Optophysiology: Illuminating cell physiology with optogenetics.

Optogenetics combines light and genetics to enable precise control of living cells, tissues, and organisms with tailored functions. Optogenetics has the advantages of noninvasiveness, rapid responsiveness, tunable reversibility, and superior spatiotemporal resolution. Following the initial discovery of microbial opsins as light-actuated ion channels, a plethora of naturally occurring or engineered photoreceptors or photosensitive domains that respond to light at varying wavelengths has ushered in the next chapter of optogenetics. Through protein engineering and synthetic biology approaches, genetically encoded photoswitches can be modularly engineered into protein scaffolds or host cells to control a myriad of biological processes, as well as to enable behavioral control and disease intervention in vivo. Here, we summarize these optogenetic tools on the basis of their fundamental photochemical properties to better inform the chemical basis and design principles. We also highlight exemplary applications of opsin-free optogenetics in dissecting cellular physiology (designated "optophysiology") and describe the current progress, as well as future trends, in wireless optogenetics, which enables remote interrogation of physiological processes with minimal invasiveness. This review is anticipated to spark novel thoughts on engineering next-generation optogenetic tools and devices that promise to accelerate both basic and translational studies.

Engineering of NEMO as calcium indicators with large dynamics and high sensitivity.

Genetically encoded calcium indicators (GECIs) are indispensable tools for real-time monitoring of intracellular calcium signals and cellular activities in living organisms. Current GECIs face the challenge of suboptimal peak signal-to-baseline ratio (SBR) with limited resolution for reporting subtle calcium transients. We report herein the development of a suite of calcium sensors, designated NEMO, with fast kinetics and wide dynamic ranges (>100-fold). NEMO indicators report Ca2+ transients with peak SBRs around 20-fold larger than the top-of-the-range GCaMP6 series. NEMO sensors further enable the quantification of absolution calcium concentration with ratiometric or photochromic imaging. Compared with GCaMP6s, NEMOs could detect single action potentials in neurons with a peak SBR two times higher and a median peak SBR four times larger in vivo, thereby outperforming most existing state-of-the-art GECIs. Given their high sensitivity and resolution to report intracellular Ca2+ signals, NEMO sensors may find broad applications in monitoring neuronal activities and other Ca2+-modulated physiological processes in both mammals and plants.

LncRNA CamK-A Regulates Ca2+-Signaling-Mediated Tumor Microenvironment Remodeling.

Cancer cells entail metabolic adaptation and microenvironmental remodeling to survive and progress. Both calcium (Ca2+) flux and Ca2+-dependent signaling play a crucial role in this process, although the underlying mechanism has yet to be elucidated. Through RNA screening, we identified one long noncoding RNA (lncRNA) named CamK-A (lncRNA for calcium-dependent kinase activation) in tumorigenesis. CamK-A is highly expressed in multiple human cancers and involved in cancer microenvironment remodeling via activation of Ca2+-triggered signaling. Mechanistically, CamK-A activates Ca2+/calmodulin-dependent kinase PNCK, which in turn phosphorylates IκBα and triggers calcium-dependent nuclear factor κB (NF-κB) activation. This regulation results in the tumor microenvironment remodeling, including macrophage recruitment, angiogenesis, and tumor progression. Notably, our human-patient-derived xenograft (PDX) model studies demonstrate that targeting CamK-A robustly impaired cancer development. Clinically, CamK-A expression coordinates with the activation of CaMK-NF-κB axis, and its high expression indicates poor patient survival rate, suggesting its role as a potential biomarker and therapeutic target.

Optogenetics for transcriptional programming and genetic engineering.

Optogenetics combines genetics and biophotonics to enable noninvasive control of biological processes with high spatiotemporal precision. When engineered into protein machineries that govern the cellular information flow as depicted in the central dogma, multiple genetically encoded non-opsin photosensory modules have been harnessed to modulate gene transcription, DNA or RNA modifications, DNA recombination, and genome engineering by utilizing photons emitting in the wide range of 200-1000 nm. We present herein generally applicable modular strategies for optogenetic engineering and highlight latest advances in the broad applications of opsin-free optogenetics to program transcriptional outputs and precisely manipulate the mammalian genome, epigenome, and epitranscriptome. We also discuss current challenges and future trends in opsin-free optogenetics, which has been rapidly evolving to meet the growing needs in synthetic biology and genetics research.

Differential Lipid Binding Specificities of RAP1A and RAP1B are Encoded by the Amino Acid Sequence of the Membrane Anchors.

RAP1 proteins belong to the RAS family of small GTPases that operate as molecular switches by cycling between GDP-bound inactive and GTP-bound active states. The C-terminal anchors of RAP1 proteins are known to direct membrane localization, but how these anchors organize RAP1 on the plasma membrane (PM) has not been investigated. Using high-resolution imaging, we show that RAP1A and RAP1B form spatially segregated nanoclusters on the inner leaflet of the PM, with further lateral segregation between GDP-bound and GTP-bound proteins. The C-terminal polybasic anchors of RAP1A and RAP1B differ in their amino acid sequences and exhibit different lipid binding specificities, which can be modified by single-point mutations in the respective polybasic domains (PBD). Molecular dynamics simulations reveal that single PBD mutations substantially reduce the interactions of the membrane anchors with the PM lipid phosphatidylserine. In summary, we show that aggregate lipid binding specificity encoded within the C-terminal anchor determines PM association and nanoclustering of RAP1A and RAP1B. Taken together with previous observations on RAC1 and KRAS, the study reveals that the PBD sequences of small GTPase membrane anchors can encode distinct lipid binding specificities that govern PM interactions.

TRIM14 Inhibits cGAS Degradation Mediated by Selective Autophagy Receptor p62 to Promote Innate Immune Responses.

Cyclic GMP-AMP synthase (cGAS) is an essential DNA virus sensor that triggers type I interferon (IFN) signaling by producing cGAMP to initiate antiviral immunity. However, post-translational regulation of cGAS remains largely unknown. We report that K48-linked ubiquitination of cGAS is a recognition signal for p62-depdendent selective autophagic degradation. The induction of TRIM14 by type I IFN accelerates cGAS stabilization by recruiting USP14 to cleave the ubiquitin chains of cGAS at lysine (K) 414. Knockout of TRIM14 impairs herpes simplex virus type 1 (HSV-1)-triggered antiviral responses in a cGAS-dependent manner. Due to impaired type I IFN production, Trim14-/- mice are highly susceptible to lethal HSV-1 infection. Taken together, our findings reveal a positive feedback loop of cGAS signaling generated by TRIM14-USP14 and provide insights into the crosstalk between autophagy and type I IFN signaling in innate immunity.

Optical control of protein delivery and partitioning in the nucleolus.

The nucleolus is a subnuclear membraneless compartment intimately involved in ribosomal RNA synthesis, ribosome biogenesis and stress response. Multiple optogenetic devices have been developed to manipulate nuclear protein import and export, but molecular tools tailored for remote control over selective targeting or partitioning of cargo proteins into subnuclear compartments capable of phase separation are still limited. Here, we report a set of single-component photoinducible nucleolus-targeting tools, designated pNUTs, to enable rapid and reversible nucleoplasm-to-nucleolus shuttling, with the half-lives ranging from milliseconds to minutes. pNUTs allow both global protein infiltration into nucleoli and local delivery of cargoes into the outermost layer of the nucleolus, the granular component. When coupled with the amyotrophic lateral sclerosis (ALS)-associated C9ORF72 proline/arginine-rich dipeptide repeats, pNUTs allow us to photomanipulate poly-proline-arginine nucleolar localization, perturb nucleolar protein nucleophosmin 1 and suppress nascent protein synthesis. pNUTs thus expand the optogenetic toolbox by permitting light-controllable interrogation of nucleolar functions and precise induction of ALS-associated toxicity in cellular models.

Targeting epigenetic regulatory machinery to overcome cancer therapy resistance.

Drug resistance, either intrinsic or acquired, represents a major hurdle to achieving optimal therapeutic outcomes during cancer treatment. In addition to acquisition of resistance-conferring genetic mutations, accumulating evidence suggests an intimate involvement of the epigenetic machinery in this process as well. Recent studies have revealed that epigenetic reprogramming, such as altered expression or relocation of DNA/histone modulators accompanied with chromatin structure remodeling, can lead to transcriptional plasticity in tumor cells, thereby driving their transformation towards a persistent state. These "persisters" represent a pool of slow-growing cells that can either re-expand when treatment is discontinued or acquire permanent resistance. Targeting epigenetic reprogramming or plasticity represents a new strategy to prevent the emergence of drug-refractory populations and to enable more consistent clinical responses. With the growing numbers of drugs or drug candidates developed to target epigenetic regulators, more and more epigenetic therapies are under preclinical evaluation, early clinical trials or approved by FDA as single agent or in combination with existing antitumor drugs. In this review, we highlight latest discoveries in the mechanistic understanding of epigenetically-induced drug resistance. In parallel, we discuss the potential of combining epigenetic drugs with existing anticancer regimens as a promising strategy for overcoming cancer drug resistance.

Ten-Eleven Translocation Ablation Impairs Cardiac Differentiation of Mouse Embryonic Stem Cells.

Ten-eleven Translocation (TET) dioxygenases mediated DNA methylation oxidation plays an important role in regulating the embryonic stem cells (ESCs) differentiation. Herein, we utilized a CRISPR/Cas9 based genome editing method to generate single, double, and triple Tet-deficient mouse ESCs (mESCs) and differentiated these cells toward cardiac progenitors. By using emerald green fluorescent protein (GFP; emGFP) expression under the control of Nkx2.5 promoter as marker for cardiac progenitor cells, we discovered that Tet1 and Tet2 depletion significantly impaired mESC-to-cardiac progenitor differentiation. Single-cell RNA-seq analysis further revealed that Tet deletion resulted in the accumulation of mesoderm progenitors to hamper cardiac differentiation. Re-expression of the Tet1 catalytic domain (Tet1CD) rescued the differentiation defect in Tet-triple knockout mESCs. Dead Cas9 (dCas9)-Tet1CD mediated loci-specific epigenome editing at the Hand1 loci validated the direct involvement of Tet-mediated epigenetic modifications in transcriptional regulation during cardiac differentiation. Our study establishes that Tet-mediated epigenetic remodeling is essential for maintaining proper transcriptional outputs to safeguard mESC-to-cardiac progenitor differentiation.

Baseline serum tumor markers predict the survival of patients with advanced non-small cell lung cancer receiving first-line immunotherapy: a multicenter retrospective study.

BACKGROUND: This study aimed to investigate the association between baseline serum tumor markers (STMs) (carcinoembryonic antigen [CEA], neuron-specific enolase [NSE], cytokeratin-19 fragment [CYFRA21-1], carbohydrate antigen 19-9 [CA19-9], and carbohydrate antigen 125 [CA125]) and the efficacy of first-line immunotherapy in patients with advanced non-small cell lung cancer. METHODS: This multicenter retrospective study evaluated patients who received first-line immunotherapy between July 2017 and July 2022. The endpoints were progression-free survival (PFS) and overall survival (OS), as defined by the Response Evaluation Criteria in Solid Tumors version 1.1. We divided the patients into three groups based on STM levels: Group A ≥ threefold upper limit of normal, threefold upper limit of normal > Group B > upper limit of normal, and Group C ≤ upper limit of normal. RESULTS: In total, 716 patients were included in this study. In Cox proportional hazards analyses, the STM levels in Group C were independently associated with superior PFS and OS in patients with lung adenocarcinoma (LUAD). Except for CA19-9 level, the STM levels in Group C were independently associated with superior PFS and OS in patients with lung squamous carcinoma (LUSC). Except for CEA and CA19-9 levels, the levels in Group A were independently associated with inferior PFS and OS in patients with LUAD and LUSC. CONCLUSIONS: Serum CEA, NSE, CYFRA21-1, and CA125 levels can predict PFS and OS in patients with LUAD and LUSC, and serum CA19-9 levels can predict PFS and OS in patients with LUAD. The higher the serum NSE, CYFRA21-1, and CA125 levels, the worse the PFS and OS in patients with LUAD and LUSC. In addition, the higher the serum CA19-9 level, the worse the OS in patients with LUAD.

Circularly permuted LOV2 as a modular photoswitch for optogenetic engineering.

Plant-based photosensors, such as the light-oxygen-voltage sensing domain 2 (LOV2) from oat phototropin 1, can be modularly wired into cell signaling networks to remotely control protein activity and physiological processes. However, the applicability of LOV2 is hampered by the limited choice of available caging surfaces and its preference to accommodate the effector domains downstream of the C-terminal Jα helix. Here, we engineered a set of LOV2 circular permutants (cpLOV2) with additional caging capabilities, thereby expanding the repertoire of genetically encoded photoswitches to accelerate the design of optogenetic devices. We demonstrate the use of cpLOV2-based optogenetic tools to reversibly gate ion channels, antagonize CRISPR-Cas9-mediated genome engineering, control protein subcellular localization, reprogram transcriptional outputs, elicit cell suicide and generate photoactivatable chimeric antigen receptor T cells for inducible tumor cell killing. Our approach is widely applicable for engineering other photoreceptors to meet the growing need of optogenetic tools tailored for biomedical and biotechnological applications.

Probing the Substrate Requirements of the In†Vitro Geranylation Activity of Selenouridine Synthase (SelU).

Natural RNA modifications diversify the structures and functions of existing nucleic acid building blocks. Geranyl is one of the most hydrophobic groups recently identified in bacterial tRNAs. Selenouridine synthase (SelU, also called mnmH) is an enzyme with a dual activity which catalyzes selenation and geranylation in tRNAs containing 2-thiouridine using selenophosphate or geranyl-pyrophosphate as cofactors. In this study, we explored the in vitro geranylation process of tRNA anticodon stem loops (ASL) mediated by SelU and showed that the geranylation activity was abolished when U35 was mutated to A35 (ASL-tRNALys (s2U)UU to ASL-tRNAIle (s2U)AU ). By examining the SelU cofactor geranyl-pyrophosphate (gePP) and its analogues, we found that only the geranyl group, but not dimethylallyl- and farnesyl-pyrophosphate with either shorter or longer terpene chains, could be incorporated into ASL. The degree of tRNA geranylation in the end-point analysis for SelU follows the order of ASLLys (s2UUU) ≃ ASLGln (s2UUG) >ASLGlu (s2UUC) . These findings suggest a putative mechanism for substrate discrimination by SelU and reveal key factors that might influence its enzymatic activity. Given that SelU plays an important role in bacterial translation systems, inhibiting this enzyme and targeting its geranylation and selenation pathways could be exploited as a promising strategy to develop SelU-based antibiotics.

Metallic phase enabling MoS2 nanosheets as an efficient sonosensitizer for photothermal-enhanced sonodynamic antibacterial therapy.

Two-dimensional (2D) transition metal dichalcogenide (TMD) nanosheets (e.g., MoS2) with metallic phase (1T or 1T´ phase) have been proven to exhibit superior performances in various applications as compared to their semiconducting 2H-phase counterparts. However, it remains unclear how the crystal phase of 2D TMD nanosheets affects their sonodynamic property. In this work, we report the preparation of MoS2 nanosheets with different phases (metallic 1T/1T´ or semiconducting 2H) and exploration of its crystal-phase effect on photothermal-enhanced sonodynamic antibacterial therapy. Interestingly, the defective 2D MoS2 nanosheets with high-percentage metallic 1T/1T´ phase (denoted as M-MoS2) present much higher activity towards the ultrasound-induced generation of reactive oxygen species (ROS) as compared to the semiconducting 2H-phase MoS2 nanosheets. More interestingly, owing to its metallic phase-enabled strong absorption in the near-infrared-II (NIR-II) regime, the ultrasound-induced ROS generation performance of the M-MoS2 nanosheets can be further enhanced by the photothermal effect under a 1064 nm laser irradiation. Thus, after modifying with polyvinylpyrrolidone, the M-MoS2 nanosheets can be used as an efficient sonosensitizer for photothermal-enhanced sonodynamic bacterial elimination under ultrasound treatment combining with NIR-II laser irradiation. This study demonstrates that metallic MoS2 nanosheets can be used as a promising sonosensitizer for antibacterial therapy, which might be also promising for cancer therapies.

Ion-Conductive Hydrogel-Based Stretchable, Self-Healing, and Transparent NO2 Sensor with High Sensitivity and Selectivity at Room Temperature.

Here stretchable, self-healable, and transparent gas sensors based on salt-infiltrated hydrogels for high-performance NO2 sensing in both anaerobic environment and air at room temperature, are reported. The salt-infiltrated hydrogel displays high sensitivity to NO2 (119.9%/ppm), short response and recovery time (29.8 and 41.0 s, respectively), good linearity, low theoretical limit of detection (LOD) of 86 ppt, high selectivity, stability, and conductivity. A new gas sensing mechanism based on redox reactions occurring at the electrode-hydrogel interface is proposed to understand the sensing behaviors. The gas sensing performance of hydrogel is greatly improved by incorporating calcium chloride (CaCl2 ) in the hydrogel via a facile salt-infiltration strategy, leading to a higher sensitivity (2.32 times) and much lower LOD (0.06 times). Notably, both the gas sensing ability, conductivity, and mechanical deformability of hydrogels are readily self-healable after cutting off and reconnection. Such large deformations as 100% strain do not deprive the gas sensing capability, but rather shorten the response and recovery time significantly. The CaCl2 -infiltrated hydrogel shows excellent selectivity of NO2 , with good immunity to the interference gases. These results indicate that the salt-infiltrated hydrogel has great potential for wearable electronics equipped with gas sensing capability in both anaerobic and aerobic environments.

TRIM59 promotes breast cancer motility by suppressing p62-selective autophagic degradation of PDCD10.

Cancer cells adopt various modes of migration during metastasis. How the ubiquitination machinery contributes to cancer cell motility remains underexplored. Here, we report that tripartite motif (TRIM) 59 is frequently up-regulated in metastatic breast cancer, which is correlated with advanced clinical stages and reduced survival among breast cancer patients. TRIM59 knockdown (KD) promoted apoptosis and inhibited tumor growth, while TRIM59 overexpression led to the opposite effects. Importantly, we uncovered TRIM59 as a key regulator of cell contractility and adhesion to control the plasticity of metastatic tumor cells. At the molecular level, we identified programmed cell death protein 10 (PDCD10) as a target of TRIM59. TRIM59 stabilized PDCD10 by suppressing RING finger and transmembrane domain-containing protein 1 (RNFT1)-induced lysine 63 (K63) ubiquitination and subsequent phosphotyrosine-independent ligand for the Lck SH2 domain of 62 kDa (p62)-selective autophagic degradation. TRIM59 promoted PDCD10-mediated suppression of Ras homolog family member A (RhoA)-Rho-associated coiled-coil kinase (ROCK) 1 signaling to control the transition between amoeboid and mesenchymal invasiveness. PDCD10 overexpression or administration of a ROCK inhibitor reversed TRIM59 loss-induced contractile phenotypes, thereby accelerating cell migration, invasion, and tumor formation. These findings establish the rationale for targeting deregulated TRIM59/PDCD10 to treat breast cancer.

Identification of molecular determinants that govern distinct STIM2 activation dynamics.

The endoplasmic reticulum (ER) Ca2+ sensors stromal interaction molecule 1 (STIM1) and STIM2, which connect ER Ca2+ depletion with extracellular Ca2+ influx, are crucial for the maintenance of Ca2+ homeostasis in mammalian cells. Despite the recent progress in unraveling the role of STIM2 in Ca2+ signaling, the mechanistic underpinnings of its activation remain underexplored. We use an engineering approach to direct ER-resident STIMs to the plasma membrane (PM) while maintaining their correct membrane topology, as well as Förster resonance energy transfer (FRET) sensors that enabled in cellulo real-time monitoring of STIM activities. This allowed us to determine the calcium affinities of STIM1 and STIM2 both in cellulo and in situ, explaining the current discrepancies in the literature. We also identified the key structural determinants, especially the corresponding G residue in STIM1, which define the distinct activation dynamics of STIM2. The chimeric E470G mutation could switch STIM2 from a slow and weak Orai channel activator into a fast and potent one like STIM1 and vice versa. The systemic dissection of STIM2 activation by protein engineering sets the stage for the elucidation of the regulation and function of STIM2-mediated signaling in mammals.

Sub-chronic low-dose arsenic in rice exposure induces gut microbiome perturbations in mice.

Long-term consumption of arsenic-contaminated rice has become a public health issue that urgently needs to be addressed. In this study, mice were exposed to arsenic in rice (low dose, 0.91 mg/kg; medium dose, 9.1 mg/kg) for 30 days and 60 days, respectively, and the effects on pathological structures of spleen and skin, as well as the structure of the fecal microbiome were examined. The findings revealed dose/time cumulative effects on pathological changes, with even a low dose exposure for 30 days causing destruction of splenic follicular structure and thickening of dermal keratinized and epidermal layers. The Firmicutes/Bacteroidetes ratio in the community and the positive/negative ratio in network links were higher in arsenic groups, suggesting that arsenic resulted in a less healthy and unstable microbiome for the host. Thus lifetime consumption of arsenic in rice may have potential health effects on humans and must be carefully assessed to safeguard human health. Furthermore, in arsenic groups, arsenic-resistant bacteria or arsenic hazards remediation bacteria changed to be the dominant bacteria and acted as the core bacteria in the network modules. Some microbial arsenic transforming genes (arsC, arsR, arsA, ACR3, and aoxB) differed, indicating that the gut microbiome changed to withstand arsenic stress. Furthermore, Faecalibaculum, Lachnospiraceae_NK4A136_group, Angelakisella, Ruminiclostridium, and Desulfovibrionaceae are positively associated with arsenic dosage and may be useful in the early detection of arsenicals.

A molecular toolbox for interrogation of membrane contact sites.

Membrane contact sites (MCSs) are specialized subcellular compartments formed by closely apposed membranes from two organelles. The intermembrane gap is separated by a distance ranging from 10 to 35 nm. MCSs are typically maintained through dynamic protein-protein and protein-lipid interactions. These intermembrane contact sites constitute important intracellular signalling hotspots to mediate a plethora of cellular processes, including calcium homeostasis, lipid metabolism, membrane biogenesis and organelle remodelling. In recent years, a series of genetically encoded probes and chemogenetic or optogenetic actuators have been invented to aid the visualization and interrogation of MCSs in both fixed and living cells. These molecular tools have greatly accelerated the pace of mechanistic dissection of membrane contact sites at the molecular level. In this review, we present an overview on the latest progress in this endeavour, and provide a general guide to the selection of methods and molecular tools for probing interorganellar membrane contact sites.