Genetic Modification of KNAT7 Transcription Factor Expression Enhances Saccharification and Reduces Recalcitrance of Woody Biomass in Poplars

The precise role of KNAT7 transcription factors (TFs) in regulating secondary cell wall (SCW) biosynthesis in poplars has remained unknown, while our understanding of KNAT7 functions in other plants is continuously evolving. To study the impact of genetic modifications of homologous and heterologous KNAT7 gene expression on SCW formation in transgenic poplars, we prepared poplar KNAT7 (PtKNAT7) overexpression (PtKNAT7-OE) and antisense suppression (PtKNAT7-AS) vector constructs for the generation of transgenic poplar lines via Agrobacterium-mediated transformation. Since the overexpression of homologous genes can sometimes result in co-suppression, we also overexpressed Arabidopsis KNAT7 (AtKNAT7-OE) in transgenic poplars. In all these constructs, the expression of KNAT7 transgenes was driven by developing xylem (DX)-specific promoter, DX15. Compared to wild-type (WT) controls, many SCW biosynthesis genes downstream of KNAT7 were highly expressed in poplar PtKNAT7-OE and AtKNAT7-OE lines. Yet, no significant increase in lignin content of woody biomass of these transgenic lines was observed. PtKNAT7-AS lines, however, showed reduced expression of many SCW biosynthesis genes downstream of KNAT7 accompanied by a reduction in lignin content of wood compared to WT controls. Syringyl to Guaiacyl lignin (S/G) ratios were significantly increased in all three KNAT7 knockdown and overexpression transgenic lines than WT controls. These transgenic lines were essentially indistinguishable from WT controls in terms of their growth phenotype. Saccharification efficiency of woody biomass was significantly increased in all transgenic lines than WT controls. Overall, our results demonstrated that developing xylem-specific alteration of KNAT7 expression affects the expression of SCW biosynthesis genes, impacting at least the lignification process and improving saccharification efficiency, hence providing one of the powerful tools for improving bioethanol production from woody biomass of bioenergy crops and trees.

Sex-Specific Alterations in Inflammatory MicroRNAs in Mouse Brain and Bone Marrow CD11b+ Cells Following Traumatic Brain Injury

Sex is a key biological variable in traumatic brain injury (TBI) and plays a significant role in neuroinflammatory responses. However, the molecular mechanisms contributing to this sexually dimorphic neuroinflammatory response remain elusive. Here we describe a significant and previously unreported tissue enrichment and sex specific alteration of a set of inflammatory microRNAs (miRNAs) in CD11b + cells of brain and bone marrow isolated from naïve mice as well as mice subjected to TBI. Our data from naïve mice demonstrated that expression levels of miR-146a-5p and miR-150-5p were relatively higher in brain CD11b + cells, and that miR-155-5p and miR-223-3p were highly enriched in bone marrow CD11b + cells. Furthermore, while miR-150-5p and miR-155-5p levels were higher in male brain CD11b + cells, no significant sexual difference was observed for miR-146a-5p and miR-223-3p. However, TBI resulted in sex specific differential responses of these miRNAs in brain CD11b + cells. Specifically, miR-223 levels in brain CD11b + cells were markedly elevated in both sexes in response to TBI at 3 and 24 hr, with levels in females being significantly higher than males at 24 hr. We then focused on analyzing several miR-223-3p targets and inflammation-related marker genes following injury. Corresponding to the greater elevation of miR-223-3p in females, the miR-223-3p targets, TRAF6 and FBXW7 were significantly reduced in females compared to males. Interestingly, anti-inflammatory genes ARG1 and IL4 were higher in females after TBI than in males. These observations suggest miR-223-3p and other inflammatory responsive miRNAs may play a key role in sex-specific neuroinflammatory response following TBI.

Expression of IL-20 Receptor Subunit β Is Linked to EAE Neuropathology and CNS Neuroinflammation

Considerable clinical evidence supports that increased blood–brain barrier (BBB) permeability is linked to immune extravasation of CNS parenchyma during neuroinflammation. Although BBB permeability and immune extravasation are known to be provoked by vascular endothelial growth factor-A (i.e., VEGF-A) and C-X-C motif chemokine ligand 12 (CXCL12), respectively, the mechanisms that link both processes are still elusive. The interleukin-20 (i.e., IL-20) cytokine signaling pathway was previously implicated in VEGF-mediated angiogenesis and is known to induce cellular response by way of signaling through IL-20 receptor subunit β (i.e., IL-20RB). Dysregulated IL-20 signaling is implicated in many inflammatory pathologies, but it’s contribution to neuroinflammation has yet to be reported. We hypothesize that the IL-20 cytokine, and the IL cytokine subfamily more broadly, play a key role in CNS neuroinflammation by signaling through IL-20RB, induce VEGF activity, and enhance both BBB-permeability and CXCL12-mediated immune extravasation. To address this hypothesis, we actively immunized IL-20RB–/– mice and wild-type mice to induce experimental autoimmune encephalomyelitis (EAE) and found that IL-20RB–/– mice showed amelioration of disease progression compared to wild-type mice. Similarly, we passively immunized IL-20RB–/– mice and wild-type mice with myelin-reactive Th1 cells from either IL-20RB–/– and wild-type genotype. Host IL-20RB–/– mice showed lesser disease progression than wild-type mice, regardless of the myelin-reactive Th1 cells genotype. Using multianalyte bead-based immunoassay and ELISA, we found distinctive changes in levels of pro-inflammatory cytokines between IL-20RB–/– mice and wild-type mice at peak of EAE. We also found detectable levels of all cytokines of the IL-20 subfamily within CNS tissues and specific alteration to IL-20 subfamily cytokines IL-19, IL-20, and IL-24, expression levels. Immunolabeling of CNS region-specific microvessels confirmed IL-20RB protein at the spinal cord microvasculature and upregulation during EAE. Microvessels isolated from macaques CNS tissues also expressed IL-20RB. Moreover, we identified the expression of all IL-20 receptor subunits: IL-22 receptor subunit α-1 (IL-22RA1), IL-20RB, and IL-20 receptor subunit α (IL-20RA) in human CNS microvessels. Notably, human cerebral microvasculature endothelial cells (HCMEC/D3) treated with IL-1β showed augmented expression of the IL-20 receptor. Lastly, IL-20-treated HCMEC/D3 showed alterations on CXCL12 apicobasal polarity consistent with a neuroinflammatory status. This evidence suggests that IL-20 subfamily cytokines may signal at the BBB via IL-20RB, triggering neuroinflammation.

Spatiotemporal changes in along-tract profilometry of cerebellar peduncles in cerebellar mutism syndrome

Cerebellar mutism syndrome, characterised by mutism, emotional lability and cerebellar motor signs, occurs in up to 40% of children following resection of medulloblastoma, the most common malignant posterior fossa tumour of childhood. Its pathophysiology remains unclear, but prior studies have implicated damage to the superior cerebellar peduncles. In this study, the objective was to conduct high-resolution spatial profilometry of the cerebellar peduncles and identify anatomic biomarkers of cerebellar mutism syndrome. Twenty-eight children with medulloblastoma (mean age 8.8, s.d. 3.8 years) underwent diffusion MRI at four timepoints over one year. Forty-nine healthy children (mean age 9.0, s.d. 4.2 years), scanned at a single timepoint, served as age- and sex-matched controls. Automated Fibre Quantification was used to segment cerebellar peduncles and compute fractional anisotropy at 30 nodes along each tract. Thirteen patients developed cerebellar mutism syndrome. Fractional anisotropy was significantly lower in the distal segments of the superior cerebellar peduncle pre-operatively in all patients (p=0.01). Pre-operative changes in fractional anisotropy did not predict cerebellar mutism syndrome. However, post-operative reductions in fractional anisotropy were highly specific to the distal left superior cerebellar peduncle, and were most pronounced at follow-up timepoints (p<0.04), in those that developed cerebellar mutism syndrome compared to patients that did not. High spatial resolution cerebellar profilometry identifies a site-specific alteration of the distal segment of the superior cerebellar peduncle unique to cerebellar mutism syndrome with important surgical implications in the treatment of these devastating tumours of childhood.

A Pilot Study to Determine the Impact of Adipose Tissue Attachment to Polypropylene Fibers In Hernia Mesh

Introduction: Prior publications have demonstrated chemical and physical alteration of hernia mesh analyzed after explantation from the body. The specific alteration documented is oxidative degradation of polypropylene mesh fibers. An animal study recently published has demonstrated that adipose tissue attachment is present instead of reparative fibrous tissue infiltration in an average of 10.9–18.9% of the intramesh healing for a variety of clinically used knitted polypropylene mesh products; 8.0% for knitted polyester meshes. This study also found that in comparison to the knitted mesh products, non-woven polypropylene mesh reduced adipose tissue attachment to 1% or less, which was a statistically significant difference. Materials and Methods: Samples of explanted polypropylene mesh from eight patients were analyzed for the presence of adipose tissue attachment, reparative fibrous tissue infiltration, and oxidative changes. Greater adipose tissue attachment areas were compared with areas of greater reparative fibrous tissue infiltration for evidence of oxidative changes in the mesh to determine if the areas of higher adipose tissue attachment correlated with an increase in oxidative changes. Results: Intra mesh healing of clinically explanted knitted meshes demonstrated adipose tissue content from 0.0% to 49.1% per analyzed segment. The oxidation index, a measure of the degree of oxidative degradation in that portion of the mesh, was higher in seven of the eight areas of greater adipose tissue attachment than areas of greater reparative fibrous tissue infiltration. Conclusion: Adipose tissue attachment does occur in knitted and woven polypropylene hernia meshes. The presence of adipose tissue may contribute to an increase in oxidative changes in knitted polypropylene hernia mesh fibers.

751 Heart Rate and Heart Rate Variability During Sleep As Biomarkers for Depression

Introduction Evidence suggests a high prevalence of depression in subjects with Sleep-Wake Disorders, with impaired sleep being both a risk factor and a symptom of depression. However, depression currently remains for the most undiagnosed in this population, which can lead to a lack or delay in the treatment, and ultimately contribute to chronicity, recurrence of depression, and increase risk of suicide. Depression is characterized by alteration in sleep architecture and imbalanced autonomic nervous system function, and specific alteration may serve as biomarkers to identify ongoing depression in subjects with Sleep-Wake Disorders undergoing polysomnography. Thus, the aim of this study is to investigate differences in sleep architecture and autonomic modulation, measured by heart rate and heart rate variability throughout sleep stages, in subjects undergoing polysomnography in a sleep clinic. Methods A preliminary sample of forty subjects undergoing polysomnography was recruited in three different sleep clinics. The Patient Health Questionnaire–9 was administered to participants before the beginning of the sleep study. A cut-off of 10 was applied to identify subjects with possible current depression. The polysomnography recordings were processed with the MEBsleep software (Medibio Limited) which automatically calculats sleep architecture indices, and heart rate and heart rate variability parameters throughout sleep stages. The Mann-Whitney U test was used to investigate differences between the depressed and non-depressed groups. Results Possible current depression was found in fourteen subjects (35%). These Subjects had statistically significant higher heart rate (median depressed=78.01, median non-depressed=64.61, p=0.01) and lower Root Mean Square of the Successive Difference (RMSSD; median depressed=18.41 ms, median non-depressed=26.52 ms, p=0.02), number of pairs of successive NN intervals that differ by more than 50 ms (pNN50. Median depressed=1.62%; median non-depressed=5.64%; p=0.03), and High Frequency (absolute power) in REM (median depressed=104.17 ms2; median non-depressed=214.58 ms2; p=0.03) than those without depression. No significant differences resulted in the sleep architecture indices. Conclusion These results preliminary indicates a decreased parasympathetic activity in subjects with possible depression during REM, suggesting that heart rate and heart rate variability during sleep may be used as biomarkers to identify current depression in subjects undergoing polysomnography in sleep clinics. Support (if any):

Quantitative Phosphoproteomic Analysis Reveals Dendritic Cell- Specific STAT Signaling After α2-3–Linked Sialic Acid Ligand Binding

Dendritic cells (DCs) are key initiators of the adaptive immunity, and upon recognition of pathogens are able to skew T cell differentiation to elicit appropriate responses. DCs possess this extraordinary capacity to discern external signals using receptors that recognize pathogen-associated molecular patterns. These can be glycan-binding receptors that recognize carbohydrate structures on pathogens or pathogen-associated patterns that additionally bind receptors, such as Toll-like receptors (TLRs). This study explores the early signaling events in DCs upon binding of α2-3 sialic acid (α2-3sia) that are recognized by Immune inhibitory Sialic acid binding immunoglobulin type lectins. α2-3sias are commonly found on bacteria, e.g. Group B Streptococcus, but can also be expressed by tumor cells. We investigated whether α2-3sia conjugated to a dendrimeric core alters DC signaling properties. Through phosphoproteomic analysis, we found differential signaling profiles in DCs after α2-3sia binding alone or in combination with LPS/TLR4 co-stimulation. α2-3sia was able to modulate the TLR4 signaling cascade, resulting in 109 altered phosphoproteins. These phosphoproteins were annotated to seven biological processes, including the regulation of the IL-12 cytokine pathway. Secretion of IL-10, the inhibitory regulator of IL-12 production, by DCs was found upregulated after overnight stimulation with the α2-3sia dendrimer. Analysis of kinase activity revealed altered signatures in the JAK-STAT signaling pathway. PhosphoSTAT3 (Ser727) and phosphoSTAT5A (Ser780), involved in the regulation of the IL-12 pathway, were both downregulated. Flow cytometric quantification indeed revealed de- phosphorylation over time upon stimulation with α2-3sia, but no α2-6sia. Inhibition of both STAT3 and -5A in moDCs resulted in a similar cytokine secretion profile as α-3sia triggered DCs. Conclusively, this study revealed a specific alteration of the JAK-STAT pathway in DCs upon simultaneous α2-3sia and LPS stimulation, altering the IL10:IL-12 cytokine secretion profile associated with reduction of inflammation. Targeted control of the STAT phosphorylation status is therefore an interesting lead for the abrogation of immune escape that bacteria or tumors impose on the host.

Age-dependent and region-specific alteration of parvalbumin neurons, perineuronal nets and microglia in the mouse prefrontal cortex and hippocampus following obesogenic diet consumption

Emergent evidence demonstrates that excessive consumption of high fat and high sugar (HFHS) diets has negative consequences on hippocampal and prefrontal cortex (PFC) function. Moreover, the delayed maturation of the PFC including the late development of parvalbumin-expressing (PV) interneurons and perineuronal nets (PNNs) may promote vulnerability to HFHS diet-induced nutritional stress. However, the young brain may have some resistance to diet-induced neuroinflammation. Thus, we examined the impact of a HFHS diet commencing either in adolescence or adulthood in male mice. PV interneurons, PNNs and microglia were assessed using immunohistochemistry. We observed greater numbers of PV neurons and PNNs in the hippocampus and the prelimbic and infralimbic PFC in adult mice in comparison to our younger cohort. Mice that consumed HFHS diet as adults had reduced numbers of hippocampal PV neurons and PNNs, which correlated with adiposity. However, we saw no effects of diet on PV and PNNs in the PFC. HFHS diet increased microgliosis in the adult cohort, and morphological changes to microglia were observed in the PFC and hippocampus of the adolescent cohort, with a shift to activated microglia phenotypes. Taken together, these findings demonstrate different regional and age-specific effects of obesogenic diets on PV neurons, PNNs and microglia.

‘Terrestrial lava tubes as analogues for Mars – a review of the mineralogy and biosignatures of lava tubes from Iceland and the Terceira islands.’

&lt;p&gt;Lava tubes have been detected on Mars and because of their subsurface nature are shielded from the harsh conditions at the surface. If water intersects with the Martian lava tubes, a life nurturing environment may exist locally in these tubes. Lava tubes on Iceland and the Azores may support similar conditions as lava tubes on Mars and have been shown to contain a wide variety of microbes. [Planetary Analogues and Lava Tube ] (PELE) field expeditions have been setup to understand the relationship between microbes and susbtrate and the preservation of microbes in deeptime within these systems. Within such systems biogenic and hydrothermal alteration processes are not necessarily mutually exclusive and a good understanding of the mineralogy helps distinguish one from the other. Here, I have performed an analytical study analysing basalt mineralogy from recent lava flows from Iceland and Azores islands, attempting to distinguish between biogenic and hydrothermal signatures. I used a workflow of semi quantitative analysis using viewing thin sections under a light microscope to understand textural information. This was supplemented by &amp;#160;ImageJ software and using SEM+EDX for point analysis of regions of interest to shed light on our areas of interest. My results showed some ambiguous features linked to alteration in a sample in the north of Iceland related to clays or spherulites, in the Azores vesicle infill of clays or devitrified glass were seen with potential bio signatures including carbon,calcum and phosphorous. These results may indicate environmental factors leading to location specific alteration or related to lava rock mineralogy. Contamination effects cannot be ignored and must be taken into consideration when reviewing these results. Overall these analyses will contribute to the larger PELE outcome by providing a complimentary workflow that can be used to assess biosignatures and specific regions of interest within lava tube rocks.&lt;/p&gt;&lt;p&gt;&amp;#160;&lt;/p&gt;