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Pancreatic β cells are responsible for insulin secretion and are important for glucose regulation in a healthy body and diabetic disease patient without prelabeling of islets. While the conventional biomarkers for diabetes have been glucose and insulin concentrations in the blood, the direct determination of the pancreatic β cell mass would provide critical information for the disease status and progression. By combining fluorination and diversity-oriented fluorescence library strategy, we have developed a multimodal pancreatic β cell probe PiF for both fluorescence and for PET (positron emission tomography). By simple tail vein injection, PiF stains pancreatic β cells specifically and allows intraoperative fluorescent imaging of pancreatic islets. PiF-injected pancreatic tissue even facilitated an antibody-free islet analysis within 2 h, dramatically accelerating the day-long histological procedure without any fixing and dehydration step. Not only islets in the pancreas but also the low background of PiF in the liver allowed us to monitor the intraportal transplanted islets, which is the first in vivo visualization of transplanted human islets without a prelabeling of the islets. Finally, we could replace the built-in fluorine atom in PiF with radioactive 18F and successfully demonstrate in situ PET imaging for pancreatic islets.

The use of intact cells in medical research offers a number of advantages over employing cell-free systems. In diagnostic, cells isolated from liquid biopsies can be directly used, speeding up the time of analysis and diminishing the risk of protein degradation by sample manipulation. In drug discovery, studies in live cells take into account aspects neglected in cell-free systems, such as uptake, metabolization, and subcellular concentration by compartmentalization of potential drug candidates. Therefore, probes for studies in cellulo are of paramount importance. Acid ceramidase (AC) is a lysosomal enzyme that hydrolyses ceramides into sphingoid bases and fatty acids. The essential role of this enzyme in the outburst and progress of several diseases, some of them still incurable, is well sustained. Despite the great clinical relevance of AC as biomarker and therapeutic target, the specific monitoring of AC activity in live cells has remained elusive due to the concomitant existence of neutral and alkaline ceramidases. In this work, we report that 1 deoxydihydroceramides are exclusively hydrolysed by AC. Using N-octanoyl-18-azidodeoxysphinganine as probe and a BODIPY substituted bicyclononyne, we show the click-reliant predominant staining of lysosomes, with extra lysosomal labeling also occurring in some cells. Importantly, using pharmacological and genetic tools together with high resolution mass spectrometry, we demonstrate that both lysosomal and extra lysosomal staining are AC dependent. These findings are translated into the specific flow cytometry monitoring of AC activity in intact cells, which fills an important gap in the field of diseases linked to altered AC activity., This article is dedicated to the memory of Professor Lina M. Obeid. We are very grateful to Dr. Ignacio Alfonso (IQAC-CSIC) and Dr. Gemma Triola (IQAC-CSIC) for their through critical reading of a previous version of the manuscript. We thank Alexandre Garcia (IQAC−CSIC), Ignacio Pérez-Pomeda (IQAC−CSIC), Eva Dalmau (IQAC−CSIC), and Dr. Manel Bosch (CCiT-UB) for their excellent technical assistance.

Brown and brown-like beige/brite adipocytes dissipate energy and have been proposed as therapeutic targets to combat metabolic disorders. However, the therapeutic effects of cell-based therapy in humans remain unclear. Here, we created human brown-like (HUMBLE) cells by engineering human white preadipocytes using CRISPR/Cas9-SAM-gRNA to activate endogenous uncoupling protein 1 expression. Obese mice that received HUMBLE cell transplants showed a sustained improvement in glucose tolerance and insulin sensitivity, as well as increased energy expenditure. Mechanistically, increased arginine/nitric oxide (NO) metabolism in HUMBLE adipocytes promoted the production of NO that was carried by S-nitrosothiols and nitrite in red blood cells to activate endogenous brown fat and improved glucose homeostasis in recipient animals. Taken together, these data demonstrate the utility of using CRISPR/Cas9 technology to engineer human white adipocytes to display brown fat-like phenotypes and may open up cell-based therapeutic opportunities to combat obesity and diabetes.

Human and mouse embryonic stem cells (ESCs) differ in terms of their pluripotency status, i.e., naive vs. primed. This affects various biological properties and leads to several technical hurdles for future clinical applications, such as difficulties in chimera formation, single-cell passaging, and gene editing. In terms of generating functional human tissues and organs via mammalian interspecies chimerism, a fluorescent chemical probe that specifically labels naive ESCs would help to isolate these cells and monitor their conversion. This study demonstrates that the fluorescent chemical probe compound of designation yellow 9 (CDy9) selectively stains naive, but not primed, mouse ESCs (mESCs). CDy9 entered cells via Slc13a5, a highly expressed membrane transporter in naive mESCs. Fluorescence-based cell sorting based on CDy9 staining successfully separated naive mESCs from primed mESCs. Mice generated using CDy9+ cells isolated during the conversion of mouse epiblast stem cells into naive mESCs exhibited coat color chimerism. Furthermore, CDy9 specifically stained cells in the inner cell mass of mouse embryos. These findings suggest that CDy9 is a useful tool to isolate functional naive mESCs.

We lost one of the great maritime scholars in 2017—Professor Richard Goss, who is considered one of the founders of maritime economics. He was a pioneer and a most influential leader in establishin...

An efficient and wash-free method to conjugate a fluorescent tag to a target membrane protein is developed, using engineered Npu DnaE split-inteins. This approach allowed fast labeling while avoiding the strenuous synthesis of a long polypeptide. Two different modes of labeling, namely specific binding and covalent conjugation, are observed. The covalent labeling was monitored within 5 min, without background staining.

Kakeromamide A (1), a new cyclic pentapeptide encompassing a thiazole ring moiety and a β-amino acid, was isolated from the marine cyanobacterium Moorea bouillonii. Its structure was elucidated by the spectral analysis and the modified Marfey’s method. Compound 1 induced differentiation of neural stem cells into astrocytes at the concentration of 10 µM.

Recent trends in regulating maritime vessel emissions have negative effects on the competitiveness of many ports as regulations increase costs for shipping operators calling the ports. This paper develops analytical models to examine the emission standards set by governments for ports in their jurisdictions. Given the emission standards set by governments, which affects fuel cost experienced by shipping operators, ports determine charges for shipping operators. Unilateral, bilateral, and single-country regulation cases are investigated. Specifically, our analysis focuses on how increase in the maximum reservation price of shipping operators, port capacity, and environmental damage costs of ports affect optimal emission standards.

Summary Elastic fibers are present in a variety of tissues and are responsible for their resilience. Until now, no optical contrast agent in the near-infrared (NIR) wavelength range of 700–900 nm has been reported for the imaging of elastic fibers. Here, we report the discovery of a NIR zwitterionic elastin probe ElaNIR (elastin NIR) through fluorescent-image-based screening. The probe was successfully applied for in vitro , ex vivo , and in vivo imaging by various imaging modalities. Age-related elastin differences shown by in vivo fluorescent and photoacoustic imaging indicated that ElaNIR can be a potentially convenient tool for uncovering changes of elastin in live models.

본 연구에서는 베이지안(Bayesian) 접근법을 활용하여 세계 58개 주요 항만에서 추출한 표본 데이터를 토대로 항만민영화가 항만효율성에 미치는 영향을 실증적으로 분석하였다. 동시에 기존의 고전적 분석과 베이지안 분석 간 결과의 차이를 비교·검토하였다. 연구 결과 항만민영화 자체는 항만효율성에 유의한 영향을 미치지 않음을 확인할 수 있었다. 이러한 결과는 항만민영화보다 항만 효율성을 결정하는 다른 중요한 요인들이 존재하고 있으며, 항만이 민영화 정책과 상관없이 항만의 기능과 효율성을 충분히 향상시킬 수 있음을 보여준다.

Efficiency gains in transport operation benefit transport operators by increasing competitive advantage. Moreover, they increase social welfare through reduction in trade costs. A popular method to measure efficiency is data envelopment analysis (DEA), a programming-based technique. This chapter summarizes key DEA models used in transportation and international trade research. The authors explain divergent DEA models, such as CCR, BCC, and directional distance function. They go on to cover some of the recent applications, for example bootstrapped truncated regression, to find determinants of efficiency. In each section, they review relevant studies in the transportation or international trade sector. Lastly, they suggest directions for future research.

Small fluorescent molecules have been an important tool for fluorescence-based imaging, thanks to their technical simplicity, sensitivity and structural flexibility. However, probing specific intracellular systems with a small fluorescent molecule is not an easy task owing to the intricate nature of the cells. Ideal imaging probes should be highly permeable and be without background noise to allow undisrupted observations. These probes will unquestionably be more advantageous for interrogating intracellular architectures and dynamics without compromising the cellular integrity, and could therefore be applied for applications in the native cellular environment. This review will highlight the advances in design strategies for cell-permeable fluorescent probes through a diversity-oriented fluorescence library approach and rational design via computational-aided predictive models. Here, we discuss a series of cell-permeable probe applications for imaging specific cell types and intracellular biomolecules, as well as the cellular environment.

A multi-color labelling technique for visualizing multiple intracellular apparatuses in their native environment using small fluorescent probes remains challenging. This approach requires both orthogonal and biocompatible coupling reactions in heterogeneous biological systems with minimum fluorescence background noise. Here, we present a palette of BODIPY probes containing azide and cyclooctyne moieties for copper-free click chemistry in living cells. The probes, referred to as ‘tame probes’, are highly permeable and specific in nature, leaving no background noise in cells. Such probes, which are rationally designed through optimized lipophilicity, water solubility and charged van der Waals surface area, allow us to demonstrate rapid and efficient concurrent multi-labelling of intracellular target components. We show that these probes are capable of not only labelling organelles and engineered proteins, but also showing the intracellular glycoconjugates’ dynamics, through the use of metabolic oligosaccharide engineering technology in various cell types. The results demonstrated in this study thus provide flexibility for multi-spectral labelling strategies in native systems in a high spatiotemporal manner.

Ports in the European Union and North America have enforced environmental regulations on controlling SOx and NOx emissions from ships in their coastal areas known as Emission Control Areas (ECAs). This study uses two-stage approaches to examine whether ECA regulations impact the efficiency of ports operating in such areas. First, port efficiencies are estimated using non-radial slacks-based measure (SBM) Data Envelopment Analysis (DEA) models. The efficiency scores estimated by the SBM DEA models are then regressed on explanatory variables, including the ECA factor, and macroeconomic indicators using bootstrapped truncated regression (BTR) models. Panel data is collected on countries in EU ECAs and non-ECAs regarding such input variables as capital and labor, with cargo as an output variable. The results indicate that ECA regulations can harm port efficiency, reflecting concerns of policy-makers and industrial managers: the average efficiency loss from an ECA designation amounts to 0.058–0.066 on a scale of 0–1, accounting for a 15–18% loss from ECA ports’ average efficiency scores.

Few tools are available for noninvasive imaging of synapses in the living mammalian brain. Current paradigms require the use of genetically modified mice or viral delivery of genetic material to the brain. To develop an alternative chemical approach, utilizing the recognition of synaptic components by organic small molecules, we designed an imaging-based, high-content screen in cultured cortical neurons to identify molecules based on their colocalization with fluorescently tagged synaptic proteins. We used this approach to screen a library of ~7000 novel fluorescent dyes, and identified a series of compounds in the xanthone family that exhibited consistent synaptic labeling. Follow-up studies with one of these compounds, CX-G3, demonstrated its ability to label acidic organelles and in particular synaptic vesicles at glutamatergic synapses in cultured neurons and murine brain tissue, indicating the potential of this screening approach to identify promising lead compounds for use as synaptic markers, sensors, and targeting devices.

Since the term “bioorthogonal” was first demonstrated in 2003, new tools for bioorthogonal chemistry have been rapidly developed. Bioorthogonal chemistry has now been widely utilized for applications in imaging various biomolecules, such as proteins, glycoconjugates, nucleic acids, and lipids. Contrasting the chemical reactions or synthesis that are typically executed in vitro with organic solvents, bioorthogonal reactions can occur inside cells under physiological conditions. Functional groups or chemical reporters for bioorthogonal chemistry are highly selective and will not perturb the native functions of biological systems. Advances in azide-based bioorthogonal chemical reporters make it possible to perform chemical reactions in living systems for wide-ranging applications. This review discusses the milestones of azide-based bioorthogonal reactions, from Staudinger ligation and copper(I)-catalyzed azide-alkyne cycloaddition to strain-promoted azide-alkyne cycloaddition. The development of bioorthogonal reporters and their capability of being built into biomolecules in vivo have been extensively applied in cellular imaging. We focus on strategies used for metabolic incorporation of chemically tagged molecular building blocks (e.g., amino acids, carbohydrates, nucleotides, and lipids) into cells via cellular machinery systems. With the aid of exogenous bioorthogonally compatible small fluorescent probes, we can selectively visualize intracellular architectures, such as protein, glycans, nucleic acids, and lipids, with high specificity to help in answering complex biological problems.

The practical application of biosensors can be determined by evaluating the sensing ability of fluorophore-modified derivatives of a receptor with appropriate recognition characteristics for target molecules. One of the key determinants for successfully obtaining a useful biosensor is wide variation in the fluorophores attached to a given receptor. Thus, using a larger fluorophore-modified receptor library provides a higher probability of obtaining a practically useful biosensor. However, no effective method has yet been developed for constructing such a diverse library of fluorophore-modified receptors. Herein, we report a method for constructing fluorophore-modified receptors by using a chemical library of synthetic fluorophores with a thiol-reactive group. This library was converted into a library of fluorophore-modified adenosine-binding ribonucleopeptide (RNP) receptors by introducing the fluorophores to the Rev peptide of the RNP complex by alkylation of the thiol group. This method enabled the construction of 263 fluorophore-modified ATP-binding RNP receptors and allowed the selection of suitable receptor-based fluorescent sensors that target ATP.

Ports create harmful effects on their adjacent population because ships discharge noxious gases like SOX, NOX, and particulate matter (PM). To tackle this problem, some ports started to control emission through regulations such as Emission Control Areas (ECA) and Reduced Speed Zone (RSZ). This paper estimates the social cost of ship emission and eco-efficiency at the Port of Incheon (POI). We further examine how the ECA and RSZ designation can reduce the social cost. The estimation is based on the activity-based approach, where ship type, engine, and movement are used to measure fuel consumption and then emission. Results suggest that the social cost of ship emission at the POI amounts to $90,805,478. The eco-efficiency of the POI, compared to the one at the Port of Las Palmas in another study, is substantially better. Under RSZ, the corresponding emission abatement values are $4,485,308, $2,642,009 and $21,932,435 from SO2, NOX and PM reduction, respectively. If 1.0% and 0.1% sulfur fuel are used complying with rules of the ECA, the social cost savings amount to $8,174,947 and $12,868,842 from SO2 reduction.

About 63% of the world’s shipping accidents are recurrent—they occur to ships that have already experienced at least one prior accident. Therefore, reducing recurrent accidents can contribute significantly to maritime safety. We study the factors affecting both first and recurrent accidents, by focusing on the duration between two accidents. Cox proportional hazard models are applied to ship accident data from 1996 to 2015, and the results identify which ships have a high risk of recurrent accidents, based on ship attributes, ship supply and market conditions, shipbuilding country, previous accident type, and ship type. The recurrent rate is high when the ship involved in the accident is old, small, flies a flag of convenience, and has no detention record. In addition, the accident risk increases when the shipping market faces a high bunker price, overcapacity in supply, a high time charter rate, or low newbuilding price. On the other hand, ships built in China and Japan have lower recurrent accid...

Drug toxicity is a long-standing concern of modern medicine. A typical anti-pain/fever drug paracetamol often causes hepatotoxicity due to peroxynitrite ONOO- . Conventional blood tests fail to offer real-time unambiguous visualization of such hepatotoxicity in vivo. Here we report a luminescent approach to evaluate acute hepatotoxicity in vivo by chromophore-conjugated upconversion nanoparticles. Upon injection, these nanoprobes mainly accumulate in the liver and the luminescence of nanoparticles remains suppressed owing to energy transfer to the chromophore. ONOO- can readily bleach the chromophore and thus recover the luminescence, the presence of ONOO- in the liver leads to fast restoring of the near-infrared emission. Taking advantages of the high tissue-penetration capability of near-infrared excitation/emission, these nanoprobes achieve real-time monitoring of hepatotoxicity in living animals, thereby providing a convenient screening strategy for assessing hepatotoxicity of synthetic drugs.

This article analyzes the carbon efficiency of the transportation sectors in China and Korea. It also investigates the effect of environmental regulations on efficiency and the opportunity costs of complying with these regulations. The main methodology employed is the Slacks-Based Measure Data Envelopment Analysis (SBM-DEA). The results show that Korea has improved its environmental efficiency in the transportation sector in recent years and it seems to have been relatively less affected by environmental regulations, whereas China has slightly deteriorated in its environmental efficiency for the same period and it has been relatively more affected by environmental regulations. The average opportunity cost of China’s provincial transportation industry, in complying with environmental regulations, tripled from US$590 million in 2004 to $1.8 billion in 2009. The unit loss per ton of Carbon dioxide (CO2) emission doubled from $64 in 2004 to $115 in 2009. In contrast, the average opportunity cost in Korea’s provincial transportation industry slightly increased for the same period. Similarly, the unit loss per ton of CO2 emission in Korea increased from $44 in 2004 to $57 in 2007, but then gradually dropped to $48 in 2009. In implementing its visionary low-carbon economy, the Chinese government needs to consider not only ways to improve overall environmental efficiency at a national level, but also the opportunity costs of complying with environmental regulations at a provincial level.

Insulin-secreting beta cells together with glucagon-producing alpha cells play an essential role in maintaining the optimal blood glucose level in the body, so the development of selective probes for imaging of these cell types in live islets is highly desired. Herein we report the development of a 2-glucosamine-based two-photon fluorescent probe, TP-β, that is suitable for imaging of beta cells in live pancreatic islets from mice. Flow cytometry studies confirmed that TP-β is suitable for isolation of primary beta cells. Moreover, two-photon imaging of TP-β-stained pancreatic islets showed brightly stained beta cells in live islets. Insulin enzyme-linked immunosorbent assays revealed that TP-β has no effect on glucose-stimulated insulin secretion from the stained islet. Finally, to develop a more convenient islet imaging application, we combined our recently published alpha-cell-selective probe TP-α with TP-β to make a "TP islet cocktail". This unique dye cocktail enabled single excitation (820 nm) and simultaneous dual-color imaging of alpha cells (green) and beta cells (red) in live pancreatic islets. This robust TP islet cocktail may serve as a valuable tool for basic diabetic studies.

Three major cruise lines account for 82% of the total market share. Their financial statements show different results between the operating incomes and net incomes over time. To examine the major causes of the differences, this study measured the efficiency of the top three cruise lines to develop a network DEA model to analyze the cruise operations at two stages, namely operating and non-operating stages. In addition, the determinants of the efficiencies were examined using a bootstrapped-truncated regression model. Overall, cruise lines were efficient at the operating stage, but varied widely in the efficiency of the non-operating stage. Cruise lines attempting high capacity expansion were relatively inefficient because of the heavy interest payments arising from the high debt-to-capital ratio. Moreover, the neglected hedging policy regarding the financial risks also contributed to the inefficiency., Highlights • This study measured the efficiency of the top three cruise lines • Network DEA and bootstrapped-truncated regression were used • They were efficient at the operating stage, but varied at the non-operating stage • Cruise lines attempting high capacity expansion were relatively inefficient • Neglected hedging policy over financial risks also contributed to the inefficiency

Mild heat stimulation of muscle cells within the physiological range represents an intriguing approach for the modulation of their functions. In this work, photothermal conversion was exploited to remotely stimulate striated muscle cells by using gold nanoshells (NSs) in combination with near-infrared (NIR) radiation. Temperature increments of approximately 5 °C were recorded by using an intracellular fluorescent molecular thermometer and were demonstrated to efficiently induce myotube contraction. The mechanism at the base of this phenomenon was thoroughly investigated and was observed to be a Ca2+-independent event directly involving actin–myosin interactions. Finally, chronic remote photothermal stimulations significantly increased the mRNA transcription of genes encoding heat shock proteins and sirtuin 1, a protein which in turn can induce mitochondrial biogenesis. Overall, we provide evidence that remote NIR + NS muscle excitation represents an effective wireless stimulation technique with great pote...

Previous studies have not considered one important aspect of port efficiency; seaports in different groups operate under different technologies. This study examines the technical efficiency (TE) of 43 ports in Vietnam and their influential factors using metafrontier analysis. The results show that cargo handling facilities and information technology are the most important inputs for ports, despite their varying contribution to port performance across regions. Land is important for the TE of ports in the North, whereas the cargo storage capacity is important to ports in the Central areas, and information technology is important to ports in the South. The majority of Vietnamese ports are operating under increasing returns to scale. On the other hand, each region needs a different approach. For example, in ideal situations, ports in the North could reduce berth length by up to 57% without affecting throughput. National and regional reference networks are developed to identify the leading ports at the regional and national levels and their connections with other ports.

A novel design strategy for the fluorescence sensing of l-DOPA is reported. Resa-Sulf displays a significant turn-on fluorescence response to l-DOPA due to its reduction properties; this sensing mechanism was fully confirmed by mechanistic studies. Furthermore, Resa-Sulf was successfully utilized to quantitatively detect l-DOPA concentrations from a commercially available source.

The need for detecting and labelling environmentally and biologically important analytes has driven considerable research efforts in developing fluorescent probes. During the sensing process, molecular motions (i.e., molecular rotations or vibrations) of a flexible fluorescent probe can be significantly altered by its embedding micro-environment or analyte, thereby leading to substantial changes in readout signals. Motion-induced change in emission (MICE) can be utilized as an effective sensing mechanism. However, in comparison to the well-understood sensing mechanisms, such as photo-induced electron transfer (PET), intramolecular charge transfer (ICT), aggregation-induced emission (AIE) and disaggregation-induced emission (DIE), MICE has not been systematically discussed to date. In this tutorial review, we will summarize the concept and mechanisms of MICE for developing single-molecular fluorescent probes, present unique advantages of MICE based sensors, demonstrate their various applications, and discuss technical challenges in this field. We expect that this review will promote a deeper understanding of MICE and facilitate the development of novel MICE based probes.

A series of push-pull type meso-ester substituted BODIPY dyes 1-4 with intense near-infrared absorption, largely enhanced photoacoustic (PA) activity and excellent photo-stability were synthesized. The impact of the electronic structure on the PA activity was also discussed. Moreover, the in vitro and in vivo PA imaging were investigated, which suggested a passive targeting capacity in the tumor site.

Numerous countries have implemented or are considering a range of policies to lower emissions from transportation. An analysis of the impacts of environmental regulation is a crucial issue, which has not been properly highlighted in the transportation literature, particularly in terms of the foregone output by such regulation. This study develops a novel DEA model that measures the foregone output when the industry emissions target is imposed. This model reflects the real regulatory process more appropriately than other models in that the authority in charge sets the emission reduction target first, and the transport operators respond to it. In addition, the model can test the industrywide impacts over a wide range of emission target values, which can help policy makers determine the optimal emission target. Finally, the proposed model was applied to the port industry in Korea. The results suggest that Korean ports can reduce their emissions by a maximum of 239,850 tons of CO2, which accounts for 13% of the total emissions in 2010. The 13% reduction in emission, however, would result in $ 91,109,000 of foregone cargo traffic to the Korean economy. In addition, the foregone cargo traffic increases at much faster rate than the emission reduction rate. For example, the shadow price of emission differs by 2.25 times between the most moderate and strictest emission targets. This suggests that the government needs to impose moderate emission targets at the initial stages if it decides to minimize the regulatory impacts on the industry.

Tumor initiating cells (TIC) are resistant to conventional anticancer therapy and associated with metastasis and relapse in cancer. Although various TIC markers and their antibodies have been proposed, it is limited to the use of antibodies for in vivo imaging or treatment of TIC. In this study, we discovered heme oxygenase 2 (HMOX2) as a novel biomarker for TIC and developed a selective small molecule probe TiNIR (tumor initiating cell probe with near infrared). TiNIR detects and enriches the functionally active TIC in human lung tumors, and through the photoacoustic property, TiNIR also visualizes lung TIC in the patient-derived xenograft (PDX) model. Furthermore, we demonstrate that TiNIR inhibits tumor growth by blocking the function of HMOX2, resulting in significantly increased survival rates of the cancer model mice. The novel therapeutic target HMOX2 and its fluorescent ligand TiNIR will open a new path for the molecular level of lung TIC diagnosis and treatment.

Detection of the biofilm of bacteria would be a counter strategy to detect hidden bacteria in their camouflage. Through unbiased screening of bacteria biofilm, we discovered a long wavelength probe CDr15 with extracellular DNA as the molecular target. CDr15 revealed a real-time geometric distribution of eDNA in a 3D bacterial colony.

Small molecule imaging probes are powerful tools to understand complex biological systems. The mainstreams of imaging probe developments have been focused on the target holding of the probes; the holding targets are often cell-type-specific biomarkers. This type of the probe mechanism can be designated as holding-oriented live-cell distinction (HOLD). Our group has worked on the development of cell-type-selective probes using a diversity-oriented fluorescence library approach (DOFLA), where unbiased phenotypic screening is employed using fluorescent library compounds. Through the conventional target identification methods such as an affinity-based analysis, we elucidated that some of the probe mechanisms are HOLD. However, we also realized that sometimes there is no specific holding target for probes or the holding targets are ubiquitous. The observation led us to test an alternative mechanism of cell-type-specific probes as gating-oriented live-cell distinction (GOLD). We started to examine the gating mechanism of probes, which is mainly based on transporters but which does not necessarily require probe holding to cellular targets. Transporters can control the in and out movement of various nutrients and chemicals. Different expression levels of transporters in various cell types could provide the molecular mechanism of differential staining of cells by regulating the intracellular accumulation of a certain specific probe. A number of GOLD probes have been developed by modifying or mimicking endogenous substrates of transporters such as inorganic ions, glucose, amino acids, or neurotransmitters, utilizing broad substrate specificity of transporters. The radiolabeled or fluorophore-conjugated substrate mimetics have been widely used for live cell distinction and various applications such as disease-related cell or tissue imaging. In humans, there are about 400 solute carrier (SLC) transporters and 50 ATP-binding cassette (ABC) transporters. Since some transporters have broad substrate specificity, they can transport not only derivatives of endogenous natural substrates but also totally synthetic diverse imaging probes, such as DOFLA probes. Without preconsidering the structure of endogenous substrates, we recently demonstrated a series of live-cell imaging probes and elucidated their molecular mechanism as a gating one, either by SLC or ABC transporters. Transporter inhibitor panel and CRISPR-based transporter libraries could provide a systematic gating target elucidation platform. Considering the generality of DOFLA and the CRISPR-based genomic tool for transporter systems (>450 in humans), the GOLD approach will offer new insight and promise for unprecedented levels of novel cell imaging probe development.

A colorimetric/fluorometric dual-signal assay is described for the determination of dopamine (DA). A nanoprobe was obtained by linking a 4-(4-dialkylaminostyryl)pyridinium derived fluorophore to citrate-capped gold nanoparticles (AuNPs). The fluorescence of the fluorophore is quenched by the AuNPs via fluorescence resonance energy transfe. In the presence of DA, the catechol group of DA can absorb on the surface of AuNPs to induce aggregation, which is accompanied by a color change from red to blue. The yellow fluorescence of the fluorophore with excitation/emission maximum at 365/570 nm is recovered. The dual-signal detection allows the quantitative analysis of DA within 300 μM by the colorimetric method and 80 μM by the fluorometric method. The detection limits for the colorimetric/fluorometric methods are 1.85 μM and 0.29 μM, respectively. Quantitative determination of DA in spiked urine samples was successfully demonstrated, with recoveries ranging from 98.2 to 106.0%. Graphical abstract A colorimetric/fluorometric dual-signal assay is described for the determination of dopamine by linking a fluorophore to gold nanoparticles. The dopamine causes aggregation of the nanoparticles to induce color change, which is followed by the recovery of the fluorescence.

The rapid and sensitive classification of bacteria is the first step of bacterial community research and the treatment of infection. Herein, a fluorescent probe BacGO is presented, which shows the best universal selectivity for Gram-positive bacteria among known probes with a minimum staining procedure for sample detection and enrichment of the live bacteria. BacGO could also be used to assess of the Gram status in the bacterial community from wastewater sludge. Furthermore, BacGO could sensitively and selectively detect a Gram-positive bacterial infection, not only in vitro but also using an in vivo keratitis mouse model. BacGO provides an unprecedented research tool for the study of dynamic bacterial communities and for clinical application.

When diabetes is diagnosed, the majority of insulin-secreting pancreatic β cells are already dysfunctional or destroyed. This β cell dysfunction/destruction usually takes place over many years, making timely detection and clinical intervention difficult. For this reason, there is immense interest in developing tools to bioimage β cell mass and/or function noninvasively to facilitate early diagnosis of diabetes as well as to assist the development of novel antidiabetic therapies. Recent years have brought significant progress in β cell imaging that is now inching towards clinical applicability. We explore here the need to bioimage human β cells noninvasively in various types of diabetes, and we discuss current and emerging tools for bioimaging β cells. Further developments in this field are expected to facilitate β cell imaging in diabetes.

Obesity is a medical condition that impacts on all levels of society and causes numerous comorbidities, such as diabetes, cardiovascular disease, and cancer. We assessed the suitability of targeting enolase, a glycolysis pathway enzyme with multiple, secondary functions in cells, to treat obesity. Treating adipocytes with ENOblock, a novel modulator of these secondary ‘moonlighting’ functions of enolase, suppressed the adipogenic program and induced mitochondrial uncoupling. Obese animals treated with ENOblock showed a reduction in body weight and increased core body temperature. Metabolic and inflammatory parameters were improved in the liver, adipose tissue and hippocampus. The mechanism of ENOblock was identified as transcriptional repression of master regulators of lipid homeostasis (Srebp-1a and Srebp-1c), gluconeogenesis (Pck-1) and inflammation (Tnf-α and Il-6). ENOblock treatment also reduced body weight gain, lowered cumulative food intake and increased fecal lipid content in mice fed a high fat diet. Our results support the further drug development of ENOblock as a therapeutic for obesity and suggest enolase as a new target for this disorder.

3131 Background: The molecules of the cell are compartmentalized into membrane- and non-membrane-bound organelles. Many non-membrane-bound organelles are phase-separated biomolecular condensates with distinct physicochemical properties that can absorb and concentrate specific proteins and nucleic acids involved in discrete biochemical processes. We reasoned that selective condensate partitioning might also occur with small molecule drugs whose targets occur within condensates, and that the therapeutic index and efficacy of such compounds might therefore relate to their ability to partition into condensates. Methods: To study the behavior of drugs within condensates, these were modeled in cells and in vitro with purified proteins and visualized by fluorescent confocal microscopy. The functional outcomes of condensate partitioning were queried in cells. Results: We found that cisplatin, tamoxifen, JQ1, THZ1, and mitoxantrone are concentrated in specific protein condensates in vitro, and that this occurs through physicochemical properties independent of the drug target. A screen of a chemically diverse fluorescent probes and mutant-protein condensates demonstrated that pi-system interactions between aromatic moieties in the protein and small molecule govern concentration in condensates. These results show that clinically important drugs partition into specific protein condensates in vitro by virtue of defined chemical properties, thereby altering their local concentration. In vitro droplet assays revealed that cisplatin is selectively concentrated in transcriptional condensates, and that this ability is required for efficient platination of target DNA. In cell studies revealed that cisplatin preferentially targets DNA contained within MED1 condensates, and disrupts the genetic regulatory elements that compose phase-separated transcriptional condensates. Live cell imaging demonstrated that transcriptional condensates are dissolved by cisplatin, whereas other condensates remain intact. Conclusions: Our results show that antineoplastic drugs partition selectively into biomolecular condensates, that this can occur through physicochemical properties independent of their molecular targets, and that drug activity may occur through condensate-related mechanisms. These results have implications for development of efficacious cancer therapeutics; effective target engagement will depend on factors such as drug partitioning in condensates. Assays of the type described here may thus help optimize condensate partitioning, target engagement, and the therapeutic index of drugs for cancer treatment.

The transportation sector is under high pressure to reduce their carbon emissions. Emission trading is widely used as a market-oriented policy instrument to reduce emissions and increase social and economic benefits. The present study introduces a new model for estimating the potential gains of CO2 emission trading for China’s transportation sector by simulating a trading market. This hypothesized trading market considers both price information and underlying emission trading process. The marginal abatement cost of transport sector’s CO2 emission is derived by designing a constrained maximum likelihood model with partial quantile order-α frontiers. Results showed an average marginal abatement cost of CO2 emission for China’s transport sector at around 1009$/tonne. The estimated potential gains of emission trading measured in monetary terms range between 2 and 22 billion dollars, accounting for 0.5% of the total profit generated by the transportation sector.

Selection of a specific neural stem/progenitor cells (NSPCs) has attracted broad attention in regenerative medicine for neurological disorders. Here, we report a fluorescent probe, CDg13, and its application for isolating strong neurogenic NSPCs. In comparison to the NSPCs isolated by other biomarkers, CDg13-stained NSPCs showed higher capability to differentiate into neurons. Target identification revealed that the fluorescence intensity of the probe within cells is inversely proportional to the expression levels of mouse and human Abcg2 transporters. These findings suggest that low Abcg2 expression is a biomarker for neurogenic NSPCs in mouse brain. Furthermore, CDg13 can be used to isolate Abcg2low cells from heterogeneous cell populations.

Pluripotent stem cells (PSC) are promising resources for regeneration therapy, but teratoma formation is one of the critical problems for safe clinical application. After differentiation, the precise detection and subsequent elimination of undifferentiated PSC is essential for teratoma-free stem cell therapy, but a practical procedure is yet to be developed. CDy1, a PSC specific fluorescent probe, was investigated for the generation of reactive oxygen species (ROS) and demonstrated to induce selective death of PSC upon visible light irradiation. Importantly, the CDy1 and/or light irradiation did not negatively affect differentiated endothelial cells. The photodynamic treatment of PSC with CDy1 and visible light irradiation confirmed the inhibition of teratoma formation in mice, and suggests a promising new approach to safe PSC-based cell therapy., Stain, shine, and kill: CDy1 selectively stains pluripotent stem cells and, upon shining visible light, kills only stem cells without affecting differentiated cells, opening the possibility of teratoma-free stem cell therapy.

Ohne genau durchdachte Fluoreszenzmarker/-sonden kann auch das fortschrittlichste hochauflosende Mikroskopieverfahren keinen Einblick in subzellulare Matrizes geben. Die Entwicklung der Biologie wurde in zunehmendem Mase durch Fortschritte im Bereich der Chemie vorangetrieben. Ein prominentes Beispiel hierfur sind niedermolekulare Fluoreszenzsonden, die nicht nur die Bildgebung auf zellularer Ebene ermoglichen, sondern auch subzellulare Abbildungen. Die meisten chemischen/biologischen Ereignisse finden innerhalb von Zellorganellen statt, sodass diese Substrukturen zunehmend mithilfe von Fluoreszenzverfahren untersucht wurden. Dieser Aufsatz fasst die vorhandenen Fluoreszenzsonden zusammen, die auf chemische/biologische Geschehnisse innerhalb einzelner Organellen zielen. Daruber hinaus werden Strategien zur Verankerung an den Organellen vorgestellt, um das Design neuer Fluoreszenzsonden anzuregen. Abschliesend werden die Zukunftsaussichten fur weitere Entwicklungen in der chemischen Biologie diskutiert.