Profound Effects of Aggregatibacter actinomycetemcomitans Leukotoxin Mutation on Adherence Properties Are Clarified in in vitro Experiments

Leukotoxin (Ltx) is a prominent virulence factor produced by Aggregatibacter actinomycetemcomitans, an oral microorganism highly associated with aggressive periodontitis. Ltx compromises host responsiveness by altering the viability of neutrophils, lymphocytes, and macrophages. Previously, we developed a Rhesus (Rh) monkey colonization model designed to determine the effect of virulence gene mutations on colonization of A. actinomycetemcomitans. Unexpectedly, an A. actinomycetemcomitans leukotoxin (ltxA) mutant (RhAa-VS2) failed to colonize in the Rh model. No previous literature suggested that Ltx was associated with A. actinomycetemcomitans binding to tooth surfaces. These results led us to explore the broad effects of the ltxA mutation in vitro. Results indicated that LtxA activity was completely abolished in RhAa-VS2 strain, while complementation significantly (P<0.0001) restored leukotoxicity compared to RhAa-VS2 strain. RT-PCR analysis of ltx gene expression ruled out polar effects. Furthermore, binding of RhAa-VS2 to salivary-coated hydroxyapatite (SHA) was significantly decreased (P<0.0001) compared to wild type RhAa3 strain. Real time RT-PCR analysis of the genes related to SHA binding in RhAa-VS2 showed that genes related to binding were downregulated [rcpA (P = 0.018), rcpB (P = 0.02), tadA (P = 0.002)] as compared to wild type RhAa3. RhAa-VS2 also exhibited decreased biofilm depth (P = 0.008) and exo-polysaccharide production (P<0.0001). Buccal epithelial cell (BEC) binding of RhAa-VS2 was unaffected. Complementation with ltxA restored binding to SHA (P<0.002) but had no effect on biofilm formation when compared to RhAa3. In conclusion, mutation of ltxA diminished hard tissue binding in vitro, which helps explain the previous in vivo failure of a ltxA knockout to colonize the Rh oral cavity. These results suggest that; 1) one specific gene knockout (in this case ltxA) could affect other seemingly unrelated genes (such as rcpA, rcpB tadA etc), and 2) some caution should be used when interpreting the effect attributed to targeted gene mutations when seen in a competitive in vivo environment.     

Protoplast formation and regeneration in <Emphasis Type=Italic>Lactobacillus delbrueckii</Emphasis>

Method for production and regeneration of Lactobacillus delbrueckii protoplasts are described. The protoplasts were obtained by treatment with a mixture of lysozyme and mutanolysin in protoplast buffer at pH 6.5 with different osmotic stabilizers. The protoplasts were regenerated on deMan, Rogosa and Sharpe (MRS) with various osmotic stabilizers. Maximum protoplast formation was obtained in protoplast buffer with sucrose as an osmotic stabilizer using a combination of lysozyme (1 mg/ml) and mutanolysin (10 μg/ml). Maximum protoplast regeneration was obtained on MRS medium with sucrose (0.5 M) as an osmotic stabilizer. The regeneration medium was also applicable to other species of lactobacilli as well. This is, to our knowledge, the first report on protoplast formation and efficient regeneration in case of L. delbrueckii.     

A study of ethanol tolerance in yeast

The ethanol tolerance of yeast and other microorganisms has remained a controversial area despite the many years of study. The complex inhibition mechanism of ethanol and the lack of a universally accepted definition and method to measure ethanol tolerance have been prime reasons for the controversy. A number of factors such as plasma membrane composition, media composition, mode of substrate feeding, osmotic pressure, temperature, intracellular ethanol accumulation, and byproduct formation have been shown to influence the ethanol tolerance of yeast. Media composition was found to have a profound effect upon the ability of a yeast strain to ferment concentrated substrates (high osmotic pressure) and to ferment at higher temperatures. Supplementation with peptone-yeast extract, magnesium, or potassium salts has a significant and positive effect upon overall fermentation rates. An intracellular accumulation of ethanol was observed during the early stages of fermentation. As fermentation proceeds, the intracellular and extracellular ethanol concentrations become similar. In addition, increases in osmotic pressure are associated with increased intracellular accumulation of ethanol. However, it was observed that nutrient limitation, not increased intracellular accumulation of ethanol, is responsible to some extent for the decreases in growth and fermentation activity of yeast cells at higher osmotic pressure and temperature.     

Evaluation of basic,heterocyclic ring systems as templates for use as potassium competitive acid blockers (pCABs)

A variety of basic, heterocyclic templates has been reported as potassium-competitive, acid pump antagonists. Herein, we report a comparison of potencies of these templates and others to establish which offers the best start point for further systematic optimisation. Modifications were carried out to improve the developability profile of the more potent 1H-pyrrolo[2,3-c]pyridine template, affording molecules with improved overall in vitro characteristics versus the reported clinical candidate AR-H047108, and comparable to the clinically efficacious AZD-0865.     

Sagittal craniosynostosis outcome assessment for two methods and timings of intervention.

A retrospective quantitative analysis of 40 infants who underwent surgery for sagittal craniosynostosis was conducted to determine whether any difference in outcome, with respect to cranial index (cranial width/cranial length x 100), could be associated with either the age at surgery or the extent of the operation. Children < or = 13 months old at surgery and for whom there were archived computed tomography digital data preoperatively, perioperatively, and 1 year postoperatively were studied. For statistical analysis, the operation was classified as either extended strip craniectomy or subtotal calvarectomy, and the age at operation was either < or = 4 months or > 4 months. Twenty-eight patients underwent extended strip craniectomy at a mean age of 5.1 months. Their mean cranial index preoperatively was 67 versus 71 at 1 year postoperatively (p < 0.0001). Of extended strip craniectomy patients, 15 were operated on at age < or = 4 months (mean = 2.9 months) and 13 at age > 4 months (mean = 7.6 months). Mean cranial indices for age at operation groups did not achieve age-appropriate normal range values 1 year postoperatively for either group, and there was no significant difference between the mean percentages of improvement achieved (p = 0.143). Twelve patients underwent subtotal calvarectomy at a mean age of 5.2 months. Their mean cranial index preoperatively was 66 versus 74 at 1 year postoperatively (p < 0.0001). The mean cranial index in this group reached age-appropriate normal range values 1 year postoperatively. The percentage improvement in cranial index 1 year after subtotal calvarectomy was greater than after extended strip craniectomy (p = 0.003). Extended strip craniectomy for sagittal craniosynostosis does not achieve normal cranial width:length proportions, even when performed before 4 months of age. Subtotal calvarectomy for sagittal craniosynostosis does achieve normal cranial width:length proportions in the majority of the children, at least when performed within the first 13 months of life.     

Developmental and neurobehavioural toxicity study of arsenic on rats following gestational exposure

To characterize developmental and behavioral alterations induced by arsenic exposure, Albino rats were exposed to arsenic (0, 1.5, 3.0 and 4.5 mg/kg/day/po) from gestation day 8 to till parturition and the offspring were observed over the first 3 postnatal weeks, until they were weaned on post-natal day (PND) 21. Once the pups were delivered (PND0), the treatment was discontinued. All pups were assessed for physical development, reflex development, strength and motor coordination from standard neurobehavioural developmental test batteries beginning on PND1. Gestational administration of arsenic at tested dose levels, showed no significant changes in the day of appearance of eye opening, startle reflex, negative geotaxis and spontaneous alteration performance in comparison to the control group. The number of live fetuses, mean fetal body weight and percentages of resorptions or malformations per litter were not affected by arsenic exposure. No treatment-related malformations or developmental variations were noted at any exposure level, suggesting that arsenic exposure at this dose level did not adversely affect behavioural endpoints of developmental toxicity.     

Sialic acid attenuates puromycin aminonucleoside-induced desialylation and oxidative stress in human podocytes

Sialoglycoproteins make a significant contribution to the negative charge of the glomerular anionic glycocalyx—crucial for efficient functioning of the glomerular permselective barrier. Defects in sialylation have serious consequences on podocyte function leading to the development of proteinuria. The aim of the current study was to investigate potential mechanisms underlying puromycin aminonucleosisde (PAN)-induced desialylation and to ascertain whether they could be corrected by administration of free sialic acid.     

Phosphatase Inhibitors Function as Novel,Broad Spectrum Botulinum Neurotoxin Antagonists in Mouse and Human Embryonic Stem Cell-Derived Motor Neuron-Based Assays

There is an urgent need to develop novel treatments to counter Botulinum neurotoxin (BoNT) poisoning. Currently, the majority of BoNT drug development efforts focus on directly inhibiting the proteolytic components of BoNT, i.e. light chains (LC). Although this is a rational approach, previous research has shown that LCs are extremely difficult drug targets and that inhibiting multi-serotype BoNTs with a single LC inhibitor may not be feasible. An alternative approach would target neuronal pathways involved in intoxication/recovery, rather than the LC itself. Phosphorylation-related mechanisms have been implicated in the intoxication pathway(s) of BoNTs. However, the effects of phosphatase inhibitors upon BoNT activity in the physiological target of BoNTs, i.e. motor neurons, have not been investigated. In this study, a small library of phosphatase inhibitors was screened for BoNT antagonism in the context of mouse embryonic stem cell-derived motor neurons (ES-MNs). Four inhibitors were found to function as BoNT/A antagonists. Subsequently, we confirmed that these inhibitors protect against BoNT/A in a dose-dependent manner in human ES-MNs. Additionally, these compounds provide protection when administered in post-intoxication scenario. Importantly, the inhibitors were also effective against BoNT serotypes B and E. To the best of our knowledge, this is the first study showing phosphatase inhibitors as broad-spectrum BoNT antagonists.     

Schiff base conjugates of p-aminosalicylic acid as antimycobacterial agents

Schiff base conjugates of p-aminosalicylic acid have been synthesized and characterized by elemental analysis, IR and (1)H NMR spectroscopy and cyclic voltammetry. Compounds containing hydroxyl-rich side chains show enhanced antimycobacterial activity against Mycobacterium smegmatis and Mycobacterium bovis BCG. Higher ClogP values and superior radical scavenging activities are thought to be the contributing factors for their enhanced antimycobacterial activities.