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We have read the insightful comments on our article "RNA-Seq analysis reveals sex-dependent transcriptomic profiles of human subacromial bursa stratified by tear etiology" published in the Journal of Orthopaedic Research.1 We thank the authors of the I Letter to the Editor i 2 for their interest in our article and have decided to carefully respond to their concerns. The plot indicates moderate to highly up- and downregulated genes with log2 fold-changes >= |2| in females with traumatic injuries over females with degenerative injuries with modest levels of statistical significance p-value <= 0.05 are distinct from those genes with low to no statistical significance and log2 fold-changes <= |2|. Mean log2 counts-per-million to log2 fold-change amplitude plot illustrating the range of statistically significant genes with log2 fold-changes >= |2|. [Color figure can be viewed at wileyonlinelibrary.com] gl Second, the authors of letter noted that samples #15 (female degenerative) and #29 (female traumatic) expressed high levels of genes located on the Y chromosome which were expressed at the same level as their male counterparts. [Extracted from the article]

Background Current diagnostic tests for pharyngitis do not distinguish between symptomatic group A Streptococcus (GAS) infection and asymptomatic colonization, resulting in over-diagnosis and unnecessary use of antibiotics. We assessed whether measures of host response could make this distinction. Methods We enrolled 18 children with pharyngitis having Centor scores of 4 or 5 and 21 controls without pharyngitis or other acute infections. Both groups had throat cultures, molecular tests for GAS and respiratory viruses and IgM serology for Epstein–Barr virus. Host response was evaluated with white blood cell count (WBC), C-reactive protein (CRP), procalcitonin (PCT), and sequencing of RNA from peripheral blood leukocytes. Results Of 18 cases, 11 had GAS pharyngitis, 3 had adenovirus pharyngitis and 4 had other pharyngitis. Among asymptomatic controls, 5 were positive for GAS. WBC, CRP, and PCT were higher in subjects with pharyngitis compared to asymptomatic controls including those with GAS. Transcriptional profiles from children with symptomatic GAS were clearly distinct from those of children in all other groups. The levels of two genes, CD177 and TLR5 each individually accurately distinguished between symptomatic and asymptomatic GAS. Optimal diagnostic sensitivity and specificity were achieved by the combination of CRP and PCT, and by each of the two gene markers. Conclusion In this exploratory study, we showed that traditional measures of inflammation and markers of host gene expression distinguish between symptomatic and asymptomatic GAS. These results point to future rapid molecular approaches for improving the diagnosis of GAS pharyngitis, that may help reduce unnecessary antibiotic use. [ABSTRACT FROM AUTHOR]

The mammalian eye lens expresses a high concentration of crystallins (α, β and γ-crystallins) to maintain the refractive index essential for lens transparency. Crystallins are long-lived proteins that do not turnover throughout life. The structural destabilization of crystallins by UV exposure, glycation, oxidative stress and mutations in crystallin genes leads to protein aggregation and development of cataracts. Several destabilizing mutations in crystallin genes are linked with human autosomal dominant hereditary cataracts. To investigate the mechanism by which the α-crystallin mutations Cryaa-R49C and Cryab-R120G lead to cataract formation, we determined whether these mutations cause an altered expression of specific transcripts in the lens at an early postnatal age by RNA-seq analysis. Using knock-in mouse models previously generated in our laboratory, in the present work, we identified genes that exhibited altered abundance in the mutant lenses, including decreased transcripts for Clic5, an intracellular water channel in Cryaa-R49C heterozygous mutant lenses, and increased transcripts for Eno1b in Cryab-R120G heterozygous mutant lenses. In addition, RNA-seq analysis revealed increased histones H2B, H2A, and H4 gene expression in Cryaa-R49C mutant lenses, suggesting that the αA-crystallin mutation regulates histone expression via a transcriptional mechanism. Additionally, these studies confirmed the increased expression of histones H2B, H2A, and H4 by proteomic analysis of Cryaa-R49C knock-in and Cryaa;Cryab gene knockout lenses reported previously. Taken together, these findings offer additional insight into the early transcriptional changes caused by Cryaa and Cryab mutations associated with autosomal dominant human cataracts, and indicate that the transcript levels of certain genes are affected by the expression of mutant α-crystallin in vivo. [ABSTRACT FROM AUTHOR]

ABSTRACT: Microarrays and RNA‐seq are at the forefront of high throughput transcriptome analyses. Since these methodologies are based on different principles, there are concerns about the concordance of data between the two techniques. The concordance of RNA‐seq and microarrays for genome‐wide analysis of differential gene expression has not been rigorously assessed in clinically derived ligament tissues. To demonstrate the concordance between RNA‐seq and microarrays and to assess potential benefits of RNA‐seq over microarrays, we assessed differences in transcript expression in anterior cruciate ligament (ACL) tissues based on time‐from‐injury. ACL remnants were collected from patients with an ACL tear at the time of ACL reconstruction. RNA prepared from torn ACL remnants was subjected to Agilent microarrays (N = 24) and RNA‐seq (N = 8). The correlation of biological replicates in RNA‐seq and microarrays data was similar (0.98 vs. 0.97), demonstrating that each platform has high internal reproducibility. Correlations between the RNA‐seq data and the individual microarrays were low, but correlations between the RNA‐seq values and the geometric mean of the microarrays values were moderate. The cross‐platform concordance for differentially expressed transcripts or enriched pathways was linearly correlated (r = 0.64). RNA‐Seq was superior in detecting low abundance transcripts and differentiating biologically critical isoforms. Additional independent validation of transcript expression was undertaken using microfluidic PCR for selected genes. PCR data showed 100% concordance (in expression pattern) with RNA‐seq and microarrays data. These findings demonstrate that RNA‐seq has advantages over microarrays for transcriptome profiling of ligament tissues when available and affordable. Furthermore, these findings are likely transferable to other musculoskeletal tissues where tissue collection is challenging and cells are in low abundance. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:484–497, 2018. [ABSTRACT FROM AUTHOR]

Background: Anterior cruciate ligament (ACL) tears are a common injury. The healing potential of the injured ACL is poorly understood and is considered limited. Therefore, most ACL tears that are treated surgically undergo reconstruction rather than repair. However, there has been renewed interest recently in repairing ACL tears despite unanswered questions regarding the healing capacity of the ACL. Hypothesis: Gene expression in the injured ACL varies with time from injury. Study Design: Descriptive laboratory study. Methods: Transcriptome-wide expression profiles of 24 human ACL remnants recovered at the time of surgical reconstruction were analyzed using the Agilent human 8x60K microarray platform. Gene ontology was performed on differentially expressed transcripts based on time from injury (acute, <3 months; intermediate, 3-12 months; chronic, >12 months). A subset of transcripts with large fold changes in expression between any 2 categories was validated via microfluidic digital polymerase chain reaction. Results: Numerous transcripts representing important biological processes were differentially expressed by time from injury. The most significant changes were noted between the acute and chronic groups. Expression of several extracellular matrix genes— namely, POSTN, COL5A1, COL1A1, and COL12A1—was lower in the chronic tears compared with acute and intermediate tears. In acute tears, processes representing angiogenesis and stem cell differentiation were affected. In intermediate tears, processes representing stem cell proliferation concomitant with cellular component organization/cellular localization were altered. In ACL tears more than 12 months out from injury, processes denoting myosin filament organization, cellular component organization/cell localization, and extracellular matrix organization were affected. Conclusion: These findings are consistent with initial repair activity in the injured ACL, which declines with time from injury. Individual genes identified in this study, such as periostin, deserve further investigation into their role in tissue repair. Clinical Relevance: The decreased healing capacity of ACL tears over time is relevant to the development of effective techniques for repairing ACL tears and may have some significance for ACL reconstruction techniques as well. The potential for healing appears to be greatest in acute ACL tears, suggesting this window should be the focus of research for ACL repair. [ABSTRACT FROM AUTHOR]

Rotator cuff tendinopathy, a major cause of shoulder disability, occurs due to trauma or degeneration. Our molecular understanding of traumatic and degenerative tears remains elusive. Here, we probed transcript level differences between traumatic and degenerative tears. Subacromial bursa tissues were collected from patients with traumatic or degenerative tears during arthroscopy (N = 32). Transcripts differentially expressed by tear etiology were detected by RNA‐seq. RNA‐seq results were validated by real‐time quantitative polymerase chain reaction. We identified 334 protein‐coding transcripts differentially expressed between traumatic and degenerative tears in females and 167 in males at a fold‐change greater than 2. In females, XIRP2, MYL1, MYBPC1, TNNT1, and LMOD2, were highly expressed in traumatic tears whereas TPSD1, CDSN, RCVRN, LTBP4, and PTGS1 were elevated in degen tears. Transcripts elevated in traumatic tears represented muscle cell differentiation and development, and muscle contraction whereas those elevated in degenerative tears represented cell activation and immune response. In males, AZGP1, CNTFR, COL9A1, ZNF98, and EREG were highly elevated in traumatic tears whereas MYL2, HOXD11, SLC6A7, CADM1, and MMP17 were highly expressed in degenerative tears. Transcripts elevated in traumatic tears represented metabolic/catabolic processes, and transmembrane protein transport while processes related to cell cycle were mainly enriched in degenerative tears. Numerous long noncoding RNAs were differentially expressed between traumatic and degenerative tears in both sexes. In summary, this study provides insights into molecular biology of bursa in patients with rotator cuff tendon disease based on tear acuity and novel sex‐based transcript differences that could inform clinical decision making in treating patients with traumatic or degenerative shoulder injuries. [ABSTRACT FROM AUTHOR]

Rotator cuff tears are a common pathology in the shoulder and generally have two underlying etiologies: traumatic and degenerative. Little is known about the molecular underpinning of these etiologies. Here we queried transcript level differences in tear etiology stratified by sex in 31 patients with rotator cuff tears. Tendon tissues were isolated from females (N = 16) and males (N = 15) with traumatic (N = 16) or degenerative (N = 15) tears during arthroscopy. Differentially expressed transcripts were identified by RNA‐seq and biological processes were probed computationally. Expression of some transcripts was validated by real‐time quantitative polymerase chain reaction (qPCR). We identified 339 and 336 transcripts differentially expressed by tear etiology in females and males, respectively, at a fold‐change greater than |2|. In females, GSTM1, MT1G, S1008A, ACSM3, DSC, FAM110C, and VNN2 were elevated in traumatic tears representing metabolic/catabolic processes, and immune response whereas CHAD, CLEC3A, IBSP, TNMD, APLNR, and CPA3 were elevated in degenerative tears representing tissue morphogenesis and developmental processes, angiogenesis, and extracellular matrix organization. In males, ELOA3B, CXCL8, ADM, TNS4, and SPOCK1 were elevated in traumatic tears representing localization of endoplasmic reticulum, chromosome organization, leukocyte/neutrophil degranulation, and protein transport whereas MYL2, TNNC1, MB, CPA3, APLNR, and CA3 were highly upregulated in degenerative tears representing muscle cell differentiation and development and angiogenesis. Numerous novel lncRNAs were identified to be differentially expressed by tear etiology in both sexes. Real‐time qPCR confirmed RNA‐seq data. This study improves our understanding of tendon biology based on underlying etiology (trauma or degeneration) in a sex‐specific manner. These findings may help drive clinical decision‐making in females and males with traumatic and degenerative shoulder injuries. [ABSTRACT FROM AUTHOR]

Background: Sex differences in the brain may play an important role in sex-differential prevalence of neuropsychiatric conditions. Methods: In order to understand the transcriptional basis of sex differences, we analyzed multiple, large-scale, human postmortem brain RNA-Seq datasets using both within-region and pan-regional frameworks. Results: We find evidence of sex-biased transcription in many autosomal genes, some of which provide evidence for pathways and cell population differences between chromosomally male and female individuals. These analyses also highlight regional differences in the extent of sex-differential gene expression. We observe an increase in specific neuronal transcripts in male brains and an increase in immune and glial function-related transcripts in female brains. Integration with single-nucleus data suggests this corresponds to sex differences in cellular states rather than cell abundance. Integration with case–control gene expression studies suggests a female molecular predisposition towards Alzheimer's disease, a female-biased disease. Autism, a male-biased diagnosis, does not exhibit a male predisposition pattern in our analysis. Conclusion: Overall, these analyses highlight mechanisms by which sex differences may interact with sex-biased conditions in the brain. Furthermore, we provide region-specific analyses of sex differences in brain gene expression to enable additional studies at the interface of gene expression and diagnostic differences. Highlights: Numerous but small autosomal sex differences in expression exist in all brain regions tested. Autosomal genes with enriched expression in males are enriched in neuronal pathways. Autosomal genes with enriched expression in females are enriched with immune system functions. Integration with single-nucleus datasets suggest these differences are more likely related to cell state differences than cell number differences. The female cortex shows an enrichment of genes expressed in Alzheimer's disease brains. Plain English Summary: We sought to understand why females have higher rates of Alzheimer's disease, and males have higher rates of autism. One idea was that the female brain at baseline may be more similar to an Alzheimer's brain, so it is easier for them to shift into that state (likewise, males may be more similar to autism). To test this, we examined gene expression differences between brains of biological males and biological females. While all people have the same ~ 25,000 genes, each gene can be on or off ('expressed') to different extents. Overall, we found that there were differences in gene expression between males and females in all brain regions tested but more differences in some brain regions than others. By looking at the role of these genes we estimate that female immune system processes might be more active in the brain. We also found female brain gene expression looked slightly more like people with Alzheimer's compared to people without Alzheimer's, which may explain why females get Alzheimer's disease more easily. However, the male brain gene expression did not look more like autism, suggesting that the reason males have higher rates of autism is complex and needs further investigation. [ABSTRACT FROM AUTHOR]

Pioglitazone (Pio) is a thiazolidinedione (TZD) insulin-sensitizing drug whose effects result predominantly from its modulation of the transcriptional activity of peroxisome proliferator-activated-receptor-gamma (PPARγ). Pio is used to treat human insulin-resistant diabetes and also frequently considered for treatment of nonalcoholic steatohepatitis (NASH). In both settings, Pio's beneficial effects are believed to result primarily from its actions on adipose PPARγ activity, which improves insulin sensitivity and reduces the delivery of fatty acids to the liver. Nevertheless, a recent clinical trial showed variable efficacy of Pio in human NASH. Hepatocytes also express PPARγ, and such expression increases with insulin resistance and in nonalcoholic fatty liver disease (NAFLD). Furthermore, mice that overexpress hepatocellular PPARγ and Pio-treated mice with extrahepatic PPARγ gene disruption develop features of NAFLD. Thus, Pio's direct impact on hepatocellular gene expression might also be a determinant of this drug's ultimate influence on insulin resistance and NAFLD. Previous studies have characterized Pio's PPARγ-dependent effects on hepatic expression of specific adipogenic, lipogenic, and other metabolic genes. However, such transcriptional regulation has not been comprehensively assessed. The studies reported here address that consideration by genome-wide comparisons of Pio's hepatic transcriptional effects in wildtype (WT) and liver-specific PPARγ-knockout (KO) mice given either control or high-fat (HFD) diets. The results identify a large set of hepatic genes for which Pio's liver PPARγ-dependent transcriptional effects are concordant with its effects on RXR-DNA binding in WT mice. These data also show that HFD modifies Pio's influence on a subset of such transcriptional regulation. Finally, our findings reveal a broader influence of Pio on PPARγ-dependent hepatic expression of nuclear genes encoding mitochondrial proteins than previously recognized. Taken together, these studies provide new insights about the tissue-specific mechanisms by which Pio affects hepatic gene expression and the broad scope of this drug's influence on such regulation. [ABSTRACT FROM AUTHOR]

Periostin (POSTN), a secretory matricellular matrix protein, plays a multitude of biologic functions. Various splice variants of POSTN have been described; however, their expression pattern and functional implications are not completely understood. This study was undertaken to decipher the differential expression pattern of POSTN and its splice variants in various tissues and cell types. We show that POSTN was more highly expressed in anterior cruciate ligament (ACL) remnants compared with articular cartilage at the cellular and tissue level. Isoforms 1 and 8 were highly expressed only in articular chondrocytes, suggesting their splice-specific regulation in chondrocytes. To discern the role of total POSTN and full-length human POSTN isoform 1 (hPOSTN-001), we stably transfected human chondrosarcoma 1 (hCh-1) cell line with hPOSTN-001 using a pcDNA3.1-hPOSTN-001 construct. RNA-sequencing analysis of hCh-1 cells identified differentially expressed genes with a known role in chondrocyte function and osteoarthritis. Similar expression of a subset of candidate genes was revealed in ACL progenitor cells and chondrocytes as well as in ACL progenitor cells in which POSTN activity was altered by overexpression and by small interfering RNA gene knockdown. Cells expressing total POSTN, not isoform 1, exhibited increased cell adhesion potential. These findings suggest an important role for POSTN in the knee.--Cai, L., Brophy, R. H., Tycksen, E. D., Duan, X., Nunley, R. M., Rai, M. F. Distinct expression pattern of periostin splice variants in chondrocytes and ligament progenitor cells. [ABSTRACT FROM AUTHOR]

MicroRNAs are small non-coding RNAs that play important roles in many cellular processes including proliferation, metabolism and differentiation. They function by binding to specific regions within the 3′UTR of target mRNAs resulting in suppression of protein synthesis and modulation of potentially many cellular pathways. We previously showed that miRNA expression levels differed between cells from distinct regions of developing human embryonic long bones. Specifically, we found that miR-181a-1 was significantly more highly expressed in hypertrophic chondrocytes compared to proliferating differentiated or progenitor chondrocytes, suggesting a potential role in regulating chondrocyte hypertrophy and/or endochondral bone formation. The goal of this study was to determine how miR-181a-1 together with its clustered miRNA, miR-181b-1, regulates osteogenesis. We show that over-expression of the miR-181a/b-1 cluster enhanced osteogenesis and that cellular pathways associated with protein synthesis and mitochondrial metabolism were significantly up-regulated. Metabolic assays revealed that the oxygen consumption rate and ATP-linked respiration were increased by miR-181a/b-1. To further decipher a potential mechanism causing these metabolic changes, we showed that PTEN (phosphatase and tensin homolog) levels were suppressed following miR-181a/b-1 over-expression, and that PI3K/AKT signaling was subsequently increased. Over-expression of PTEN was found to attenuate the enhancing effects of miR-181a/b-1, providing further evidence that miR-181a/b-1 regulates the PTEN/PI3K/AKT axis to enhance osteogenic differentiation and mitochondrial metabolism. These findings have important implications for the design of miR-181a/b targeting strategies to treat bone conditions such as fractures or heterotopic ossification. • Over-expression of miR-181a/b-1 induces osteogenic differentiation of human primary dedifferentiated chondrocytes. • Enhanced osteogenesis occurs by PTEN suppression and activation of PI3K/AKT signaling and mitochondrial respiration. • Targeting miR-181a/b-1 in vivo will be important to test effects on bone fracture healing or heterotopic ossification. [ABSTRACT FROM AUTHOR]

Aberrant chondrocyte metabolism significantly contributes to cartilage degeneration and osteoarthritis (OA) genesis. However, the mechanisms driving the metabolic shift in OA chondrocytes remain unclear. Interestingly, carbonic anhydrase 2 (CA2) is implicated in metabolic regulation, and its expression dramatically increases in OA chondrocytes, but its exact role and mechanism are poorly understood. This study investigates the mechanistic role of CA2 in chondrocyte metabolic homeostasis under hypoxic and inflammatory conditions. RNA-seq was performed on CA2-deficient C28/I2 cells to identify pathways affected by the loss of CA2 function. We examined CA2's impact on chondrocyte metabolism, anabolism, and catabolism using C28/I2 cells and primary chondrocytes under normoxia and hypoxia and in a model of inflammatory OA. RNA-seq revealed enrichment of glycolysis, apoptosis, and TNF signaling pathways in CA2-deficient cells. Under hypoxia, CA2 expression increased 10-fold in a HIF-1α-independent manner. Knockdown of CA2 reduced extracellular lactate production, increased ADP/ATP ratio, impaired glycolysis, reduced glycolytic capacity, and lowered expression of glycolysis rate-limiting enzymes but did not disrupt pH i and ROS production. CA2 deficiency altered chondrocyte anabolic and catabolic equilibrium by affecting PI3K/AKT and RELA/p65 signaling. CA2-deficient chondrocytes displayed impeded migration, suppressed proliferation, and cell cycle arrest at the G0/G1 phase. Forced expression of CA2 stabilized chondrocyte metabolism and restored cellular functions. Our research uncovered a hitherto unknown mechanistic role for CA2 in regulating chondrocyte energy metabolism and inflammation, underscoring its potential as a critical mediator in OA pathogenesis. Further research using a murine model of experimental OA is warranted to capture the functional implications of CA2. • CA2 deficiency disrupts glycolysis and impairs extracellular lactate production in chondrocytes. • CA2 deficiency alters chondrocyte anabolic and catabolic balance via PI3K/AKT and RELA/p65 signaling pathways. • CA2-deficient chondrocytes exhibit impaired migration, suppressed proliferation, and cell cycle arrest at the G0/G1 phase. • Forced expression of CA2 stabilizes chondrocyte metabolism and restores normal cellular functions. [ABSTRACT FROM AUTHOR]

Despite the widespread use of oxytocin for induction of labor, mechanistic insights into fetal/neonatal wellbeing are lacking because of the absence of an animal model that recapitulates modern obstetric practice. Here, we create and validate a hi-fidelity pregnant rat model that mirrors labor induction with oxytocin in laboring women. The model consists of an implantable preprogrammed microprocessor-controlled infusion pump that delivers a gradually escalating dose of intravenous oxytocin to induce birth at term gestation. We validated the model with molecular biological experiments on the uterine myometrium and telemetry-supported assessment of changes in intrauterine pressure. Finally, we applied this model to test the hypothesis that labor induction with oxytocin would be associated with oxidative stress in the newborn brain. Analysis of biomarkers of oxidative stress and changes in the expression of associated genes were no different between oxytocin-exposed and saline-treated pups, suggesting that oxytocin-induced labor was not associated with oxidative stress in the developing brain. Collectively, we provide a viable and realistic animal model for labor induction and augmentation with oxytocin that would enable new lines of investigation related to the impact of perinatal oxytocin exposure on the mother-infant dyad. [ABSTRACT FROM AUTHOR]

Pathogenic variants in surfactant proteins SP-B and SP-C cause surfactant deficiency and interstitial lung disease. Surfactant proteins are synthesized as precursors (proSP-B, proSP-C), trafficked, and processed via a vesicular-regulated secretion pathway; however, control of vesicular trafficking events is not fully understood. Through the Undiagnosed Diseases Network, we evaluated a child with interstitial lung disease suggestive of surfactant deficiency. Variants in known surfactant dysfunction disorder genes were not found in trio exome sequencing. Instead, a de novo heterozygous variant in RAB5B was identified in the Ras/Rab GTPases family nucleotide binding domain, p.Asp136His. Functional studies were performed in Caenorhabditis elegans by knocking the proband variant into the conserved position (Asp135) of the ortholog, rab-5. Genetic analysis demonstrated that rab-5[Asp135His] is damaging, producing a strong dominant negative gene product. rab-5[Asp135His] heterozygotes were also defective in endocytosis and early endosome (EE) fusion. Immunostaining studies of the proband's lung biopsy revealed that RAB5B and EE marker EEA1 were significantly reduced in alveolar type II cells and that mature SP-B and SP-C were significantly reduced, while proSP-B and proSP-C were normal. Furthermore, staining normal lung showed colocalization of RAB5B and EEA1 with proSP-B and proSP-C. These findings indicate that dominant negative-acting RAB5B Asp136His and EE dysfunction cause a defect in processing/trafficking to produce mature SP-B and SP-C, resulting in interstitial lung disease, and that RAB5B and EEs normally function in the surfactant secretion pathway. Together, the data suggest a noncanonical function for RAB5B and identify RAB5B p.Asp136His as a genetic mechanism for a surfactant dysfunction disorder. [ABSTRACT FROM AUTHOR]

• The delivery of IL-1β by CNS infiltrating myeloid cells is succeeded by inflammasome-mediated production of IL-1β by astrocytes during viral recovery. • IL-1R1 signaling in NSC during acute encephalitis maintains a proinflammatory environment via generation of proinflammatory astrocytes throughout recovery. • Adult hippocampal NSC downregulate neurogenic and upregulate astrogenic differentiation pathways in the setting of CNS viral infection and recovery. • Only newly generated astrocytes prevent synapse repair and promote spatial learning and memory deficits via their production of IL-1β. Innate immune responses to emerging RNA viruses are increasingly recognized as having significant contributions to neurologic sequelae, especially memory disorders. Using a recovery model of West Nile virus (WNV) encephalitis, we show that, while macrophages deliver the antiviral and anti-neurogenic cytokine IL-1β during acute infection; viral recovery is associated with continued astrocyte inflammasome-mediated production of inflammatory levels of IL-1β, which is maintained by hippocampal astrogenesis via IL-1R1 signaling in neural stem cells (NSC). Accordingly, aberrant astrogenesis is prevented in the absence of IL-1 signaling in NSC, indicating that only newly generated astrocytes exert neurotoxic effects, preventing synapse repair and promoting spatial learning deficits. Ex vivo evaluation of IL-1β-treated adult hippocampal NSC revealed the upregulation of developmental differentiation pathways that derail adult neurogenesis in favor of astrogenesis, following viral infection. We conclude that NSC-specific IL-1 signaling within the hippocampus during viral encephalitis prevents synapse recovery and promotes spatial learning defects via altered fates of NSC progeny that maintain inflammation. [ABSTRACT FROM AUTHOR]

Calcium signals are initiated in immune cells by the process of store-operated calcium entry (SOCE), where receptor activation triggers transient calcium release from the endoplasmic reticulum, followed by opening of plasma-membrane calcium-release activated calcium (CRAC) channels. ORAI1, ORAI2, and ORAI3 are known to comprise the CRAC channel; however, the contributions of individual isoforms to neutrophil function are not well understood. Here, we show that loss of ORAI1 partially decreases calcium influx, while loss of both ORAI1 and ORAI2 completely abolishes SOCE. In other immune-cell types, loss of ORAI2 enhances SOCE. In contrast, we find that ORAI2-deficient neutrophils display decreased calcium influx, which is correlated with measurable differences in the regulation of neutrophil membrane potential via KCa3.1. Decreased SOCE in ORAI1-, ORAI2-, and ORAI1/2-deficient neutrophils impairs multiple neutrophil functions, including phagocytosis, degranulation, leukotriene, and reactive oxygen species (ROS) production, rendering ORAI1/2-deficient mice highly susceptible to staphylococcal infection. This study demonstrates that ORAI1 and ORAI2 are the primary components of the neutrophil CRAC channel and identifies subpopulations of neutrophils where cell-membrane potential functions as a rheostat to modulate the SOCE response. These findings have implications for mechanisms that modulate neutrophil function during infection, acute and chronic inflammatory conditions, and cancer. [ABSTRACT FROM AUTHOR]

Stem cell systems are essential for the development and maintenance of polarized tissues. Intercellular signaling pathways control stem cell systems, where niche cells signal stem cells to maintain the stem cell fate/self-renewal and inhibit differentiation. In the C. elegans germline, GLP-1 Notch signaling specifies the stem cell fate, employing the sequence-specific DNA binding protein LAG-1 to implement the transcriptional response. We undertook a comprehensive genome-wide approach to identify transcriptional targets of GLP-1 signaling. We expected primary response target genes to be evident at the intersection of genes identified as directly bound by LAG-1, from ChIP-seq experiments, with genes identified as requiring GLP-1 signaling for RNA accumulation, from RNA-seq analysis. Furthermore, we performed a time-course transcriptomics analysis following auxin inducible degradation of LAG-1 to distinguish between genes whose RNA level was a primary or secondary response of GLP-1 signaling. Surprisingly, only lst-1 and sygl-1, the two known target genes of GLP-1 in the germline, fulfilled these criteria, indicating that these two genes are the primary response targets of GLP-1 Notch and may be the sole germline GLP-1 signaling protein-coding transcriptional targets for mediating the stem cell fate. In addition, three secondary response genes were identified based on their timing following loss of LAG-1, their lack of a LAG-1 ChIP-seq peak and that their glp-1 dependent mRNA accumulation could be explained by a requirement for lst-1 and sygl-1 activity. Moreover, our analysis also suggests that the function of the primary response genes lst-1 and sygl-1 can account for the glp-1 dependent peak protein accumulation of FBF-2, which promotes the stem cell fate and, in part, for the spatial restriction of elevated LAG-1 accumulation to the stem cell region. Author summary: Stem cell systems are central to tissue development, homeostasis and regeneration, where niche to stem cell signaling pathways promote the stem cell fate/self-renewal and inhibit differentiation. The evolutionarily conserved GLP-1 Notch signaling pathway in the C. elegans germline is an experimentally tractable system, allowing dissection of control of the stem cell fate and inhibition of meiotic development. However, as in many systems, the primary molecular targets of the signaling pathway in stem cells is incompletely known, as are secondary molecular targets, and this knowledge is essential for a deep understanding of stem cell systems. Here we focus on the identification of the primary transcriptional targets of the GLP-1 signaling pathway that promotes the stem cell fate, employing unbiased multilevel genomic approaches. We identify only lst-1 and sygl-1, two of a number of previously reported targets, as likely the sole primary mRNA transcriptional targets of GLP-1 signaling that promote the germline stem cell fate. We also identify secondary GLP-1 signaling RNA and protein targets, whose expression shows dependence on lst-1 and sygl-1, where the protein targets reinforce the importance of posttranscriptional regulation in control of the stem cell fate. [ABSTRACT FROM AUTHOR]

Apolipoprotein E (APOE) is a strong genetic risk factor for late-onset Alzheimer's disease (LOAD). APOE4 increases and APOE2 decreases risk relative to APOE3. In the P301S mouse model of tauopathy, ApoE4 increases tau pathology and neurodegeneration when compared with ApoE3 or the absence of ApoE. However, the role of ApoE isoforms and lipid metabolism in contributing to tau-mediated degeneration is unknown. We demonstrate that in P301S tau mice, ApoE4 strongly promotes glial lipid accumulation and perturbations in cholesterol metabolism and lysosomal function. Increasing lipid efflux in glia via an LXR agonist or Abca1 overexpression strongly attenuates tau pathology and neurodegeneration in P301S/ApoE4 mice. We also demonstrate reductions in reactive astrocytes and microglia, as well as changes in cholesterol biosynthesis and metabolism in glia of tauopathy mice in response to LXR activation. These data suggest that promoting efflux of glial lipids may serve as a therapeutic approach to ameliorate tau and ApoE4-linked neurodegeneration. [Display omitted] • Lipidomic profiling reveals changes in cholesterol metabolism in P301S/ApoE4 mice • LXR agonist GW3965 decreases tauopathy and neuroinflammation in P301S/E4 mice • LXR agonist upregulates Abca1 and reduces lipid accumulation in vivo and in vitro • Abca1 overexpression mitigates tauopathy and neuroinflammation in P301S/ApoE4 mice Using lipidomics coupled with immunostaining, Litvinchuk et al. demonstrate that ApoE promotes changes in cholesterol metabolism and lipid accumulation in glia of 9.5-month-old P301S mice. LXR agonist GW3965 diet and Abca1 overexpression markedly decrease tauopathy, neurodegeneration, synapse loss and behavioral deficits, neuroinflammation, and glial lipid accumulation in 9.5-month-old P301S/ApoE4 mice. [ABSTRACT FROM AUTHOR]

Anterior cruciate ligament (ACL) tears occur in isolation or in tandem with other intra‐articular injuries such as meniscus tears. The impact of injury pattern on the molecular biology of the injured ACL is unknown. Here, we tested the hypothesis that the biological response of the ACL to injury varies based on the presence or absence of concomitant meniscus tear. We performed RNA‐seq on 28 ACL tears remnants (12 isolated, 16 combined). In total, 16,654 transcripts were differentially expressed between isolated and combined injury groups at false discovery rate of 0.05. Due to the large number of differentially expressed transcripts, we undertook an Ensembl approach to discover features that acted as hub genes that did not necessarily have large fold changes or high statistical significance, but instead had high biological significance. Our data revealed a negatively correlated module containing 5,960 transcripts (down‐regulated in combined injury) and a positively correlated module containing 2,260 transcripts (up‐regulated in combined injury). TNS1, MEF2D, NOTCH3, SOGA1, and MLXIP were highly‐connected hub genes in the negatively correlated module and SCN2A, CSMD3, LRC44, USH2A, and LRP1B were critical hub genes in the positively correlated module. Transcripts in the negatively correlated module were associated with biological adhesion, actin‐filament organization, cell junction assembly, and cell matrix adhesion. The positively correlated module transcripts were enriched for neuron migration and exocytosis regulation. These findings indicate genes and pathways reflective of healing deficiency and gain of neurogenic signaling in combined ACL and meniscus tears, suggesting their diminished repair potential. The biological response of ACL to injury could have implications for healing potential of the ligament and the long term health of the knee. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2612–2621, 2018. Anterior cruciate ligament (ACL) tears occur in isolation or with other intra‐articular injuries such as meniscus tears. The impact of injury pattern on the molecular biology of ACL tears is unknown. RNA‐seq identifies genes and pathways demonstrating significant healing deficiency in combined ACL and meniscus tears compared to isolated ACL tears. The impact of injury pattern on the biological response of ACL to injury could have implications for healing potential of the ligament and long‐term knee health. [ABSTRACT FROM AUTHOR]

Obesity and diabetes modulate macrophage activation, often leading to prolonged inflammation and dysfunctional tissue repair. Increasing evidence suggests that the NLRP3 inflammasome plays an important role in obesity-associated inflammation. We have previously shown that activation of the lipotoxic inflammasome by excess fatty acids in macrophages occurs via a lysosome-dependent pathway. However, the mechanisms that link cellular lipid metabolism to altered inflammation remain poorly understood. PPARγ is a nuclear receptor transcription factor expressed by macrophages that is known to alter lipid handling, mitochondrial function, and inflammatory cytokine expression. To undercover novel links between metabolic signaling and lipotoxic inflammasome activation, we investigated mouse primary macrophages deficient in PPARγ. Contrary to our expectation, PPARγ knockout (KO) macrophages released significantly less IL-1β and IL-1α in response to lipotoxic stimulation. The suppression occurred at the transcriptional level and was apparent for multiple activators of the NLRP3 inflammasome. RNA sequencing revealed upregulation of IFN-β in activated PPARγKO macrophages, and this was confirmed at the protein level. A blocking Ab against the type 1 IFNR restored the release of IL-1β to wild type levels in PPARγKO cells, confirming the mechanistic link between these events. Conversely, PPARγ activation with rosiglitazone selectively suppressed IFN-β expression in activated macrophages. Loss of PPARγ also resulted in diminished expression of genes involved in sterol biosynthesis, a pathway known to influence IFN production. Together, these findings demonstrate a cross-talk pathway that influences the interplay between metabolism and inflammation in macrophages. [ABSTRACT FROM AUTHOR]

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by an abnormal expansion of polyglutamine repeats in the huntingtin protein (Htt). Transcriptional dysregulation is an early event in the course of HD progression and is thought to contribute to disease pathogenesis, but how mutant Htt causes transcriptional alterations and subsequent cell death in neurons is not well understood. RNA-Seq analysis revealed that expression of a mutant Htt fragment in primary cortical neurons leads to robust gene expression changes before neuronal death. Basic helix-loop-helix transcription factor Twist1, which is essential for embryogenesis and is normally expressed at low levels in mature neurons, was substantially up-regulated in mutant Htt-expressing neurons in culture and in the brains of HD mouse models. Knockdown of Twist1 by RNAi in mutant Htt-expressing primary cortical neurons reversed the altered expression of a subset of genes involved in neuronal function and, importantly, abrogated neurotoxicity. Using brain-derived neurotrophic factor (Bdnf), which is known to be involved in HD pathogenesis, as a model gene, we found that Twist1 knockdown could reverse mutant Htt-induced DNA hypermethylation at the Bdnf regulatory region and reactivate Bdnf expression. Together, these results suggest that Twist1 is an important upstream mediator of mutant Htt-induced neuronal death and may in part operate through epigenetic mechanisms. [ABSTRACT FROM AUTHOR]

Background: The arrival of RNA-seq as a high-throughput method competitive to the established microarray technologies has necessarily driven a need for comparative evaluation. To date, cross-platform comparisons of these technologies have been relatively few in number of platforms analyzed and were typically gene name annotation oriented. Here, we present a more extensive and yet precise assessment to elucidate differences and similarities in performance of numerous aspects including dynamic range, fidelity of raw signal and fold-change with sample titration, and concordance with qRT-PCR (TaqMan). To ensure that these results were not confounded by incompatible comparisons, we introduce the concept of probe mapping directed "transcript pattern". A transcript pattern identifies probe(set)s across platforms that target a common set of transcripts for a specific gene. Thus, three levels of data were examined: entire data sets, data derived from a subset of 15,442 RefSeq genes common across platforms, and data derived from the transcript pattern defined subset of 7,034 RefSeq genes. Results: In general, there were substantial core similarities between all 6 platforms evaluated; but, to varying degrees, the two RNA-seq protocols outperformed three of the four microarray platforms in most categories. Notably, a fourth microarray platform, Agilent with a modified protocol, was comparable, or marginally superior, to the RNA-seq protocols within these same assessments, especially in regards to fold-change evaluation. Furthermore, these 3 platforms (Agilent and two RNA-seq methods) demonstrated over 80 % fold-change concordance with the gold standard qRT-PCR (TaqMan). Conclusions: This study suggests that microarrays can perform on nearly equal footing with RNA-seq, in certain key features, specifically when the dynamic range is comparable. Furthermore, the concept of a transcript pattern has been introduced that may minimize potential confounding factors of multi-platform comparison and may be useful for similar evaluations. [ABSTRACT FROM AUTHOR]

Substantial regressions of metastatic lesions have been observed in up to 70% of patients with melanoma who received adoptively transferred autologous tumor-infiltrating lymphocytes (TILs) in phase 2 clinical trials. In addition, 40% of patients treated in a recent trial experienced complete regressions of all measurable lesions for at least 5 years following TIL treatment. To evaluate the potential association between the ability of TILs to mediate durable regressions and their ability to recognize potent antigens that presumably include mutated gene products, we developed a new screening approach involving mining whole-exome sequence data to identify mutated proteins expressed in patient tumors. We then synthesized and evaluated candidate mutated T cell epitopes that were identified using a major histocompatibility complex-binding algorithm for recognition by TILs. Using this approach, we identified mutated antigens expressed on autologous tumor cells that were recognized by three bulk TIL lines from three individuals with melanoma that were associated with objective tumor regressions following adoptive transfer. This simplified approach for identifying mutated antigens recognized by T cells avoids the need to generate and laboriously screen cDNA libraries from tumors and may represent a generally applicable method for identifying mutated antigens expressed in a variety of tumor types. [ABSTRACT FROM AUTHOR]

Correction to: I Scientific Reports i https://doi.org/10.1038/s41598-022-07236-x, published online 24 February 2022 The HTML version of the original version of this Article contained an error in the legend of Figure 4, where a portion of the text was incorrectly split and captured as an image credit. The original article can be found online at https://doi.org/10.1038/s41598-022-07236-x. [Extracted from the article]

The original article can be found online at https://doi.org/10.1038/s41598-022-07236-x. Correction to: I Scientific Reports i https://doi.org/10.1038/s41598-022-07236-x, published online 24 February 2022 The HTML version of the original version of this Article contained an error in the legend of Figure 4, where a portion of the text was incorrectly split and captured as an image credit. [Extracted from the article]

The phytohormone auxin plays crucial roles in nearly every aspect of plant growth and development. The auxin response factor (ARF) transcription factor family regulates auxin-responsive gene expression and exhibits nuclear localization in regions of high auxin responsiveness. Here we show that the ARF7 and ARF19 proteins accumulate in micron-sized assemblies within the cytoplasm of tissues with attenuated auxin responsiveness. We found that the intrinsically disordered middle region and the folded PB1 interaction domain of ARFs drive protein assembly formation. Mutation of a single lysine within the PB1 domain abrogates cytoplasmic assemblies, promotes ARF nuclear localization, and results in an altered transcriptome and morphological defects. Our data suggest a model in which ARF nucleo-cytoplasmic partitioning regulates auxin responsiveness, providing a mechanism for cellular competence for auxin signaling. • Condensation of transcription factors to attenuate hormone response • Mechanism for nucleo-cytoplasmic partitioning of transcription factors ARF transcription factors mediate the activity of the phytohormone auxin, regulating every aspect of plant development. Powers et al. determine that some ARFs undergo phase transition to form large-order cytoplasmic protein assemblies that limit auxin responsiveness in a developmentally relevant context, illustrating a strong link between condensate formation and biological function. [ABSTRACT FROM AUTHOR]