Ribosomal translation of fluorinated non-canonical amino acids for de novo biologically active fluorinated macrocyclic peptides.

Fluorination has emerged as a promising strategy in medicinal chemistry to improve the pharmacological profiles of drug candidates. Similarly, incorporating fluorinated non-canonical amino acids into macrocyclic peptides expands chemical diversity and enhances their pharmacological properties, from improved metabolic stability to enhanced cell permeability and target interactions. However, only a limited number of fluorinated non-canonical amino acids, which are canonical amino acid analogs, have been incorporated into macrocyclic peptides by ribosomes for de novo construction and target-based screening of fluorinated macrocyclic peptides. In this study, we report the ribosomal translation of a series of distinct fluorinated non-canonical amino acids, including mono-to tri-fluorinated variants, as well as fluorinated l-amino acids, d-amino acids, ß-amino acids, etc. This enabled the de novo discovery of fluorinated macrocyclic peptides with high affinity for EphA2, and particularly the identification of those exhibiting broad-spectrum activity against Gram-negative bacteria by targeting the BAM complex. This study not only expands the scope of ribosomally translatable fluorinated amino acids but also underscores the versatility of fluorinated macrocyclic peptides as potent therapeutic agents.

Self-Assembled MXene Supported on Carbonization-Free Wood for a Symmetrical All-Wood Eco-Supercapacitor.

As an emerging two-dimensional (2D) material, MXene has garnered significant interest in advanced energy storage systems, yet the stackable structure, poor mechanical stability, and lack of moldability limit its large-scale applications. To address this challenge, herein, the self-assembly of MXene on carbonization-free wood was obtained to serve as high-performance electrodes for symmetrical all-wood eco-supercapacitors by a steam-driven self-assembly method. This method can be implemented in a low-temperature environment, significantly simplifying traditional high-temperature annealing processes and generating minimal impact on the environment, human health, and resource consumption. The environmentally friendly steam-driven self-assembly strategy can be further extended into various wood-based electrodes, regardless of the types and structures of wood. As a typical platform electrode, the optimized MXene@delignified balsa wood (MDBW) achieves high areal capacitance and specific capacitance values of 2.99 F cm-2 and 580.55 F g-1 at an extensive mass loading of 5.16 mg cm-2, respectively, with almost loss-free capacitance after 10,000 cycles at 50 mA cm-2. In addition, an all-solid-state symmetrical all-wood eco-supercapacitor was further assembled based on MDBW-20 as both positive and negative electrodes to achieve a high energy density of 19.22 µWh cm-2 at a power density of 0.58 mW cm-2. This work provides an effective strategy to optimize wood-based electrodes for the practical application of biomass eco-supercapacitors.

Heat shock potentiates aminoglycosides against gram-negative bacteria by enhancing antibiotic uptake, protein aggregation, and ROS.

The potentiation of antibiotics is a promising strategy for combatting antibiotic-resistant/tolerant bacteria. Herein, we report that a 5-min sublethal heat shock enhances the bactericidal actions of aminoglycoside antibiotics by six orders of magnitude against both exponential- and stationary-phase Escherichia coli. This combined treatment also effectively kills various E. coli persisters, E. coli clinical isolates, and numerous gram-negative but not gram-positive bacteria and enables aminoglycosides at 5% of minimum inhibitory concentrations to eradicate multidrug-resistant pathogens Acinetobacter baumannii and Klebsiella pneumoniae. Mechanistically, the potentiation is achieved comprehensively by heat shock-enhanced proton motive force that thus promotes the bacterial uptake of aminoglycosides, as well as by increasing irreversible protein aggregation and reactive oxygen species that further augment the downstream lethality of aminoglycosides. Consistently, protonophores, chemical chaperones, antioxidants, and anaerobic culturing abolish heat shock-enhanced aminoglycoside lethality. We also demonstrate as a proof of concept that infrared irradiation- or photothermal nanosphere-induced thermal treatments potentiate aminoglycoside killing of Pseudomonas aeruginosa in a mouse acute skin wound model. Our study advances the understanding of the mechanism of actions of aminoglycosides and demonstrates a high potential for thermal ablation in curing bacterial infections when combined with aminoglycosides.

CCCP Facilitates Aminoglycoside to Kill Late Stationary-Phase Escherichia coli by Elevating Hydroxyl Radical.

Improving the efficacy of existing antibiotics is significant for combatting antibiotic resistance that poses a major threat to human health. Carbonyl cyanide m-chlorophenylhydrazine (CCCP), a well-known protonophore for dissipating proton motive force (PMF), has been widely used to block the PMF-dependent uptake of aminoglycoside antibiotics and thus suppress aminoglycoside lethality. Here, we report that CCCP and its functional analog FCCP, but not other types of protonophores, unprecedently potentiate aminoglycosides (e.g., tobramycin and gentamicin) by 3-4 orders of magnitude killing of Escherichia coli, Staphylococcus aureus, Shigella flexneri, and Vibrio alginolyticus cells in stationary phase but not these cells in exponential phase nor other 12 bacterial species we examined. Overall, the effect of CCCP on aminoglycoside lethality undergoes a gradual transition from suppression against E. coli exponential-phase cells to potentiation against late stationary-phase cells, with the cell growth status and culture medium being crucial. Consistently, disturbance of the PMF by changing transmembrane proton gradient (ΔpH) or electric potential (ΔΨ) also potentiates tobramycin. Nevertheless, CCCP neither increases the intracellular concentration of tobramycin nor decreases the MIC of the antibiotic, thus excluding that CCCP acts as an efflux pump inhibitor to potentiate aminoglycosides. Rather, we show that the combined treatment dramatically enhances the cellular level of hydroxyl radical under both aerobic and anaerobic culturing conditions, under which the antioxidant N-acetyl cysteine fully suppresses both hydroxyl radical accumulation and cell death. Together, these findings open a new avenue to develop certain protonophores as aminoglycoside adjuvants against pathogens in stationary phase and also illustrate an essential role of hydroxyl radical in aminoglycoside lethality regardless of aerobic respiration.

5-Methylindole kills various bacterial pathogens and potentiates aminoglycoside against methicillin-resistant Staphylococcus aureus.

Antibiotic resistance of bacterial pathogens has become a severe threat to human health. To counteract antibiotic resistance, it is of significance to discover new antibiotics and also improve the efficacy of existing antibiotics. Here we show that 5-methylindole, a derivative of the interspecies signaling molecule indole, is able to directly kill various Gram-positive pathogens (e.g., Staphylococcus aureus and Enterococcus faecalis) and also Gram-negative ones (e.g., Escherichia coli and Pseudomonas aeruginosa), with 2-methylindole being less potent. Particularly, 5-methylindole can kill methicillin-resistant S. aureus, multidrug-resistant Klebsiella pneumoniae, Mycobacterium tuberculosis, and antibiotic-tolerant S. aureus persisters. Furthermore, 5-methylindole significantly potentiates aminoglycoside antibiotics, but not fluoroquinolones, killing of S. aureus. In addition, 5-iodoindole also potentiates aminoglycosides. Our findings open a new avenue to develop indole derivatives like 5-methylindole as antibacterial agents or adjuvants of aminoglycoside.

n-Butanol Potentiates Subinhibitory Aminoglycosides against Bacterial Persisters and Multidrug-Resistant MRSA by Rapidly Enhancing Antibiotic Uptake.

Potentiation of traditional antibiotics is of significance for combating antibiotic-resistant bacteria that have become a severe threat to human and animal health. Here, we report that 1 min co-treatment with n-butanol greatly and specifically enhances the bactericidal action of aminoglycosides by 5 orders of magnitude against stationary-phase Staphylococcus aureus cells, with n-propanol and isobutanol showing less potency. This combined treatment also rapidly kills various S. aureus persisters, methicillin-resistant S. aureus (MRSA) cells, and numerous Gram-positive and -negative pathogens including some clinically isolated multidrug-resistant pathogens (e.g., S. aureus, Staphylococcus epidermidis, and Enterococcus faecalis) in vitro, as well as S. aureus in mice. Mechanistically, the potentiation results from the actions of aminoglycosides on their conventional target ribosome rather than the antiseptic effect of n-butanol and is achieved by rapidly enhancing the bacterial uptake of aminoglycosides, while salts and inhibitors of proton motive force (e.g., CCCP) can diminish this uptake. Importantly, such n-butanol-enhanced antibiotic uptake even enables subinhibitory concentrations of aminoglycosides to rapidly kill both MRSA and conventional S. aureus cells. Given n-butanol is a non-metabolite in the pathogens we tested, our work may open avenues to develop a metabolite-independent strategy for aminoglycoside potentiation to rapidly eliminate antibiotic-resistant/tolerant pathogens, as well as for reducing the toxicity associated with aminoglycoside use.

Mechanosensitive Channels Mediate Hypoionic Shock-Induced Aminoglycoside Potentiation against Bacterial Persisters by Enhancing Antibiotic Uptake.

Improving the efficacy of existing antibiotics is a promising strategy for combating antibiotic-resistant/tolerant bacterial pathogens that have become a severe threat to human health. We previously reported that aminoglycoside antibiotics could be dramatically potentiated against stationary-phase Escherichia coli cells under hypoionic shock conditions (i.e., treatment with ion-free solutions), but the underlying molecular mechanism remains unknown. Here, we show that mechanosensitive (MS) channels, a ubiquitous protein family sensing mechanical forces of cell membrane, mediate such hypoionic shock-induced aminoglycoside potentiation. Two-minute treatment under conditions of hypoionic shock (e.g., in pure water) greatly enhances the bactericidal effects of aminoglycosides against both spontaneous and triggered E. coli persisters, numerous strains of Gram-negative pathogens in vitro, and Pseudomonas aeruginosa in mice. Such potentiation is achieved by hypoionic shock-enhanced bacterial uptake of aminoglycosides and is linked to hypoionic shock-induced destabilization of the cytoplasmic membrane in E. coli. Genetic and biochemical analyses reveal that MscS-family channels directly and redundantly mediate aminoglycoside uptake upon hypoionic shock and thus potentiation, with MscL channel showing reduced effect. Molecular docking and site-directed mutagenesis analyses reveal a putative streptomycin-binding pocket in MscS, critical for streptomycin uptake and potentiation. These results suggest that hypoionic shock treatment destabilizes the cytoplasmic membrane and thus changes the membrane tension, which immediately activates MS channels that are able to effectively transport aminoglycosides into the cytoplasm for downstream killing. Our findings reveal the biological effects of hypoionic shock on bacteria and can help to develop novel adjuvants for aminoglycoside potentiation to combat bacterial pathogens via activating MS channels.

The Caenorhabditis elegans 12-kDa small heat shock proteins with little in vitro chaperone activity play crucial roles for its dauer formation, longevity, and reproduction.

Small heat shock proteins (sHSPs) are known to exhibit in vitro chaperone activity by suppressing the aggregation of misfolded proteins. The 12-kDa sHSPs (Hsp12s) subfamily members from Caenorhabditis elegans, including Hsp12.2, Hsp12.3, and Hsp12.6, however, are devoid of such chaperone activity, and their in vivo functions are poorly understood. Here we verified that Hsp12.1, similar to its homologs Hsp12.2, Hsp12.3, and Hsp12.6, hardly exhibited any chaperone activity. Strikingly, we demonstrated that these Hsp12s seem to play crucial physiological roles in C. elegans, for suppressing dauer formation and promoting both longevity and reproduction. A unique sHSP gene from Filarial nematode worm Brugia malayi was identified such that it encodes two products, one as a full-length Hsp12.6 protein and the other one having an N-terminal arm of normal length but lacks the C-terminal extension. This gene may represent an intermediate form in evolution from a common sHSP to a Hsp12. Together, our study offers insights on what biological functions the chaperone-defective sHSPs may exhibit and also implicates an evolutionary scenario for the unique Hsp12s subfamily.

Gentamicin Combined With Hypoionic Shock Rapidly Eradicates Aquaculture Bacteria in vitro and in vivo.

Bacterial pathogens are a major cause of infectious diseases in aquatic animals. The abuse of antibiotics in the aquatic industry has led to the proliferation of antibiotic resistance. It is therefore essential to develop more effective and safer strategies to increase the efficacy and extend the life span of the antibiotics used in aquaculture. In this study, we show that six aquaculture bacterial pathogens (i.e., Aeromonas hydrophila, Vibrio alginolyticus, Edwardsiella tarda, Streptococcus iniae, Vibrio harveyi, and Vibrio fluvialis) in the stationary phase can be rapidly killed after immersion in gentamicin- or neomycin-containing, ion-free solutions for a few minutes. Such hypoionic shock treatment enhances the bacterial uptake of gentamicin in an ATP-dependent manner. Importantly, we demonstrate, as a proof of concept, that gentamicin under hypoionic shock conditions can effectively kill A. hydrophila in vivo in a skin infection model of zebrafish (Danio rerio), completely curing the infected fish. Given that pathogenic bacteria generally adhere to the skin surface and gills of aquatic animals, our strategy is of potential significance for bacterial infection control, especially for small-scale economic fish farming and ornamental fish farming. Further, the combined treatment can be completed within 5 min with a relatively small volume of solution, thus minimizing the amount of residual antibiotics in both animals and the environment.

A high-throughput genetically directed protein crosslinking analysis reveals the physiological relevance of the ATP synthase 'inserted' state.

ATP synthase, a highly conserved protein complex that has a subunit composition of α3 ß3 γδεab2 c8-15 for the bacterial enzyme, is a key player in supplying energy to living organisms. This protein complex consists of a peripheral F1 sector (α3 ß3 γδε) and a membrane-integrated Fo sector (ab2 c8-15 ). Structural analyses of the isolated protein components revealed that, remarkably, the C-terminal domain of its ε-subunit seems to adopt two dramatically different structures, but the physiological relevance of this conformational change remains largely unknown. In an attempt to decipher this, we developed a high-throughput in vivo protein photo-cross-linking analysis pipeline based on the introduction of the unnatural amino acid into the target protein via the scarless genome-targeted site-directed mutagenesis technique, and probing the cross-linked products via the high-throughput polyacrylamide gel electrophoresis technique. Employing this pipeline, we examined the interactions involving the C-terminal helix of the ε-subunit in cells living under a variety of experimental conditions. These studies enabled us to uncover that the bacterial ATP synthase exists as an equilibrium between the 'inserted' and 'noninserted' state in cells, maintaining a moderate but significant level of net ATP synthesis when shifting to the former upon exposing to unfavorable energetically stressful conditions. Such a mechanism allows the bacterial ATP synthases to proportionally and instantly switch between two reversible functional states in responding to changing environmental conditions. Importantly, this high-throughput approach could allow us to decipher the physiological relevance of protein-protein interactions identified under in vitro conditions or to unveil novel physiological context-dependent protein-protein interactions that are unknown before.

Global analysis of daily new COVID-19 cases reveals many static-phase countries including the United States potentially with unstoppable epidemic.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic is hitting many countries. It is hypothesized the epidemic is differentially progressing in different countries. AIM: To investigate how the COVID-19 epidemic is going on in different countries by analyzing representative countries. METHODS: The status of COVID-19 epidemic in over 60 most affected countries was characterized. The data of daily new cases of each country were collected from Worldometer. The data of daily tests for the United States, Italy, and South Korea were collected from the Website of One World Data. Levels of daily positive COVID-19 tests in the two most affected states of the United States (New York and New Jersey) were collected from the website of the COVID Tracking Project. Statistics were analyzed using Microcal Origin software with ANOVA algorithm, and significance level was set at a P value of 0.05. RESULTS: The COVID-19 epidemic was differentially progressing in different countries. Comparative analyses of daily new cases as of April 19, 2020 revealed that 61 most affected countries can be classified into four types: Downward (22), upward (20), static-phase (12), and uncertain ones (7). In particular, the 12 static-phase countries including the United States were characterized by largely constant numbers of daily new cases in the past over 14 d. Furthermore, these static-phase countries were overall significantly lower in testing density (P = 0.016) but higher in the level of positive COVID-19 tests than downward countries (P = 0.028). These findings suggested that the testing capacity in static-phase countries was lagging behind the spread of the outbreak, i.e., daily new cases (confirmed) were likely less than daily new infections and the remaining undocumented infections were thus still expanding, resulting in unstoppable epidemic. CONCLUSION: Increasing the testing capacity and/or reducing the COVID-19 transmission are urgently needed to stop the potentially unstoppable, severing crisis in static-phase countries.

Forecasting the cumulative number of COVID-19 deaths in China: a Boltzmann function-based modeling study.

The COVID-19 outbreak is ongoing in China. Here, Boltzmann function-based analyses reveal the potential total numbers of COVID-19 deaths: 3,260 (95% confidence interval [CI], 3187-3394) in China; 110 (95% CI, 109-112) in Hubei Province; 3,174 (95% CI, 3095-3270) outside Hubei; 2,550 (95% CI, 2494-2621) in Wuhan City; and 617 (95% CI, 607-632) outside Wuhan.

Negligible risk of the COVID-19 resurgence caused by work resuming in China (outside Hubei): a statistical probability study.

The COVID-19 outbreak in China appears to reach the late stage since late March 2020, and a stepwise restoration of economic operations is implemented. Risk assessment for such economic restoration is of significance. Here, we estimated the probability of COVID-19 resurgence caused by work resuming in typical provinces/cities and found that such probability is very limited (<5% for all the regions except Beijing). Our work may inform provincial governments to make risk level-based, differentiated control measures.

5-Methylindole Potentiates Aminoglycoside Against Gram-Positive Bacteria Including Staphylococcus aureus Persisters Under Hypoionic Conditions.

Antibiotic resistance/tolerance has become a severe threat to human and animal health. To combat antibiotic-resistant/tolerant bacteria, it is of significance to improve the efficacy of traditional antibiotics. Here we show that indole potentiates tobramycin to kill stationary-phase Staphylococcus aureus cells after a short, combined treatment, with its derivative 5-methylindole being the most potent compound tested and with the absence of ions as a prerequisite. Consistently, this combined treatment also kills various types of S. aureus persister cells as induced by the protonophore CCCP, nutrient shift, or starvation, as well as methicillin-resistant S. aureus (MRSA) cells. Importantly, 5-methylindole potentiates tobramycin killing of S. aureus persisters in a mouse acute skin wound model. Furthermore, 5-methylindole facilitates killing of many strains of gram-positive pathogens such as Staphylococcus epidermidis, Enterococcus faecalis, and Streptococcus pyogenes by aminoglycoside antibiotics, whereas it suppresses the action of aminoglycoside against the gram-negative pathogens Escherichia coli and Shigella flexneri. In conclusion, our work may pave the way for the development of indole derivatives as adjuvants to potentiate aminoglycosides against gram-positive pathogens.

Rapid Freezing Enables Aminoglycosides To Eradicate Bacterial Persisters via Enhancing Mechanosensitive Channel MscL-Mediated Antibiotic Uptake.

Bacterial persisters exhibit noninherited antibiotic tolerance and are linked to the recalcitrance of bacterial infections. It is very urgent but also challenging to develop antipersister strategies. Here, we report that 10-s freezing with liquid nitrogen dramatically enhances the bactericidal action of aminoglycoside antibiotics by 2 to 6 orders of magnitude against many Gram-negative pathogens, with weaker potentiation effects on Gram-positive bacteria. In particular, antibiotic-tolerant Escherichia coli and Pseudomonas aeruginosa persisters-which were prepared by treating exponential-phase cells with ampicillin, ofloxacin, the protonophore cyanide m-chlorophenyl hydrazone (CCCP), or bacteriostatic antibiotics-can be effectively killed. We demonstrated, as a proof of concept, that freezing potentiated the aminoglycosides' killing of P. aeruginosa persisters in a mouse acute skin wound model. Mechanistically, freezing dramatically increased the bacterial uptake of aminoglycosides regardless of the presence of CCCP, indicating that the effects are independent of the proton motive force (PMF). In line with these results, we found that the effects were linked to freezing-induced cell membrane damage and were attributable, at least partly, to the mechanosensitive ion channel MscL, which was able to directly mediate such freezing-enhanced aminoglycoside uptake. In view of these results, we propose that the freezing-induced aminoglycoside potentiation is achieved by freezing-induced cell membrane destabilization, which, in turn, activates the MscL channel, which is able to effectively take up aminoglycosides in a PMF-independent manner. Our work may pave the way for the development of antipersister strategies that utilize the same mechanism as freezing but do so without causing any injury to animal cells.IMPORTANCE Antibiotics have long been used to successfully kill bacterial pathogens, but antibiotic resistance/tolerance usually has led to the failure of antibiotic therapy, and it has become a severe threat to human health. How to improve the efficacy of existing antibiotics is of importance for combating antibiotic-resistant/tolerant pathogens. Here, we report that 10-s rapid freezing with liquid nitrogen dramatically enhanced the bactericidal action of aminoglycoside antibiotics by 2 to 6 orders of magnitude against many bacterial pathogens in vitro and also in a mouse skin wound model. In particular, such combined treatment was able to effectively kill persister cells of Escherichia coli and Pseudomonas aeruginosa, which are per se tolerant of conventional treatment with bactericidal antibiotics for several hours. We also demonstrated that freezing-induced aminoglycoside potentiation was apparently linked to freezing-induced cell membrane damage that may have activated the mechanosensitive ion channel MscL, which, in turn, was able to effectively uptake aminoglycoside antibiotics in a proton motive force-independent manner. Our report sheds light on the development of a new strategy against bacterial pathogens by combining existing antibiotics with a conventional physical treatment or with MscL agonists.

Degp degrades a wide range of substrate proteins in Escherichia coli under stress conditions.

DegP, a periplasmic dual-functional protease and chaperone in Gram-negative bacteria, is critical for bacterial stress resistance, but the precise underlying mechanisms are not fully understood. Here, we show that the protease function of DegP is critical for Escherichia coli cells to maintain membrane integrity, particularly under heat shock conditions (42°C). Site-directed photo-cross-linking, mass spectrometry and immunoblotting analyses reveal that both periplasmic proteins (e.g. OppA and MalE) and ß-barrel outer membrane proteins (OMPs) are DegP-interacting proteins and that OppA is degraded by DegP in vitro and in vivo at 42°C. In addition, OmpA and BamA, chimeric ß-barrel OMPs containing a soluble periplasmic domain, are bound to DegP in both unfolded and folded forms, whereas only the unfolded forms are degradable by DegP. The presence of folded OmpA as a substrate of DegP is attributed to its periplasmic domain, which is resistant to DegP degradation and even generally protects pure ß-barrel OMPs from degradation in an intra-molecular way. Furthermore, a pair of residues (R262 and V328) in the PDZ domain-1 of DegP play important roles for binding unfolded and folded ß-barrel OMPs, with R262 being critical. Our study, together with earlier reports, indicates that DegP plays a critical role in protein quality control in the bacterial periplasm by degrading both periplasmic proteins and ß-barrel OMPs under stress conditions and likely also by participating in the folding of chimeric ß-barrel OMPs. A working model is proposed to illustrate the finely tuned functions of DegP with respect to different substrate proteins.

Hypoionic Shock Facilitates Aminoglycoside Killing of Both Nutrient Shift- and Starvation-Induced Bacterial Persister Cells by Rapidly Enhancing Aminoglycoside Uptake.

Bacterial persister cells are phenotypic variants that exhibit transient antibiotic tolerance and play a leading role in chronic infections and the development of antibiotic resistance. Determining the mechanism that underlies persister formation and developing anti-persister strategies, therefore, are clinically important goals. Here, we report that many gram-negative and gram-positive bacteria become highly tolerant to typical bactericidal antibiotics when the carbon source for their antibiotic-sensitive exponential growth phase is shifted to fumarate, suggesting a role for fumarate in persister induction. Nutrient shift-induced Escherichia coli but not Staphylococcus aureus persister cells can be killed by aminoglycosides upon hypoionic shock (i.e., the absence of ions), which is achieved by suspending the persisters in aminoglycoside-containing pure water for only 1 or 2 min. Such potentiation can be abolished by inhibitors of the electron transport chain (e.g., NaN3) or proton motive force (e.g., CCCP). Additionally, we show that hypoionic shock facilitates the eradication of starvation-induced E. coli but not S. aureus persisters by aminoglycosides, and that such potentiation can be significantly suppressed by NaN3 or CCCP. Mechanistically, hypoionic shock dramatically enhances aminoglycoside uptake by both nutrient shift- and starvation-induced E. coli persisters, whereas CCCP can diminish this uptake. Results of our study illustrate the general role of fumarate in bacterial persistence and may open new avenues for persister eradication and aminoglycoside use.