Biosynthesis of benzyliasoquinoline alkaloids

This research was carried out to obtain a convenient route for the synthesis of [7_ 14C]-p-hydroxy benzaldehyde. Section 1 of the thesis includes a route involving intermediates with protecting groups like benzyl and methyl ethers of the phenols. The benzyl ethers afforded the product in relatively better yield. The overall synthesis involves four steps. Section 2 describes the reactions carried out directly on phenols, and a three step pathway is obtained for the synthesis of p-hydroxy benzaldehyde, which was repeated on labelled compounds to obtain [7_14C]p- hydroxy benzaldehyde. The synthesis involves the reaction of p-bromophenol with Cu14CN to yield [7_ 14C]-p-cyano phenol, which is then reduced to the aldehyde by means of a simple and clean photolysis method. The same route was tried out to get 3,4-dihydroxybenzaldehyde and was found to work equally well for the synthesis of this compound. Section 3 deals with the isolation of labelled alkaloids, corydaline, protopine and reticu1ine from [2-3H,1-14C]-dopamine (3H/ 14C ratio = 4) fed Corydalis solida. 3H/14C ratios in the labelled alkaloids were determined. The uncorrected values showed almost 50% loss of 3H relative to 14C in reticuline, and roughly 75% loss of the 3H relative to 14C in corydaline and protopine

The biochemical consequences of ascorbate deficiency in Arabidopsis thaliana

Biochemical consequences of ascorbate deficiency were studied in the leaf tissue of Arabidopsis thaliana ascorbate-deficient vtc mutants with a view of understanding the relationship between ascorbate, stress response and metabolism. Ascorbate is an important antioxidant and is also a cofactor for 2-oxoglutarate-dependent dioxygenases, which are involved in the biosynthesis of a number of metabolites. The response of wild type (Col-0) and vtc1, vtc2-1, vtc2-2 and vtc3-1 mutants to high light intensity, wounding and salinity was investigated using a metabolomics and proteomics approach. Metabolite profiling and comparative proteomics were performed by liquid chromatography-quadrupole time of flight mass spectrometry (LC-QToF MS) and targeted analysis of plant hormones and flavonoids by liquid chromatography triple-quadrupole mass spectrometry (LC-QQQ-MS). These combined analyses revealed the effect of ascorbate deficiency and stress on metabolites and cell wall proteins. LC-QToF-MS based untargeted metabolite profiling methodologies were developed for analysis of metabolites on a large scale. Using this method about 3000-5000 metabolites (mass-retention time pairs) could be reproducibly detected in A. thaliana leaf extract and aligned between samples. Approximately 1000 metabolites were differentially expressed between WT and vtc mutants in different experiments. Of these, twenty eight compounds were confirmed to be differentially expressed by LC-QQQ-MS between WT and vtc mutants, and eight of these compounds were positively identified and validated with standards. The plant hormones abscisic acid (ABA), salicylic acid (SA) and jasmonic acid (JA) have all been implicated in plant stress responses and differences in their accumulation in some of the vtc mutants have been reported. A systematic study of the response to stress of these hormones in several vtc mutants was carried out using LC- QQQ- MS. While some of the mutants showed increased SA and SA-glycoside accumulation, stress-induced ABA and JA accumulation was generally unaffected. Methods for identifying the metabolites in a targeted manner by LC- QQQ-MS was developed and were shown that all vtc mutants were impaired in the accumulation of anthocyanin in response to HL treatment. In strong contrast to anthocyanin, flavonol glycosides were not affected by ascorbate deficiency. Therefore, ascorbate deficiency has a specific effect on the anthocyanin biosynthesis. Ascorbate occurs in the plant cell wall and isolation of apoplastic fluid showed that all vtc mutants have decreased apoplastic ascorbate compared to WT. Ionically-bound proteins were from the cell wall of A. thaliana leaves. Peroxidase specific activity in this fraction tended to be higher in vtc mutants than WT. High light intensity also increased peroxidase activity in WT and vtc mutants. To determine which peroxidase isoenzyme caused increased peroxidase activity, ionically-bound cell wall N-glycosylated proteins were isolated by Concanavalin A chromatography and analysed by LC-QToF-MS. Comparison of WT and vtc2-2 grown in low light and high light identified 937 peptides significantly different between WT and vtc2-2 and some are also affected by light intensity. Specifically, peroxidases 33 and 34 had increased abundance in vtc2-2. The results show that ascorbate deficiency causes a detectable change in the metabolome of A. thaliana leaves, with specific effects on anthocyanin accumulation being detected. Ascorbate deficiency also influences the expression of cell wall proteins. Peroxidase activity is increased, and this response could be related to the increased pathogen resistance reported in vtc mutants

Effect of Homofermentative Inoculant on Fermentation Characteristics and Nutritive Values of Corn Silage

An in vitro study was planned to assess the effects of a homofermentative microbial inoculant on the fermentation parameters and nutritive value of corn silage. The inoculant was applied at concentrations of 5x104cfu/g of forage (T1), 1x105cfu/g of forage (T2) 1.5x105cfu/g of forage (T3) and a negative control group (T0) without bacterial inoculant in three replicates each. At day 3, 7, 45 and 90 of the experiment individual buckets were opened to characterize the material, quick acidification, dry matter recovery, and aerobic stability of silage respectively. The temperature of the trial samples was 32.75±1.92 throughout the trial duration. A rapid and significant reduction in pH even at third day of trial from 6.5 to 3.61 in the treatment (T2 and T3) groups and remained consistent till 90 day of experiment (with non-significant fluctuations) when compared with control group (6.5 to 5.0). The levels of lactic acid, acetic acid and propionic acids were significantly (P5cfu/g and 1.5x105cfu/g inclusion levels of inoculant

Spectrum of Complications in Children with Moderate to Severe DKA Admitted in Pediatric Intensive Care Unit of a Tertiary Care Hospital

Background: Diabetic Ketoacidosis (DKA) is the major source of mortality and morbidity in type 1 Diabetes Mellitus. Cautious fluid resuscitation, insulin and electrolyte replacement along with observation for complications is the mainstay of treatment. Early identification of complications lead to better management and outcome. Objective: To find out the complications of moderate to severe DKA in pediatric patients admitted in Pediatric Intensive Care Unit (PICU). Methods: Cross-sectional Study was conducted in PICU of University of Child Health Sciences & The Children’s Hospital, Lahore from Jan, 2015 to Dec, 2018.Data was collected from retrospective records of patients admitted in PICU. All patients with moderate/severe DKA were included. Patients with developmental delay were excluded. Data was analyzed using SPSS version 25. Chi-square test was used to find out p-value. Results: A total of 152 patients with moderate/severe DKA were included in the study, having mean age of 7.86 ± 4.06 years. Most patients 42% (n=64) were more than 10 years of age. Mean weight of the children was 19.89 ± 8.70 kg. Majority 59% (n= 89) were of new onset diabetes. Severe DKA was seen in 74% while moderate DKA in 26% patients. The complications seen were: hypokalemia 33% (n=50), cerebral edema 28% (n=42), hypernatremia 18% (n=27), hyponatremia 8% (n=12) and acute kidney injury (AKI) in 7% (n=11). Survival rate was 85% while mortality rate was 15%. Conclusion: Hypokalemia and cerebral edema were the most common complications observed. Complications were more common in those patients who presented with DKA in first presentation. Key words: complications, DKA severity, children, PICU

The influence of ascorbate on anthocyanin accumulation during high light acclimation in Arabidopsis thaliana: further evidence for redox control of anthocyanin synthesis

PublishedArticleThis is the peer reviewed version of the following article: AGE, M., SULTANA, N., PASZKIEWICZ, K., FLORANCE, H. and SMIRNOFF, N. (2012), The influence of ascorbate on anthocyanin accumulation during high light acclimation in Arabidopsis thaliana: further evidence for redox control of anthocyanin synthesis. Plant, Cell & Environment, 35: 388–404. , which has been published in final form at http://dx.doi.org/10.1111/j.1365-3040.2011.02369. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.Ascorbate and anthocyanins act as photoprotectants during exposure to high light (HL). They accumulate in Arabidopsis leaves in response to HL on a similar time-scale, suggesting a potential relationship between them. Flavonoids and related metabolites were identified and profiled by LC-MS/MS. The ascorbate deficient mutants vtc1, vtc2 and vtc3 accumulated less anthocyanin than wild-type during HL acclimation. In contrast, kaempferol glycoside accumulation was less affected by light and not decreased by ascorbate deficiency, while sinapoyl malate levels decreased during HL acclimation. Comparison of six Arabidopsis ecotypes showed a positive correlation between ascorbate and anthocyanin accumulation in HL. mRNA-Seq analysis showed that all flavonoid biosynthesis transcripts were increased by HL acclimation in wild-type. RT-PCR analysis showed that vtc1 and vtc2 were impaired in HL induction of transcripts of anthocyanin biosynthesis enzymes, and the transcription factors PAP1, GL3 and EGL3 that activate the pathway. Abscisic acid and jasmonic acid, hormones that could affect anthocyanin accumulation, were unaffected in vtc mutants. It is concluded that HL induction of anthocyanin synthesis involves a redox-sensitive process upstream of the known transcription factors. Because anthocyanins accumulate in preference to kaempferol glycosides and sinapoyl malate in HL, they might have specific properties that make them useful in high light acclimation.Biotechnology and Biological SciencesResearch Council (BBSRC)Exeter University ScienceStrategy FundHazara UniversityMansehra (NWFP) PakistanHigher Education Commission (Pakistan

Spectrum of Complications in Children with Moderate to Severe DKA Admitted in Pediatric Intensive Care Unit of a Tertiary Care Hospital

Background: Diabetic Ketoacidosis (DKA) is the major source of mortality and morbidity in type 1 Diabetes Mellitus. Cautious fluid resuscitation, insulin and electrolyte replacement along with observation for complications is the mainstay of treatment. Early identification of complications lead to better management and outcome. Objective: To find out the complications of moderate to severe DKA in pediatric patients admitted in Pediatric Intensive Care Unit (PICU). Methods: Cross-sectional Study was conducted in PICU of University of Child Health Sciences & The Children’s Hospital, Lahore from Jan, 2015 to Dec, 2018.Data was collected from retrospective records of patients admitted in PICU. All patients with moderate/severe DKA were included. Patients with developmental delay were excluded. Data was analyzed using SPSS version 25. Chi-square test was used to find out p-value. Results: A total of 152 patients with moderate/severe DKA were included in the study, having mean age of 7.86 ± 4.06 years. Most patients 42% (n=64) were more than 10 years of age. Mean weight of the children was 19.89 ± 8.70 kg. Majority 59% (n= 89) were of new onset diabetes. Severe DKA was seen in 74% while moderate DKA in 26% patients. The complications seen were: hypokalemia 33% (n=50), cerebral edema 28% (n=42), hypernatremia 18% (n=27), hyponatremia 8% (n=12) and acute kidney injury (AKI) in 7% (n=11). Survival rate was 85% while mortality rate was 15%. Conclusion: Hypokalemia and cerebral edema were the most common complications observed. Complications were more common in those patients who presented with DKA in first presentation. Key words: complications, DKA severity, children, PICU

Effect of D-chiro-inositol on hormonal parameters and insulin resistance in women with polycystic ovary syndrome

Background: Polycystic ovary syndrome (PCOS) is an endocrine disorder among women of reproductive age, characterized by hormonal imbalance and insulin resistance. D-chiro-inositol, a naturally occurring inositol isomer, has been suggested as a potential treatment option for PCOS. This study aimed to investigate the effects of D-chiro-inositol supplementation on hormonal parameters, and insulin resistance in women with PCOS. Methods: This randomized controlled study was conducted among 60 women of PCOS with insulin resistance, who were assigned to either Group A (D-chiro-inositol) or Group B (placebo) for 12 weeks. S. FSH, LH, S. total testosterone, fasting blood glucose, fasting insulin, and insulin resistance (HOMA-IR) were measured at baseline and after 12 weeks of treatment. Statistical analyses were performed using SPSS version 23.0 for Windows. Results: After 12 weeks of treatment, significant reductions in serum luteinizing hormone, serum total testosterone, fasting insulin, and HOMA-IR were observed in the D-chiro-inositol group compared to the placebo group. However, no significant changes were observed in fasting blood glucose levels. D-chiro-inositol was well-tolerated, with no significant differences in side effects between the two groups. Conclusions: D-chiro-inositol supplementation for 12 weeks significantly improved hormonal parameters, and insulin resistance in women with PCOS. The treatment was well-tolerated, suggesting that D-chiro-inositol can be an effective therapeutic option for patients with PCOS

Effect of estradiol valerate on endometrial thickness in polycystic ovary syndrome having ovulation induction with letrozole

Background: PCOS is a common endocrine disorder in women of reproductive age. Letrozole is an orally active aromatase inhibitor and as effective as chlomiphene citrate for induction of ovulation. Estrogen is important in the regeneration and growth of the endometrium prior to ovulation prepare the tissue to respond to progesterone post ovulation in PCOS patients. Aim of the study was to assess the effects of estradiol valerate on endometrial thickness in PCOS having ovulation induction with letrozole. Methods: This randomized controlled study was conducted in the department of reproductive endocrinology and infertility, BSMMU, Dhaka, with 1 year duration. A total 80 diagnosed cases of PCOS patients with subfertility were included in this study. Among them 40 patients received letrozole and estradiol valerate and 40 patients received letrozole and placebo. Results: On day 8, mean endometrial thickness was not statistically significant between two groups (p=0.436). On day of triggering, mean endometrial thickness was significantly higher in intervention group 9.2±1.4 mm than control group 8.2±1.4 mm (p=0.004). Mean changes of endometrial thickness on day of triggering compared with on day 8 was significantly higher in intervention group 3.2±1.5 mm than control group 2.5±1.6 mm (p=0.043). Pregnancy rate was higher in intervention group 13 (38.2%) than control group 8 (22.2%) with relative risk 1.72, 95% CI (0.82-3.63%), that was not statistically significant between two groups (p=0.144). Conclusions: Mean changes of endometrial thickness on day of triggering were significantly higher in intervention group than control group. The pregnancy rate achieved with letrozole+estradiol valerate combination was higher than that achieved with letrozole and placebo group

Comparative Chloroplast Genomics in Phyllanthaceae Species

Family Phyllanthaceae belongs to the eudicot order Malpighiales, and its species are herbs, shrubs, and trees that are mostly distributed in tropical regions. Here, we elucidate the molecular evolution of the chloroplast genome in Phyllanthaceae and identify the polymorphic loci for phylogenetic inference. We de novo assembled the chloroplast genomes of three Phyllanthaceae species, i.e., Phyllanthus emblica, Flueggea virosa, and Leptopus cordifolius, and compared them with six other previously reported genomes. All species comprised two inverted repeat regions (size range 23,921–27,128 bp) that separated large single-copy (83,627–89,932 bp) and small single-copy (17,424–19,441 bp) regions. Chloroplast genomes contained 111–112 unique genes, including 77–78 protein-coding, 30 tRNAs, and 4 rRNAs. The deletion/pseudogenization of rps16 genes was found in only two species. High variability was seen in the number of oligonucleotide repeats, while guanine-cytosine contents, codon usage, amino acid frequency, simple sequence repeats, synonymous and non-synonymous substitutions, and transition and transversion substitutions were similar. The transition substitutions were higher in coding sequences than in non-coding sequences. Phylogenetic analysis revealed the polyphyletic nature of the genus Phyllanthus. The polymorphic protein-coding genes, including rpl22, ycf1, matK, ndhF, and rps15, were also determined, which may be helpful for reconstructing the high-resolution phylogenetic tree of the family Phyllanthaceae. Overall, the study provides insight into the chloroplast genome evolution in Phyllanthaceae

Clinical outcome of tetanus based on dakar tetanus severity score in paediatric intensive care unit of a tertiary care hospital

Objective: To evaluate the validity of  Dakar scoring system in predicting the outcome of tetanus in children of post neonatal age. Method: A prospective single center study conducted at ICU (Intensive Care Unit) of –removed for blind review-- 131 children with diagnosis of tetanus from January 2020 to June 2021 by analyzing demographic and clinical parameters. Results: Mean age of children was 8.11 ± 3.45 years with male predominance 91(69.5%). Major proportion of children were in range of 5-10 years comprising 65(50%). Period of onset of spasm was < 2 days in 92(70%) that correlate well with shorter incubation period (p-value <0.001). Mean duration of ICU stay was (18.81 ± 11.73) days. Only 14(10.7%) received three doses of vaccination but no one received booster dose. In majority of patients trauma due to road traffic accident 86(65.6%) was the predisposing factor followed by ear discharge 31(23.7%) and other causes 14(10.7%). Out of 131 patients 104(79.3%) received mechanical ventilation because of disease severity. Regarding outcome mild cases 2(1.5%), moderate 61(46.6%) and severe were 68(51.9%) according to Dakar scoring system and out of these severe cases 18(13.7%) expired (p-value < 0.001). However, 53(40.4%) discharged, 50(38.1%) shifted out to other wards after stabilization and 10(7.6%) left against medical advice. (p-value 0.001) Conclusion: Dakar scoring system correlates well with disease severity and should be adopted to aid clinical triage and management as with effective and appropriate treatment patients can have good prognosis. However, preventive aspects should be emphasized regarding booster dose of tetanus. Keywords: Tetanus, Outcome, Dakar scoring syste