Nek7 Protects Telomeres from Oxidative DNA Damage by Phosphorylation and Stabilization of TRF1.

Telomeric repeat binding factor 1 (TRF1) is essential to the maintenance of telomere chromatin structure and integrity. However, how telomere integrity is maintained, especially in response to damage, remains poorly understood. Here, we identify Nek7, a member of the Never in Mitosis Gene A (NIMA) kinase family, as a regulator of telomere integrity. Nek7 is recruited to telomeres and stabilizes TRF1 at telomeres after damage in an ATM activation-dependent manner. Nek7 deficiency leads to telomere aberrations, long-lasting γH2AX and 53BP1 foci, and augmented cell death upon oxidative telomeric DNA damage. Mechanistically, Nek7 interacts with and phosphorylates TRF1 on Ser114, which prevents TRF1 from binding to Fbx4, an Skp1-Cul1-F box E3 ligase subunit, thereby alleviating proteasomal degradation of TRF1, leading to a stable association of TRF1 with Tin2 to form a shelterin complex. Our data reveal a mechanism of efficient protection of telomeres from damage through Nek7-dependent stabilization of TRF1.

Neural Representations in Visual and Parietal Cortex Differentiate between Imagined, Perceived, and Illusory Experiences.

Humans constantly receive massive amounts of information, both perceived from the external environment and imagined from the internal world. To function properly, the brain needs to correctly identify the origin of information being processed. Recent work has suggested common neural substrates for perception and imagery. However, it has remained unclear how the brain differentiates between external and internal experiences with shared neural codes. Here we tested this question in human participants (male and female) by systematically investigating the neural processes underlying the generation and maintenance of visual information from voluntary imagery, veridical perception, and illusion. The inclusion of illusion allowed us to differentiate between objective and subjective internality: while illusion has an objectively internal origin and can be viewed as involuntary imagery, it is also subjectively perceived as having an external origin like perception. Combining fMRI, eye-tracking, multivariate decoding, and encoding approaches, we observed superior orientation representations in parietal cortex during imagery compared with perception, and conversely in early visual cortex. This imagery dominance gradually developed along a posterior-to-anterior cortical hierarchy from early visual to parietal cortex, emerged in the early epoch of imagery and sustained into the delay epoch, and persisted across varied imagined contents. Moreover, representational strength of illusion was more comparable to imagery in early visual cortex, but more comparable to perception in parietal cortex, suggesting content-specific representations in parietal cortex differentiate between subjectively internal and external experiences, as opposed to early visual cortex. These findings together support a domain-general engagement of parietal cortex in internally generated experience.SIGNIFICANCE STATEMENT How does the brain differentiate between imagined and perceived experiences? Combining fMRI, eye-tracking, multivariate decoding, and encoding approaches, the current study revealed enhanced stimulus-specific representations in visual imagery originating from parietal cortex, supporting the subjective experience of imagery. This neural principle was further validated by evidence from visual illusion, wherein illusion resembled perception and imagery at different levels of cortical hierarchy. Our findings provide direct evidence for the critical role of parietal cortex as a domain-general region for content-specific imagery, and offer new insights into the neural mechanisms underlying the differentiation between subjectively internal and external experiences.

Oxidative stress in hair follicle development and hair growth: Signalling pathways, intervening mechanisms and potential of natural antioxidants.

Hair follicle development and hair growth are regulated by multiple factors and multiple signalling pathways. The hair follicle, as an important skin appendage, is the basis for hair growth, and it has the functions of safeguarding the body, perceiving the environment and regulating body temperature. Hair growth undergoes a regular hair cycle, including anagen, catagen and telogen. A small amount of physiological shedding of hair occurs under normal conditions, always in a dynamic equilibrium. Hair loss occurs when the skin or hair follicles are stimulated by oxidative stress, inflammation or hormonal disorders that disrupt the homeostasis of the hair follicles. Numerous researches have indicated that oxidative stress is an important factor causing hair loss. Here, we summarize the signalling pathways and intervention mechanisms by which oxidative stress affects hair follicle development and hair growth, discuss existing treatments for hair loss via the antioxidant pathway and provide our own insights. In addition, we collate antioxidant natural products promoting hair growth in recent years and discuss the limitations and perspectives of current hair loss prevention and treatment.

Effect of blood collection tube containing protease inhibitors on the pre-analytical stability of Alzheimer's disease plasma biomarkers.

The reliability of plasma biomarkers of Alzheimer's disease (AD) can be compromised by protease-induced degradation. This can limit the feasibility of conducting plasma biomarker studies in environments that lack the capacity for immediate processing and appropriate storage of blood samples. We hypothesized that blood collection tube supplementation with protease inhibitors can improve the stability of plasma biomarkers at room temperatures (RT). In this study, we conducted a comparative analysis of blood biomarker stability in traditional ethylenediaminetetraacetic acid (EDTA) tubes versus BD™ P100 collection tubes, the latter being coated with a protease inhibitor cocktail. The stability of six plasma AD biomarkers was evaluated over time under RT conditions. We evaluated three experimental approaches. In Approach 1, pooled plasma samples underwent storage at RT for up to 96 h. In Approach 2, plasma samples isolated upfront from whole blood collected into EDTA or P100 tubes were stored at RT for 0 h or 24 h before biomarker measurements. In Approach 3, whole blood samples were collected into paired EDTA and P100 tubes, followed by storage at RT for 0 h or 24 h before isolating the plasma for analyses. Biomarkers were measured with Single Molecule Array (Simoa) and immunoprecipitation-mass spectrometry (IP-MS) assays. Both the IP-MS and Simoa methods revealed that the use of P100 tubes significantly improves the stability of Aß42 and Aß40 across all approaches. However, the Aß42/Aß40 ratio levels were significantly stabilized only in the IP-MS assay in Approach 3. No significant differences were observed in the levels of plasma p-tau181, GFAP, and NfL for samples collected using either tube type in any of the approaches. Supplementation of blood collection tubes with protease inhibitors could reduce the protease-induced degradation of plasma Aß42 and Aß40, and the Aß42/40 ratio for the IP-MS assay. These findings have crucial implications for preanalytical procedures, particularly in resource-limited settings.

Large-Scale Atomic Strain Defects on Palladium Surfaces for Enhanced Oxygen Reduction and Zinc-Air Batteries.

Designing nano-electrocatalysts rich in surface defects is critical to improve their catalytic performance. However, prevailing synthesis techniques rely heavily on complex procedures that compromise defect extensiveness and uniformity, casting a high demand for methods capable of synthesizing large-scale crystalline defects. An innovative design strategy is herein proposed that induces ample strain/dislocation defects during the growth of palladium (Pd), which is well-known as a good oxygen reduction reaction (ORR) catalyst. The controlled defect engineering on Pd core is achieved by the tensile stress exerted from an intentionally applied Fe3O4 skin layer during synthesis, which changes the surface free energy of Pd to stabilize the defect presence. With such large-scale crystalline defects, this Pd catalyst exhibits significantly higher ORR activity than commercial Pt/C, enabling its promising future in zinc-air battery catalysis. Additionally, the protective Fe3O4 skin covering the catalyst also enhances its catalytic stability. Theoretical calculations show that the superior catalytic property of such defect-engineered Pd is associated with the correspondingly modified adsorption energy of *O intermediates onto its surface, which further improves the reaction rate and thus boosts ORR kinetics. Findings here are expected to provide a paradigm for designing efficient and stable metal catalysts with plentiful large-scale strain defects.

Where Do Plasma Biomarkers fit in With Current Alzheimer's Disease Detection?

OBJECTIVES: We examine the clinical utility of plasma-based detection for Alzheimer's disease (AD) pathophysiology in older adults with mild cognitive impairment (MCI) and whether cognitive screening can inform when to use plasma-based AD tests. METHODS: Seventy-four community-dwelling older adults with MCI had testing with plasma phosphorylated tau (p-tau) 217 and 181, positron emission tomography (PET) imaging for amyloid beta (Aß), and cognitive assessment. Receiver operating characteristic (ROC) analysis was used to assess the diagnostic value of plasma p-tau. RESULTS: Plasma p-tau217 distinguished MCI participants who had PET imaging evidence of Aß accumulation from those without (AUC of 0.92, specificity of 0.96, and sensitivity of 0.90), outperforming plasma p-tau181 (AUC of 0.76, specificity of 0.87 and sensitivity of 0.59) for the same purpose. Of the 60 MCI participants that were amnestic, 22 were Aß+. The 14 participants that were nonamnestic were all Aß-. CONCLUSIONS: Our findings support the clinical use of plasma p-tau, particularly p-tau217, for patient detection of AD pathophysiology in older adults with amnestic MCI, but not in those who are nonamnestic.

Iron (II)-based metal-organic framework nanozyme for boosting tumor ferroptosis through inhibiting DNA damage repair and system Xc.

Development of ferroptosis-inducible nanoplatforms with high efficiency and specificity is highly needed and challenging in tumor ferrotherapy. Here, we demonstrate highly effective tumor ferrotherapy using iron (II)-based metal-organic framework (FessMOF) nanoparticles, assembled from disulfide bonds and ferrous ions. The as-prepared FessMOF nanoparticles exhibit peroxidase-like activity and pH/glutathione-dependent degradability, which enables tumor-responsive catalytic therapy and glutathione depletion by the thiol/disulfide exchange to suppress glutathione peroxidase 4, respectively. Upon PEGylation and Actinomycin D (ActD) loading, the resulting FessMOF/ActD-PEG nanoplatform induces marked DNA damage and lipid peroxidation. Concurrently, we found that ActD can inhibit Xc- system and elicit ferritinophagy, which further boosts the ferrotherapeutic efficacy of the FessMOF/ActD-PEG. In vivo experiments demonstrate that our fabricated nanoplatform presents excellent biocompatibility and a high tumor inhibition rate of 91.89%.

Metal ions-anchored bacterial outer membrane vesicles for enhanced ferroptosis induction and immune stimulation in targeted antitumor therapy.

The activation of ferroptosis presents a versatile strategy for enhancing the antitumor immune responses in cancer therapy. However, developing ferroptosis inducers that combine high biocompatibility and therapeutic efficiency remains challenging. In this study, we propose a novel approach using biological nanoparticles derived from outer membrane vesicles (OMVs) of Escherichia coli for tumor treatment, aiming to activate ferroptosis and stimulate the immune responses. Specifically, we functionalize the OMVs by anchoring them with ferrous ions via electrostatic interactions and loading them with the STING agonist-4, followed by tumor-targeting DSPE-PEG-FA decoration, henceforth referred to as OMV/SaFeFA. The anchoring of ferrous ions endows the OMVs with peroxidase-like activity, capable of inducing cellular lipid peroxidation by catalyzing H2O2 to •OH. Furthermore, OMV/SaFeFA exhibits pH-responsive release of ferrous ions and the agonist, along with tumor-targeting capabilities, enabling tumor-specific therapy while minimizing side effects. Notably, the concurrent activation of the STING pathway and ferroptosis elicits robust antitumor responses in colon tumor-bearing mouse models, leading to exceptional therapeutic efficacy and prolonged survival. Importantly, no acute toxicity was observed in mice receiving OMV/SaFeFA treatments, underscoring its potential for future tumor therapy and clinical translation.

Toxoplasma gondii association with host mitochondria requires key mitochondrial protein import machinery.

Host mitochondrial association (HMA) is a well-known phenomenon during Toxoplasma gondii infection of the host cell. The T. gondii locus mitochondrial association factor 1 (MAF1) is required for HMA and MAF1 encodes distinct paralogs of secreted dense granule effector proteins, some of which mediate the HMA phenotype (MAF1b paralogs drive HMA; MAF1a paralogs do not). To identify host proteins required for MAF1b-mediated HMA, we performed unbiased, label-free quantitative proteomics on host cells infected with type II parasites expressing MAF1b, MAF1a, and an HMA-incompetent MAF1b mutant. Across these samples, we identified ∼1,360 MAF1-interacting proteins, but only 13 that were significantly and uniquely enriched in MAF1b pull-downs. The gene products include multiple mitochondria-associated proteins, including those that traffic to the mitochondrial outer membrane. Based on follow-up endoribonuclease-prepared short interfering RNA (esiRNA) experiments targeting these candidate MAF1b-targeted host factors, we determined that the mitochondrial receptor protein TOM70 and mitochondria-specific chaperone HSPA9 were essential mediators of HMA. Additionally, the enrichment of TOM70 at the parasitophorous vacuole membrane interface suggests parasite-driven sequestration of TOM70 by the parasite. These results show that the interface between the T. gondii vacuole and the host mitochondria is characterized by interactions between a single parasite effector and multiple target host proteins, some of which are critical for the HMA phenotype itself. The elucidation of the functional members of this complex will permit us to explain the link between HMA and changes in the biology of the host cell.

Coupling effects of irrigation and nitrogen on spring maize yield and greenhouse gas emissions in Northwestern China.

BACKGROUND: This study explored the mechanism of irrigation and nitrogen (N) coupling on spring maize yield and soil greenhouse gas (GHG) emissions, with the objective of achieving water saving, high yield and emission reduction. Field experiments were conducted to analyze the effects of multiple irrigation and N management strategies on GHG emissions and to determine the optimal balance between GHG, water conservation and grain yield. The experiments were conducted on spring maize with three irrigation levels (low, IL; medium, IM; and high, IH) and 4 N application levels (N40, N80, N120 and N160 kg N ha-1). RESULTS: The IL treatment exhibited the lowest N2O and CO2 emission fluxes and the lowest CH4 uptake fluxes. The N40 treatment exhibited the lowest N2O and CO2 emission fluxes and the highest CH4 uptake flux. Significant positive correlations were observed among N2O and CO2 emission fluxes, CH4 uptake fluxes, and soil moisture and inorganic N content. Maize yield initially increased and then decreased with rising levels of irrigation and N management. By employing the TOPSIS method to assess yield and greenhouse effects, we identified the IMN120 treatment as optimal given that this treatment achieved the highest yield (14 686.26 kg ha-1) and water use efficiency (3.51 kg m-3) while maintaining relatively low global warming potential (573.30 kg CO2 eq ∙ ha-1) and GHG intensity (0.0390 kg CO2 eq ∙ kg-1). CONCLUSION: Irrigation optimization and N management are key to reducing GHG emissions, enhancing yield, and promoting both the sustainable development of agriculture and environmental protection. © 2024 Society of Chemical Industry.

Clinical application of serum-based proteomics technology in human tumor research.

The rapid development of proteomics technology in the past decades has led to further human understanding of tumor research, and in some ways, the technology plays a very important supporting role in the early detection of tumors. Human serum has been shown to contain a variety of proteins closely related to life activities, and the dynamic change in proteins can often reflect the physiological and pathological conditions of the body. Serum has the advantage of easy extraction, so the application of proteomics technology in serum has become a hot spot and frontier area for the study of malignant tumors. However, there are still many difficulties in the standardized use of proteomic technologies, which inevitably limit the clinical application of proteomic technologies due to the heterogeneity of human proteins leading to incomplete whole proteome populations, in addition to most serum protein markers being now not highly specific in aiding the early detection of tumors. Nevertheless, further development of proteomics technologies will greatly increase our understanding of tumor biology and help discover more new tumor biomarkers with specificity that will enable medical technology.

On-chip-angiogenesis based on a high-throughput biomimetic three-dimensional cell spheroid culture system.

Angiogenesis is one of the most essential developmental processes and plays a key role in organogenesis and tumorigenesis in which epithelial cells proliferate and migrate, thus resulting in sprouting and extension of the existing vasculature. The study of angiogenesis in vivo is limited by difficulties related to imaging of the fine structure of vascular sprouting within non-transparent bulk tissue. Thus, many model systems have been proposed in recent years. However, to meet the urgent need for high-throughput studies and screening, further improvements are still required, particularly in terms of scaling-up. In this study, we combined microchip fabrication with the culture of three-dimensional (3D) spheroids, thus providing a platform for 3D multilayer angiogenesis-on-a-chip. Using this platform, we investigated the precise effects of vascular endothelial growth factor (VEGF) on angiogenesis. In comparison with two-dimensional (2D) angiogenesis assays, our 3D angiogenesis platform demonstrated superior sprouting and provided proof of concept that our 3D biomimetic angiogenesis-on-a-chip could serve as a powerful tool for pro- or anti-angiogenesis candidate drug screening.

Rapid Determination of Phase Diagrams for Biomolecular Liquid-Liquid Phase Separation with Microfluidics.

Biomolecular phase separation is currently emerging in both the medical and life science fields. Meanwhile, the application of liquid-liquid phase separation has been extended to many fields including drug discovery, fibrous material fabrication, 3D printing, and polymer design. Although more than 8600 proteins and other synthetic macromolecules are capable of phase separation as recently reported, there is still a lack of a high-throughput approach to quantitatively characterize its phase behaviors. To meet this requirement, here, we proposed fast and high-resolution acquisition of biomolecular phase diagrams using microfluidic chips. Using this platform, we demonstrated the phase behavior of polyU/RRASLRRASLRRASL in a quantitative manner. Up to 1750 concentration conditions can be generated in 140 min. The detection limitation of our device to capture the saturation concentration for phase separation is about 5 times lower than that of the traditional turbidity method. Thus, our results provide a basis for the rapid acquisition of phase diagrams with high-throughput and pave the way for its wide application.

The Capture of a Disabled Proteasome Identifies Erg25 as a Substrate for Endoplasmic Reticulum Associated Degradation.

Studies in the yeast Saccharomyces cerevisiae have helped define mechanisms underlying the activity of the ubiquitin-proteasome system (UPS), uncover the proteasome assembly pathway, and link the UPS to the maintenance of cellular homeostasis. However, the spectrum of UPS substrates is incompletely defined, even though multiple techniques-including MS-have been used. Therefore, we developed a substrate trapping proteomics workflow to identify previously unknown UPS substrates. We first generated a yeast strain with an epitope tagged proteasome subunit to which a proteasome inhibitor could be applied. Parallel experiments utilized inhibitor insensitive strains or strains lacking the tagged subunit. After affinity isolation, enriched proteins were resolved, in-gel digested, and analyzed by high resolution liquid chromatography-tandem MS. A total of 149 proteasome partners were identified, including all 33 proteasome subunits. When we next compared data between inhibitor sensitive and resistant cells, 27 proteasome partners were significantly enriched. Among these proteins were known UPS substrates and proteins that escort ubiquitinated substrates to the proteasome. We also detected Erg25 as a high-confidence partner. Erg25 is a methyl oxidase that converts dimethylzymosterol to zymosterol, a precursor of the plasma membrane sterol, ergosterol. Because Erg25 is a resident of the endoplasmic reticulum (ER) and had not previously been directly characterized as a UPS substrate, we asked whether Erg25 is a target of the ER associated degradation (ERAD) pathway, which most commonly mediates proteasome-dependent destruction of aberrant proteins. As anticipated, Erg25 was ubiquitinated and associated with stalled proteasomes. Further, Erg25 degradation depended on ERAD-associated ubiquitin ligases and was regulated by sterol synthesis. These data expand the cohort of lipid biosynthetic enzymes targeted for ERAD, highlight the role of the UPS in maintaining ER function, and provide a novel tool to uncover other UPS substrates via manipulations of our engineered strain.

Uncovering the Metabolic Origin of Aspartate for Tumor Growth Using an Integrated Molecular Deactivator.

Reprogrammed glucose metabolism is vital for cancer cells, but aspartate, an intermediate metabolic product, is the limiting factor for cancer cell proliferation. However, due to the complexity of metabolic pathways, it remains unclear whether glucose is the primary source of endogenous aspartate. Here, we report the design of an innovative molecular deactivator, based on a multifunctional upconversion nanoprobe, to explore the link between glucose and aspartate. This molecular deactivator mainly works in the acidic, hypoxic tumor microenvironment and deactivates multiple types of glucose transporters on cancer cell membranes upon illumination at 980 nm. Cancer cell proliferation in vivo is strongly inhibited by blocking glucose transporters. Our experimental data confirm that the cellular synthesis of aspartate for tumor growth is glucose-dependent. This work also demonstrates the untapped potential of molecularly engineered upconversion nanoprobes for discovering hidden metabolic pathways and improving therapeutic efficacy of conventional antitumor drugs.

Genome-Wide Identification and Expression Analysis of the 14-3-3 Gene Family in Mango (Mangifera indica L.).

Members of the Mi14-3-3 gene family interact with target proteins that are widely involved in plant hormone signal transduction and physiology-related metabolism and play important roles in plant growth, development and stress responses. In this study, 14-3-3s family members are identified by the bioinformatic analysis of the mango (Mangifera indica L.) genome. The gene structures, chromosomal distributions, genetic evolution, and expression patterns of these genes and the physical and chemical properties and conserved motifs of their proteins are analysed systematically. The results identified 16 members of the 14-3-3 genes family in the mango genome. The members were not evenly distributed across the chromosomes, and the gene structure analysis showed that the gene sequence length and intron number varied greatly among the different members. Protein sequence analysis showed that the Mi14-3-3 proteins had similar physical and chemical properties and secondary and tertiary structures, and protein subcellular localization showed that the Mi14-3-3 family proteins were localized to the nucleus. The sequence analysis of the Mi14-3-3s showed that all Mi14-3-3 proteins contain a typical conserved PFAM00244 domain, and promoter sequence analysis showed that the Mi14-3-3 promoters contain multiple hormone-, stress-, and light-responsive cis-regulatory elements. Expression analysis showed that the 14-3-3 genes were expressed in all tissues of mango, but that their expression patterns were different. Drought, salt and low temperature stresses affected the expression levels of 14-3-3 genes, and different 14-3-3 genes had different responses to these stresses. This study provides a reference for further studies on the function and regulation of Mi14-3-3 family members.

The N-cadherin interactome in primary cardiomyocytes as defined using quantitative proximity proteomics.

The junctional complexes that couple cardiomyocytes must transmit the mechanical forces of contraction while maintaining adhesive homeostasis. The adherens junction (AJ) connects the actomyosin networks of neighboring cardiomyocytes and is required for proper heart function. Yet little is known about the molecular composition of the cardiomyocyte AJ or how it is organized to function under mechanical load. Here, we define the architecture, dynamics and proteome of the cardiomyocyte AJ. Mouse neonatal cardiomyocytes assemble stable AJs along intercellular contacts with organizational and structural hallmarks similar to mature contacts. We combine quantitative mass spectrometry with proximity labeling to identify the N-cadherin (CDH2) interactome. We define over 350 proteins in this interactome, nearly 200 of which are unique to CDH2 and not part of the E-cadherin (CDH1) interactome. CDH2-specific interactors comprise primarily adaptor and adhesion proteins that promote junction specialization. Our results provide novel insight into the cardiomyocyte AJ and offer a proteomic atlas for defining the molecular complexes that regulate cardiomyocyte intercellular adhesion. This article has an associated First Person interview with the first authors of the paper.

Albumin Protects Lung Cells against Acrolein Cytotoxicity and Acrolein-Adducted Albumin Increases Heme Oxygenase 1 Transcripts.

Albumin is an abundant protein in the lung lining fluid that forms an interface between lung epithelial cells and the external environment. In the lung, albumin can be targeted for adduction by inhaled acrolein. Acrolein, an α,ß-unsaturated aldehyde, reacts with biomolecules via Michael addition at the ß-carbon or Schiff base formation at the carbonyl carbon. To gain insight into acrolein's mode of action, we investigated in vitro albumin-acrolein reactivity and the consequence of albumin adduction by acrolein on cytotoxicity and transcript changes in NCI-H441 and human airway epithelial cells (HAEC). Albumin protected NCI-H441 cells from acrolein toxicity. In addition, albumin inhibited acrolein-induced increase of transcripts associated with cellular stress response, activating transcription factor 3 (ATF3), and antioxidant response, heme oxygenase 1 (HMOX1) in HAEC cells. Acrolein-adducted albumin itself increased HMOX1 transcripts but not ATF3 transcripts. The HMOX1 transcript increase was inhibited by hydralazine, a carbonyl scavenger, suggesting that the carbonyl group of acrolein-adducted albumin mediated HMOX1 transcript increase. In acutely exposed C57BL/6J mice, bronchoalveolar lavage protein carbonylation increased. Acrolein-adducted albumin Cys34 was identified by nLC-MS/MS. These findings indicate that adduction of albumin by acrolein confers a cytoprotective function by scavenging free acrolein, decreasing a cellular stress response, and inducing an antioxidant gene response. Further, these results suggest that ß-carbon reactivity may be required for acrolein's cytotoxicity and ATF3 transcript increase, and the carbonyl group of acrolein-adducted albumin can induce HMOX1 transcript increase.

Dynamic Microfluidic Cytometry for Single-Cell Cellomics: High-Throughput Probing Single-Cell-Resolution Signaling.

Cell signaling is a fast, dynamic, and complex process, which controls a variety of critical physiological functions. Methods to investigate such dynamic information, however, suffer from limited throughput in the single-cell level and a lack of precise fluid manipulation. Herein, we present a new strategy, termed dynamic microfluidic cytometry (DMC), for high-throughput probing of G protein-coupled receptor (GPCR) signaling in single-cell resolution (single-cell cellomics analysis) by creatively applied cyclical cell trapping, stimulating, and releasing automatically. Dose-response curves and half-maximal effective concentration (EC50) values for HeLa cells treated with adenosine triphosphate (ATP), histamine (HA), and acetylcholine chloride (ACH) were successfully obtained in the single-cell level. High-throughput single-cell dynamic signaling was further implemented by sequential or simultaneous stimulation, which revealed that different mechanisms were working in triggering intracellular calcium release. In addition, simultaneous stimulation to two different types of cells, HeLa and NIH-3T3 cells, was also successfully realized, which was crucial for online comparison of dynamic signaling of different types of cells. We believe that the proposed DMC provides a versatile means for high-throughput probing single-cell dynamic signaling, which is potentially useful in chemical biology, cell biology, and pharmacology.

UBE3B Is a Calmodulin-regulated, Mitochondrion-associated E3 Ubiquitin Ligase.

Recent genome-wide studies found that patients with hypotonia, developmental delay, intellectual disability, congenital anomalies, characteristic facial dysmorphic features, and low cholesterol levels suffer from Kaufman oculocerebrofacial syndrome (KOS, also reported as blepharophimosis-ptosis-intellectual disability syndrome). The primary cause of KOS is autosomal recessive mutations in the gene UBE3B However, to date, there are no studies that have determined the cellular or enzymatic function of UBE3B. Here, we report that UBE3B is a mitochondrion-associated protein with homologous to the E6-AP Cterminus (HECT) E3 ubiquitin ligase activity. Mutating the catalytic cysteine (C1036A) or deleting the entire HECT domain (amino acids 758-1068) results in loss of UBE3B's ubiquitylation activity. Knockdown of UBE3B in human cells induces changes in mitochondrial morphology and physiology, a decrease in mitochondrial volume, and a severe suppression of cellular proliferation. We also discovered that UBE3B interacts with calmodulin via its N-terminal isoleucine-glutamine (IQ) motif. Deletion of the IQ motif (amino acids 29-58) results in loss of calmodulin binding and a significant increase in the in vitro ubiquitylation activity of UBE3B. In addition, we found that changes in calcium levels in vitro disrupt the calmodulin-UBE3B interaction. These studies demonstrate that UBE3B is an E3 ubiquitin ligase and reveal that the enzyme is regulated by calmodulin. Furthermore, the modulation of UBE3B via calmodulin and calcium implicates a role for calcium signaling in mitochondrial protein ubiquitylation, protein turnover, and disease.