Rap1 small GTPase is essential for maintaining pulmonary endothelial barrier function in mice.

Vascular permeability is dynamically but tightly controlled by vascular endothelial (VE)-cadherin-mediated endothelial cell-cell junctions to maintain homeostasis. Thus, impairments of VE-cadherin-mediated cell adhesions lead to hyperpermeability, promoting the development and progression of various disease processes. Notably, the lungs are a highly vulnerable organ wherein pulmonary inflammation and infection result in vascular leakage. Herein, we showed that Rap1, a small GTPase, plays an essential role for maintaining pulmonary endothelial barrier function in mice. Endothelial cell-specific Rap1a/Rap1b double knockout mice exhibited severe pulmonary edema. They also showed vascular leakage in the hearts, but not in the brains. En face analyses of the pulmonary arteries and 3D-immunofluorescence analyses of the lungs revealed that Rap1 potentiates VE-cadherin-mediated endothelial cell-cell junctions through dynamic actin cytoskeleton reorganization. Rap1 inhibits formation of cytoplasmic actin bundles perpendicularly binding VE-cadherin adhesions through inhibition of a Rho-ROCK pathway-induced activation of cytoplasmic nonmuscle myosin II (NM-II). Simultaneously, Rap1 induces junctional NM-II activation to create circumferential actin bundles, which anchor and stabilize VE-cadherin at cell-cell junctions. We also showed that the mice carrying only one allele of either Rap1a or Rap1b out of the two Rap1 genes are more vulnerable to lipopolysaccharide (LPS)-induced pulmonary vascular leakage than wild-type mice, while activation of Rap1 by administration of 007, an activator for Epac, attenuates LPS-induced increase in pulmonary endothelial permeability in wild-type mice. Thus, we demonstrate that Rap1 plays an essential role for maintaining pulmonary endothelial barrier functions under physiological conditions and provides protection against inflammation-induced pulmonary vascular leakage.

Predicting Peptide Permeability Across Diverse Barriers: A Systematic Investigation.

Peptide-based therapeutics hold immense promise for the treatment of various diseases. However, their effectiveness is often hampered by poor cell membrane permeability, hindering targeted intracellular delivery and oral drug development. This study addressed this challenge by introducing a novel graph neural network (GNN) framework and advanced machine learning algorithms to build predictive models for peptide permeability. Our models offer systematic evaluation across diverse peptides (natural, modified, linear and cyclic) and cell lines [Caco-2, Ralph Russ canine kidney (RRCK) and parallel artificial membrane permeability assay (PAMPA)]. The predictive models for linear and cyclic peptides in Caco-2 and RRCK cell lines were constructed for the first time, with an impressive coefficient of determination (R2) of 0.708, 0.484, 0.553, and 0.528 in the test set, respectively. Notably, the GNN framework behaved better in permeability prediction with larger data sets and improved the accuracy of cyclic peptide prediction in the PAMPA cell line. The R2 increased by about 0.32 compared with the reported models. Furthermore, the important molecular structural features that contribute to good permeability were interpreted; the influence of cell lines, peptide modification, and cyclization on permeability were successfully revealed. To facilitate broader use, we deployed these models on the user-friendly KNIME platform (https://github.com/ifyoungnet/PharmPapp). This work provides a rapid and reliable strategy for systematically assessing peptide permeability, aiding researchers in drug delivery optimization, peptide preselection during drug discovery, and potentially the design of targeted peptide-based materials.

Development of stapled NONO-associated peptides reveals unexpected cell permeability and nuclear localisation.

The non-POU domain-containing octamer-binding protein (NONO) is a nucleic acid-binding protein with diverse functions that has been identified as a potential cancer target in cell biology studies. Little is known about structural motifs that mediate binding to NONO apart from its ability to form homodimers, as well as heterodimers and oligomers with related homologues. We report a stapling approach to macrocyclise helical peptides derived from the insulin-like growth factor binding protein (IGFBP-3) that NONO interacts with, and also from the dimerisation domain of NONO itself. Using a range of chemistries including Pd-catalysed cross-coupling, cysteine arylation and cysteine alkylation, we successfully improved the helicity and observed modest peptide binding to the NONO dimer, although binding could not be saturated at micromolar concentrations. Unexpectedly, we observed cell permeability and preferential nuclear localisation of various dye-labelled peptides in live confocal microscopy, indicating the potential for developing peptide-based tools to study NONO in a cellular context.

Sensing chemical-induced DNA damage using CRISPR/Cas9-mediated gene-deletion yeast-reporter strains.

Microorganism-based genotoxicity assessments are vital for evaluating potential chemical-induced DNA damage. In this study, we developed both chromosomally integrated and single-copy plasmid-based reporter assays in budding yeast using a RNR3 promoter-driven luciferase gene. These assays were designed to compare the response to genotoxic chemicals with a pre-established multicopy plasmid-based assay. Despite exhibiting the lowest luciferase activity, the chromosomally integrated reporter assay showed the highest fold induction (i.e., the ratio of luciferase activity in the presence and absence of the chemical) compared with the established plasmid-based assay. Using CRISPR/Cas9 technology, we generated mutants with single- or double-gene deletions, affecting major DNA repair pathways or cell permeability. This enabled us to evaluate reporter gene responses to genotoxicants in a single-copy plasmid-based assay. Elevated background activities were observed in several mutants, such as mag1Δ cells, even without exposure to chemicals. However, substantial luciferase induction was detected in single-deletion mutants following exposure to specific chemicals, including mag1Δ, mms2Δ, and rad59Δ cells treated with methyl methanesulfonate; rad59Δ cells exposed to camptothecin; and mms2Δ and rad10Δ cells treated with mitomycin C (MMC) and cisplatin (CDDP). Notably, mms2Δ/rad10Δ cells treated with MMC or CDDP exhibited significantly enhanced luciferase induction compared with the parent single-deletion mutants, suggesting that postreplication and for nucleotide excision repair processes predominantly contribute to repairing DNA crosslinks. Overall, our findings demonstrate the utility of yeast-based reporter assays employing strains with multiple-deletion mutations in DNA repair genes. These assays serve as valuable tools for investigating DNA repair mechanisms and assessing chemical-induced DNA damage. KEY POINTS: • Responses to genotoxic chemicals were investigated in three types of reporter yeast. • Yeast strains with single- and double-deletions of DNA repair genes were tested. • Two DNA repair pathways predominantly contributed to DNA crosslink repair in yeast.

Discovery of Cell-Permeable Allosteric Inhibitors of Liver Pyruvate Kinase: Design and Synthesis of Sulfone-Based Urolithins.

Metabolic dysfunction-associated fatty liver disease (MAFLD) presents a significant global health challenge, characterized by the accumulation of liver fat and impacting a considerable portion of the worldwide population. Despite its widespread occurrence, effective treatments for MAFLD are limited. The liver-specific isoform of pyruvate kinase (PKL) has been identified as a promising target for developing MAFLD therapies. Urolithin C, an allosteric inhibitor of PKL, has shown potential in preliminary studies. Expanding upon this groundwork, our study delved into delineating the structure-activity relationship of urolithin C via the synthesis of sulfone-based urolithin analogs. Our results highlight that incorporating a sulfone moiety leads to substantial PKL inhibition, with additional catechol moieties further enhancing this effect. Despite modest improvements in liver cell lines, there was a significant increase in inhibition observed in HepG2 cell lysates. Specifically, compounds 15d, 9d, 15e, 18a, 12d, and 15a displayed promising IC50 values ranging from 4.3 µM to 18.7 µM. Notably, compound 15e not only demonstrated a decrease in PKL activity and triacylglycerol (TAG) content but also showed efficient cellular uptake. These findings position compound 15e as a promising candidate for pharmacological MAFLD treatment, warranting further research and studies.

Large Libraries of Structurally Diverse Macrocycles Suitable for Membrane Permeation.

Macrocycles offer an attractive format for drug development due to their good binding properties and potential to cross cell membranes. To efficiently identify macrocyclic ligands for new targets, methods for the synthesis and screening of large combinatorial libraries of small cyclic peptides were developed, many of them using thiol groups for efficient peptide macrocyclization. However, a weakness of these libraries is that invariant thiol-containing building blocks such as cysteine are used, resulting in a region that does not contribute to library diversity but increases molecule size. Herein, we synthesized a series of structurally diverse thiol-containing elements and used them for the combinatorial synthesis of a 2,688-member library of small, structurally diverse peptidic macrocycles with unprecedented skeletal complexity. We then used this library to discover potent thrombin and plasma kallikrein inhibitors, some also demonstrating favorable membrane permeability. X-ray structure analysis of macrocycle-target complexes showed that the size and shape of the newly developed thiol elements are key for binding. The strategy and library format presented in this work significantly enhance structural diversity by allowing combinatorial modifications to a previously invariant region of peptide macrocycles, which may be broadly applied in the development of membrane permeable therapeutics.

Stapled Peptides as Inhibitors of mRNA Deadenylation.

Therapeutic intervention targeting mRNA typically aims at reducing the levels of disease-causing sequences. Achieving the opposite effect of blocking the destruction of beneficial mRNA remains underexplored. The degradation of mRNA starts with the removal of poly(A) tails reducing their stability and translational activity which is mainly regulated by the CCR4-NOT complex. The subunit NOT9 binds various RNA binding proteins which recruit mRNA in a sequence-specific manner to the CCR4-NOT complex to promote their deadenylation. These RNA binding proteins interact with NOT9 through a helical NOT9 binding motif which we used as a starting point for development of the hydrocarbon stapled peptide NIP-2. The peptide (KD = 60.4 nM) was able to inhibit RNA-binding (IC50 = 333 nM) as well as the deadenylation activity of the CCR4-NOT complex in vitro while being cell-permeable (EC50 = 2.44 µM). A co-crystal structure of NIP-2 bound to NOT9 allowed further optimization of the peptide through point mutation leading to NIP-2-H27A-N3(KD = 122 nM) with high cell permeability (cell-permeability EC50 = 0.34 µM). The optimized peptide was able to inhibit deadenylation of target mRNAs when used in HeLa cells at a concentration of 100 µM demonstrating the feasibility of increasing mRNA stability.

Exocyclic and Linker Editing of Lys63-linked Ubiquitin Chains Modulators Specifically Inhibits Non-homologous End-joining Repair.

Despite the great advances in discovering cyclic peptides against protein targets, their reduced aqueous solubility, cell permeability, and activity of the cyclic peptide restrict its utilization in advanced biological research and therapeutic applications. Here we report on a novel approach of structural alternation of the exocyclic and linker parts that led to a new derivative with significantly improved cell activity allowing us to dissect its mode of action in detail. We have identified an effective cyclic peptide (CP7) that induces approximately a 9-fold increase in DNA damage accumulation and a remarkable increase in apoptotic cancer cell death compared to the reported molecule. Notably, treating cells with CP7 leads to a dramatic decrease in the efficiency of non-homologous end joining (NHEJ) repair of DNA double-strand breaks (DSBs), which is accompanied by an increase in homologous recombination (HR) repair. Interestingly, treating BRCA1-deficient cells with CP7 restores HR integrity, which is accompanied by increased resistance to CP7. Additionally, CP7 treatment increases the sensitivity of cancer cells to ionizing radiation. Collectively, our findings demonstrate that CP7 is a selective inhibitor of NHEJ, offering a potential strategy to enhance the effectiveness of radiation therapy.

Membrane Permeant Analogs for Independent Cellular Introduction of the Terpene Precursors Isopentenyl- and Dimethylallyl-Pyrophosphate.

Isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) are the central five-carbon precursors to all terpenes. Despite their significance, exogenous, independent delivery of IPP and DMAPP to cells is impossible as the negatively charged pyrophosphate makes these molecules membrane impermeant. Herein, we demonstrate a facile method to circumvent this challenge through esterification of the ß-phosphate with two self-immolative esters (SIEs) that neutralize the negatively charged pyrophosphate to yield membrane-permeant analogs of IPP and DMAPP. Following cellular incorporation, general esterase activity initiates cleavage of the SIEs, resulting in traceless release of IPP and DMAPP for metabolic utilization. Addition of the synthesized IPP and DMAPP precursor analogs rescued cell growth of glioblastoma (U-87MG) cancer cells concurrently treated with the HMG-CoA reductase inhibitor pitavastatin, which otherwise abrogates cell growth via blocking production of IPP and DMAPP. This work demonstrates a new application of a prodrug strategy to incorporate a metabolic intermediate and promises to enable future interrogation of the distinct biological roles of IPP and DMAPP.

Discovery of novel protein degraders based on bioorthogonal reaction-driven intracellular self-assembly strategy.

Proteolysis targeting chimera (PROTAC) is a promising therapeutic modality capable of degrading undruggable proteins and overcoming the shortcomings of traditional inhibitors. However, the molecular weight and pharmaceutical properties of PROTACs fall outside of a reasonable range. To overcome the inherent poor druggability of PROTACs, an intracellular self-assembly strategy based on bio-orthogonal reaction was proposed and applied in this study. Herein, two novel classes of intracellular precursors that can self-assemble into protein degraders through bio-orthogonal reactions were explored, including a novel class of E3 ubiquitin ligase ligands bearing tetrazine (E3L-Tz) and target protein ligands incorporated with norbornene (TPL-Nb). These two types of precursors could spontaneously undergo bio-orthogonal reactions in living cells, affording novel PROTACs. Among these precursors, the biological activities of PROTACs formed by target protein ligand with norbornene group (S4N-1) were more potent than others and degrade VEGFR-2, PDGFR-ß and EphB4. The results demonstrated that a highly specific bio-orthogonal reaction driven intracellular self-assembly strategy in living cells could be utilized to improve the degradation activity of PROTACs.

Design, synthesis and bioactivity evaluation of self-assembled PROTACs based on multi-target kinase inhibitors.

Proteolysis targeting chimera (PROTAC) is a heterobifunctional molecule with enormous potential for its ability to overcome the limitations of traditional inhibitors. However, its inherent disadvantages have been increasingly revealed, such as poor cell permeability caused by large molecule weight. Herein, to overcome the inherent shortcomings, intracellular self-assembly was proposed based on bioorthogonal reaction and molecular fragments, affording a novel type of self-assembled PROTACs. Two types of precursors incorporated with tetrazine and norbornene as bioorthogonal groups were designed and synthesized, and they could subsequently be conjugated in cells to generate novel PROTACs. Fortunately, ultrafast HRMS and HPLC assays indicated that self-assembled PROTACs driven by the bio-orthogonal reaction were detected in living U87 cells. Biological evaluation suggested that the precursor molecule LN-1 could degrade PDGFR-ß protein in a concentration-dependent manner, while cancer cells were co-treated with another precursor molecule, TzB. Our findings verified the feasibility of a self-assembly strategy in future development of novel PROTACs.

Effects of cross-linking of rice protein with ferulic acid on digestion and absorption of ferulic acid.

Though rice proteins have been applied to improve the stability of phenolic compounds, it is unclear how rice proteins affect phenolic acid's digestion and bioavailability. This study investigated the consequences of protein-ferulic acid interactions in the gastrointestinal environment. Ferulic acid and rice proteins formed complexes at room temperature, both with and without laccase. Rice protein was reported to be able to prevent ferulic acid from degrading in simulated oral fluid and remain stable in gastrointestinal fluids. With the hydrolysis of pepsin and pancreatin, rice protein-ferulic acid complexes degraded and released ferulic acid. While digested ferulic acid's DPPH scavenging activity was dramatically reduced, it was retained for the rice protein-ferulic acid complex. Moreover, the permeability coefficient of ferulic acid was not affected. Thus, rice protein is a promising food matrix to protect ferulic acid in the digestive tract and maintain the antioxidant functions of ferulic acid.

Organic Anion Transporting Polypeptide 3A1 (OATP3A1)-Gated Bio-Orthogonal Labeling of Intracellular Proteins.

Organic anion transporting polypeptides (OATPs) were found to readily deliver membrane impermeable, tetrazine bearing fluorescent probes into cells. This feature was explored in OATP3A1 conditioned bio-orthogonal labeling schemes of various intracellular proteins in live cells. Confocal microscopy and super-resolution microscopy (STED) studies have shown that highly specific and efficient staining of the selected intracellular proteins can be achieved with the otherwise non-permeable probes when OATP3A1 is present in the cell membrane of cells. Such a transport protein linked bio-orthogonal labeling scheme is believed to be useful in OATP3A1 activity-controlled protein expression studies in the future.

Cyclic helix B peptide ameliorated the sepsis-induced injury in human HPMEC cells through regulating NF-κB.

BACKGROUND: Sepsis is a life-threatening condition. The incidence of severe sepsis is increasing. Sepsis is often complicated with organ dysfunctions. Cyclic helix B peptide (CHBP) is a peptide derivant of erythropoietin with powerful tissue-protective efficacies. However, the role of CHBP in sepsis-induced injury remains unclear. MATERIAL AND METHODS: Lyso-phosphatidylserine (LPS) was used to induce sepsis in human pulmonary microvascular endothelial cells (HPMECs). Cell growth was detected using Cell Counting Kit-8. Cell permeability was measured using fluorescein isothiocyanate (FITC)-dextran. Cecal ligation and puncture (CLP) method was applied to induce sepsis and CHBP was provided to test its efficacy. Western blot assays were used to evaluate gene expression. RESULTS: Administration of CHBP ameliorated LPS-induced injury in HPMECs dose-dependently. Administration of CHBP decreased the permeability of LPS-treated HPMEC cells in a same way as well. Furthermore, we identified that recombinant CHBP protein (Re-CHBP) ameliorated CLP-induced injury in vivo. Finally, we found that administration of NF-κB activator, TNF-α, abolished the function of Re-CHBP in LPS-treated HPMEC cells. CONCLUSION: CHBP ameliorated sepsis-induced injury dose dependently both in vitro and in vivo through decreasing the permeability of HPMEC cells via suppressing NF-κB signaling and inflammation. Present findings highlight the importance of CHBP/NF-κB signaling in septic injury and provide new insights into therapeutic strategies for sepsis-induced injury.

Cell-permeable fluorescent indicator for imaging formaldehyde activity in living systems.

Formaldehyde (FA), as a reactive signaling molecule, plays an important role in living systems through a diverse array of cellular pathways. However, no systematic investigation for detection and imaging of FA by rendering cells transiently permeable has been reported yet. Specifically, we developed a new cell-permeable fluorescence probe functionality that was enhanced cellular entry efficiency and well retained intracellularly after activation for visualizing endogenous FA changes. Moreover, a smart "multi-lock system -key-and-lock" strategy，which have provoked a starting point for the use of probe and related biochemical tools to monitor FA in lysosomes. The versatile "latent" fluorophore that can undergo a subsequent self-immolative spacer for interrogating the roles and functions of FA in living systems as well as related biomedical applications.

Toxic effects and molecular mechanisms of sulfamethoxazole on Scenedesmus obliquus.

The antibiotic sulfamethoxazole (SMX) is a pollutant that is widely distributed in the global water environment.This substance has toxic effects on various aquatic organisms. Previous studies on SMX have focused on its acute toxicity towards algae and the changes induced at biological and cellular levels, rather than its biotoxicity and mechanisms at the molecular level. In this study, we investigated the effects of SMX on Scenedesmus obliquus as the model organism by performing transmission electron microscopy and transcriptome sequencing analyses. Exposure to SMX promoted gene expression, resulting in changes to algal cell ultrastructure. The cell walls became blurred, the chloroplast structure was seriously damaged, and the number and volume of mitochondria per cell increased. These changes were related to the inhibition of cell growth, decrease in chlorophyll content, increase in cell membrane permeability, and increased production of reactive oxygen species, which led to increased amounts of the lipid peroxidation product malondialdehyde, and higher activities of antioxidant enzymes. Our results suggest that SMX affects gene expression by influencing non-coding RNA metabolic processes, leading to changes in nuclear structures. Abnormally expressed long non-coding RNAs extensively regulate downstream gene expression through various mechanisms, such as chromatin recombination, thereby promoting tumor occurrence, invasion, and metastasis. This abnormal expression may be an important mechanism underlying the carcinogenic effects of SMX.

Transcriptional and Post-Translational Regulation of Junctional Adhesion Molecule-B (JAM-B) in Leukocytes under Inflammatory Stimuli.

Junctional adhesion molecules (JAMs; comprising JAM-A, -B and -C) act as receptors for viruses, mediate cell permeability, facilitate leukocyte migration during sterile and non-sterile inflammation and are important for the maintenance of epithelial barrier integrity. As such, they are implicated in the development of both communicable and non-communicable chronic diseases. Here, we investigated the expression and regulation of JAM-B in leukocytes under pathogen- and host-derived inflammatory stimuli using immunoassays, qPCR and pharmacological inhibitors of inflammatory signalling pathways. We show that JAM-B is expressed at both the mRNA and protein level in leukocytes. JAM-B protein is localised to the cytoplasm, Golgi apparatus and in the nucleus around ring-shaped structures. We also provide evidence that JAM-B nuclear localisation occurs via the classical importin-α/ß pathway, which is likely mediated through JAM-B protein nuclear localisation signals (NLS) and export signals (NES). In addition, we provide evidence that under both pathogen- and host-derived inflammatory stimuli, JAM-B transcription is regulated via the NF-κB-dependent pathways, whereas at the post-translational level JAM-B is regulated by ubiquitin-proteosome pathways. Anaphase-promoting ubiquitin ligase complex (APC/C) and herpes simplex virus-associated ubiquitin-specific protease (HAUSP/USP) were identified as candidates for JAM-B ubiquitination and de-ubiquitination, respectively. The expression and regulation of JAM-B in leukocytes reported here is a novel observation and contrasts with previous reports. The data reported here suggest that JAM-B expression in leukocytes is under the control of common inflammatory pathways.

Improved Cathepsin Probes for Sensitive Molecular Imaging.

Cysteine cathepsin proteases are found under normal conditions in the lysosomal compartments of cells, where they play pivotal roles in a variety of cellular processes such as protein and lipid metabolism, autophagy, antigen presentation, and cell growth and proliferation. As a consequence, aberrant localization and activity contribute to several pathologic conditions such as a variety of malignancies, cardiovascular diseases, osteoporosis, and other diseases. Hence, there is a resurgence of interest to expand the toolkit to monitor intracellular cathepsin activity and better ascertain their functions under these circumstances. Previous fluorescent activity-based probes (ABPs) that target cathepsins B, L, and S enabled detection of their activity in intact cells as well as non-invasive detection in animal disease models. However, their binding potency is suboptimal compared to the cathepsin inhibitor on which they were based, as the P1 positive charge was capped by a reporter tag. Here, we show the development of an improved cathepsin ABP that has a P1 positive charge by linking the tag on an additional amino acid at the end of the probe. While enhancing potency towards recombinant cathepsins, the new probe had reduced cell permeability due to additional peptide bonds. At a second phase, the probe was trimmed; the fluorophore was linked to an extended carbobenzoxy moiety, leading to enhanced cell permeability and superb detection of cathepsin activity in intact cells. In conclusion, this work introduces a prototype design for the next generation of highly sensitive ABPs that have excellent detection of cellular cathepsin activity.

Hepatocyte growth factor prevents pericyte loss in diabetic retinopathy.

Diabetic retinopathy (DR) is a disease that causes blindness due to vascular leakage or abnormal angiogenesis. Hepatocyte growth factor (HGF) is increased in the serum or vitreous fluid in proliferative diabetic retinopathy (PDR) patients, although the effect of HGF on the blood vessels remains unclear. This study focused on the effect of HGF on pericyte (PC) survival and endothelial cell (EC) permeability. It was demonstrated that HGF was increased in the diabetic mouse retina. However, HGF prevented PC apoptosis caused by TNF-α, which increased in the diabetic retinas both in vitro and in vivo. In addition, HGF was involved in PC survival by increasing the Akt signaling pathway. Moreover, HGF strengthened the EC tight junction in co-cultures of PCs and ECs by promoting PC survival, thereby reducing EC permeability. These results suggest that HGF may play a role in the prevention of increased vascular leakage by inhibiting the PC loss that occurs in DR to some extent. However, careful HGF reduction in DR might avoid an increase in PC loss.

A copper-specific microbial fuel cell biosensor based on riboflavin biosynthesis of engineered Escherichia coli.

Copper pollution poses a serious threat to the aquatic environment; however, in situ analytical methods for copper monitoring are still scarce. In the current study, Escherichia coli Rosetta was genetically modified to express OprF and ribB with promoter Pt7 and PcusC , respectively, which could synthesize porin and senses Cu2+ to produce riboflavin. The cell membrane permeability of this engineered strain was increased and its riboflavin production (1.45-3.56 µM) was positively correlated to Cu2+ (0-0.5 mM). The biosynthetic strain was then employed in microbial fuel cell (MFC) based biosensor. Under optimal operating parameters of pH 7.1 and 37°C, the maximum voltage (248, 295, 333, 352, and 407 mV) of the constructed MFC biosensor showed a linear correlation with Cu2+ concentration (0.1, 0.2, 0.3, 0.4, 0.5 mM, respectively; R2 = 0.977). The continuous mode testing demonstrated that the MFC biosensor specifically senses Cu2+ with calculated detection limit of 28 µM, which conforms to the common Cu2+ safety standard (32 µM). The results obtained with the developed biosensor system were consistent with the existing analytical methods such as colorimetry, flame atomic absorption spectrometry, and inductively coupled plasma optical emission spectrometry. In conclusion, this MFC-based biosensor overcomes the signal conversion and transmission problems of conventional approaches, providing a fast and economic analytical alternative for in situ monitoring of Cu2+ in water.