DNA extraction from crayfish exoskeleton

Crayfish exoskeleton (CE) samples are generally less invasive and easy to be collected. However, it is difficult to extract DNA from them. This study was intended to investigate CE as a DNA source and design an easy and efficient DNA extraction protocol for polymerase chain reactions. Specific primer pair (PPO-F, PPO-R) was used to amplify extracted DNA from CE, and compared to crayfish tail muscle DNA sample. Moreover, seven microsatellites markers were used to amplify the CE DNA samples set. Since the extracted DNA from CE is suitable for gene amplification, the results present usefulness of CE as an easy and convenient DNA source for PCR-based population genetic research.     

Phase resetting analysis of high potassium epileptiform activity in CA3 region of the rat hippocampus

The theory of phase resetting can reveal important information about the dynamic behavior of a periodic system when a single brief stimulus is applied to that system at the appropriate time. Phase resetting studies have revealed the existence in some biological systems of a vulnerable stimulus window generating desynchronization and suppression of the activity. The objective of this study was to test the hypothesis that a "singular" stimulus could annihilate this activity. Perfusion with the high-K solution produced synchronous, quasi-periodic population bursts with inter-burst interval of ~0.8-1.5 seconds. A single 0.1 ms duration anodic pulse of programmable delay and magnitude was applied to the somatic layer of the CA3 pyramidal cells. Three types of phase-resetting behavior were observed: (1) Weak resetting with little or no effect on the timing of the subsequent burst, (2) Strong resetting where the applied current pulse delayed the next event by one time period, (3) Singular behavior where the applied pulse partially or completely suppressed the subsequent bursting. The singular stimulus parameter window, however, was very narrow making it difficult to generate the singular behavior reliably. Nevertheless, the results indicate that singularities exist for high potassium neural activity and that a well timed pulse applied with the right amplitude can suppress this activity. This study suggests that phase resetting of a population of neurons is possible for quasi-periodic interictal activity and similar techniques could be applied to the control of epileptic seizures.     

Constructions of expression vectors of polyhydroxybutyrate-co-hydroxyvalerate (PHBV) and transient expression of transgenes in immature oil palm embryos

Polyhydroxybutyrate-co-hydroxyvalerate (PHBV) is a polyhydroxyalkanoate (PHA) bioplastic group with thermoplastic properties is thus high in quality and can be degradable. PHBV can be produced by bacteria, but the process is not economically competitive with polymers produced from petrochemicals. To overcome this problem, research on transgenic plants has been carried out as one of the solutions to produce PHBV in economically sound alternative manner. Four different genes encoded with the enzymes necessary to catalyze PHBV are bktB, phaB, phaC and tdcB. All the genes came with modified CaMV 35S promoters (except for the tdcB gene, which was promoted by the native CaMV 35S promoter), nos terminator sequences and plastid sequences in order to target the genes into the plastids. Subcloning resulted in the generation of two different orientations of the tdcB, pLMIN (left) and pRMIN (right), both 17.557 and 19.967 kb in sizes. Both plasmids were transformed in immature embryos (IE) of oil palm via Agrobacterium tumefaciens. Assays of GUS were performed on one-week-old calli and 90% of the calli turned completely blue. This preliminary test showed positive results of integration. Six-months-old calli were harvested and RNA of the calli were isolated. RT-PCR was used to confirm the transient expression of PHBV transgenes in the calli. The bands were 258, 260, 315 and 200 bp in size for bktB, phaB, phaC and tdcB transgenes respectively. The data obtained showed that the bktB, phaB, phaC and tdcB genes were successfully integrated and expressed in the oil palm genome.     

In vitro selection of DNA-cleaving deoxyribozyme with site-specific thymidine excision activity

Single-nucleotide polymorphisms, either inherited or due to spontaneous DNA damage, are associated with numerous diseases. Developing tools for site-specific nucleotide modification may one day provide a way to alter disease polymorphisms. Here, we describe the in vitro selection and characterization of a new deoxyribozyme called F-8, which catalyzes nucleotide excision specifically at thymidine. Cleavage by F-8 generates 3′- and 5′-phosphate ends recognized by DNA modifying enzymes, which repair the targeted deoxyribonucleotide while maintaining the integrity of the rest of the sequence. These results illustrate the potential of DNAzymes as tools for DNA manipulation.     

Differential expression of specific cellular defense proteins in rat hypothalamus under simulated microgravity induced conditions: Comparative proteomics

Microgravity severely halts the structural and functional cerebral capacity of astronauts especially affecting their brains due to the stress produced by cephalic fluid shift. We employed a rat tail suspension model to substantiate simulated microgravity (SM) in brain. In this study, comparative mass spectrometry was applied in order to demonstrate the differential expression of 17 specific cellular defense proteins. Gamma‐enolase, peptidyl‐prolyl cis‐trans isomerase A, glial fibrillary acidic protein, heat shock protein HSP 90‐alpha, 10 kDa heat shock protein, mitochondrial, heat shock cognate 71 kDa protein, superoxide dismutase 1 and dihydropyrimidinase‐related protein 2 were found to be upregulated by HPLC/ESI‐TOF. Furthermore, five differentially expressed proteins including 60 kDa heat shock protein, mitochondrial, heat shock protein HSP 90‐beta, peroxiredoxin‐2, stress‐induced‐phosphoprotein, and UCHL‐1 were found to be upregulated by HPLC/ESI‐Q‐TOF MS. In addition, downregulated proteins include cytochrome C, superoxide dismutase 2, somatic, and excitatory amino acid transporter 1 and protein DJ‐1. Validity of MS results was successfully performed by Western blot analysis of DJ‐1 protein. This study will not only help to understand the neurochemical responses produced under microgravity but also will give future direction to cure the proteomic losses and their after effects in astronauts.     

Climate-Induced Elevational Range Shifts and Increase in Plant Species Richness in a Himalayan Biodiversity Epicentre

Global average temperature increase during the last century has induced species geographic range shifts and extinctions. Montane floras, in particular, are highly sensitive to climate change and mountains serve as suitable observation sites for tracing climate-induced biological response. The Himalaya constitute an important global biodiversity hotspot, yet studies on species’ response to climate change from this region are lacking. Here we use historical (1849–50) and the recent (2007–2010) data on temperature and endemic species’ elevational ranges to perform a correlative study in the two alpine valleys of Sikkim. We show that the ongoing warming in the alpine Sikkim Himalaya has transformed the plant assemblages. This study lends support to the hypothesis that changing climate is causing species distribution changes. We provide first evidence of warmer winters in the region compared to the last two centuries, with mean temperatures of the warmest and the coldest months may have increased by 0.76±0.25°C and 3.65±2°C, respectively. Warming-driven geographical range shifts were recorded in 87% of 124 endemic plant species studied in the region; upper range extensions of species have resulted in increased species richness in the upper alpine zone, compared to the 19^th century. We recorded a shift of 23–998 m in species’ upper elevation limit and a mean upward displacement rate of 27.53±22.04 m/decade in the present study. We infer that the present-day plant assemblages and community structure in the Himalaya is substantially different from the last century and is, therefore, in a state of flux under the impact of warming. The continued trend of warming is likely to result in ongoing elevational range contractions and eventually, species extinctions, particularly at mountaintops.     

Characterization of centchroman binding protein in plasma of rhesus monkey (Macaca mulatta)

Centchroman, a nonsteroidal antifertility agent was studied for its binding to monkey (Macaca mulatta) plasma proteins using charcoal adsorption and electrophoretic techniques. ¹⁴C-centchroman showed a low affinity binding and did not compete for ³H-cortisol or ³H-DHT binding sites in plasma. ¹⁴C-centchroman binding protein was heat stable in nature and showed the electrophoretic pattern similar to that of albumin (Rf 0.70). Thus centchroman binds to albumin in monkey plasma which can be suggested as carrier protein for this contraceptive agent.     

Breeding lines of the Indian mega-rice variety,MTU 1010, possessing protein kinase <Emphasis Type="Italic">OsPSTOL</Emphasis> (<Emphasis Type="Italic">Pup1</Emphasis>), show better root system architecture and higher yield in soils with low phosphorus

MTU 1010 is a high-yielding mega-variety of rice grown extensively in India. However, it does not perform well in soils with low phosphorus (P) levels. With an objective to improve MTU 1010 for tolerance to low soil P, we have transferred Pup1, a major quantitative trait locus (QTL) associated with tolerance from another mega-variety, Swarna, through marker-assisted backcross breeding (MABB). Foreground selection of the F₁ and backcross plants was performed with the co-dominant, closely linked CAPS marker, K20-2, while two flanking markers RM28011 and RM28157 were utilized for recombinant selection. At each backcross generation, positive plants were also analyzed with a set of 85 parental polymorphic SSR markers to identify the QTL-positive plants possessing maximum introgression of MTU 1010 genome. At BC₂F₁, the best backcross plant was selfed to generate BC₂F₂s. Among them, the plants homozygous for Pup1 (n?=?22) were reconfirmed using the functional marker for Pup1, viz., K46-1, and they were advanced through pedigree method of selection until BC₂F₆ generation. A total of five elite BC₂F₆ lines, possessing Pup1 and phenotypically similar to MTU 1010, were screened in the low soil P plot and normal plot (with optimum soil P levels) during wet season, 2016. All the selected lines showed better performance under low P soil with more number of productive tillers, better root system architecture, and significantly higher yield (>?390%) as compared to MTU 1010. Further, under normal soil, the lines were observed to be similar to or better than MTU 1010 for most of the agro-morphological traits and yield. This study represents the successful application of marker-assisted selection for improvement of tolerance to low soil P in a high-yielding Indian rice variety.     

5-Aminolevulinic Acid-Induced Heavy Metal Stress Tolerance and Underlying Mechanisms in Plants

Journal of Plant Growth Regulation - Plants face different types of biotic and abiotic stresses during their life span. Heavy metal (HM) stress is considered as one of the most challenging and...     

Improvement of blast resistance of the popular high-yielding,medium slender-grain type,bacterial blight resistant rice variety,Improved Samba Mahsuri by marker-assisted breeding

Improved Samba Mahsuri (ISM) is a popular, high-yielding, bacterial blight resistant rice variety possessing medium-slender grain type. As ISM is highly susceptible to blast disease of rice, through the present study we have transferred two major blast resistance genes, Pi2 and Pi54 into the elite variety by marker-assisted backcross breeding. The two blast resistance genes were transferred to ISM through sets of backcrosses. In every backcross generation, PCR-based markers, specific for the blast resistance genes (Pi2 and Pi54) and bacterial blight resistance genes (Xa21, xa13 and xa5) were utilized for foreground selection, while a set of 144 parental polymorphic SSR markers were used for background selection and backcrossing was carried out until BC₂ generation. A solitary BC₂F₁ plant possessing Pi2 or Pi54 along with Xa21, xa13 and xa5 and >?90% recovery of ISM genome was selected from the two sets of backcrosses were crossed and the intercross F₁s (ICF₁s) thus obtained were selfed to generate ICF₂s. Homozygous ICF₂ plants carrying all the five resistance genes were identified through markers and advanced through selfing till ICF₅ generation by adopting pedigree method of selection. Three best lines at ICF₅, possessing excellent resistance against bacterial blight and blast and closely resembling or superior to ISM in terms of grain quality: yield and agro-morphological traits have been identified and advanced for multi-location trials.