Injury tolerance and moment response of the knee joint to combined valgus bending and shear loading

Valgus bending and shearing of the knee have been identified as primary mechanisms of injuries in a lateral loading environment applicable to pedestrian-car collisions. Previous studies have reported on the structural response of the knee joint to pure valgus bending and lateral shearing, as well as the estimated injury thresholds for the knee bending angle and shear displacement based on experimental tests. However, epidemiological studies indicate that most knee injuries are due to the combined effects of bending and shear loading. Therefore, characterization of knee stiffness for combined loading and the associated injury tolerances is necessary for developing vehicle countermeasures to mitigate pedestrian injuries. Isolated knee joint specimens (n=40) from postmortem human subjects were tested in valgus bending at a loading rate representative of a pedestrian-car impact. The effect of lateral shear force combined with the bending moment on the stiffness response and the injury tolerances of the knee was concurrently evaluated. In addition to the knee moment-angle response, the bending angle and shear displacement corresponding to the first instance of primary ligament failure were determined in each test. The failure displacements were subsequently used to estimate an injury threshold function based on a simplified analytical model of the knee. The validity of the determined injury threshold function was subsequently verified using a finite element model. Post-test necropsy of the knees indicated medial collateral ligament injury consistent with the clinical injuries observed in pedestrian victims. The moment-angle response in valgus bending was determined at quasistatic and dynamic loading rates and compared to previously published test data. The peak bending moment values scaled to an average adult male showed no significant change with variation in the superimposed shear load. An injury threshold function for the knee in terms of bending angle and shear displacement was determined by performing regression analysis on the experimental data. The threshold values of the bending angle (16.2 deg) and shear displacement (25.2 mm) estimated from the injury threshold function were in agreement with previously published knee injury threshold data. The continuous knee injury function expressed in terms of bending angle and shear displacement enabled injury prediction for combined loading conditions such as those observed in pedestrian-car collisions.     

Aluminium-induced production of oxygen radicals, lipid peroxidation and DNA damage in seedlings of rice (Oryza sativa)

The effect of aluminium (Al) on seedlings of two rice cultivars, Pusa Basmati and Vikas was investigated after different hours of exposure to 80 mol/L of external Al supply. With increasing time of exposure, the growing seedlings readily absorbed Al and its localization was greater in roots than shoots. Prolonged exposure to Al intensified lipid peroxidation, changed the activities of SOD and peroxidase and caused DNA damage. However, differential responses were observed between the seedlings of two rice cultivars under Al stress. A close inverse relationship existed between decreased root growth and increased Al accumulation, lipid peroxidation, SOD, peroxidase activities and DNA damage. The results demonstrate that roots are the major sites of Al localization and accumulation of Al promoted oxygen free radicals mediated peroxidation of membranes as evidenced by increased MDA levels and the activities of SOD and peroxidase. Our results for the first time showed that Al can cause DNA damage in rice.     

Enhanced production of plumbagin in immobilized cells of Plumbago rosea by elicitation and in situ adsorption

Cell cultures of Plumbago rosea were immobilized in calcium alginate and cultured in Murashige and Skoog's basal medium containing 10 mM CaCl(2) for the production of plumbagin, an important medicinal compound. Studies were carried to find out the impact of immobilization on the increased accumulation of this secondary metabolite. Immobilization in calcium alginate enhanced the production of plumbagin by three, two and one folds compared to that of control, un-crosslinked alginate and CaCl(2) treated cells respectively. Cell loading at a level of 20% to the polymer volume (Na-alginate) was optimal and maximum plumbagin was obtained. At higher cell loading (40-50%), lower plumbagin accumulation was noticed. Addition of 200 mg l(-1) chitosan as an elicitor to the immobilized cells resulted in eight and two folds higher accumulation of plumbagin over control and immobilized cells. Also, more than 70% of the plumbagin was released into the medium, which is highly desirable for easy recovery of the product. Sucrose utilization rate of the cells was higher when cells were subjected to in situ product removal using Amberlite XAD-7. This may indicate that the toxicity of plumbagin was reduced on cells when it was removed from the medium. Cells subjected to combined treatments of chitosan, immobilization and in situ extraction showed a synergistic effect and yielded 92.13 mg g(-1) DCW of plumbagin which is 21, 5.7, 2.5 times higher than control, immobilized, immobilized and elicited cells respectively.     

Poly(D,L-Lactide-Co-Glycolide) microspheres containing 5-fluorouracil: Optimization of process parameters

The objective of this research was to optimize the processing parameters for poly(D,L-lactide-coglycolide) (PLGA) microspheres of 5-fluorouracil (5-FU) and to mathematically relate the process parameters and properties of microspheres. Microspheres were prepared by a water-in-oil-in-water emulsion solvent evaporation technique. A 3² factorial design was employed to study the effect of the volume of the internal phase of the primary emulsion and the volume of the external phase of the secondary emulsion on yield, particle size, and encapsulation efficiency of microspheres. An increase in the volume of the internal phase of the primary emulsion resulted in a decrease in yield and encapsulation efficiency and an increase in particle size of microspheres. When the volume of the external phase of the secondary emulsion was increased, a decrease in yield, particle size, and encapsulation efficiency was observed. Microspheres with good batch-to-batch reproducibility could be produced. Scanning electron microscopic study indicated that microspheres existed as aggregates.     

Production of pyrroloquinazoline alkaloid from leaf and petiole-derived callus cultures of <Emphasis Type="Italic">Adhatoda zeylanica</Emphasis>

Summary Callus cultures of Adhatoda zeylanica Medicus were established from leaf and petiole explants. Accumulation of a bioactive pyrroloquinazoline alkaloid, vasicine, in callus cultures was detected and confirmed by thin layer chromatography, electron-ionization mass spectra, ¹³C NMR and high-pressure liquid chromatography analysis. The mass of vasicine obtained from leaf-derived callus cultures was found as 188 and this is comparable to that of the authentic sample. The retention time for leaf-derived extract was 10.065 and for the petiole-derived extract was 9.78 (authentic sample had 9.6 retention time) on high-performance liquid chromatography. The mass and NMR spectra were compared with the spectra obtained from the authentic sample of vasicine. Different growth regulators greatly influenced the growth of callus cultures. The accumulation of vasicine was more in leaf-derived callus grown on Murashige and Skoog (MS) medium with 2.3 μM kinetin, and 4.5 μM 2,4-dichlorophenoxyacetic acid. This is the first report on in vitro production of a pharmacologically important compound vasicine and its characterization by mass spectrometry and ¹³C NMR studies from callus cultures of Adhatoda zeylanica.     

In vitro propagation of guayule (Parthenium argentatum) — a rubber yielding shrub

Nodal explants (0.5 to 0.8 cm long) isolated from 2-year old shrubs of guayule, Parthenium argentatum Gray, when cultured on MS medium supplemented with different concentrations of KN, BAP, 2,4-D, 2,4-D + BAP, NAA and NAA + BAP produced callus tissues and shoots simultaneously with varying frequencies. Shoots were regenerated with a high frequency (80–88%) from callus on MS medium containing NAA + BAP with or without glutamine. Addition of glutamine to these media improved considerably the number of shoots formed from a known amount of callus. Shoots could be regenerated from 200 day old callus cultures with a very high frequency but the organogenetic capacity declined thereafter. Increase in the concentration of sucrose (upto 4%) significantly enhanced the shoot forming ability of callus, but higher concentrations (6%) suppressed it. Rooting was induced only in dark when IAA, IBA and NAA were used, but 2,4-D could induce them both in light and dark. The system is suitable for the mass propagation of this important rubber yielding plant.Abbreviations MS Murashige and Skoog (1962) - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - NAA -Naphthaleneacetic acid - 2,4-D 2,4-Dichlorophenoxyacetic acid - KN Kinetin - BAP 6-Benzylaminopurine     

Circadian Assessment of Lipids in the Hyperphagic Obese Rat Compared with Lean Litter-Mates

Time and feeding influences on cholesterol, triglyceride, glucose and insulin levels, and serum cholinesterase activity were assessed in a genetically-hyperlipidemic hyperphagic obese rat model, and compared with its lean litter-mate. Following a 28-day acclimation to a 12-hr light/dark cycle, blood samples were obtained every 2 hr from rats via tail bleed for a 24-hr period. Synchronization with other animal studies was established by endogenous serum Cortisol levels [acrophase 18-20 hr after light onset (HALO) in both groups]. Triglycerides cholesterol, insulin and glucose levels were significantly elevated in obese versus lean rats. Obese rats were observed to feed throughout the 24-hr cycle, whereas lean litter-mates ate only during the dark cycle. No circadian rhythmicity was found in glucose levels with either rat group. Insulin levels were not correlated. Although triglyceride levels peaks at 13 HALO in lean rats, no pattern was observed in obese rats. Cholesterol levels were unchanged with time in either group. Cholinesterase activity followed a circadian rhythm in the lean, but not obese, rats with an acrophase estimated at 8 HALO. In contrast to previous reports, enzyme activity was not correlated with triglyceride levels in either rat group. Circadian similarities in insulin levels between rat groups suggest changes in insulin metabolism and/or secretion which are likely to be independent of feeding or activity. Conversely, triglyceride levels remained elevated throughout the 24-hr period in obese rats, whereas significant increases were observed in lean rats during the dark active cycle. These data suggest that triglyceride levels, and not insulin and cholesterol levels, are most likely dependent on feeding patterns.     

A Simple and Novel Strategy for the Production of a Pan-specific Antiserum against Elapid Snakes of Asia

Snakebite envenomation is a serious medical problem in many tropical developing countries and was considered by WHO as a neglected tropical disease. Antivenom (AV), the rational and most effective treatment modality, is either unaffordable and/or unavailable in many affected countries. Moreover, each AV is specific to only one (monospecific) or a few (polyspecific) snake venoms. This demands that each country to prepare AV against its local snake venoms, which is often not feasible. Preparation of a ‘pan-specific’ AV against many snakes over a wide geographical area in some countries/regions has not been possible. If a ‘pan-specific’ AV effective against a variety of snakes from many countries could be prepared, it could be produced economically in large volume for use in many countries and save many lives. The aim of this study was to produce a pan-specific antiserum effective against major medically important elapids in Asia. The strategy was to use toxin fractions (TFs) of the venoms in place of crude venoms in order to reduce the number of antigens the horses were exposed to. This enabled inclusion of a greater variety of elapid venoms in the immunogen mix, thus exposing the horse immune system to a diverse repertoire of toxin epitopes, and gave rise to antiserum with wide paraspecificity against elapid venoms. Twelve venom samples from six medically important elapid snakes (4 Naja spp. and 2 Bungarus spp.) were collected from 12 regions/countries in Asia. Nine of these 12 venoms were ultra-filtered to remove high molecular weight, non-toxic and highly immunogenic proteins. The remaining 3 venoms were not ultra-filtered due to limited amounts available. The 9 toxin fractions (TFs) together with the 3 crude venoms were emulsified in complete Freund’s adjuvant and used to immunize 3 horses using a low dose, low volume, multisite immunization protocol. The horse antisera were assayed by ELISA and by in vivo lethality neutralization in mice. The findings were: a) The 9 TFs were shown to contain all of the venom toxins but were devoid of high MW proteins. When these TFs, together with the 3 crude venoms, were used as the immunogen, satisfactory ELISA antibody titers against homologous/heterologous venoms were obtained. b) The horse antiserum immunologically reacted with and neutralized the lethal effects of both the homologous and the 16 heterologous Asian/African elapid venoms tested. Thus, the use of TFs in place of crude venoms and the inclusion of a variety of elapid venoms in the immunogen mix resulted in antiserum with wide paraspecificity against elapid venoms from distant geographic areas. The antivenom prepared from this antiserum would be expected to be pan-specific and effective in treating envenomations by most elapids in many Asian countries. Due to economies of scale, the antivenom could be produced inexpensively and save many lives. This simple strategy and procedure could be readily adapted for the production of pan-specific antisera against elapids of other continents.     

Single nucleotide polymorphism in sugar pathway and disease resistance genes in sugarcane

Key message

Single nucleotide polymorphism in sugar pathway and disease resistance genes showing genetic association with sugar content and red rot resistance would be useful in marker-assisted genetic improvement of sugarcane.

Abstract

Validation and genotyping of potential sequence variants in candidate genes are necessary to understand their functional significance and trait association potential. We discovered, characterized, validated and genotyped SNPs and InDels in sugar pathway and disease resistance genes of Saccharum complex and sugarcane varieties using amplicon sequencing and CAPS assays. The SNPs were abundant in the non-coding 3′UTRs than 5′UTRs and coding sequences depicting a strong bias toward C to T transition substitutions than transversions. Sequencing of cloned amplicons validated 61.6 and 45.2 % SNPs detected in silico in 21 sugar pathway and 16 disease resistance genes, respectively. Sixteen SNPs in four sugar pathway genes and 10 SNPs in nine disease resistance genes were validated through cost-effective CAPS assay. Functional and adaptive significance of SNP and protein haplotypes identified in sugar pathway and disease resistance genes was assessed by correlating their allelic variation with missense amino acid substitutions in the functional domains, alteration in protein structure models and possible modulation of catalytic enzyme activity in contrasting high and low sugar and moderately red rot resistant and highly susceptible sugarcane genotypes. A strong genetic association of five SNPs in the sugar pathway and disease resistance genes, and an InDel marker in the promoter sequence of sucrose synthase-2 gene, with sugar content and red rot resistance, was evident. The functionally relevant SNPs and InDels, detected and validated in sugar pathway and disease resistance genes, and genic CAPS markers designed, would be of immense use in marker-assisted genetic improvement of sugarcane for sugar content and disease resistance.

     

Mucosal surface ferricyanide reductase activity in mouse duodenum

Mouse duodenum possesses mucosal surface ferricyanide reductase activity. The reducing activity, determined in vitro by measuring ferrocyanide production from ferricyanide, was found to be greater in duodenal fragments when compared with ileal fragments. Experiments with right-side out tied-off duodenal sacs show that reduction occurs mainly on the mucosal side and indicates that the reducing activity is associated with the brush border membrane. Experiments using mice with increased levels of iron absorption (hypoxic, iron-deficient) showed corresponding increases in reducing activity. The increase was present in duodenal but not ileal fragments. Inhibitor studies showed no effect of several compounds which inhibit other, more characterized, transplasma membrane reductases. In particular, doxorubicin (10 m) and quinacrine (1 mm) were without effect on duodenal mucosal transplasma membrane reducing activity. Depolarization of the membrane potential with high medium K ⁺ inhibited reducing activity. N-ethyl malemide (1 mm) was a potent inhibitor, but iodoacetate was found to be less inhibitory. Comparision with inhibitory effects on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) demonstrated that the effect of N-ethyl malemide on reducing activity was not secondary to GAPDH. Collectively these results indicate that mouse duodenum possesses mucosal surface transplasma membrane ferricyanide reductase activity and that the activity is correlated with the process of intestinal iron absorption. Furthermore, the reducing activity appears to be distinct from other reported transplasma membrane reductases.