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Despite growing evidence of the importance of melatonin and serotonin in the plant life, there is still much debate over the stability of melatonin, with extraction and analysis methods varying greatly from lab to lab with respect to time, temperature, light levels, extraction solvents, and mechanical disruption. The variability in methodology has created conflicting results that confound the comparison of studies to determine the role of melatonin in plant physiology. We here describe a fully validated method for the quantification of melatonin, serotonin and their biosynthetic precursors: tryptophan, tryptamine and N-acetylserotonin by liquid chromatography single quadrupole mass spectrometry (LC-MS) in diverse plant species and tissues. This method can be performed on a simple and inexpensive platform, and is both rapid and simple to implement. The method has excellent reproducibility and acceptable sensitivity with percent relative standard deviation (%RSD) in all matrices between 1 and 10% and recovery values of 82–113% for all analytes. Instrument detection limits were 24.4 ng/mL, 6.10 ng/mL, 1.52 ng/mL, 6.10 ng/mL, and 95.3 pg/mL, for serotonin, tryptophan, tryptamine, N-acetylserotonin and melatonin respectively. Method detection limits were 1.62 μg/g, 0.407 μg/g, 0.101 μg/g, 0.407 μg/g, and 6.17 ng/g respectively. The optimized method was then utilized to examine the issue of variable stability of melatonin in plant tissue culture systems. Media composition (Murashige and Skoog, Driver and Kuniyuki walnut or Lloyd and McCown's woody plant medium) and light (16 h photoperiod or dark) were found to have no effect on melatonin or serotonin content. A Youden trial suggested temperature as a major factor leading to degradation of melatonin. Both melatonin and serotonin appeared to be stable across the first 10 days in media, melatonin losses reached a mean minimum degradation at 28 days of approximately 90%; serotonin reached a mean minimum value of approximately 60% at 28 days. These results suggest that melatonin and serotonin show considerable stability in plant systems and these indoleamines and related compounds can be used for investigations that span over 3 weeks.

BackgroundProtoplast technologies offer unique opportunities for fundamental research and to develop novel germplasm through somatic hybridization, organelle transfer, protoclonal variation, and direct insertion of DNA. Applying protoplast technologies to develop Dutch elm disease resistant American elms (Ulmus americana L.) was proposed over 30 years ago, but has not been achieved. A primary factor restricting protoplast technology to American elm is the resistance of the cell walls to enzymatic degradation and a long lag phase prior to cell wall re-synthesis and cell division.ResultsThis study suggests that resistance to enzymatic degradation in American elm was due to water soluble phenylpropanoids. Incubating tobacco (Nicotiana tabacum L.) leaf tissue, an easily digestible species, in aqueous elm extract inhibits cell wall digestion in a dose dependent manner. This can be mimicked by p-coumaric or ferulic acid, phenylpropanoids known to re-enforce cell walls. Culturing American elm tissue in the presence of 2-aminoindane-2-phosphonic acid (AIP; 10-150 μM), an inhibitor of phenylalanine ammonia lyase (PAL), reduced flavonoid content, decreased tissue browning, and increased isolation rates significantly from 11.8% (±3.27) in controls to 65.3% (±4.60). Protoplasts isolated from callus grown in 100 μM AIP developed cell walls by day 2, had a division rate of 28.5% (±3.59) by day 6, and proliferated into callus by day 14. Heterokaryons were successfully produced using electrofusion and fused protoplasts remained viable when embedded in agarose.ConclusionsThis study describes a novel approach of modifying phenylpropanoid biosynthesis to facilitate efficient protoplast isolation which has historically been problematic for American elm. This isolation system has facilitated recovery of viable protoplasts capable of rapid cell wall re-synthesis and sustained cell division to form callus. Further, isolated protoplasts survived electrofusion and viable heterokaryons were produced. Together, these results provide the first evidence of sustained cell division, callus regeneration, and potential application of somatic cell fusion in American elm, suggesting that this source of protoplasts may be ideal for genetic manipulation of this species. The technological advance made with American elm in this study has potential implications in other woody species for fundamental and applied research which require availability of viable protoplasts.

Background: Hirschsprung disease (HD) is a developmental disorder characterized by absence of ganglia in the distal colon, resulting in a functional obstruction. It is a common cause of pediatric intestinal obstruction. Objective of present study was to evaluate a checklist of radiologic and clinical signs to determine the probability of HD in suspicious patients.Methods: In a diagnostic accuracy study, 19 children with clinical manifestations of HD attended pediatric OPD in a tertiary care teaching hospital, Haldia from January 2014 to December 2016 were assessed. A checklist was used to evaluate the items proposed by contrast enema (CE), based on six subscales, including transitional zone, rectosigmoid index (RSI), irregular contractions in aganglionic region, cobblestone appearance, filling defect due to fecaloid materials and lack of meconium defecation during the first 48 hours after birth. The patients were classified as high score and low score. Sensitivity and specificity were calculated for identifying HD, in comparison with pathologically proved or ruled out HD.Results: Of the 19 patients, 11 (57.89%) cases had HD and 08 (42.11%) cases were without HD. The mean age was 2.793 ± 4.21 months. Abdominal distension, lack of meconium defecation, and constipation were the most common clinical symptoms with frequencies of 15 (78.9%), 11 (57.8%), and 14 (73.68%), respectively. In summary, the mean sensitivity of detecting the radiological signs of transition zone, spastic colon, reversed recto-sigmoid index and the overall impression in histological confirmed HD patients are 59.09%, 49.99%, 59.09% and 56.06% respectively.Conclusions: The mean specificity of detecting the absence of the radiological signs of transition zone, spastic colon, reversed recto-sigmoid index and the overall impression in histological confirmed non-HD patients are 68.75%, 81.25%, 87.5% and 79.17% respectively. This would in turn give an overall mean specificity rate of 79.17% in successfully excluding HD with the above mentioned radiological signs from the contrast enema.

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<p class="abstract"><strong>Background:</strong> <span lang="EN-IN">Distal radius fracture is one of the most common fractures. It may be sustained due to low-energy trauma or high-energy trauma. Objectives: To compare the clinical effectiveness of Kirschner wire fixation with and plaster immobilization for patients with fracture of the distal radius.</span></p><p class="abstract"><strong>Methods:</strong> <span lang="EN-IN">Interventions Kirschner wire fixation: wires are passed through the skin over the dorsal aspect of the distal radius and into the bone to hold the fracture in the correct anatomical position. About 79 patients with Distal Radial Fractures presented to MGM Medical College and LSK Hospital, Kishanganj between November 2012 and June 2014 were included in the study</span>.<strong></strong></p><p class="abstract"><strong>Results:</strong> <span lang="EN-IN">The majority were men (60.4%). Majority of the patients (69.8%) sustained the injury due to fall. The side of involvement was nearly equal and that there was no predominance of the either sides. In our study, according to AO classification, 31 cases were of Type A, 17 were of Type B and 5 were of Type C. The Anatomical evaluation by Sarmiento’s Criteria showed 33 patients with excellent result, 15 patients with good result and 5 with a fair result. At final follow-up by ‘The Gartland and Werley criteria for functional outcome’ 37 patients had excellent result, 13 had good result, 2 had fair result and 1 had a poor result. </span></p><p class="abstract"><strong>Conclusions:</strong> <span lang="EN-IN">This study demonstrates that percutaneous Kirschner<strong> </strong>wire pinning is a minimally invasive technique that provides an effective means of maintaining the anatomical<strong> </strong>fracture reduction. It does not required highly skilled<strong> </strong>personnel or sophisticated tools for application. It is a<strong> </strong>suitable method for fixation of displaced Colles fracture with minimal intra-articular involvement. The technique involves a minimal procedure that provides anatomic reduction, fracture fixation, and maintenance of reduction with an adequate method of immobilization.</span></p>

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<p class="abstract"><strong>Background:</strong> <span lang="EN-IN">Anterior cruciate ligament (ACL) injuries remain a common orthopaedic disease, particularly in young adults. The treatment of choice for ACL injuries is ACL reconstruction (ligamentoplasty). </span><span lang="EN-IN">The present study was conducted to observe and evaluate the outcome results of arthroscopic ACL reconstruction by hamstring grafts using Endobutton-CL technique for femoral fixation and Bioabsorbable Intrafix Screw technique for tibial fixation in ACL injury.</span></p><p class="abstract"><strong>Methods:</strong> <span lang="EN-IN">This prospective comparative observational study related data were captured between January 2014 to December 2015 in a tertiary care teaching hospital, Haldia. About 36 patients with arthroscopic anatomic ACL reconstruction using hamstring tendon graft were evaluated and followed up for functional outcome. Patients were evaluated for pain, functioning and stability of knee using validated knee scoring systems which included tegner activity scale and IKDC score</span>.<strong></strong></p><p class="abstract"><strong>Results:</strong> Out of 36 patients 31 patients (86.11%) were male and 5 patients (13.88%) were females. About 25 patients (69.44%) had right sided ACL injury and remaining 11 patients (30.55%) had left sided ACL injury. The mean of the pre-op IKDC scoring was 42.45 with SD of 9.68 and the mean of the post-operative IKDC scoring was 81.87 with SD of 13.40, so improvement was statistically significant<span lang="EN-IN">. </span></p><p class="abstract"><strong>Conclusions:</strong> <span lang="EN-IN">The technique of arthroscopic ACL reconstruction offers an excellent knee function, knee stability and restoration of preoperative functional status with minimal complications.</span></p>