Traditional Biochemical Analysis Research Articles

Formation Mechanism of Novel Chips and Application Research in Biochemistry

It is crucial to systematically study the interaction between samples and components at different concentrations in biochemical analysis. Microfluidic chip technology can complete precise liquid control in micron-scale channels and has been widely used in the biochemical analysis in recent years. The microfluidic concentration gradient chip is a tool that can rapidly construct a stable biochemical concentration gradient, which can be combined with most cell culture, chemical analysis, and other technologies to provide a new platform for traditional biochemical analysis. This paper reviews the formation mechanism of microfluidic concentration gradient chips and their applications in biochemistry and other fields and provides new ideas for broadening the application research of concentration gradients.

Label-Free Uric Acid Estimation of Spot Urine Using Portable Device Based on UV Spectrophotometry.

The maintenance of uric acid levels is crucial for the human body. In this study, the feasibility of using portable ultraviolet (UV) spectrophotometry to measure the uric acid of spot urine without the need to add reagents has been demonstrated for the first time. UV spectral analysis has been used to inspect the uric acid concentration in urine. It is found that the absorption spectrum of urine has a high correlation with the concentration of uric acid at a wavelength of around 290–300 nm. Uric acid levels measured with a spectral analyzer compared to uric acid concentrations measured with a traditional biochemical analysis showed good agreement. The portable prototype is label-free and capable of displaying the inspection result of each measurement within 10 s. In the long run, this device can assist people in checking uric acid levels of spot urine with higher frequency and can adjust diet or medication in real time for more efficient health management.

A small molecule high throughput screening platform to profile conformational properties of nascent, ribosome-bound proteins

Genetic mutations cause a wide spectrum of human disease by disrupting protein folding, both during and after synthesis. Transient de-novo folding intermediates therefore represent potential drug targets for pharmacological correction of protein folding disorders. Here we develop a FRET-based high-throughput screening (HTS) assay in 1,536-well format capable of identifying small molecules that interact with nascent polypeptides and correct genetic, cotranslational folding defects. Ribosome nascent chain complexes (RNCs) containing donor and acceptor fluorophores were isolated from cell free translation reactions, immobilized on Nickel-NTA/IDA beads, and imaged by high-content microscopy. Quantitative FRET measurements obtained from as little as 0.4 attomole of protein/bead enabled rapid assessment of conformational changes with a high degree of reproducibility. Using this assay, we performed a pilot screen of ~ 50,000 small molecules to identify compounds that interact with RNCs containing the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) harboring a disease-causing mutation (A455E). Screen results yielded 133 primary hits and 1 validated hit that normalized FRET values of the mutant nascent peptide. This system provides a scalable, tractable, structure-based discovery platform for screening small molecules that bind to or impact the folding of protein substrates that are not amenable to traditional biochemical analyses.

The Impulse of Force as an Effective Indicator of Exercise Capacity in Competitive Rowers and Canoeists.

The aim of the research was to present the importance of measuring the impulse of force in assessing exercise capacity in professional rowers and canoeists rather than conducting traditional physiological and biochemical analyses. A group of 20 athletes (12 rowers and 8 canoeists) underwent progressive intensity exercise tests in two testing sessions (before and after a training period). During the tests, maximal aerobic capacity, impulse of strength, metabolic indices, and markers of skeletal muscle damage were assessed. Total testosterone, free testosterone, cortisol, interleukin 1,6, and creatine kinase were evaluated in venous blood. The impulse of force at the ventilatory threshold and at the maximum load was correlated with free testosterone and a total testosterone/cortisol ratio during exercise (p ≤ 0.05) and was negatively correlated with cortisol concentration (p ≤ 0.05) in the first testing session (before training intervention). Values were positively correlated with concentration of total testosterone, free testosterone and total testosterone/cortisol, and free testosterone/cortisol ratios during exercise (p ≤ 0.05) in the second testing session (after training intervention). Biochemical indices of overtraining were correlated with maximum oxygen uptake in each session and with the impulse of force at the ventilatory threshold and the maximum load of exercise. Thus, there is an indirect relationship between maximal aerobic capacity and the impulse of force. The measurement of the impulse of force can become a simple and effective method for evaluating the overall exercise capacity of rowers because of its relationship with indices of the load and changes in hormonal indices of anabolic and catabolic processes during exercise. Therefore, it may constitute a replacement for the traditional laboratory measurement of VO2max in highly qualified rowers and canoeists.

Correlation of Kidney Size on Computed Tomography with GFR, Creatinine and HbA1C for an Accurate Diagnosis of Patients with Diabetes and/or Chronic Kidney Disease.

Diabetes is considered one of the major causes of chronic kidney disease (CKD), affecting renal blood vessels and nerves. Diagnosis of CKD by traditional biochemical serum and blood analyses is insufficient and insensitive, thus requiring the development of a more robust technique. This novel study aims to propose a new method for the accurate diagnosis of CKD, quantification of kidney damage, and its prognosis by physicians by measuring the kidney volume on computed tomography (CT). In total, 251 patients were enrolled in this retrospective study. They were divided into four groups: control, patients having diabetes, patients having CKD, and patients having both diabetes and CKD. Results showed that kidney volume correlated negatively with both GFR and HbA1C on CT images, in addition to decreasing faster in males than females. Moreover, HbA1C was shown to correlate positively with creatinine and negatively with GFR. Finally, GFR was more robust than creatinine when correlated with age. The association between kidney volume with GFR and HbA1c can be used to accurately anticipate kidney volume in established CKD on CT scan, especially in resource-poor settings. Furthermore, HbA1C can serve as a powerful biomarker for studying renal function in diabetic CKD patients as it correlates with creatinine and GFR.

UPLC/MS/MS-Based Metabolomics Study of the Hepatotoxicity and Nephrotoxicity in Rats Induced by Polygonum multiflorum Thunb.

Polygonum multiflorum Thunb. (PM) is one of the most frequently used natural products in China. Its hepatotoxicity has been proven and reported. However, chronic PM toxicity is a dynamic process, and a few studies have reported the long-term hepatotoxic mechanism of PM or its nephrotoxicity. To elucidate the mechanism of hepatotoxicity and nephrotoxicity induced by PM after different administration times, different samples from rats were systematically investigated by traditional biochemical analysis, histopathological observation, and nontargeted metabolomics. The concentrations of direct bilirubin (DBIL) at 4 weeks and total bile acid, DBIL, uric acid, and blood urea nitrogen at 8 weeks were significantly increased in the treatment group compared with those in the control group. Approximately, 12 metabolites and 24 proteins were considered as unique toxic biomarkers and targets. Metabolic pathway analysis showed that the primary pathways disrupted by PM were phenylalanine and tyrosine metabolism, which resulted in liver injury, accompanied by chronic kidney injury. As the administration time increased, the toxicity of PM gradually affected vitamin B6, bile acid, and bilirubin metabolism, leading to aggravated liver injury, abnormal biochemical indicators, and marked nephrotoxicity. Our results suggest that the hepatotoxicity and nephrotoxicity caused by PM are both dynamic processes that affect different metabolic pathways at different administration times, which indicated that PM-induced liver and kidney injury should be treated differently in the clinic according to the degree of injury.

Simultaneous, hybrid single-molecule method by optical tweezers and fluorescence

As studies on life sciences progress toward the single-molecule level, new experiments have put forward more requirements for simultaneously displaying the mechanical properties and conformational changes of biomolecules. Optical tweezers and fluorescence microscopy have been combined to solve this problem. The combination of instruments forms a new generation of hybrid single-molecule technology that breaks through the limitations of traditional biochemical analysis. Powerful manipulation and fluorescence visualization have been widely used, and these techniques provide new possibilities for studying complex biochemical reactions at the single-molecule level. This paper explains the features of this combined technique, including the application characteristics of single-trap and dual-traps, the anti-bleaching method, and optical tweezers combined with epi-fluorescence, confocal fluorescence, total internal reflection fluorescence, and other fluorescence methods. Using typical experiments, we analyze technical solutions and explain the factors and principles that instrument designers should consider. This review aims to give an introduction to this novel fusion technology process and describe important biological results.

Radial variation in biochemical composition of the bovine caudal intervertebral disc.

Bovine caudal discs have been widely used in spine research due to their increased availability, large size, and mechanical and biochemical properties that are comparable to healthy human discs. However, despite their extensive use, the radial variations in bovine disc composition have not yet been rigorously quantified with high spatial resolution. Previous studies were limited to qualitative analyses or provided limited spatial resolution in biochemical properties. Thus, the main objective of this study was to provide quantitative measurements of biochemical composition with higher spatial resolution than previous studies that employed traditional biochemical techniques. Specifically, traditional biochemical analyses were used to measure water, sulfated glycosaminoglycan, collagen, and DNA contents. Gravimetric water content was compared to data obtained through Raman spectroscopy and differential scanning calorimetry. Additionally, spatial distribution of lipids in the disc's collagen network was visualized and quantified, for the first time, using multi‐modal second harmonic generation (SHG) and Coherent anti‐Stokes Raman (CARS) microscopy. Some heterogeneity was observed in the nucleus pulposus, where the water content and water‐to‐protein ratio of the inner nucleus were greater than the outer nucleus. In contrast, the bovine annulus fibrosus exhibited a more heterogeneous distribution of biochemical properties. Comparable results between orthohydroxyproline assay and SHG imaging highlight the potential benefit of using SHG microscopy as a less destructive method for measuring collagen content, particularly when relative changes are of interest. CARS images showed that lipid deposits were distributed equally throughout the disc and appeared either as individual droplets or as clusters of small droplets. In conclusion, this study provided a more comprehensive assessment of spatial variations in biochemical composition of the bovine caudal disc.

Single-Stranded DNA Curtains for Studying the Srs2 Helicase Using Total Internal Reflection Fluorescence Microscopy.

Helicases are crucial participants in many types of DNA repair reactions, including homologous recombination. The properties of these enzymes can be assayed by traditional bulk biochemical analysis; however, these types of assays cannot directly access some types of information. In particular, bulk biochemical assays cannot readily access information that may be obscured in population averages. Single-molecule assays offer the potential advantage of being able to visualize the molecules in question in real time, thus providing direct access to questions relating to translocation velocity, processivity, and insights into how helicases may behave on different types of substrates. Here, we describe the use of single-stranded DNA (ssDNA) curtains as an assay for directly viewing the behavior of the Saccharomyces cerevisiae Srs2 helicase on single molecules of ssDNA. When used with total internal reflection fluorescence microscopy, these methods can be used to track the binding and movements of individual helicase complexes, and allow new insights into helicase behaviors at the single-molecule level.

Development and preliminary validation of a multi-trophic metabarcoding biotic index for monitoring benthic organic enrichment

Sea-based fish farms are associated with strong, benthic enrichment gradients and routine monitoring is usually required by regulation. This has traditionally been based on benthic macrofaunal communities, which is reliant on taxonomic expertise, and turn-around times can be slow, limiting opportunities for adaptive management. Environmental metabarcoding is a powerful high-throughput sequencing-based technique that can identify and quantify benthic assemblages. Previous studies have demonstrated relationships between specific taxonomic groups (e.g., bacteria, foraminifera) and anthropogenic effects. However, the absence of fixed categorical scales makes the use of data challenging for routine biomonitoring. In this study, we analysed 105 sediment samples collected over three years from three salmon farms spanning two separate bioregions. Environmental DNA and RNA (eDNA/eRNA) metabarcoding of three taxonomic groups (foraminifera, bacteria, and general eukaryotes) was undertaken in parallel with traditional macrofaunal and biochemical analyses, which were used to calculate an Enrichment Stage index for each sample. The most abundant 200–250 Operational Taxonomic Units in each taxonomic group were assessed and assigned to Eco-Groups. These bioindicator taxa were then used to develop a Metabarcoding Biotic Index for each taxonomic group individually and in combination (multi-trophic Metabarcoding Biotic Index). The weakest relationship was between the foraminiferal Metabarcoding Biotic Index and Enrichment Stage (eDNA/eRNA, R2=0.731–0.850), whereas strong correspondence was obtained between the multi-trophic Metabarcoding Biotic Index and Enrichment Stage, or when just bacteria and eukaryotes data were combined (eDNA/RNA, R2>0.900). The strong relationships obtained between the multi-trophic Metabarcoding Biotic Index and Enrichment Stage confirms that this method has the potential to complement or even replace current fish farm biomonitoring techniques in the near future.

PII: S0928-1258(17)30185-7

Pharmaceutical industrialization in Bangladesh, both by multinational and local companies, has increased significantly in the last two decades. Most of the pharmaceutical products are found to be therapeutically competent to meet the demands of general population satisfactorily. However, complaints regarding the compromised quality of the products stored in markets are also reported very often. In order to ensure the overall drug user safety, the present review discussed the prime requirements for maintaining the desired microbiological quality of the commonly used pharmaceutical products in Bangladesh. Information relating to the prevalence of bacteria, fungi and the specific pathogens has been collected mostly from the recent researches conducted on an array of finished pharmaceutical medicaments. All data, achieved by means of traditional microbiological and biochemical analyses as described in the published papers cited in this review, have been further critically analyzed in context to the recommended microbial limits, user safety and the legislative aspects. Microbial contamination usually degrades the product quality as well as the product stability, which in turn creates treatment complications. Moreover, through microbial spoilage within the medicament, active drug constituents may be transformed to less potent or chemically inactive forms. Contamination by bacteria and fungi generally occur either from raw materials, manufacturing environment or at the storage stage which may hinder the consumer safety. This review article presented some valid evidences for microbial contamination in the pharmaceutical finished products in Bangladesh and discusses about possible remedies to improve the overall management of public health.

Estimation of synergistic effect of humic fertilizer and Bacillus subtilis on lettuce plants by reflectance measurements

ABSTRACTPrevious research showed that the use of plant growth promoting rhizobacteria helped to increase nutrient use efficiency. The individual and combined effects of combined action of humic fertilizer and rhizobacteria Bacillus subtilis No. 2 on the lettuce yield, chlorophyll, total nitrogen and nitrate-(N) contents in lettuce leaves was studied. Traditional biochemical analysis and crop reflectance method were compared. Vegetation Indices and key spectrum characteristics - a median of frequency spectrum and bandwidth of frequency spectrum were used to estimate chlorophyll content in plant leaves.The synergistic effect of bacteria and humic fertilizer was evidenced by increase in N and chlorophyll contents and in decreased nitrates content in lettuce leaves. Humic fertilizer resulted in decreased nitrates concentration in plants, whereas bacillus (B). subtilis No. 2 increased total N and chlorophyll contents. Results indicated that the application rate of humic fertilizer may be reduced when B. subtilis No. 2 is also applied.

Detection of single bacteria – causative agents of meningitis using raman microscopy

Early diagnostics of meningitis is a very topical problem as it is a fulminant disease with a high level of mortality. The progress of this disease is, as a rule, accompanied by the appearance of bacteria in the cerebrospinal fluid (CSF) composition. The examination of the CSF is well known to be the only reliable approach to the identification of meningitis. However, the traditional biochemical analyses are time consuming and not always reliable, simple, and inexpensive, whereas the optical methods are poorly developed. This work is devoted to the study of Raman spectra of several bacterial cultures which are mainly present during meningitis. Raman microscopy is a prompt and noninvasive technique capable of providing reliable information about molecular-level alterations of biological objects at their minimal quantity and size. It was shown that there are characteristic lines in Raman spectra which can be the reliable markers for determination of bacterial form of meningitis at a level of a single bacterium.

Application of Bioinformatic Tools for the Identification and Characterization of Microbes in the Medical Microbiology Laboratory

Genome sequencing technologies have provided information that enables a faster, more precise characterization of bacteria, according to DNA and RNA sequences. Additionally, comparison of genomes from closely related bacteria makes it possible to determine why some strains are pathogenic, to predict clinical outcomes of infections, and to develop therapeutic strategies. Application of this technology to the clinical laboratory provides reliable and accurate means of reducing turnaround time and identification of pathogenic bacteria that might be incorrectly or not readily identified by traditional methods. In this class unit, undergraduate Medical Laboratory Science students used genome databases and online computer algorithms with simulated sequence data to identify a bacterium, as an alternative to traditional biochemical analysis. In addition, students used the genome sequence to search for virulence genes and antibiotic resistance genes. These analytical processes are applicable to molecular protocols currently in use. The students gained familiarity with bioinformatics analysis and deepened their understanding of genome structure and function. ABBREVIATIONS:DNA – deoxyribonucleic acid, RNA – ribonucleic acid, MLS – medical laboratory science, rRNA – ribosomal ribonucleic acid, NCBI – National Center for Biotechnology Information, BLAST – Basic Local Alignment Search Tool, RAST – Rapid Annotation using Subsystem Technology, IDNS – Integrated Database Network Service, FASTA – a text based nucleotide and/or protein sequence format, RefSeq – reference sequence, NIH – National Institutes of Health

The potential of label-free nonlinear optical molecular microscopy to non-invasively characterize the viability of engineered human tissue constructs

Nonlinear optical molecular imaging and quantitative analytic methods were developed to non-invasively assess the viability of tissue-engineered constructs manufactured from primary human cells. Label-free optical measures of local tissue structure and biochemistry characterized morphologic and functional differences between controls and stressed constructs. Rigorous statistical analysis accounted for variability between human patients. Fluorescence intensity-based spatial assessment and metabolic sensing differentiated controls from thermally-stressed and from metabolically-stressed constructs. Fluorescence lifetime-based sensing differentiated controls from thermally-stressed constructs. Unlike traditional histological (found to be generally reliable, but destructive) and biochemical (non-invasive, but found to be unreliable) tissue analyses, label-free optical assessments had the advantages of being both non-invasive and reliable. Thus, such optical measures could serve as reliable manufacturing release criteria for cell-based tissue-engineered constructs prior to human implantation, thereby addressing a critical regulatory need in regenerative medicine.

Proteomic Applications in the Study of Human Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are undifferentiated cells with an unlimited capacity for self-renewal and able to differentiate towards specific lineages under appropriate conditions. MSCs are, a priori, a good target for cell therapy and clinical trials as an alternative to embryonic stem cells, avoiding ethical problems and the chance for malignant transformation in the host. However, regarding MSCs, several biological implications must be solved before their application in cell therapy, such as safe ex vivo expansion and manipulation to obtain an extensive cell quantity amplification number for use in the host without risk accumulation of genetic and epigenetic abnormalities. Cell surface markers for direct characterization of MSCs remain unknown, and the precise molecular mechanisms whereby growth factors stimulate their differentiation are still missing. In the last decade, quantitative proteomics has emerged as a promising set of techniques to address these questions, the answers to which will determine whether MSCs retain their potential for use in cell therapy. Proteomics provides tools to globally analyze cellular activity at the protein level. This proteomic profiling allows the elucidation of connections between broad cellular pathways and molecules that were previously impossible to determine using only traditional biochemical analysis. However; thus far, the results obtained must be orthogonally validated with other approaches. This review will focus on how these techniques have been applied in the evaluation of MSCs for their future applications in safe therapies.

Comparative Study on Microfluidic Performance of ZnO Surface Acoustic Wave Devices on Various Substrates

ZnO thin film-based surface acoustic wave (SAW) devices were fabricated on Si, glass and polyimide (PI) substrates, and their microfluidic performances were compared. The Rayleigh and Sezawa mode waves were observed from the ZnO/Si devices, the Rayleigh and Lamb modes from the ZnO/PI, and only the Rayleigh mode from the ZnO/glass devices. The ZnO/Si devices have the best performance with the highest acoustic streaming velocity of about 10 cm/s and the shortest particle concentration time of less than 10 sec. The ZnO/glass SAW devices deliver comparable performances to that on Si substrate, while the ZnO/PI devices perform not as good as the devices on other two types of substrates due to the large acoustic attenuation, but still can deliver a streaming velocity up to 1.0 cm/s and reasonable particle concentration function with a particle concentration time of ∼70 sec. Owing to its low cost, easy fabrication and compatible with traditional glass-based biochemical analysis systems, ZnO/glass SAW device is believed to have better potential for lab-on-a-chip application.

Understanding the metabolic fate and assessing the biosafety of MnO nanoparticles by metabonomic analysis

Recently, some types of MnO nanoparticle (Mn-NP) with favorable imaging capacity have been developed to improve the biocompatible profile of the existing Mn-based MRI contrast agent Mn-DPDP; however, the overall bio-effects and potential toxicity remain largely unknown. In this study, 1H NMR-based metabolic profiling, integrated with traditional biochemical analysis and histopathological examinations, was used to investigate the absorption, distribution, metabolism, excretion and toxicity of Mn-NPs as candidates for MRI contrast agent. The metabolic responses in biofluids (plasma and urine) and tissues (liver, spleen, kidney, lung and brain) from rats could be divided into four classes following Mn-NP administration: Mn biodistribution-dependent, time-dependent, dose-dependent and complicated metabolic variations. The variations of these metabolites involved in lipid, energy, amino acid and other nutrient metabolism, which disclosed the metabolic fate and biological effects of Mn-NPs in rats. The changes of metabolic profile implied that the disturbance and impairment of biological functions induced by Mn-NP exposure were correlated with the particle size and the surface chemistry of nanoparticles. Integration of metabonomic technology with traditional methods provides a promising tool to understand the toxicological behavior of biomedical nanomaterials and will result in informed decision-making during drug development.

A genetic approach for the identification of exosporium assembly determinants of Bacillus anthracis

The exosporium is the outermost layer of spores of the zoonotic pathogen Bacillus anthracis. The composition of the exosporium and its functions are only partly understood. Because this outer spore layer is refractive to traditional biochemical analysis, a genetic approach is needed in order to define the proteins which comprise this important spore layer and its assembly pathway. We have created a novel genetic screening system for the identification and isolation of mutants with defects in exosporium assembly during B. anthracis spore maturation. The system is based on the targeting sequence of the BclA exosporium nap layer glycoprotein and a fluorescent reporter. By utilizing this screening system and gene inactivation with Tn916, several novel putative exosporium-associated determinants were identified. A sampling of the mutants obtained was further characterized, confirming their exosporium defect and validating the utility of this screen to identify novel spore determinants in the genome of this pathogen.

Biomass for thermochemical conversion: targets and challenges

Bioenergy will be one component of a suite of alternatives to fossil fuels. Effective conversion of biomass to energy will require the careful pairing of advanced conversion technologies with biomass feedstocks optimized for the purpose. Lignocellulosic biomass can be converted to useful energy products via two distinct pathways: enzymatic or thermochemical conversion. The thermochemical pathways are reviewed and potential biotechnology or breeding targets to improve feedstocks for pyrolysis, gasification, and combustion are identified. Biomass traits influencing the effectiveness of the thermochemical process (cell wall composition, mineral and moisture content) differ from those important for enzymatic conversion and so properties are discussed in the language of biologists (biochemical analysis) as well as that of engineers (proximate and ultimate analysis). We discuss the genetic control, potential environmental influence, and consequences of modification of these traits. Improving feedstocks for thermochemical conversion can be accomplished by the optimization of lignin levels, and the reduction of ash and moisture content. We suggest that ultimate analysis and associated properties such as H:C, O:C, and heating value might be more amenable than traditional biochemical analysis to the high-throughput necessary for the phenotyping of large plant populations. Expanding our knowledge of these biomass traits will play a critical role in the utilization of biomass for energy production globally, and add to our understanding of how plants tailor their composition with their environment.