Novel Dual-Emission Emitters Featuring Phenothiazine-S-Oxide and Phenothiazine-S,S-Dioxide Motifs.

In this study, we have successfully designed and synthesized two novel dual-emission emitters featuring phenothiazine-5-oxide and phenothiazine-5,5-dioxide motifs, characterized by highly lopsided and asymmetric conformational states. Through rigorous spectral examinations and DFT calculations, the compounds exhibit distinctive ICT phenomena, coupled with efficient emission in solid states and AIEE characteristics under high water fractions in DMF/H2O mixtures. These non-planar luminogens exhibit vibrant green and blue solid-state luminescence, with fluorescence quantum yields of 24.1% and 15.21%, respectively. Additionally, they both emit green fluorescence in THF solution, with notable emission quantum yields 36.4% and 30.4%. Comprehensive theoretical investigations unveil well-defined electron cloud density separation between the energies of HOMO/LUMO levels within the two luminogens. Notably, the targeted molecule harboring the phenothiazine-S,S-dioxide motif also demonstrates remarkable reversible mechanofluorochromic properties. Moreover, we testify their potential in applications such as solid-state rewritable information storage and live-cell imaging in solution states. Through theoretical calculations and comparative studies, we have explored the intrinsic relationship between molecular structure and performance, effectively screening and identifying new fluorescent molecules exhibiting outstanding luminescent attributes. These discoveries establish robust theoretical and technical foundation for the synthesis and application of efficient DSE-based MFC materials, opening new avenues in the realm of advanced luminescent materials.

GATA3 functions downstream of BRCA1 to promote DNA damage repair and suppress dedifferentiation in breast cancer.

BACKGROUND: Inadequate DNA damage repair promotes aberrant differentiation of mammary epithelial cells. Mammary luminal cell fate is mainly determined by a few transcription factors including GATA3. We previously reported that GATA3 functions downstream of BRCA1 to suppress aberrant differentiation in breast cancer. How GATA3 impacts DNA damage repair preventing aberrant cell differentiation in breast cancer remains elusive. We previously demonstrated that loss of p18, a cell cycle inhibitor, in mice induces luminal-type mammary tumors, whereas depletion of either Brca1 or Gata3 in p18 null mice leads to basal-like breast cancers (BLBCs) with activation of epithelial-mesenchymal transition (EMT). We took advantage of these mutant mice to examine the role of Gata3 as well as the interaction of Gata3 and Brca1 in DNA damage repair in mammary tumorigenesis. RESULTS: Depletion of Gata3, like that of Brca1, promoted DNA damage accumulation in breast cancer cells in vitro and in basal-like breast cancers in vivo. Reconstitution of Gata3 improved DNA damage repair in Brca1-deficient mammary tumorigenesis. Overexpression of GATA3 promoted homologous recombination (HR)-mediated DNA damage repair and restored HR efficiency of BRCA1-deficient cells. Depletion of Gata3 sensitized tumor cells to PARP inhibitor (PARPi), and reconstitution of Gata3 enhanced resistance of Brca1-deficient tumor cells to PARP inhibitor. CONCLUSIONS: These results demonstrate that Gata3 functions downstream of BRCA1 to promote DNA damage repair and suppress dedifferentiation in mammary tumorigenesis and progression. Our findings suggest that PARP inhibitors are effective for the treatment of GATA3-deficient BLBCs.

A Sensitive Technique Unravels the Kinetics of Activation and Trans-Cleavage of CRISPR-Cas Systems.

Activation of the CRISPR-Cas13a system requires the formation of a crRNA-Cas13a ribonucleoprotein (RNP) complex and the binding of an RNA activator to the RNP. These two binding processes play a crucial role in the performance of the CRISPR-Cas13a system. However, the binding kinetics remain poorly understood, and a main challenge is the lack of a sensitive method for real-time measurements of the dynamically formed active CRISPR-Cas13a enzyme. We describe here a new method to study the binding kinetics and report the rate constants (kon and koff) and dissociation constant (Kd) for the binding between Cas13a and its activator. The method is able to unravel and quantify the kinetics of binding and cleavage separately, on the basis of measuring the real-time trans-cleavage rates of the CRISPR-Cas system and obtaining the real-time concentrations of the active CRISPR-Cas ternary complex. We further discovered that once activated, the Cas13a system operates at a wide range of temperatures (7-37 °C) with fast trans-cleavage kinetics. The new method and findings are important for diverse applications of the Cas13a system, such as the demonstrated quantification of microRNA at ambient temperatures (e.g., 25 °C).

Glutamatergic Projection from the Ventral Tegmental Area to the Zona Incerta Regulates Fear Response.

Innate defensive behavior is important for animal survival. The Vglut2+ neurons in the ventral tegmental area (VTA) have been demonstrated to play important roles in innate defensive behaviors, but the neural circuit mechanism is still unclear. Here, we find that VTA - zona incerta (ZI) glutamatergic projection is involved in regulating innate fear responses. Combining calcium signal recording and chemogentics, we find that VTA-Vglut2+ neurons respond to foot shock stimulus. Inhibition of VTA-Vglut2+ neurons reduces foot shock-evoked freezing, while chemogentic activation of these neurons results in an enhanced fear response. Using viral tracing and immunofluorescence, we show that VTA - Vglut2+ neurons send direct excitatory outputs to the ZI. Moreover, we find that the activity of VTAVglut2 - ZI projection is pivotal in modulating fear response. Together, our study reveals a new VTA - ZI glutamatergic circuit in mediating innate fear response and provides a potential target for treating post-traumatic stress disorder.

Deficiency of FRMD5 results in neurodevelopmental dysfunction and autistic-like behavior in mice.

The pathophysiology of autism spectrum disorders (ASDs) is causally linked to postsynaptic scaffolding proteins, as evidenced by numerous large-scale genomic studies [1, 2] and in vitro and in vivo neurobiological studies of mutations in animal models [3, 4]. However, due to the distinct phenotypic and genetic heterogeneity observed in ASD patients, individual mutation genes account for only a small proportion (<2%) of cases [1, 5]. Recently, a human genetic study revealed a correlation between de novo variants in FERM domain-containing-5 (FRMD5) and neurodevelopmental abnormalities [6]. In this study, we demonstrate that deficiency of the scaffolding protein FRMD5 leads to neurodevelopmental dysfunction and ASD-like behavior in mice. FRMD5 deficiency results in morphological abnormalities in neurons and synaptic dysfunction in mice. Frmd5-deficient mice display learning and memory dysfunction, impaired social function, and increased repetitive stereotyped behavior. Mechanistically, tandem mass tag (TMT)-labeled quantitative proteomics revealed that FRMD5 deletion affects the distribution of synaptic proteins involved in the pathological process of ASD. Collectively, our findings delineate the critical role of FRMD5 in neurodevelopment and ASD pathophysiology, suggesting potential therapeutic implications for the treatment of ASD.

POH1 induces Smad3 deubiquitination and promotes lung cancer metastasis.

Smad3 is the key mediator of TGF-ß1-triggered signal transduction and the related biological responses, promoting cell invasion and metastasis in various cancers, including lung cancer. However, the deubiquitinase stabilizing Smad3 remains unknown. In this study, we present a paradigm in which POH1 is identified as a novel deubiquitinase of Smad3 that plays a tumor-promoting role in lung adenocarcinoma (LUAD) by regulating Smad3 stability. POH1 markedly increased Smad3 protein levels and prolonged its half-life. POH1 directly interacted and colocalized with Smad3, leading to the removal of poly-deubiquitination of Smad3. Functionally, POH1 facilitated cell proliferation, migration, and invasion by stabilizing Smad3. Importantly, POH1 also promoted liver metastasis of lung cancer cells. The protein levels of both POH1 and Smad3 were raised in the tumor tissues of patients with LUAD, which predicts poor prognosis. Collectively, we demonstrate that POH1 acts as an oncoprotein by enhancing TGF-ß1/Smad3 signaling and TGF-ß1-mediated metastasis of lung cancer.

Systematic pan-cancer analysis of RNF186 with potential implications in progression and prognosis in human cancer.

AIMS: Cancer remains a significant global public health issue. There is growing proof that Ring Finger Protein 186 (RNF186) may play a function in pan-cancer, however, this has not yet been thoroughly determined. This study aims to analyze RNF186 with potential implications in progression and prognosis in human cancer. MATERIALS AND METHODS: A comprehensive bioinformatics approaches combined with experimental verification were used across 33 types of cancers in this study to conduct a pan-cancer investigation of RNF186 from the perspectives of gene expression, prognosis, genomic alterations, immunological markers, gene set, and function. KEY FINDINGS: RNF186 is a valuable prognostic biomarker in several cancer types, especially breast invasive carcinoma (BRCA) and uterine corpus endometrial carcinoma (UCEC). The levels of RNF186 promoter methylation and genetic alterations may be responsible for some cancers' abnormal expression. Furthermore, RNF186 expression was determined to be associated with immune checkpoint genes. Analysis of RNF186-related genes revealed that proteasome and PI3K-AKT signaling pathway were primarily involved in the cellular function of RNF186. Additionally, our research first confirmed that RNF186 may function as an oncogene and contribute to cancer proliferation, migration and invasion in UCEC. In contrast, RNF186 may play an inhibitory role in BRCA progression. This function depends on the ligase activity of RNF186. SIGNIFICANCE: This study suggests that RNF186 is a novel critical target for tumor progression in BRCA and UCEC. It reveals that RNF186 may be associated with tumor immunotherapy, which may provide an effective predictive evaluation of the prognosis of immunotherapy.

Diallyl trisulfide improves spinal cord ischemia-reperfusion injury damage by activating AMPK to stabilize mitochondrial function.

BACKGROUND: Spinal cord ischemia-reperfusion injury (SCII) is a catastrophic event, which can cause paraplegia in severe cases. In the reperfusion stage, oxidative stress was up-regulated, which aggravated the injury and apoptosis of neurons. As the main active ingredient of garlic, diallyl trisulfide (DATS) displays strong antioxidant capacity. However, it is unknown whether DATS can protect the neurons of SCII. MATERIALS AND METHODS: In this study, the descending aorta at the distal end of the left subclavian artery was ligated and perfused again after 14 min. Samples including blood and spinal cord (L2-L5) were taken 24 h later for morphological and biochemical examination. RESULTS: After SCII, the rats showed motor dysfunction, increase apoptosis, malondialdehyde content, mitochondrial biogenesis and dynamic balance disorder. After the application of DATS, the adenosine monophosphate activated protein kinase (AMPK) was activated, the mitochondrial damage was improved, the oxidative stress was weakened, and the neuronal damage was recovered to some extent. However, the addition of compound C significantly weakened the protective effect of DATS. CONCLUSION: Oxidative stress caused by mitochondrial damage was one of the important mechanisms of neuronal damage in SCII. DATS could activate AMPK, stabilize mitochondrial biogenesis and dynamic balance, and reduce neuronal damage caused by oxidative stress.

Kindlin-2 in myoepithelium controls luminal progenitor commitment to alveoli in mouse mammary gland.

Myoepithelium plays an important role in mammary gland development, but less is known about the molecular mechanism underlying how myoepithelium controls acinus differentiation during gestation. Herein, we found that loss of Kindlin-2 in myoepithelial cells impaired mammary morphogenesis, alveologenesis, and lactation. Using five genetically modified mouse lines combined with single-cell RNA sequencing, we found a Kindlin-2-Stat3-Dll1 signaling cascade in myoepithelial cells that inactivates Notch signaling in luminal cells and consequently drives luminal progenitor commitment to alveolar cells identity. Single-cell profiling revealed that Kindlin-2 loss significantly reduces the proportion of matured alveolar cells. Mechanistically, Kindlin-2 depletion in myoepithelial cells promotes Stat3 activation and upregulates Dll1, which activates the Notch pathway in luminal cells and inhibits luminal progenitor differentiation and maturation during gestation. Inhibition of Notch1 with tangeretin allowed luminal progenitors to regain commitment ability in the pregnant mice with Kindlin-2 depletion in myoepithelium. Taken together, we demonstrated that Kindlin-2 is essential to myoepithelium-controlled luminal progenitors to alveoli transition during gestation.

Development of a DNAzyme Walker for the Detection of APE1 in Living Cancer Cells.

DNAzyme walker technology is a compelling option for bioanalytical and drug delivery applications. While nucleic acid and protein targets have been used to activate DNAzyme walkers, investigations into enzyme-triggered DNAzyme walkers in living cells are still in their early stages. The base excision repair (BER) pathway presents an array of enzymes that are overexpressed in cancer cells. Here, we introduce a DNAzyme walker system that sensitively and specifically detects the BER enzyme apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1). We constructed the DNAzyme walker on the surface of 20 nm-diameter gold nanoparticles. We achieved a detection limit of 160 fM of APE1 in a buffer and in whole cell lysate equivalent to the amount of APE1 in a single HeLa cell in a sample volume of 100 µL. Confocal imaging of the DNAzyme walking reveals a cytoplasmic distribution of APE1 in HeLa cells. Walking activity is tunable to exogenous Mn2+ concentrations and the uptake of the DNAzyme walker system does not require transfection assistance. We demonstrate the investigative potential of the DNAzyme walker for up-regulated or overactive enzyme biomarkers of the BER pathway in cancer cells.

FRMD8 targets both CDK4 activation and RB degradation to suppress colon cancer growth.

Cyclin-dependent kinase 4 (CDK4) and retinoblastoma protein (RB) are both important cell-cycle regulators that function in different scenarios. Here, we report that FERM domain-containing 8 (FRMD8) inhibits CDK4 activation and stabilizes RB, thereby causing cell-cycle arrest and inhibiting colorectal cancer (CRC) cell growth. FRMD8 interacts separately with CDK7 and CDK4, and it disrupts the interaction of CDK7 with CDK4, subsequently inhibiting CDK4 activation. FRMD8 competes with MDM2 to bind RB and attenuates MDM2-mediated RB degradation. Frmd8 deficiency in mice accelerates azoxymethane/dextran-sodium-sulfate-induced colorectal adenoma formation. The FRMD8 promoter is hypermethylated, and low expression of FRMD8 predicts poor prognosis in CRC patients. Further, we identify an LKCHE-containing FRMD8 peptide that blocks MDM2 binding to RB and stabilizes RB. Combined application of the CDK4 inhibitor and FRMD8 peptide leads to marked suppression of CRC cell growth. Therefore, using an LKCHE-containing peptide to interfere with the MDM2-RB interaction may have therapeutic value in CDK4/6 inhibitor-resistant patients.

Recent advances in RNA sample preparation techniques for the detection of SARS-CoV-2 in saliva and gargle.

Molecular detection of SARS-CoV-2 in gargle and saliva complements the standard analysis of nasopharyngeal swabs (NPS) specimens. Although gargle and saliva specimens can be readily obtained non-invasively, appropriate collection and processing of gargle and saliva specimens are critical to the accuracy and sensitivity of the overall analytical method. This review highlights challenges and recent advances in the treatment of gargle and saliva samples for subsequent analysis using reverse transcription polymerase chain reaction (RT-PCR) and isothermal amplification techniques. Important considerations include appropriate collection of gargle and saliva samples, on-site inactivation of viruses in the sample, preservation of viral RNA, extraction and concentration of viral RNA, removal of substances that inhibit nucleic acid amplification reactions, and the compatibility of sample treatment protocols with the subsequent nucleic acid amplification and detection techniques. The principles and approaches discussed in this review are applicable to molecular detection of other microbial pathogens.

Polymer optical fiber detector adapted for low-dose-rate gamma-ray detection.

In this Letter, a polymer optical fiber (POF) detector with a convex spherical aperture microstructure probe is designed for low-energy and low-dose rate gamma-ray detection. Simulation and experimental results demonstrate that this structure has a higher optical coupling efficiency and that the angular coherence of the detector depends strongly on the depth of the probe micro-aperture. By modeling the relationship between angular coherence and micro-aperture depth, the optimal depth of the micro-aperture is determined. The sensitivity of the fabricated POF detector is 701 cps at 59.5-keV gamma-ray of 2.78 µSv/h and the maximum percentage error of the average count rate at different angles is 5.16%.

FERM domain-containing protein FRMD6 activates the mTOR signaling pathway and promotes lung cancer progression.

FRMD6, a member of the 4.1 ezrin-radixin-moesin domain-containing protein family, has been reported to inhibit tumor progression in multiple cancers. Here, we demonstrate the involvement of FRMD6 in lung cancer progression. We find that FRMD6 is overexpressed in lung cancer tissues relative to in normal lung tissues. In addition, the enhanced expression of FRMD6 is associated with poor outcomes in patients with lung squamous cell carcinoma (n = 75, P = 0.0054) and lung adenocarcinoma (n = 94, P = 0.0330). Cell migration and proliferation in vitro and tumor formation in vivo are promoted by FRMD6 but are suppressed by the depletion of FRMD6. Mechanistically, FRMD6 interacts and colocalizes with mTOR and S6K, which are the key molecules of the mTOR signaling pathway. FRMD6 markedly enhances the interaction between mTOR and S6K, subsequently increasing the levels of endogenous pS6K and downstream pS6 in lung cancer cells. Furthermore, knocking out FRMD6 inhibits the activation of the mTOR signaling pathway in Frmd6-/- gene KO MEFs and mice. Altogether, our results show that FRMD6 contributes to lung cancer progression by activating the mTOR signaling pathway.

CRISPR techniques and potential for the detection and discrimination of SARS-CoV-2 variants of concern.

The continuing evolution of the SARS-CoV-2 virus has led to the emergence of many variants, including variants of concern (VOCs). CRISPR-Cas systems have been used to develop techniques for the detection of variants. These techniques have focused on the detection of variant-specific mutations in the spike protein gene of SARS-CoV-2. These sequences mostly carry single-nucleotide mutations and are difficult to differentiate using a single CRISPR-based assay. Here we discuss the specificity of the Cas9, Cas12, and Cas13 systems, important considerations of mutation sites, design of guide RNA, and recent progress in CRISPR-based assays for SARS-CoV-2 variants. Strategies for discriminating single-nucleotide mutations include optimizing the position of mismatches, modifying nucleotides in the guide RNA, and using two guide RNAs to recognize the specific mutation sequence and a conservative sequence. Further research is needed to confront challenges in the detection and differentiation of variants and sublineages of SARS-CoV-2 in clinical diagnostic and point-of-care applications.

Effects of Electronic Irradiation on the Characteristics of the Silicon Magnetic Sensitive Transistor.

This work researched the effects of irradiation on the current-voltage characteristics and voltage magnetic sensitivity of the silicon magnetic sensitive transistor (SMST). The 1-MeV electron irradiation source was used to irradiate the SMST. The irradiation fluences were 1 × 1012 e/cm2, 1 × 1013 e/cm2 and 1 × 1014 e/cm2, respectively (the irradiation flux was 1 × 1010 cm-2·s-1). The experimental results demonstrate that the collector current (IC) of the SMST occurs attenuation after irradiation under the same collector voltage (VCE) and the base current (IB). The attenuated rate of the IC increases obviously with the enhance of electron irradiation fluence when the IB is the same. Moreover, the attenuated rate of the IC increases slight with the rise of the IB when the electron irradiation fluence is the same. When the supply voltage is 5.0 V (RL = 1.5 kΩ) and the IB is 4.0 mA, the voltage magnetic sensitivity (SV) of the SMST occurs attenuate after irradiation. The attenuated rate of the SV increases with the enhance of electron irradiation fluence.

Synthesis, structure and mechanofluorochromic properties of phenothiazine-S-oxide and phenothiazine-S,S-dioxide derivatives.

In this work, two novel luminescent molecules containing distorted phenothiazine-S-oxide and phenothiazine-S,S dioxide skeletons were synthesized by oxidation reactions using different oxidants (m-chloroperoxybenzoic acid, acetic acid /hydrogen peroxide). The target compounds were all confirmed by 1H NMR, 13C NMR and EI-MS. Combined with the results of UV-vis absorption spectra and fluorescence emission spectra, we found that the different oxidation states of S-atom, from sulfide (+2) to sulfoxide (+4) and sulfone (+6), led to the blue, yellow-green and yellowish fluorescence of these compounds in the solid states. Subsequent studies showed that the molecule containing the phenothiazine-S-oxide skeleton exhibited obvious solvatochromism, and the increase of solvent polarity induced a red-shift in the emission wavelength. Moreover, this molecule also exhibited a rare self-recovery mechanochromatic behavior. In addition, these properties were further confirmed by theoretical calculations and X-ray single-crystal diffraction analysis.

Single cell profiling of primary and paired metastatic lymph node tumors in breast cancer patients.

The microenvironment of lymph node metastasized tumors (LNMT) determines tumor progression and response to therapy, but a systematic study of LNMT is lacking. Here, we generate single-cell maps of primary tumors (PTs) and paired LNMTs in 8 breast cancer patients. We demonstrate that the activation, cytotoxicity, and proliferation of T cells are suppressed in LNMT compared with PT. CD4+CXCL13+ T cells in LNMT are more likely to differentiate into an exhausted state. Interestingly, LAMP3+ dendritic cells in LNMT display lower T cell priming and activating ability than in PT. Additionally, we identify a subtype of PLA2G2A+ cancer-associated fibroblasts enriched in HER2+ breast cancer patients that promotes immune infiltration. We also show that the antigen-presentation pathway is downregulated in malignant cells of the metastatic lymph node. Altogether, we characterize the microenvironment of LNMT and PT, which may shed light on the individualized therapeutic strategies for breast cancer patients with lymph node metastasis.