A new fusion strategy for rapid strain differentiation based on MALDI-TOF MS and Raman spectra.

Typing of bacterial subspecies is urgently needed for the diagnosis and efficient treatment during disease outbreaks. Physicochemical spectroscopy can provide a rapid analysis but its identification accuracy is still far from satisfactory. Herein, a novel feature-extractor-based fusion-assisted machine learning strategy has been developed for high accuracy and rapid strain differentiation using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and Raman spectroscopy. Based on this fusion approach, rapid and reliable identification and analysis can be performed within 24 hours. Validation on a panel of important pathogens comprising Staphylococcus aureus, Klebsiella pneumoniae, Escherichia coli, and Acinetobacter baumannii showed that the identification accuracies of k-nearest neighbors (KNNs), support vector machines (SVMs) and artificial neural networks (ANNs) were 100%. In particular, when benchmarked against a MALDI-TOF MS spectral dataset, the new approach improved the identification accuracy of Acinetobacter baumannii from 87.67% to 100%. This work demonstrates the effectiveness of combining MALDI-TOF MS and Raman spectroscopy fusion data in pathogenic bacterial subtyping.

Preadsorbed Chymotrypsin Modulated the Composition of Protein Corona and Immunological Response.

It is well-known that proteins after administration into biological environments adsorb on the surface of nanoparticles (NPs). The biological identity could be determined by protein corona, but whether and how the preadsorbed molecules impact the composition of the corona and immunological response have rarely been reported. Here, the effects of preadsorbed chymotrypsin (Chy) on forming protein corona and subsequent immunological response are reported. We find that preadsorbed Chy on the surface of AuNPs results in a protein corona with enriched immunoglobulins and reduced human serum albumin protein, which further affect the polarization of macrophages into specific phenotypes. Our study suggests that the protein surrounding the nanoparticles could affect the protein corona and immunological response, which may direct the preparation of multifunctional nanomedicine for future studies.

Protocol for bacterial typing using Fourier transform infrared spectroscopy.

The Fourier transform infrared (FT-IR) signals obtained from bacterial samples are specific and reproducible, making FT-IR an efficient tool for bacterial typing at a subspecies level. However, the typing accuracy could be affected by many factors, including sample preparation and spectral acquisition. Here, we present a unified protocol for bacterial typing based on FT-IR spectroscopy. We describe sample preparation from bacterial culture and FT-IR spectrum collection. We then detail FT-IR spectrum preprocessing and multivariate analysis of spectral data for bacterial typing.

Ultrathin Two-Dimensional BiOCl-Bi2 S3 -Cu2 S Ternary Heterostructures with Enhanced LSPR effect for NIR Photonic Bacterial Disinfection.

Photonic disinfection, particularly near-infrared (NIR) light triggered antibacterial, has emerged as a highly promising solution for combating pathogenic microbes due to its spatiotemporal operability, safety, and low cost of apparatus. However, it remains challenging to construct NIR-responsive antibacterial agents with high light-converting efficacy and elucidate synergistic mechanisms. In this work, ultrathin two-dimensional (2D) BiOCl-Bi2 S3 -Cu2 S ternary heterostructures that can efficiently kill drug-resistant bacteria were synthesized by doping 0D Bi2 S3 and Cu2 S nanoparticles in the 2D BiOCl nanosheets via a facile one-pot hydrothermal method. Notably, the incorporation of Cu2 S nanoparticles bestows strong NIR light-harvesting capability to the composite nanosheets due to their localized surface plasmon resonance (LSPR). Upon NIR light illumination, the BiOCl-Bi2 S3 -Cu2 S nanosheets can achieve enhanced photonic hyperthermia and reaction oxygen species (ROS) generation, serving as single light-activated bi-functional photothermal/photodynamic therapeutics. High-speed hot electrons and large local electronic fields caused by LSPR might play an important role in thermal vibrations and effective carrier separations, respectively. Benefiting from the unique ternary heterostructures, both the photothermal conversion and ROS generation efficacy of BiOCl-Bi2 S3 -Cu2 S nanosheets are significantly improved compared to the binary BiOCl-Cu2 S or BiOCl-Bi2 S3 nanosheets. Accordingly, the ternary composite nanosheets can effectively kill bacteria via the NIR-driven photonic disinfection mechanism. This work presents a new type of 2D composite nanosheets with ternary heterostructures for NIR photonic disinfection.

Heterojunction structured BiOCl-Bi2S3 nanosheets as mitochondria-targeted near-infrared photothermal and photodynamic therapy agent.

Mitochondria-targeted phototherapy, especially combined photothermal therapy (PTT) and photodynamic therapy (PDT), has been regarded as an attractive strategy for the treatment of tumor. In this study, a facile approach to prepare two-dimensional (2D) BiOCl-Bi2S3 nanostructures was developed, where Bi2S3 quantum dots were doped in/on the ultrathin BiOCl nanosheets, forming a p-n heterojunction. The BiOCl-Bi2S3 shows favorable photothermal conversion efficiency (32%) and synergistically reactive oxygen species (ROS) generating capability under near-infrared (NIR) irradiation. Moreover, the conjugation of synthetic targeting ligand to the surface of BiOCl-Bi2S3 endows the heterojunction effective tumor targeting ability and selective mitochondrial accumulation. The combined cancer targeting ability and synergistic PTT/PDT permit enhanced cooperative phototherapeutic efficiency of the 2D heterojunction. This study provides an attractive way for designing new class of heterostructure materials for potential applications in subcellular-targeted phototherapy.

Efficient classification of Escherichia coli and Shigella using FT-IR spectroscopy and multivariate analysis.

Accurate and effective discrimination of E. coli and Shigella is an important clinical issue, and there are many limitations in traditional methods of analysis. FT-IR shows great potential in the classification of bacteria with high specificity and low cost. In this study, we evaluated the efficiency of this technique when combined with multivariate analysis for rapid classification of E. coli and Shigella, which is difficult using traditional analytical methods. Machine learning and statistical tools were employed in combination with FT-IR to classify 14 E. coli and 9 Shigella strains. The classification accuracies for select E. coli and Shigella strains from blood agar were 0.7826, 0.8696, and 0.9565 at the genus, species, and strain levels, respectively. In addition, we used the FT-IR data of select strains from three different culture media for cross-validation, yielding an accuracy of 0.3681 at the strain level. These results indicate that the bacterial culture conditions have a significant impact on the FT-IR patterns. Based on this, an improved strategy for training an ensemble classifier model considering bacterial culture factors was constructed, resulting in almost perfect separation with an accuracy of 0.9394 for strain-level classification. These results show the potential of FT-IR combined with multivariate analysis for more reliable bacterial classification.

Progress in infrared spectroscopy as an efficient tool for predicting protein secondary structure.

Infrared (IR) spectroscopy is a highly sensitive technique that provides complete information on chemical compositions. The IR spectra of proteins or peptides give rise to nine characteristic IR absorption bands. The amide I bands are the most prominent and sensitive vibrational bands and widely used to predict protein secondary structures. The interference of H2O absorbance is the greatest challenge for IR protein secondary structure prediction. Much effort has been made to reduce/eliminate the interference of H2O, simplify operation steps, and increase prediction accuracy. Progress in sampling and equipment has rendered the Fourier transform infrared (FTIR) technique suitable for determining the protein secondary structure in broader concentration ranges, greatly simplifying the operating steps. This review highlights the recent progress in sample preparation, data analysis, and equipment development of FTIR in A/T mode, with a focus on recent applications of FTIR spectroscopy in the prediction of protein secondary structure. This review also provides a brief introduction of the progress in ATR-FTIR for predicting protein secondary structure and discusses some combined IR methods, such as AFM-based IR spectroscopy, that are used to analyze protein structural dynamics and protein aggregation.

Discovery of 1-Amino-1H-imidazole-5-carboxamide Derivatives as Highly Selective, Covalent Bruton's Tyrosine Kinase (BTK) Inhibitors.

Bruton's tyrosine kinase (BTK) inhibitors suppressing the aberrant activation of BTK have led to a paradigm shift in the therapy of B-cell malignancies. However, there is an urgent need to discover more selective covalent BTK inhibitors owing to the off-target adverse effects of the approved inhibitor, ibrutinib. Herein, we disclose the discovery and preliminary activity studies of novel BTK inhibitors carrying 1-amino-1H-imidazole-5-carboxamide as a hinge binder. The most potent BTK inhibitor 26 demonstrates impressive selectivity, favorable pharmacokinetic properties, and robust antitumor efficacy in vivo, which indicates its potential as a novel therapeutic option for B-cell lymphomas. Importantly, to the best of our knowledge, this is the first example of a 1-amino-1H-imidazole-5-carboxamide scaffold used as the hinge binder of kinase inhibitors, which will largely expand the chemical diversity of kinase inhibitors.

Evolution of the protein corona affects macrophage polarization.

When nanoparticles (NPs) come into contact with bioenvironments, a protein corona forms on the NP surface. Previous reports showed that the constituents of the corona change with time. However, how different protein corona compositions influence cells, especially immune cells, has received less attention. Macrophages are important immune cells that can be polarized into a pro-inflammatory (M1) or anti-inflammatory (M2) phenotype. In this study, AuNPs were incubated with human plasma for different periods to obtain time-related AuNP-coronas, and the influences of time-related AuNP-coronas on macrophage polarization were investigated. The macrophage morphology, biomarkers, cytokine secretion studies show that the pristine AuNPs and 4 h-AuNP-corona induced macrophage cells into M2 phenotype, while the co-incubation of 12 h-AuNP-corona and macrophage cells result in M1 phenotype. Further proteomic analysis showed that the compositions of protein corona were changing constantly after AuNPs contacted with plasma. When the incubation time increased to 12 h, the immune proteins in protein corona were increased significantly, which play a key role in modulation of the different macrophages polarization. Our findings demonstrated that plasma incubation time is an important parameter that needs to be taken into account in the study of nano-immune interactions and safe use of NPs in biological systems. Moreover, our finding can be a new efficient strategy for activating inflammatory or anti-inflammatory in medical treatment.

Structure-activity relationship investigation for imidazopyrazole-3-carboxamide derivatives as novel selective inhibitors of Bruton's tyrosine kinase.

BTK (Bruton's tyrosine kinase) inhibitors are the most promising drugs for the treatment of hematological tumors. A high selectivity of BTK inhibitors ensures reduced side effects from off-targeting. Accordingly, here, based on Zanubrutinib, we designed and synthesized a new range of imidazopyrazole-3-carboxamide derivatives as novel BTK inhibitors that retained the amide group for improved selectivity. These compounds revealed potent inhibitory activity against BTK in vitro. Remarkably, compounds 12a (IC50 5.2 nM) and 18a (IC50 4.9 nM) possessed the highest kinase selectivity. Both of these effectively inhibited the auto-phosphorylation of BTK, blocked the cell cycle in G0/G1 phase, and induced apoptosis in the TMD8 cells. In a TMD8 cells xenograft model, a twice-daily dose of compound 12a at 25 mg/kg and a thrice-daily dose of compound 18a at 15 mg/kg significantly suppressed the tumor growth without obvious toxicity. Collectively, 12a and 18a are the potential selective BTK inhibitors that can be developed further.

CaCO3-Encapsulated Au Nanoparticles Modulate Macrophages toward M1-like Phenotype.

Macrophage cells are plastic and can be polarized into opposing phenotypes, pro-inflammatory (M1-like cells) or anti-inflammatory (M2-like cells). Reprograming of M2-like cells into M1 phenotype will contribute significantly to combatting cancer. Gold nanoparticles (AuNPs) are intensively studied in various fields for their distinctive photo-chemical properties. However, the immune response of AuNPs is still unclear. In this study, AuNPs and CaCO3-encapsulated Au nanoparticles (Au@CaCO3 NPs) were synthesized as stimuli for macrophage modulation. Co-incubation of AuNPs and macrophages leads to a dramatically elongated macrophage cell morphology. Moreover, increased expression of M2 biomarker and M2-inducing cytokines suggests that AuNPs induce macrophage polarization toward M2 phenotype. More interestingly, the co-incubation of Au@CaCO3 NPs and macrophage cells resulted in a round cellular morphology and induced the secretion of M1 biomarker and inflammatory cytokines. Our studies demonstrate that the strategy of CaCO3-encapsulated Au nanoparticles can be used in modulating the polarization of M1 macrophages. Our strategy provides an efficient method for activating inflammation in macrophages, which will be useful for the application of nanoparticles in cancer therapy.

Labeled-protein corona-coated Bi2S3 nanorods targeted to lysosomes for bioimaging and efficient photothermal cancer therapy.

One of the main diseases contributing to human death are malignant tumors. Phototherapy is a promising approach for cancer therapy, and functional nanoparticles with targeting ligands are commonly used to improve the therapeutic efficiency. However, recent studies have shown that nanoparticles in contact with a biological fluid can rapidly form a "protein corona" on their surface, which will remarkably decrease the targeting ability. Here, we describe the preparation of hybrid nanomaterials with Bi2S3 nanorods as the core, and fluorescein-isothiocyanate and folic acid-modified human serum albumin (HSA-FITC-FA) as the shell. By using fluorescent binding label (FITC) and imaging techniques, we discovered the image of the cell lysosomes, indicating that the photothermal therapy agent was predominantly targeted to and accumulated in lysosomes. Combined with photothermal therapy agent (Bi2S3 nanorods) and targeting ligand (FA), the obtained product shows enhanced photothermal therapy under near-infrared region laser irradiation. Additionally, SDS-PAGE shows that the modified HSA shell could remarkably reduce the reabsorption of protein corona from blood serum, minimized the adverse effect of protein corona on targetability. Taken together, the results indicate that our strategy has the potential for preparing efficient photothermal nanomaterials with image-guided subcellular organelle-targeting cancer cell ablation ability.

Cross-Linked Polyamide Chains Enhanced the Fluorescence of Polymer Carbon Dots.

Carbon dots (CDs) have attracted tremendous attention for their outstanding advantages in luminescence. Here, α-amino-substituted lysine derivatives with the determined chemical structure were employed as precursors to obtain bright and highly stable fluorescent CDs through a facile hydrothermal route. The relationships among the chemical structure of precursors, CD fluorescence, and particle size were investigated. The results indicated that increased numbers of functional groups in precursors could promote the degree of cross-linking and lead to a smaller size, better fluorescent properties, and stronger stability of CDs. The C-CDs that were prepared from lysine derivatives with most functional groups showed excitation-dependent dual excitation and dual emission (DE2), high-stability luminescence, strong resistance to photobleaching, and high selectivity to Fe3+ and could be used as a sensitive probe for Fe3+ detection.

Detailed insight into the formation of protein corona: Conformational change, stability and aggregation.

In a physiological fluid (e.g., blood), nanomaterials will strongly interact with proteins to form "protein corona". The structure and aggregation of protein corona around the nanoparticles are of vital importance in the safe application of nanomaterials in living organisms. Here, we combined systematic methods, including transmission electron microscopy, scanning electron microscopy equipped with energy dispersive X-ray spectroscopy, Fourier transform infrared spectroscopy, hydrogen/deuterium exchange techniques and fluorescence quenching to explore the conformational change, stability and aggregation of protein corona bound on magnetic nanoparticles. For the first time we observed that the conformational change of protein corona could induce proteins to aggregate. We believe that these findings will deepen our understanding of the protein corona.

Conformational-transited protein corona regulated cell-membrane penetration and induced cytotoxicity of ultrasmall Au nanoparticles.

Nanoparticles (NP) in biological fluids almost invariably become coated with proteins to form protein coronas. It is the NP-protein corona rather than the bare nanoparticle that determines the nanoparticle's bio-behavior. Here, ultrasmall gold nanoparticles (AuNPs) coated by a human serum albumin (HSA) corona were studied by Fourier transform infrared spectroscopy, denature experiments, fluorescence quenching. Moreover, the intracellular fate of AuNPs and the AuNP-HSA corona has also been investigated. The results show that HSA corona undergo a conformational transition (partial ß-sheet changed to α-helicity) when they adsorb on AuNPs, which lead to an enhanced thermal stability. Importantly, we observed that the conformation-transited protein corona-AuNP complex could induce cell apoptosis. Meanwhile, for the first time, the conformation-transited HSA on the AuNPs surface are shown to disrupt living cell membranes. The results obtained here not only provide the detailed conformational behavior of HSA molecules on nanoparticles, but also reveal the structure-function relationship of protein corona, which is of utmost importance in the safe application of nanoscale objects in living organisms.

Obtaining information about protein secondary structures in aqueous solution using Fourier transform IR spectroscopy.

Fourier transform IR (FTIR) spectroscopy is a nondestructive technique for structural characterization of proteins and polypeptides. The IR spectral data of polymers are usually interpreted in terms of the vibrations of a structural repeat. The repeat units in proteins give rise to nine characteristic IR absorption bands (amides A, B and I-VII). Amide I bands (1,700-1,600 cm(-1)) are the most prominent and sensitive vibrational bands of the protein backbone, and they relate to protein secondary structural components. In this protocol, we have detailed the principles that underlie the determination of protein secondary structure by FTIR spectroscopy, as well as the basic steps involved in protein sample preparation, instrument operation, FTIR spectra collection and spectra analysis in order to estimate protein secondary-structural components in aqueous (both H2O and deuterium oxide (D2O)) solution using algorithms, such as second-derivative, deconvolution and curve fitting. Small amounts of high-purity (>95%) proteins at high concentrations (>3 mg ml(-1)) are needed in this protocol; typically, the procedure can be completed in 1-2 d.

Synthesis, antibacterial activity, and biological evaluation of formyl hydroxyamino derivatives as novel potent peptide deformylase inhibitors against drug-resistant bacteria.

Peptide deformylase (PDF) has been identified as a promising target for novel antibacterial agents. In this study, a series of novel formyl hydroxyamino derivatives were designed and synthesized as PDF inhibitors and their antibacterial activities were evaluated. Among the potent PDF inhibitors (1o, 1q, 1o', 1q', and 1x), in vivo studies showed that compound 1q possesses mild toxicity, a good pharmacokinetic profile and protective effects. The good in vivo efficacy and low toxicity suggest that this class of compounds has potential for development and use in future antibacterial drugs.