Structural and functional changes of bovine carbonic anhydrase as a consequence of temperature

The temperature dependence of the activity and structure of the enzyme carbonic anhydrase was studied. The Arrhenius plot shows a jump which is seen usually in proteins with more than one subunit or with one subunit but more than one domain. Since carbonic anhydrase has only one subunit with one domain, the fine conformational changes of the protein motifs could only be detected through circular dichroism polarimetry. It seems that the jump in Arrhenius plot is a result of some slight structural changes in the secondary and tertiary structures of the enzyme.     

Stability, structural and suicide inactivation changes of mushroom tyrosinase after acetylation by N-acetylimidazole

Modification (acetylation) of Tyr residues with N-acetylimidazole protects outstandingly mushroom tyrosinase (MT) from the suicide inactivation in the presence of its catecholic substrate, 4-[(4-methylbenzo) azo]-1,2-benzenediol. UV spectrophotometric experiments and differential scanning calorimetry (DSC) studies indicated a decrease in kinetic stability of the enzyme alongside with increase in its thermal stability as well as its stability against n-dodecyl trimethylammonium bromide as a denaturizing agent. Pace analysis resulted in standard Gibbs free energy values of 46.54 and 52.09 kJ/mol in the absence of denaturant for native and modified enzyme, respectively. Structural studies by circular dichroism (CD) spectrophotometry showed that modification did not have major impact on the secondary structure of MT; however, induced some changes in its tertiary structure. The near-UV CD results revealed that the modification had enhanced intramolecular van der Waals interactions in the enzyme structure, which was in coincidence with its thermodynamic stability.     

Rising the Persian Gulf Black-Lip Pearl Oyster to the Species Level: Fragmented Habitat and Chaotic Genetic Patchiness in <Emphasis Type="Italic">Pinctada persica</Emphasis>

Marine organisms with long pelagic larval stages are expected to exhibit low genetic differentiation due to their potential to disperse over large distances. Growing body of evidence, however, suggests that marine populations can differentiate over small spatial scales. Here we focused on black-lip pearl oysters from the Persian Gulf that are thought to belong to the Pinctada margaritifera complex given their morphological affinities. This species complex includes seven lineages that show a wide distribution ranging from the Persian Gulf (Pinctada margaritifera persica) and Indian Ocean (P. m. zanzibarensis) to the French Polynesia (P. m. cumingii) and Hawai’i (P. m. galtsoffi). Despite the long pelagic larval phase of P. m. persica, this lineage is absent from continental locations and can only be found on a few islands of the Persian Gulf. Mitochondrial COI-based analyses indicated that P. m. persica belongs to a clearly divergent ESU and groups with specimens from Mauritius (P. m. zanzibarensis). Microsatellite data, used here to assess the spatial scale of realized dispersal of Persian Gulf black-lip pearl oysters, revealed significant genetic structure among islands distant of only a few dozen kilometres. The scantiness of suitable habitats most likely restricted the distribution of this lineage originating the observed chaotic genetic patchiness. The hatchery-based enhancement performed in one of the sampled islands may also have affected population genetic structure. The long-term accumulation of genetic differences likely resulted from the allopatric divergence between P. m. persica and the neighbouring Indian Ocean black-lip pearl oysters.     

Bioproduction of phenylethanoid glycosides by plant cell culture of Scrophularia striata Boiss.: from shake-flasks to bioreactor

Phenylethanoid glycosides (PeG) are a class of polyphenols found in some plants that have pharmaceutical effects as anti-inflammatories and anti-oxidants. The presence of PeG (acteoside) in the aerial parts of Scrophularia striata Boiss. has been demonstrated. Considerable progress has been made using plant cell cultures to stimulate formation and accumulation of secondary metabolites. The present study optimized phenylethanoid production from shake flasks to bioreactor using a cell culture of S. striata. The optimal conditions for production of cell biomass by scale-up to a bioreactor were determined to be a pH of 4.8, air flow rate of 0.5–1.5 l min⁻¹, and mixing speed of 110–170 rpm at 25 ± 1 °C in darkness. Growth parameters and PeG production were measured and compared with the results from the shake flasks. The results showed that cell biomass was high in the bioreactor (15.64 g l⁻¹ DW) and in the shake flasks (14.16 g l⁻¹ DW). The acteoside content in the bioreactor was 1404.20 μg g⁻¹ DW, which is threefold higher than in the shake flasks (459.71 μg g⁻¹ DW). The echinacoside concentration in the bioreactor was 1449.39 μg g⁻¹, 1.36-fold lower than in the shake flasks (1973.03 μg g⁻¹ DW). This study established an efficient way for production of acteoside, the major PeG, in a bioreactor.

     

An integrated wavelength-shifting strategy for enhancement of microalgal growth rate in PMMA- and polycarbonate-based photobioreactors

This study investigates the spectral shifting of UV-A radiation, using fluorescent material, as a tool for enhancing Chlorella sp. growth rate in simulated photobioreactors made of UV-stabilized polycarbonate (PC) and poly (methyl methacrylate) (PMMA). The feasibility of using a fluorescent coating as a wavelength shifter layer, to shift UV-A radiation to the PAR range, was explored. For this purpose, a variety of concentrations of fluorescent dye dissolved in thermoplastic acrylic resin were prepared and used to coat PMMA and PC sheets which then were placed between the radiation source and the culture flask. Compared with the uncoated sheets, the panels coated with the wavelength shifter layer exhibited 74% and 45% (for PC and PMMA substrates, respectively) increase in biomass productivity during the same culture period. It was also found that the elimination of UV-A radiation increased chlorophyll a content of the cells.     

Correlation between the papanicolaou stain and the Wright-Giemsa stain in body fluids: a quality assurance study

OBJECTIVE: To compare the use of Papanicolaou and Wright-Giemsa stains for the evaluation of body fluids in cytology and hematology laboratories and determine whether other factors account for discrepancies in diagnosis. STUDY DESIGN: We retrospectively reviewed cytopathology reports of peritoneal, pleural, and cerebrospinal fluids received by hematology and cytology laboratories for 1 year. Cases were divided into 3 categories-benign, atypical, and malignant--and slides of discrepant diagnoses were reviewed. RESULTS: During this period, 198 of 3212 (0.61%) cases received by the hematology laboratory and 252 of 4402 (0.57%) cases received by the cytology laboratory were diagnosed as malignant or atypical. Of 3212 cases simultaneously received by the cytology and hematology laboratories, 17 diagnosed as malignant by hematology were diagnosed benign by cytology (sensitivity 96%). Sixteen cases diagnosed as malignant by cytology were diagnosed as benign by hematology (sensitivity 97%). No benign cases were diagnosed as malignant (specificity 100%). Review of the glass slides of the discrepant cases revealed 8 cases undercalled by hematology and 7 cases undercalled by cytology. CONCLUSION: Papanicolaou stain is superior for carcinoma and Wright-Giemsa stain for hematopoietic disorders, but used together they may reduce false negative results. Delays in processing, staining technique, and interobserver variability contribute to discrepancy.     

Increased lignan biosynthesis in the suspension cultures of Linum album by fungal extracts

Linum album accumulates anti-tumor podophyllotoxin (PTOX) and its related lignans, which were originally isolated from an endangered species Podophyllum. In the present study, we examined the effects of five fungal extracts on the production of lignans in L. album cell cultures. Fusarium graminearum extract induced the highest increase of PTOX [143 μg g⁻¹ dry weight (DW) of the L. album cell culture], while Rhizopus stolonifer extract enhanced the accumulation of lariciresinol up to 364 μg g⁻¹ DW, instead of PTOX. Typical elicitors, such as chitin, chitosan, or methyl jasmonate (MeJA), were shown to be less effective in lignan production in L. album cell cultures. These results verified the advantages of fungal extracts to increase lignan production in L. album cell culture, and suggested potential on-demand metabolic engineering of lignan biosynthesis using differential fungal extracts.     

Unraveling salinity stress responses in ancestral and neglected wheat species at early growth stage: A baseline for utilization in future wheat improvement programs

In this study, we analyzed the behavior of several neglected, ancestral, and domesticated wheat genotypes, including Ae. triuncialis, Ae. neglecta, Ae. caudata, Ae. umbellulata, Ae. tauschii, Ae. speltoides, T. boeoticum, T. urartu, T. durum, and T. aestivum under control and salinity stress to assess the mechanisms involved in salinity tolerance. Physiological and biochemical traits including root/shoot biomasses, root/shoot ion concentrations, activity of antioxidant enzymes APX, SOD, and GXP, and the relative expression of TaHKT1;5, TaSOS1, APX, GXP, and MnSOD genes were measured. Analysis of variance (ANOVA) revealed significant effects of the salinity treatments and genotypes for all evaluated traits. Salinity stress (350 mM NaCl) significantly decreased root/shoot biomasses, K+ concentration in root/shoot, and root/shoot K+/Na+ ratios. In contrast, salinity stress significantly increased Na+ concentration in root and shoot, activity of antioxidant enzymes (APX, SOD, and GPX) and relative expression of salt tolerance-related genes (TaHKT1;5, TaSOS1, APX, GPX, and MnSOD). Based on heat map and principal component analysis, the relationships among physiological traits and relative expression of salt-responsive genes were investigated. Remarkably, we observed a significant association between the relative expression of TaHKT1;5 with root K+ concentration and K+/Na+ ratio and with TaSOS1. Taken together, our study revealed that two neglected (Ae. triuncialis) and ancestral (Ae. tauschii) wheat genotypes responded better to salinity stress than other genotypes. Further molecular tasks are therefore essential to specify the pathways linked with salinity tolerance in these genotypes.     

Gaussian process regression model for normalization of LC-MS data using scan-level information

Background

Differences in sample collection, biomolecule extraction, and instrument variability introduce bias to data generated by liquid chromatography coupled with mass spectrometry (LC-MS). Normalization is used to address these issues. In this paper, we introduce a new normalization method using the Gaussian process regression model (GPRM) that utilizes information from individual scans within an extracted ion chromatogram (EIC) of a peak. The proposed method is particularly applicable for normalization based on analysis order of LC-MS runs. Our method uses measurement variabilities estimated through LC-MS data acquired from quality control samples to correct for bias caused by instrument drift. Maximum likelihood approach is used to find the optimal parameters for the fitted GPRM. We review several normalization methods and compare their performance with GPRM.

Results

To evaluate the performance of different normalization methods, we consider LC-MS data from a study where metabolomic approach is utilized to discover biomarkers for liver cancer. The LC-MS data were acquired by analysis of sera from liver cancer patients and cirrhotic controls. In addition, LC-MS runs from a quality control (QC) sample are included to assess the run to run variability and to evaluate the ability of various normalization method in reducing this undesired variability. Also, ANOVA models are applied to the normalized LC-MS data to identify ions with intensity measurements that are significantly different between cases and controls.

Conclusions

One of the challenges in using label-free LC-MS for quantitation of biomolecules is systematic bias in measurements. Several normalization methods have been introduced to overcome this issue, but there is no universally applicable approach at the present time. Each data set should be carefully examined to determine the most appropriate normalization method. We review here several existing methods and introduce the GPRM for normalization of LC-MS data. Through our in-house data set, we show that the GPRM outperforms other normalization methods considered here, in terms of decreasing the variability of ion intensities among quality control runs.