Methodical Considerations and Resistance Evaluation against F. graminearum and F. culmorum Head Blight in Wheat. The Influence of Mixture of Isolates on Aggressiveness and Resistance Expression.

In resistance tests to Fusarium head blight (FHB), the mixing of inocula before inoculation is normal, but no information about the background of mixing was given. Therefore, four experiments (2013-2015) were made with four independent isolates, their all-possible (11) mixtures and a control. Four cultivars with differing FHB resistance were used. Disease index (DI), Fusarium damaged kernels (FDK) and deoxynivalenol (DON) were evaluated. The isolates used were not stable in aggressiveness. Their mixtures did not also give a stable aggressiveness; it depended on the composition of mix. The three traits diverged in their responses. After the mixing, the aggressiveness was always less than that of the most pathogenic component was. However, in most cases it was significantly higher than the arithmetical mean of the participating isolates. A mixture was not better than a single isolate was. The prediction of the aggressiveness level is problematic even if the aggressiveness of the components was tested. Resistance expression is different in the mixing variants and in the three traits tested. Of them, DON is the most sensitive. More reliable resistance and toxin data can be received when instead of one more independent isolates are used. This is important when highly correct data are needed (genetic research or cultivar registration).

The Influence of the Dilution Rate on the Aggressiveness of Inocula and the Expression of Resistance against Fusarium Head Blight in Wheat.

In previous research, conidium concentrations varying between 10,000 and 1,000,000/mL have not been related to any aggressiveness test. Therefore, two Fusarium graminearum and two Fusarium culmorum isolates were tested in the field on seven genotypes highly differing in resistance at no dilution, and 1:1, 1:2, 1:4, 1:8, and 1:16 dilutions in two years (2013 and 2014). The isolates showed different aggressiveness, which changed significantly at different dilution rates for disease index (DI), Fusarium-damaged kernels (FDK), and deoxynivalenol (DON). The traits also had diverging responses to the infection. The effect of the dilution could not be forecasted. The genotype ranks also varied. Dilution seldomly increased aggressiveness, but often lower aggressiveness occurred at high variation. The maximum and minimum values varied between 15% and 40% for traits and dilutions. The reductions between the non-diluted and diluted values (total means) for DI ranged from 6% and 33%, for FDK 8.3-37.7%, and for DON 5.8-44.8%. The most sensitive and most important trait was DON. The introduction of the aggressiveness test provides improved regulation compared to the uncontrolled manipulation of the conidium concentration. The use of more isolates significantly increases the credibility of phenotyping in genetic and cultivar registration studies.

Methodical Considerations and Resistance Evaluation Against Fusarium graminearum and F. culmorum Head Blight in Wheat. Part 3. Susceptibility Window and Resistance Expression.

Flowering is the most favorable host stage for Fusarium infection in wheat, which is called the susceptibility window (SW). It is not known how long it takes, how it changes in different resistance classes, nor how stable is the plant reaction in the SW. We have no information, how the traits disease index (DI), Fusarium-damaged kernel rate (FDK), and deoxynivalenol (DON) respond within the 16 days period. Seven winter wheat genotypes differing in resistance were tested (2013-2014). Four Fusarium isolates were used for inoculation at mid-anthesis, and 4, 8, 11, 13, and 16 days thereafter. The DI was not suitable to determine the length of the SW. In the Fusarium-damaged kernels (FDK), a sharp 50% decrease was found after the 8th day. The largest reduction (above 60%) was recorded for DON at each resistance level between the 8th and 11th day. This trait showed the SW most precisely. The SW is reasonably stable in the first 8-9 days. This fits for all resistance classes. The use of four isolates significantly improved the reliability and credit of the testing. The stable eight-day long SW helps to reduce the number of inoculations. The most important trait to determine the SW is the DON reaction and not the visual symptoms.

The terminal pathway of the complement system is activated in focal penetrating but not in mild diffuse traumatic brain injury.

The complement system plays an important role in the inflammatory response activated by many central nervous system disorders. However, its significance in traumatic diffuse traumatic axonal injury (TAI) is not fully known. Here we analyze the complement activity in two rat models of traumatic brain injury (TBI); a focal penetration injury (pen-TBI) and a rotational acceleration injury (rot-TBI) that leads to a mild TAI. We used in situ hybridization to examine the distribution of mRNA for C1q and C3 and immunohistochemistry to examine the presence of the C3 protein and C5b-9 complex at 1-5 days after injury. We found a time-dependent complement activity in both models. However, the responses caused by the two models were different. We detected C5b-9 surrounding the cavity in pen-TBI, but C5b-9 was not found in the rot-TBI. Our findings suggest that the terminal complement pathway is progressed to the formation of the C5b-9 membrane attack complex only in the penetrating TBI but not in isolated TAI model. This indicates that the complement activation does not lead to membrane-damaging effects and a subsequent secondary axotomy in TAI by the terminal complex C5b-9. The role of complement activation in TAI is unclear, but might indicate an alternative function following rot-TBI, such as opsonizing the synapses for elimination.

Time-dependent changes of protein biomarker levels in the cerebrospinal fluid after blast traumatic brain injury.

Time-dependent changes of protein biomarkers in the cerebrospinal fluid (CSF) can be used to identify the pathological processes in traumatic brain injury (TBI) as well as to follow the progression of the disease. We obtained CSF from a large animal model (swine) of blast-induced traumatic brain injury prior to and at 6, 24, 72 h, and 2 wk after a single exposure to blast overpressure, and determined changes in the CSF levels of neurofilament-heavy chain, neuron-specific enolase, brain-specific creatine kinase, glial fibrillary acidic protein, calcium-binding protein ß (S100ß), Claudin-5, vascular endothelial growth factor, and von Willebrand factor using reverse phase protein microarray. We detected biphasic temporal patterns in the CSF concentrations of all tested protein markers except S100ß. The CSF levels of all markers were significantly increased 6 h after the injury compared to preinjury levels. Values were then decreased at 24 h, prior to a second increase in all markers but S100ß at 72 h. At 2 wk postinjury, the CSF concentrations of all biomarkers were decreased once again; brain-specific creatine kinase, Claudin-5, von Willebrand factor, and S100ß levels were no longer significantly higher than their preinjury values while neurofilament-heavy chain, neuron-specific enolase, vascular endothelial growth factor, and glial fibrillary acidic protein levels remained significantly elevated compared to baseline. Our findings implicate neuronal and glial cell damage, compromised vascular permeability, and inflammation in blast-induced traumatic brain injury, as well as demonstrate the value of determining the temporal pattern of biomarker changes that may be of diagnostic value.

A Model for Mild Traumatic Brain Injury that Induces Limited Transient Memory Impairment and Increased Levels of Axon Related Serum Biomarkers.

Mild traumatic brain injury (mTBI) is one of the most common neuronal insults and can lead to long-term disabilities. mTBI occurs when the head is exposed to a rapid acceleration-deceleration movement triggering axonal injuries. Our limited understanding of the underlying pathological changes makes it difficult to predict the outcome of mTBI. In this study we used a scalable rat model for rotational acceleration TBI, previously characterized for the threshold of axonal pathology. We have analyzed whether a TBI just above the defined threshold would induce any detectable behavioral changes and/or changes in serum biomarkers. The effect of injury on sensory motor functions, memory and anxiety were assessed by beam walking, radial arms maze and elevated plus maze at 3-7 days following TBI. The only behavioral deficits found were transient impairments in working and reference memory. Blood serum was analyzed at 1, 3, and 14 days after injury for changes in selected protein biomarkers. Serum levels of neurofilament heavy chain and Tau, as well as S100B and myelin basic protein showed significant increases in the injured animals at all time points. No signs of macroscopic injuries such as intracerebral hematomas or contusions were found. Amyloid precursor protein immunostaining indicated axonal injuries at all time points analyzed. In summary, this model mimics some of the key symptoms of mTBI, such as transient memory impairment, which is paralleled by an increase in serum biomarkers. Our findings suggest that serum biomarkers may be used to detect mTBI. The model provides a suitable foundation for further investigation of the underlying pathology of mTBI.

The effect of enriched environment on the outcome of traumatic brain injury; a behavioral, proteomics, and histological study.

De novo hippocampal neurogenesis contributes to functional recovery following traumatic brain injury (TBI). Enriched environment (EEN) can improve the outcome of TBI by positively affecting neurogenesis. Blast induced traumatic brain injury (bTBI) characterized by memory impairment and increased anxiety levels, is a leading cause of chronic disability among soldiers. Using a rodent model of bTBI we asked: (a) whether long-term exposure to EEN after injury can ameliorate behavioral abnormalities and (b) what the effects of EEN are at the molecular and cellular levels and on de novo neurogenesis. We found that housing injured animals in EEN resulted in significantly improved spatial memory while animals in normal housing (NH) showed persistent memory impairment. VEGF and Tau protein but not Interleukin-6 (IL-6) levels were normalized in the dorsal hippocampus (DHC) of EEN rats while all three markers remained elevated in NH rats. Interestingly, after peaking at 6 weeks post-injury, anxiety returned to normal levels at 2 months independent of housing conditions. Housing animals in EEN had no significant effect on VEGF and Tau protein levels in the ventral hippocampus (VHC) and the amygdala (AD). We also found that EEN reduced IL-6 and IFNγ levels in the VHC; these markers remained elevated following NH. We observed an increase in GFAP and DCX immunoreactivities in the VHC of NH animals at 2 months post-injury. Conversely, injured animals housed in EEN showed no increase in GFAP or DCX immunoreactivity in their VHC. In summary, long-term exposure of injured animals to EEN appears to play a positive role in the restoration of memory functions but not on anxiety, which returned to normal levels after a significant period of time. Cellular and molecular changes in response to EEN appear to be a part of neurogenesis-independent as well as dependent recovery processes triggered by bTBI.

Time-dependent changes in serum biomarker levels after blast traumatic brain injury.

Neuronal and glial proteins detected in the peripheral circulating blood after injury can reflect the extent of the damage caused by blast traumatic brain injury (bTBI). The temporal pattern of their serum levels can further predict the severity and outcome of the injury. As part of characterizing a large-animal model of bTBI, we determined the changes in the serum levels of S100B, neuron-specific enolase (NSE), myelin basic protein (MBP), and neurofilament heavy chain (NF-H). Blood samples were obtained prior to injury and at 6, 24, 72 h, and 2 weeks post-injury from animals with different severities of bTBI; protein levels were determined using reverse phase protein microarray (RPPM) technology. Serum levels of S100B, MBP, and NF-H, but not NSE, showed a time-dependent increase following injury. The detected changes in S100B and MBP levels showed no correlation with the severity of the injury. However, serum NF-H levels increased in a unique, rapid manner, peaking at 6 h post-injury only in animals exposed to severe blast with poor clinical and pathological outcomes. We conclude that the sudden increase in serum NF-H levels following bTBI may be a useful indicator of injury severity. If additional studies verify our findings, the observed early peak of serum NF-H levels can be developed into a useful diagnostic tool for predicting the extent of damage following bTBI.

Stress and traumatic brain injury: a behavioral, proteomics, and histological study.

Psychological stress and traumatic brain injury (TBI) can both result in lasting neurobehavioral abnormalities. Post-traumatic stress disorder and blast induced TBI (bTBI) have become the most significant health issues in current military conflicts. Importantly, military bTBI virtually never occurs without stress. In this experiment, we assessed anxiety and spatial memory of rats at different time points after repeated exposure to stress alone or in combination with a single mild blast. At 2 months after injury or sham we analyzed the serum, prefrontal cortex (PFC), and hippocampus (HC) of all animals by proteomics and immunohistochemistry. Stressed sham animals showed an early increase in anxiety but no memory impairment at any measured time point. They had elevated levels of serum corticosterone (CORT) and hippocampal IL-6 but no other cellular or protein changes. Stressed injured animals had increased anxiety that returned to normal at 2 months and significant spatial memory impairment that lasted up to 2 months. They had elevated serum levels of CORT, CK-BB, NF-H, NSE, GFAP, and VEGF. Moreover, all of the measured protein markers were elevated in the HC and the PFC; rats had an increased number of TUNEL-positive cells in the HC and elevated GFAP and Iba1 immunoreactivity in the HC and the PFC. Our findings suggest that exposure to repeated stress alone causes a transient increase in anxiety and no significant memory impairment or cellular and molecular changes. In contrast, repeated stress and blast results in lasting behavioral, molecular, and cellular abnormalities characterized by memory impairment, neuronal and glial cell loss, inflammation, and gliosis. These findings may have implications in the development of diagnostic and therapeutic measures for conditions caused by stress or a combination of stress and bTBI.

Reverse phase protein microarray technology in traumatic brain injury.

Antibody based, high throughput proteomics technology represents an exciting new approach in understanding the pathobiologies of complex disorders such as cancer, stroke and traumatic brain injury. Reverse phase protein microarray (RPPA) can complement the classical methods based on mass spectrometry as a high throughput validation and quantification method. RPPA technology can address problematic issues, such as sample complexity, sensitivity, quantification, reproducibility and throughput, which are currently associated with mass spectrometry-based approaches. However, there are technical challenges, predominantly associated with the selection and use of antibodies, preparation and representation of samples and with analyzing and quantifying primary RPPA data. Here we present ways to identify and overcome some of the current issues associated with RPPA. We believe that using stringent quality controls, improved bioinformatics analysis and interpretation of primary RPPA data, this method will significantly contribute in generating new level of understanding about complex disorders at the level of systems biology.

Proteomic biomarkers for blast neurotrauma: targeting cerebral edema, inflammation, and neuronal death cascades.

Proteomics for blast traumatic brain injury (bTBI) research represents an exciting new approach that can greatly help to address the complex pathology of this condition. Antibody-based platforms, antibody microarrays (AbMA), and reverse capture protein microarrays (RCPM) can complement the classical methods based on 2D gel electrophoresis and mass spectrometry (2DGE/MS). These new technologies can address problematic issues, such as sample complexity, sensitivity, quantitation, reproducibility, and analysis time, which are typically associated with 2DGE/MS. Combined with bioinformatics analysis and interpretation of primary microarray data, these methods will generate a new level of understanding about bTBI at the level of systems biology. As biological and clinical knowledge and the availability of these systems become more widely established, we expect that AbMA and RCPM will be used routinely in clinical diagnostics, and also for following therapeutic progress. At the technical level, we anticipate that these platforms will evolve to accommodate comprehensive, high-speed, label-free analysis on a human proteome-wide scale.

Members of the NuRD chromatin remodeling complex interact with AUF1 in developing cortical neurons.

Chromatin remodeling plays an important role in coordinating gene expression during cortical development, however the identity of molecular complexes present in differentiating cortical neurons that mediate the process remains poorly understood. The A + U-rich element-binding factor 1 (AUF1) is a known regulator of messenger RNA stability and also acts as a transcription factor upon binding to AT-rich DNA elements. Here we show that AUF1 is specifically expressed in subsets of proliferating neural precursors and differentiating postmitotic neurons of the developing cerebral cortex. Moreover, AUF1 is coexpressed with histone deacetylase 1 (HDAC1) and metastasis-associated protein 2 (MTA2), members of the nucleosome remodeling and histone deacetylase complex. AUF1 specifically and simultaneously binds to HDAC1, MTA2, and AT-rich DNA element, its gene regulatory function is modulated by the extent of histone acetylation and in animals lacking AUF1, the composition of the complex is modified. These results suggest that AUF1 is involved in integrating genetic and epigenetic signals during cortical development through recruiting HDAC1 and MTA2 to AT-rich DNA elements.

SATB2 interacts with chromatin-remodeling molecules in differentiating cortical neurons.

During our search for developmental regulators of neuronal differentiation, we identified special AT-rich sequence-binding protein (SATB)2 that is specifically expressed in the developing rat neocortex and binds to AT-rich DNA elements. Here we investigated whether the regulatory function of SATB2 involves chromatin remodeling at the AT-rich DNA site. In-vitro and in-vivo assays using a DNA affinity pre-incubation specificity test of recognition and chromatin immunoprecipitation showed that SATB2 specifically binds to histone deacetylase 1 and metastasis-associated protein 2, members of the nucleosome-remodeling and histone deacetylase complex. Double immunohistochemistry showed that, in the developing rat neocortex, SATB2 is coexpressed with both proteins. Using a cell culture model, we showed that trichostatin A treatment, which blocks the activities of histone deacetylases, reverses the AT-rich dsDNA-dependent repressor effect of SATB2. These findings suggested that the molecular regulatory function of SATB2 involves modification of the chromatin structure. Semi-quantitative chromatin immunoprecipitation analysis of cortices from SATB2 mutant and wild-type animals indicated that, in the knock-out brains, SATB2 is replaced in the chromatin-remodeling complex by AU-rich element RNA binding protein 1, another AT-rich DNA binding protein also expressed in differentiating cortical neurons. These results suggested that an altered chromatin structure, due to the presence of different AT-rich DNA binding proteins in the chromatin-remodeling complex, may contribute to the developmental abnormalities observed in the SATB2 mutant animals. These findings also raised the interesting possibility that SATB2, along with other AT-rich DNA binding proteins, is involved in mediating epigenetic influences during cortical development.

Satb2 is a postmitotic determinant for upper-layer neuron specification in the neocortex.

Pyramidal neurons of the neocortex can be subdivided into two major groups: deep- (DL) and upper-layer (UL) neurons. Here we report that the expression of the AT-rich DNA-binding protein Satb2 defines two subclasses of UL neurons: UL1 (Satb2 positive) and UL2 (Satb2 negative). In the absence of Satb2, UL1 neurons lose their identity and activate DL- and UL2-specific genetic programs. UL1 neurons in Satb2 mutants fail to migrate to superficial layers and do not contribute to the corpus callosum but to the corticospinal tract, which is normally populated by DL axons. Ctip2, a gene required for the formation of the corticospinal tract, is ectopically expressed in all UL1 neurons in the absence of Satb2. Satb2 protein interacts with the Ctip2 genomic region and controls chromatin remodeling at this locus. Satb2 therefore is required for the initiation of the UL1-specific genetic program and for the inactivation of DL- and UL2-specific genes.

Effect of penetrating brain injury on aquaporin-4 expression using a rat model.

Cerebral edema (CE) is a frequent and potentially lethal consequence of various neurotraumas, including penetrating brain injury (PBI). Aquaporin-4 (AQP4) water channel is predominantly expressed by astrocytes and plays an important role in regulating water balance in the normal and injured brain. Using a rat model of PBI, we show that AQP4 immunoreactivity was substantially increased in the peri-injury area at both 24 and 72 h after PBI. The increase in AQP4 expression was paralleled by increased GFAP expression. The two proteins were co-expressed by peri-vascular astrocytes, whereas reactive astroglia identified by their stellar morphology did not express AQP4 at either time points after injury. Western analysis confirmed the increase in AQP4 immunoreactivity observed in the injured tissue. The apparent increase in AQP4 immunoreactivity was likely due to de novo AQP4 protein synthesis, as most of the increased AQP4 immunoreactivity was found in the soluble (cytosolic) fraction. Our results demonstrate dynamic spatial and temporal changes in AQP4 expression that contribute to the molecular pathophysiology of PBI.

Ikaros is expressed in developing striatal neurons and involved in enkephalinergic differentiation.

The Ikaros (Ik) gene encodes alternatively spliced zinc-finger proteins originally identified in developing hematopoietic organs and acts as master regulator of lymphoid development. During our search for transcription factors that control the developmental expression of the enkephalin (ENK) gene we found that Ik-1 and Ik-2 isoforms are specifically expressed in the embryonic striatum and bind the Ik-like cis-regulatory DNA element present on the ENK gene. Ik proteins are expressed by both proliferating (BrdU+/nestin+) and by post-mitotic differentiating (MAP2+) cells in the developing striatum between embryonic day 12 and post-natal day 2 and mRNAs encoding for the Ik and ENK genes are co-expressed by a subset of differentiating striatal neurons. Blocking the DNA binding of Ik proteins in differentiating embryonic striatal neuronal cultures resulted in decreased ENK expression and mutant animals lacking the DNA-binding domain of Ik had a deficit in the number of ENK but not in dynorphin or substance P mRNA+ cells. Animals lacking the protein interaction domain of Ik showed no deficit. These results demonstrate that Ik-1 and Ik-2 proteins through their DNA binding act as positive regulators of ENK gene expression in the developing striatum and participate in regulating enkephalinergic differentiation.

AUF1 is expressed in the developing brain, binds to AT-rich double-stranded DNA, and regulates enkephalin gene expression.

During our search for transcriptional regulators that control the developmentally regulated expression of the enkephalin (ENK) gene, we identified AUF1. ENK, a peptide neurotransmitter, displays precise cell-specific expression in the adult brain. AUF1 (also known as heterogeneous nuclear ribonucleoprotein D) has been known to regulate gene expression through altering the stability of AU-rich mRNAs. We show here that in the developing brain AUF1 proteins are expressed in a spatiotemporally defined manner, and p37 and p40/42 isoforms bind to an AT-rich double-stranded (ds) DNA element of the rat ENK (rENK) gene. This AT-rich dsDNA sequence acts as a cis-regulatory DNA element and is involved in regulating the cell-specific expression of the ENK gene in primary neuronal cultures. The AT-rich dsDNA elements are present at approximately 2.5 kb 5'upstream of the rat, human, and mouse ENK genes. AUF1 proteins are shown here to provide direct interaction between these upstream AT-rich DNA sequences and the TATA region of the rENK gene. Double immunohistochemistry demonstrated that in the developing brain AUF1 proteins are expressed by proliferating neural progenitors and by differentiating neurons populating brain regions, which will not express the ENK gene in the adult, suggesting a repressor role for AUF1 proteins during enkephalinergic differentiation. Their subnuclear distribution and interactions with AT-rich DNA suggest that in the developing brain they can be involved in complex nuclear regulatory mechanisms controlling the development- and cell-specific expression of the ENK gene.

Role of secondary attachment sites in changing the specificity of site-specific recombination.

We previously proposed that lambdoid phages change their insertion specificity by adapting their integrases to sequences found in secondary attachment sites. To test this model, we quantified recombination between partners that carried sequences from secondary attachment sites catalyzed by wild-type and by mutant integrases with altered specificities. The results are consistent with the model, and indicate differential core site usage in excision and integration.

Isolation and characterization of SATB2, a novel AT-rich DNA binding protein expressed in development- and cell-specific manner in the rat brain.

AT-rich DNA elements play an important role in regulating cell-specific gene expression. One of the AT-rich DNA binding proteins, SATB1 is a novel type of transcription factor that regulates gene expression in the hematopoietic lineage through chromatin modification. Using DNA-affinity purification followed by mass spectrometry we identified and isolated a related protein, SATB2 from the developing rat cerebral cortex. SATB2 shows homology to SATB1 and the rat protein is practically identical to the mouse and human SATB2. Using competitive EMSA, we show that recombinant SATB2 protein binds with high affinity and specificity to AT-rich dsDNA. Using RT-PCR, Western analysis and immunohistochemistry we demonstrate that SATB2 expression is restricted to a subset of postmitotic, differentiating neurons in the rat neocortex at ages E16 and P4. We suggest that similar to its homologue SATB1, SATB2 is also involved in regulating gene expression through altering chromatin structure in differentiating cortical neurons.

A peptide of human muscarinic acetylcholine receptor 3 is antigenic in primary Sjogren's syndrome.

To evaluate the antigenicity of a peptide representing a part of the second extracellular loop of the human muscarinic acetylcholine receptor 3 (m3AChR) with autoimmune sera from primary Sjogren's syndrome (pSS), enzyme-linked immunosorbent assays (ELISAs) were developed. On the basis of the computer-predicted data, a 16-mer synthetic peptide KRTVPPGECFIQFLSE (KRSE213-228) was produced by solid-phase peptide synthesis. cDNA coding for the KRSE peptide was chemically synthetized and utilized to express the recombinant glutathione S-transferase (GST)-KRSE fusion protein. The immunoreactivities of the two antigens were tested in ELISAs with the sera of 40 pSS patients and 40 healthy controls. The specificity of the reaction was confirmed by inhibition assays and immunoblottings. The pSS sera resulted in significantly higher mean optical densities than those of the healthy controls (KRSE: 0.4149 vs 0.1494, p<0.0001; GST-KRSE 0.4765 vs 0.1764, p<0.0001). The immunological recognition with the recombinant fusion antigen was significantly better than that for the free peptide (p=0.0068). The sensitivities of the assays were 77.5% (KRSE) and 97% (GST-KRSE). The results of the concentration-dependent inhibition assays by the two systems of peptide presentation indicated that the KRSE sequence is specific for pSS sera. This is the first demonstration of the antigenicity of a novel peptide fragment of the human m3AChR in pSS. The analysed peptide could be of diagnostic relevance.