Reprogramming Hypoxic Tumor-Associated Macrophages by Nanoglycoclusters for Boosted Cancer Immunotherapy.

The tumor-associated macrophages (TAMs) in intratumoral hypoxic regions are key drivers of immune escape. Reprogramming the hypoxic TAMs to antitumor phenotype holds great therapeutic benefits but remains challenging for current drugs. Here, an in situ activated nanoglycocluster is reported to realize effective tumor penetration and potent repolarization of hypoxic TAMs. Triggered by the hypoxia-upregulated matrix metalloproteinase-2 (MMP-2), the nanoglycocluster is self-assembled from the administered mannose-containing precursor glycopeptides and presents densely-arrayed mannoses to multivalently engage with mannose receptors on M2-like TAMs for efficient phenotype switch. By virtue of the high diffusivity of precursor glycopeptides due to their low molecular mass and weak affinity with TAMs in perivascular regions, the nanoglycoclusters are capable of substantially accumulating in hypoxic areas to strongly interact with local TAMs. This enables the efficient repolarization of overall TAMs with a higher rate than the small-molecule drug R848 and CD40 antibody, and beneficial therapeutic effects in mouse tumor models especially when combining with PD-1 antibody. This on-demand activated immunoagent is endowed with tumor-penetrating properties and inspires the design of diverse intelligent nanomedicines for hypoxia-related cancer immunotherapy.

Single-Molecule Imaging Reveals Differential AT1R Stoichiometry Change in Biased Signaling.

G protein-coupled receptors (GPCRs) represent promising therapeutic targets due to their involvement in numerous physiological processes mediated by downstream G protein- and ß-arrestin-mediated signal transduction cascades. Although the precise control of GPCR signaling pathways is therapeutically valuable, the molecular details for governing biased GPCR signaling remain elusive. The Angiotensin II type 1 receptor (AT1R), a prototypical class A GPCR with profound implications for cardiovascular functions, has become a focal point for biased ligand-based clinical interventions. Herein, we used single-molecule live-cell imaging techniques to evaluate the changes in stoichiometry and dynamics of AT1R with distinct biased ligand stimulations in real time. It was revealed that AT1R existed predominantly in monomers and dimers and underwent oligomerization upon ligand stimulation. Notably, ß-arrestin-biased ligands induced the formation of higher-order aggregates, resulting in a slower diffusion profile for AT1R compared to G protein-biased ligands. Furthermore, we demonstrated that the augmented aggregation of AT1R, triggered by activation from each biased ligand, was completely abrogated in ß-arrestin knockout cells. These findings furnish novel insights into the intricate relationship between GPCR aggregation states and biased signaling, underscoring the pivotal role of molecular behaviors in guiding the development of selective therapeutic agents.

MdZAT5 regulates drought tolerance via mediating accumulation of drought-responsive miRNAs and mRNAs in apple.

Drought limits apple yield and fruit quality. However, the molecular mechanism of apple in response to drought is not well known. Here, we report a Cys2/His2 (C2H2)-type zinc-finger protein, MdZAT5, that positively regulates apple drought tolerance by regulating drought-responsive RNAs and microRNAs (miRNAs). DNA affinity purification and sequencing and yeast-one hybrid analysis identified the binding motifs of MdZAT5, T/ACACT/AC/A/G. Chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP-qPCR) and electrophoretic mobility shift assays (EMSAs) showed that MdZAT5 directly binds to the promoters of the drought-responsive genes including MdRHA2a, MdLEA14, MdTPX1, and MdCAT3, and activates their expression under drought stress. MdZAT5 interacts with and directly targets HYPONASTIC LEAVES1 (MdHYL1). MdZAT5 may facilitate the interaction of MdHYL1 with pri-miRNAs or MdDCL1 by activating MdHYL1 expression, thereby regulating the biogenesis of drought-responsive miRNAs. Genetic dissection showed that MdHYL1 is essential for MdZAT5-mediated drought tolerance and miRNA biogenesis. In addition, ChIP-qPCR and EMSA revealed that MdZAT5 binds directly to the promoters of some MIR genes including Mdm-miR171i and Mdm-miR172c, and modulates their transcription. Taken together, our findings improve our understanding of the molecular mechanisms of drought response in apple and provide a candidate gene for the breeding of drought-tolerant cultivars.

Surface-Engineered Gold Nanoclusters for Stimulated Emission Depletion and Correlated Light and Electron Microscopy Imaging.

Stimulated emission depletion (STED) nanoscopy is an emerging super-resolution imaging platform for the study of the cellular structure. Developing suitable fluorescent probes of small size, good photostability, and easy functionalization is still in demand. Herein, we introduce a new type of surface-engineered gold nanoclusters (Au NCs) that are ultrasmall (1.7 nm) and ultrabright (QY = 60%) for STED bioimaging. A rigid shell formed by l-arginine (l-Arg) and 6-aza-2-thiothymine (ATT) on the Au NC surface enables not only its strong fluorescence in aqueous solution but also its easy chemical modification for specific biomolecule labeling. Au NCs show remarkable performance as STED nanoprobes, including high depletion efficiency, good photobleaching resistance, and low saturation intensity. Super-resolution imaging has been achieved with these Au NCs, and targeted nanoscopic imaging of cellular tubulin has been demonstrated. Moreover, the circular structure of lysosomes in live cells has been revealed. As a Au NC is also an ideal probe for electron microscopy, dual imaging of Aß42 aggregates with the single labeling probe of Au NCs has been realized in correlative light and electron microscopy (CLEM). This work reports, for the first time, the application of Au NCs as a novel probe in STED and CLEM imaging. With their excellent properties, Au NCs show promising potential for nanoscale bioimaging.

The positive feedback regulatory loop of miR160-Auxin Response Factor 17-HYPONASTIC LEAVES 1 mediates drought tolerance in apple trees.

Drought stress tolerance is a complex trait regulated by multiple factors. Here, we demonstrate that the miRNA160-Auxin Response Factor 17 (ARF17)-HYPONASTIC LEAVES 1 module is crucial for apple (Malus domestica) drought tolerance. Using stable transgenic plants, we found that drought tolerance was improved by higher levels of Mdm-miR160 or MdHYL1 and by decreased levels of MdARF17, whereas reductions in MdHYL1 or increases in MdARF17 led to greater drought sensitivity. Further study revealed that modulation of drought tolerance was achieved through regulation of drought-responsive miRNA levels by MdARF17 and MdHYL1; MdARF17 interacted with MdHYL1 and bound to the promoter of MdHYL1. Genetic analysis further suggested that MdHYL1 is a direct downstream target of MdARF17. Importantly, MdARF17 and MdHYL1 regulated the abundance of Mdm-miR160. In addition, the Mdm-miR160-MdARF17-MdHYL1 module regulated adventitious root development. We also found that Mdm-miR160 can move from the scion to the rootstock in apple and tomato (Solanum lycopersicum), thereby improving root development and drought tolerance of the rootstock. Our study revealed the mechanisms by which the positive feedback loop of Mdm-miR160-MdARF17-MdHYL1 influences apple drought tolerance.

Single-Molecule Fluorescence Imaging Reveals GABAB Receptor Aggregation State Changes.

The GABAB receptor is a typical G protein-coupled receptor, and its functional impairment is related to a variety of diseases. While the premise of GABAB receptor activation is the formation of heterodimers, the receptor also forms a tetramer on the cell membrane. Thus, it is important to study the effect of the GABAB receptor aggregation state on its activation and signaling. In this study, we have applied single-molecule photobleaching step counting and single-molecule tracking methods to investigate the formation and change of GABAB dimers and tetramers. A single-molecule stoichiometry assay of the wild-type and mutant receptors revealed the key sites on the interface of ligand-binding domains of the receptor for its dimerization. Moreover, we found that the receptor showed different aggregation behaviors at different conditions. Our results offered new evidence for a better understanding of the molecular basis for GABAB receptor aggregation and activation.

Single-Molecule Imaging of Protein Interactions and Dynamics.

Live-cell single-molecule fluorescence imaging has become a powerful analytical tool to investigate cellular processes that are not accessible to conventional biochemical approaches. This has greatly enriched our understanding of the behaviors of single biomolecules in their native environments and their roles in cellular events. Here, we review recent advances in fluorescence-based single-molecule bioimaging of proteins in living cells. We begin with practical considerations of the design of single-molecule fluorescence imaging experiments such as the choice of imaging modalities, fluorescent probes, and labeling methods. We then describe analytical observables from single-molecule data and the associated molecular parameters along with examples of live-cell single-molecule studies. Lastly, we discuss computational algorithms developed for single-molecule data analysis.

Increased CO2 and light intensity regulate growth and leaf gas exchange in tomato.

Carbon dioxide concentration (CO2 ) and light intensity are known to play important roles in plant growth and carbon assimilation. Nevertheless, the underlying physiological mechanisms have not yet been fully explored. Tomato seedlings (Solanum lycopersicum Mill. cv. Jingpeng No. 1) were exposed to two levels of CO2 and three levels of light intensity and the effects on growth, leaf gas exchange and water use efficiency were investigated. Elevated CO2 and increased light intensity promoted growth, dry matter accumulation and pigment concentration and together the seedling health index. Elevated CO2 had no significant effect on leaf nitrogen content but did significantly upregulate Calvin cycle enzyme activity. Increased CO2 and light intensity promoted photosynthesis, both on a leaf-area basis and on a chlorophyll basis. Increased CO2 also increased light-saturated maximum photosynthetic rate, apparent quantum efficiency and carboxylation efficiency and, together with increased light intensity, it raised photosynthetic capacity. However, increased CO2 reduced transpiration and water consumption across different levels of light intensity, thus significantly increasing both leaf-level and plant-level water use efficiency. Among the range of treatments imposed, the combination of increased CO2 (800 µmol CO2 mol-1 ) and high light intensity (400 µmol m-2 s-1 ) resulted in optimal growth and carbon assimilation. We conclude that the combination of increased CO2 and increased light intensity worked synergistically to promote growth, photosynthetic capacity and water use efficiency by upregulation of pigment concentration, Calvin cycle enzyme activity, light energy use and CO2 fixation. Increased CO2 also lowered transpiration and hence water usage.

Automated Stoichiometry Analysis of Single-Molecule Fluorescence Imaging Traces via Deep Learning.

The stoichiometry of protein complexes is precisely regulated in cells and is fundamental to protein function. Singe-molecule fluorescence imaging based photobleaching event counting is a new approach for protein stoichiometry determination under physiological conditions. Due to the interference of the high noise level and photoblinking events, accurately extracting real bleaching steps from single-molecule fluorescence traces is still a challenging task. Here, we develop a novel method of using convolutional and long-short-term memory deep learning neural network (CLDNN) for photobleaching event counting. We design the  convolutional layers to accurately extract features of steplike photobleaching drops and long-short-term memory (LSTM) recurrent layers to distinguish between photobleaching and photoblinking events. Compared with traditional algorithms, CLDNN shows higher accuracy with at least 2 orders of magnitude improvement of efficiency, and it does not require user-specified parameters. We have verified our CLDNN method using experimental data from imaging of single dye-labeled molecules in vitro and epidermal growth factor receptors (EGFR) on cells. Our CLDNN method is expected to provide a new strategy to stoichiometry study and time series analysis in chemistry.

Molecular detection and characterization of Theileria infection in cattle and yaks from Tibet Plateau Region, China.

Theileriosis continues to threaten the livestock industry worldwide, but comprehensive epidemiological surveys for this disease have not been conducted in the Tibet Plateau Region, China. In this study, we screened 154 cattle blood samples from the Tibet Plateau Region (Lhasa, Lhoka, and Tianzhu), China, for detection of Theileria pathogens by polymerase chain reaction (PCR) with species-specific primers. The results revealed that the prevalence was 6.9 % (2/29) for Theileria orientalis and 27.6 % (8/29) for Theileria sinensis in Lhasa, 0 % (0/30) for T. orientalis and 26.7 % (8/30) for T. sinensis in Lhoka, and 0 % (0/95) for T. orientalis and 30.5 % (29/95) for T. sinensis in Tianzhu. Interestingly, Theileria luwenshuni, which was a previously reported pathogenic Theileria sp. in sheep and goats, was detected in blood samples from cattle and yaks for the first time, with a prevalence of 10 % (3/30) in Lhoka and 1.1 % (1/95) in Tianzhu. No other Theileria sp. was detected in these samples. T. sinensis and T. orientalis infections were detected in cattle and yaks, and T. luwenshuni was discovered for the first time in cattle and yaks in the Tibet Plateau Region, China.

Anaplasma infection of Bactrian camels (Camelus bactrianus) and ticks in Xinjiang, China.

BACKGROUND: To date, anaplasmosis has been reported to be a subclinical disease in Indian and Arabian one-humped camels (Camelus dromedarius) and llamas (Lama glama). However, no information on Anaplasma infection in two-humped Bactrian camels (Camelus bactrianus) in China has been published to date. The aim of this study was to investigate the prevalence of Anaplasma spp. in domestic Bactrian camels and ticks in Xinjiang, China. FINDINGS: A total of 382 ticks were collected from the Bactrian camels and from environmental sources. Of these, 84 were morphologically identified as belonging to the Rhipicephalus sanguineus group and genetically identified (12S rDNA, 16S rDNA and the cytochrome c oxidase 1 genes) as R. sanguineus group ticks (temporally designated as Rhipicephalus sp. Xinjiang). PCR testing showed that 7.2% (20/279) of the camels harbored Anaplasma platys DNA. However, microscopic examination revealed no A. platys inclusions in blood smears from the camels. The PCR prevalence of A. platys DNA was 9.5% (6/63) in Rhipicephalus sp. Xinjiang from the Bactrian camels and 14.3% (3/21) in Rhipicephalus sp. Xinjiang from the vegetation. A. platys DNA was not detected by PCR in other tick species (Hyalomma asiaticum, Dermacentor niveus and Hyalomma dromedarii), and no other Anaplasma species were detected in these samples. CONCLUSIONS: This is the first report of A. platys in Bactrian camels in Xinjiang, China. The moderate positivity observed indicates that these animals might be a natural host for this pathogen in China.

Report of Theileria luwenshuni and Theileria sp. RSR from cervids in Gansu, China.

Theileria parasites are important tick-borne pathogens of animals and cause huge economic losses worldwide. Here, we undertook to assess the prevalence of Theileria spp. in cervids in Gansu, China, based on PCR analysis and sequencing of the 18S rRNA genes. Molecular survey showed that Theileria luwenshuni and Theileria sp. RSR were frequently found in Gansu cervids, and the prevalence of T. luwenshuni was 66.7% (n = 9, 6/9) in roe deer in Zhengning County, 58.3% (n = 12, 7/12) in sika deer in Ningxian County, 50% (n = 10, 5/10) in sika deer and 42.9% (n = 14, 6/14) in red deer in Weiyuan County. The prevalence of Theileria sp. RSR was 77.8% (n = 9, 7/9) in roe deer in Zhengning County, 75% (n = 12, 9/12) in sika deer in Ningxian County, 60.0% (n = 10, 6/10) in sika deer, and 50.0% (n = 14, 7/14) in red deer in Weiyuan County. The co-prevalence of the two T. luwenshuni and Theileria sp. RSR was 55.6% (n = 5, 5/9) in roe deer in Zhengning County, 50% (n = 12, 6/12) in sika deer in Ningxian county, 50% (n = 10, 5/10) in sika deer and 35.7% (n = 14, 5/14) in red deer in Weiyuan County, respectively. No other Theileria species was found in these samples. T. luwenshuni and Theileria sp. RSR were detected for the first time in cervids in Gansu, China. Animal experiments showed that four sheep were inoculated with roe deer blood from Zhengning, sika deer blood from Ningxian, sika deer blood, and red deer blood from Weiyuan, respectively, and the Theileria isolated from these inoculated sheep was identified as T. luwenshuni, but Theileria sp. RSR was not detected in these sheep. Our results extend our understanding of the epidemiology of cervine theileriosis in Gansu, China, and will facilitate the implementation of measures to control theileriosis in cervids and small ruminants. This was the first report to demonstrate the occurrence of T. luwenshuni from roe deer, sika deer, and red deer worldwide.