Physiological methods to study biofilm disinfection

This report reviews the development of a rapidin situ approach to study the physiological responses of bacteria within biofilms to disinfectants. One method utilized direct viable counts (DVC) to assess the disinfection efficacy when thin biofilms were exposed to chlorine or monochloramine. Results obtained using the DVC method were one log higher than plate count (PC) estimates of the surviving population after disinfection. Other methods incorporated the use of fluorogenic stains, a cryotomy technique to yield thin (5-m) sections of biofilm communities and examination by fluorescence microscopy. The fluorogenic stains used in this approach included 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), which indicates cellular electron transport activity and Rhodamine 123, which responds specifically to proton motive force. The use of these stains allowed the microscopic discrimination of physiologically active bacteria as well as heterogeneities of active cells within thicker biofilms. The results of experiments using these techniques with pure culture and binary population biofilms on stainless steel coupons indicated biocidal activity of chlorine-based disinfectants occurred initially at the bulk-fluid interface of the communities and progressed toward the substratum. This approach provided a unique opportunity to describe the spatial response of bacteria within biofilms to antimicrobial agents and address mechanisms explaining their comparative resistance to disinfection in a way that has not been possible using traditional approaches. Results obtained using this alternative approach were also consistently higher than PC data following disinfection. These observations suggest that traditional methods involving biofilm removal and bacterial enumeration by colony formation overestimate biocide efficacy. Hence the alternative approach described here more accurately indicates the ability of bacteria surviving disinfection to recover and grow as well as demonstrate spatial heterogeneities in cellular physiological activities within biofilms.     

Variability of water supply affected shoot biomass and root depth distribution of four temperate grassland species in monocultures and mixtures

极端降雨事件对生态系统功能影响的研究主要集中在干旱或洪水事件，通常包括全年或某个季度的平均或总降雨量的变化。然而，在不改变平均量或总量的情况下，增加降雨变异性的影响还很少被研究。本研究调查了增加水分供应变异性对单一栽培和混合栽培下4种草原植物的地上和根系生物量以及根系生物量分布的影响。将多年生黑麦草(Lolium perenne L., 浅根)，菊苣(Cichorium intybus L., 深根)，白车轴草(Trifolium repens L., 浅根)和红车轴草(Trifolium pratense L., 深根)种植在中型实验生态系统中。单一栽培条件下种植4株同种植物，混合栽 培条件下每种植物各1株。水分供应变异性处理设置低中高3种水平：与低变异水分供应处理相比，另设置一个中度变异处理(±40%)和高度变 异处理(±80%)，测定了地上和根的生物量，拟合了根系垂直分布模型。研究结果表明，与低变异处理相比，白车轴草，红车轴草和4种植物 混种群落的茎生物量在高变异处理下显著减少。在所有处理中，4种植物混种群落产生的茎和根生物量比单一栽培(超产)中物种表现所预测的要多。在水分变异增加的情况下，菊苣在单一栽培下根系分配了更多的生物量到更深的土层，而白车轴草在单一栽培下根系总生物量显著降 低。这些结果表明，水分供应变异的增加会对单一栽培和混合栽培草地的地上和根的生物量产生负面影响。在野外尺度上，水分变异对多物种 草地系统功能的影响需要进一步研究。     

Tumor necrosis factor-α-induced neutral sphingomyelinase-2 modulates synaptic plasticity by controlling the membrane insertion of NMDA receptors

The insertion and removal of NMDA receptors from the synapse are critical events that modulate synaptic plasticity. While a great deal of progress has been made on understanding the mechanisms that modulate trafficking of NMDA receptors, we do not currently understand the molecular events required for the fusion of receptor containing vesicles with the plasma membrane. Here, we show that sphingomyelin phosphodiesterase 3 (also known as neutral sphingomyelinase-2) is critical for tumor necrosis factor (TNF) α-induced trafficking of NMDA receptors and synaptic plasticity. TNFα initiated a rapid increase in ceramide that was associated with increased surface localization of NMDA receptor NR1 subunits and a specific clustering of NR1 phosphorylated on serines 896 and 897 into lipid rafts. Brief applications of TNFα increased the rate and amplitude of NMDA-evoked calcium bursts and enhanced excitatory post-synaptic currents. Pharmacological inhibition or genetic mutation of neutral sphingomyelinase-2 prevented TNFα-induced generation of ceramide, phosphorylation of NR1 subunits, clustering of NR1, enhancement of NMDA-evoked calcium flux and excitatory post-synaptic currents.     

HIV-1 Tat through phosphorylation of NMDA receptors potentiates glutamate excitotoxicity

Toxic effects of HIV-1 proteins contribute to altered function and decreased survival of select populations of neurons in HIV-1-infected brain. One such HIV-1 protein, Tat, can activate calcium release from IP3-sensitive intracellular pools, induce calcium influx in neural cells, and, as a result, can increase neuronal cell death. Here, we provide evidence that Tat potentiates excitatory amino acid (glutamate and NMDA) triggered calcium flux, as well as glutamate- and staurosporine-mediated neurotoxicity. Calcium flux in cultured rat hippocampal neurons triggered by the transient application of glutamate or NMDA was facilitated by pre-exposure to Tat. Facilitation of glutamate-triggered calcium flux by Tat was prevented by inhibitors of ADP-ribosylation of G(i)/G(o) proteins (pertussis toxin), protein kinase C (H7 and bisindolymide), and IP3-mediated calcium release (xestospongin C), but was not prevented by an activator of G(s) (cholera toxin) or an inhibitor of protein kinase A (H89). Facilitation of NMDA-triggered calcium flux by Tat was reversed by inhibitors of tyrosine kinase (genestein and herbimycin A) and by an inhibitor of NMDA receptor function (zinc). Tat increased 32P incorporation into NMDA receptor subunits NR2A and NR2B and this effect was blocked by genestein. Subtoxic concentrations of Tat combined with subtoxic concentrations of glutamate or staurosporine increased neuronal cell death significantly. Together, these findings suggest that NMDA receptors play an important role in Tat neurotoxicity and the mechanisms identified may provide additional therapeutic targets for the treatment of HIV-1 associated dementia.     

Matrix metalloproteinase-1 activates a pertussis toxin-sensitive signaling pathway that stimulates the release of matrix metalloproteinase-9

The matrix metalloproteinases (MMPs) are a family of structurally related metalloendopeptidases so named due to their propensity to target extracellular matrix (ECM) proteins. Accumulating evidence, however, suggests that these proteases cleave numerous non-ECM substrates including enzymes and cell surface receptors. MMPs may also bind to cell surface receptors, though such binding has typically been thought to mediate internalization and degradation of the bound protease. More recently, it has been shown that MMP-1 coimmunoprecipitates with the alpha2beta1 integrin, a receptor for collagen. This association may serve to localize the enzymatic activity of MMP-1 so that collagen is cleaved and cell migration is facilitated. In other studies, however, it has been shown that integrin engagement may be linked to the activation of signaling cascades including those mediated by Gialpha containing heterotrimers. As an example, alpha2beta1 can form a complex with CD47 that may associate with Gialpha. In the present study we have therefore investigated the possibility that MMP-1 may affect intracellular changes that are linked to the activation of a Gi protein-coupled receptor. We show that treatment of neural cells with MMP-1 is followed by a rapid reduction in cytosolic levels of cAMP. Moreover, MMP-1 potentiates proteinase activated receptor-1 (PAR-1) agonist-linked increases in intracellular calcium, an effect which is often observed when an agonist of a Gi protein-coupled receptor is administered in association with an agonist of a Gq coupled receptor. In addition, MMP-1 stimulates pertussis toxin sensitive release ofMMP-9 both from cultured neural cells and monocyte/macrophages. Together, these results suggest that MMP-1 signals through a pertussis toxin-sensitive G protein-coupled receptor.     

Ceramide metabolism analysis in a model of binge drinking reveals both neuroprotective and toxic effects of ethanol

Binge drinking is a common form of alcohol abuse that involves repeated rounds of intoxication followed by withdrawal. The episodic effects of binge drinking and withdrawal on brain resident cells are thought to contribute to neural remodeling and neurological damage. However, the molecular mechanisms for these neurodegenerative effects are not understood. Ethanol (EtOH) regulates the metabolism of ceramide, a highly bioactive lipid that is enriched in brain. We used a mouse model of binge drinking to determine the effects of EtOH intoxication and withdrawal on brain ceramide metabolism. Intoxication and acute alcohol withdrawal were each associated with distinct changes in ceramide regulatory genes and metabolic products. EtOH intoxication was accompanied by decreased concentrations of multiple ceramides, coincident with reductions in the expression of enzymes involved in the production of ceramides, and increased expression of ceramide‐degrading enzymes. EtOH withdrawal was associated with specific increases in ceramide C16:0, C18:0, and C20:0 and increased expression of enzymes involved with ceramide production. These data suggest that EtOH intoxication may evoke a ceramide phenotype that is neuroprotective, whereas EtOH withdrawal results in a metabolic shift that increases the production of potentially toxic ceramide species.

     

Attenuated neuroprotective effect of riboflavin under UV-B irradiation via miR-203/c-Jun signaling pathway in vivo and in vitro

Background

Riboflavin (RF) or vitamin B2 is known to have neuroprotective effects. In the present study, we report the attenuation of the neuroprotective effects of RF under UV-B irradiation. Preconditioning of UV-B irradiated riboflavin (UV-B-RF) showed attenuated neuroprotective effects compared to that of RF in SH-SY5Y neuroblostoma cell line and primary cortical neurons in vitro and a rat model of cerebral ischemia in vivo.

Results

Results indicated that RF pretreatment significantly inhibited cell death and reduced LDH secretion compared to that of the UV-B-RF pretreatment in primary cortical neuron cultures subjected to oxygen glucose deprivation in vitro and cortical brain tissue subjected to ischemic injury in vivo. Further mechanistic studies using cortical neuron cultures revealed that RF treatment induced increased miR-203 expression which in turn inhibited c-Jun expression and increased neuronal cell survival. Functional assays clearly demonstrated that the UV-B-RF preconditioning failed to sustain the increased expression of miR-203 and the decreased levels of c-Jun, mediating the neuroprotective effects of RF. UV-B irradiation attenuated the neuroprotective effects of RF through modulation of the miR-203/c-Jun signaling pathway.

Conclusion

Thus, the ability of UV-B to serve as a modulator of this neuroprotective signaling pathway warrants further studies into its role as a regulator of other cytoprotective/neuroprotective signaling pathways.     

Human gamma-aminobutyric acid A receptor alpha2 gene moderates the acute effects of alcohol and brain mRNA expression

γ-aminobutyric acid A (GABA_A) receptors moderate several of the behavioral effects of alcohol. In fact, recent studies have shown an association between the gene for the α2-subunit of the GABA_A receptor ( GABRA2 ) and alcoholism. In the present study, we examined the functional relevance of the GABRA2 gene in alcohol dependence by assessing brain GABRA2 mRNA and GABA_Aα2-subunit protein levels in post-mortem prefrontal cortical tissue collected from control and alcohol-dependent individuals. In addition, using an endophenotype approach, we tested whether the GABRA2 gene moderates sensitivity to the acute effects of alcohol in two independent samples from distinct human alcohol challenge studies. Results indicated that GABRA2 mRNA levels significantly differed by GABRA2 genotype. GABRA2 single nucleotide polymorphisms (rs573400, rs279871 and rs279858) were significantly associated with sensitivity to the acute effects of alcohol. Specifically, there was a significant main effect of GABRA2  × breath alcohol concentration on several measures of subjective responses to alcohol, including the hedonic value of alcohol. Importantly, reanalysis of a previous intravenous alcohol administration study confirmed the results of the oral alcohol challenge study. In summary, these results extend previous findings and provide new insights into the putative biobehavioral mechanisms that may moderate the association between the GABRA2 gene, sensitivity to the acute effects of alcohol and ultimately alcohol dependence.