between host and pathogen are involving various dynamic changes in transcript expression and critical for understanding host immunity against infections and its associated pathogenesis. Herein, we established a model of channel catfish infected with Aeromonas veronii. The infected fish had prominent body surface bleeding, and the spleen showed hyperemia and swelling. Then, the spleen of channel catfish infected with A. veronii was analyzed by dual RNA sequencing (RNA-seq), and the transcriptome data were compared with uninfected channel catfish spleen or bacteria cultured in vitro. The transcript expression profile of pathogen-host interaction between A. veronii and channel catfish was successfully studied. During infection, the host was enriched for multiple immune-related signaling pathways, such as the Toll-like receptor signaling pathway, Cytokine-cytokine receptor interaction, and T cell receptor signaling pathway; and significantly upregulated for many innate immune-related genes, including IL-8. At the same time, we found that A. veronii mainly harmed the host spleen through hemolysin. Our current findings are of great significance in clarifying the pathogenesis of channel catfish induced by A. veronii and provide gene targets for developing preventive measures.

The plasma and tissue kinetics of sulfadiazine and its metabolite in Ictalurus punctatus after oral gavage at two temperatures.

A plasma and tissue kinetic study of sulfadiazine (SDZ) and its metabolite, N4 -acetyl sulfadiazine (ACT-SDZ), was characterized in channel catfish (Ictalurus punctatus) following a single oral dose of 50 mg/kg at 18 and 24°C. Samples were collected at predetermined time points and determined by ultra-performance liquid chromatography. The classical one-compartmental method was used to estimate the pharmacokinetic parameters. Results showed that the changing of temperature was markedly influential on the kinetics of SDZ and ACT-SDZ in plasma and tissues. When the temperature was increased from 18 to 24°C, the elimination half-life (K10_HF) of SDZ was decreased in gill, kidney, and muscle + skin, but increased in liver and plasma. The K10_HF of ACT-SDZ also had a decreased trend in gill, liver, and plasma but had comparable values in kidney and muscle + skin. The absorption half-life (K01_HF), time to peak concentration (Tmax ), and area under concentration-time curve (AUC0-∞ ) of SDZ and ACT-SDZ all exhibited declined tendencies in plasma and tissues. The apparent volume of distribution (V_F) of SDZ in plasma was increased from 0.53 to 1.48 L/kg, and the apparent systemic total body clearance (Cl_F) was increased from 0.028 to 0.060 L/h/kg. In a word, K01_HF, Tmax , and AUC0-∞ of SDZ and ACT-SDZ were decreased in plasma and tissues with the increase of temperature, whereas the V_F and Cl_F of SDZ were increased. Meanwhile, we calculated the percentage of time profile of SDZ concentration more than minimum inhibitory concentration to total time (%T > MIC) to guide clinical usage of SDZ. When the dosage interval was 24 h, the values of %T > MIC were all >90% in plasma and most tissues. Therefore, we recommend an oral dose of SDZ at 50 mg/kg once per 24 h at 18-24°C against the fish pathogens with an MIC value of ≤6.4 µg/mL.

Enhanced Reactive Brilliant Blue Removal Using Chitosan-Biochar Hydrogel Beads.

To address the challenges associated with the weak affinity and difficult separation of biochar, we developed chitosan-biochar hydrogel beads (CBHBs) as an efficient solution for removing reactive brilliant blue (RBB KN-R) from wastewater. The adsorption behavior and mechanism of RBB KN-R onto CBHBs were extensively studied. Notably, the adsorption capacity of RBB KN-R showed pH-dependence, and the highest adsorption capacity was observed at pH 2. The adsorption process was well fitted with the pseudo-second-order kinetic model and the intraparticle diffusion model. Film diffusion and intraparticle diffusion were both responsible for the adsorption of RBB KN-R onto CBHBs. At 298.15 K, the maximum adsorption capacity qm was determined to be 140.74 mg/g, with higher temperatures favoring the adsorption process. A complex mechanism involving π-π interactions, electrostatic attraction, hydrophobic interaction, and hydrogen bonding was found to contribute to the overall adsorption process. The experimental data discovered the coexisting substances and elevated ionic strength hindered the adsorption capacity. Significantly, after three cycles of adsorption-desorption, the CBHBs maintained an adsorption capacity above 95% for RBB KN-R. These promising results imply that CBHBs are a durable and cost-effective adsorbent for efficient removal of dyes from wastewater.

CD30 Regulation of IL-13-STAT6 Pathway in Breast Implant-Associated Anaplastic Large Cell Lymphoma.

BACKGROUND: Breast implant-associated anaplastic large cell lymphoma (BIA-ALCL) is a rare, usually indolent CD30+ T-cell lymphoma with tumor cells, often surrounded by eosinophils, expressing IL-13 and pSTAT6. OBJECTIVES: The aim of this study was to understand the unique tumor pathology and growth regulation of BIA-ALCL, leading to potential targeted therapies. METHODS: We silenced CD30 and analyzed its effect on IL-13 signaling and tumor cell viability. IL-13 signaling receptors of BIA-ALCL cell lines were evaluated by flow cytometry and pSTAT6 detected by immunohistochemistry. CD30 was deleted by CRISPR/Cas9 editing. Effects of CD30 deletion on transcription of IL-13 and IL-4, and phosphorylation of STAT6 were determined by real-time polymerase chain reaction and western blotting. The effect of CD30 deletion on p38 mitogen-activated protein kinase (MAPK) phosphorylation was determined. Suppression of IL-13 transcription by a p38 MAPK inhibitor was tested. Tumor cell viability following CD30 deletion and treatment with a pSTAT6 inhibitor were measured in cytotoxicity assays. RESULTS: BIA-ALCL lines TLBR1 and TLBR2 displayed signaling receptors IL-4Rα, IL-13Rα1 and downstream pSTAT6. Deletion of CD30 by CRISPR/Cas9 editing significantly decreased transcription of IL-13, less so Th2 cytokine IL-4, and phosphorylation of STAT6. Mechanistically, we found CD30 expression is required for p38 MAPK phosphorylation and activation, and IL-13-STAT6 signaling was reduced by an inhibitor of p38 MAPK in BIA-ALCL tumor cells. Tumor cell viability was decreased by silencing of CD30, and a specific inhibitor of STAT6, indicating STAT6 inhibition is cytotoxic to BIA-ALCL tumor cells. CONCLUSIONS: These findings suggest reagents targeting the IL-13 pathway, pSTAT6 and p38 MAPK, may become useful for treating BIA-ALCL patients.

Non-Layered Te/In2 S3 Tunneling Heterojunctions with Ultrahigh Photoresponsivity and Fast Photoresponse.

A photodetector based on 2D non-layered materials can easily utilize the photogating effect to achieve considerable photogain, but at the cost of response speed. Here, a rationally designed tunneling heterojunction fabricated by vertical stacking of non-layered In2 S3 and Te flakes is studied systematically. The Te/In2 S3 heterojunctions possess type-II band alignment and can transfer to type-I or type-III depending on the electric field applied, allowing for tunable tunneling of the photoinduced carriers. The Te/In2 S3 tunneling heterojunction exhibits a reverse rectification ratio exceeding 104 , an ultralow forward current of 10-12 A, and a current on/off ratio over 105 . A photodetector based on the heterojunctions shows an ultrahigh photoresponsivity of 146 A W-1 in the visible range. Furthermore, the devices exhibit a response time of 5 ms, which is two and four orders of magnitude faster than that of its constituent In2 S3 and Te. The simultaneously improved photocurrent and response speed are attributed to the direct tunneling of the photoinduced carriers, as well as a combined mechanism of photoconductive and photogating effects. In addition, the photodetector exhibits a clear photovoltaic effect, which can work in a self-powered mode.

A Multicancer Malignant Pleural Effusion Diagnostic Test Using Hexokinase 2 and Single-Cell Sequencing.

BACKGROUND: Malignant pleural effusion (MPE) represents advanced malignant disease with poor prognosis. To date, pleural effusion cytology remains the best test to diagnose MPE but suffers from limited diagnostic sensitivity and high variation. We report a hexokinase 2-based method (HK2-seq) as a novel diagnostic method for multicancer MPE diagnosis. METHODS: HK2-seq employed HK2 as a new metabolic function-associated marker to detect disseminated tumor cells engaging increased glycolysis in pleural effusion from many cancer types. Single-cell sequencing was used to confirm the malignancy of HK2-derived high glycolytic tumor cells (hgTCs) at the single-cell level via surveying genome-wide copy number alterations (CNAs), leading to establishment of definitive MPE diagnosis. RESULTS: In a prospective cohort study including 111 patients with pleural effusion, the HK2 test showed diagnostic sensitivity, diagnostic specificity, positive predictive value, and negative predictive value of 91% (95% CI: 80%-97%), 84% (95% CI: 68%-93%), 90% (95% CI: 79%-96%), and 86% (95% CI: 70%-95%), respectively, in MPE diagnosis across 12 different cancer types. In contrast, pleural effusion cytology exhibits an overall diagnostic sensitivity of 45%. In addition to confirming the tumor origin of hgTCs, single-cell sequencing allowed identification of prognostic or targetable CNAs in hgTCs, especially CNAs found in liquid biopsies but absent in solid biopsies. CONCLUSIONS: HK2-seq establishes definitive MPE diagnosis across many cancer types with high diagnostic performance. It has the potential to be used for multicancer detection of circulating tumor cells in blood and other types of body fluids, as well as liquid biopsy-based genomic characterization for informative diagnosis.