Atypical behaviour and connectivity in SHANK3-mutant macaques.

Mutation or disruption of the SH3 and ankyrin repeat domains 3 (SHANK3) gene represents a highly penetrant, monogenic risk factor for autism spectrum disorder, and is a cause of Phelan-McDermid syndrome. Recent advances in gene editing have enabled the creation of genetically engineered non-human-primate models, which might better approximate the behavioural and neural phenotypes of autism spectrum disorder than do rodent models, and may lead to more effective treatments. Here we report CRISPR-Cas9-mediated generation of germline-transmissible mutations of SHANK3 in cynomolgus macaques (Macaca fascicularis) and their F1 offspring. Genotyping of somatic cells as well as brain biopsies confirmed mutations in the SHANK3 gene and reduced levels of SHANK3 protein in these macaques. Analysis of data from functional magnetic resonance imaging revealed altered local and global connectivity patterns that were indicative of circuit abnormalities. The founder mutants exhibited sleep disturbances, motor deficits and increased repetitive behaviours, as well as social and learning impairments. Together, these results parallel some aspects of the dysfunctions in the SHANK3 gene and circuits, as well as the behavioural phenotypes, that characterize autism spectrum disorder and Phelan-McDermid syndrome.

A bimodal time-gated luminescence-magnetic resonance imaging nanoprobe based on a europium(III) complex anchored on BSA-coated MnO2 nanosheets for highly selective detection of H2O2.

A novel nanocomposite, [Eu(BTD)3(DPBT)]-BSA@MnO2, is reported to serve as an effective nanoprobe for bimodal time-gated luminescence (TGL) and magnetic resonance (MR) imaging of H2O2in vitro and in vivo. The nanoprobe was fabricated by immobilizing visible-light-excitable Eu3+ complexes in bovine serum albumin (BSA)-coated lamellar MnO2 nanosheets. The TGL of the Eu3+ complex was effectively quenched by the MnO2 nanosheets. Upon exposure to H2O2, the MnO2 nanosheets underwent reduction to Mn2+, which simultaneously triggered rapid, selective and sensitive "turn-on" responses toward H2O2 in both TGL and MR detection modes. The presence of a protective "corona" formed by BSA enables the nanoprobe to withstand high concentrations of glutathione (GSH), a strong reducing agent of MnO2 nanosheets. This capability allows the nanoprobe to be utilized for detecting H2O2 in living biosamples. The combined utilization of TGL and MR detection modes enables the nanoprobe to image H2O2 across a wide range of resolutions, from the subcellular level to the whole body, without any depth limitations. The results obtained from these modes can be cross-validated, enhancing the accuracy of the detection. The capability of the nanoprobe was validated by TGL imaging of endogenous and exogenous H2O2 in live HeLa cells, as well as bimodal TGL-MR imaging of H2O2 in tumor-bearing mice. The research achievements suggest that the integration of luminescent lanthanide complexes with protein-coated MnO2 nanosheets offers a promising bimodal TGL-MR sensing platform for H2O2in vitro and in vivo.

Responsive ß-Diketonate-europium(III) Complex-Based Probe for Time-Gated Luminescence Detection and Imaging of Hydrogen Sulfide In Vitro and In Vivo.

As a crucial biological gasotransmitter, hydrogen sulfide (H2S) plays important roles in many pathological and physiological processes. Highly selective and sensitive detection of H2S is significant for the precise diagnosis and evaluation of diverse diseases. Nevertheless, challenges remain in view of the interference of autofluorescence in organisms and the stronger reactivity of H2S itself. Herein, we report the design and synthesis of a novel H2S-responsive ß-diketonate-europium(III) complex-based probe, [Eu(DNB-Npketo)3(terpy)], for background-free time-gated luminescence (TGL) detection and imaging of H2S in autofluorescence-rich biological samples. The probe, consisting of a 2,4-dinitrobenzenesulfonyl (DNB) group coupled to a ß-diketonate-europium(III) complex, shows almost no luminescence owing to the existence of intramolecular photoinduced electron transfer. The cleavage of the DNB group by a H2S-triggered reaction results in the recovery of the long-lived luminescence of the Eu3+ complex, allowing the detection of H2S in complicated biological samples to be performed in TGL mode. The probe showed a fast response, high specificity, and high sensitivity toward H2S, which enabled it to be successfully used for the quantitative TGL detection of H2S in tissue homogenates of mouse organs. Additionally, the low cytotoxicity of the probe allowed it to be further used for the TGL imaging of H2S in living cells and mice under different stimuli. All of the results suggested the potential of the probe for the investigation and diagnosis of H2S-related diseases.

Tracking Plasma Membrane Damage Using a Ruthenium(II) Complex Phosphorescent Indicator Paired with Cholesterol.

Long-term in situ plasma membrane-targeted imaging is highly significant for investigating specific biological processes and functions, especially for the imaging and tracking of apoptosis processes of cells. However, currently developed membrane probes are rarely utilized to monitor the in situ damage of the plasma membrane. Herein, a transition-metal complex phosphorescent indicator, Ru-Chol, effectively paired with cholesterol, exhibits excellent properties on staining the plasma membrane, with excellent antipermeability, good photostability, large Stokes shift, and long luminescence lifetime. In addition, Ru-Chol not only has the potential to differentiate cancerous cells from normal cells but also tracks in real time the entire progression of cisplatin-induced plasma membrane damage and cell apoptosis. Therefore, Ru-Chol can serve as an efficient tool for the monitoring of morphological and physiological changes in the plasma membrane, providing assistance for drug screening and early diagnosis and treatment of diseases, such as immunodeficiency, diabetes, cirrhosis, and tumors.

Ratiometric lysosome-targeting luminescent probe based on a coumarin-ruthenium(II) complex for formaldehyde detection and imaging in living cells and mouse brain tissues.

Ratiometric luminescence probes have attracted widespread attention because of their self-calibration capability. However, some defects, such as small emission shift, severe spectral overlap and poor water solubility, limit their application in the field of biological imaging. In this study, a unique luminescence probe, Ru-COU, has been developed by combining tris(bipyridine)ruthenium(II) complex with coumarin derivative through a formaldehyde-responsive linker. The probe exhibited a large emission shift (Δλ > 100 nm) and good water solubility, achieving ratiometric emission responses at 505 nm and 610 nm toward formaldehyde under acidic conditions. Besides, ratiometric luminescence imaging of formaldehyde in living cells and Alzheimer disease mouse's brain slices demonstrates the potential value of Ru-COU for the diagnosis and treatment of formaldehyde related diseases.

FRET Luminescent Probe for the Ratiometric Imaging of Peroxynitrite in Rat Brain Models of Epilepsy-Based on Organic Dye-Conjugated Iridium(III) Complex.

Epilepsy is a chronic neurological disorder characterized by recurrent seizures globally, imposing a substantial burden on patients and their families. The pathological role of peroxynitrite (ONOO-), which can trigger oxidative stress, inflammation, and neuronal hyperexcitability, is critical in epilepsy. However, the development of reliable, in situ, and real-time optical imaging tools to detect ONOO- in the brain encounters some challenges related to the depth of tissue penetration, background interference, optical bleaching, and spectral overlapping. To address these limitations, we present Ir-CBM, a new one-photon and two-photon excitable and long-lived ratiometric luminescent probe designed specifically for precise detection of ONOO- in epilepsy-based on the Förster resonance energy transfer mechanism by combining an iridium(III) complex with an organic fluorophore. Ir-CBM possesses the advantages of rapid response, one-/two-photon excitation, and ratiometric luminescent imaging for monitoring the cellular levels of ONOO- and evaluating the effects of different therapeutic drugs on ONOO- in the brain of an epilepsy model rat. The development and utilization of Ir-CBM offer valuable insights into the design of ratiometric luminescent probes. Furthermore, Ir-CBM serves as a rapid imaging and screening tool for antiepileptic drugs, thereby accelerating the exploration of novel antiepileptic drug screening and improving preventive and therapeutic strategies in epilepsy research.

Mitochondria-Targetable Ratiometric Time-Gated Luminescence Probe Activated by Selenocysteine for the Visual Monitoring of Liver Injuries.

Liver injury can result from various risk factors including diabetes, virus, alcohol, drugs, and other toxins, which is mainly responsible for global mortality and morbidity. Selenocysteine (Sec), as the main undertaker of selenium function in the life system, features prominently in a series of hepatic injuries and has close association with the pathological progression of liver injuries. Here, we report a mitochondria-targetable lanthanide complex-based probe, Mito-NPTTA-Tb3+/Eu3+, that can be used for accurately determining Sec in live cells and laboratory animals via the ratiometric time-gated luminescence (TGL) technique. This probe is composed of 2,2':6',2″-terpyridine-Tb3+/Eu3+ mixed complexes as the luminophore, 2,4-dinitrophenyl (DNP) as the responsive moiety and a lipophilic triphenylphosphonium cation (PPh3+) as the mitochondria-targeting moiety. Upon reaction with Sec, accompanied by the cleavage of DNP from the probe molecule, the I540/I690 ratio of the probe increased by 55 times, which enabled Sec to be detected with the ratiometric TGL method. After being incubated with living cells, the probe molecules were selectively accumulated in mitochondria to allow the mitochondrial Sec to be successfully imaged under the ratiometric TGL mode. Importantly, using this probe coupled with the ratiometric TGL imaging technique, the fluctuations of liver Sec in various liver injuries of model mice induced by diabetes, drug, toxin, and alcohol were precisely monitored, revealing that Sec plays an important antioxidant role during the oxidative stress process in liver injury, and the Sec levels have a close interrelationship with the degree of liver injury. All the results suggest that the new probe Mito-NPTTA-Tb3+/Eu3+ could be a potential tool for the accurate diagnosis of liver injury.

An activatable nanoprobe based on nanocomposites of visible-light-excitable europium(III) complex-anchored MnO2 nanosheets for bimodal time-gated luminescence and magnetic resonance imaging of tumor cells.

Bimodal imaging probes that combine magnetic resonance imaging (MRI) and photoluminescence imaging are quite appealing since they can supply both anatomical and molecular information to effectively ameliorate the accuracy of detection. In this study, an activatable nanoprobe, [Eu(BTD)3(DPBT)]@MnO2, for bimodal time-gated luminescence imaging (TGLI) and MRI has been constructed by anchoring visible-light-excitable Eu3+ complexes on lamellar MnO2 nanosheets. Due to the luminescence quenching effect and non-magnetic resonance (MR) activity of MnO2 nanosheets, the developed nanoprobe presents quite weak TGL and MR signals. After exposure to H2O2 or GSH, accompanied by the transformation from MnO2 to Mn2+, the nanoprobe exhibits rapid, sensitive, and selective "turn-on" responses towards GSH and H2O2 in TGL and MR detection modes. Furthermore, the nanoprobe displays high stability, low cytotoxicity, good biocompatibility and water dispersion. Given the high contents of GSH and H2O2 in cancer cells, the nanoprobe was used for the identification of cancer cells by TGLI of intracellular GSH and H2O2, as well as for the tracing of tumor cells in tumor-bearing mice by tumor-targeting in vivo MRI and TGLI of tumor tissues. The research outcomes proved the potential of [Eu(BTD)3(DPBT)]@MnO2 as a useful nanoprobe for the tracing and accurate detection of cancer cells in vitro and in vivo via bimodal TGLI and MRI.

Comparative transcriptome analysis of experimental cryptorchidism: of mice and cynomolgus monkeys.

In most mammalian species, the testis descends from the abdomen into the scrotum during fetal or neonatal life. The failure of testicular descent, a pathological condition known as cryptorchidism, has long been the subject of scientific interest in a wide range of fields, including medicine, developmental biology, and evolutionary biology. In this study, we analyzed global gene expression changes associated with experimental cryptorchidism in mice by using RNA-seq. A total of 453 differentially expressed genes were identified, of which 236 genes were upregulated, and 217 genes were downregulated. Gene ontology, pathway, and gene network analysis highlighted the activation of inflammatory response in experimental cryptorchidism. By examining the promoter regions of differentially expressed genes, we identified 12 causal transcription factors. In addition, we also induced experimental cryptorchidism in two cynomolgus monkeys and performed RNA-seq. A cross-species comparison was performed at the gene expression level. Our study provides a valuable resource for further understanding the molecular mechanisms of cryptorchidism in mammals.

Responsive Metal Complex Probes for Time-Gated Luminescence Biosensing and Imaging.

The development of reliable bioanalytical probes for selective and sensitive detection of particular analytes in biological systems is essential for better understanding the roles of the analytes in their native contexts. In the last two decades, luminescent metal complexes have greatly contributed to the development of such probes for biosensing and imaging due to their unique spectral and temporal properties, controllable cell membrane permeability, and cytotoxicity. Conjugating an analyte-activatable moiety to the metal complex luminophores allows the production of responsive metal complex probes for this analyte detection. Owing to their long-lifetime emissions, the responsive metal complex probes are accessible to the technique of time-gated luminescence (TGL) detection and imaging. With a delay time after pulsed excitation, the TGL technique allows for collection of only long-lived luminescence from responsive metal complex probes, while filtering out short-lived background autofluorescence, providing a background-free approach for the detection and imaging of the analyte at subcellular and/or molecular levels. Responsive metal complex probes, therefore, have emerged as complementary sensing and imaging tools of organic dye-based fluorescent probes for the in situ detection of analytes in complicated biological environments.In this Account, we describe the advances in the development of metal complex probes and their applications for TGL bioassays with particular focus on our efforts made in this field. We first introduce the photophysical/-chemical properties of luminescent metal complexes, including lanthanide (europium and terbium) and transition metal (ruthenium and iridium) complexes. The luminescence lifetimes (τ) of lanthanide and transition metal complexes are at micro/millisecond (µs/ms) and hundreds/thousands nanosecond (ns) levels, respectively. The emission lifetimes are significantly longer than the autofluorescence lifetime (τ < 10 ns) of biological samples. Such long-lived luminescence of these metal complexes enables our research on demonstrating responsive probes for background-free TGL detection of some reactive biomolecules, such as reactive oxygen/nitrogen species (ROS/RNS) and biothiols.We conclude this Account by outlining the future directions to further develop new generation responsive TGL probes for promoting their practical applications. The responsive TGL probes are expected to be translated for biomedical and/or (pre)clinical investigations of biomolecules in situ. Reversibility, lower toxicity, ability of excitation at longer wavelength, and potential to be translated are key criteria for the development of next-generation probes. We also anticipate that further development of responsive TGL probes will contribute to the bioassay in more challenging biological systems, such as plants that have significant higher background autofluorescence than animals.

Smart Bimodal Imaging of Hypochlorous Acid In Vivo Using a Heterobimetallic Ruthenium(II)-Gadolinium(III) Complex Probe.

A unique heterobimetallic Ru(II)-Gd(III) complex, Ru-AN-Gd, is reported to serve as an effective probe for bimodal phosphorescence-magnetic resonance (MR) imaging of hypochlorous acid (HClO) in vitro and in vivo. The probe was designed by incorporating a MR contrast agent, Gd-DOTA, into a HClO-responsive bipyridine-Ru(II) complex derivative. The specific reaction between Ru-AN-Gd and HClO triggers the cleavage of an ether bond in the probe molecule, resulting in phosphorescence turn-on and MR turn-off responses to HClO. The integration of MR and phosphorescence detection modes allows the probe to be employed for detecting HClO in a quite wide concentration range (0.6-2000 µM) and for imaging HClO at various resolutions ranging from the subcellular level to the whole body without a depth limit. Its applicability was demonstrated by phosphorescence imaging of lysosomal HClO in live cells, visualization of HClO generation in a mouse arthritis model, and bimodal phosphorescence-MR imaging of HClO in drug-induced acute liver and kidney injury of a mouse. The research achievements suggested the potential of Ru-AN-Gd for diagnosis and treatment monitoring of HClO-related disease.

Correction to: Functional analysis of GhCHS, GhANR and GhLAR in colored fiber formation of Gossypium hirsutum L.

In the original publication of this article [1], the authors pointed out the Fig. 4b was same with Fig. 4c. The correct Fig. 4b should be below.

A folic acid-functionalized dual-emissive nanoprobe for "double-check" luminescence imaging of cancer cells.

Development of luminescent probes for rapid and effective discrimination and detection of cancer cells has the potential to address the current challenges in early diagnosis and treatment monitoring of cancer diseases. In this work, we report the preparation of a unique folic acid (FA)-functionalized dual-emissive nanoprobe, CTMR@BHHBCB-Eu-FA, for steady-state and time-gated luminescence "double-check" imaging of cancer cells. The nanoprobe was engineered by covalently doping two luminescent dyes, 5-carboxytetramethylrhodamine (CTMR) and BHHBCB-Eu3+, in core and shell of silica nanoparticles, followed by surface modification of the nanoparticles with FA, a cancer cell-targeting molecule. As-prepared nanoprobe is monodisperse and highly stable in buffer displaying two strong emissions, short-lived emission from CTMR at 584 nm and long-lived emission from BHHBCB-Eu3+ at 612 nm. The nanoprobe is biocompatible, and can specifically recognize folate receptor (FR)-overexpressed cancer cells through the FA-FR binding interaction. Using the nanoprobe, the "double-check" imaging of HeLa cells was successfully achieved at steady-state and time-gated luminescence modes, indicating the capability of the nanoprobe for cancer cell imaging.

Effect of thoracic paravertebral nerve block on the early postoperative rehabilitation in patients undergoing thoracoscopic radical lung cancer surgery.

OBJECTIVE: To evaluate the effect of thoracic paravertebral nerve block on early postoperative rehabilitation in patients undergoing radical thoracoscopic surgery for lung cancer. METHODS: Ninety patients scheduled for elective video-assisted thoracoscopic lobectomy of lung cancer were divided into 2 groups: the general anesthesia group (GA group, n = 45) and the TPVB group (TP group, n = 45). The primary outcome was the decline rate of the 6-min walking test (6MWT); the second outcomes were as follows: absolute value and the completion rate of 6MWT, postoperative analgesia deficiency and pain scores, oxycodone consumption, sleep quality, the incidence of postoperative pulmonary complications, and the hospital stay. RESULTS: Compared with the GA group, the TP group had a lower decline rate of the 6MWT on POD1 and POD2. The walking distance on POD1 and POD2 in the TP group was significantly longer than that in the GA group; the completion rate at POD1 in the TP group was higher than that in the GA group. The pain scores and oxycodone consumption at POD1 in the TP group were lower than the GA group. The sleep quality in the TP group was higher than the GA group. CONCLUSIONS: TPVB can significantly improve postoperative rehabilitation in patients undergoing thoracoscopic radical lung cancer surgery, which is helpful for promoting the early recovery of patients. TRIAL REGISTRATION: Chinese Clinical Trial Registry, ChiCTR1900026213. Registered 26 Sept. 2019, http://www.chictr.org.cn/showproj.aspx?proj=43733 .

"Dual-Key-and-Lock" Ruthenium Complex Probe for Lysosomal Formaldehyde in Cancer Cells and Tumors.

Biomedical investigations reveal that excessive formaldehyde generation is possibly a critical factor for tissue cancerization, cancer progression, and metastasis. Responsive molecular probes that can detect lysosomal formaldehyde in live cells and tumors and monitor drug-triggered formaldehyde scavenging contribute potentially to future cancer diagnosis and treatment monitoring. Herein, a novel "dual-key-and-lock" strategy-based ruthenium(II) complex probe, Ru-FA, is reported as an effective tool for formaldehyde detection in vitro and in vivo. Ru-FA shows weak luminescence due to photon-induced electron transfer (PET) process from Ru(II) center to electron withdrawing group 2,4-dinitrobenzene (DNB). Triggered by the specific reaction with formaldehyde (first "key") in an acidic microenvironment (second "key"), DNB is cleaved from Ru-FA, affording an emissive Ru(II) complex derivative, Ru-NR. Spectrometric analysis including steady-state and time-gated luminescence indicates that Ru-FA is favorable to be used as the probe for quantification of formaldehyde in human sera and mouse organs. Ru-FA is biocompatible and cell membrane permeable. Together with its smart "dual-key-and-lock" response to formaldehyde, luminescence imaging of lysosomal formaldehyde in live cells, visualization of tumor-derived endogenous formaldehyde, and monitoring of formaldehyde scavenging in mice were achieved, followed by the successful demonstration on detection of formaldehyde in tumors and other organs. These in vivo and in vitro detection confirm not only the excessive formaldehyde generation in tumors, but also the efficient drug administration to scavenge formaldehyde, demonstrating the potential application of Ru-FA in cancer diagnosis and treatment monitoring through lysosomal formaldehyde detection.

Precise Monitoring of Drug-Induced Kidney Injury Using an Endoplasmic Reticulum-Targetable Ratiometric Time-Gated Luminescence Probe for Superoxide Anions.

Drug-induced acute kidney injury (AKI), caused by renal drug metabolism, has been considered to be a major barrier in drug development and clinical treatment. Among various drugs, anticancer drugs, cisplatin, and aminoglycoside antibiotic gentamicin, are known to be able to induce excessive or unfolded accumulation of proteins in the endoplasmic reticulum (ER) of cells, leading to ER stress. Meanwhile, reactive oxygen species (ROS) are formed, and superoxide anion (O2•-), the first produced ROS, is a key species to induce the AKI. Due to the lack of appropriate tools, the early diagnosis of AKI induced by cisplatin, gentamicin, or other drugs is still a crucial challenge. Herein, we report a lanthanide complex-based ER-targetable luminescence probe for O2•-, ER-(4'-trifluoromethanesulfonyloxy-2,2':6',2''-terpyridine-6,6''-diyl)bis(methylenenitrilo)tetrakis (aceticacid) (NFTTA)-Eu3+/Tb3+, for the sensitive monitoring of drug-induced AKI via mapping the generation of O2•- in live cells and laboratory animals. Using this probe coupled with the ratiometric time-gated luminescence (RTGL) imaging technique, the changes of O2•- level in the ER of live cells induced by different stimuli were precisely monitored. More importantly, the substantial increases in O2•- levels were observed in the cisplatin- and gentamicin-induced kidney injury of mice. In addition, the protective effects of l-carnitine (LC) and epigallocatechin-3-gallate (EGCG) against cisplatin- and gentamicin-induced nephrotoxicity were visualized and elucidated for the first time. The results demonstrated the potential of ER-NFTTA-Eu3+/Tb3+ for examining and monitoring O2•- in drug-induced AKI and for providing a diagnosis and treatment of nephrotoxicity diseases.

Mitochondria-Targetable Ratiometric Time-Gated Luminescence Probe for Carbon Monoxide Based on Lanthanide Complexes.

As a critical gasotransmitter, carbon monoxide (CO) has been demonstrated to be related with mitochondrial respiration, but the monitoring of CO in mitochondria remains a great challenge. In this work, a unique ratiometric time-gated luminescence (TGL) probe, Mito-NBTTA-Tb3+/Eu3+, that can specifically respond to mitochondrial CO has been developed. The probe was designed by incorporating a mitochondria-targeting moiety, triphenylphosphonium, into a CO-activatable terpyridine polyacid derivative, 4'-(4-nitrobenzyloxy-2,2':6',2''-terpyridine-6,6''-diyl) bis(methylenenitrilo) tetrakis(acetic acid), for coordinating to Eu3+ and Tb3+ ions to construct lanthanide complex-based probe for ratiometric TGL detection of CO. Upon reaction with CO, accompanied by the conversion of nitro group to amino group, a 1,6-rearrangement-elimination reaction occurs, which leads to the cleavage of 4-nitrobenzyl group from Mito-NBTTA-Tb3+/Eu3+, resulting in the significant increase of Tb3+ emission at 540 nm and moderate decrease of Eu3+ emission at 610 nm. After the reaction, the I540/ I610 ratio was found to be 48-fold enhanced. This feature allowed Mito-NBTTA-Tb3+/Eu3+ to be employed as a ratiometric TGL probe for CO detection with the I540/ I610 ratio as a signal. In addition, the probe showed outstanding mitochondria-localization characteristic, which enabled the probe to be successfully applied to imaging CO within mitochondria of living cells under TGL and ratiometric modes. The application of Mito-NBTTA-Tb3+/Eu3+ was demonstrated by the visualization and quantitative detection of exogenous and endogenous CO in living cells and mouse liver tissue slices, as well as in living Daphnia magna and mice. All of the results suggested the potential of Mito-NBTTA-Tb3+/Eu3+ for the quantitative monitoring of CO in vitro and in vivo.

Functional analysis of GhCHS, GhANR and GhLAR in colored fiber formation of Gossypium hirsutum L.

BACKGROUND: The formation of natural colored fibers mainly results from the accumulation of different anthocyanidins and their derivatives in the fibers of Gossypium hirsutum L. Chalcone synthase (CHS) is the first committed enzyme of flavonoid biosynthesis, and anthocyanidins are transported into fiber cells after biosynthesis mainly by Anthocyanidin reductase (ANR) and Leucoanthocyanidin reductase (LAR) to present diverse colors with distinct stability. The biochemical and molecular mechanism of pigment formation in natural colored cotton fiber is not clear. RESULTS: The three key genes of GhCHS, GhANR and GhLAR were predominantly expressed in the developing fibers of colored cotton. In the GhCHSi, GhANRi and GhLARi transgenic cottons, the expression levels of GhCHS, GhANR and GhLAR significantly decreased in the developing cotton fiber, negatively correlated with the content of anthocyanidins and the color depth of cotton fiber. In colored cotton Zongxu1 (ZX1) and the GhCHSi, GhANRi and GhLARi transgenic lines of ZX1, HZ and ZH, the anthocyanidin contents of the leaves, cotton kernels, the mixture of fiber and seedcoat were all changed and positively correlated with the fiber color. CONCLUSION: The three genes of GhCHS, GhANR and GhLAR were predominantly expressed early in developing colored cotton fibers and identified to be a key genes of cotton fiber color formation. The expression levels of the three genes affected the anthocyanidin contents and fiber color depth. So the three genes played a crucial part in cotton fiber color formation and has important significant to improve natural colored cotton quality and create new colored cotton germplasm resources by genetic engineering.

Iridium(III) Complex-Based Activatable Probe for Phosphorescent/Time-Gated Luminescent Sensing and Imaging of Cysteine in Mitochondria of Live Cells and Animals.

This study reports an activatable iridium(III) complex probe for phosphorescence/time-gated luminescence detection of cysteine (Cys) in vitro and in vivo. The probe, [Ir(ppy)2 (NTY-bpy)](PF6 ) [ppy: 2-phenylpyridine; NTY-bpy: 4-methyl-4'-(2-nitrovinyl)-2,2'-bipyridine], is developed by incorporating a strong electron-withdrawing group, nitroolefin, into a bipyridine ligand of the IrIII complex. The luminescence of the probe is quenched owing to the intramolecular charge transfer (ICT) process, but switched on by a specific recognition reaction between the probe and Cys. [Ir(ppy)2 (NTY-bpy)](PF6 ) shows high sensitivity and selectivity for Cys detection and good biocompatibility. The long-lived emission of [Ir(ppy)2 (NTY-bpy)](PF6 ) allows time-gated luminescence analysis of Cys in cells and human sera. These properties make it convenient for the phosphorescence and time-gated luminescence imaging and flow cytometry analysis of Cys in live samples. The Cys images in cancer cells and inflamed macrophage cells reveal that [Ir(ppy)2 (NTY-bpy)](PF6 ) is distributed in mitochondria after cellular internalization. Visualizations and flow cytometry analysis of mitochondrial Cys levels and Cys-mediated redox activities of live cells are achieved. By using [Ir(ppy)2 (NTY-bpy)](PF6 ) as a probe, in vivo sensing and imaging of Cys in D. magna, zebrafish, and mice are then demonstrated.

Time-Gated Luminescent In Situ Hybridization (LISH): Highly Sensitive Detection of Pathogenic Staphylococcus aureus.

We describe simple direct conjugation of a single TEGylated Europium chelate to DNA that binds to intracellular rRNA and is then detected using a homogeneous luminescent in situ hybridisation (LISH) technique. As a proof-of-principle, Staphylococcus aureus (S. aureus) was selected as a model for our study to show the ability of this probe to bind to intracellular 16S ribosomal rRNA. A highly purified Europium chelate conjugated oligonucleotide probe complementary to an rRNA sequence-specific S. aureus was prepared and found to be soluble and stable in aqueous solution. The probe was able to bind specifically to S. aureus via in situ hybridisation to differentiate S. aureus from a closely related but less pathogenic Staphylococcus species (S. epidermidis). A time-gated luminescent (TGL) microscope system was used to generate the high signal-to-noise ratio (SNR) images of the S. aureus. After excitation (365 nm, Chelate λmax = 335 nm), the long-lived (Eu3+) luminescent emission from the probe was detected without interference from natural background autofluorescence typically seen in biological samples. The luminescent images were found to have 6 times higher SNR or sensitivity compared to the fluorescent images using conventional fluorophore Alexa Fluor 488. The TEGylated Europium chelate -oligo probe stained S. aureus with mean signal intensity 3.5 times higher than the threshold level of signal from S. epidermidis (with SNR 8 times higher). A positive control probe (EUB338-BHHTEGST-Eu3+) has mean signal intensity for S. aureus and S. epidermidis equally 3.2 times higher than the threshold of signal for a negative NON-EUB338 control probe. The direct conjugation of a single Europium chelate to DNA provides simplicity and improvement over existing bovine serum albumin (BSA)/streptavidin/biotinylated DNA platforms for multi-attachment of Europium chelate per DNA and more importantly makes it feasible for hybridisation to intracellular RNA targets. This probe has great potential for highly sensitive homogeneous in situ hybridisation detection of the vast range of intracellular DNA targets.