Application of NIR Spectroscopy Coupled with PLS Regression for Quantification of Total Polyphenol Contents from the Fruit and Aerial Parts of Citrullus colocynthis

Introduction

Citrullus colocynthis (L.) Schrad is extensively used to treat diabetes, obesity, fever, cancer, amenorrhea, jaundice, leukemia, rheumatism, and respiratory diseases. Chemical studies have indicated the presence of several cucurbitacins, flavones, and other polyphenols in this plant. These phytochemical constituents are responsible for the interesting antioxidant and other biological activities of C. colocynthis.

Objective

In the present study, for the first time, near infrared (NIR) spectroscopy coupled with partial least square (PLS) regression analysis was used to quantify the polyphenolic phytochemicals of C. colocynthis.

Methodology

The fruit and aerial parts of the C. colocynthis were extracted individually in methanol followed by fractionation in n‐hexane, chloroform, ethyl acetate, n‐butanol, and water. Near infrared (NIR) spectra were obtained in absorption mode in the wavelength range 700–2500 nm. The PLS regression model was then built from the obtained spectral data to quantify the total polyphenol contents in the selected plant samples.

Results

The PLS regression model obtained had a R² value of 99% with a 0.98 correlationship value and a good prediction with a root mean square error of prediction (RMSEP) value of 1.89% and correlation of 0.98. These results were further confirmed through UV–vis spectroscopy and it is found that the ethyl acetate fraction has the maximum value for polyphenol contents (101.7 mg/100 g; NIR, 100.4 mg/100 g; UV–vis).

Conclusions

The polyphenolic phytochemicals of the fruit and aerial parts of C. colocynthis have been quantified successfully by using multivariate analysis in a non‐destructive, economical, precise, and highly sensitive method, which uses very simple sample preparation. Copyright © 2017 John Wiley & Sons, Ltd.     

Biodegradation of Phenol Using Immobilized Nocardia hydrocarbonoxydans in a Pulsed Plate Bioreactor: Effect of Packed Stages,Cell Carrier Loading,and Cell Acclimatization on Startup and Steady-State Behavior

The effect of the number of stages and cell carrier loading on the steady-state and startup performance of a continuous pulsed plate bioreactor with glass beads as the cell carrier material for biodegradation of phenol in wastewater using immobilized Nocardia hydrocarbonoxydans has been studied. It was found that the performance of the pulsed plate bioreactor during startup and at steady state can be improved by an increase in cell carrier loading, number of stages, total plate stack height, and with a decrease in plate spacing. The startup time for the continuous bioreactor can be decreased by increasing the number of preacclimatization steps for the cells. The attainment of steady effluent phenol concentration can be considered as an indication of steady state of the continuous bioreactor, as when phenol concentration attained a steady value, biofilm thickness, and the attached biomass dry weight also attained a constant value.     

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.     

桥接组合式内固定系统结合Nice结与锁定钢板治疗锁骨中段粉碎性骨折疗效比较研究

摘要 目的：对比桥接组合式内固定系统（OBS）结合Nice结与锁定钢板治疗锁骨中段粉碎性骨折的疗效。方法：回顾性选取2021年6月至2022年1月间在我院接受治疗的锁骨中段粉碎性骨折患者（n=66）的临床资料。根据手术方式的不同将患者分为A组（锁定钢板治疗,32例）和B组（桥接组合式内固定系统结合Nice结,34例）,对比两组临床症状恢复情况、并发症发生率、视觉疼痛模拟评分（VAS）、Constant-Murley评分和肩关节活动度。结果：两组骨折愈合时间对比无差异（P>0.05）。B组术中出血量少于A组,手术时间短于A组（P<0.05）。两组术后3个月Constant-Murley评分升高,VAS评分下降（P<0.05）,B组术后3个月VAS评分低于A组,Constant-Murley评分高于A组（P<0.05）。两组术后3个月前屈、后伸、内旋、外旋的肩关节活动度增大（P<0.05）,且B组术后3个月前屈、后伸、内旋、外旋的肩关节活动度均大于A组（P<0.05）。两组并发症发生率组间比较无统计学差异（P>0.05）。结论：OBS与锁定钢板治疗锁骨中段粉碎性骨折相比,OBS结合Nice结治疗可减少术中出血量,缩短手术时间,促进骨折愈合,扩大肩关节活动度,改善肩关节功能,疗效更好。     

Antimicrobial Susceptibility Testing of Mycobacterium Tuberculosis Complex for First and Second Line Drugs by Broth Dilution in a Microtiter Plate Format

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium tuberculosis (Mtb). Antimicrobial susceptibility testing (AST) of Mtb has traditionally been performed by the agar method of proportion or by macrobroth testing on an instrument such as the BACTEC (Becton Dickinson, Sparks, MD), VersaTREK (TREK Diagnostics, Cleveland, OH) or BacT/ALERT (bioMérieux, Hazelwood, MO). The agar proportion method, while considered the “gold” standard of AST, is labor intensive and requires calculation of resistance by performing colony counts on drug-containing agar as compared to drug-free agar. If there is ≥1% growth on the drug-containing medium as compared to drug-free medium, the organism is considered resistant to that drug. The macrobroth methods require instrumentation and test break point ("critical") drug concentrations for the first line drugs (isoniazid, ethambutol, rifampin, and pyrazinamide). The method described here is commercially available in a 96 well microtiter plate format [MYCOTB (TREK Diagnostics)] and contains increasing concentrations of 12 antimicrobials used for treatment of tuberculosis including both first (isoniazid, rifampin, ethambutol) and second line drugs (amikacin, cycloserine, ethionamide, kanamycin, moxifloxacin, ofloxacin, para-aminosalicylic acid, rifabutin, and streptomycin). Pyrazinamide, a first line drug, is not included in the microtiter plate due to its need for acidic test conditions. Advantages of the microtiter system include both ease of set up and faster turn around time (14 days) compared with traditional agar proportion (21 days). In addition, the plate can be set up from inoculum prepared using either broth or solid medium. Since the microtiter plate format is new and since Mtb presents unique safety challenges in the laboratory, this protocol will describe how to safely setup, incubate and read the microtiter plate.     

Analysis of Volatile and Oxidation Sensitive Compounds Using a Cold Inlet System and Electron Impact Mass Spectrometry

This video presents a protocol for the mass spectrometrical analysis of volatile and oxidation sensitive compounds using electron impact ionization. The analysis of volatile and oxidation sensitive compounds by mass spectrometry is not easily achieved, as all state-of-the-art mass spectrometric methods require at least one sample preparation step, e.g., dissolution and dilution of the analyte (electrospray ionization), co-crystallization of the analyte with a matrix compound (matrix-assisted laser desorption/ionization), or transfer of the prepared samples into the ionization source of the mass spectrometer, to be conducted under atmospheric conditions. Here, the use of a sample inlet system is described which enables the analysis of volatile metal organyls, silanes, and phosphanes using a sector field mass spectrometer equipped with an electron impact ionization source. All sample preparation steps and the sample introduction into the ion source of the mass spectrometer take place either under air-free conditions or under vacuum, enabling the analysis of compounds highly susceptible to oxidation. The presented technique is especially of interest for inorganic chemists, working with metal organyls, silanes, or phosphanes, which have to be handled using inert conditions, such as the Schlenk technique. The principle of operation is presented in this video.     

Motif-Independent Prediction of a Secondary Metabolism Gene Cluster Using Comparative Genomics: Application to Sequenced Genomes of Aspergillus and Ten Other Filamentous Fungal Species

Despite their biological importance, a significant number of genes for secondary metabolite biosynthesis (SMB) remain undetected due largely to the fact that they are highly diverse and are not expressed under a variety of cultivation conditions. Several software tools including SMURF and antiSMASH have been developed to predict fungal SMB gene clusters by finding core genes encoding polyketide synthase, nonribosomal peptide synthetase and dimethylallyltryptophan synthase as well as several others typically present in the cluster. In this work, we have devised a novel comparative genomics method to identify SMB gene clusters that is independent of motif information of the known SMB genes. The method detects SMB gene clusters by searching for a similar order of genes and their presence in nonsyntenic blocks. With this method, we were able to identify many known SMB gene clusters with the core genes in the genomic sequences of 10 filamentous fungi. Furthermore, we have also detected SMB gene clusters without core genes, including the kojic acid biosynthesis gene cluster of Aspergillus oryzae. By varying the detection parameters of the method, a significant difference in the sequence characteristics was detected between the genes residing inside the clusters and those outside the clusters.     

Expression and Purification of Human PAG, a Transmembrane Adapter Protein Using an Insect Cell Expression System and its Structure Basis

In this study, we report the purification and structure basis of human phosphoprotein associated with glycosphingolipid-enriched microdomains (PAG), a C-SRC tyrosine kinase (CSK)-binding protein. Human PAG was produced using an insect cell expression system. The PAG was purified by metal affinity, ion exchange, and gel filtration chromatographies. The final purity of gel-purified PAG was evaluated by SDS-PAGE and mass spectrometry. Recombinant human PAG migrates to 60 kDa on SDS-PAGE gel, while native PAG is a 46 kDa transmembrane adapter protein in lipid rafts. Recombinant human PAG has a difference of 2590.7 Da with a calculated mass (47803.41 Da) and an observed mass (50394.1 Da) by mass spectrometry. Consequently, although human PAG sequence shares well-known sites for modifications such as myristoylation, palmitoylation, and tyrosine phosphorylation sites, perhaps the difference suggests the existence of unknown modification sites. We show the high PAG-binding ability with CSK in vitro as well as the human PAG structure characterized by 11 α-helix structures including a 3 kDa transmembrane domain.     

Monitoring the Differentiation and Migration Patterns of Neural Cells Derived from Human Embryonic Stem Cells Using a Microfluidic Culture System

Microfluidics can provide unique experimental tools to visualize the development of neural structures within a microscale device, which is followed by guidance of neurite growth in the axonal isolation compartment. We utilized microfluidics technology to monitor the differentiation and migration of neural cells derived from human embryonic stem cells (hESCs). We co-cultured hESCs with PA6 stromal cells, and isolated neural rosette-like structures, which subsequently formed neurospheres in suspension culture. Tuj1-positive neural cells, but not nestin-positive neural precursor cells (NPCs), were able to enter the microfluidics grooves (microchannels), suggesting that neural cell-migratory capacity was dependent upon neuronal differentiation stage. We also showed that bundles of axons formed and extended into the microchannels. Taken together, these results demonstrated that microfluidics technology can provide useful tools to study neurite outgrowth and axon guidance of neural cells, which are derived from human embryonic stem cells.     

A Simple Method for Determining Antitubulinic Activities of Ansamitocins and Related Compounds Using a Cilia Regeneration System with Deciliated Tetrahymena

Cilia regeneration with deciliated Tetrahymena pyriformis W was tested to determine the antitubulinic activities of ansamitocins and related compounds in a microbial system. Various factors interfered with the regeneration process, i.e. excess shearing force in deciliation procedure, high temperature (32°C or above), high or low pH (pH 9 or 5), and exogenous divalent cations, such as Zn²⁺, Mn²⁺, Cu²⁺ and Co²⁺. Under suitable conditions, cilia regeneration was completed in about 60 min of incubation, and a number of newly formed cilia were observed around the cell surface. When ansamitocins were added to the recovery solution, cilia regeneration was completely suppressed without alterations in cell shape or the surface structure of deciliated Tetrahymena. In this assay system, the inhibitory activity of ansamitocins was slightly stronger than that of maytansine.     

Cloning and Overexpression of a Tagged CMP-N-Acetylneuraminic Acid Synthetase from E. coli Using a Lambda Phage System and Application of the Enzyme to the Synthesis of CMP-N-Acetylneuraminic Acid

The gene coding from CMP-N-acetylneuraminic acid (CMP-NeuAc) synthetase (Ec 2.7.7.43) was amplified from total DNA of E. coli strain K-235 through a primer-directed polymerase chain reaction. The gene was fused with a modified ribosome binding site of the original CMP-NeuAc synthetase gene and a decapeptide tag sequence which served as a marker for screening of expressed proteins. The gene was cloned into lambda ZAP vector at EcoRI and XbaI sites and overexpressed in E. coli Sure at a level approximately 1000 times that of the wild type. The decapeptide-containing enzyme retained almost the same specificity as indicated by the V_max and K_m values using CTP and NeuAc as substrates. A preparative synthesis of CMP-NeuAc based on the recombinant enzyme was demonstrated.     

Statistical and Economical Efficiency in Assessment of Liver Regeneration Using Defined Sample Size and Selection in Combination With a Fully Automated Image Analysis System

Quantification of liver regeneration is frequently based on determining the 5-bromo-2-deoxyuridine labeling index (BrdU-LI). The quantitative result is influenced by preanalytical, analytical, and postanalytical variables such as the region of interest (ROI). We aimed to present our newly developed and validated automatic computer-based image analysis system (AnalySIS-Macro), and to standardize the selection and sample size of ROIs. Images from BrdU-labeled and immunohistochemically stained liver sections were analyzed conventionally and with the newly developed AnalySIS-Macro and used for validation of the system. Automatic quantification correlated well with the manual counting result (r=0.9976). Validation of our AnalySIS-Macro revealed its high sensitivity (>90%) and specificity. The BrdU-LI ranged from 11% to 57% within the same liver (32.96 ± 11.94%), reflecting the highly variable spatial distribution of hepatocyte proliferation. At least 2000 hepatocytes (10 images at 200× magnification) per lobe were required as sample size for achieving a representative BrdU-LI. Furthermore, the number of pericentral areas should be equal to that of periportal areas. The combination of our AnalySIS-Macro with rules for the selection and size of ROIs represents an accurate, sensitive, specific, and efficient diagnostic tool for the determination of the BrdU-LI and the spatial distribution of proliferating hepatocytes. (J Histochem Cytochem 57:1075–1085, 2009)