NeuM: A Neuron-Selective Probe Incorporates into Live Neuronal Membranes via Enhanced Clathrin-Mediated Endocytosis in Primary Neurons.

The development of a small-molecule probe designed to selectively target neurons would enhance the exploration of intricate neuronal structures and functions. Among such probes, NeuO stands out as the pioneer and has gained significant traction in the field of research. Nevertheless, neither the mechanism behind neuron-selectivity nor the cellular localization has been determined. Here, we introduce NeuM, a derivative of NeuO, designed to target neuronal cell membranes. Furthermore, we elucidate the mechanism behind the selective neuronal membrane trafficking that distinguishes neurons. In an aqueous buffer, NeuM autonomously assembles into micellar structures, leading to the quenching of its fluorescence (Φ=0.001). Upon exposure to neurons, NeuM micelles were selectively internalized into neuronal endosomes via clathrin-mediated endocytosis. Through the endocytic recycling pathway, NeuM micelles integrate into neuronal membrane, dispersing fluorescent NeuM molecules in the membrane (Φ=0.61). Molecular dynamics simulations demonstrated that NeuM, in comparison to NeuO, possesses optimal lipophilicity and molecular length, facilitating its stable incorporation into phospholipid layers. The stable integration of NeuM within neuronal membrane allows the prolonged monitoring of neurons, as well as the visualization of intricate neuronal structures.

Novel Inhibitor of Mixed-Lineage Kinase Domain-Like Protein: The Antifibrotic Effects of a Necroptosis Antagonist.

The pseudokinase mixed-lineage kinase domain-like protein plays a crucial role in programmed cell death via necroptosis. We developed a novel mixed-lineage kinase domain-like inhibitor, P28, which demonstrated potent necroptosis inhibition and antifibrotic effects. P28 treatment directly inhibited mixed-lineage kinase domain-like phosphorylation and oligomerization after necroptosis induction, inhibited immune cell death after necroptosis, and reduced the expression of adhesion molecules. Additionally, P28 treatment reduced the level of activation of hepatic stellate cells and the expression of hepatic fibrosis markers induced by necroptosis stimulation. Unlike the necrosulfonamide treatment, the P28 treatment did not induce cytotoxicity. Finally, the cysteine covalent bonding of P28 was confirmed by liquid chromatography-tandem mass spectrometry.

Multimodal Imaging Probe Development for Pancreatic ß Cells: From Fluorescence to PET.

Pancreatic ß 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 ß 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 ß cell probe PiF for both fluorescence and for PET (positron emission tomography). By simple tail vein injection, PiF stains pancreatic ß 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.

Visualizing biofilm by targeting eDNA with long wavelength probe CDr15.

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.

Visualizing Microglia with a Fluorescence Turn-On Ugt1a7c Substrate.

Microglia, the brain-resident macrophage, are involved in brain development and contribute to the progression of neural disorders. Despite the importance of microglia, imaging of live microglia at a cellular resolution has been limited to transgenic mice. Efforts have therefore been dedicated to developing new methods for microglia detection and imaging. Using a thorough structure-activity relationships study, we developed CDr20, a high-performance fluorogenic chemical probe that enables the visualization of microglia both in vitro and in vivo. Using a genome-scale CRISPR-Cas9 knockout screen, the UDP-glucuronosyltransferase Ugt1a7c was identified as the target of CDr20. The glucuronidation of CDr20 by Ugt1a7c in microglia produces fluorescence.

Development of a Universal Fluorescent Probe for Gram-Positive Bacteria.

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.

Imaging inflammation using an activated macrophage probe with Slc18b1 as the activation-selective gating target.

Activated macrophages have the potential to be ideal targets for imaging inflammation. However, probe selectivity over non-activated macrophages and probe delivery to target tissue have been challenging. Here, we report a small molecule probe specific for activated macrophages, called CDg16, and demonstrate its application to visualizing inflammatory atherosclerotic plaques in vivo. Through a systematic transporter screen using a CRISPR activation library, we identify the orphan transporter Slc18b1/SLC18B1 as the gating target of CDg16.

CDy14: a novel biofilm probe targeting exopolysaccharide Psl.

Detection of biofilm bacteria would be an ideal method for the physicians to diagnose chronic bacterial infections directly, but there are few imaging probes available so far. Here, we report the development of a novel biofilm detecting fluorescent probe, CDy14, through an unbiased screening of a fluorescence library and elucidated its binding partner Psl, an exopolysaccharide of the biofilm.

A palette of background-free tame fluorescent probes for intracellular multi-color labelling in live cells.

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.

Effects of an applied voltage on direct interspecies electron transfer via conductive materials for methane production.

Direct interspecies electron transfer (DIET) between exoelectrogenic bacteria and methanogenic archaea via conductive materials is reported as an efficient method to produce methane in anaerobic organic waste digestion. A voltage can be applied to the conductive materials to accelerate the DIET between two groups of microorganisms to produce methane. To evaluate this hypothesis, two sets of anaerobic serum bottles with and without applied voltage were used with a pair of graphite rods as conductive materials to facilitate DIET. Initially, the methane production rate was similar between the two sets of serum bottles, and later the serum bottles with an applied voltage of 0.39V showed a 168% higher methane production rate than serum bottles without an applied voltage. In cyclic voltammograms, the characteristic redox peaks for hydrogen and acetate oxidation were identified in the serum bottles with an applied voltage. In the microbial community analyses, hydrogenotrophic methanogens (e.g. Methanobacterium) were observed to be abundant in serum bottles with an applied voltage, while methanogens utilizing carbon dioxide (e.g., Methanosaeta and Methanosarcina) were dominant in serum bottles without an applied voltage. Taken together, the applied voltage on conductive materials might not be effective to promote DIET in methane production. Instead, it appeared to generate a condition for hydrogenotrophic methanogenesis.

Optical visualisation of thermogenesis in stimulated single-cell brown adipocytes.

The identification of brown adipose deposits in adults has led to significant interest in targeting this metabolically active tissue for treatment of obesity and diabetes. Improved methods for the direct measurement of heat production as the signature function of brown adipocytes (BAs), particularly at the single cell level, would be of substantial benefit to these ongoing efforts. Here, we report the first application of a small molecule-type thermosensitive fluorescent dye, ERthermAC, to monitor thermogenesis in BAs derived from murine brown fat precursors and in human brown fat cells differentiated from human neck brown preadipocytes. ERthermAC accumulated in the endoplasmic reticulum of BAs and displayed a marked change in fluorescence intensity in response to adrenergic stimulation of cells, which corresponded to temperature change. ERthermAC fluorescence intensity profiles were congruent with mitochondrial depolarisation events visualised by the JC-1 probe. Moreover, the averaged fluorescence intensity changes across a population of cells correlated well with dynamic changes such as thermal power, oxygen consumption, and extracellular acidification rates. These findings suggest ERthermAC as a promising new tool for studying thermogenic function in brown adipocytes of both murine and human origins.

Specific Triazine Herbicides Induce Amyloid-ß42 Production.

Proteolytic cleavage of the amyloid-ß protein precursor (AßPP) by secretases leads to extracellular release of amyloid-ß (Aß) peptides. Increased production of Aß42 over Aß40 and aggregation into oligomers and plaques constitute an Alzheimer's disease (AD) hallmark. Identifying products of the 'human chemical exposome' (HCE) able to induce Aß42 production may be a key to understanding some of the initiating causes of AD and to generate non-genetic, chemically-induced AD animal models. A cell model was used to screen HCE libraries for Aß42 inducers. Out of 3500+ compounds, six triazine herbicides were found that induced a ß- and γ-secretases-dependent, 2-10 fold increase in the production of extracellular Aß42 in various cell lines, primary neuronal cells, and neurons differentiated from human-induced pluripotent stem cells (iPSCs). Immunoprecipitation/mass spectrometry analyses show enhanced production of Aß peptides cleaved at positions 42/43, and reduced production of peptides cleaved at positions 38 and lower, a characteristic of AD. Neurons derived from iPSCs obtained from a familial AD (FAD) patient (AßPP K724N) produced more Aß42 versus Aß40 than neurons derived from healthy controls iPSCs (AßPP WT). Triazines enhanced Aß42 production in both control and AD iPSCs-derived neurons. Triazines also shifted the cleavage pattern of alcadeinα, another γ-secretase substrate, suggesting a direct effect of triazines on γ-secretase activity. In conclusion, several widely used triazines enhance the production of toxic, aggregation prone Aß42/Aß43 amyloids, suggesting the possible existence of environmental "Alzheimerogens" which may contribute to the initiation and propagation of the amyloidogenic process in late-onset AD.

Development of background-free tame fluorescent probes for intracellular live cell imaging.

Fluorescence labelling of an intracellular biomolecule in native living cells is a powerful strategy to achieve in-depth understanding of the biomolecule's roles and functions. Besides being nontoxic and specific, desirable labelling probes should be highly cell permeable without nonspecific interactions with other cellular components to warrant high signal-to-noise ratio. While it is critical, rational design for such probes is tricky. Here we report the first predictive model for cell permeable background-free probe development through optimized lipophilicity, water solubility and charged van der Waals surface area. The model was developed by utilizing high-throughput screening in combination with cheminformatics. We demonstrate its reliability by developing CO-1 and AzG-1, a cyclooctyne- and azide-containing BODIPY probe, respectively, which specifically label intracellular target organelles and engineered proteins with minimum background. The results provide an efficient strategy for development of background-free probes, referred to as 'tame' probes, and novel tools for live cell intracellular imaging.

Enrichment of specific electro-active microorganisms and enhancement of methane production by adding granular activated carbon in anaerobic reactors.

Direct interspecies electron transfer (DIET) via conductive materials can provide significant benefits to anaerobic methane formation in terms of production amount and rate. Although granular activated carbon (GAC) demonstrated its applicability in facilitating DIET in methanogenesis, DIET in continuous flow anaerobic reactors has not been verified. Here, evidences of DIET via GAC were explored. The reactor supplemented with GAC showed 1.8-fold higher methane production rate than that without GAC (35.7 versus 20.1±7.1mL-CH4/d). Around 34% of methane formation was attributed to the biomass attached to GAC. Pyrosequencing of 16S rRNA gene demonstrated the enrichment of exoelectrogens (e.g. Geobacter) and hydrogenotrophic methanogens (e.g. Methanospirillum and Methanolinea) from the biomass attached to GAC. Furthermore, anodic and cathodic currents generation was observed in an electrochemical cell containing GAC biomass. Taken together, GAC supplementation created an environment for enriching the microorganisms involved in DIET, which increased the methane production rate.

Membrane distillation combined with an anaerobic moving bed biofilm reactor for treating municipal wastewater.

A fermentative strategy with an anaerobic moving bed biofilm reactor (AMBBR) was used for the treatment of domestic wastewater. The feasibility of using a membrane separation technique for post-treatment of anaerobic bio-effluent was evaluated with emphasis on employing a membrane distillation (MD). Three different hydrophobic 0.2 µm membranes made of polytetrafluoroethylene (PTFE), polyvinylidene fluoride (PVDF), and polypropylene (PP) were examined in this study. The initial permeate flux of the membranes ranged from 2.5 to 6.3 L m(-2) h(-1) when treating AMBBR effluent at a temperature difference between the feed and permeate streams of 20 °C, with the permeate flux increasing in the order PP < PVDF < PTFE. The permeate flux of the PTFE membrane gradually decreased to 84% of the initial flux after the 45 h run for distillation, while a flux decline in MD with either the PVDF or PP membrane was not found under the identical distillation conditions. During long-term distillation with the PVDF membrane, total phosphorus was completely rejected and >98% rejection of dissolved organic carbon was also achieved. The characterization of wastewater effluent organic matter (EfOM) using an innovative suite of analytical tools verified that almost all of the EfOM was rejected via the PVDF MD treatment.

Effects of volatile solid concentration and mixing ratio on hydrogen production by co-digesting molasses wastewater and sewage sludge.

Co-digesting molasses wastewater and sewage sludge was evaluated for hydrogen production by response surface methodology (RSM). Batch experiments in accordance with various dilution ratios (40- to 5-fold) and waste mixing composition ratios (100:0, 80:20, 60:40, 40:60, 20:80, and 0:100, on a volume basis) were conducted. Volatile solid (VS) concentration strongly affected the hydrogen production rate and yield compared with the waste mixing ratio. The specific hydrogen production rate was predicted to be optimal when the VS concentration ranged from 10 to 12 g/l at all the mixing ratios of molasses wastewater and sewage sludge. A hydrogen yield of over 50 ml H2/g VS(removed) was obtained from mixed waste of 10% sewage sludge and 10 g/l VS (about 10-fold dilution ratio). The optimal chemical oxygen demand/ total nitrogen ratio for co-digesting molasses wastewater and sewage sludge was between 250 and 300 with a hydrogen yield above 20 ml H2/g VS(removed).

Membrane fouling control using a rotary disk in a submerged anaerobic membrane sponge bioreactor.

Despite significant research efforts over the last few decades, membrane fouling in anaerobic membrane bioreactors (AnMBRs) remains an unsolved problem that increases the overall operational costs and obstructs the industrial applications. Herein, we developed a method for effectively controlling the membrane fouling in a sponge-submerged AnMBRs using an anaerobic rotary disk MBR (ARMBR). The disk rotation led the effective collision between the sponge and membrane surface; thus successfully enhanced the membrane permeability in the ARMBR. The effect of the disk rotational speed and sponge volume fraction on the membrane permeability and the relationship between the water flow direction and membrane permeability were investigated. The long-term feasibility was tested over 100days of synthetic wastewater treatment. As a result, stable and economical performance was observed without membrane replacement and washing. The proposed integrated rotary disk-supporting media appears to be a feasible and even beneficial option in the AnMBR technology.

Two-stage biogas production by co-digesting molasses wastewater and sewage sludge.

We evaluated the feasibility of co-digesting molasses wastewater and sewage sludge in a two-stage hydrogen- and methane-producing system. The highest energy was recovered at the 21-h hydraulic retention time (HRT) of the first hydrogenic reactor and at 56-h HRT of the secondary methanogenic reactor. Hence, the two-stage system recovered 1,822 kJ from 1 L of the mixed wastes (19.7: hydrogenic reactor plus, 1,802 kJ L(-1): methanogenic reactor). Despite the overloaded VFA-run with a short HRT of 56 h, the GAC-CH4 reactor increased methane production rate and yields due to enhanced pH buffer capacity. An RNA-based community analysis showed that the Ethanoligenens and Methanosaeta dominated the hydrogen and methane bioreactor, respectively. The two-stage system of co-digesting molasses and sewage sludge is particularly cost-effective due to non-pretreatment of sewage sludge.

Removal of pharmaceutical residue in municipal wastewater by DAF (dissolved air flotation)-MBR (membrane bioreactor) and ozone oxidation.

Growing attention is given to pharmaceutical residue in the water environment. It is known that pharmaceuticals are able to survive from a series of wastewater treatment processes. Concerns regarding pharmaceutical residues are attributed to the fact that they are being detected in water and sediment environment ubiquitously. Pharmaceutical treatment using a series of wastewater treatment processes of the DAF (dissolved air flotation)-MBR (membrane bioreactor)-ozone oxidation was conducted in the study. DAF, without addition of coagulant, could remove COD(cr) (chemical oxygen demand by Cr) up to over 70%, BOD 73%, SS 83%, T-N 55%, NH4(+) 23%, and T-P 65% in influent of municipal wastewater. Average removal rates of water quality parameters by the DAF-MBR system were very high, e.g. COD(cr) 95.88%, BOD5 99.66%, COD(mn) (chemical oxygen demand by Mn) 93.63%, T-N 69.75%, NH4-N 98.46%, T-P 78.23%, and SS 99.51%, which satisfy effluent water quality standards. Despite the high removal rate of the wastewater treatment system, pharmaceuticals were eliminated to be about 50-99% by the MBR system, depending on specific pharmaceuticals. Ibuprofen was well removed by MBR system up to over 95%, while removal rate of bezafibrate ranged between 50 and 90%. With over 5 mg/l of ozone oxidation, most pharmaceuticals which survived the DAF-MBR process were removed completely or resulted in very low survival rate within the range of few micrograms per litre. However, some pharmaceuticals such as bezafibrate and naproxen tended to be resistant to ozone oxidation.

Characteristics of flux and gel layer on microfilter and non-woven fabric filter surface based on anoxic-aerobic MBRs.

Non-woven fabric filter- (NWFF) and microfilter-MBR modules were made using 100 µm polypropylene and 0.25 µm polyethylene materials, respectively. The performances and mechanisms of the two processes were investigated, including additional batch filtration tests to find the function of the dynamic gel layer on the membrane surface. The HRT of both MBRs was 9 h and the operating permeate flux was 13 L/m(2)/h. The two MBRs consisted of an anoxic and aerobic reactor. The NWFF or microfilter (MF) was submerged in each of the aerobic reactors. The two MBRs showed similar performances for the removal of organic matters, suspended solids and nitrogen. Cake formation on the NWFF contributed to major resistance, while the gel layer on the microfilter or internal fouling of the pores played a key role in the fouling of the membrane surface. The amount of soluble extracellular polymer substances (EPS) (13 mg/L) of the attached sludge on the NWFF surface was larger than that (11 mg/L) of that suspended sludge. Consequently, the functional gel layer for the coarse and microfilter is established based on the relationship among the EPS, transmembrane pressure and MLSS.