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Preterm infants have a high risk of neonatal white matter injury (WMI). WMI leads to reduced myelination, inflammation, and clinical neurodevelopmental deficits for which there are no effective treatments. Ionotropic glutamate receptor (iGluR) induced excitotoxicity contributes to oligodendrocyte (OL) lineage cell loss and demyelination in brain models of neonatal and adult WMI. Here, we hypothesized that simulated ischemia (oxygen–glucose deprivation) damages white matter via activation of iGluR signaling, and that iGluR inhibition shortly after WMI could mitigate OL loss, enhance myelination, and suppress inflammation in an ex vivo cerebellar slice model of developing WMI. Inhibition of iGluR signaling by a combined block of AMPA and NMDA receptors, shortly after simulated ischemia, restored myelination, reduced apoptotic OLs, and enhanced OL precursor cell proliferation and maturation as well as upregulated expression of transcription factors regulating OL development and remyelination. Our findings demonstrate that iGluR inhibition post–injury alleviates OL lineage cell loss and inflammation and promotes myelination upon developing WMI. The findings may help to develop therapeutic interventions for the WMI treatment.

Reactive gliosis and inflammation are risk factors for white matter injury (WMI) development, which are correlated with the development of many neurodevelopmental deficits with no treatment. This study aimed to understand the mechanisms correlated with WMI, with a particular focus on the role of nuclear factor‑kappa B (NF‑kB) and p38 mitogen‑activated protein kinases (MAPKs) pathways. Seven‑day‑old Wistar rats were used to generate cerebellar tissue slices. Slices were cultured and randomly allocated to one of 3 groups and treated as follows: group‑I (control); group‑II (WMI), slices were subjected to 20 min of oxygen‑glucose deprivation (OGD); group‑III (WMI+ blockers), slices were subjected to OGD and treated with the blockers. Results showed that OGD insult triggered a marked increase in the apoptosis among WM elements, as confirmed by TUNEL assay. Immunocytochemical experiments revealed that there was a significant decrease in the percent of MBP+ OLs and NG2+ OPCs, and myelin integrity. There was also a significant increase in the percent of reactive microglia and astrocytes. BrdU immunostaining revealed there was an increase in the percent of proliferating microglia and astrocytes. Q‑RT‑PCR results showed OGD upregulated the expression levels of cytokines (TNF‑α, IL‑1, IL‑6, and IL‑1β) and inducible nitric oxide synthase (iNOS). On the other hand, treatment with BAY11 or SB203580 significantly enhanced the OL survival, restored myelin loss, and reduced microglia and astrocyte reactivity, and downregulated the iNOS and cytokine expression. Our findings demonstrate that blocking of NF‑KB/p38 MAPK pathways alleviated reactive gliosis, inflammation, and OL loss upon WMI. The findings may help to develop therapeutic interventions for WMI.

Summary Microbes can invade as whole communities, but the ecology of whole community invasions is poorly understood. Here, we investigate how invader propagule pressure (the number of invading organisms) affects the composition and function of invaded laboratory methanogenic communities. An invading community was equally successful at establishing itself in a resident community regardless of propagule pressure, which varied between 0.01 and 10% of the size resident community. Invasion resulted in enhanced biogas production (to the level of the pure invading community) but only when propagule pressure was 1% or greater. This inconsistency between invasion success and changes in function can be explained by a lower richness of invading taxa at lower propagule pressures, and an important functional role of the taxa that were absent. Our results highlight that whole community invasion ecology cannot simply be extrapolated from our understanding of single species invasions. Moreover, we show that methane production can be enhanced by invading poorly performing reactors with a better performing community at levels that may be practical in industrial settings., Graphical abstract, Highlights • Complex communities can successfully invade other communities • Invasions change the community composition independently of propagule pressure • Number of taxa invading increases with increased propagule pressure • Larger propagule pressure impacts both the community structure and function, Ecology; Microbiology; Applied microbiology

Microbes can invade as whole communities, but the ecology of whole community invasions are poorly understood. Here, we investigate how invader frequency affects the composition and function of invaded laboratory methanogenic communities. An invading community was equally successful at establishing itself in a resident community regardless of initial invader frequency, which varied between 0.01 and 10%. Invasion resulted in enhanced biogas production (to the level of the pure invading community), but only when invader frequency was 1% or greater. This inconsistency between invasion success and changes in function can be explained by a lower number of invading taxa (but not individuals) at lower initial invader frequencies, and an important functional role of the taxa that were absent. Our results highlight that whole community invasion ecology cannot simply be extrapolated from our understanding of single species invasions. Moreover, we show that methane production can be enhanced by invading poorly performing reactors with a better performing community at levels that may be practical in industrial settings.

Background: Astrocyte pH regulation is essential for brain function. Results: Bicarbonate-independent acid load recovery in whole-mount astrocytes was achieved primarily via V-ATPase, which also held astrocytes at a basic resting pH. Conclusion: V-ATPase is the major bicarbonate-independent H+ extruder in a whole-mount astrocyte population. Significance: High levels of V-ATPase expression will isolate astrocyte pH regulation from cytoplasm changes in other ion species., The mechanisms of HCO3−-independent intracellular pH (pHi) regulation were examined in fibrous astrocytes within isolated neonatal rat optic nerve (RON) and in cultured cortical astrocytes. In agreement with previous studies, resting pHi in cultured astrocytes was 6.82 ± 0.06 and inhibition of the V-ATPase H+ pump by Cl− removal or via the selective inhibitor bafilomycin had only a small effect upon resting pHi and recovery following an acid load. In contrast, resting pHi in RON astrocytes was 7.10 ± 0.04, significantly less acidic than that in cultured cells (p < 0.001), and responded to inhibition of V-ATPase with profound acidification to the 6.3–6.5 range. Fluorescent immuno-staining and immuno-gold labeling confirmed the presence V-ATPase in the cell membrane of RON astrocyte processes and somata. Using ammonia pulse recovery, pHi recovery in RON astrocyte was achieved largely via V-ATPase with sodium-proton exchange (NHE) playing a minor role. The findings indicate that astrocytes in a whole-mount preparation such as the optic nerve rely to a greater degree upon V-ATPase for HCO3−-independent pHi regulation than do cultured astrocytes, with important functional consequences for the regulation of pH in the CNS.

We report first results on developing smart sensor systems for the automatic and frequent collection of animal weight and gait data, under the hostile conditions of a livestock farm. The novelty in our approach is to sense frequently the animals’ floor contact, in suitably chosen locations, under natural and unobtrusive conditions. We demonstrate a pilot low profile rubberized mat sensor heads, delivering a large number of plastic optical fiber transmission measurements taken frequently from individual deformation sensors. The acquired data allows gait analysis from guided-path tomography images or by machine learning. We demonstrate weight estimation with accuracy better than 1% over a range suitable for deployment in pig farms.

Hydrogen and methane were simultaneously produced in a two-phase reactor, op- erated to separate the reactions of hydrogen and methanogen production. Each reactor was inoculated with a seed enriched with different microbial consortia. The first phase was operated with a hydraulic retention time of 7 days and at an organic loading rate of 7.7 g VS L−1 d−1 that produced a stable pH of 5.5. This suppressed the growth of methanogens and as a result, the off gas contained up to 27% hydrogen. The second phase was operated with a hydraulic retention time of 12 days and at an organic loading rate of 3.6 g VS L−1 d−1. This permitted the growth of hydrogenotrophs and methanogens to produce methane at a concen- tration of 60%. Examination of the microbial population of the two reactors both microscopically and using PCR, showed an effective separation of hydrogen- and methane-producing microbial communities. The study revealed that the suppression of hydrogentrophs and methanogens can be achieved by adopting rapid method that leads the growth of hydrogen- and methane-producing granules in phase-separated anaerobic environment.

The total energy produced from co-digested food waste and sewage sludge was compared for single phase mesophilic anaerobic digestion producing methane and two-phase hydrogen production followed by methane production. Both single and two-phase reactors were operated at close to optimum conditions. The single phase methaniser had a methane yield of 0.48 m 3 methane/kg VS destroyed. The two-phase system had a hydrogen yield of 0.13 m 3 hydrogen/kg VS destroyed, and a methane yield of 0.67 m 3 methane/kg VS destroyed. Introduction of a hydrogen producing, pre-treatment phase increased the overall VS destruction 69–89%, however the total energy yield decreased by 13.4% due to the low hydrogen yield obtained in the first stage. The release of ammonia in the hydrogeniser was low and so with less alkalinity available, pH control was necessary. It was much higher in the methaniser and adequate to buffer any pH change. This also ensures more nitrogen in the digestate to enhance its value for recycling. The two-stage process is an attractive option where solids destruction is an important consideration but further optimisation of the hydrogen production stage is still required.

The basic division between white matter ‘fibrous’ astrocytes and grey matter ‘protoplasmic’ astrocytes is well established in terms of their morphological differences. The availability of transgenic animals with green fluorescent protein (GFP) expression restricted to specific glial cell types now provides an approach for looking at changes in cell number and morphology in the two astrocyte types in whole mount preparations. This is an important goal, as the ease of generating astrocyte cultures has led to a proliferation of studies that have examined ischaemic effects on astrocytes in vitro. This has in turn engendered a belief that astrocytes have an extraordinary resistance to ischaemic injury, a belief that runs counter to almost all the data available from in vivo and whole-mount preparations. One possible source of this confusion is the reactive changes that occur in astrocytes following injury, which include an increase in cell number that may obscure early astrocyte cell death and which has been reported to initiate within hours of an ischaemic event. However, we show here that neither white matter nor grey matter GFP(+) astrocytes exhibit any feature of reactive astrocytosis within a 180-min period of reperfusion following modelled ischaemia in neonatal whole-mount preparations. We also show that white matter astrocytes are much more sensitive to ischaemia-reperfusion injury than are grey matter astrocytes, a feature that may have high significance for developmental disorders of white matter tracts such as cerebral palsy.

Controlled oxidation reactions catalyzed by the large, proton-pumping complexes of the respiratory chain generate an electrochemical gradient across the mitochondrial inner membrane that is harnessed for ATP production. However, several alternative respiratory pathways in plants allow the maintenance of substrate oxidation while minimizing the production of ATP. We have investigated the role of light in the regulation of these energy-dissipating pathways by transcriptional profiling of the alternative oxidase, uncoupling protein, and type II NAD(P)H dehydrogenase gene families in etiolated Arabidopsis seedlings. Expression of the nda1 and ndc1 NAD(P)H dehydrogenase genes was rapidly up-regulated by a broad range of light intensities and qualities. For both genes, light induction appears to be a direct transcriptional effect that is independent of carbon status. Mutant analyses demonstrated the involvement of two separate photoreceptor families in nda1 and ndc1 light regulation: the phytochromes (phyA and phyB) and an undetermined blue light photoreceptor. In the case of the nda1 gene, the different photoreceptor systems generate distinct kinetic induction profiles that are integrated in white light response. Primary transcriptional control of light response was localized to a 99-bp region of the nda1 promoter, which contains an I-box flanked by two GT-1 elements, an arrangement prevalent in the promoters of photosynthesis-associated genes. Light induction was specific to nda1 and ndc1. The only other substantial light effect observed was a decrease in aox2 expression. Overall, these results suggest that light directly influences the respiratory electron transport chain via photoreceptor-mediated transcriptional control, likely for supporting photosynthetic metabolism.

Cerebral palsy is a common birth disorder that frequently involves ischemic-type injury to developing white matter (WM). Dead glial cells are a common feature of this injury and here we describe a novel form of acute ischemic cell death in developing WM astrocytes. Ischemia, modeled by the withdrawal of oxygen and glucose, evoked [Ca 2+ ] i increases and cell death in astrocytes in post-natal day 10 (P10) rat optic nerve (RON). Removing extracellular Ca 2+ prevented increases in [Ca 2+ ] i but increased the amount of cell death. Astrocytes showed rapid [Na + ] i increases during ischemia and cell death was reduced to control levels by substitution of extracellular Na + or Cl or by perfusion with bumetanide, a selective Na-K-Cl cotransport (NKCC) blocker. Astrocytes showed marked swelling during ischemia in the absence of extracellular Ca 2+ , which was blocked by bumetanide. Raising the extracellular osmolarity to limit water uptake reduced ischemic astrocyte death to control levels. Ultrastructural examination showed that post-ischemic astrocytes had lost their processes and frequently were necrotic, effects partially prevented by bumetanide. At this point in development, therefore. NKCC activation in astrocytes during ischemia produces an osmo-regulatory challenge. Astrocytes can subsequently regulate their cell volume in a Ca 2+ -dependent fashion but this will require ATP hydrolysis and does not protect the cells against acute cell death.

The phytochromes are a family of plant photoreceptor proteins that control several adaptive developmental strategies1,2. For example, the phytochromes perceive far-red light (wavelengths between 700 and 800 nm) reflected or scattered from the leaves of nearby vegetation. This provides an early warning of potential shading, and triggers a series of ‘shade-avoidance’ responses, such as a rapid increase in elongation3, by which the plant attempts to overgrow its neighbours3. Other, less immediate, responses include accelerated flowering and early production of seeds. However, little is known about the molecular events that connect light perception with increased growth in shade avoidance. Here we show that the circadian clock gates this rapid shade-avoidance response. It is most apparent around dusk and is accompanied by altered expression of several genes. One of these rapidly responsive genes encodes a basic helix–loop–helix protein, PIL1, previously shown to interact with the clock protein TOC1 (ref. 4). Furthermore PIL1 and TOC1 are both required for the accelerated growth associated with the shade-avoidance response.

In Arabidopsis flowering is accelerated by reduced red:far-red (R:FR) ratio which signals the presence of neighbouring vegetation. Hastened flowering is one component of the shade-avoidance syndrome of responses, which alter many aspects of development in response to the threat of potential competition. Of the red/far-red-absorbing photoreceptors it is phyB that plays the most prominent role in shade-avoidance, although other related phytochromes act redundantly with phyB. It is well established that the phyB mutant has a constitutively early flowering phenotype. However, we have shown that the early flowering phenotype of phyB is temperature-dependent. We have established that this temperature-sensitive flowering response defines a pathway that appears to be independent of the autonomous-FLC pathway. Furthermore, we have demonstrated that the phytochromes control the expression of the floral promoter FT. We have also shown that other phyB-controlled responses, including petiole elongation, are not sensitive to the same temperature change. This suggests that discrete pathways control flowering and petiole elongation, components of the shade-avoidance response. This work provides an insight into the phytochrome and temperature interactions that maintain flowering control.

Summary We describe a chemically induced gene control mechanism for plants based on the ALCR transcription factor and alcA promoter of Aspergillus nidulans, which we have called the alc system. Ethanol, the chemical inducer, is not toxic at levels required for induction, and can be applied to the plants by spraying, root drenching and addition to liquid growth media. The alc system is very sensitive to ethanol and the induction is rapid; 0.01% (1.7 mM) ethanol in liquid growth media initiates chloramphenicol acetyl transferase (CAT) reporter gene expression within 4 h, with maximal expression occurring after 4 days. In the complete absence of ethanol, there is no detectable expression of CAT, nor do we observe induction in plants subjected to wound, cold or drought stress, or following treatment with either salicylic acid or methyl jasmonate. However, extreme anoxia resulting in elevated levels of alcohol dehydrogenase activity in both roots and leaves gave substantial induction of CAT in leaves but not in roots. We believe that the alc system will have broad utility in the exogenous control of plant gene expression in pure science and that it also has considerable potential in agriculture.

Astrocytes are fundamentally important to the maintenance and proper functioning of the central nervous system. During the period of development when myelination is occurring, white matter astrocytes are particularly sensitive to ischemic injury and their failure to regulate glutamate during ischemic conditions may be an important factor in excitotoxic injury. Here, we have identified key mechanisms of injury that operate on the processes of immature white matter astrocytes during oxygen-glucose deprivation (OGD) using GFAP-GFP mice. Oxygen-glucose deprivation produced a parallel loss of astrocyte processes and somata, assessed by both the retention of GFP fluorescence within these structures and by quantitative electron microscopy. Oxygen-glucose deprivation-induced process loss was Ca2+ independent and had two distinct mechanisms. Substituting either extracellular Na+ or Cl−, or perfusion with the Na—K–Cl co-transport blocker bumetanide, provided protection up to 40 mins of OGD but not beyond that point. HCO−3 substitution or perfusion with 4,4′-diisothiocyanostilbene-2,2′-disulphonic acid provided complete protection of the processes up to 60 mins of OGD. Zero-Na+/zero-K+ conditions provided complete protection from OGD-induced injury of processes and somata at all time points. We conclude that acute ischemic-type injury of immature astrocytes follows a cytotoxic ion influx mediated in part by Na—K–Cl co-transport and in part by Na+- and K+-dependent HCO−3 transport, a mechanism that is common to both cell processes and somata. This work provides a basis on which preventative strategies may be developed to protect white matter astrocytes from ischemic injury in susceptible individuals.

This protocol details an RNA preparation for medium-scale, high-purity RNA production from higher plants. It uses hot acid phenol with standard sodium acetate ethanol precipitation and is suitable for producing RNA for both Northern blotting and enzyme-based downstream applications such as RT-PCR and microarray studies.

A method is presented for the extraction of total protein from Arabidopsis thaliana tissue. The protocol was designed for the solubilization of a range of proteins and their efficient and quantitative recovery. It is especially compatible with the small quantities of available tissue often associated with this species and was originally intended for Western blot preparations. Samples extracted using this method can be quantitated directly using a commercially available kit.

A method is presented for the extraction of total protein from Arabidopsis thaliana tissue. The protocol was designed for the solubilization of a range of proteins and their efficient and quantitative recovery. It is especially compatible with the small quantities of available tissue often associated with this species and was originally intended for Western blot preparations. Samples extracted using this method can be quantitated directly using a commercially available kit.

This protocol details an RNA preparation for medium-scale, high-purity RNA production from higher plants. It uses hot acid phenol with standard sodium acetate ethanol precipitation and is suitable for producing RNA for both Northern blotting and enzyme-based downstream applications such as RT-PCR and microarray studies.

Injury to oligodendrocyte processes, the structures responsible for myelination, is implicated in many forms of brain disorder. Here we show NMDA (N-methyl-D-aspartate) receptor subunit expression on oligodendrocyte processes, and the presence of NMDA receptor subunit messenger RNA in isolated white matter. NR1, NR2A, NR2B, NR2C, NR2D and NR3A subunits showed clustered expression in cell processes, but NR3B was absent. During modelled ischaemia, NMDA receptor activation resulted in rapid Ca2+-dependent detachment and disintegration of oligodendroglial processes in the white matter of mice expressing green fluorescent protein (GFP) specifically in oligodendrocytes (CNP-GFP mice). This effect occurred at mouse ages corresponding to both the initiation and the conclusion of myelination. NR1 subunits were found mainly in oligodendrocyte processes, whereas AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)/kainate receptor subunits were mainly found in the somata. Consistent with this observation, injury to the somata was prevented by blocking AMPA/kainate receptors, and preventing injury to oligodendroglial processes required the blocking of NMDA receptors. The presence of NMDA receptors in oligodendrocyte processes explains why previous studies that have focused on the somata have not detected a role for NMDA receptors in oligodendrocyte injury. These NMDA receptors bestow a high sensitivity to acute injury and represent an important new target for drug development in a variety of brain disorders.

The efficacy of the ethanol-inducible alc transgene expression system, derived from the filamentous fungus Aspergillus nidulans, has been demonstrated in transgenic tomato. Two direct comparisons have been made. First, this study has utilized two transgenic lines carrying distinct reporter genes (chloramphenicol acetyltransferase and beta-glucuronidase) to distinguish aspects of induction determined by the nature of the gene/gene product rather than that of the plant. Second, comparisons have been made to data generated in other species in order to identify any species-specific effects. The induction profiles for different genes in different species have shown remarkable similarity indicating the broad applicability of this gene switch. While there are minor differences observed between species, these probably arise from diversity in their metabolism. A series of potential alternative inducers have also been tested, revealing that ethanol (through metabolism to acetaldehyde) is better than other alcohols and ketones included in this study. Expression driven by alc was demonstrated to vary spatially, the upper younger leaves having higher activity than the lower older leaves; this will be important for some applications, and for experimental design. The highest levels of activity from ethanol-inducible transgene expression were determined to be the equivalent of those from the constitutive Cauliflower Mosaic Virus 35S promoter. This suggests that the alc system could be an important tool for plant functional genomics.

In Arabidopsis flowering is accelerated by reduced red:far-red (R:FR) ratio which signals the presence of neighbouring vegetation. Hastened flowering is one component of the shade-avoidance syndrome of responses, which alter many aspects of development in response to the threat of potential competition. Of the red/far-red-absorbing photoreceptors it is phyB that plays the most prominent role in shade-avoidance, although other related phytochromes act redundantly with phyB. It is well established that the phyB mutant has a constitutively early flowering phenotype. However, we have shown that the early flowering phenotype of phyB is temperature-dependent. We have established that this temperature-sensitive flowering response defines a pathway that appears to be independent of the autonomous-FLC pathway. Furthermore, we have demonstrated that the phytochromes control the expression of the floral promoter FT. We have also shown that other phyB-controlled responses, including petiole elongation, are not sensitive to the same temperature change. This suggests that discrete pathways control flowering and petiole elongation, components of the shade-avoidance response. This work provides an insight into the phytochrome and temperature interactions that maintain flowering control.

Environmental cues play a crucial role in regulating plant growth and development. Given the sedentary nature of plants, this close coupling between environment and development is essential for survival under conditions that are ever changing. The light environment is a particularly important determinant of plant development and the periodicity, direction, quantity and quality of incident light are continuously monitored by plants. Regulatory light signals are perceived by a number of specialised photoreceptor systems, each of which displays maximal light absorption in a specific region of the light spectrum. The photoreversible phytochromes absorb predominantly in the red (R) and far-red (FR) region of the spectrum, the cryptochromes and phototropin absorb light in the blue/UV-A region of the spectrum, and other, as yet unknown, photoreceptors are involved in perception of UV-A and UV-B light. The phytochromes are perhaps the best characterised of these photoreceptor systems. In the higher plants, phytochromes comprise a number of closely related photoreceptor proteins, the apoproteins of which are encoded by a small family of genes. In Arabidopsis thaliana there are five apophytochrome-encoding genes, PHYA, PHYB, PHYC, PHYD and PHYE (Sharrock and Quail 1989, Clack et al 1994). In the last few years genetic approaches, mainly focussed on Arabidopsis, have had a major impact in increasing our understanding of the functions of individual members of the phytochrome family and in identifying components of the signal transduction networks that couple light reception to alteration in gene expression and changes in growth and development.

Many transgenic plant studies use constitutive promoters to express transgenes. For certain genes, deleterious effects arise from constant expression in all tissues throughout development. We describe a chemically inducible plant gene expression system, with negligible background activity, that obviates this problem. We demonstrate its potential by showing inducible manipulation of carbon metabolism in transgenic plants. Upon rapid induction of yeast cytosolic invertase, a marked phenotype appears in developing leaves that is absent from leaves that developed before induction or after it has ceased.