In vitro investigation of protein assembly by combined microscopy and infrared spectroscopy at the nanometer scale.

The nanoscale structure and dynamics of proteins on surfaces has been extensively studied using various imaging techniques, such as transmission electron microscopy and atomic force microscopy (AFM) in liquid environments. These powerful imaging techniques, however, can potentially damage or perturb delicate biological material and do not provide chemical information, which prevents a fundamental understanding of the dynamic processes underlying their evolution under physiological conditions. Here, we use a platform developed in our laboratory that enables acquisition of infrared (IR) spectroscopy and AFM images of biological material in physiological liquids with nanometer resolution in a cell closed by atomically thin graphene membranes transparent to IR photons. In this work, we studied the self-assembly process of S-layer proteins at the graphene-aqueous solution interface. The graphene acts also as the membrane separating the solution containing the proteins and Ca2+ ions from the AFM tip, thus eliminating sample damage and contamination effects. The formation of S-layer protein lattices and their structural evolution was monitored by AFM and by recording the amide I and II IR absorption bands, which reveal the noncovalent interaction between proteins and their response to the environment, including ionic strength and solvation. Our measurement platform opens unique opportunities to study biological material and soft materials in general.

Genome Mining from Agriculturally Relevant Fungi Led to a d-Glucose Esterified Polyketide with a Terpene-like Core Structure.

Comparison of biosynthetic gene clusters (BGCs) found in devastating plant pathogens and biocontrol fungi revealed an uncharacterized and conserved polyketide BGC. Genome mining identified the associated metabolite to be treconorin, which has a terpene-like, trans-fused 5,7-bicyclic core that is proposed to derive from a (4 + 3) cycloaddition. The core is esterified with d-glucose, which derives from the glycosidic cleavage of a trehalose ester precursor. This glycomodification strategy is different from the commonly observed glycosylation of natural products.

Investigation of Indigoidine Synthetase Reveals a Conserved Active-Site Base Residue of Nonribosomal Peptide Synthetase Oxidases.

Nonribosomal peptide synthetase (NRPS) oxidase (Ox) domains oxidize protein-bound intermediates to install crucial structural motifs in bioactive natural products. The mechanism of this domain remains elusive. Here, by studying indigoidine synthetase, a single-module NRPS involved in the biosynthesis of indigoidine and several other bacterial secondary metabolites, we demonstrate that its Ox domain utilizes an active-site base residue, tyrosine 665, to deprotonate a protein-bound l-glutaminyl residue. We further validate the generality of this active-site residue among NRPS Ox domains. These findings not only resolve the biosynthetic pathway mediated by indigoidine synthetase but enable mechanistic insight into NRPS Ox domains.

Residents' satisfaction with primary medical and health services in Western China.

BACKGROUND: Currently, China is in the process of medical and health care reform, and the establishment of primary medical and health services covering urban and rural residents is an important aspect of this process. Studying the satisfaction of residents of underdeveloped areas with their primary medical and health services and identifying the factors that can increase the satisfaction of different groups may improve patient compliance and ultimately improve health. Moreover, such research may provide a reference for the development of medical and health undertakings in similarly underdeveloped areas. METHODS: A face-to-face survey was conducted on a stratified random sample of 2200 residents in Gansu by using structured questionnaires. Demographic characteristics were collated, and questionnaires were factor-analysed and weighted using SPSS software to obtain scores for each factor, as well as total satisfaction scores. The characteristics of poorly satisfied populations were determined by a multiple linear regression analysis using SAS software. A cluster analysis was performed using SAS software for classification and a separate discussion of populations. RESULTS: The hypertension self-awareness rate (11.29%) of the sampled population was lower than the average hypertension prevalence (23.85%), as recorded in the 2014 Health Statistical Yearbook of the region. The disease knowledge awareness factor was the lowest factor (2.857), whereas the policy awareness factor was the highest factor (4.772). The overall satisfaction was moderate (3.898). The multivariate linear regression model was significant (p <0.05). The regression coefficients were -0.041 for minors; 0.065 for unemployed people; and 0.094 for people with an elementary school educational level, a value lower than that of other population groups. A cluster analysis was used to divide the respondents into five groups. The overall satisfaction was lowest in the second population group (rural, middle-aged)(Fz = 3.64) and was highest in the fourth population group(minors) (Fz = 4.13). Different population groups showed different satisfaction rates in F1 to F6. CONCLUSION: Hypertensive patients had low self-awareness, and residents had a poor grasp of disease and limited health knowledge. Their overall satisfaction was moderate. Residents expressed comparatively high satisfaction with the current policy. Minors, adults with low level of education, unemployed people and other vulnerable groups expressed low overall satisfaction. The degree of satisfaction varied greatly among the different groups. Targeted medical and health practices should be implemented for different groups; additionally, the public health practice should be strengthened.

Methane detection using scattering material as the gas cell.

A compact methane (CH4) detection system is presented and developed by using an alumina ceramic scattering material as its gas cell. Due to the material's high scattering performance, the optical path length of the gas cell at 1653.7 nm can reach 15.96 cm although its physical length along the light transmission direction is only 0.50 cm. The wavelength modulation spectroscopy technique is employed to enhance the detection sensitivity, and the second harmonic gas absorption signal with low noise is detected and processed. The long-term stability of the system is investigated by the Allan deviation analysis method. Detection limits of 4.5 and 2.6 ppm are achieved at averaging times of 20 s and 200 s, respectively. The dynamic gas exchange performance is also experimentally studied. The experimental results indicate that our system is a good choice for practical applications owing to its small volume, high sensitivity, and stability.

Fast two-dimensional fluorescence correlation spectroscopy technique for tea quality detection.

A fast two-dimensional fluorescence correlation spectroscopy technique based on light emitting diodes is developed, which uses light intensity and excitation wavelength as quickly changeable and easily controllable external perturbations. A compact and automatic system is set up to detect tea quality. A partial least square regression method is used to create predictive models for tea grades. Compared to the traditional fluorescence spectroscopy method, this convenient two-dimensional correlation spectroscopy technique is more accurate according to our experimental results and is promising for practical applications.

Gas-self-filter-based erbium-doped fiber loop laser for gas detection.

An erbium-doped fiber (EDF) loop laser, based on a gas-self-filter (GSF), is developed with single or multiple wavelength emission. The GSF is a type of Mach-Zehnder interferometer with a gas cell in one arm. By matching the destructive wavelength of the interferometer with the gas absorption line, the self-filtering function is achieved. A GSF-based multi-wavelength laser with a side-mode suppression ratio of ~50 dB is performed. As an example, C2H2 gas is detected using a single-wavelength GSF-based laser with correlation spectroscopy, and a good linearity of the measurement is obtained. The present laser has the potential advantage for multiple gas detection, e.g., being free of wavelength calibration.

Enhanced 3-D asynchronous correlation data preprocessing method for Raman spectroscopy of Chinese handmade paper.

We have developed a novel 3D asynchronous correlation method (3D-ACM) designed for the classification and identification of Chinese handmade paper samples using Raman spectra and machine learning. The 3D-ACM approach involves two rounds of tensor product and Hilbert transform operations. In the tensor product process, the outer product of the spectral data from different samples within the same category is computed, establishing inner connections among all samples within that category. The Hilbert transform introduces a 90-degree phase shift, resulting in a true three-dimensional spectral data structure. This expansion significantly increases the number of equivalent frequency points and samples within each category. This enhancement substantially boosts spectral resolution and reveals more hidden information within the spectral data. To maximize the potential of 3D-ACM, we employed six machine learning models: principal component analysis (PCA) with linear regression (LR), support vector machine (SVM) with LR, k-Nearest Neighbors (KNN), random forest (RF), and convolutional neural network (CNN). When applied to the 3D-ACM data preprocessing method, R-squared values of PLS-LR, KNN, RF and CNN supervised models, approached or equaled 1. This indicates exceptional performance comparable to unsupervised models like PCA. 3D-ACM stands as a versatile mathematical technique not confined to spectral data. It also eliminates the necessity for additional experimental setups or external control conditions, distinct from traditional two-dimensional correlation spectroscopy. Moreover, it preserves the original experimental data, setting it apart from conventional data preprocessing methods. This positions 3D-ACM as a promising tool for future material classification and identification in conjunction with machine learning.

Tensor product based 2-D correlation data preprocessing methods for Raman spectroscopy of Chinese handmade paper.

The paper introduces two new methods, namely the cross correlation method (CCM) and two-dimensional correlation method (TDCM), for preprocessing Raman spectroscopy data for analyzing Chinese handmade paper samples. CCM expands the spectral dimension from 1×N to 1×2N-1 by taking cross-correlation between two spectral data of the same category. TDCM includes two-dimensional synchronous correlation method (TDSCM) and two-dimensional asynchronous correlation method (TDACM), which expand the spectral dimension from 1×N to N×N by taking tensor products between two spectral data and between one spectral data and the Hilbert transformation of the other spectral data of the same category, respectively. The experimental data were preprocessed using baseline removal, CCM, TDSCM, and TDACM methods. Four machine learning models were employed to evaluate the effects of these methods: principal component analysis (PCA) combined with linear regression (LR), support vector machine (SVM) combined with LR, k-Nearest Neighbors (KNN), and random forest (RF). The results show that the R-squared values for the PCA model were nearly 1 for all types of data, indicating high accuracy. However, for SVM-LR, KNN, and RF models, the R-squared values were sorted in the order of raw data, baseline removal data, CCM, TDSCM, and TDACM preprocessed data. The R-squared values of KNN and RF machine learning models for TDACM preprocessed data were approaching 1, indicating that the accuracy of machine learning was significantly improved by nearly 100%. This has led to a remarkable improvement in the accuracy of supervised models such as KNN and RF, bringing them closer to the level of unsupervised models such as PCA.

Tuning a high performing multiplexed-CRISPRi Pseudomonas putida strain to further enhance indigoidine production.

In this study, a 14-gene edited Pseudomonas putida KT2440 strain for heterologous indigoidine production was examined using three distinct omic datasets. Transcriptomic data indicated that CRISPR/dCpf1-interference (CRISPRi) mediated multiplex repression caused global gene expression changes, implying potential undesirable changes in metabolic flux. 13C-metabolic flux analysis (13C-MFA) revealed that the core P. putida flux network after CRISPRi repression was conserved, with moderate reduction of TCA cycle and pyruvate shunt activity along with glyoxylate shunt activation during glucose catabolism. Metabolomic results identified a change in intracellular TCA metabolites and extracellular metabolite secretion profiles (sugars and succinate overflow) in the engineered strains. These omic analyses guided further strain engineering, with a random mutagenesis screen first identifying an optimal ribosome binding site (RBS) for Cpf1 that enabled stronger product-substrate pairing (1.6-fold increase). Then, deletion strains were constructed with excision of the PHA operon (ΔphaAZC-IID) resulting in a 2.2-fold increase in indigoidine titer over the optimized Cpf1-RBS construct at the end of the growth phase (∼6 h). The maximum indigoidine titer (at 72 h) in the ΔphaAZC-IID strain had a 1.5-fold and 1.8-fold increase compared to the optimized Cpf1-RBS construct and the original strain, respectively. Overall, this study demonstrated that integration of omic data types is essential for understanding responses to complex metabolic engineering designs and directly quantified the effect of such modifications on central metabolism.

Protective effect of polysaccharide from Sophora japonica L. flower buds against UVB radiation in a human keratinocyte cell line (HaCaT cells).

Natured botanical extract has attracted considerable attention recently in the field of skin anti-ultraviolet (UV) radiation. As a medicinal herb, Sophora japonica flower buds contained several components such as flavonoids, isoflavonoids, triterpenes, alkaloids and polysaccharides, which have multiple pharmacological properties except hemostatic agents which have been used in China and Korea for centuries. The purpose of our study was to investigate whether polysaccharide extracted from Sophora japonica L. flower buds (PS) was able to attenuate UVB-induced damage using a human keratinocyte cell line (HaCaT cells). HaCaT cells were pretreated with PS in a serum-free medium for 2 h and then irradiated with different doses of UVB rays. The results showed that the PS attenuated UVB-induced cytotoxicity which was verified by MTT method and morphology feature assay. UVB exposure (30-120 mJ/cm2) reduced HaCaT cells viability significantly following with the increased irradiation dose 24 h later, while pretreatment with PS (0.25-2.0 mg/mL) attenuated UVB-induced cytotoxicity significantly and increased cell viability in a dose-dependent manner except 30 mJ/cm2 group. The PS reduced the ROS generation, down-regulated the expression of phosphor-JNK and phosphor-p38 MAPK proteins significantly through MAPK pathway in UVB-irradiated HaCaT cells. It also decreased the apoptosis rate at low dose of UVB ray and protected the cells from apoptosis which had been identified by the down-regulated level of active-caspase3 in UVB-irradiated HaCaT cells. In conclusion, PS pretreatment protected HaCaT keratinocytes from UVB irradiation-induced skin injuries effectively, and the underlying mechanism may involve MAPK signaling pathway which contribute to apoptotic cell death. However, further studies especially whose using human systems are needed to determine efficacy of PS in vivo.

Interrogation technique for a fiber Bragg grating sensing array based on a Sagnac interferometer and an acousto-optic modulator.

We propose and experimentally demonstrate a novel real-time interrogation technique for a fiber Bragg grating (FBG) sensing system that is based on a frequency-shifted asymmetric Sagnac interferometer. FBG sensors are connected to the Sagnac loop by an optical coupler, and an acousto-optic modulator (AOM) is asymmetrically placed in the Sagnac loop. By linearly sweeping the driving frequency of the AOM, the environmental variation around each FBG sensor can be determined by measuring the spectrum of the interference signals of the two counterpropagating light beams reflected by the corresponding FBG. The system has the advantages of low cost and real-time sensing.