Protein Profiling Studies Research Articles

Characterization of a Fatty Acid Amide Hydrolase (FAAH) in Hirudo Verbana

The endocannabinoid system plays a critical role in modulating both peripheral and central nervous system function. Despite being present throughout the animal kingdom, there has been relatively little investigation of the endocannabinoid system beyond traditional animal models. In this study, we report on the identification and characterization of a putative fatty acid amide hydrolase (FAAH) in the medicinal leech, Hirudo verbana. FAAH is the primary enzyme responsible for metabolizing the endocannabinoid signaling molecule arachidonoyl ethanolamide (anandamide or AEA) and therefore plays a critical role in regulating AEA levels in the nervous system. mRNA encoding Hirudo FAAH (HirFAAH) is expressed in the leech central nervous system (CNS) and sequence analysis suggests that this is an orthologue of FAAH-2 observed in vertebrates. Functionally, HirFAAH has serine hydrolase activity based on activity-based protein profiling (ABPP) studies using the fluorophosphonate probe TAMRA-FP. HirFAAH also hydrolyzes arachidonyl 7-amino, 4-methyl coumarin amide (AAMCA), a substrate specific to FAAH. Hydrolase activity during both the ABPP and AAMCA assays was eliminated by a mutation at a conserved catalytic serine. Activity was also blocked by the known FAAH inhibitor, URB597. Treatment of Hirudo ganglia with URB597 potentiated synapses made by the pressure-sensitive mechanosensory neuron (P cell), mimicking the effects of exogenously applied AEA. The Hirudo CNS has been a useful system in which to study properties of endocannabinoid modulation of nociception relevant to vertebrates. Therefore, this characterization of HirFAAH is an important contribution to comparative studies of the endocannabinoid system.

Comparative Brain Proteomic Analysis between Sham and Cerebral Ischemia Experimental Groups.

Sham control groups are essential in experimental animal studies to reduce the influence of surgical intervention. The intraluminal filament procedure is one of the most common models of middle cerebral artery occlusion (MCAO) used in the study of brain ischemia. However, a sham group is usually not included in the experimental design of these studies. In this study, we aimed to evaluate the relevance of the sham group by analyzing and comparing the brain protein profiles of the sham and MCAO groups. In the sham group, 98 dysregulated proteins were detected, compared to 171 in the ischemic group. Moreover, a comparative study of protein profiles revealed the existence of a pool of 57 proteins that appeared to be dysregulated in both sham and ischemic animals. These results indicated that the surgical procedure required for the intraluminal occlusion of the middle cerebral artery (MCA) induces changes in brain protein expression that are not associated with ischemic lesions. This study highlights the importance of including sham control groups in the experimental design, to ensure that surgical intervention does not affect the therapeutic target under study.

ROLE OF HIGH-TEMPERATURE PROTEIN G IN THE VIRULENCE AND DEVELOPMENT OF ANTIMICROBIAL RESISTANCE IN SOME ORAL BACTERIAL ISOLATES

A marked increase in antimicrobial resistance among common bacterial pathogens has made the search for new therapeutic targets highly imperative. The present study was aimed to analyse the potential of bacterial chaperone high-temperature protein G (HtpG) as a therapeutic target by determining its role in virulence and antibiotic resistance. Two bacterial isolates obtained from the oral cavity of healthy human subjects were characterized and tentatively identified as Staphylococcus aureus and Streptococcus mutans. Survival assays of these isolates showed induction of thermotolerance with around 24% increase in survival at lethal temperature after preconditioning. Protein profile studies revealed prominent expression of proteins of 99, 77 and 40 kDa in thermo tolerance group as compared to the control. HtpG inhibition was achieved by using a pharmacological inhibitor of human Hsp90, geldanamycin (GA). In disc diffusion assays, there was no significant inhibitory impact of HtpG on antibiotic susceptibility to six antimicrobial compounds in both the isolates as compared to the control. An 18-19% reduction in biofilm-forming capacity was observed for both isolates after GA treatment. This study lays strong experimental evidence to the involvement of HtpG in the development of anti-microbial resistance through biofilm formation.

Unraveling the surface marker signature of cell-derived vesicles via proteome analysis and nanoparticle flow cytometry

The cell-derived vesicles (CDVs) obtained using a proprietary extrusion process are the foundation of BioDrone platform technology. With superior productivity and versatility, this technology has garnered increasing attention in broad applications, particularly as a drug delivery vehicle. Previously, we showed that CDVs exhibited varying levels of expression for tetraspanin and organelle membrane markers while revealing no discernible differences in physical characteristics compared to naturally produced extracellular vesicles (EVs). To further understand and utilize the therapeutic potentials of CDVs, a more comprehensive study of membrane protein profiles is necessary. In addition, it is crucial to validate that the CDVs produced from extrusion are indeed intact lipid vesicles rather than other impurities. Here, we produced multiple batches of CDVs and EVs from HEK293 cells. CDVs and EVs were subjected to the same purification processes for subsequent proteome and particle analyses. The proteome analyses revealed unique proteome signatures between CDVs, EVs, and parental cells. Extensive proteome analyses identified the nine most prominent membrane markers that are abundant in CDVs compared to cells and EVs. Subsequent western blotting and nanoparticle flow cytometry analyses confirmed that CD63, lysosome-associated membrane glycoprotein 1 (LAMP1), and nicastrin (NCSTN) are highly enriched in CDVs, whereas CD81, CD9, and prostaglandin F2 receptor negative regulator (PTGFRN) are more abundant in EVs. This highlights the unique membrane composition and marker signature of CDVs that are distinct from EVs. Lastly, we demonstrated that more than 90% of the CDVs are genuine lipid vesicles by combining two different classes of vesicle labeling dyes and detergents to disrupt lipid membranes. This indicates that our proprietary extrusion technology is highly compatible with other well-characterized EV production methods. The robust CDV markers identified in this study will also facilitate the engineering of CDVs to achieve enhanced therapeutic effects or tissue-selective cargo delivery.

Nanomolar Protein Thermal Profiling with Modified Cyanine Dyes.

Protein properties and interactions have been widely investigated by using external labels. However, the micromolar sensitivity of the current dyes limits their applicability due to the high material consumption and assay cost. In response to this challenge, we synthesized a series of cyanine5 (Cy5) dye-based quencher molecules to develop an external dye technique to probe proteins at the nanomolar protein level in a high-throughput one-step assay format. Several families of Cy5 dye-based quenchers with ring and/or side-chain modifications were designed and synthesized by introducing organic small molecules or peptides. Our results showed that steric hindrance and electrostatic interactions are more important than hydrophobicity in the interaction between the luminescent negatively charged europium-chelate-labeled peptide (Eu-probe) and the quencher molecules. The presence of substituents on the quencher indolenine rings reduces their quenching property, whereas the increased positive charge on the indolenine side chain improved the interaction between the quenchers and the luminescent compound. The designed quencher structures entirely altered the dynamics of the Eu-probe (protein-probe) for studying protein stability and interactions, as we were able to reduce the quencher concentration 100-fold. Moreover, the new quencher molecules allowed us to conduct the experiments using neutral buffer conditions, known as the peptide-probe assay. These improvements enabled us to apply the method in a one-step format for nanomolar protein-ligand interaction and protein profiling studies instead of the previously developed two-step protocol. These improvements provide a faster and simpler method with lower material consumption.

Using soil nitrogen amendments in mitigating ozone stress in agricultural crops: a case study of cluster beans.

The climate change scenario in the coming years is liable to have serious negative consequences on agricultural productivity. Increasing tropospheric ozone concentration is an important aspect of climate change, which, due to its oxidative nature, is injurious to the plants. Due to the multifarious nature and continuously increasing concentration of tropospheric ozone, it is prerequisite to develop strategies to manage ozone stress in plants. Present study not only evaluates the potential of soil nitrogen amendments in ameliorating ozone stress in plants, but also focuses upon the mechanistic approaches adopted by the different plant cultivars to combat ozone stress. Three doses of nitrogen amendments, recommended (N1), 1.5× recommended (N2) and 2× recommended (N3), were given to two cultivars (S-151 and PUSA-N) of Cymopsis tetragonoloba exposed to ambient ozone stress. Control plants were also maintained in which no nitrogen treatment was given. Nitrogen supplementation reduced the root nodulation frequency and leghaemoglobin content, which subsequently increased the cellular nitrogen metabolism as evident through increase in the activities of nitrate reductase and nitrite reductase in both the test cultivars. The positive effects of nitrogen amendments are clearly evident in the 1D protein profile studies which showed a greater accumulation of larger sub-units of RuBisCO in nitrogen amended plants. The results clearly indicate that N2 treatment effectively enhanced the yield of both the cultivars (84.8% and 76.37%, in S-151 and PUSA-N, respectively); however, the mechanistic approach adopted by the two cultivars was different. Whereas the yield quantity showed higher increments in S-151, the yield quality parameters (carbohydrates and nitrogen contents) responded more positively in PUSA-N.

A study of lipid and protein profiles and liver enzyme levels in neonatal diarrhea calves based on clinical severity of the disease

The purpose of this study was to investigate the serum lipid and protein profile as well as liver enzyme levels in neonatal calves with diarrhea. The study included 40 calves, 30 with diarrhea and 10 healthy (control). Calves with diarrhea were divided into three groups based on clinical findings: mild, moderate, and severe. Blood samples were taken from the vena jugularis after routine clinical examinations of the calves to determine the lipid profile, protein profile, liver enzyme levels, and glucose levels. Serum total cholesterol (TC) (P<0.01), HDL (P<0.01), and LDL (P<0.05) levels were all found to be lower in the calves in the mild, moderate, and severe groups compared to the control group. No significant difference in TP and ALB values was found between the groups. Additionally, serum aspartate aminotransferase (AST) (P<0.01), alkaline phosphatase (ALP), and glucose (P<0.05) levels were also greater in calves with diarrhea compared to the control group. The current study concluded that there was no change in the protein profile in calves with neonatal diarrhea, but the lipid profile was negatively affected and liver function was impaired, and the impairment in liver function increased as the clinical severity of the disease increased.

Serum protein profile study of myocardial infarction using a LED induced fluorescence based HPLC system

Cardiovascular diseases (CVDs) are the major health conditions for high mortality and morbidity in humans. Delay in the diagnosis of CVDs effect patients long and short-term health condition. In -house assembled UV-light emitting diode (LED) based fluorescence detector for high -performance liquid chromatography (HPLC) (HPLC-LED-IF) system is used to record serum chromatograms of three categories of samples namely, before medicated- myocardial infarction (B-MI), after medicated- MI (A-MI), and normal. The sensitivity and performance of HPLC-LED-IF system is estimated using commercial serum proteins. Statistical analysis tools like, descriptive statistics, principal component analysis (PCA), and Match/ No Match test were applied to visualize the variation in three groups of samples. Statistical analysis of the protein profile data showed fairly good discrimination among the three categories. The receiver operating characteristic (ROC) curve also supported the reliability of the method to diagnose MI.

Biochemical properties and heavy metal analysis of a commercial fish Opisthopterus tardoore

Aim: The study was designed to evaluate various biochemical parameters, protein expression, antioxidant enzymes and heavy metals by testing an edible fish Opisthopterus tardoore. Methodology: The experimental fish were collected from five major fish markets of Bhubaneswar, India and distribution of carbohydrate, lipid and protein content were assessed in muscle tissues. To enhance the understanding of molecular mechanism, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of crude proteins was conducted. Catalase (CAT) and guaiacol peroxidase (GPX) activities were determined both spectrophotometrically and by native gel electrophoresis. For heavy metal analysis, three toxic elements viz.,chromium, cadmium and lead were examined by atomic absorption spectrophotometer. Results: The muscle tissues showed variation in the concentrations of carbohydrate (0.17 ± 0.02 to 0.28 ± 0.04 % f.wt.), lipid (3.48 ± 0.27 to 6.62 ± 0.44 % d. wt.) and protein (1.56 ± 0.11 to 2.92 ± 0.16 % f. wt.) with respect to the markets from where fish samples were collected. The protein profiling study revealed the disappearance as well as de novo synthesis of some proteins. The activities of CAT and GPX were determined for establishing a possible bioindicator of metal toxicity in fish. A rapid disturbance in their enzymatic actions was noticed which was also confirmed through native polyacrylamide gel electrophoresis with respect to fish samples. The heavy metals analysis manifested a varying level of accumulation of Cr whereas Pb and Cd were found below the detection limits. Interpretation: The biochemical disturbances and accumulation of Cr in muscle tissues of O. tardoore reflect the pollution status of nearby coastal areas of the Bay of Bengal, Odisha. Key words: Biochemical analysis, Chromium toxicity, Guaiacol peroxidase, Opisthopterus tardoore, Protein expression

Proteomic Approaches in the Study of Placenta of Pregnancy Complicated by Gestational Diabetes Mellitus.

Gestational diabetes mellitus (GDM), a glucose intolerance developing or first recognized during pregnancy, leads to a series of short- and long-term maternal and fetal complications, somehow related to placenta structural and functional changes. The focus and the objective of the present review are to discuss the results which can be obtained by different mass spectrometric approaches in the study of placenta protein profile. Thus, matrix-assisted laser desorption/ionization mass spectrometry (MALDI) has been applied on placenta omogenates before and after one-dimensional electrophoretic separation, followed by tryptic digestion. MALDI imaging was used for direct investigation on the placenta tissue (both maternal and fetal sides). The results showed that some differences among the absolute abundances of some proteins are present for placenta samples from GDM patients. The majority of investigations were carried out by two-dimensional electrophoresis (2DE) followed by LC-MS/MS or, directly by the label-free LC-MSE approach. It should be emphasized that all these techniques were showed differences in the protein expression between the placenta samples from healthy or GDM subjects. 2DE was also employed to separate and compare placental protein levels from GDM and the control groups: differentially expressed proteins between the two groups were identified by MALDI-TOF/TOF mass spectrometry and were further confirmed by Western blotting. The physiopathological significance of the obtained results are reported and discussed in this narrative review. The experimental data obtained until now show that the newest, mass spectrometric approaches can be considered a valid tool to investigate the possible changes of placenta in the presence of GDM.

Antitumor Activity of Choerospondias axillaris Fruit Extract by Regulating the Expression of SNCAIP and SNCA on MDA-MB-231 Cells.

Objective:Cancer is a huge problem of disease globally. Today, the percentage of people die from cancer is more than a combination of various diseases. In females, most common types of malignancies that occur are breast and cervical. The present focus has been shifted on medicinal plants as a form of therapy and there is a constant need to identify new therapeutic agents. Choerospondias axillaris (C. axillaris), an underutilized fruit, has been used in the remedy of various diseases. In the present communication, we evaluated the molecular mechanism of C. axillaris methanol extract in regulating cell death in human breast cancer cells (MDA-MB-231). Methods:Methanol extract of C. axillaris was prepared and compounds were screened by Gas chromatography-mass spectrometry. The effect of fruit extract was determined on MDA-MB-231 cells by MTT ((3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay and to analyse the molecular mechanism of human breast cancer cells after treating with fruit extract, protein profiling study was performed by two-dimensional gel electrophoresis. Results:A total 9 differentially expressed proteins were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS/MS) analysis. Among 9 identified proteins, synphilin-1 protein was found to be significantly downregulated, validated by western blot and RT-qPCR analysis. Possible interacting partners of synphilin-1 (SNCAIP) were analyzed for their possible role in cancer by the in-silico method. Conclusion:Our data implicate that the presence of bioactive compound(s) in C. axillaris fruits might play an important role in inhibiting the proliferation of breast carcinoma cells and Synphilin-1 protein may play a role of apoptotic function.

PIWI-interacting RNA (piRNA): a narrative review of its biogenesis, function, and emerging role in lung cancer.

Cancer remains elusive in many aspects, especially in its causes and control. After protein profiling, genetic screening, and mutation studies, scientists now have turned their attention to epigenetic modulation. This new arena has brought to light the world of noncoding RNA (ncRNA). Although very complicated and often confusing, ncRNA domains are now among the most attractive molecular markers for epigenetic control of cancer. Long ncRNA and microRNA (miRNA) have been studied best among the noncoding genome and huge data have accumulated regarding their inhibitory and promoting effects in cancer. Another sector of ncRNAs is the world of PIWI-interacting RNAs (piRNAs). Initially discovered with the asymmetric division of germline stem cells in the Drosophila ovary, piRNAs have a unique capability to associate with mammalian proteins analogous to P-element induced wimpy testis (PIWI) in Drosophila and are capable of silencing transposons. After a brief introduction to its discovery timelines, the present narrative review covers the biogenesis, function, and role of piRNAs in lung cancer. The effects on lung cancer are highlighted under sections of cell proliferation, stemness maintenance, metastasis, and overall survival, and the review concludes with a discussion of recent discoveries of another class of small ncRNAs, the piRNA-like RNAs (piR-Ls).

Circular RNA, microRNA and Protein Profiles of the Longissimus Dorsi of Germany ZIKA and Sichuan White Rabbits.

Due to the dietetic properties and remarkable nutritive value of rabbit meat, its industry is increasing rapidly. However, the association between circular RNAs, microRNAs, and proteins and muscle fiber type, and meat quality of rabbit is still unknown. Here, using deep sequencing and iTRAQ proteomics technologies we first identified 3159 circRNAs, 356 miRNAs, and 755 proteins in the longissimus dorsi tissues from Sichuan white (SCWrabs) and Germany great line ZIKA rabbits (ZIKArabs). Next, we identified 267 circRNAs, 3 miRNAs, and 29 proteins differentially expressed in the muscle tissues of SCWrabs and ZIKArabs. Interaction network analysis revealed some key regulation relationships between noncoding RNAs and proteins that might be associated with the muscle fiber type and meat quality of rabbit. Further, miRNA isoforms and gene variants identified in SCWrabs and ZIKArabs revealed some pathways and biological processes related to the muscle development. This is the first study of noncoding RNA and protein profiles for the two rabbit breeds. It provides a valuable resource for future studies in rabbits and will improve our understanding of the molecular regulation mechanisms in the muscle development of livestock. More importantly, the output of our study will benefit the researchers and producers in the rabbit breeding program.

New Directions in Research on Aging.

Biology of aging is an active and rapidly expanding area of biomedical research. Over the years, focus of work in this field has been gradually shifting from studying the effects and symptoms of aging to searching for mechanisms of the aging process. Progress of this work led to an additional shift from looking for “the mechanism” of aging and formulating the corresponding “theories of aging” to appreciation that aging represents a net result of multiple physiological changes and their intricate interactions. It was also shown that mechanisms of aging include nutrient-dependent signaling pathways which have been remarkably conserved in the course of the evolution. Another important development in this field is increased emphasis on searching for pharmacological and environmental interventions that can extend healthspan or influence other aspects of aging. Progress in understanding the key role of aging as a risk factor for chronic disease provides impetus for these studies. Data from the recent pandemic provided additional evidence for the impact of age on resilience. Progress of work in this area also was influenced by major analytical and technological advances, including greatly improved methods for the study of gene expression, protein, lipids, and metabolites profiles, enhanced ability to produce various genetic modifications and novel approaches to assessment of biological age. Progress in research on the biology of aging provides reasons for optimism about the chances that safe and widely applicable anti-aging interventions with significant benefits for both individual and public health will be developed in the not too distant future.Graphical abstract

Affinity-based protein profiling identifies vitamin D3 as a heat shock protein 70 antagonist that regulates hedgehog transduction in murine basal cell carcinoma

Vitamin D3 (VD3) is a seco-steroid that inhibits the Hedgehog (Hh) signaling pathway. Initial studies suggested its anti-Hh activity results from direct inhibition of Smoothened, a seven-transmembrane cell surface receptor that is a key regulator of the Hh signaling cascade. More recently, a role for the Vitamin D Receptor in mediating inhibition of Hh-signaling by seco-steroid has been suggested. Herein, an affinity-based protein profiling study was carried out to better understand the cellular proteins that govern VD3-mediated anti-Hh activity. We synthesized a novel biotinylated VD3 analogue (8) for use as a chemical probe to explore cellular binding targets of the seco-steroidal scaffold. Through a series of pull-down experiments and follow up mass spectrum analyses, heat shock protein 70 (Hsp70) was identified as a primary binding protein of VD3. Hsp70 was validated as a binding target of VD3 through a series of biochemical and cellular assays. VD3 bound with micromolar affinity to Hsp70. In addition, both selective knockdown of Hsp70 expression and pharmacological inhibition of its activity with known Hsp70 inhibitors suppressed Hh-signaling transduction in murine basal cell carcinoma cells, suggesting that Hsp70 regulates proper Hh-signaling. Additional cellular assays suggest that VD3 and its seco-steroidal metabolites inhibit Hh-signaling through different mechanisms.

Comparative study of herbal and non-herbal egg protein profile using High Performance Liquid Chromatography (HPLC)

The purpose of this research is to compare the protein profile of herbals egg and non herbal egg using High Performance Liquid Chromatography (HPLC). This research used HPLC Shimadzu 6.1 with column C18, flow rate mobile phase 1 ml/min, wavelenght detector UV Vis 220 nm and temperature 50o C. Mobile fase that used in this research was 10 and 60 % acetonitril (CH3CN) in water containing 0,05% triflouroacetic acid. Supplementation of herb containing bioactive antioxidant compounds affect the formation of egg yolk protein containing immunoglobulin. In this study, herbal eggs and non-herbal eggs were seen from their protein profile using the high performance liquid chromatography (HPLC) method and then analyzed using descriptive qualitative analysis. The result show that herbal egg yolk sample has a dominant protein with a molecular weight of 50.41 kDa. Herbal egg yolk protein appears at a retention time (RT) of 56.87 minutes, an area of 3303488 units of area, and the peak height of the graph / peak at 50,974 µAU. Meanwhile, non-herbal egg yolk sample has a dominant protein with a molecular weight of 49.94 kDa. This protein appears at a retention time of 1.307 minutes, an area of 149445550 units of area, and the peak height of the graph / peak at 402.6026 µAU. The results showed that the the peak of HPLC indicated an antioxidants were bound to the bioactive protein fractions of egg yolk. It could be concluded that bioactive herbal bound to egg yolk igY, but the bioactive compounds have not been identified yet.

A resample-replace lasso procedure for combining high-dimensional markers with limit of detection

In disease screening, a biomarker combination developed by combining multiple markers tends to have a higher sensitivity than an individual marker. Parametric methods for marker combination rely on the inverse of covariance matrices, which is often a non-trivial problem for high-dimensional data generated by modern high-throughput technologies. Additionally, another common problem in disease diagnosis is the existence of limit of detection (LOD) for an instrument – that is, when a biomarker's value falls below the limit, it cannot be observed and is assigned an NA value. To handle these two challenges in combining high-dimensional biomarkers with the presence of LOD, we propose a resample-replace lasso procedure. We first impute the values below LOD and then use the graphical lasso method to estimate the means and precision matrices for the high-dimensional biomarkers. The simulation results show that our method outperforms alternative methods such as either substitute NA values with LOD values or remove observations that have NA values. A real case analysis on a protein profiling study of glioblastoma patients on their survival status indicates that the biomarker combination obtained through the proposed method is more accurate in distinguishing between two groups.

Biological Synthesis of PbS, As3S4, HgS, CdS Nanoparticles using Pseudomonas aeruginosa and their Structural, Morphological, Photoluminescence as well as Whole Cell Protein Profiling Studies.

Metal sulfide nanoparticles are semi-conductors that possess many applications in optics, optoelectronics and magnetic devices. There are physical and chemical methods for their synthesis but such methods involve toxic precursors as well as many obnoxious by-products. Hence, biological synthesis of metal sulfide nanoparticles are efficient enough to transform toxic metals to non-toxic ones. Pseudomonas aeruginosa, isolated from textile effluent and tolerant of high levels of heavy metals, was used for the green synthesis of metal sulfide (HgS, As3S4, CdS and PbS) nanoparticles. The optical, structural and morphological nature of metal sulfide nanoparticles was also determined. FTIR (Fourier Transform Infra-red) analysis showed spectral changes when P. aeruginosa was grown in medium containing heavy metals viz. Hg, As, Pb and Cd indicating that there are functional groups viz. carboxyl, hydroxyl, phosphate, amino and amide, that exists on the surface of the bacteria, thus facilitating binding of metals on its surface. The bacterial samples which were treated with different metals at different concentrations, were subjected to whole cell protein analysis using SDS-PAGE (Sodium dodecyl Sulphate- Polyacrylamide gel electrophoresis) and protein profiling. The total protein estimation revealed that there was an increase in the protein concentration in the presence of heavy metals and a significant change in the banding pattern was observed which showed induction of a set of proteins under heavy metal stress especially mercury.

Atrazine detoxification by intracellular crude enzyme extracts derived from epiphytic root bacteria associated with emergent hydrophytes

The present study demonstrated atrazine detoxification by intracellular crude enzyme extracts of Pseudomonas spp. strains ACB and TLB. Indigenous bacterial protein-based remediation techniques could be an alternative to bioaugmentation which pose multiple challenges when applied to the field. Intracellular enzymes were extracted from strains ACB and TLB and their degradation potential of 10 mg L−1 was determined using Gas Chromatography; further, enzyme extracts were subjected to protein profiling studies. In span of 6 h, enzyme extracts of strain ACB showed maximum degradation at 30 °C and 40 °C (71%) and enzyme extracts of strain TLB showed maximum degradation at 40 °C (48%). Atrazine degradation by enzyme extracts of strain ACB showed maximum degradation at pH 7 (71%) and pH 6 (69%) in 6 h. Similarly, enzyme extracts of strain TLB showed maximal degradation at pH 6 (46%) in 6 h. The present study demonstrated, for the first time, efficient atrazine remediation by intracellular crude enzyme extracts from epiphytic root bacteria at a range of temperature and pH conditions. Protein profiling studies indicated that atrazine induced expression of CoA ester lyase and alkyl hydroperoxide reductase in the strains ACB and TLB respectively. Expressions of these proteins have never been associated with atrazine exposure.

Предикторы перекисного окисления липидов как критерий диагностики первичной открытоугольной глаукомы

This review of modern literature presents the issue of early diagnosis of primary open-angle glaucoma (POAG) which is one of the most important problems in ophthalmology. According to the World Health Organization (WHO), the number of glaucoma patients all over the world ranges from 60.5 to 105 million people, and in the next 10 years it will increase by another 10 million. The subtle onset of the disease usually leads to late diagnosis, since visual field defects are often first detectable by visual field testing when 25–35% of retinal ganglion cells have already been lost. In modern studies, it has been established that the cell death in POAG is accompanied by oxidative stress. It is considered a key factor indicating the pathological process of a fairly large number of chronic diseases of the whole body, including complicated eye pathologies and inflammatory processes. The resulting free radicals oxidize lipids and phospholipids which leads to their peroxidation and triggers damage to cell membranes. The lipid peroxidation is one of obligatory processes occurring in neurodegenerative conditions including POAG. Modern technologies analyzing lipid peroxidation may accelerate the diagnosis, to identify glaucoma biomarkers at an earlier stage, and as a result to maintain quality of vision more efficiently. These studies were carried out in different countries and using different methods such as serological proteomic analysis, chromatography and mass spectrometry, and new technological approaches such as MultiOmics. The study of the protein profile, lipase activity, nitric oxide concentration, activity of carbonic anhydrase enzyme, malondialdehyde (MDA) and other compounds are also very informative. Various fluids were taken as a basis for the study: tears, aqueous humor of the anterior chamber, blood and its components. Together these technologies enable to evaluate the oxidative process in different ways. Earlier diagnosis of glaucoma is very important as it will enable to maintain the quality of vision for a longer time and reduce the rate of the disease progression. Keywords: primary open-angle glaucoma, lipid peroxidation, lacrimal fluid, serum.