DNA Persistence and Preservation Following Environmental Insult

This research was conducted to provide empirical evidence to supplement advice available to the forensic community for the collection of muscle tissue for forensic analysis. This type of collection is normally carried out to determine the identity of individuals following mass disasters, such as plane crashes or natural disasters. DNA degradation was assessed in two model organisms, pig and rabbit (with human DNA as a control), over various time points. Rabbit recombination activating gene (RAG 1) was aligned to identify conserved regions in pig, rabbit and human. Primers were designed and optimised to create a 4-plex PCR multiplex that can amplify 70 bp, 194 bp, 305 bp and 384 bp in three species. The 4-plex multiplex was found to work efficiently in all three species down to 0.3 ng of DNA template. The multiplex was used to assess whether DNA degradation can be predicted by accumulated degree-days (ADD), which provides a measure of both time and temperature. A series of field studies were performed to assess DNA persistence in pig and rabbit soft muscle tissues using a combination of whole animals, suspended muscle tissues (insect activity free) and muscle fragments. Field studies were carried out in: August-September 2009; February-May 2010; May-June 2010; June-July 2010 and September-November 2010. Soft muscle tissue samples were collected at different ADD. 4-plex multiplex results showed that DNA was more persistent in pig tissues compared to rabbit tissues. In the September 2010 experiments, full multiplex amplification was obtained from rabbit until 137 ADD (whole carcases) and 210 ADD (body fragments and suspended tissues), while in the August 2009 experiments, full multiplex amplification was obtained until 112 ADD (whole carcases and body fragments) and until 141 ADD (suspended tissues). In the June 2010 experiments, full multiplex amplification was possible until 64 ADD. Pig whole carcases which were placed in the field in February 2010, showed multiplex amplification until day 90 (603 ADD), followed by September 2010 (until day 44 (490 ADD)) and May 2010 (until day 27 (338 ADD)). During the September 2010 project, body fragments produced full amplification until muscles were collected (342 ADD), while in case of whole carcases and suspended tissues; the amplification was possible until 490 ADD. There was complete failure of amplification of 305 bp and 384 bp in pig whole carcases after 342 ADD, while in suspended tissues, the amplification of 305 bp and 384 bp was possible until 420 ADD. The statistical analysis showed that amplification success of larger amplicons (194 bp, 305 bp and 384 bp) reduces with increase in ADD in pig and rabbit whole carcases, body fragments and suspended tissues while 70 bp was more persistence. The results showed that there was no significant difference in DNA persistence between whole carcases verses suspended tissues (Z=0.57, p>0.05) and whole carcases verses body fragments (Z=1.71, p>0.05), There was however a significant difference (Z=2.31, p<0.05) in DNA persistence in suspended tissues and body fragments with increase in ADD. The results from field experiments suggested that muscle tissues, if available, should be collected for DNA profiling, since even if degraded, a profile can be obtained. The results also suggested that the isolation of tissues from insect activity as quickly as possible (even if immediate storage is not possible) may be beneficial for DNA persistence. Seasonal variation in DNA persistence was observed due to maggot mass growth which increases carcase decomposition and ultimately effect on DNA persistence. Controlled incubation experiments were also performed at 27 °C, 37 °C and 47 °C until 21 days to assess DNA persistence, as these temperatures were not available under field conditions. The results showed that the amplification of 70 bp was more persistent compared to larger amplicons (194 bp, 305 bp and 384 bp). The drop-out in amplification of larger amplicons occurred more rapidly in samples incubated under laboratory conditions compared to the field samples. The statistical analysis showed species, ADD and temperature have strong effect (p<0.05) on DNA persistence under controlled conditions. The appearance of 70 bp amplicons in all samples collected from field and in most samples from controlled incubation experiments suggested that soft muscle tissues exposed to different environments can be used to perform SNP analysis. The full 4-plex multiplex amplification obtained from rabbit and pig preserved and dehydrated samples suggested that 96% ethanol, cell lysis solution (with and without 1% sodium azide) and dehydration can be used to preserve fresh and partially decomposed soft muscle tissues at room temperature for one year. The drop-out in amplification of larger amplicons in tissues preserved in 10% buffered formalin suggested that formalin was not suitable for long term storage. This system should therefore be considered as an additional method during Disaster victim identification (DVI) work to preserve fresh and partially decomposed samples. This study also suggested that the developed multiplex (4-plex) can be used to assess DNA persistence in human decomposing bodies and in experimental studies

Microscopic detection of chloroplast transgenic plastids using fluorescent probe

Background: Fluorescent marker genes have modernized many areas of molecular biology, specifically in plant biotechnology and genetic engineering studies. The use of fluorescent proteins permits the scientists to purify the desired clones visually in transformation work. Green fluorescent protein (gfp) derived from Aequorea victoria has been the most common and favorite fluorescent marker which is being widely used as a visual selection marker gene. It can be easily visualized under UV light without the involvement of any substrate and is non-destructive as well.Method: A species-specific chloroplast transformation vector was constructed with gfp as a fluorescent marker gene. The recombinant vector was biolistically integrated in tobacco plastome and transgenic cells were initially screened on spectinomycin containing regeneration medium.Results: The successful plastome integration was verified by using cellular DNA from drug resistant clones in PCR and southern blotting. The expression of gfp in transplastomic clones was microscopically investigated using simple florescent as well as confocal laser scanning microscopes.Conclusion: Regeneration of transgenic plants was significantly helped by visual identification of fluorescent at different stages of development, also enabling to identify the homozygous and heterozygous tissues. No toxic effect of the gfp was observed and lack of toxicity as maintained by normal phenotypic performance of plants.

Time Management and its Application in Academic Life: A Study of Omani Students at Dhofar University, Sultanate of Oman

The basic aim of this study is to identify and discuss the awareness of Time Management (TM) and its application in the academic life of Omani Students in English Unit, Dhofar University, Sultanate of Oman. This paper also inculcates and indoctrinates the importance of TM in students’ practical life. It has been observed by the researchers that students complain about the scarcity of time rather than lack of time. The study upholds that pedagogically, TM and its applications in EFL scenario are crucial. Thus the paper advocates that students’ sufferings can be subsided by enhancing their TM skills. This paper finds out that the most of the Omani students at the University of Dhofar suffer from Mismanagement of Time due to lack of focus on study skills.The researchers also give fruitful suggestions in order to overcome the confronted problems such as procrastination, distractions and so on.Keywords: TM: Time Management, Study Skills, Omani Students, EFL, Procrastination, Dhofar University

Synthons for supramolecular assemblies: Synthesis of new triazine-core polyhydroxylated and multi-N-donor compounds

The synthesis of new triazine-core polyhydroxylated and multi-N-donor compounds are being reported. The reaction of 2,4,6-tris(4-n-butylaminophenyl)triazine (1) with cyanuric chloride provided star-shaped fragment (2) with six reactive chloro-substituents. Upon treatment of this fragment with bis(2-hydroxyethyl)amine (3a) and bis(2-pyridylmethyl)amine (3b), functionalized polyhydroxylated (4a) and multi-N-donor compounds (4b) were obtained in high yields through nucleophilic aromatic substitution. These functionalized compounds are expected to have unique applications in supramolecular self-assembly and material chemistry

SODIUM AND CALCIUM LEVELS IN PATIENTS WITH CIRRHOSIS OF THE LIVER AND ITS CORRELATION WITH HEPATIC ENCEPHALOPATHY

Introduction: Patients with chronic liver disease have a specific susceptibility to an electrolyte imbalance that can deteriorate their condition to hepatic encephalopathy. It occursin about 57% of hospitalized patients with cirrhosis. Hyponatremia in cirrhosis is an in dependent risk factor for mortality and is common in patients with end-stage liver disease. These patients had a significantly higher incidence of hepatic encephalopathy, hepatorenal syndrome, and spontaneous bacterial peritonitis. Hepatic encephalopathy is worsened by the presence of hyponatremia and hypocalcemia. Aim:The aim of this study is to evaluate sodium and calcium levels in CLD patients and its correlation with hepatic encephalopathy. Methods: This cross-sectional study was conducted in SMHS Hospital,Govt.Medical College Srinagar, Jammu Kashmir,India, during a three-year period. 150 patients with chronic liver disease admitted to the general medicine and gastroenterology department were included. Data was entered in SPSS software version 20. Qualitative data were analyzed using chi-square, and quantitative data were analyzed using independent t-tests. A P-value <0.05 was considered significant. Results: Our study on 150 CLD patients showed that the serum sodium level and serum calcium level were significantly lower in patients with hepatic encephalopathy with P-Values of 0.0006 and 0.002, respectively. Conclusion: It seems that reducing serum sodium and calcium level can be predictive of hepatic encephalopathy inpatients with chronic liver disease

Molecular Identification of Genetically Modified Crops for Biosafety and Legitimacy of Transgenes

Crops undergo artificially DNA modifications for improvements are considered as genetically modified (GM) crops. These modifications could be in indigenous DNA or by introduction of foreign DNA as transgenes. There are 29 different crops and fruit trees in 42 countries, which have been successfully modified for various traits like herbicide tolerance, insect/pest resistance, disease resistance and quality improvement. GM crops are grown worldwide and its area is significantly increasing every year. Many countries have very strict rules and regulations for GM crops and are also a trade barrier in some situations. Hence, identification and testing of crops for GM contents is important for identity and legitimacy of transgene to simplify the international trade. Normally, molecular identification is performed at three different levels, i.e., DNA, RNA and protein, and each level has its own importance in testing about the nature and type of GM crops. In this chapter, current scenario of GM crops and different molecular testing tools are described in brief

Invasive retroperitoneal infection due to basidiobolus ranarum with response to potassium lodide: Case report and review of the literature

We report a case of invasive retroperitoneal zygomycotic infection caused by Basidiobolus ranarum in a healthy 8-year-old boy. The youngster responded dramatically to potassium iodide. The clinical and pathological features are reviewed to highlight the problems encountered in the management of this rare infection

crossed cerebellar diaschisis in partial status epilepticus

Crossed cerebellar diaschisis is a phenomena described as decrease in metabolism in the cerebellar hemisphere contralateral to the supratentorial lesion. A 61 year old man presented with status epilepticus. CT Brain showed small acute hemorrhage in left parietal lobe.MRI revealed diffusion restriction in right cerebral and left cerebellar hemispheres with hyperintensities on T2WI and FLAIR. However, CT perfusion was suggestive of increased perfusion in these areas. Periictal EEG showed frequent bursts of higher voltage spike and wave discharges over the right hemisphere. He was treated with IV antiepileptics. But he continued to deteriorate, and expired at 10th day of admission

Stability analyses of sunflower (Helianthus annuus L.) hybrids for oleic acid and yield traits under multi location trials in Pakistan

The development of a hybrid with high oleic acid is an important breeding goal for sunflower. High oleic acid sunflower has better cooking quality due to low oxidation and rancidity. Hence, inbred lines differing for oleic acid content were selected, alongside the development of hybrids where one or both parents exhibited high oleic acid content in edible oil, and then evaluated at various sites (i.e. with comparatively low temperature during sunflower reproductive phase at Sargodha and Faisalabad; while high temperature and low humidity at Bhawalpur and Multan) in Pakistan during spring season. Moreover, autumn season was relatively cool and high humid for sites (Faisalabad and Sargodha, Pakistan). DNA profiling of hybrids differing for oleic acid content using N1-3F/N2-1R confirmed the presence of a high oleic acid allele in the hybrids. Oleic acid content and seed yield components were increased at high temperature and low humidity to a greater extent in spring than in autumn season. Among the hybrids, one (H5) had stable high oleic acid content during the spring season with higher seed yield and kernel to seed percentage than the check cultivars (Hysun-33 and FH-331). Analysis of the combining ability of two locations revealed a relationship between mean oleic acid contents and combining ability, thereby suggesting the effectiveness of selection in developing high oleic acid inbred lines. Newly developed inbred C.112.P was a positive combiner for oleic acid at all sites except Sargodha, while restorer populations such as RH.344, RH.345 and RH.347 were positive male combiners

(E)-4-(4-Fluoro­styr­yl)benzoic acid

The title compound, C15H11FO2, is an important inter­mediate in the synthesis of side-chain ligands for polymeric liquid crystals. The vinyl group is almost coplanar with both the aromatic rings. The crystal structure is stabilized by inter­molecular O—H⋯O hydrogen bonding