Photothermal treatment-based heat stress regulates function of myeloid-derived suppressor cells.

Photothermal therapy is an alternative cancer therapy that uses a photothermal agent with light irradiation to induce fatal hyperthermia in cancer cells. In a previous study, we found that ex vivo photothermal (PT) treatment induced expression of heat shock proteins (HSPs), such as HSP70, HSP27, and HSP90, in cancer cells; moreover, immunization with lysates from PT-treated tumor cells resulted in significant tumor growth inhibition in tumor-bearing mice. In this study, we hypothesized that sublethal PT treatment of antigen-presenting cells regulates their immunogenicity. We observed the upregulation of expression of intracellular HSP70 and surface activation markers, such as CD40, CD80, CD86, and MHC class II, in sublethal PT-treated cells. The protumoral activity of myeloid-derived suppressor cells (MDSCs) was reduced by sublethal hyperthermia. Furthermore, poorly immunogenic MDSCs were converted into immunogenic antigen-presenting cells by PT treatment. The differences in immunogenicity between MDSCs untreated or treated with the PT technique were evaluated using the Student's t-test or Mann-Whitney rank sum test. Collectively, direct hyperthermic treatment resulted in phenotypic changes and the functional regulation of immune cells.

HOS15-mediated turnover of PRR7 enhances freezing tolerance.

Arabidopsis PSEUDORESPONSE REGULATOR7 (PRR7) is a core component of the circadian oscillator which also plays a crucial role in freezing tolerance. PRR7 undergoes proteasome-dependent degradation to discretely phase maximal expression in early evening. While its repressive activity on downstream genes is integral to cold regulation, the mechanism of the conditional regulation of the PRR7 abundance is unknown. We used mutant analysis, protein interaction and ubiquitylation assays to establish that the ubiquitin ligase adaptor, HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE 15 (HOS15), controls the protein accumulation pattern of PRR7 through direct protein-protein interactions at low temperatures. Freezing tolerance and electrolyte leakage assays show that PRR7 enhances cold temperature sensitivity, supported by ChIP-qPCR at C-REPEAT BINDING FACTOR1 (CBF1) and COLD-REGULATED 15A (COR15A) promoters where PRR7 levels were higher in hos15 mutants. HOS15 mediates PRR7 turnover through enhanced ubiquitylation at low temperature in the dark. Under the same conditions, increased PRR7 association with the promoters of CBFs and COR15A in hos15 correlates with decreased CBF1 and COR15A transcription and enhanced freezing sensitivity. We propose a novel mechanism whereby HOS15-mediated degradation of PRR7 provides an intersection between the circadian system and other cold acclimation pathways that lead to increased freezing tolerance.

HOS15-mediated turnover of PRR7 enhances freezing tolerance.

Arabidopsis PSEUDO RESPONSE REGULATOR7 (PRR7) is a core component of the circadian oscillator which also plays a crucial role in freezing tolerance. PRR7 undergoes proteasome-dependent degradation to discretely phase maximal expression in early evening. While its transcriptional repressive activity on downstream genes is integral to cold regulation, the mechanism of the conditional regulation of the PRR7 protein activity is unknown. We used double mutant analysis, protein interaction and ubiquitylation assays to establish that the ubiquitin ligase adaptor, HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE 15 (HOS15), controls the protein accumulation pattern of PRR7 through direct protein-protein interactions. Freezing tolerance and electrolyte leakage assays show that PRR7 enhances cold temperature sensitivity, supported by ChIP-qPCR at C-REPEAT BINDING FACTOR (CBF) and COLD REGULATED 15A (COR15A) promoters where PRR7 levels were higher in hos15 mutants. We establish that HOS15 mediates PRR7 protein turnover through enhanced ubiquitylation at low temperature in the dark. Under the same conditions, increased PRR7 association with the promoter regions of CBFs and COR15A in hos15 correlates with decreased CBF1 and COR15A transcription and enhanced freezing sensitivity. We propose a novel mechanism whereby HOS15-mediated regulation of PRR7 provides an intersection between the circadian system and other cold acclimation pathways leading to freezing tolerance through upregulation of CBF1 and COR15A.

Genetic and clinical landscape of Chinese frontotemporal dementia: dominance of TBK1 and OPTN mutations.

BACKGROUND: Our study aims to evaluate the genetic and phenotypic spectrum of Frontotemporal dementia (FTD) gene variant carriers in Chinese populations, investigate mutation frequencies, and assess the functional properties of TBK1 and OPTN variants. METHODS: Clinically diagnosed FTD patients underwent genetic analysis through exome sequencing, repeat-primed polymerase chain reaction, and Sanger sequencing. TBK1 and OPTN variants were biologically characterized in vitro using immunofluorescence, immunoprecipitation, and immunoblotting analysis. The frequencies of genes implicated in FTD in China were analyzed through a literature review and meta-analysis. RESULTS: Of the 261 Chinese FTD patients, 61 (23.4%) carried potential causative variants in FTD-related genes, including MAPT (n = 17), TBK1 (n = 7), OPTN (n = 6), GRN (n = 6), ANXA11 (n = 4), CHMP2B (n = 3), C9orf72 GGGGCC repeats (n = 2), CYLD (n = 2), PRNP (n = 2), SQSTM1 (n = 2), TARDBP (n = 2), VCP (n = 1), CCNF (n = 1), CHCHD10 (n = 1), SIGMAR1 (n = 1), CHCHD2 (n = 1), FUS (n = 1), TMEM106B (n = 1), and UBQLN2 (n = 1). 29 variants can be considered novel, including the MAPT p.D54N, p.E342K, p.R221P, p.T263I, TBK1 p.E696G, p.I37T, p.E232Q, p.S398F, p.T78A, p.Q150P, p.W259fs, OPTN p.R144G, p.F475V, GRN p.V473fs, p.C307fs, p.R101fs, CHMP2B p.K6N, p.R186Q, ANXA11 p.Q155*, CYLD p.T157I, SQSTM1 p.S403A, UBQLN2 p.P509H, CCNF p.S160N, CHCHD10 p.A8T, SIGMAR1 p.S117L, CHCHD2 p.P53fs, FUS p.S235G & p.S236G, and TMEM106B p.L144V variants. Patients with TBK1 and OPTN variants presented with heterogeneous clinical phenotypes. Functional analysis demonstrated that TBK1 I37T and E232Q mutants showed decreased autophosphorylation, and the OPTN phosphorylation was reduced by the TBK1 I37T mutant. The OPTN-TBK1 complex formation was enhanced by the TBK1 E696G mutant, while OPTN R144G and F475V mutants exhibited reduced recruitment to autophagosomes compared to the wild-type. The overall frequency of TBK1 and OPTN in Chinese FTD patients was 2.0% and 0.3%, respectively. CONCLUSIONS: Our study demonstrates the extensive genetic and phenotypic heterogeneity of Chinese FTD patients. TBK1 mutations are the second most frequent cause of clinical FTD after MAPT in the Chinese.

A light-controlled phospholipase C for imaging of lipid dynamics and controlling neural plasticity.

Phospholipase C (PLC) is a key enzyme that regulates physiological processes via lipid and calcium signaling. Despite advances in protein engineering, no tools are available for direct PLC control. Here, we developed a novel optogenetic tool, light-controlled PLCß (opto-PLCß). Opto-PLCß uses a light-induced dimer module, which directs an engineered PLC to the plasma membrane in a light-dependent manner. Our design includes an autoinhibitory capacity, ensuring stringent control over PLC activity. Opto-PLCß triggers reversible calcium responses and lipid dynamics in a restricted region, allowing precise spatiotemporal control of PLC signaling. Using our system, we discovered that phospholipase D-mediated phosphatidic acid contributes to diacylglycerol clearance on the plasma membrane. Moreover, we extended its applicability in vivo, demonstrating that opto-PLCß can enhance amygdala synaptic plasticity and associative fear learning in mice. Thus, opto-PLCß offers precise spatiotemporal control, enabling comprehensive investigation of PLC-mediated signaling pathways, lipid dynamics, and their physiological consequences in vivo.

Machine learning model for circulating tumor DNA detection in chronic obstructive pulmonary disease patients with lung cancer.

Background: Patients with chronic obstructive pulmonary disease (COPD) have a high risk of developing lung cancer. Due to the high rates of complications from invasive diagnostic procedures in this population, detecting circulating tumor DNA (ctDNA) as a non-invasive method might be useful. However, clinical characteristics that are predictive of ctDNA mutation detection remain incompletely understood. This study aimed to investigate factors associated with ctDNA detection in COPD patients with lung cancer. Methods: Herein, 177 patients with COPD and lung cancer were prospectively recruited. Plasma ctDNA was genotyped using targeted deep sequencing. Comprehensive clinical variables were collected, including the emphysema index (EI), using chest computed tomography. Machine learning models were constructed to predict ctDNA detection. Results: At least one ctDNA mutation was detected in 54 (30.5%) patients. After adjustment for potential confounders, tumor stage, C-reactive protein (CRP) level, and milder emphysema were independently associated with ctDNA detection. An increase of 1% in the EI was associated with a 7% decrease in the odds of ctDNA detection (adjusted odds ratio =0.933; 95% confidence interval: 0.857-0.999; P=0.047). Machine learning models composed of multiple clinical factors predicted individuals with ctDNA mutations at high performance (AUC =0.774). Conclusions: ctDNA mutations were likely to be observed in COPD patients with lung cancer who had an advanced clinical stage, high CRP level, or milder emphysema. This was validated in machine learning models with high accuracy. Further prospective studies are required to validate the clinical utility of our findings.

Ex vivo photothermal treatment-induced immunogenic cell death for anticancer vaccine development.

Photothermal therapy is an anti-cancer strategy that induce cell death by converting light energy into heat energy. During photothermal therapy, cancer cells were treated with photothermal agents, such as indocyanine green, and irradiated with a laser. Heat stress in cancer cells results in cellular death and inflammatory responses. In the present study, we demonstrated how ex vivo photothermal (PT)-treated cells underwent immunogenic cell death. PT treatment caused significant expression of heat shock protein (HSP) 27, HSP70, and HSP90 in murine tumor cells. To evaluate the immunogenicity of heat-stressed cells, lysate from PT-treated tumor cells or water-based heated cells was pulsed to syngeneic bone-marrow-derived dendritic cells (DCs) to generate a DC-based vaccine. Administration with PT-treated tumor lysates-pulsed DC vaccine resulted in significant inhibition of tumor growth in BALB/c and C57BL/6 syngeneic tumor-bearing mice. The immunogenicity of PT-treated cancer cells was reduced in the presence of HSP inhibitors, J2, VER-155008 or 17-AAG. Our study elucidates how PT techniques have distinct mechanisms from water-based heating and might be a potentially robust and efficient solution to developing an anti-cancer vaccine.