Clostridial neurotoxins as a drug delivery vehicle targeting nervous system

Several neuronal disorders require drug treatment using drug delivery systems for specific delivery of the drugs for the targeted tissues, both at the peripheral and central nervous system levels. We describe a review of information currently available on the potential use of appropriate domains of clostridial neurotoxins, tetanus and botulinum, for effective drug delivery to neuronal systems. While both tetanus and botulinum neurotoxins are capable of delivering drugs the neuronal cells, tetanus neurotoxin is limited in clinical use because of general immunization of population against tetanus. Botulinum neurotoxin which is also being used as a therapeutic reagent has strong potential for drug delivery to nervous tissues.     

Frequency of ��-thalassemia trait and other hemoglobinopathies in northern and western India

INTRODUCTION:

India is an ethnically diverse country with an approximate population of 1.2 billion. The frequency of beta-thalassemia trait (βTT) has variously been reported from <1% to 17% and an average of 3.3%. Most of these studies have been carried out on small population groups and some have been based on hospital-based patients. There is also a variation in the prevalence of hemoglobinopathies in different regions and population groups in the country. A high frequency of Hb D has been reported from the North in the Punjabi population, Hb E in the eastern region of India and Hb S is mainly reported from populations of tribal origin from different parts of the country.

OBJECTIVES:

To study the gene frequency of βTT and other hemoglobinopathies in three regions East (Kolkata), West (Mumbai) and North (Delhi) in larghe population group (schoolchildren) for a more accurate assessment of gene frequency for planning of control programmes for haemoglobinopathies.

MATERIALS AND METHODS:

This study included 5408 children from 11 schools in Delhi, 5682 from 75 schools in Mumbai and 957 schoolchildren from Kolkata who were screened for βTT and haemoglobinopathies. These included 5684 children from 75 schools in Mumbai and 5408 children from 11 schools in Delhi. Children were 11-18 years of age of both sexes. The final report is, however, only on 11090 schoolchildren from Mumbai and Delhi as data from Kolkata was restricted both in numbers and objectives and could not be included for comparison.

RESULTS:

The overall gene frequency of βTT in Mumbai and Delhi was 4.05% being 2.68% and 5.47% in children of the two cities respectively. In Mumbai, the gene frequency was evenly distributed. Majority of the children with βTT from Mumbai were from Marathi (38.9%) and Gujarati (25%) speaking groups. Gene frequency was >5% in Bhatias, Khatris, Lohanas and Schedule Castes. In Delhi, a higher incidence was observed in schoolchildren of North and West Delhi (5.8-9.2%). The schoolchildren of North and West Delhi comprised predominantly of Punjabi origin compared to children in the South of the city (2.2%, 2.3%). When analyzed state-wise, the highest incidence was observed in children of Punjabi origin (7.6%) and was >4% from several other states. Majority of the traits from Mumbai were anemic (95.1% male and 85.6% in female). The prevalence of anemia was lower (62.7% male and 58.4% female) children with βTT from Delhi. This was a reflection of the higher prevalence of anemia in children without hemoglobinopathy in Mumbai than in Delhi. Nutritional deficiency was probably more severe and rampant in children Mumbai. Gene frequency of Hb D was greater in schoolchildren from Delhi (1.1%) than in Mumbai (0.7%). Hb S trait (0.2%) was observed exclusively in children from Mumbai. A low incidence of Hb E trait (0.04%) was seen in children in Mumbai. A higher incidence is reported from the East. The number of cases studied from the eastern region was small as the data from the East (Kolkata) could not be included in the analysis.

CONCLUSION:

This study comprises a larger number of children studied for the gene frequency of βTT and other hemoglobinopathies from India. Population groups with higher gene frequencies require screening programmes and facilities for antenatal diagnosis as well as increased awareness and educational programmes to control the birth of thalassemic homozygotes. The overall carrier frequency of βTT was 4.05% and reinforces the differential frequency of β-thalassemia trait in schoolchildren from Delhi and Mumbai and the higher incidence of hemoglobin D in Punjabis as reported previously. The birth incidence calculated thereof for homozygous thalassemics would be 11,316 per year which are added each year to the existing load of homozygous thalassemics. This is much higher than the previously reported number of births annually. Hence suitable control measures need to be undertaken urgently in India.     

A new on-line, in-tube pre-column derivatization technique for high performance liquid chromatographic determination of azithromycin in human serum

Pre-column derivatization methods for high performance liquid chromatographic assay of specific pharmaceutical agents using 9-fluorenylmethyl chloroformate (FMOC-Cl) have received special attention because highly fluorescent and stable adducts are provided by these methods. However, unlike the post-column on-line techniques, long derivatization time is needed and the reaction cannot be well controlled. A new, sensitive and fast pre-column on-line derivatization technique coupled with high-performance liquid chromatography using FMOC-Cl as labeling agent is described and validated for determination of azithromycin in human serum. After extraction of the drug from serum, the residue was reconstituted in mixture of acetonitrile-phosphate buffer (3:1, v/v; pH 8.5) and directly injected onto the chromatographic system. Continuous on-line derivatization and analysis of the compounds were successfully performed using in-tube elution of FMOC-Cl. The total time needed for derivatization and chromatographic analysis of the drug was 13 min. The assay was reliable and reproducible, with limit of quantification of 10 ng/ml. The described technique may offer significant advantages over existing off-line derivatization methods using FMOC-Cl.     

Novel strategies targeting pathogen transmission reduction in insect vectors: Tsetse-transmitted trypanosomiasis control

Insect vectors are essential for the transmission of important human diseases such as malaria, leishmaniasis, Chagas and sleeping sickness. Insects are also responsible for the transmission of agricultural diseases that affect livestock and crops. Traditionally, control of the vector populations has been an effective disease management strategy. Recently, vector control strategies have been fortified by research in insect biology and in insect–pathogen interactions as well as by the development of transgenic technologies. In addition to insect population reduction methods, disease control via selective elimination of pathogens in insects can now be explored. Here we explore the tsetse vectors of African trypanosomes and describe the application of recent knowledge gained in their symbiotic, reproductive and vectorial biology to develop novel disease control strategies.     

A GLYCOPROTEIN NONCOVALENTLY ASSOCIATED WITH CELL‐WALL POLYSACCHARIDE OF THE RED MICROALGA PORPHYRIDIUM SP. (RHODOPHYTA)1

The cells of the red microalga Porphyridium sp. (UTEX 637) are encapsulated in a cell wall of a negatively charged mucilaginous polysaccharide complex composed of 10 different sugars, sulfate, and proteins. In this work, we studied the proteins associated with the cell‐wall polysaccharide. A number of noncovalently associated proteins were resolved by SDS‐PAGE, but no covalently bound proteins were detected. The most prominent protein detected was a 66‐kDa glycoprotein consisting of a polypeptide of approximately 58 kDa and a glycan moiety of approximately 8 kDa containing N‐linked terminal mannose. In size‐exclusion chromatography, the 66‐kDa protein was coeluted with the polysaccharide and could be separated from the polysaccharide only after denaturation of the protein, indicating that the 66‐kDa protein was tightly bound to the polysaccharide. Western blot analysis revealed that the 66‐kDa protein was specific to Porphyridium sp. and P. cruentum, because it was not detected in the other species of red microalgae examined. Indirect immunofluorescence assay confirmed the location of the protein in the algal cell wall. The sequence of cDNA clone encoding the 66‐kDa glycoprotein, detected in our in‐house expressed sequence tag database of Porphyridium sp., revealed that this is a novel protein with no similarity to any protein in the public domain databases and our in‐house expressed sequence tag database of the red microalga Rhodella reticulata. The 66‐kDa protein bound polysaccharides from red algae but not from those of other origins tested. Possible roles of the 66‐kDa protein in the biosynthesis of the polysaccharide are discussed.     

Evaluating the impact of biodiversity offsetting on native vegetation

Biodiversity offsetting is a globally influential policy mechanism for reconciling trade-offs between development and biodiversity loss. However, there is little robust evidence of its effectiveness. We evaluated the outcomes of a jurisdictional offsetting policy (Victoria, Australia). Offsets under Victoria's Native Vegetation Framework (2002–2013) aimed to prevent loss and degradation of remnant vegetation, and generate gains in vegetation extent and quality. We categorised offsets into those with near-complete baseline woody vegetation cover (“avoided loss”, 2702 ha) and with incomplete cover (“regeneration”, 501 ha), and evaluated impacts on woody vegetation extent from 2008 to 2018. We used two approaches to estimate the counterfactual. First, we used statistical matching on biophysical covariates: a common approach in conservation impact evaluation, but which risks ignoring potentially important psychosocial confounders. Second, we compared changes in offsets with changes in sites that were not offsets for the study duration but were later enrolled as offsets, to partially account for self-selection bias (where landholders enrolling land may have shared characteristics affecting how they manage land). Matching on biophysical covariates, we estimated that regeneration offsets increased woody vegetation extent by 1.9%–3.6%/year more than non-offset sites (138–180 ha from 2008 to 2018) but this effect weakened with the second approach (0.3%–1.9%/year more than non-offset sites; 19–97 ha from 2008 to 2018) and disappeared when a single outlier land parcel was removed. Neither approach detected any impact of avoided loss offsets. We cannot conclusively demonstrate whether the policy goal of ‘net gain’ (NG) was achieved because of data limitations. However, given our evidence that the majority of increases in woody vegetation extent were not additional (would have happened without the scheme), a NG outcome seems unlikely. The results highlight the importance of considering self-selection bias in the design and evaluation of regulatory biodiversity offsetting policy, and the challenges of conducting robust impact evaluations of jurisdictional biodiversity offsetting policies.     

Targeted editing of multiple homologues of GTR1 and GTR2 genes provides the ideal low-seed,high-leaf glucosinolate oilseed mustard with uncompromised defence and yield

Glucosinolate content in the two major oilseed Brassica crops—rapeseed and mustard has been reduced to the globally accepted Canola quality level (<30 μmoles/g of seed dry weight, DW), making the protein-rich seed meal useful as animal feed. However, the overall lower glucosinolate content in seeds as well as in the other parts of such plants renders them vulnerable to biotic challenges. We report CRISPR/Cas9-based editing of glucosinolate transporter (GTR) family genes in mustard (Brassica juncea) to develop ideal lines with the desired low seed glucosinolate content (SGC) while maintaining high glucosinolate levels in the other plant parts for uncompromised plant defence. Use of three gRNAs provided highly efficient and precise editing of four BjuGTR1 and six BjuGTR2 homologues leading to a reduction of SGC from 146.09 μmoles/g DW to as low as 6.21 μmoles/g DW. Detailed analysis of the GTR-edited lines showed higher accumulation and distributional changes of glucosinolates in the foliar parts. However, the changes did not affect the plant defence and yield parameters. When tested against the pathogen Sclerotinia sclerotiorum and generalist pest Spodoptera litura, the GTR-edited lines displayed a defence response at par or better than that of the wild-type line. The GTR-edited lines were equivalent to the wild-type line for various seed yield and seed quality traits. Our results demonstrate that simultaneous editing of multiple GTR1 and GTR2 homologues in mustard can provide the desired low-seed, high-leaf glucosinolate lines with an uncompromised defence and yield.     

High performance liquid chromatographic determination of topiramate in human serum using UV detection

Topiramate has no ultraviolet, visible or fluorescence absorption. Analysis of the drug in human serum has been reported by high performance liquid chromatography (HPLC) with either mass detector or fluorescence detection after precolumn derivatization using 9-fluorenylmethyl chloroformate as fluorescent labeling agent. This study was aimed to validate derivatization and analysis of topiramate in human serum with HPLC using UV detection. The drug was extracted from human serum by liquid-liquid extraction and subjected to derivatization with 9-fluorenylmethyl chloroformate. Analysis was performed on a phenyl column using of spectrophotometer detection operated at wavelength of 264 nm. A mixture of phosphate buffer (0.05M) containing triethylamine (1 ml/l, v/v; pH 2.3) and methanol (28:72, v/v) at a flow rate of 2.5 ml/min was used as mobile phase. No interference was found with endogenous substances. Validity of the method was studied and the method was precise and accurate with a linearity range from 40 ng/ml to 40 microg/ml. The limit of quantification was 40 ng/ml of serum. The correlation coefficient between HPLC methods using fluorescence and UV detections was studied and found to be 0.992.     

Glutamine decreases lipopolysaccharide-induced intestinal inflammation in infant rats

Using a gastrostomy-fed (GF) rat infant "pup-in-a-cup" model, the effects of protein deprivation and supplemental glutamine (Gln) and glutamate (Glu) were examined to test the hypothesis that Gln decreases the proinflammatory response induced by LPS in the developing infant rat small intestine. Four groups of 6- to 7-day-old pups were fed a rat milk substitute (RMS), one providing 100% and three providing 25% of normal protein intake for another 6 days. Two of the 25% protein-fed groups received supplemental Gln or Glu. GF and LPS treatment blunted body growth and intestinal villus height and increased intestinal cytokine-induced neutrophil chemoattractant (CINC) mRNA in the protein-deprived, non-Gln-treated group compared with mother-fed pups (P < 0.05). Gln blunted intestinal CINC mRNA (P < 0.05), but Glu did not. Intestinal CINC peptide in the LPS-treated pups provided 100 and 25% protein was elevated approximately 13-fold compared with the mother-reared pups (P < 0.001). Gln and Glu decreased intestinal CINC peptide by 73 and 80%, respectively. GF, LPS-treated pups also had a higher level of plasma CINC peptide (P < 0.05). Gln but not Glu decreased plasma CINC peptide (P < 0.05). An approximate sixfold elevation of intestinal MPO activity in the GF, LPS-treated rats was decreased by Gln and Glu by 92% (P < 0.001) and 54% (P < 0.05), respectively. Intestinal and plasma TNF-alpha were increased in GF, LPS-treated pups (P < 0.01), and Gln and Glu both blunted this increase (P < 0.05) in the intestine but not in the plasma. The results indicate that Gln decreases the LPS-induced inflammatory response in infant rat intestine under different conditions of protein intake.