Increased Transcript Abundance Research Articles

Transcriptomic analysis implicates ABA signaling and carbon supply in the differential outgrowth of petunia axillary buds

BackgroundShoot branching of flowering plants exhibits phenotypic plasticity and variability. This plasticity is determined by the activity of axillary meristems, which in turn is influenced by endogenous and exogenous cues such as nutrients and light. In many species, not all buds on the main shoot develop into branches despite favorable growing conditions. In petunia, basal axillary buds (buds 1–3) typically do not grow out to form branches, while more apical axillary buds (buds 6 and 7) are competent to grow.ResultsThe genetic regulation of buds was explored using transcriptome analyses of petunia axillary buds at different positions on the main stem. To suppress or promote bud outgrowth, we grew the plants in media with differing phosphate (P) levels. Using RNA-seq, we found many (> 5000) differentially expressed genes between bud 6 or 7, and bud 2. In addition, more genes were differentially expressed when we transferred the plants from low P to high P medium, compared with shifting from high P to low P medium. Buds 6 and 7 had increased transcript abundance of cytokinin and auxin-related genes, whereas the basal non-growing buds (bud 2 and to a lesser extent bud 3) had higher expression of strigolactone, abscisic acid, and dormancy-related genes, suggesting the outgrowth of these basal buds was actively suppressed. Consistent with this, the expression of ABA associated genes decreased significantly in apical buds after stimulating growth by switching the medium from low P to high P. Furthermore, comparisons between our data and transcriptome data from other species suggest that the suppression of outgrowth of bud 2 was correlated with a limited supply of carbon to these axillary buds. Candidate genes that might repress bud outgrowth were identified by co-expression analysis.ConclusionsPlants need to balance growth of axillary buds into branches to fit with available resources while allowing some buds to remain dormant to grow after the loss of plant parts or in response to a change in environmental conditions. Here we demonstrate that different buds on the same plant with different developmental potentials have quite different transcriptome profiles.

6-Gingerol via overexpression of miR-322-5p impede lipopolysaccharide-caused inflammatory response in RAW264.7 cells.

Acute lung injury (ALI) and sepsis are complicated syndromes that are often left untreated in critically ill patients. 6-Gingerol is a phenolic phytochemical compound that is found in fresh ginger, has pharmacological effects against inflammation. This study explored the roles of 6-gingerol in a mouse model of acute lung injury caused by lipopolysaccharide (LPS) and RAW-264.7 cells inflammation. The LPS-induced animal model underwent histopathological examinations, and RAW-264.7 cells viability was determined by Cell counting Kit-8 (CCk-8) assay. Additionally, qRT-PCR, Immunofluorescence, Western blot, and ELISA were used in vivo and in vitro to identify inflammatory factors and proteins associated with NF-κB and MAPK signaling pathways. In a histological examination 6-gingerol exhibited protective effects. Moreover, 6-gingerol elevated cell viability and downregulated inflammatory factors Interlukin-1β (IL-1β), Interlukin-6 (IL-6) and Tumor necrosis factor-α (TNF-α) in LPS-treated RAW-264.7 cells. Furthermore, 6-gingerol decreased phosphorylation of P65, P38 and the level of JNK in NF-κB and MAPK pathways. Importantly, 6-gingerol increased transcript abundance of miR-322-5p which suppressed by LPS and miR-322-5p downregulation negated the protective functions of 6-gingerol. The protective activity of 6-gingerol was mediated by miR-322-5p up-regulation.

Regulation of TRIB1 abundance in hepatocyte models in response to proteasome inhibition

Tribbles related homolog 1 (TRIB1) contributes to lipid and glucose homeostasis by facilitating the degradation of cognate cargos by the proteasome. In view of the key metabolic role of TRIB1 and the impact of proteasome inhibition on hepatic function, we continue our exploration of TRIB1 regulation in two commonly used human hepatocyte models, transformed cell lines HuH-7 and HepG2. In both models, proteasome inhibitors potently upregulated both endogenous and recombinant TRIB1 mRNA and protein levels. Increased transcript abundance was unaffected by MAPK inhibitors while ER stress was a weaker inducer. Suppressing proteasome function via PSMB3 silencing was sufficient to increase TRIB1 mRNA expression. ATF3 was required to sustain basal TRIB1 expression and support maximal induction. Despite increasing TRIB1 protein abundance and stabilizing bulk ubiquitylation, proteasome inhibition delayed but did not prevent TRIB1 loss upon translation block. Immunoprecipitation experiments indicated that TRIB1 was not ubiquitylated in response to proteasome inhibition. A control bona fide proteasome substrate revealed that high doses of proteasome inhibitors resulted in incomplete proteasome inhibition. Cytoplasm retained TRIB1 was unstable, suggesting that TRIB1 lability is regulated prior to its nuclear import. N-terminal deletion and substitutions were insufficient to stabilize TRIB1. These findings identify transcriptional regulation as a prominent mechanism increasing TRIB1 abundance in transformed hepatocyte cell lines in response to proteasome inhibition and provide evidence of an inhibitor resistant proteasome activity responsible for TRIB1 degradation.

Cadmium exposure is associated with increased transcript abundance of multiple heavy metal associated transporter genes in roots of hemp (Cannabis sativa L.).

Industrial hemp (Cannabis sativa L.) has demonstrated promise for phytoremediation due to an extensive root system, large biomass, and ability to survive under relatively high levels of heavy metals. However, little research has been conducted to determine the impact of heavy metal uptake in hemp grown for medicinal use. This study evaluated the potential for cadmium (Cd) uptake and its impact on growth, physiological responses, and transcript expression of metal transporter genes in a hemp variety grown for flower production. The cultivar 'Purple Tiger' was exposed to 0, 2.5, 10, and 25 mg·L-1 Cd in a greenhouse hydroponic study in two independent experiments. Plants exposed to 25 mg·L-1 Cd displayed stunted plant growth characteristics, reduced photochemical efficiency, and premature senescence suggesting Cd toxicity. At the two lower concentrations of Cd (2.5 and 10 mg·L-1 Cd), plant height, biomass, and photochemical efficiency were not affected, with chlorophyll content index (CCI) being slightly lower at 10 mg·L-1 Cd, compared to 2.5 mg·L-1 Cd. There were no consistent differences between the two experiments in total cannabidiol (CDB) and tetrahydrocannabinol (THC) concentrations in flower tissues at 2.5 and 10 mg·L-1 Cd, compared to the control treatment. Root tissue accumulated the highest amount of Cd compared to other tissues for all the Cd treatments, suggesting preferential root sequestration of this heavy metal in hemp. Transcript abundance analysis of heavy metal-associated (HMA) transporter genes suggested that all seven members of this gene family are expressed in hemp, albeit with higher expression in the roots than in the leaves. In roots, CsHMA3 was up-regulated at 45 and 68d after treatment (DAT), and CsHMA1, CsHMA4, and CsHMA5 were upregulated only under long term Cd stress at 68 DAT, at 10 mg·L-1 Cd. Results suggest that expression of multiple HMA transporter genes in the root tissue may be upregulated in hemp exposed to 10 mg·L-1 Cd in a nutrient solution. These transporters could be involved in Cd uptake in the roots via regulating its transport and sequestration, and xylem loading for long distance transport of Cd to shoot, leaf, and flower tissues.

Screening of barley germplasm for drought tolerance based on root architecture, agronomic traits and identification of novel allelic variants of HVA1

AbstractDrought is a major constraint for barley production as it is normally cultivated in rainfed and marginal areas lacking optimum productivity. The domestication bottleneck and further selection pressure have resulted in reduced genetic diversity in barley. Genebank germplasm holds a huge potential for identifying new alleles for stress tolerance. In the present study, a diverse set of 214 accessions from Indian National Genebank were screened for drought tolerance in hydroponics and field conditions. Analysis of variance revealed a significant effect of drought on root architecture, relative water content, membrane stability index, chlorophyll content, plant height, and yield attributes. Cumulative stress response in terms of better root phenotype, physiological and agronomic traits showed accessions IC113045, EC578521, IC582699, EC492318, EC578711, EC667420, IC393980 and IC594943 as most promising donors for breeding programmes in drought‐prone areas. Further allelic variation of candidate gene, Hordeum vulgare aleurone 1 (HVA1), and its promoter sequence was studied in a subset of drought‐tolerant and ‐susceptible accessions. The HVA1 gene showed six SNPs and one indel in the genic regions whereas three SNPs and one indel in promoter. Two alleles of HVA1 gene, one in exotic and other in indigenous accession, were found to be associated with drought tolerance. These results were confirmed by qRT‐PCR analysis exhibiting significant increase in transcript abundance of HVA1 in drought‐tolerant accessions in comparison with susceptible accessions, thereby highlighting its possible role in imparting drought tolerance. The study helped identify genetic resources for drought tolerance in barley and unravelled new alleles of HVA1.

A multi-level study of the effects of glucocorticoids on human T cells

Abstract Glucocorticoids (GCs) have been the cornerstone of immunosuppressive therapies for the past 70 years, yet the mechanisms by which they regulate human immunity remain poorly understood. Specifically, there remains a need for direct insight into how GCs regulate the functions of human T cells. We have previously demonstrated that response to GCs in humans is highly cell type-dependent. In this project, we combine high-throughput techniques to study the effects of GCs on human CD4+ and CD8+ T cells, in vivo and in vitro. As part of a clinical study in 20 healthy volunteers, T cells were purified before and 2 and 4 hours after a dose of the GC methylprednisolone. RNA-seq was performed to identify GC-responsive genes. We identified a strong transcriptional response in T cells, where increased transcript abundance predominates. While there is a high degree of overlap of GC-responsive genes and in response directionality, CD4+ T cells show a stronger and more unique response than CD8+ T cells. To understand the functional genomic consequences of GC treatment in primary T cells, we developed a protocol to simultaneously assay gene expression (RNA-seq), chromatin accessibility (ATAC-seq), and glucocorticoid receptor binding (CUT&RUN) on cells treated in vitro. We are also performing experiments using tandem mass tagging (TMT) and mass spectrometry to identify which of the differentially expressed transcripts correspond to protein-level changes. Integrating data obtained at the transcript, chromatin, and protein levels will provide us with a means to identify biologically relevant effects of glucocorticoids and will guide the selection of assays to determine the functional consequences of those effects on human T cells.

Responses of flavonoids to solar UV radiation and gradual soil drying in two Medicago truncatula accessions

Ground level UV-B (290–315 nm) and UV-A (315–400 nm) radiation regulates multiple aspects of plant growth and development. In a natural environment, UV radiation interacts in a complex manner with other environmental factors (e.g., drought) to regulate plants’ morphology, physiology, and growth. To assess the interactive effects of UV radiation and soil drying on plants’ secondary metabolites and transcript abundance, we performed a field experiment using two different accessions of Medicago truncatula (F83005-5 French origin and Jemalong A17 Australian origin). Plants were grown for 37 days under long-pass filters to assess the effects of UV short wavelength (290–350 nm, UVsw) and UV-A long wavelength (350–400 nm, UV-Alw). Soil–water deficit was induced by not watering half of the plants during the last seven days of the experiment. The two accessions differed in the concentration of flavonoids in the leaf epidermis and in the whole leaf: F83005-5 had higher concentration than Jemalong A17. They also differed in the composition of the flavonoids: a greater number of apigenin derivatives than tricin derivatives in Jemalong A17 and the opposite in F83005-5. Furthermore, UVsw and soil drying interacted positively to regulate the biosynthesis of flavonoids in Jemalong A17 through an increase in transcript abundance of CHALCONE SYNTHASE (CHS). However, in F83005-5, this enhanced CHS transcript abundance was not detected. Taken together the observed metabolite and gene transcript responses suggest differences in mechanisms for acclimation and stress tolerance between the accessions.Graphical abstract

Integrated comparative transcriptome and weighted gene co-expression network analysis provide valuable insights into the response mechanisms of crayfish (Procambarus clarkii) to copper stress

One of the significant limitations of aquaculture worldwide is the prevalence of divalent copper (Cu). Crayfish (Procambarus clarkii) are economically important freshwater species adapted to a variety of environmental stimuli, including heavy metal stresses; however, large-scale transcriptomic data of the hepatopancreas of crayfish in response to Cu stress are still scarce. Here, integrated comparative transcriptome and weighted gene co-expression network analyses were initially applied to investigate gene expression profiles of the hepatopancreas of crayfish subjected to Cu stress for different periods. As a result, 4662 significant differentially expressed genes (DEGs) were identified following Cu stress. Bioinformatics analyses revealed that the "focal adhesion" pathway was one of the most significantly upregulated response pathways following Cu stress, and seven DEGs mapped to this pathway were identified as hub genes. Furthermore, the seven hub genes were examined by quantitative PCR, and each was found to have a substantial increase in transcript abundance, suggesting a critical role of the "focal adhesion" pathway in the response of crayfish to Cu stress. Our transcriptomic data can be a good resource for the functional transcriptomics of crayfish, and these results may provide valuable insights into the molecular response mechanisms underlying crayfish to Cu stress.

Manganese Deficiency Suppresses Growth and Photosynthetic Processes but Causes an Increase in the Expression of Photosynthetic Genes in Scots Pine Seedlings

Manganese deficiency is a serious plant nutritional disorder, resulting in the loss of crop productivity in many parts of the world. Despite the progress made in the study of angiosperms, the demand for Mn in gymnosperms and the physiological responses to Mn deficiency remain unexplored. We studied the influence of Mn deficiency for 24 weeks on Pinus sylvestris L. seedling growth, ion homeostasis, pigment contents, lipid peroxidation, chlorophyll fluorescence indices and the transcript levels of photosynthetic genes and genes involved in chlorophyll biosynthesis. It was shown that Mn-deficient plants demonstrated suppressed growth when the Mn content in the needles decreased below 0.34 µmol/g DW. The contents of photosynthetic pigments decreased when the Mn content in the needles reached 0.10 µmol/g DW. Mn deficiency per se did not lead to a decrease in the nutrient content in the organs of seedlings. Photoinhibition of PSII was observed in Mn-deficient plants, although this was not accompanied by the development of oxidative stress. Mn-deficient plants had an increased transcript abundance of genes (psbO, psbP, psbQ, psbA and psbC), encoding proteins directly associated with the Mn cluster also as other proteins involved in photosynthesis, whose activities do not depend on Mn directly. Furthermore, the transcript levels of the genes encoding the large subunit of Rubisco, light-dependent NADPH-protochlorophyllide oxidoreductase and subunits of light-independent protochlorophyllide reductase were also increased in Mn-deficient plants.

A comparative study on photosynthetic characteristics and flavonoid metabolism between Camellia petelotii (Merr.) Sealy and Camellia impressinervis Chang &Liang.

Camellia petelotii (Merr.) Sealy and Camellia impressinervis Chang & Liang belong to the golden subgroup of Camellia (Theaceae). This subgroup contains the yellow-flowering species of the genus, which have high medicinal and ornamental value and a narrow geographical distribution. These species differ in their tolerance to high light intensity. This study aimed to explore the differences in their light-stress responses and light damage repair processes, and the effect of these networks on secondary metabolite synthesis. Two-year-old plants of both species grown at 300 µmol·m-2·s-1 photosynthetically active radiation (PAR) were shifted to 700 µmol·m-2·s-1 PAR for 5 days shifting back to 300 µmol·m-2·s-1 PAR for recovery for 5 days. Leaf samples were collected at the start of the experiment and 2 days after each shift. Data analysis included measuring photosynthetic indicators, differential transcriptome expression, and quantifying plant hormones, pigments, and flavonoids. Camellia impressinervis showed a weak ability to recover from photodamage that occurred at 700 µmol·m-2·s-1 compared with C. petelotii. Photodamage led to decreased photosynthesis, as shown by repressed transcript abundance for photosystem II genes psbA, B, C, O, and Q, photosystem I genes psaB, D, E, H, and N, electron transfer genes petE and F, and ATP synthesis genes ATPF1A and ATPF1B. High-light stress caused more severe damage to C. impressinervis, which showed a stronger response to reactive oxygen species than C. petelotii. In addition, high-light stress promoted the growth and development of high zeatin signalling and increased transcript abundance of adenylate dimethylallyl transferase (IPT) and histidine-containing phosphotransferase (AHP). The identification of transcriptional differences in the regulatory networks that respond to high-light stress and activate recovery of light damage in these two rare species adds to the resources available to conserve them and improve their value through molecular breeding.

Soil metatranscriptome demonstrates a shift in C, N, and S metabolisms of a grassland ecosystem in response to elevated atmospheric CO2.

Soil organisms play an important role in the equilibrium and cycling of nutrients. Because elevated CO2 (eCO2) affects plant metabolism, including rhizodeposition, it directly impacts the soil microbiome and microbial processes. Therefore, eCO2 directly influences the cycling of different elements in terrestrial ecosystems. Hence, possible changes in the cycles of carbon (C), nitrogen (N), and sulfur (S) were analyzed, alongside the assessment of changes in the composition and structure of the soil microbiome through a functional metatranscriptomics approach (cDNA from mRNA) from soil samples taken at the Giessen free-air CO2 enrichment (Gi-FACE) experiment. Results showed changes in the expression of C cycle genes under eCO2 with an increase in the transcript abundance for carbohydrate and amino acid uptake, and degradation, alongside an increase in the transcript abundance for cellulose, chitin, and lignin degradation and prokaryotic carbon fixation. In addition, N cycle changes included a decrease in the transcript abundance of N2O reductase, involved in the last step of the denitrification process, which explains the increase of N2O emissions in the Gi-FACE. Also, a shift in nitrate () metabolism occurred, with an increase in transcript abundance for the dissimilatory reduction to ammonium () (DNRA) pathway. S metabolism showed increased transcripts for sulfate () assimilation under eCO2 conditions. Furthermore, soil bacteriome, mycobiome, and virome significantly differed between ambient and elevated CO2 conditions. The results exhibited the effects of eCO2 on the transcript abundance of C, N, and S cycles, and the soil microbiome. This finding showed a direct connection between eCO2 and the increased greenhouse gas emission, as well as the importance of soil nutrient availability to maintain the balance of soil ecosystems.

Stress-related expression of the chloroplast EGY3 pseudoprotease and its possible impact on chloroplasts’ proteome composition

The EGY3 is a pseudoprotease, located in the thylakoid membrane, that shares homology with the family of site-2-proteases (S2P). Although S2P proteases are present in the cells of all living organisms, the EGY3 was found only in plant cells. The sequence of the pseudoprotease is highly conserved in the plant kingdom; however, little is known about its physiological importance. Results obtained with real-time PCR indicated that the expression of the EGY3 gene is dramatically induced during the first few hours of exposure to high light and high-temperature stress. The observed increase in transcript abundance correlates with protein accumulation level, which indicates that EGY3 participates in response to both high-temperature and high light stresses. The lack of the pseudoprotease leads, in both stresses, to lower concentrations of hydrogen peroxide. However, the decrease of chloroplast copper/zinc superoxide dismutase 2 level was observed only during the high light stress. In both analyzed stressful conditions, proteins related to RubisCO folding, glycine metabolism, and photosystem I were identified as differently accumulating in egy3 mutant lines and WT plants; however, the functional status of PSII during analyzed stressful conditions remains very similar. Our results lead to a conclusion that EGY3 pseudoprotease participates in response to high light and high-temperature stress; however, its role is associated rather with photosystem I and light-independent reactions of photosynthesis.

MRNA N6 -methyladenosine is critical for cold tolerance in Arabidopsis.

SUMMARYPlants respond to low temperatures by altering the mRNA abundance of thousands of genes contributing to numerous physiological and metabolic processes that allow them to adapt. At the post‐transcriptional level, these cold stress‐responsive transcripts undergo alternative splicing, microRNA‐mediated regulation and alternative polyadenylation, amongst others. Recently, m6A, m5C and other mRNA modifications that can affect the regulation and stability of RNA were discovered, thus revealing another layer of post‐transcriptional regulation that plays an important role in modulating gene expression. The importance of m6A in plant growth and development has been appreciated, although its significance under stress conditions is still underexplored. To assess the role of m6A modifications during cold stress responses, methylated RNA immunoprecipitation sequencing was performed in Arabidopsis seedlings esposed to low temperature stress (4°C) for 24 h. This transcriptome‐wide m6A analysis revealed large‐scale shifts in this modification in response to low temperature stress. Because m6A is known to affect transcript stability/degradation and translation, we investigated these possibilities. Interestingly, we found that cold‐enriched m6A‐containing transcripts demonstrated the largest increases in transcript abundance coupled with increased ribosome occupancy under cold stress. The significance of the m6A epitranscriptome on plant cold tolerance was further assessed using the mta mutant in which the major m6A methyltransferase gene was mutated. Compared to the wild‐type, along with the differences in CBFs and COR gene expression levels, the mta mutant exhibited hypersensitivity to cold treatment as determined by primary root growth, biomass, and reactive oxygen species accumulation. Furthermore, and most importantly, both non‐acclimated and cold‐acclimated mta mutant demonstrated hypersensitivity to freezing tolerance. Taken together, these findings suggest a critical role for the epitranscriptome in cold tolerance of Arabidopsis.

A previously uncharacterized gene, PA2146, contributes to biofilm formation and drug tolerance across the ɣ-Proteobacteria

Transcriptomic studies have revealed a large number of uncharacterized genes that are differentially expressed in biofilms, which may be important in regulating biofilm phenotypes such as resistance to antimicrobial agents. To identify biofilm genes of unknown function in P. aeruginosa, we made use of RNA-seq and selected 27 uncharacterized genes that were induced upon biofilm growth. Biofilms by respective mutants were subsequently analyzed for two biofilm characteristics, the biofilm architecture and drug susceptibility. The screen revealed 12 out of 27 genes to contribute to biofilm formation and 13 drug susceptibility, with 8 genes affecting both biofilm phenotypes. Amongst the genes affecting both biofilm phenotypes was PA2146, encoding a small hypothetical protein that exhibited some of the most substantial increases in transcript abundance during biofilm growth by P. aeruginosa PAO1 and clinical isolates. PA2146 is highly conserved in ɣ-proteobacteria. Inactivation of PA2146 affected both biofilm phenotypes in P. aeruginosa PAO1, with inactivation of homologs in Klebsiella pneumoniae and Escherichia coli having similar effects. Heterologous expression of PA2146 homologs complemented the P. aeruginosa ∆PA2146, suggesting that PA2146 homologs substitute for and play a similar role as PA2146 in P. aeruginosa.

Atypical Climacteric and Functional Ethylene Metabolism and Signaling During Fruit Ripening in Blueberry (Vaccinium sp.).

Climacteric fruits display an increase in respiration and ethylene production during the onset of ripening, while such changes are minimal in non-climacteric fruits. Ethylene is a primary regulator of ripening in climacteric fruits. The ripening behavior and role of ethylene in blueberry (Vaccinium sp.) ripening is controversial. This work aimed to clarify the fruit ripening behavior and the associated role of ethylene in blueberry. Southern highbush (Vaccinium corymbosum hybrids) and rabbiteye (Vaccinium ashei) blueberry displayed an increase in the rate of respiration and ethylene evolution, both reaching a maxima around the Pink and Ripe stages of fruit development, consistent with climacteric fruit ripening behavior. Increase in ethylene evolution was associated with increases in transcript abundance of its biosynthesis genes, AMINOCYCLOPROPANE CARBOXYLATE (ACC) SYNTHASE1 (ACS1) and ACC OXIDASE2 (ACO2), implicating them in developmental ethylene production during ripening. Blueberry fruit did not display autocatalytic system 2 ethylene during ripening as ACS transcript abundance and ACC concentration were not enhanced upon treatment with an ethylene-releasing compound (ethephon). However, ACO transcript abundance was enhanced in response to ethephon, suggesting that ACO was not rate-limiting. Transcript abundance of multiple genes associated with ethylene signal transduction was upregulated concomitant with developmental increase in ethylene evolution, and in response to exogenous ethylene. As these changes require ethylene signal transduction, fruit ripening in blueberry appears to involve functional ethylene signaling. Together, these data indicate that blueberry fruit display atypical climacteric ripening, characterized by a respiratory climacteric, developmentally regulated but non-autocatalytic increase in ethylene evolution, and functional ethylene signaling.

Anterior Cruciate Ligament Reconstruction Upregulates Vitamin D Activating Pathways within the Quadriceps

Anterior cruciate ligament (ACL) tears are prevalent sport‐related injuries that result in protracted quadriceps atrophy and weakness despite surgical reconstruction and physical therapy. Vitamin D and its receptor are integral for skeletal muscle development and homeostasis. In addition, vitamin D status is positively associated with muscle size and strength. If the vitamin D receptor and related enzymes are upregulated with ACL injury and reconstruction, then overall vitamin D status may be an important effector of recovery. Concentrations of vitamin D needed to support optimal recovery may differ from typical clinical cut points used to diagnose vitamin D deficiency, which could have powerful therapeutic implications following ACL injury and reconstruction. This is especially important given recent research showing over 50% of athletes have vitamin D inadequacy. We hypothesized that subjects with circulating vitamin D sufficient to suppress parathyroid hormone secretion (≥ 40 ng/mL) would be better protected against quadriceps atrophy at the conclusion of rehabilitation. Muscle biopsies (vastus lateralis) were obtained from ACL‐injured and Healthy limbs of 16 participants (6M,10F; 19±5yr, body mass index: 25.1±3.7kg/m2) prior to surgical reconstruction, 1 week following reconstruction (Recon), and following the completion of post‐surgical rehabilitation (6 months following surgery, Post‐Rehab). RNA was isolated from all quadriceps biopsies, followed by library construction, sequencing, and bioinformatics analysis. Transcript abundance of vitamin D activating pathway regulators (vitamin D receptor [VDR], and Cytochrome P450 2R1 [CYP2R1] and Family 27 Subfamily B Member 1 [CYP27B1]) was assessed following analysis using DESeq2 and false discovery rate (FDR) adjustment of p‐values. Additionally, 25‐hydroxy vitamin D (25(OH)D) was assessed in serum samples obtained at the time of reconstruction surgery. Quadriceps fiber cross‐sectional area was measured via laminin immunohistochemistry. Recon (1 week post‐surgery) muscle showed increased transcript abundance of the vitamin D receptor, VDR(elevated 7.8 fold, FDR<0.05), and vitamin D‐activating pathway regulators CYP2R1 (elevated 1.5 fold, FDR<0.05) and CYP27B1 (elevated 2.6 fold, FDR<0.05) when compared to the Healthy limb. Following the completion of rehabilitation at the 6‐month follow‐up, participants with 25(OH)D above 40 ng/mL showed 6.4% atrophy of quadriceps fiber size compared to 22.8% atrophy in participants with 25(OH)D below 40 ng/mL (p=0.06). These findings provide evidence for surgically induced elevations in the vitamin D activating pathway within skeletal muscle. Upregulation of the vitamin D activating pathway may support quadriceps mitochondrial health and satellite cell activity and self‐renewal capacity, both of which are compromised following ACL reconstruction. In addition, elevated vitamin D concentrations may protect against protracted quadriceps atrophy. Future research should investigate if manipulation of vitamin D concentrations can enhance quadriceps functional recovery following ACL reconstruction.

Understanding the Mechanism of Diabetes Mellitus in a LRBA-Deficient Patient.

Simple SummaryDeficiency of lipopolysaccharide responsive beige like anchoring protein (LRBA) has been reported to cause immunological complications that can be fatal in children. Diabetes mellitus (DM) has been reported in some patients with LRBA deficiency. However, the underlying mechanism of the DM is not known. The current study provides potential novel insights into the underlying mechanism of DM in a LRBA-deficient patient. Additionally, in mouse pancreatic β-cells, we show that LRBA plays a role in the dynamics of insulin secretion and biosynthesis.The scope of this study is to show that DM in a LRBA-deficient patient with a stop codon mutation (c.3999 G > A) was not mediated through autoimmunity. We have evaluated the ability of the proband’s T cells to be activated by assessing their CTLA-4 expression. A nonsignificant difference was seen in the CTLA-4 expression on CD3+ T cells compared to the healthy control at basal level and after stimulation with PMA/ionomycin. Blood transcriptomic analysis have shown a remarkable increase in abundance of transcripts related to CD71+ erythroid cells. There were no differences in the expression of modules related to autoimmunity diseases between the proband and pooled healthy controls. In addition, our novel findings show that siRNA knockdown of LRBA in mouse pancreatic β-cells leads reduced cellular proinsulin, insulin and consequently insulin secretion, without change in cell viability in cultured MIN6 cells.

Molecular and Chemical Screening for Inherent Disease Resistance Factors of Norway Spruce (Picea abies) Clones Against Conifer Stem Rot Pathogen Heterobasidion parviporum

Root and stem rot of conifer trees caused by Heterobasidion annosum species complex leads to huge economic losses in Europe, yet not much is known about the molecular and chemical basis for host resistance. To identify inherent chemical or molecular markers in clones found to be either resistant or susceptible, we sampled needle tissues of all the clones before pathogen inoculation. We conducted a short-term resistance screening by using the pathogen H. parviporum to inoculate 70 Norway spruce clones. Based on lesion size, subsets of highly susceptible and resistant clones were further analyzed. Terpene detection and RNA sequencing were performed to explore inherent variations in genotypes differing in resistance to pathogenic challenge at chemical and transcriptional levels. A negative correlation emerged between resistance and growth. Terpene profiles of resistant clones showed higher content of monoterpenes and sesquiterpenes, with concomitant increased transcript abundance of genes involved in the terpenoid pathway. A set of upregulated genes relevant to flavonoid biosynthesis was observed in resistant genotypes, whereas higher transcripts of lignin biosynthetic genes were prevalent in susceptible clones. Genes involved in flavonoid and lignin biosynthesis as well as terpene content may have a role in facilitating resistance of Norway spruce against H. parviporum. Our results provide strong support on the feasibility of sampling needle tissues before pathogen inoculation, and the approach could be of value for large-scale screening of novel biomarkers for durable resistance. The additional insights could form a basis for further research on resistance screening in this pathosystem.

Physiological, molecular, and immune responses to milk thistle extract administration in goats during peripartum period

Background Using natural compounds as additives in livestock nutrition could be a new goal in livestock production. Milk thistle extract is rich in bioactive compounds such as silymarin, which act as a strong antioxidant agent.Objective The current study aimed to investigate the metabolic profile, oxidative statue, and immune response after milk thistle extract administration in goats during the peripartum period.Materials and methods Multiparous pregnant Egyptian Nubian goats (n=16) were allocated into four experimental groups. The first group was kept as the control group. The second group was administrated milk thistle extract (10 g/day), whereas third and fourth groups were administrated 20 and 30 g/day for 4 months, respectively. Blood biochemical parameters were measured using colorimetric and enzyme-linked immunosorbent assay. Gene expressions of antioxidant genes [catalase (CAT), superoxide dismutase (SOD1, SOD2), glutathione peroxidase (GPX1), and peroxiredoxin 2] and transcription factor (nuclear factor erythroid 2 related factor 2) were evaluated using quantitative real-time PCR.Results and conclusion Biochemical parameters (total protein, glucose, total lipids, total cholesterol, triglycerides, high-density lipoprotein, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, urea, creatinine, triiodothyronine, and thyroxine) in plasma of groups administrated with milk thistle extract did not significantly differ compared with the control group. Milk thistle extract at high levels (20 and 30 g/day) significantly increased the level of activity of antioxidant enzymes (SOD, CAT, and GPX), total antioxidant capacity, and total immunoglobulin in cases compared with the control group. Moreover, milk thistle extract (20 or 30 g/day) significantly decreased the level of malondialdehyde (lipid peroxidation biomarker) and tumor necrosis factor alpha (inflammatory biomarker) in cases compared with the control group. The results indicated a significant increase in transcript abundance of CAT, GPX1, and SOD1 mRNA in the three groups administrated with milk thistle extract compared with the control group. However, mRNA expressions of SOD2, peroxiredoxin 2, and nuclear factor erythroid 2 related factor 2 were significantly up-regulated after administration with milk thistle extract at high levels (20 and 30 g/day). Milk thistle extract exerts antioxidant, anti-inflammatory, and immune-modulator effects during pregnancy and lactation in goat and maintained normal physiological functions.

Escherichia coli and Staphylococcus aureus Differentially Regulate Nrf2 Pathway in Bovine Mammary Epithelial Cells: Relation to Distinct Innate Immune Response.

Escherichia coli and Staphylococcus aureus are major mastitis causing pathogens in dairy cattle but elicit distinct immune and an inflammatory response in the udder. However, the host determinants responsible for this difference remains largely unknown. Our initial studies focused on the global transcriptomic response of primary bovine mammary epithelial cells (pbMECs) to heat-killed E. coli and S. aureus. RNA-sequencing transcriptome analysis demonstrates a significant difference in expression profiles induced by E. coli compared with S. aureus. A major differential response was the activation of innate immune response by E. coli, but not by S. aureus. Interestingly, E. coli stimulation increased transcript abundance of several genes downstream of Nrf2 (nuclear factor erythroid 2-related factor 2) that were enriched in gene sets with a focus on metabolism and immune system. However, none of these genes was dysregulated by S. aureus. Western blot analysis confirms that S. aureus impairs Nrf2 activation as compared to E. coli. Using Nrf2-knockdown cells we demonstrate that Nrf2 is necessary for bpMECs to mount an effective innate defensive response. In support of this notion, nuclear Nrf2 overexpression augmented S. aureus-stimulated inflammatory response. We also show that, unlike E. coli, S. aureus disrupts the non-canonical p62/SQSTM1-Keap1 pathway responsible for Nrf2 activation through inhibiting p62/SQSTM1 phosphorylation at S349. Collectively, our findings provide important insights into the contribution of the Nrf2 pathway to the pathogen-species specific immune response in bovine mammary epithelial cells and raise a possibility that impairment of Nrf2 activation contributes to, at least in part, the weak inflammatory response in S. aureus mastitis.