Covalent Inhibition of a Host-Pathogen Protein-Protein Interaction Reduces the Infectivity of Streptococcus pneumoniae.

The ever-expanding antibiotic resistance urgently calls for novel antibacterial therapeutics, especially those with a new mode of action. We report herein our exploration of protein-protein interaction (PPI) inhibition as a new mechanism to thwart bacterial pathogenesis. Specifically, we describe potent and specific inhibitors of the pneumococcal surface protein PspC, an important virulence factor that facilitates the infection of Streptococcus pneumoniae. Specifically, PspC has been documented to recruit human complement factor H (hFH) to suppress host complement activation and/or promote the bacterial attachment to host tissues. The CCP9 domain of hFH was recombinantly expressed to inhibit the PspC-hFH interaction as demonstrated on live pneumococcal cells. The inhibitor allowed for the first pharmacological intervention of the PspC-hFH interaction. This PPI inhibition reduced pneumococci's attachment to epithelial cells and also resensitized the D39 strain of S. pneumoniae for opsonization. Importantly, we have further devised covalent versions of CCP9, which afforded long-lasting PspC inhibition with low nanomolar potency. Overall, our results showcase the promise of PPI inhibition for combating bacterial infections as well as the power of covalent inhibitors.

PI3K/HSCB axis facilitates FOG1 nuclear translocation to promote erythropoiesis and megakaryopoiesis.

Erythropoiesis and megakaryopoiesis are stringently regulated by signaling pathways. However, the precise molecular mechanisms through which signaling pathways regulate key transcription factors controlling erythropoiesis and megakaryopoiesis remain partially understood. Herein, we identified heat shock cognate B (HSCB), which is well known for its iron-sulfur cluster delivery function, as an indispensable protein for friend of GATA 1 (FOG1) nuclear translocation during erythropoiesis of K562 human erythroleukemia cells and cord-blood-derived human CD34+CD90+hematopoietic stem cells (HSCs), as well as during megakaryopoiesis of the CD34+CD90+HSCs. Mechanistically, HSCB could be phosphorylated by phosphoinositol-3-kinase (PI3K) to bind with and mediate the proteasomal degradation of transforming acidic coiled-coil containing protein 3 (TACC3), which otherwise detained FOG1 in the cytoplasm, thereby facilitating FOG1 nuclear translocation. Given that PI3K is activated during both erythropoiesis and megakaryopoiesis, and that FOG1 is a key transcription factor for these processes, our findings elucidate an important, previously unrecognized iron-sulfur cluster delivery independent function of HSCB in erythropoiesis and megakaryopoiesis.

Strategies for the Construction of Multicyclic Phage Display Libraries.

Peptide therapeutics have gained great interest due to their multiple advantages over small molecule and antibody-based drugs. Peptide drugs are easier to synthesize, have the potential for oral bioavailability, and are large enough to target protein-protein interactions that are undruggable by small molecules. However, two major limitations have made it difficult to develop novel peptide therapeutics not derived from natural products, including the metabolic instability of peptides and the difficulty of reaching antibody-like potencies and specificities. Compared to linear and disulfide-monocyclized peptides, multicyclic peptides can provide increased conformational rigidity, enhanced metabolic stability, and higher potency in inhibiting protein-protein interactions. The identification of novel multicyclic peptide binders can be difficult, however, recent advancements in the construction of multicyclic phage libraries have greatly advanced the process of identifying novel multicyclic peptide binders for therapeutically relevant protein targets. This review will describe the current approaches used to create multicyclic peptide libraries, highlighting the novel chemistries developed and the proof-of-concept work done on validating these libraries against different protein targets.

The Optimized Design of Soil-Touching Parts of a Greenhouse Humanoid Weeding Shovel Based on Strain Sensing and DEM-ADAMS Coupling Simulation.

To overcome the shortcomings of plowing and rotary tillage, a human-like weeding shoveling machine was designed. The machine's various moving rods were analyzed using Matlab R2019b(9.7.0.1190202) software to determine the appropriate entry and cutting conditions, as well as non-cutting conditions. It was concluded that a θ2 of 90° was optimal for cutting the soil and that the shoveling depth was suitable for greenhouse weeding. The Adams and DEM coupled discrete element simulation system was developed for this machine and was used to analyze the rotating shaft torque and shovel bending moment. A strain measurement system based on strain gauges was designed to measure the rotating shaft torque and shovel bar bending moment. A bending moment and torque measurement system was designed to perform field measurement tests for comparison with simulation results. The simulation system's rotating shaft had an average torque error of 6.26%, while the shovel rod's bending moment had an average error of 5.43%. The simulation accuracy was within the acceptable error range. Table U8 (81 × 44) of the Uniform Design of the Mixing Factor Level for the Homogeneous Virtual Simulation Test includes eight levels of forward machine speed ranging from 0.1 to 0.45 m/s and four levels of output shaft speed ranging from 90 to 165 r/min. Crank lengths were set at four levels ranging from 155 to 185 mm, while shovel lengths were set at four levels ranging from 185 to 230 mm. Four types of shovel shapes were proposed, including pointed curved shovels, pointed straight shovels, straight-edged curved shovels, and straight-edged straight shovels. A mathematical model was created via a regression analysis of the results of coupled simulation tests to establish the relationship between shaft torque and shovel rod bending moment, tool advance speed, shaft speed, crank length, tool length, and tool shape. The model was used to determine the optimum working parameters.

Rapid Grapevine Health Diagnosis Based on Digital Imaging and Deep Learning.

Deep learning plays a vital role in precise grapevine disease detection, yet practical applications for farmer assistance are scarce despite promising results. The objective of this research is to develop an intelligent approach, supported by user-friendly, open-source software named AI GrapeCare (Version 1, created by Osama Elsherbiny). This approach utilizes RGB imagery and hybrid deep networks for the detection and prevention of grapevine diseases. Exploring the optimal deep learning architecture involved combining convolutional neural networks (CNNs), long short-term memory (LSTM), deep neural networks (DNNs), and transfer learning networks (including VGG16, VGG19, ResNet50, and ResNet101V2). A gray level co-occurrence matrix (GLCM) was employed to measure the textural characteristics. The plant disease detection platform (PDD) created a dataset of real-life grape leaf images from vineyards to improve plant disease identification. A data augmentation technique was applied to address the issue of limited images. Subsequently, the augmented dataset was used to train the models and enhance their capability to accurately identify and classify plant diseases in real-world scenarios. The analyzed outcomes indicated that the combined CNNRGB-LSTMGLCM deep network, based on the VGG16 pretrained network and data augmentation, outperformed the separate deep network and nonaugmented version features. Its validation accuracy, classification precision, recall, and F-measure are all 96.6%, with a 93.4% intersection over union and a loss of 0.123. Furthermore, the software developed through the proposed approach holds great promise as a rapid tool for diagnosing grapevine diseases in less than one minute. The framework of the study shows potential for future expansion to include various types of trees. This capability can assist farmers in early detection of tree diseases, enabling them to implement preventive measures.

Chlamydia psittaci causing severe pneumonia with an initial complaint of massive watery sputum: a case report.

Background: With the widespread application of metagenomic next-generation sequencing (mNGS) in pathogen detection, the reports of severe Chlamydia psittaci (C. psittaci) pneumonia are increasing. It is essential to determine the best management of severe C. psittaci pneumonia. Case Description: This report describes a 51-year-old male patient who presented with symptoms of expectoration, relative bradycardia, and dyspnea. Lung computed tomography (CT) on day 1 (D1) showed consolidation of the left lower lobe. He was intubated and transferred to the intensive care unit (ICU). The symptoms of high fever and progressive dyspnea [the lowest level of arterial partial pressure of oxygen/fractional inspired oxygen (PaO2/FiO2): 52 mmHg] persisted on D3. Meanwhile, he produced a large volume of golden-yellow, watery sputum, due to which endotracheal suction was repeatedly performed to maintain patency of the airway. The repeat radiography showed extensive deterioration of diffuse exudation in bilateral lobes. An early treatment with methylprednisolone was initiated on D3, after which the watery sputum decreased and turned viscous. The mNGS of the bronchoalveolar lavage fluid (BALF) identified C. psittaci on D7, and combined targeted antimicrobial therapy (azithromycin and doxycycline) was subsequently initiated. After 1 week of treatment, the patient was extubated on D14. He was transferred to the respiratory department on D17 and discharged on D25 with oral medications (azithromycin and doxycycline for 2 weeks). The repeat chest CT on D68 showed that the bilateral exudation and left lower lobe consolidation had almost disappeared, without pleural effusion. Conclusions: In severe C. psittaci pneumonia, although the presentations differ, the rapid pathogen identification through BALF mNGS may facilitate the early use of effective antibiotics. Timely and comprehensive treatment is important for improving outcomes in severe C. psittaci pneumonia.

Functional study of a lytic polysaccharide monooxygenase MsLPMO3 from Morchella sextelata in the oxidative degradation of cellulose.

Lytic polysaccharide monooxygenases (LPMOs) can improve the effectiveness with which agricultural waste is utilized. This study described the potent AA9 family protein MsLPMO3, derived from Morchella sextelata. It exhibited strong binding to phosphoric acid swollen cellulose (PASC), and had the considerable binding ability to Cu2+ with a Kd value of 2.70 µM by isothermal titration calorimetry (ITC). MsLPMO3 could also act on PASC at the C1 carbon via MALDI-TOF-MS results. Moreover, MsLPMO3 could boost the hydrolysis efficiency of corncob and wheat bran in combination with glycoside hydrolases. MsLPMO3 also exhibited strong oxidizing ability for 2,6-dimethoxyphenol (2,6-DMP), achieving the best Vmax value of 443.36 U·g-1 for pH 7.4 with a H2O2 concentration of 300 µM. The structure of MsLPMO3 was obtained using AlphaFold2, and the molecular docking results elucidated the specific interactions and key residues involved in the recognition process between MsLPMO3 and cellulose. Altogether, this study expands the knowledge of AA9 family proteins in cellulose degradation, providing valuable insights into the mechanisms of synergistic degradation of lignocellulose with cellulases.

Nanoarchitectonics on residual carbon from gasification fine slag upon two step low temperature activation for application in supercapacitors.

In this paper, the carbon electrode materials were prepared by the KOH-HNO3 low-temperature activation technique using cheap residual carbon from gasification fine slag (CK) as raw materials. The results showed that the prepared material (CKN-2) which obtained by dry-wet sequential activation at 500°C for 1.5 h at carbon to KOH ratio of 1:2 and further at 80°C for 1 h in 2 mol/L HNO3 solution. The specific capacitance of CKN-2 reached 142 F/g at a current density of 0.5 A/g. CKN-2 was used to assemble a symmetrical (CKN-2//CKN-2) supercapacitor, which exhibited an energy density of 6.80 Wh/kg at a power density of 244.8 W/kg. The CKN-2//CKN-2 capacitor was tested for stability after 10,000 cycles, with a capacitance retention rate of 97%. These results demonstrate that residual carbon from gasification fine slag can be effectively used to produce high-performance carbon electrode materials for supercapacitors using the KOH-HNO3 low-temperature sequential co-activation technique.

Phage Display of Two Distinct Warheads to Inhibit Challenging Proteins.

Falling in between traditional small molecules and antibodies in size, peptides are emerging as a privileged therapeutic modality, one that can harness the benefits of both small molecule and antibody drugs. To discover potential peptide therapeutics, it is highly desirable to have high throughput screening platforms that can assess peptides with diverse and non-natural functional motifs. With this contribution, we present a novel phage library that incorporates two distinct designer groups. As an example, a pair of reversible covalent warheads was installed onto phage-displayed peptides to target a cysteine and a lysine. The double modification is realized by sequential modification of an N-terminal cysteine and then an internal cysteine using chemoselective chemistry. Screening of this double-warhead-presenting library against TEV protease readily revealed peptide inhibitors with single-digit micromolar potency. Importantly, our structure-activity studies demonstrate that both covalent warheads make important contributions to TEV protease inhibition. We envision that our strategy of double phage modification can be readily extended to build phage libraries with diverse structural motifs, allowing facile expansion of the chemical space coverable by phage display.

Fluorosulfate as a Latent Sulfate in Peptides and Proteins.

Sulfation widely exists in the eukaryotic proteome. However, understanding the biological functions of sulfation in peptides and proteins has been hampered by the lack of methods to control its spatial or temporal distribution in the proteome. Herein, we report that fluorosulfate can serve as a latent precursor of sulfate in peptides and proteins, which can be efficiently converted to sulfate by hydroxamic acid reagents under physiologically relevant conditions. Photocaging the hydroxamic acid reagents further allowed for the light-controlled activation of functional sulfopeptides. This work provides a valuable tool for probing the functional roles of sulfation in peptides and proteins.

Optimized Design of Touching Parts of Soil Disinfection Machine Based on Strain Sensing and Discrete Element Simulation.

With the increasing level in the intensification of agricultural production in China, continuous cropping obstacles have become a problem that needs to be solved. The use of vertical rotary tillage technology and soil disinfection technology is an effective solution. In this paper, a vertical rotary soil-tilling variable disinfection combine was developed and an on-board control system with STM32 as the control core was designed to realize the real-time acquisition of powder monopoly torque information and the variable application of soil disinfection chemicals. Based on the obtained experimental soil parameters, a discrete element soil particle model was established, and orthogonal experiments were conducted to analyze the single-blade roller tillage process, and the optimal operating parameters were finally selected as 500 mm powder monopoly depth, 320 r/min knife roller speed, and 0.26 m/s forward speed, respectively. The field experiment found that the average tillage depth of the implement was 489 mm, the stability coefficient of tillage depth was 94.50%, the uniformity coefficient of soil disinfection was 85.57%, and the applied amount and the speed ratio coefficient of the given flow were linearly related, respectively. This research provides a technical reference for the deep tillage and soil disinfection of the powder monopoly.

A Cysteine-Directed Proximity-Driven Crosslinking Method for Native Peptide Bicyclization.

Efficient and site-specific modification of native peptides and proteins is desirable for synthesizing antibody-drug conjugates as well as for constructing chemically modified peptide libraries using genetically encoded platforms such as phage display. In particular, there is much interest in efficient multicyclization of native peptides due to the appeals of multicyclic peptides as therapeutics. However, conventional approaches for multicyclic peptide synthesis require orthogonal protecting groups or non-proteinogenic clickable handles. Herein, we report a cysteine-directed proximity-driven strategy for the constructing bicyclic peptides from simple natural peptide precursors. This linear to bicycle transformation initiates with rapid cysteine labeling, which then triggers proximity-driven amine-selective cyclization. This bicyclization proceeds rapidly under physiologic conditions, yielding bicyclic peptides with a Cys-Lys-Cys, Lys-Cys-Lys or N-terminus-Cys-Cys stapling pattern. We demonstrate the utility and power of this strategy by constructing bicyclic peptides fused to proteins as well as to the M13 phage, paving the way to phage display of novel bicyclic peptide libraries.

Diazaborine-Mediated Bicyclization of Native Peptides with Inducible Reversibility.

Multicyclic peptides are appealing candidates for peptide-based drug discovery. While various methods are developed for peptide cyclization, few allow multicyclization of native peptides. Herein we report a novel cross-linker DCA-RMR1, which elicits facile bicyclization of native peptides via N-terminus Cys-Cys cross-linking. The bicyclization is fast, affords quantitative conversion, and tolerates various side chain functionalities. Importantly, the resulting diazaborine linkage, while stable at a neutral pH, can readily reverse upon mild acidification to give pH-responsive peptides.

Study on the Discoloration Mechanism of Eucalyptus Wood during Thermal Treatment in Different Media.

Chromophore structures in wood are the core elements for regulating wood color. Thermal treatment can regulate the color of wood, thus increasing its added value. In this study, conventional thermal treatment was used to regulate the color of Eucalyptus, in order to make its color close to the precious wood species Burma padauk. The color change in Eucalyptus wood was analyzed using the chromaticity index and UV-Vis. The chromophore structures in the treated wood and their discoloration mechanisms were characterized via FTIR, XPS, NMR, etc. The results showed that the color of eucalyptus could be regulated via thermal treatment to become more similar to the color of Burma padauk under both saturated steam and hot air. The treated wood showed a color difference in the 400~500 nm region in spectral absorption. The changes in the chromophore structures of wood were accompanied by the degradation of hemicelluloses. Meanwhile, demethoxylation occurred in the syringyl structure G of lignin, which led to the polymerization of lignin and decreased the lightness value of wood. Moreover, the number of conjugated structures in the chromophore groups increased, which caused the color of the wood to tend toward red. This study provides a reference for the color regulation of wood, and the mechanisms are also discussed.

Research on NO generation characteristics of ammonia-premixed flame.

Ammonia is a promising fuel with high energy density, accessible storage, and no CO2 production by combustion, but its combustion produces the pollutant NO. In this study, a Bunsen burner experimental bench was selected to investigate the concentration of NO generated by ammonia combustion at different initial oxygen concentrations. Further, the reaction pathways of NO were analyzed in depth, and sensitivity analysis was performed. The results show that the Konnov mechanism has an excellent predictive effect on NO generated by ammonia combustion. In the ammonia-premixed laminar flame at atmospheric pressure, the NO concentration peaked at an equivalence ratio of 0.9. The high initial oxygen concentration enhanced the combustion of ammonia-premixed flame and increased the conversion of NH3 to NO. NO was not only a product but a contribution to the combustion of NH3. As the equivalence ratio increases, NH2 consumes a large amount of NO and reduces NO production. The high initial oxygen concentration enhanced NO production, and the effect was more pronounced at low equivalents. The study results provide theoretical guidance for the utilization of ammonia combustion and pollutant reduction and help to drive the process of ammonia combustion toward practicality.

N-Terminal cysteine mediated backbone-side chain cyclization for chemically enhanced phage display.

Phage display, an ingenious invention for evaluating peptide libraries, has been limited to natural peptides that are ribosomally assembled with proteinogenic amino acids. Recently, there has been growing interest in chemically modifying phage libraries to create nonnatural cyclic and multicyclic peptides, which are appealing for use as inhibitors of protein-protein interactions. While earlier reports largely focused on side-chain side-chain cyclization, we report herein a novel strategy for creating backbone-side chain cyclized peptide libraries on phage. Our strategy capitalizes on the unique reactivity of an N-terminal cysteine (NCys) with 2-cyanobenzothiazole (CBT) which, in conjugation with another thiol-reactive group, can elicit rapid cyclization between an NCys and an internal cysteine. The resulting library was screened against two model proteins, namely Keap1 and Sortase A. The screening readily revealed potent inhibitors for both proteins with certain Keap1 ligands reaching low nanomolar potency. The backbone-side chain cyclization strategy described herein presents a significant addition to the toolkit of creating nonnatural macrocyclic peptide libraries for phage display.

Fast Cysteine Bioconjugation Chemistry.

Cysteine bioconjugation serves as a powerful tool in biological research and has been widely used for chemical modification of proteins, constructing antibody-drug conjugates, and enabling cell imaging studies. Cysteine conjugation reactions with fast kinetics and exquisite selectivity have been under heavy pursuit as they would allow clean protein modification with just stoichiometric amounts of reagents, which minimizes side reactions, simplifies purification and broadens functional group tolerance. In this concept, we summarize the recent advances in fast cysteine bioconjugation, and discuss the mechanism and chemical principles that underlie the high efficiencies of the newly developed cysteine reactive reagents.

Lysine-Targeted Reversible Covalent Ligand Discovery for Proteins via Phage Display.

Binding via reversible covalent bond formation presents a novel and powerful mechanism to enhance the potency of synthetic inhibitors for therapeutically important proteins. Work on this front has yielded the anticancer drug bortezomib as well as the antisickling drug voxelotor. However, the rational design of reversible covalent inhibitors remains difficult even when noncovalent inhibitors are available as a scaffold. Herein, we report chemically modified phage libraries, both linear and cyclic, that incorporate 2-acetylphenylboronic acid (APBA) as a warhead to bind lysines via reversible iminoboronate formation. To demonstrate their utility, these APBA-presenting phage libraries were screened against sortase A of Staphylococcus aureus, as well as the spike protein of SARS-CoV-2. For both protein targets, peptide ligands were readily identified with single-digit micromolar potency and excellent specificity, enabling live-cell sortase inhibition and highly sensitive spike protein detection, respectively. Furthermore, our structure-activity studies unambiguously demonstrate the benefit of the APBA warhead for protein binding. Overall, this contribution shows for the first time that reversible covalent inhibitors can be developed via phage display for a protein of interest. The phage display platform should be widely applicable to proteins including those involved in protein-protein interactions.

A genome-wide atlas of antibiotic susceptibility targets and pathways to tolerance.

Detailed knowledge on how bacteria evade antibiotics and eventually develop resistance could open avenues for novel therapeutics and diagnostics. It is thereby key to develop a comprehensive genome-wide understanding of how bacteria process antibiotic stress, and how modulation of the involved processes affects their ability to overcome said stress. Here we undertake a comprehensive genetic analysis of how the human pathogen Streptococcus pneumoniae responds to 20 antibiotics. We build a genome-wide atlas of drug susceptibility determinants and generated a genetic interaction network that connects cellular processes and genes of unknown function, which we show can be used as therapeutic targets. Pathway analysis reveals a genome-wide atlas of cellular processes that can make a bacterium less susceptible, and often tolerant, in an antibiotic specific manner. Importantly, modulation of these processes confers fitness benefits during active infections under antibiotic selection. Moreover, screening of sequenced clinical isolates demonstrates that mutations in genes that decrease antibiotic sensitivity and increase tolerance readily evolve and are frequently associated with resistant strains, indicating such mutations could be harbingers for the emergence of antibiotic resistance.

Is Travel Time Associated with Health Service Utilization in Northwest China? Evidence from Shaanxi Province.

Purpose: Northwest China has a large area, low population density, and few health resources, which makes the utilization of health resources in this region difficult. The objective of this study was to assess utilization of health services and its association with travel time in Shaanxi Province. Patients and Methods: Data were obtained from the fifth Household Health Service Survey of Shaanxi Province conducted in 2013. Binary logistic regression was used to assess the relationship between travel time and health service utilization, and negative binomial regression was conducted to assess the relationship between travel time and the frequency of health service utilization. Results: A total of 42.6% of patients used health services, with a higher use rate among rural residents than among urban residents (47.0% and 27.4%, respectively). A total of 30.9% of patients traveled more than 15 min to the nearest medical facility (33.3% in rural areas and 22.6% in urban areas). A total of 12.4% of patients traveled more than 30 min to the nearest medical facility (15.1% in rural areas and 3.0% in urban areas). Urban residents living farthest from health care facilities (more than 30 min) had a 2.12-fold higher probability of health service utilization and expected to have a health service utilization rate 1.77 times greater than that of residents with a travel time of less than 5 min. Among the rural population, there was no significant correlation between travel time and health service utilization. Conclusion: Urban patients living farthest from hospitals were more likely to use health services and used health services more frequently. This suggests that more attention should be given to urban patients who live far away from health service providers in Shaanxi Province.