In vitro and in vivo model systems to study microbial biofilm formation

Biofilm formation is often considered the underlying reason why treatment with an antimicrobial agent fails and as an estimated 65-80% of all human infections is thought to be biofilm-related, this presents a serious challenge. Biofilm model systems are essential to gain a better understanding of the mechanisms involved in biofilm formation and resistance. In this review a comprehensive overview of various in vitro and in vivo systems is presented, and their advantages and disadvantages are discussed.     

Pro-fibrotic activity of lysophosphatidic acid in adipose tissue: In vivo and in vitro evidence

Lysophosphatidic acid (LPA) is a pro-fibrotic mediator acting via specific receptors (LPARs) and is synthesized by autotaxin, that increases with obesity. We tested whether LPA could play a role in adipose tissue (AT)-fibrosis associated with obesity. Fibrosis [type I, III, and IV collagens (COL), fibronectin (FN), TGFβ, CTGF and αSMA] and inflammation (MCP1 and F4/80) markers were quantified: (i) in vivo in inguinal (IAT) and perigonadic (PGAT) AT from obese-diabetic db/db mice treated with the LPAR antagonist Ki16425 (5 mg/kg/day ip for 7 weeks); and (ii) in vitro in human AT explants in primary culture for 72 h in the presence of oleoyl-LPA (10 μM) and/or Ki16425 (10 μM) and/or the HIF-1α inhibitor YC-1 (100 μM). Treatment of db/db mice with Ki16425 reduced Col I and IV mRNAs in IAT and PGAT while Col III mRNAs were only reduced in IAT. This was associated with reduction of COL protein staining in both IAT and PGAT. AT explants showed a spontaneous and time-dependent increase in ATX expression and production of LPA in the culture medium, along with increased levels of Col I and III, TGFβ and αSMA mRNAs and of COL protein staining. In vitro fibrosis was blocked by Ki16425 and was further amplified by oleoyl-LPA. LPA-dependent in vitro fibrosis was blocked by co-treatment with YC1. Our results show that endogenous and exogenous LPA exert a pro-fibrotic activity in AT in vivo and in vitro. This activity could be mediated by an LPA1R-dependent pathway and could involve HIF-1α.     

Amelioration of intracerebroventricular streptozotocin induced cognitive impairment by Evolvulus alsinoides in rats: In vitro and in vivo evidence

Evolvulus alsinoides, also known as Shankpushapi, is a commonly used traditional medicine for enhancing memory. We evaluated the in vitro free radical scavenging and enzymes [acetylcholinesterase, butyrylcholinestrase, glycogen synthase kinase-3-β (GSK-3-β), rho kinase (ROCK II), prolyl endopeptidase (PEP), catechol-O-methyl transferase (COMT) and lipoxygenase (LOX)] inhibitory activities of aqueous and hydro-alcoholic extracts of E. alsinoides. Hydro-alcoholic extract of E. alsinoides demonstrated more free radical scavenging activity as compared to aqueous extract. Hydro-alcoholic extract also showed higher cholinesterase, GSK-3-β, ROCK II, PEP, COMT and LOX enzyme inhibitory activities as compared to aqueous extract. Phytochemical analysis revealed more flavanoids in hydro-alcoholic extract as compared to aqueous extract but no significant difference in phenolic content of the two extracts was observed. Based on in vitro data, hydro-alcoholic extract (100, 300 and 500 mg/kg, p.o.) was selected for in vivo study in intracerebroventricularly injected streptozotocin (STZ) induced cognitive impairment in male Wistar rats. Elevated plus maze, passive avoidance and Morris water maze were used for assessment of cognitive function on 14th, 21st and 28th day after STZ injection. Oxidative stress parameters (malondialdehyde, reduced glutathione, nitric oxide levels and superoxide dismutase activity), cholinergic dysfunction and rho kinase (ROCK II) expression were studied in cerebral cortex and hippocampus of rat brain at the end of the study. Hydro-alcoholic extract of E. alsinoides dose dependently prevented STZ induced cognitive impairment by reducing the oxidative stress, improving cholinergic function and preventing the increase in rho kinase expression. The results suggest an anti-Alzheimer potential of hydro-alcoholic extract of E. alsinoides.     

In vitro growth of Trypanosoma musculi in cellfree medium conditioned by rodent macrophages and mercaptoethanol

Studies of the roles of certain components of an in vitro culture system for Trypanosoma musculi were conducted. Mouse macrophages were shown to be highly effective in supporting trypanosome growth; the magnitude of growth was proportional to the number of macrophages. Addition of 2-mercaptoethanol to cultures significantly enhanced the rate of parasite growth. Rat spleen cells were only slightly less efficient than mouse spleen cells in supporting growth of T. musculi. Addition of mouse serum to cultures containing mouse spleen cells slightly enhanced growth of T. musculi but depressed the support afforded by rat spleen cells. Conditioned medium, prepared by separately culturing mouse spleen or peritoneal exudate cells, was capable of supporting extensive trypanosome growth. Conditioning was dependent upon the number of cells cultured and the time of culture, an excess of either resulting in medium containing inhibitors of trypanosome growth. Considerable importance is assigned to the future characterization of the trypanosome growth-promoting substances elaborated by macrophages.     

A possible interaction between CCKergic and GABAergic systems in the rat brain

Cholecystokinin sulfated octapeptide (CCK-8S) was given to rats i.p. at single doses of 10 and 100 nmol/kg, respectively. It produced a modification in GABA levels in several areas of the rat brain. After 30 min of injection, the lower dose (10 nmol/kg) increased GABA levels in striatum by 31% (P<0.05). The higher dose (100 nmol/kg) enhanced GABA levels either in hippocampus by 78% (P<0.05) or in frontal cerebral cortex by 81% (P<0.05) and decreased in olfactory bulbs by 57% (P<0.01). Thus, these results show that systemic injection of CCK-8S, produced regional specific changes on GABA levels in brain, and these effects were dose-dependent. Systemic pretreatment with the CCK(B) receptor antagonist, PD 135,158, 1 mg/kg i.p., on the endogenous levels of GABA in certain regions was also studied. The selective CCK(B) receptor antagonist, PD 135,158, did not have an effect per se on the endogenous levels of GABA but prevents the action induced by the neuropeptide. We suggest that the action of CCK may be mediated via a selective action on the CCK(B) receptor subtypes.     

In vitro biotechnological advancements in Malabar nut (Adhatoda vasica Nees): Achievements,status and prospects

Adhatoda vasica Nees, belonging to family Acanthaceae, is a well-known medicinal plant. It is endorsed for its pyrroloquinazoline alkaloids and its derivatives, such as vasicine and vasicinone. Germinating A. vasica seeds is a tedious task; on that account, vegetative propagation is the preferred method for its multiplication. For rapid and large-scale multiplication, germplasm conservation as well as secondary metabolites production, in vitro culture of A. vasica was preferred over conventional propagation by several researchers; however, some major applications of this tissue culture technique are still awaiting to undergo extensive research. The present review, for the first time, illustrates all the major achievements associated with in vitro regeneration of A. vasica, reported till date and highlights the future prospects.     

An analysis of GABA receptor changes in the discrete regions of mouse brain after acute and chronic treatments with morphine

Abstract: The effects of morphine on the affinity and distribution of GABA receptors in the mouse regions (striatum, medulla, diencephalon, cortex, and cerebellum) were investigated in relation to: (a) acute administration, (b) chronic administration (tolerance), (c) precipitated withdrawal by naloxone, an opiate antagonist, and (d) abrupt withdrawal for 8 and 24 h. The alterations in the affinity as reflected by the dissociation constant (K_D) and the number of receptors (B_max) in the synaptic membranes obtained from controls and various treatments were determined by radioligand binding assay using [³H]muscimol as a ligand. Significant changes were observed in striatum, medulla, and diencephalon, whereas other regions including whole brain exhibited marginal changes. In general the number of GABA receptors increased after tolerance development, which upon abrupt withdrawal returned to control levels except in the case of naloxone-induced precipitated withdrawal. The affinity changes in different regions were diverse in nature and were not evident in the whole brain membranes. These results indicate that: (a) the regional alterations in the affinity and distribution of GABA receptors may play a role in the induction, maintenance, and regression of morphine tolerance; (b) abrupt withdrawal and antagonist precipitated withdrawal affect the GABA system differently, (c) chronic morphine treatment appears to influence the GABA receptors in the cerebellum, a region generally known for its lack of opiate receptors.     

Effect of lactic acid fermentation of wheat and barley whole meal flour on carbohydrate composition and digestibility in mink (Mustela vison)

Wheat and barley whole meal flours (WMFs) were subjected to treatment by fermentation, autoclaving, and fermentation followed by autoclaving. The WMFs were analysed for chemical composition, formulated into wet diets (282 g kg⁻¹) and fed to adult mink (Mustela vison) for determination of total tract digestibility of total starch, total carbohydrate, crude protein and fat. Fermentation of WMF/water mixtures inoculated with a Lactobacillus sp. (strain AD₂) was performed at 30°C for 16 h. Autoclaving was carried out for 60 min at 120°C. Fermentation increased colony-forming units (CFUs) to about 10⁸ g⁻¹ and lowered pH to 3.7–3.8 in both WMFs. All carbohydrate parameters were affected by type of cereal, and were, except for total starch, affected by treatment. Levels of total dietary fibre and β-glucans decreased by fermentation in both WMFs. The decrease in total β-glucans from 33.5 to 18.4 g kg⁻¹ in barley WMF, was mainly restricted to the soluble fraction. Glucose levels in barley WMF increased simultaneously from 0.6 to 12.3 g kg⁻¹. The main effects of autoclaving were increased levels of total dietary fibre, maltose, and increased hydration capacity. With fermentation prior to autoclaving, increases in levels of the fibre fractions and maltose were prevented while hydration capacity prevailed as an effect of autoclaving. Compared with fermentation alone, the combined treatment increased damaged starch levels and hydration capacity. Digestibilities of total carbohydrate, crude protein and fat were significantly higher for wheat than for barley. Fermentation had no effect on digestibility of total starch or total carbohydrate of wheat, but increased digestibility of total starch of barley significantly from 0.742 to 0.880, and of total carbohydrate from 0.457 to 0.616. Autoclaving had no significant effect on digestibility of total starch and total carbohydrate of wheat. Digestibility of total starch and total carbohydrate in barley increased significantly after autoclaving. Total starch and total carbohydrate digestibility of both wheat and barley were significantly enhanced by combined fermentation and autoclaving compared with fermentation alone. Compared with autoclaving alone, combined fermentation and autoclaving promoted no significant improvement of total starch and total carbohydrate digestibility in wheat, whereas total carbohydrate digestibility in barley increased from 0.605 to 0.672. Fat digestibility was slightly improved by both fermentation and autoclaving. Autoclaving of cereals reduced significantly the faecal dry matter contents of mink. This effect could be counteracted by preceding fermentation. In conclusion, lactic acid fermentation of wheat and especially barley provided chemical changes of benefit for carbohydrate digestion in the mink.     

In vitro cytotoxic and mutagenic evaluation of thirteen commercial herbal mixtures sold in KwaZulu-Natal,South Africa

Cytotoxic and mutagenic effects of thirteen commercial herbal mixtures sold in KwaZulu-Natal, South Africa were evaluated using the neutral red uptake (NRU) assay and the Ames test. The herbal mixtures tested included Umzimba omubi, Umuthi wekukhwehlela ne zilonda, Mvusa ukunzi, Umpatisa inkosi, Imbiza ephuzwato, Vusa umzimba, Ingwe® muthi mixture, Ibhubezi™, Supreme one hundred™, Sejeso herbal mixture Ingwe®, Lion izifozonke Ingwe®, Stameta™ BODicare® and Ingwe® special muti. The relative cytotoxicity of the herbal mixtures was established by determining their NI₅₀ values (50% inhibition of neutral red uptake). The test revealed that the most toxic herbal mixture was Umpatisa inkosi with an NI₅₀ value of 0.016 mg/mL and the least toxic mixture was Stameta™ BODicare® with an NI₅₀ value of 28.00 mg/mL. The herbal mixtures showed no mutagenic effects against Salmonella typhimurium tester strains TA98, TA100, TA102, TA1535 and TA1537 when the assay was done without S9 metabolic activation. However, four herbal mixtures, Umpatisa inkosi, Imbiza ephuzwato, Vusa umzimba and Stameta™ BODicare® showed mutagenic effects against TA98 but not the rest of the tester strains after using S9 metabolic activation. Umpatisa inkosi also exhibited weak mutagenic activity against TA1535 after metabolic activation. The remaining mixtures did not show mutagenic effects against all the tester strains after S9 metabolic activation. The cytotoxic and mutagenic results reported here offer a step toward determining the safety of commercial herbal mixtures in South Africa. Herbal mixtures showing higher cytotoxic and mutagenic effects need to be further investigated for their possible effects on humans.     

Diabetic encephalopathy‐related depression: experimental evidence that insulin and clonazepam restore antioxidant status in rat brain

There is increasing evidence suggesting that oxidative stress plays an important role in the development of many chronic and degenerative conditions such as diabetic encephalopathy and depression. Considering that diabetic rats and mice present higher depressive‐like behaviour when submitted to the forced swimming test and that treatment with insulin and/or clonazepam is able to reverse the behavioural changes of the diabetic rats, the present work investigated the antioxidant status, specifically total antioxidant reactivity and antioxidant potential of insulin and clonazepam, as well as the effect of this drugs upon protein oxidative damage and reactive species formation in cortex, hippocampus and striatum from diabetic rats submitted to forced swimming test. It was verified that longer immobility time in diabetic rats and insulin plus clonazepam treatment reversed this depressive‐like behaviour. Moreover, data obtained in this study allowed to demonstrate through different parameters such as protein carbonyl content, 2′7′‐dichlorofluorescein oxidation, catalase, superoxide dismutase, glutathione peroxidase assay, total radical‐trapping antioxidant potential and total antioxidant reactivity that there is oxidative stress in cortex, hippocampus and striatum from diabetic rats under depressive‐like behaviour and highlight the insulin and/or clonazepam effect in these different brain areas, restoring antioxidant status and protein damage. Copyright © 2014 John Wiley & Sons, Ltd.     

In vitro ruminal methanogenesis of a hay-rich substrate in response to different combination supplements of nitrocompounds; pyromellitic diimide and 2-bromoethanesulphonate

Sixteen combinations of 5 treatments at 4 levels were designed in a L₁₆(4⁵) orthogonal experimental design to evaluate associative effects of five methanogenesis inhibitors at four dose levels: nitroethane (NE, 0 mM, 5 mM, 10 mM and 15 mM), 2-nitroethanol (NEOH, 0 mM, 5 mM, 10 mM and 15 mM), 2-nitro-1-propanol (NPOH, 0 mM, 5 mM, 10 mM and 15 mM), pyromellitic diimide (PMDI, 0 mM, 0.02 mM, 0.05 mM and 0.07 mM) and 2-bromoethanesulphonate (BES, 0 mM, 0.01 mM, 0.03 mM and 0.05 mM) on in vitro ruminal methane production of the mixed substrate (Chinese wildrye hay:maize meal = 4:1) using a cumulative gas production technique. After 48 h incubation, in vitro dry matter disappearance (IVDMD), total gas production (GP₄₈, ml/g DM) and total volatile fatty acids (VFA) production in various combinations of these inhibitors were decreased by 10.6-56.0, 26.5-44.5 and 20.3-47.6%, respectively (P<0.05). The molar proportion of acetate in the inhibitor combination groups was decreased by 6.6-12.5% while those of propionate and butyrate were increased by 7.0-19.2 and 21.9-56.5% (P<0.01), respectively. Methane proportion (MP) in total gas production was reduced by 79.4-98.5% (P<0.01), and the highest inhibition occurred in the combination of 10 mM NE, 10 mM NPOH, 0.07 mM PMDI and 0.01 mM BES in cultures. The partial correlation coefficients between NE, NEOH, NPOH, PMDI or BES and CH₄ proportion were −0.465 (P<0.01), −0.417 (P<0.01), −0.355 (P<0.05), −0.408 (P<0.01) and −0.345 (P<0.05), respectively, indicating that NE was the most potent inhibitor, followed by NEOH and PMDI, and finally NPOH and BES. In general, VFA production in the inhibitor combinations was substantially shifted to produce much more butyrate and propionate and less acetate. The combination of 15 mM NE, 10 mM NEOH, 5 mM NPOH, 0.07 mM PMDI and 0.01 mM BES in cultures, leading to >95% methane inhibition, may be the optimal application of these inhibitors with less depression of total VFA production. Further feeding trials to validate these combinations is still required on rumen function, methane production, growth performance and milk production.     

In vivo functional brain imaging and a therapeutic trial of l-arginine in MELAS patients

Background

Mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) is the most common type of mitochondrial disease and is characterized by stroke-like episodes (SEs), myopathy, lactic acidosis, diabetes mellitus, hearing-loss and cardiomyopathy. The causal hypotheses for SEs in MELAS presented to date are angiopathy, cytopathy and neuronal hyperexcitability. L-arginine (Arg) has been applied for the therapy in MELAS patients.

Scope of review

We will introduce novel in vivo functional brain imaging techniques such as MRI and PET, and discuss the pathogenesis of SEs in MELAS patients. We will further describe here our clinical experience with L-arg therapy and discuss the dual pharmaceutical effects of this drug on MELAS.

Major conclusions

Administration of L-arg to MELAS patients has been successful in reducing neurological symptoms due to acute strokes and preventing recurrences of SEs in the chronic phase. L-Arg has dual pharmaceutical effects on both angiopathy and cytopathy in MELAS.

General significance

In vivo functional brain imaging promotes a better understanding of the pathogenesis and potential therapies for MELAS patients. This article is part of a Special Issue entitled Biochemistry of Mitochondria, Life and Intervention 2010.     

Development and use of quantitative competitive PCR assays for relative quantifying rumen anaerobic fungal populations in both in vitro and in vivo systems

This paper describes the use of a quantitative competitive polymerase chain reaction (QC-PCR) assay; using PCR primers to the rRNA locus of rumen fungi and a standard-control DNA including design and validation. In order to test the efficiency of this method for quantifying anaerobic rumen fungi, it has been attempted to evaluate this method in in vitro conditions by comparing with an assay based on measuring cell wall chitin. The changes in fungal growth have been studied when they are grown in in vitro on either untreated (US) or sodium hydroxide treated wheat straw (TS). Results showed that rumen fungi growth was significantly higher in treated samples compared with untreated during the 12 d incubation (P < 0.05) and plotting the chitin assay's results against the competitive PCR's showed high positive correlation (R² ≥ 0.87). The low mean values of the coefficients of variance in repeatability in the QC-PCR method against the chitin assay demonstrated more reliability of this new approach. And finally, the efficiency of this method was investigated in in vivo conditions. Samples of rumen fluid were collected from four fistulated Holstein steers which were fed four different diets (basal diet, high starch, high sucrose and starch plus sucrose) in rotation. The results of QC-PCR showed that addition of these non-structural carbohydrates to the basal diets caused a significant decrease in rumen anaerobic fungi biomass. The QC-PCR method appears to be a reliable and can be used for rumen samples.     

Genetical and biochemical evidence that the hydroxylation pattern of the anthocyanin B-ring in Silene dioica is determined at the p-coumaroyl-coenzyme a stage

In petals of Silene dioica, gene P controls the 3′-hydroxylation of the anthocyanin B-ring and the hydroxylation pattern of the hydroxycinnamoyl acyl group bound to the 4″'-hydroxyl group of rhamnose of anthocyanidin 3-rhamnosyl(1→6)glucoside-5-glucoside. In this paper, experiments are presented which show that gene P is involved in the hydroxylation of p-coumaroyl-CoA to caffeoyl-CoA, which is then used both as a precursor in anthocyanin biosynthesis and as a substrate for the final acylation.     

Plasmodium knowlesi from archival blood films: Further evidence that human infections are widely distributed and not newly emergent in Malaysian Borneo

Human infections with Plasmodium knowlesi have been misdiagnosed by microscopy as Plasmodium malariae due to their morphological similarities. Although microscopy-identified P. malariae cases have been reported in the state of Sarawak (Malaysian Borno) as early as 1952, recent epidemiological studies suggest the absence of indigenous P. malariae infections. The present study aimed to determine the past incidence and distribution of P. knowlesi infections in the state of Sarawak based on archival blood films from patients diagnosed by microscopy as having P. malariae infections. Nested PCR assays were used to identify Plasmodium species in DNA extracted from 47 thick blood films collected in 1996 from patients in seven different divisions throughout the state of Sarawak. Plasmodium knowlesi DNA was detected in 35 (97.2%) of 36 blood films that were positive for Plasmodium DNA, with patients originating from all seven divisions. Only one sample was positive for P. malariae DNA. This study provides further evidence of the widespread distribution of human infections with P. knowlesi in Sarawak and its past occurrence. Taken together with data from previous studies, our findings suggest that P. knowlesi malaria is not a newly emergent disease in humans.     

Probing for missing links in the binary and ternary V(V)-citrate-(H2O2) systems: Synthetic efforts and in vitro insulin mimetic activity studies

In a pH-specific fashion, V₂O₅ and citric acid in the absence and presence of H₂O₂ reacted and afforded, in the presence of NaOH and (CH₆N₃)₂CO₃, two new dinuclear V(V) binary non-peroxo (CH₆N₃)₆[V₂O₄(C₆H₄O₇)₂] · 2H₂O (1) and ternary peroxo (CH₆N₃)₄[V₂O₂(Ο₂)₂(C₆H₅O₇)₂] · 6Η₂Ο (2) species, respectively. Complexes 1 and 2 were further characterized by elemental analysis, UV/Vis, FT-IR, NMR (solution and solid state Cross Polarization-Magic Angle Spinning (CP-MAS)) and Raman spectroscopies, cyclic voltammetry, and X-ray crystallography. Both 1 and 2 are members of the family of dinuclear V(V)-citrate species bearing citrate with a distinct coordination mode and degree of deprotonation, with 2 being the missing link in the family of pH-structural variants of the ternary V(V)-peroxo-citrate system. Given that 1 and 2 possess distinct structural features, relevant binary V(III), V(IV) and V(V), and ternary V(V) species bearing O- and N-containing ligands were tested in in vitro cell cultures to assess their cellular toxicity and insulin mimetic capacity. The results project a clear profile for all species tested, earmarking the importance of vanadium oxidation state and its ligand environment in influencing further binary and ternary interactions of vanadium arising with variable mass cellular targets, ultimately leading to a specific (non)toxic phenotype and glucose uptake ability.     

Timing of the water-to-land transition and metamorphosis in the land hermit crab Coenobita compressus H. Milne Edwards: evidence that settlement and metamorphosis are de-coupled

After metamorphosing from the last larval stage to the transitional megalopal stage in the marine plankton, the hermit crab Coenobita compressus moves ashore where it undergoes a second metamorphosis to the first juvenile instar on land. In two experiments using laboratory-reared crabs, I moved megalopae from water to land after different amounts of time at this stage and investigated the impact of this manipulation on the timing of and survival through the second metamorphosis. In the Involuntary Settlement experiment, megalopae were moved to land when they were 3, 6, 9, 12, or 15 days old. None of those moved between the ages of 3 and 6 days survived through metamorphosis, but the majority of 9-day-old megalopae survived, as did most 12- and 15-day-old megalopae. This suggests that developmental changes early in the megalopal stage prepare C. compressus for terrestrial life. Once on land, megalopae that had been moved to land at 9 days spent about nine additional days there before metamorphosing, while 12- and 15-day-old megalopae metamorphosed after spending about 5 and 4 days, respectively, on land. In the Voluntary Settlement experiment, megalopae were given access to land when they were 1, 5, 10, or 15 days old, but were not forced to make the transition. Those given access to land after 1 day voluntarily left their dishes for the first time after an average of 7 days in water. Those given access when they were 5 days old remained in water about 4 days longer, while those given access when they were 10 and 15 days old left after less than a day. In both experiments, the timing of metamorphosis relative to settlement (i.e., transition to land) showed that these events are dissociated to a degree and revealed the presence of a metamorphic clock. I discuss why the dissociation of settlement and metamorphosis may have been favored in the land hermit crab and in another anomuran crab.