Refolding and simultaneous purification by three-phase partitioning of recombinant proteins from inclusion bodies

Many recombinant eukaryotic proteins tend to form insoluble aggregates called inclusion bodies, especially when expressed in Escherichia coli. We report the first application of the technique of three-phase partitioning (TPP) to obtain correctly refolded active proteins from solubilized inclusion bodies. TPP was used for refolding 12 different proteins overexpressed in E. coli. In each case, the protein refolded by TPP gave either higher refolding yield than the earlier reported method or succeeded where earlier efforts have failed. TPP-refolded proteins were characterized and compared to conventionally purified proteins in terms of their spectral characteristics and/or biological activity. The methodology is scaleable and parallelizable and does not require subsequent concentration steps. This approach may serve as a useful complement to existing refolding strategies of diverse proteins from inclusion bodies.     

Microvascular PO2 during extreme hemodilution with hemoglobin site specifically PEGylated at Cys-93(beta) in hamster window chamber

The oxygen transport capacity of nonhypertensive polyethylene glycol (PEG)-conjugated hemoglobin solutions were investigated in the hamster chamber window model. Microvascular measurements were made to determine oxygen delivery in conditions of extreme hemodilution [hematocrit (Hct) 11%]. Two isovolemic hemodilution steps were performed with a 6% Dextran 70 (70-kDa molecular mass) plasma expander until Hct was 35% of control. Isovolemic blood volume exchange was continued using two surface-modified PEGylated hemoglobins (P5K2, P(50) = 8.6, and P10K2, P(50) = 8.3; P(50) is the hemoglobin Po(2) corresponding to its 50% oxygen saturation) until Hct was 11%. P5K2 and P10K2 are PEG-conjugated hemoglobins that maintain most of the hemoglobin allosteric properties and have a cooperativity index of n = 2.2. The effects of these molecular solutions were compared with those obtained in a previous study using MP4, a PEG-modified hemoglobin whose P(50) was 5.4 and cooperativity was 1.2 (Tsai et al., Am J Physiol Heart Circ Physiol 285: H1411-H1419, 2003). Tissue oxygen levels were higher after P5K2 (7.0 +/- 2.5 mmHg) and P10K2 (6.3 +/- 2.3 mmHg) versus MP4 (1.7 +/- 0.5 mmHg) or the nonoxygen carrier Dextran 70 (1.3 +/- 1.2 mmHg). Microvascular oxygen delivery was higher after P5K2 and P10K2 (2.22 and 2.34 ml O(2)/dl blood) compared with MP4 (1.41 ml O(2)/dl blood) or Dextran 70 (0.90 ml O(2)/dl blood); however, all these values were lower than control (7.42 ml O(2)/dl blood). The total hemoglobin in blood was similar in all cases; therefore, the improvement in tissue Po(2) and oxygen delivery appears to be due to the increased cooperativity of the new molecules.     

Carbon quality and nutrient status drive the temperature sensitivity of organic matter decomposition in subtropical peat soils

Estimates of gaseous carbon (C) fluxes in wetlands are heavily based on temperature. However, isolating specific effects of temperature on anaerobic C processing from other controls (C quality and nutrients) has proven difficult. Here, we test the hypothesis that temperature sensitivity of soil organic matter (SOM) decomposition is more influenced by C quality than nutrient availability in subtropical freshwater, sawgrass (Cladium jamaicense)-based peats. Carbon age (characterized by depth: 0–10 and 10–20 cm) was used as a surrogate of C quality while two sites were selected with contrasting levels of nutrient (P) availability. In anaerobic laboratory incubations temperature was increased in 5 °C steps to assess the proportion of C available at a given temperature (i.e. thermo-labile C) as productions of gaseous (CO₂ and CH₄) and dissolved organic C (DOC) fractions. Thermo-labile C increased 3.1–3.6 times from 15 °C to 30 °C in all soils. Disproportionate increase in the production of gaseous forms versus DOC as well as CH₄:CO₂ was observed with warming. Observed Q₁₀ values followed the trend of CH₄ (~14) ? CO₂ (~2.5) > DOC (~1.7) and temperature sensitivity was more dependent on C quality than nutrient availability over the entire temperature range. Spectral analysis indicated more bio-available DOC production at higher temperature. Regression analysis also indicated that C quality primarily influenced SOM decomposition at lower temperature, while at higher temperature nutrient limitation dominantly controlled SOM decomposition. These findings confirm the role of C quality in temperature sensitivity of warm peat soils, but also indicate an increased importance of nutrient limitation at higher temperature.     

Deciphering the Mechanisms of Endophyte-Mediated Biofortification of Fe and Zn in Wheat

An investigation was carried out to understand the mechanism(s) underlying enhanced Fe or Zn uptake in low Fe–Zn accumulator wheat genotype 4HPYT-414, due to inoculation of siderophore-producing and zinc-solubilizing endophytes—Arthrobacter sulfonivorans DS-68 and Arthrobacter sp. DS-179. Root anatomical features, using transmission electron microscopy (TEM), qualitative and quantitative aspects of production of organic acids and sugars in root exudates, and expression of TaZIP genes were analysed to relate to endophyte-mediated higher concentrations of Fe and Zn in the roots and shoots of wheat plants. TEM studies revealed that the endodermis, cortical region, root hair extension, xylem and xylem vessels, pericycle and vascular bundles were more pronounced and thicker in inoculated treatments, as compared to control. The organic acid profile of root exudates revealed five types of organic acids, with citric acid being predominant. Inoculation of A. sulfonivorans and Arthrobacter sp. brought about 5- and eightfold increases in the amounts of acids, respectively, as compared to control, particularly citric acid, succinic acid and acetic acid. Among the four TaZIP genes targeted, expression was achieved only for TaZIP3 and TaZIP7 genes, which showed 1–2 fold increases in the inoculated treatments. The results clearly indicated that the endophyte-mediated overexpression of TaZIP3 and TaZIP7 genes in roots and shoots, and the observed anatomical and exudate changes were acting synergistically in facilitating higher Fe and Zn translocation in roots and shoots.     

Horizontal acquisition of multiple mitochondrial genes from a parasitic plant followed by gene conversion with host mitochondrial genes

Background  

Horizontal gene transfer (HGT) is relatively common in plant mitochondrial genomes but the mechanisms, extent and consequences of transfer remain largely unknown. Previous results indicate that parasitic plants are often involved as either transfer donors or recipients, suggesting that direct contact between parasite and host facilitates genetic transfer among plants.     

Salicylic Acid-Mediated Regulation of Morpho-Physiological and Yield Attributes of Wheat and Barley Plants in Deferring Salinity Stress

Salinity has been observed to be a global problem that impede the physiological characteristics of plants. Salicylic acid (SA) as a phytohormone play multifaceted role in plants in terms of development as well as stress management. The current study was conducted to evaluate the effect of salinity and salicylic acid on the performance of wheat and barley plants under field experimentation followed by on-farm study to validate the results. This research was firstly conducted in a 4-year research barley field (2012–2013 and 2013–2014) and wheat (2014–2015 and 2015–2016) and subsequently in an on-farm research in four places (2017–2018). Results depicted that salinity decreased plant yield components and altered ion concentrations (Na⁺/K⁺) causing reduced grain and biological yield. However, SA foliar application induced yield components, especially grain number of plants in both years in non-saline and saline conditions. Exogenously SA application not only led to higher grain yield of barley and wheat but also significantly improved their salt tolerance. Our findings revealed that optimum SA concentrations for achieving highest barley yield were 0.85 and 0.78 mM under saline and non-saline conditions, respectively, while on-farm scale studies observed that foliar application of SA increased grain and biological yield of wheat in Ardakan, Ashkzar (saline soil and water) and Mehrabad (non-saline field) regions. There was no significant effect in Tijerd, a completely non-saline field. The grain yields were higher in SA-treated Ardakan, Ashkzar, and Mehrabad plants in field by 19, 16, and 15%, respectively. Based on present detailed studies, it was concluded that SA improved salinity tolerance and increased crop yield. So, optimum concentration (1.0–1.5 mM) with proper time application (double ridges), SA increased wheat and barley yields up to 20%. Therefore, SA priming could be used as a potent strategy to cope up salinity stress from plants.

     

The Potential of Canna lily for Wastewater Treatment Under Indian Conditions

Low cost treatment of polluted wastewater has become a serious challenge in most of the urban areas of developing countries. The present study was undertaken to investigate the potential of Canna lily towards removal of carbon, nitrogen, and phosphorus from wastewater under sub-tropical conditions. A constructed wetland (CW) cell supporting vegetative layer of Canna lily was used to treat wastewater having high strength of CNP. Removal of biological oxygen demand (BOD₃) and chemical oxygen demand (COD) varied between 69.8–96.4% and 63.6–99.1%, respectively. C. lily could efficiently remove carbon from a difficult to degrade wastewater at COD:BOD ratio of 24.4. Simultaneous reduction in TKN and nitrate pointed to good nitrification rates, and efficient plant assimilation as the dominant nutrient removal mechanism in the present study. Suitable Indian agro-climatic conditions favored plant growth and no evident stress over the Canna plant was observed. High removal rate of 809.8 mg/m²-day for TKN, 15.0 mg/m²-day for nitrate, and 164.2 mg/m²-day for phosphate suggests for a possible use of Canna-based CW for wastewater treatment for small, rural, and remote Indian communities.     

Serotonin released from amacrine neurons is scavenged and degraded in bipolar neurons in the retina

Among mental disorders, mental retardation has been shown to be caused by various factors including a large array of genetic mutations. On the basis of remarkable progress, the emerging view is that defects in the regulation of synaptic activity and morphogenesis of dendritic spines are apparently common features associated with mutations in several genes implicated in mental retardation. In this review, we will discuss X-linked MR-related gene products that are potentially involved in the normal structure and function of the synapses, with a particular focus on pre- and/or post-synaptic plasticity mechanisms. Progress in understanding the underlying conditions leading to mental retardation will undoubtedly be gained from a closer collaboration of geneticists, physiologists and cognitive neuroscientists, which should enable the establishment of standardized approaches.