Traditional detection techniques are difficult to distinguish and detect multiple /3-lactam antibiotics simultaneously. The detection performance of sensor arrays is also limited due to the poor catalytic activity of nanozymes or the preparation of multiple receptor units. Here, inspired by the microenvironment of amino acid residues in the natural enzyme catalytic active center, a histidine-functionalized copper-based nanozyme (Cu-HAc@His) was constructed through a defect engineering strategy. Compared with the parent nanozyme (Cu-HAc), the obtained Cu-HAc@His nanozyme has higher peroxidase-like (POD) activity. Given the significant difference in catalytic activity of Cu-HAc@His nanozyme under different pH, a sensor array was constructed using three different pH values for efficient identification of nine /3-lactam antibiotics. Meanwhile, relying on the improvement of various machine learning (ML) models, the precision of the concentration-irrelevant classification system built using this array has been enhanced to 93.52%. This result has played a role in promoting blind sample recognition. More importantly, combining regression algorithms with sensor arrays enables precise quantitative analysis of multiple /3-lactam antibiotics. This work is not only of great significance for improving the identification of /3-lactam antibiotics in actual samples, but supplies a point of reference and direction for developing highly active POD nanozymes down the line.

Although nanozyme sensor arrays can simultaneously recognize multiple target substances, they are currently rarely used for identifying Beta-lactam antibiotics (BLs). This may be due to the lower catalytic performance of some nanozymes in practical applications, which further limits the detection performance of nanozyme sensor arrays. Therefore, developing highly active nanozymes is particularly important. Here, we introduced histidine during the preparation of Cu-1,3,5-benzenetricarboxylic acid (Cu-BTC) to obtain Cu-BTC@His nanozymes with high laccase-like (LAC) catalytic activity. Due to the unique physicochemical properties of BLs, they can inhibit the LAC activity of Cu-BTC@His, and the degree of inhibition increases with the increase of reaction time. A three-channel nanozyme sensor array was constructed based on reaction kinetics and applied to the discrimination of nine BLs. In addition, by optimizing multiple machine learning (ML) algorithms, the accuracy of the neglected concentration detection model constructed based on this array has been improved from 31.27 % to 95.92 %, which is beneficial for identifying unknown samples in real samples. This work is not only of great significance for improving the identification of BLs in complex samples, but also provides some reference and guidance for the design of highly active laccase-like nanozymes in the future.

The development of a synchronous analysis method for multi-category antibiotics detection is crucial for ensuring public health and food safety. Here, a caffeic acid-Cu (CA-Cu) nanozyme with exceptional laccase-mimicking and peroxidase-mimicking behaviors was effectively prepared. Owing to the distinct physicochemical characteristics of antibiotics, they exert varying levels of influence on the double-enzyme mimetic activities of CA-Cu, with the extent of this effect growing as the reaction time lengthens. A sensor array comprising four channels (two enzyme-mimicking behaviors x two time points = four sensing units) was successfully constructed and utilized for distinguishing eight different antibiotics. Moreover, by fine-tuning several machine learning (ML) models, the performance of the concentration-independent analysis system grounded in this array was significantly enhanced, thereby improving its accuracy from 54.02 % to 97.70 %. This advancement greatly facilitates the recognition of unknown antibiotic compounds in real samples. This study holds significant importance in facilitating the high-efficiency establishment of array channels and enhancing the accurate identification of multi-category antibiotics in intricate samples.

Ribonucleotide reductase (RNR) catalyzes the synthesis of four deoxyribonucleoside triphosphates (dNTPs), which are essential for DNA replication. Although dNTP imbalances reduce replication fidelity and elevate mutation rates, the impact of RNR dysfunction on Mycobacterium tuberculosis (Mtb) physiology and drug resistance remains unknown. Here, we constructed inducible knockdown strains for the RNR R1 subunit NrdE in Mtb and Mycolicibacterium smegmatis (Msm). NrdE knockdown significantly impaired growth and metabolic imbalances, indirectly disrupting oxidative homeostasis and mycolic acid synthesis, while increasing levels of intracellular ROS accumulation and enhancing cell wall permeability. Additionally, we developed genomic mutant strains, Msm-Y252A and Msm-Q255A, featuring targeted point mutations in the substrate-specific site (S-site) of the RNR loop domain, which determines NDP reduction specificity. The Msm-Y252A displayed a 1.9-fold decrease in dATP and increases in dGTP (1.6-fold), dTTP (9.0-fold), and dCTP (1.3-fold). In contrast, Msm-Q255A exhibited elevated intracellular levels of dGTP (1.6-fold), dTTP (6.1-fold), and dATP (1.5-fold), while dCTP levels remained unchanged. Neither the NrdE knockdown strain nor the S-site mutants exhibited direct resistance development; however, they both showed genomic instability, enhancing the emergence of rifampicin-resistant mutants, even with a 70-fold and a 25-fold increase in mutation frequency for Msm-Y252A and Msm-Q255A, respectively. This study demonstrates that NrdE is integral to Mycobacterium survival and genomic stability and that its RNR dysfunction creates a mutagenic environment under selective pressure, indirectly contributes to the development of drug resistance, positioning NrdE as an effective target for therapeutic strategies and a valuable molecular marker for early detection of drug-resistant Mtb.

The overuse of quinolone antibiotics (QNs) seriously endangers human health and the ecological environment. In this work, a copper dihydroxosulfate (Cu2(OH)2SO4) nanosheet exhibiting notable peroxidase-like (POD) and laccase-like (LAC) activities has been developed in basic deep eutectic solvents (DES). The unique physicochemical properties of QNs allow them to enhance the POD activity of Cu2(OH)2SO4, and with the extension of reaction time, this enhancement gradually intensifies. Conversely, when QNs are introduced into the LAC reaction system of Cu2(OH)2SO4, they significantly inhibit its LAC activity, with the degree of inhibition growing increasingly evident as the reaction time increases. A nanozyme sensing array has been developed via reaction dynamics to identify eight QNs. This method cleverly achieves self-calibration through two reverse signals, further improving the sensing performance of the sensor array. Moreover, through the optimization of various machine learning (ML), the precision of the concentration-independent recognition model built upon this array has been enhanced from 39.08% to 91.95%. This improvement is advantageous for the identification of unknown samples within actual samples. This work carries significant implications for enhancing the discrimination of QNs in complex samples.

Objective·To evaluate the clearance and pharmacokinetics/pharmacodynamics (PK/PD) of antibiotics from the perspective of protein binding rates in critically ill patients undergoing intermittent hemodialysis (IHD), in order to explore the association between protein binding rate and dialysis clearance of antibiotics, and to provide theoretical basis for developing antibiotic dosing regimens during hemodialysis.Methods·Nineteen patients undergone low-flux hemodialysis and received antibiotic therapy at the Department of Nephrology, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, were enrolled and divided into the meropenem group (n=7), the vancomycin group (n=5) and the ceftriaxone group (n=7) according to the type of antibiotics. A liquid chromatography with tandem mass spectrometry (LC-MS/MS) method was established to detect meropenem, vancomycin, and ceftriaxone in human plasma/serum and dialysate. A two-compartment pharmacokinetic model was established using MATLAB. Instantaneous and total dialysis clearance rates were calculated, and PK/PD parameters were analyzed.Results·No significant differences were found in the clinical characteristics of subjects among the three groups. The dialysis clearance rates were as follows: meropenem group (5.14‒5.97 L/h) > vancomycin group (2.87‒3.77 L/h)> ceftriaxone group (1.21‒1.90 L/h), with statistically significant differences (P<0.001). All three antibiotics showed good fit in the two-compartment pharmacokinetic model with a dialysate chamber (fval%<2), and the calculated PK/PD parameters were consistent with previous literature. For meropenem, the fraction of time that the free drug concentration remained above the minimal inhibitory concentration (%fT>MIC) values were 95.2%, 60.8% and 32.4% at minimal inhibitory concentration (MIC) values of 2, 8 and 16 μg/mL, respectively. For ceftriaxone (free concentration), the %fT>MIC values were all above 45.0% at MICs of 0.25, 4 and 16 μg/mL. For vancomycin, only 14.0% of the trough concentrations reached the target range of 15‒20 mg/L.Conclusion·The three antibiotics are well described by the two-compartment model. The plasma protein binding rate has a significant effect on the dialysis clearance of antibiotics in low-flux IHD, with higher protein binding associated with lower clearance. The regimens of meropenem (0.5 g/d) and ceftriaxone (2.0 g/d) are generally effective among patients undergoing low-flux IHD, while the vancomycin regimen with a loading dose of 1.0 g and a maintenance dose of 0.5 g/2 d carries a risk of treatment failure.

Liquid-liquid phase separation (LLPS) of biomolecules is a crucial mechanism in regulating cellular functions through dynamic formation of membrane-less organelles. The assembly of nucleation seeds is a key step that triggers LLPS; however, it is challenging to precisely study its assembly mechanism due to the complexity of the condensation process. Recently, metal ions have been found to play important roles in inducing LLPS. To elucidate the assembling mechanism, a small ubiquitin-like modifier (SUMO) protein was employed as a model protein to study metal-induced condensation. The results indicate that SUMO possesses two weak Cu(II)-binding sites across the protein surface, enabling intermolecular bridging among SUMO molecules. The formation of assembling seeds is confirmed by mass photometry analysis, showing the Cu(II)-induced dynamic clusterization of SUMO at nanoscale. Increasing the Cu(II) binding affinity of SUMO significantly promotes the protein condensation, underscoring the pivotal role of Cu(II) coordination in LLPS. This work provides insights into the protein assembly mechanism through non-specific intermolecular metal coordination.

Acinetobacter baumannii is a major pathogen of nosocomial meningitis and ventriculitis. Due to very limited antibiotic treatment options, polymyxins are often used as a last-line therapy. To optimise polymyxin use in the intraventricular environment, cerebrospinal fluid (CSF) proteomics was employed to investigate host-pathogen-polymyxin interactions in a 69-year-old patient with multidrug-resistant A. baumannii ventriculitis treated with a combination of intrathecal (ITH; 50,000 IU q24h/q48h), intraventricular (IVT; 50,000 IU q48h), and intravenous (500,000 IU, q12h) polymyxin B. CSF was collected before the first ITH dose in the ICU (0 h) and at 24 h, Day 7 and Day 26. The proteome was quantified at each time point and proteins with Qvalue <0.05 and fold change >1.2 were considered differentially expressed. Within 24 h of ITH/IVT polymyxin B administration, the innate immune system and neuroimmunity were highly active, evidenced by up-regulation of various pathways related to pathogen invasion, endocytosis and neutrophil degranulation. Blood-brain barrier impairment had worsened at 24 h but signs of repair were evident on Day 7 and Day 26. This is the first CSF proteomic study with polymyxins. Our findings provide critical mechanistic insights into optimizing ITH/IVT polymyxin administration.

Objective: How to choose the appropriate antibiotics and dosage has always been a difficult issue during the treatment of multi-drug-resistant bacterial infections. Our study aims to resolve this difficulty by introducing our multi-disciplinary treatment (MDT) clinical decision-making scheme based on rigorous interpretation of antibiotic susceptibility tests and precise therapeutic drug monitoring (TDM)-guided dosage adjustment.Method: The treatment course of an elderly patient who developed a multi-drug-resistant Pseudomonas aeruginosa (MDRPA) bloodstream infection from a brain abscess was presented.Results: In the treatment process, ceftazidime-avibactam (CAZ-AVI) was used empirically for treating the infection and clinical symptoms improved. However, the follow-up bacterial susceptibility test showed that the bacteria were resistant to CAZ-AVI. Considering the low fault tolerance of clinical therapy, the treatment was switched to a 1 mg/kg maintenance dose of susceptible polymyxin B, and TDM showed that the AUC(24h, ss) of 65.5 mgh/L had been achieved. However, clinical symptoms were not improved after 6 days of treatment. Facing the complicated situation, the cooperation of physicians, clinical pharmacologists, and microbiologists was applied, and the treatment finally succeeded with the pathogen eradicated when polymyxin B dose was increased to 1.4 mg/kg, with the AUC(24h, ss) of 98.6 mgh/L.Conclusion: MDT collaboration on the premise of scientific and standardized drug management is helpful for the recovery process in patients. The empirical judgment of doctors, the medication recommendations from experts in the field of TDM and pharmacokinetics/pharmacodynamics, and the drug susceptibility results provided by the clinical microbiology laboratory all provide the direction of treatment.

BACKGROUND Rice straw (RS) is one of the largest sources of lignocellulosic, which is an abundant raw material for biofuels and chemicals. However, the natural degradation of RS under a low temperature environment is the biggest obstacle to returning straw to the field. RESULTS In the present study, one bacillus strain W118 was isolated. Strain W118 was identified as Bacillus cereus through morphological and physiological characterization and 16S rDNA sequencing. The optimum growth temperature and pH of strain W118 were 20 degrees C and 6.5, respectively. Simultaneously, it was found that the strain W118 grew well at low temperature, even at a temperature of 4 degrees C (OD600 = 1.40 +/- 0.01). The decrease of various compositions of RS after the fermentation process at a temperature of 20 degrees C and 4 degrees C for 14 days was 27.00 +/- 0.02% and 23.70 +/- 0.04%, respectively. The composition of RS decreased to 50.71 +/- 0.02% after being fermented at 4 degrees C for 25 days. The results of scanning electron microscopy, Fourier transform infrared spectroscopy and X-ray diffraction of RS showed that the compositions of RS were significant decreased. CONCLUSION This test suggests that the strain W118 is efficient for degrading RS at low temperature, which has great application potential for straw degradation in a low temperature area. (c) 2022 Society of Chemical Industry.

Deubiquitinating enzyme (DUB) abnormalities are associated with many diseases. Previous attempts have been made to introduce various chemical groups such as alkynes, unsaturated olefins and alkyl halides to the C-terminus of ubiquitin (Ub) to capture the active-site cysteine residue in DUBs for structural and biochemical studies. Here, we find that a Ub C-terminal acyl azide can capture DUBs, thereby forming thioester bonds in buffers and cell lysates. This finding not only makes ubiquitin acyl azide a chemical probe for capturing DUBs, but also extends the utility of azide groups in biological applications.

Bacterial cells were long thought to be "bags of enzymes"with minimal internal structures. In recent years, membrane-less organelles formed by liquid-liquid phase separation (LLPS) of proteins or nucleic acids have been found to be involved in many important biological processes, although most of them were studied on eukaryotic cells. Here, we report that NikR, a bacterial nickel-responsive regulatory protein, exhibits LLPS both in solution and inside cells. Analyses of cellular nickel uptake and cell growth of E. coli confirm that LLPS enhances the regulatory function of NikR, while disruption of LLPS in cells promotes the expression of nickel transporter (nik) genes, which are negatively regulated by NikR. Mechanistic study shows that Ni(II) ions induces the accumulation of nik promoter DNA into the condensates formed by NikR. This result suggests that the formation of membrane-less compartments can be a regulatory mechanism of metal trans-porter proteins in bacterial cells.

ABSTRACT The antibacterial activity of rifamycins specifically relies on the inhibition of transcription by directly binding to the β-subunit of bacterial DNA-dependent RNA polymerase (RNAP). However, its killing efficacy is substantially diminished in most gram-negative bacteria. To systematically reveal the cellular functions that counteract rifamycin-mediated killing in the gram-negative model organism Escherichia coli, we performed a genome-wide Tn5 transposon-mediated screen to identify mutants with altered susceptibility to rifampicin. Combined with targeted gene knockouts, our results showed that the β-barrel assembly machinery plays a crucial role in restricting rifampicin from entering the cell, whereas mutants deficient in other cellular permeability barriers, such as lipopolysaccharide and enterobacterial common antigen, had no such effect. At bactericidal concentrations, the killing efficacy of rifampicin was strongly influenced by cellular functions, including iron acquisition, DNA repair, aerobic respiration, and carbon metabolism. Although iron acquisition de facto has a strong impact or dependence on cellular redox, our results suggest that their effects on rifampicin efficacy do not rely on hydroxyl radical formation. We provide evidence that maintenance of DNA replication and transcription-coupled nucleotide excision repair protects E. coli cells against rifampicin killing. Moreover, our results showed that sustained aerobic respiration and carbon catabolism diminish rifampicin’s killing efficacy, and this effect relies on the inhibition of transcription but not on translation. These findings suggest that the killing efficacy of rifamycins is largely determined by cellular responses upon the inhibition of RNAP and may expand our knowledge of the action mechanisms of rifamycins. IMPORTANCE Rifamycins are a group of antibiotics with a wide antibacterial spectrum. Although the binding target of rifamycin has been well characterized, the mechanisms underlying the discrepant killing efficacy between gram-negative and gram-positive bacteria remain poorly understood. Using a high-throughput screen combined with targeted gene knockouts in the gram-negative model organism Escherichia coli, we established that rifampicin efficacy is strongly dependent on several cellular pathways, including iron acquisition, DNA repair, aerobic respiration, and carbon metabolism. In addition, we provide evidence that these pathways modulate rifampicin efficacy in a manner distinct from redox-related killing. Our findings provide insights into the mechanism of rifamycin efficacy and may aid in the development of new antimicrobial adjuvants.

Protein reactions play important roles in the mechanism of action of cisplatin. In this work, we found that cisplatin is highly reactive to the RING finger domain of RNF11, a key protein involved in tumorigenesis and metastasis. The results show that cisplatin binds to RNF11 at the zinc coordination site and leads to zinc ejection from the protein. The formation of S-Pt(II) coordination and Zn(II) ions release have been confirmed by UV-vis spectrometry using zinc dye and thiol agent, showing reducing the contents of thiol groups while forming S-Pt bonds and releasing zinc ions. Electrospray ionization-mass spectrometry measurement indicates that each RNF11 can bind up to three platinum atoms. Kinetical analysis shows a reasonable platination rate of RNF11 with t(1/2) similar to 3 h. CD, nuclear magnetic resonance, and gel electrophoresis measurements indicate that the cisplatin reaction causes protein unfolding and oligomerization of RNF11. Pull-down assay confirms that the platination of RNF11 interferes with the protein interaction of RNF11 with UBE2N, a key step of the functionalization of RNF11. Furthermore, Cu(I) was found to promote the platination of RNF11, which could lead to increased protein reactivity to cisplatin in tumor cells with high copper levels. These results indicate that the platination-induced zinc release of RNF11 disrupts the protein structure and interferes with its functions.

In order to improve the light absorbition and photovoltaic performance and to decrease costs, five novel D-A-π-A motif copolymerized sulfur coordination-metal complexes (BDTT-BBT- Ni, BDTT-BBT- Cu, BDTT-BBT-Zn, BDTT-BBT-Cd and BDTT-BBT-Hg) have been designed and synthesized for being used as photosensitizers. And the photovolataic performance test results show that the highest PCE of the copolymer complex BDTT-BBT-Hg of five copolymers has reached 11.77% and the copolymer photosensitizers are simple and inexpensive to synthesize and have good solubility.

The unique thermodynamic and kinetic coordination chemistry of ruthenium allows it to modulate key adverse aggregation and membrane interactions of α-synuclein (α-syn) associated with Parkinson's disease. We show that the low-toxic RuIII complex trans-[ImH][RuCl4 (Me2 SO)(Im)] (NAMI-A) has dual inhibitory effects on both aggregation and membrane interactions of α-syn with submicromolar affinity, and disassembles pre-formed fibrils. NAMI-A abolishes the cytotoxicity of α-syn towards neuronal cells and mitigates neurodegeneration and motor impairments in a rat model of Parkinson's. Multinuclear NMR and MS analyses show that NAMI-A binds to residues involved in protein aggregation and membrane binding. NMR studies reveal the key steps in pro-drug activation and the effect of activated NAMI-A species on protein folding. Our findings provide a new basis for designing ruthenium complexes which could mitigate α-syn-induced Parkinson's pathology differently from organic agents.

Cu( i ) can substitute Zn( ii ) in the RING-domain of RNF11, which perturbs the protein structure, disrupts protein complexes, and interferes with transcriptional regulation of RNF11 in cells.

Rapid monitoring of real bacterial metabolic perturbations to antibiotics may be helpful to better understand the mechanisms of action and more targeted treatment. In this study, the real metabolic responses to antibiotic treatment in living bacteria were profiled rapidly by induced electrospray ionization mass spectrometry. Significant metabolic perturbations were profiled after antibiotic treatment compared with untreated bacteria. Similar and unique metabolic responses were observed with different antibiotic treatments. Further multivariable analysis was performed to determine significant metabolites as potential biomarkers. Moreover, different metabolic disturbances were detected for serial dilutions of antibiotic treatments. Overall, combined with induced electrospray ionization mass spectrometry, the rapid and real bacterial metabolic status caused by antibiotics was monitored, suggesting the potential application of our method in mechanism exploration and clinical diagnosis.

Microalgal biomass is an emerging source of renewable energy and health-related compounds. However, harvesting of microalgae is a techno-economic hinder. In this research, chitosan and polyacrylamide were optimized harvesting condition for Chlorella vulgaris. Stirring at 300 rpm for 2 min is optimum for chitosan and polyacrylamide. Low-dose (10 mg/L) chitosan (flocculation efficiency (FE), 98.10 ± 1.06%) is more efficient than high-dose (25 mg/L) polyacrylamide (FE 94.57 ± 0.55%) for harvesting C. vulgaris. Chitosan resulted flocs settled more quickly than polyacrylamide, while polyacrylamide keep > 90% FE in a wider pH range (7–10) than chitosan (7–8). Chitosan and polyacrylamide both have no negative effect on biomass composition, including protein, carbohydrate, and carotenoid. C. vulgaris in flocs could successfully regrow in fresh culture media. The residual culture media was recycled with little impact on cell growth. All the results suggested that chitosan and polyacrylamide could harvest high-quality microalgal biomass.