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Background: In endemic regions of several countries, the prevalence of leprosy has not come down to the level of elimination. On the contrary, new cases are being detected in large numbers. Clinically, it is frequently noted that despite completion of multibacillary multidrug therapy for 12 months, the lesions remain active, especially in cases with high bacteriological indices. Aim: The present study focused on finding out the viable number of Mycobacterium leprae during the 12-month regimen of multibacillary multidrug therapy, at six and 12 months intervals and, attempting to determine their role in disease transmission. Methods: Seventy eight cases of multibacillary leprosy cases were recruited from leprosy patients registered at The Leprosy Mission hospitals at Shahdara (Delhi), Naini (Uttar Pradesh) and Champa (Chhattisgarh), respectively. Slit skin smears were collected from these patients which were transported to the laboratory for further processing. Ribonucleic acid was extracted by TRIzol method. Total Ribonucleic acid was used for real-time reverse transcription-polymerase chain reaction (two-step reactions). A standard sample with a known copy number was run along with unknown samples for a reverse transcription-polymerase chain reaction. Patients were further assessed for their clinical and molecular parameters during 6th month and 12th month of therapy. Results: All 78 new cases showed the presence of a viable load of bacilli at the time of recruitment, but we were able to follow up only on 36 of these patients for one year. Among these, using three different genes, 20/36 for esxA, 22/36 for hsp18 and 24/36 for 16S rRNA cases showed viability of M. leprae at the time of completion of 12 months of multidrug therapy treatment. All these positive patients were histopathologically active and had bacillary indexes ranging between 3+ and 4+. Patients with a high copy number of the Mycobacterium leprae gene, even after completion of treatment as per WHO recommended fixed-dose multidrug therapy, indicated the presence of live bacilli. Limitations: Follow up for one year was difficult, especially in Delhi because of the migratory nature of the population. Patients who defaulted for scheduled sampling were not included in the study. Conclusion: The presence of a viable load of bacilli even after completion of therapy may be one of the reasons for relapse and continued transmission of leprosy in the community
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Monitoring of host cell proteins(HCPs)during the manufacturing of monoclonal antibodies(mAb)has become a critical requirement to provide effective and safe drug products.Enzyme-linked immunosor-bent assays are still the gold standard methods for the quantification of protein impurities.However,this technique has several limitations and does,among others,not enable the precise identification of pro-teins.In this context,mass spectrometry(MS)became an alternative and orthogonal method that de-livers qualitative and quantitative information on all identified HCPs.However,in order to be routinely implemented in biopharmaceutical companies,liquid chromatography-MS based methods still need to be standardized to provide highest sensitivity and robust and accurate quantification.Here,we present a promising MS-based analytical workflow coupling the use of an innovative quantification standard,the HCP Profiler solution,with a spectral library-based data-independent acquisition(DIA)method and strict data validation criteria.The performances of the HCP Profiler solution were compared to more con-ventional standard protein spikes and the DIA approach was benchmarked against a classical data-dependent acquisition on a series of samples produced at various stages of the manufacturing process.While we also explored spectral library-free DIA interpretation,the spectral library-based approach still showed highest accuracy and reproducibility(coefficients of variation＜10％)with a sensitivity down to the sub-ng/mg mAb level.Thus,this workflow is today mature to be used as a robust and straightforward method to support mAb manufacturing process developments and drug products quality control.
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ObjectiveHenoch-Schönlein purpura（HSP） is one of the dominant diseases in Mongolian medicine. Qishun Baolier（QSBLE）， as the main prescription for the treatment of HSP， has significant clinical effect， but its mechanism is not yet clear. Baed on this， this study is intended to screen the differentially expressed proteins before and after treatment， and preliminarily explore the molecular mechanism of QSBLE in the treatment of HSP. MethodTaking oneself as the control， 30 HSP patients aged 6-45 years were collected， and QSBLE was taken orally at 12：00 and 24：00， respectively. The dose was adjusted according to age and the course of treatment was one week. The distribution of proteinuria， hematuria and skin purpura of all patients were determined before and after treatment. The serum samples of 10 patients with clinically significant remission after QSBLE treatment were randomly selected for proteomics. Isobaric tags for relative and absolute quantification（iTRAQ） combined with liquid chromatography tandem mass spectrometry（LC-MS/MS） was used to analyze the proteins in serum of HSP patients before and after treatment， and differential proteins were analyzed bioinformatically and the protein-protein interaction（PPI） networks were constructed. ResultA total of 378 proteins were identified from serum， including 18 differentially expressed proteins， of which 15 proteins were up-regulated and 3 proteins were down regulated. Bioinformatics showed that the differential proteins were mainly involved in biological processes such as immune response， immunoglobulin production， phagocytosis， adaptive immune response before and after treatment. Biological processes， pathways and proteins were used to construct the PPI network， the proteins represented by immunoglobulin heavy constant γ1（IGHG1）， immunoglobulin λ-chain 7-43（IGLV7-43）， gelsolin（GSN） and 60 kDa heat shock protein（HSPD1） were involved in biological processes and related pathways such as adaptive immune response， immunoglobulin production， leukocyte-mediated immunity， regulation of stress response， regulation of immune system processes， regulation of trauma response， and these proteins were at the center of the PPI network. ConclusionQSBLE may play a role in the treatment of HSP by regulating the expression of IGHG1， IGLV7-43， GSN， HSPD1 and other key proteins to affect immune-related biological processes.
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The present study aimed to explore the mechanism of the sweating of Dipsacus asper on content changes of triterpene sa-ponins by detecting the total triterpene saponins and the index component asperosaponin Ⅵ in the crude and sweated D. asper, and analyzing the differentially expressed proteins by isobaric tags for relative and absolute quantification(iTRAQ) combined with LC-MS/MS. After sweating, the content of total triterpene saponins decreased manifestly, while that of asperosaponin Ⅵ increased significantly. As revealed by the iTRAQ-LC-MS/MS analysis, 140 proteins with significant differential expression were figured out, with 50 up-regulated and 90 down-regulated. GO analysis indicated a variety of hydrolases, oxido-reductases, and transferases in the differential proteins. The results of activity test on two differentially expressed oxido-reductases were consistent with those of the iTRAQ-LC-MS/MS analysis. As demonstrated by the analysis of enzymes related to the triterpene saponin biosynthesis pathway, two enzymes(from CYP450 and UGT families, respectively, which are involved in the structural modification of triterpene saponins) were significantly down-regulated after sweating. The findings suggested that sweating of D. asper presumedly regulated triterpene saponins by affecting the expression of downstream CYP450 s and UGTs in the biosynthesis pathway of triterpene saponins of D. asper.
Subject(s)
Humans , Chromatography, Liquid , Dipsacaceae , Saponins , Sweating , Tandem Mass Spectrometry , TriterpenesABSTRACT
Objective To compare the differentially expressed proteins between cypermethrin-resistant and -sensitive Culex pipiens pallens, so as to unravel the mechanism underlying the resistance to cypermethrin in Cx. p. pallens. Methods A quantitative proteomic analysis was performed among cypermethrin-sensitive and -resistant isolates of Cx. p. pallens using isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with liquid chromatography with tandem mass spectrometry (LC-MS/MS). Results A total of 164 differentially expressed proteins were identified between cypermethrin-sensitive and -resistant isolates of Cx. p. pallens, including 54 up-regulated proteins and 110 down-regulated proteins. A large number of cuticular proteins, larval cuticular proteins, pupal cuticular proteins and cuticular structural constituent proteins, which are associated with cytoskeletal structure and components, were differentially expressed between cypermethrin-sensitive and -resistant isolates of Cx. p. pallens. Thirteen proteins, which were involved in energy production and conversion, translation, ribosomal structure and biogenesis, lipid transport and metabolism, post-translational modification, protein turnover, chaperones, cytoskeleton and intracellular transportation, were validated to be differentially expressed between cypermethrin-sensitive and -resistant isolates of Cx. p. pallens, which may serve as potential markers of cypermethrin resistance. Conclusion Multiple insecticide resistance mechanisms contribute to the resistance to cypermethrin in Cx. p. pallens, including cuticular resistance and metabolic resistance, and the cuticular protein genes and cytochrome P450 enzymes may play an important role in the resistance of Cx. p. pallens to cypermethrin.
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Objective: By applying the proteomic method, to analyze differences of protein expressions and signal pathways in veins at arteriovenous fistula in chronic kidney disease stage 5 (CKD5) patients with or without diabetes so as to explore the pathogenesis of high incidence of arteriovenous fistula functional incapacitation in CKD5 patients with diabetes. Methods: The protein expression RAW datasets of vascular access from CKD5 patients with or without diabetes in Proteomexchange Database were screened out and downloaded. Then the identified features were searched from UniProt/SwisssProt human proteins database through the software ProteomeDiscovery (PD). Then, the PD generated a file of quantitative proteins data. The significantly different proteins between two groups were screened out and analyzed by T-test or Adj T-test depending on homogeneity of variance. These significantly different proteins were enriched into different biological pathways through IPA analysis, GO enrichment analysis, and KEGG pathway analysis, which signifies these biological pathways are significantly different. Ultimately, the correlation of proteins in different biological pathway was analyzed by Spearman correlation test. Results: TheiTRAQ labeled proteins RAW datasets comparing the vessels from CKD5 patients with diabetes and without diabetes (PXD010883) were collected at first. After searching identified features again and disposing the data, a total of 120 significantly different proteins including 89 up-regulated proteins and 31 down-regulated proteins were screened out finally. Through GO, KEGG and IPA analyses, there were two significantly different biological pathways. On the one hand, the purine nucleoside monophosphate metabolic change and the ratio of ATP/AMP decrease were reflected in oxidative phosphorylation receding and AMP metabolism enhancing in CKD5 patients with diabetes. On the other hand, the muscle system process which played a vital role in VSM cells receded in CKD5 patients with diabetes. It was specific in the decrease of MYH11, CNN1 and so on. Excitingly, the significantly differential genes in the two pathway had a strong correlation. The results explained why CKD5 patients with diabetes always have cardiovascular disease. Conclusion: The ratio of ATP/AMP reduction caused by diabetes led to muscle function disorder of VSM cells, which explains the reason for the internal fistula functional incapacitation in CKD5 patients with diabetes.
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Objective@#To screen the deferentially expressed proteins in hippocampus and striatum in rat models of diabetes mellitus and normal SD rats, and to elucidate the effects of hyperglycemia on central nervous system.@*Methods@#SD rats were randomly divided into normal group(n=10)and hyperglycemia group(n=15); rat models of diabetes were induced by the high-fat and high-sugar diet combined with single intraperitoneal injection of streptozotocin. The hippocampus and striatum tissues from hyperglycemia rat and normal SD rat(control group) were selected as specimens. Isobaric tags for relative and absolute quantification(iTRAQ) technique and 2-dimensional liquid chromatography-tandem mass spectrometry(2D-LC-MS/MS) were used for identifying the deferentially expressed proteins. The deferentially expressed proteins were analyzed by bio-informatics for searching candidate proteins.Finally, the candidate proteins were verified by Western blotting.@*Results@#A total of 347 proteins showed significantly different expressions, which included 139 up-regulated proteins and 289 down-regulated proteins. These proteins included metabolic glutamate receptor 2, ferritin, leucine repeat protein and complex 2, which are related to Parkinson′s disease. KEEN search indicated that the pathway of dopaminergic synaptic, complement, gap junction, autophagic and Fcgamma R-mediated phagocytosis were involved with these significant differentially expressed proteins. The protein interaction network showed that ferrin, aldehyde/ketone reductase(ARK), Ca2+ -binding protein(CaBP), and protein tyrosine phosphatase(PTP) were located at the crossed nodes. The expression levels of ferrin, ARK, PTP, and CaBP, which were verified by Western blotting, and the results were consistent with those of the quantitative mass spectrometry(P<0.05).@*Conclusion@#Deferentially expressed proteins in hippocampus striatum of diabetic rats can be effectively screened by iTRAQ technique combined with 2D-LC-MS/MS. It may provide new clues for the mechanism of neurodegenerative disease.
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Objective To compare the difference of protein expression between the post-overwintering stage and the diapauses preparation stage in Culex pipiens pallens, so as to reveal the mechanisms underlying the overwintering diapause of Cx. pipienspallens. Methods A quantitative proteomic analysis was performed in Cx. pipiens pallens before and after overwintering diapause by using isobaric tags for relative and absolute quantification (iTRAQ) labeling. Results A total of 244 differentially expressed proteins were identified in Cx. pipiens pallens before and after overwintering diapause, including 126 up-regulated proteins and 118 down-regulated proteins. iTRAQ-based quantitative proteomic analysis revealed that these differentially expressed proteins were linked to function and energy production and conversion, lipid metabolism, remodeling of cytoskeleton, carbohydrate metabolism, protein transport, molecular chaperones, stress tolerance and metabolic enzymes. Conclusions This is the first study to identify the overwintering diapause-related proteins in Cx. pipiens pallens using proteomics tools, which reveals KEGG pathways and GO terms associated with the overwintering diapauses of Cx. pipiens pallens. Our findings provide additional understandings pertaining to the mechanisms underlying the overwintering diapauses of Cx. pipiens pallens.
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Objective To compare the difference of protein expression between the post-overwintering stage and the diapauses preparation stage in Culex pipiens pallens, so as to reveal the mechanisms underlying the overwintering diapause of Cx. pipienspallens. Methods A quantitative proteomic analysis was performed in Cx. pipiens pallens before and after overwintering diapause by using isobaric tags for relative and absolute quantification (iTRAQ) labeling. Results A total of 244 differentially expressed proteins were identified in Cx. pipiens pallens before and after overwintering diapause, including 126 up-regulated proteins and 118 down-regulated proteins. iTRAQ-based quantitative proteomic analysis revealed that these differentially expressed proteins were linked to function and energy production and conversion, lipid metabolism, remodeling of cytoskeleton, carbohydrate metabolism, protein transport, molecular chaperones, stress tolerance and metabolic enzymes. Conclusions This is the first study to identify the overwintering diapause-related proteins in Cx. pipiens pallens using proteomics tools, which reveals KEGG pathways and GO terms associated with the overwintering diapauses of Cx. pipiens pallens. Our findings provide additional understandings pertaining to the mechanisms underlying the overwintering diapauses of Cx. pipiens pallens.
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In this paper we discuss recent significant developments in the field of venom research, specifically the emergence of top-down proteomic applications that allow achieving compositional resolution at the level of the protein species present in the venom, and the absolute quantification of the venom proteins (the term "protein species" is used here to refer to all the different molecular forms in which a protein can be found. Please consult the special issue of Jornal of Proteomics "Towards deciphering proteomes via the proteoform, protein speciation, moonlighting and protein code concepts" published in 2016, vol. 134, pages 1-202). Challenges remain to be solved in order to achieve a compact and automated platform with which to routinely carry out comprehensive quantitative analysis of all toxins present in a venom. This short essay reflects the authors' view of the immediate future in this direction for the proteomic analysis of venoms, particularly of snakes.(AU)
Subject(s)
Animals , Poisons/analysis , Proteome , Proteomics , Snakes , Mass SpectrometryABSTRACT
Abstract In this paper we discuss recent significant developments in the field of venom research, specifically the emergence of top-down proteomic applications that allow achieving compositional resolution at the level of the protein species present in the venom, and the absolute quantification of the venom proteins (the term protein species is used here to refer to all the different molecular forms in which a protein can be found. Please consult the special issue of Jornal of Proteomics Towards deciphering proteomes via the proteoform, protein speciation, moonlighting and protein code concepts published in 2016, vol. 134, pages 1-202). Challenges remain to be solved in order to achieve a compact and automated platform with which to routinely carry out comprehensive quantitative analysis of all toxins present in a venom. This short essay reflects the authors view of the immediate future in this direction for the proteomic analysis of venoms, particularly of snakes.
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Digital PCR is an emerging analysis technology for absolute quantification after realtime-PCR. Through digital PCR, single DNA molecules are distributed into isolated reactions, and the product with fluorescence signal can be detected and analyzed after amplification. With the advantages of higher sensitivity and accuracy, digital PCR, independent of a standard curve, is developing rapidly and applied widely to the next generation sequencing and detection fields, such as gene mutation, copy number variation, microorganism, and genetically modified food. In this article, we reviewed the quantitative method and research progress of digital PCR technology in the main application fields.
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La producción del cultivo de papa en Colombia se puede afectar por infección con diferentes patógenos virales, entre ellos, el Potato yellow vein virus (PYVV) que puede reducir la producción entre el 30 % y 50%. PYVV se ha diagnosticado molecularmente usando RT-PCR convencional en hojas sintomáticas y no sintomáticas. Sin embargo, no hay reportes sobre la detección y distribución viral en diferentes órganos infectados por PYVV en las plantas que expresan síntomas y sin síntomas. El objetivo de esta investigación, fue detectar a PYVV por RT-PCR convencional con cebadores específicos y por qRT-PCR (tiempo real) utilizando Sondas TaqMan® y analizar la distribución viral en plantas de S. tuberosum grupo Phureja cv. Criolla Colombia (papa criolla). Se logró la detección del virus en todos los órganos analizados (foliolo, peciolo, tallo aéreo y subterráneo, pedúnculo floral, pétalo y antera) mediante ambas técnicas, sin embargo, qRT-PCR fue 100 veces más sensible que la técnica convencional. Adicionalmente, se realizó la cuantificación absoluta del gen de la proteína mayor de la cápside de PYVV (CP). Los resultados indican que cuando la planta no expresa síntomas (NS), hay una distribución homogénea del virus, con un promedio del número de copias del gen CP de 4.09×107±2.35×107; mientras que en plantas sintomáticas el título viral es mayor (6.82×108±1.74×108) y la distribución heterogénea en los órganos, con mayor acumulación en órganos de la zona aérea. Este es el primer informe sobre la detección de PYVV en diferentes órganos de papa por medio de tiempo real, incluyendo las anteras y pedúnculo floral. La información debe ser de utilidad para el diagnóstico de PYVV y para adelantar estudios sobre la biología del virus y la relación con el huésped y el vector. La información suministrada debe ser valiosa para agricultores y fitomejoradores, además para programas de indexado de plantas contra PYVV y en la certificación de semilla.
Potato yield in Colombia could be affected by the infection with different viral pathogens, among which, Potato vein yellow virus (PYVV) could reduce potato production by 30% to 50%. PYVV has been diagnosed molecularly in symptomatic and symptomless leaves samples by conventional RT-PCR. However, the PYVV detection and distribution in different organs of symptomatic and symptomless plants have not been reported until now. The aim of this research was to detect and analyze PYVV distribution in different organs of infected S. tuberosum group Phureja cv. Criolla Colombia (papa criolla) plants using conventional and real time qRT-PCR usindTaqMan® probes. It was achieved to detect the virus in all analyzed organs (leaflets, petiole, peduncle, anther, petals, aerial and underground stem) by both techniques; however, qRT-PCR was 100 times more sensitive than the conventional technique. Additionally, the absolute quantification of coat major protein gene (CP) was determined. The results shown that in non symptomatic plants (NS), PYVV was distributed homogenously with an average CP gene copy number of 4.09 × 107 ± 2.35 × 107, while in symptomatic moderate and severe plants (M) or (S) the viral load was greater (6.82×108±1.74×108) with an heterogeneous distribution regarding the organ and with greater accumulation in the aerial organs. The results presented in this study will be important for PYVV detection and further studies on the virus biology, host and vector relations. The information should be useful to farmers, breeders, indexing and seed certification programs.
Subject(s)
Crinivirus , Plant Diseases , Plant Viruses , Solanum tuberosum , Plants , Plants, EdibleABSTRACT
Data independent acquisition ( DIA ) is a novel MS scan mode for quantitative proteomics, acquiring all precursors as well as fragments without any loss of low abundant ions, and breaks the throughput limitation of product ion quantification by high-resolution MS. Here we developed three DIA methods on quadrupole-linear ion trap-Orbitrap ( Q-qIT-OT ) Tribrid MS, classic DIA, as well as novel wide isolation window SIM scan ( WiSIM)-DIA and full scan-DIA ( Full MS-DIA) . Quantitative analysis of 10 low abundant peptides spiked in Hela cell digest was performed by the three methods for linearity, reproducibility and sensitivity evaluation. The results showed that the LOQs reached amol level ( 14-435 amol ) with good linearity and effective MS/MS confirmation. WiSIM-DIA utilizes ultra-high resolution SIM scan for quantification complementary with classic DIA. The isolation window of Full MS-DIA was down to 3 amu, and the data could be directly used for database searching, thus realizing the integration of data dependent acquisition ( DDA) and DIA, and avoiding the limitation of using spectra library.
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The technology of Magnetic Resonance Spectroscopy(MRS) is a newly-developed mean for analyzing some specific nucleus and their compounds making use of the principles of magnetic resonance and the effects of chemical shift. Currently, among MRS applications, proton magnetic resonance spectroscopy (1HMRS) is the most widely applied one developed from single voxel to three-dimensional multi-voxel scanning technique. It provides a lot of important information for clinical studies. This article mainly reviews the methods for absolute quantification measurement of brain metabolites using multi-voxel MRS.
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This experiment was based on a full-length SAMDC sequence from the drought stress induced cDNA library of Cleistogenes songorica.Two methods,absolute quantification and relative quantification,were used to analyze the gene expression under different tissue and drought stress treatment by real-time quantitaive PCR experiments and were compared each other,while CsSAMDC gene was used as a case.Leaves and roots tissue were sampled from the plants at 0,4,6,8,and 10 d of drought stress and 1 and 4 d after rewatering.The 2-CT method was used to analyze the relative changes in gene expression from quantitative real-time PCR experiments.A standard curve was generated by the purified plasmid DNA.The primer concentration in the realtime PCR was optimized.All absolute quatification data were normalized by the normalization factor from the most stable house-keeping genes in C.songorica.Relative quantification results using 2-CT method showed that CsSAMDC transcripts increased 5.93?0.71 fold in roots,down regulated 0.62?0.13 fold in leaves,under drought stress.Absolute quantification results showed that CsSAMDC transcripts up-regulated in roots significantly under drought stress as 1ong as 8 to 1 0 days after drought stress,while down-regulated in both roots and leaves after drought stress within 6 days and 1 to 4 days after rewatering,compared with control.CsSAMDC transcipt showed similar tendency of gene expression compared by using relative and absolute quantification methods.